Methods for unlocking competence for natural transformation in lactic acid bacteria

Abstract

The present invention relates to a method for transforming a strain of a lactic acid bacterium, such as of the Lactococcus genus through competence for natural transformation. The present invention further relates to strains obtained or obtainable by this method. The present invention also relates to the use of specific strains of a lactic acid bacterium, such as of the genus Lactococcus having inherently low, reduced or disrupted activity of specific proteins for transforming these strains through natural transformation. The present invention further relates to methods for increasing the rate of natural transformation by increasing competence for natural transformation of a strain of a lactic acid bacterium, such as of the genus Lactococcus and for identifying a strain of a lactic acid bacterium, such as of the Lactococcus genus which is transformable through competence for natural transformation.

Description

[0001] METHODS FOR UNLOCKING COMPETENCE FOR NATURAL TRANSFORMATION IN LACTIC ACID BACTERIA

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a method for transforming a strain of a lactic acid bacterium, such as of the Lactococcus genus through competence for natural transformation. The present invention further relates to strains obtained or obtainable by this method. The present invention also relates to the use of specific strains of a lactic acid bacterium, such as of the genus Lactococcus having inherently low, reduced or disrupted activity of specific proteins for transforming these strains through natural transformation. The present invention further relates to methods for increasing the rate of natural transformation by competence of a strain of a lactic acid bacterium, such as of the genus Lactococcus and for identifying a strain of a lactic acid bacterium, such as of the Lactococcus genus which is spontaneously transformable through competence for natural transformation.

[0004] BACKGROUND OF THE INVENTION

[0005] The Lactococcus genus includes the Lactococcus lactis and Lactococcus cremoris species, that are amongst the most studied species of lactic acid bacteria for both basic and applied research. Initially, the food industry has used their ability to produce lactic acid for centuries to preserve a wide variety of fermented foods (e.g., cheese, buttermilk, and sour cream). In addition, species of the Lactococcus genus are now largely exploited as a safe production platform for recombinant proteins due to their easy production and downstream purification. Finally, these species are of high interest as vectors for therapeutics or vaccine antigens. Because of all these possible applications, the Lactococcus genus has become a powerful living tool of tremendous interest across the scientific community in just a few decades.

[0006] One of the commonalities shared by this large subset of applications is the ability to easily engineer its genome. Aside from commonly used techniques such as electroporation, conjugation, or transduction, the use of competence for natural DNA transformation is an appealing alternative. Natural transformation is a lateral gene transfer process in which the bacterium is able to capture, internalize, and recombine a naked DNA fragment present in the extracellular environment. This process is thus of major interest for a variety of reasons. First, natural transformation does not necessitate the involvement of a third party as required for conjugation or transduction (i.e., another cell or a bacteriophage, respectively). Second, the donor DNA could simply be a linear fragment obtained by polymerase chain reaction and added to the culture medium. Third, natural transformation provides a quick and accurate DNA engineering tool. Competence activation is typically divided into two distinct phases (i.e., early and late). During the early phase, a stimulus provokes the production of the central regulator of competence (ComX). The latter, in association with the RIMA polymerase, will then trigger the expression of the competence (com) regulon, which corresponds to the late phase. This regulon codes for all proteins responsible for DNA transformation, from DNA capture outside the cell to recombination into the genome.

[0007] No mechanism of early-phase activation has been identified in the Lactococcus genus so far. Accordingly, there remains a need for improving methods for naturally transforming strains of the genus Lactococcus using competence for DNA transformation, and in particular for Lactococcus lactis and Lactococcus cremoris species.

[0008] WO2018114983 relates to a method for transforming a strain of the Lactococcus genus through natural transformation, as well as to strains obtained by such method.

[0009] Wydau et al. in 2006 investigated the conservation, among lactococcal strains, of key genetic components of the natural competence process in streptococci, including comX, c / n-box, and dprA. They established that ComX controls the expression of the late competence genes in L. lactis, and constructed a comX overexpression system.

[0010] Ercan et al. (2015) showed that carbon starvation induces the transcription of comX and several late competence genes in Lactococcus lactis but failed to obtain spontaneous natural transformation.

[0011] David et al. (2017) demonstrated that competence for natural transformation can be induced in Lactococcus cremoris by overexpressing comX and by inactivating the Clp machinery but failed to obtain spontaneous natural transformation.

[0012] Mulder et al. (2017) demonstrated that natural transformation can be induced in Lactococcus lactis and in Lactococcus cremoris by overexpressing comX but failed to obtain spontaneous natural transformation.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1. Natural transformation by comX overexpression in multiple L. lactis strains. (A) List of genes required for natural transformation in L. lactis. Gene-associated function in DNA transformation is indicated on the left. Reg. denotes regulation. (B) Graph displaying the rates of natural transformation of 15 L. lactis strains harboring the complete set of late competence genes and containing the comX-overexpressing plasmid pGhPx / / r-com '. Transformation assays were performed in M17 supplemented with glucose 0.1% (w / v) and xylose 1% (w / v), with rpsL* as donor DNA (20 g ml-1), added at time zero. Cells were spread after ~24 hours of culture. Dots show the values for technical triplicates; mean values ± standard deviations.

[0015] Figure 2. Spontaneous natural transformation in multiple L. lactis strains. (A) Graph displaying the rates of spontaneous natural transformation of 18 L. lactis strains harboring the complete set of late competence genes in M17G medium. (B) Effect of comX or comEC deletion on transformability compared to the wild-type strain DGCC12653. Data show the rates of natural transformation observed in M17G medium after overnight culture with donor DNA. Dots show the values for technical triplicates, mean values ± standard deviations. (C) Timing of transformation events in strain DGCC12653 (bars, number of streptomycin-resistant CFUs) during cell growth (dotted line) in M17G medium. (D) Graph displaying the rates of natural transformation of f., lactis DGCC12653 strain in M17 medium supplemented either with glucose 0,5% (w / v) (M17G) or maltose 1% (w / v) (M17M). (E) Graph displaying the rates of natural transformation of f., lactis DGCC12653 strain in CDM containing different proportions of glucose (G) and maltose (M): glucose 0,21% (w / v), glucose 0,21% (w / v) + maltose 1,1% (w / v) (named CDM*), glucose 0,5% (w / v), glucose 0,5% (w / v) + maltose 1,1% (w / v), maltose 1,1% (w / v). (F) Graph displaying the rates of natural transformation of L. lactis DGCC12653 strain in CDM* and in CDM* without aspartate, glutamate and nitrogen bases (named CDM*-DEB). (G) Graph displaying the rates of natural transformation of L. lactis DGCC12653 strain in CDM supplemented with 0,21% glucose (w / v) and different secondary carbon sources at 1,1% (w / v) (maltose, xylose, galactose, cellobiose, arabinose, and trehalose). (H) Graph displaying the rates of spontaneous natural transformation of 8 L. lactis strains harboring the complete set of late competence genes in CDM*-DEB. All transformations assays were performed in CDM*-DEB with rpsL* as donor DNA (20 g ml-1), added at time zero. Cells were spread after ~24 hours of culture. Dots show the values for technical triplicates; mean values ± standard deviations.

[0016] Figure 3. Involvement of CcpA in competence repression. (A) Graph depicting the results of luciferase assays which demonstrate the impact of the sugar transition on PCOmx activation and the timing of transformation in CDM*-DEB. Luminescence kinetics (solid line - RLU / ODeoo - left axis) is displayed with cell growth (dotted line - OD6oo - upper-right axis) and timing of transformation (grey bars - downer-right axis). (B) Graph displaying the effect of ccpA deletion on PCOmx activation in CDM*-DEB. Growth (dotted lines; OD6oo) and kinetics of PCOmx specific luciferase activity (solid lines; RLU / ODeoo) monitored over time for DGCC12653 WT (dark grey) and CcpA" strains (light grey). Solid and dotted dark lines are representative of the mean of technical and biological triplicates (WT and CcpA-, respectively), and light lines are representative of standard deviation. (C) EMSA performed with a gradient of purified CcpA (rCcpA) onCy3PCOmx (left) and the CDS of dnaE from Streptococcus salivarius as negative control (right). Lanes without rCcpA are indicated by a minus sign. C and P indicate the rCcpA-DNA complex and the unbound probe, respectively. Gels displayed are representative of technical triplicates.

[0017] Figure 4. Involvement of CodY in competence repression. (A) Graph displaying the effect of codY deletion on PCOmx activation in CDM*-DEB. Growth (dotted lines; OD6oo) and kinetics of PCOmx specific luciferase activity (solid lines; RLU / ODeoo) monitored over time for DGCC12653 WT (dark grey) and CodY- strains (light grey). Solid and dotted dark lines are representative of the mean of technical and biological triplicates (WT and CodY-, respectively), and light lines are representative of standard deviation. (B) Effect of codY deletion on transformability of strain DGCC12653. Data show the rates of natural transformation observed for WT (dark grey) and CodY- strains (light grey) in CDM*-DEB after overnight culture with donor DNA. Dots show the values for biological triplicates (CodY ) or technical triplicates (WT), mean values ± standard deviations. (C) Table showing the impact of CodY on comX and late competence genes expression observed by RIMA sequencing. Only the first gene of late operons comG comG A, comGB, comGC, comGD, comGE, comGF and comGG), comE comEA and comEC) and comF comFA and comFC) are listed. The indicated values correspond to the RNA abundance ratio between DGCC12653 CodY- strain and wild type. (D) EMSA with purified CodY (rCodY) shows binding onCy3PCOmx. Effect of isoleucine on rCodY binding to PCOmx. EMSAs performed with a gradient of rCodY onCy3PCOmx without (left) or with 5 mM isoleucine (right). Lanes without rCodY are indicated by a minus sign. C and P indicate the rCodY-DNA complex and the unbound probe, respectively.

[0018] Figure 5. Involvement of CovRS in competence repression. (A) Graph displaying the effect of covRS deletion on PCOmx activation in CDM*. Growth (dotted lines; OD6oo) and kinetics of PCOmx specific luciferase activity (solid lines; RLU / ODeoo) monitored over time for DGCC12653 WT (dark grey) and CovRS- strains (light grey). Solid and dotted dark lines are representative of the mean of technical and biological triplicates (WT and CovRS-, respectively), and light lines are representative of standard deviation. (B) Effect of covRS deletion on transformability of strain DGCC12653. Data show the rates of natural transformation observed for WT (dark grey) and CovRS- strains (light grey) in CDM* after overnight culture with donor DNA. Dots show the values for biological triplicates (CovRS ) or technical triplicates (WT), mean values ± standard deviations. (C) Table showing the impact of CovRS on comX and late competence genes expression observed by RNA sequencing. Only the first gene of late operons comG comG A, comGB, comGC, comGD, comGE, comGF and comGG), comE comEA and comEC) and comF (comFA and comFC) are listed. The indicated values correspond to the RNA abundance ratio between DGCC12653 CovRS- strain and wild type. (D) EMSA performed with a gradient of purified CovR (rCovR) onCy3PCOmx (left) and the CDS of dnaE from Streptococcus salivarius as negative control (right). Lanes without rCovR are indicated by a minus sign. C and P indicate the rCovR-DNA complexes and the unbound probe, respectively. Gels displayed are representative of technical triplicates.

[0019] Figure 6. Impact of initial pH on competence repression. Data show the rates of natural transformation observed for WT (dark grey) and CovRS- strains (light grey) after overnight culture with donor DNA in CDM*-DEB buffered at different initial pH values. Dots show the values for biological triplicates (CovRS ) or technical triplicates (WT), mean values ± standard deviations. Figure 7. Involvement of MecA in competence repression. (A) Alignment of MecA protein sequences from L. lactis DGCC12653 (SEQ ID NO: 2) and DGCC12671 (SEQ ID NO: 1) strains, showing the unique substitution L125R in DGCC12653. (B) Schematic view of the swap of mecA genes between strains DGCC12653 (light grey) and DGCC12671 (dark grey). Genes encoding MecA-R125 (variant) and MecA-L125 (wild type) are colored in white and shaded grey, respectively. Straight arrows and straight stopped lines illustrate activated or repressed pathways, respectively. Curved arrows display protein production. (C) Effects of MecA-L125R and -R125L substitutions on transformability. Data show the rates of natural transformation observed for DGCC12653 WT (MecA-R125), DGCC12653* (MecA-L125), DGCC12671 WT (MecA-L125), DGCC12671* (MecA-R125), and mecA- deleted (MecA ) strains after overnight culture with donor DNA in CDM*-DEB. Dots show the values for biological triplicates (mutant strains) or technical triplicates (WT), mean values ± standard deviations. (D) Effects of mecA deletion (MecA ) on spontaneous transformability in 9 L. lactis strains. Data show the rates of natural transformation in four different media composed of CDM- DEB with glucose (Glu) 0.21% (w / v) and a second plant sugar at 1.1% (w / v) (maltose, black; galactose, dotted grey; xylose, squared grey; cellobiose, light grey). (E) Effects of mecA deletion (MecA ) on spontaneous transformability of L. cremoris KW2 strain. Data show the rates of natural transformation in four different media composed of CDM-DEB with glucose (Glu) 0.21% (w / v) and a second plant sugar at 1.1% (w / v) (maltose, black; galactose, dotted grey; xylose, squared grey; cellobiose, light grey). All transformations assays were performed with rpsL* as donor DNA (20 g ml-1), added at time zero. Cells were spread after ~24 hours of culture.

[0020] Figure 8. Comparison of competence regulation at the global level between Lactococcus, Streptococcus, and Bacillus. In Streptococcus (left panel), the proximal regulation of comX is performed by a quorum sensing (QS) system (ComRS or ComCDE, light grey). Additionally, distal regulation systems (e.g., Two Component Systems (TCSs), mid grey) responding to environmental cues are modulating the expression of the QS response regulator / sensor instead of comX as found in L. lactis. Finally, the post-translational regulation system (dark grey) plays a minor role in ComX degradation. In Bacillus (right panel), the proximal regulation of ComK, the functional equivalent of ComX, is achieved by a range of transcriptional regulators (e.g., CodY, Degll, Rok, and AbrB). However, a distal regulation involving a QS system (ComXAP) triggers the production of ComS to inhibit ComK degradation in B. subtUis. In Lactococcus (middle panel), no QS system has been identified so far and proximal regulation of comX seems to be exclusively influenced by environmental conditions. Moreover, post-translational regulation was shown to be dominant in competence repression.

[0021] Figure 9. Natural transformation by comX overexpression in repaired 1AA59 L. lactis strains. (A) Graph comparing the rates of natural transformation by comX overexpression of 1AA59 L. lactis strains harboring a repaired version of comFA-comFC genes either on a plasmid (pGhPx / / r-com '- comFAC), or in the chromosome (allelic exchange of comFA-comFC genes), corresponding to trans- and cis-repair, respectively. Transformation assays were performed in M17 supplemented with glucose 0.25% (w / v) and xylose 1% (w / v), with rpsL * as donor DNA (20 pg ml-1), added at time zero. Cells were spread after 48 hours of culture. Dots show the values for technical triplicates; mean values ± standard deviations. (B) Graph displaying the rates of natural transformation of the c / s-repaired 1AA59 L. lactis strain harboring the pGhPx / / T-com 'plasmid in M17 supplemented with xylose 1% (w / v) and glucose concentrations ranging from 0.10% to 0.35% (w / v). Transformation assays were performed with rpsL * as donor DNA (20 g ml-1), added at time zero. Cells were spread after 48 hours of culture. Dots show the values for technical triplicates; mean values ± standard deviations.

[0022] Figure 10. Natural transformation in repaired IL1403 L. lactis strains. (A) Graph displaying the effect of cis- and trans-repair of dprA on rates of natural transformation upon comX-overexpres- sion in IL1403 strain. Data show the rates of natural transformation observed in IL1403 dprA* pGhPxyrrComX, IL1403 dprA* pGhPxyn~comX-dprA, IL1403 dprA pGhPx / / r-com ' and IL1403 dprA pGhPXyn~comX-dprA. Transformation assays were performed in M17 supplemented with glucose 0.1% (w / v) and xylose 1% (w / v), with rpsLu* (dark grey) and 07535: -.spec (light grey) as donor DNA (10 pg ml-1), added at time zero. Cells were spread after 24 hours of culture. Dots show the values for technical triplicates; mean values ± standard deviations. (B) Graph displaying the effects of dprA and comX c / s-repair, along or not with mecA alteration, on spontaneous natural transformation in L. lactis IL1403. Data show the rates of spontaneous natural transformation of IL1403 dprA* comX* mecA(L125), IL1403 dprA comX mecA(L125), IL1403 dprA comX mecA(R125) and IL1403 dprA comX mecA' "DmecA" is "mecA"). Transformation assays were performed in CDM* (light grey) and CDM*-DEB (dark grey) with rpsL * as donor DNA (20 pg ml-x), added at time zero. Cells were spread after 24 hours of culture. Dots show the values for technical triplicates; mean values ± standard deviations. (C) Graph displaying the rates of natural transformation upon comX-overexpression by the ComRS overexpression system (pGh- comRS-comX)' in the fully- re paired IL1403 strain. Transformation assays were performed in M17G, CDM*, CDM*-DEB and M17* (*meaning supplemented with glucose 0.21% [w / v] and maltose 1.1% [w / v]), containing or not XIP 10 pM (dark grey and light grey, respectively), supplemented with rpsL * as donor DNA (20 pg ml-1) added at time zero. Cells were spread after 24 hours of culture. Dots show the values for technical triplicates; mean values ± standard deviations.

[0023] Figure 11. Natural transformation in repaired MG1363 L. cremoris strains. Graph displaying the rates of natural transformation upon comX-overexpression by either the ComRS or the mannitol- induced systems in not-, partially- and fully-repaired MG1363 strains. Data show the rates of natural transformation of MG1363 coiA* comEC* (white), MG1363 coiA* comEC* pGhcomRS- comX (thin vertical lines), MG1363 coiA* comEC* pGhPmtiA-comX (thick vertical lines), MG1363 coiA comEC* (black), MG1363 coiA comEC* pG \comRS-comX (dots), MG1363 coiA comEC* pGhPmtiA-comX (slash lines), MG1363 coiA comEC (squares), MG1363 coiA comEC pGhcomRS- comX (dark grey) and MG1363 coiA comEC pGhP-comX (light grey). Transformation assays were performed in M17G containing XIP 10 M (left part, first 9 values) and in M17 supplemented with glucose 0,1% (w / v) and mannitol 1% (w / v) (right part, last 9 values). Both transformation media were supplemented with rpsLMG* as donor DNA (20 g ml-1) added at time zero. Cells were spread after 24 hours of culture. Dots show the values for technical triplicates; mean values ± standard deviations.

[0024] SUMMARY OF THE INVENTION

[0025] It is an object of embodiments of the invention to provide methods and tools for increasing competence for natural transformation.

[0026] The present invention relates in a broad aspect to the use of strains of a lactic acid bacterium, such as of the genus Lactococcus with inherently low, reduced or disrupted activity of genes and / or proteins involved in competence repression.

[0027] Accordingly, in a first aspect the present invention relates to a method for transforming a strain of a lactic acid bacterium, such as of the genus Lactococcus, with an exogenous DNA polynucleotide comprising the steps of:

[0028] (a) providing a strain of the lactic acid bacterium, such as of the genus Lactococcus, which strain is transformable through natural transformation;

[0029] (b) contacting the strain of step (a) with an exogenous DNA polynucleotide in a growth medium and incubating the resulting mixture for integration of the exogenous DNA polynucleotide or parts of it into the genome of the strain; and

[0030] (c) selecting a derivative of said strain which has integrated the exogenous DNA polynucleotide or parts of it into its genome; which strain provided under step a) has any one or more of i) an inherently low, reduced or disrupted activity of the adaptor protein MecA relative to a control strain having a normal activity of the MecA protein; and / or ii) an inherently low, reduced, or disrupted activity of the global transcriptional regulator CodY and / or the two-component regulatory system CovRS relative to a control strain having a normal activity of CodY and / or CovRS; and / or iii) an inherently low, reduced, or disrupted activity of any of Clp proteins CIpP, and / or CIpC.

[0031] In a second aspect the present invention relates to a method for transforming a strain of a lactic acid bacterium, such as of the genus Lactococcus, with an exogenous DNA polynucleotide comprising the steps of:

[0032] (a) providing a strain of the lactic acid bacterium, such as of the genus Lactococcus, which strain is transformable through natural transformation;

[0033] (b) increasing the competence of the strain for natural transformation;

[0034] (c) contacting the strain of step (b) with an exogenous DNA polynucleotide in a growth medium and incubating the resulting mixture for integration of the exogenous DNA polynucleotide or part of it into the genome of the strain; and

[0035] (d) selecting a derivative of said strain which has integrated the exogenous DNA polynucleotide or part of it into its genome; wherein this increase in the ability of the strain to perform natural transformation under step (b) is induced by either of i) growing the strain in a growth medium to induce a diauxic shift in the growth of the strain; and / or ii) growing the strain in a growth medium without any one or more of glutamate, aspartate, xanthine, adenine, thymine, uracil, cytosine and guanine; and / or iii) transforming through natural transformation under step b) and c) at a pH lower than about 8.5, such as in the pH range of 5.0-8.5.

[0036] It is to be understood that if the increase in the ability of the strain to perform natural transformation under step (b) is induced by any of the means defined by i) or ii), then the pH may be anywhere in the range of 2-13, such as in the range of 5.0-8.5.

[0037] In a third aspect the present invention relates to the use of a strain of a lactic acid bacterium, such as of the genus Lactococcus having either of i) an inherently low, reduced or disrupted activity of the adaptor protein MecA relative to a control strain having a normal activity of the MecA protein; and / or ii) an inherently low, reduced, or disrupted activity of the global transcriptional regulator CodY and / or the two-component regulatory system CovRS relative to a control strain having a normal activity of CodY and / or CovRS; and / or iii) an inherently low, reduced, or disrupted activity of any of Clp proteins CIpP, and / or CIpC; for transforming the strain through natural transformation.

[0038] In a further aspect the present invention relates to a method for increasing the rate of natural transformation by competence of a strain of a lactic acid bacterium, such as of the genus Lactococcus, comprising the steps of

[0039] (a) providing a strain of the lactic acid bacterium, such as of the genus Lactococcus, which strain is transformable through natural transformation;

[0040] (b) performing any one or more of i) reducing or disrupting the activity of the adaptor protein MecA relative to a control strain having a normal activity of the MecA protein; and / or ii) reducing or disrupting the activity of the global transcriptional regulator CodY and / or the two-component regulatory system CovRS relative to a control strain having a normal activity of CodY and / or CovRS; and / or iii) reducing or disrupting the activity of any of Clp proteins CIpP, and / or CIpC; which strain has an increased rate of natural transformation by competence relative to a control strain not having reduced or disrupted activity under any of i) to iii).

[0041] In a further aspect the present invention relates to a method for identifying a strain of a lactic acid bacterium, such as of the genus Lactococcus which is transformable through natural transformation comprising the steps of:

[0042] (a) providing a strain of a lactic acid bacterium, such as of the genus Lactococcus;

[0043] (b) measuring the activity of any one or more of i) the adaptor protein MecA relative to a control strain having a normal activity of the MecA protein; and / or ii) the global transcriptional regulator CodY and / or the two-component regulatory system CovRS relative to a control strain having a normal activity of CodY and / or CovRS; and / or iii) the activity of any of Clp proteins CIpP, and / or CIpC;

[0044] (c) select a strain with a reduced or disrupted activity of any one of these proteins defined in (b) i)-iii);

[0045] (d) contacting the strain obtained in step (c) with an exogenous DNA polynucleotide encoding a marker gene in a growth medium and incubating the resulting mixture for integration of the exogenous DNA polynucleotide into the genome of the strain; and

[0046] (e) determining the rate of integration events; wherein a rate of at least 1 x IO-8transformants per g of DNA is indicative of a strain which is transformable through natural transformation.

[0047] DETAILED DESCRIPTION OF THE INVENTION

[0048] The present inventors have found spontaneous transformation by competence in multiple L. lactis strains of the Lactococcus genus after optimization of DNA transformation conditions. Notably, the present inventors identified three global nutritional / stress regulators that repress comX transcription directly and furthermore discovered a L. lactis strain capable to transform at high level due to a single substitution in the adaptor protein MecA of the Clp machinery. Together, these findings show that competence development in L. lactis is negatively controlled by (i) global regulators sensing nutritional and environmental conditions to modulate ComX abundance and (ii) the Clp machinery degrading ComX to limit its availability, both levels of regulation ensuring tight control of competence activation in this species.

[0049] Lactic acid bacteria and Lactococcus genus

[0050] The present invention relates to methods for transforming strains of a lactic acid bacterium, such as of the genus Lactococcus, in particular Lactococcus lactis.

[0051] The Lactococcus genus comprises but is not limited to the following species: Lactococcus lactis, Lactococcus cremoris, Lactococcus laudensis, Lactococcus carnosus, Lactococcus formo- sensis, Lactococcus fujiensis, Lactococcus hircilactis, Lactococcus kimchii, Lactococcus paracarno- sus, Lactococcus plantarum, Lactococcus raffinolactis, and Lactococcus taiwanensis. Any strain of one of these species may be used in the current invention, provided that this strain is transformable through competence for natural transformation as defined herein.

[0052] In a particular embodiment, the strain of the Lactococcus genus used according to the present invention is a strain of the Lactococcus lactis species.

[0053] Lactococcus lactis and Lactococcus cremoris

[0054] In some specific embodiments, the strain of the Lactococcus genus used according to the present invention is a strain of the Lactococcus lactis species, or of the Lactococcus cremoris species. Each of these two species comprises two subspecies (Li et al., 2019). Thus, when the strain of the Lactococcus genus is a strain of the Lactococcus lactis species, the strain may be selected from the group consisting of Lactococcus lactis subsp. hordniae and Lactococcus lactis subsp. lactis. Similarly, when the strain of the Lactococcus genus is a strain of the Lactococcus cremoris species, the strain may be selected from the group consisting of Lactococcus cremoris subsp. cremoris and Lactococcus cremoris subsp. tructae. As used herein a strain of the Lactococcus lactis species is understood to be a genetic variant of any L. lactis species or subspecies. Similarly, a strain of the Lactococcus cremoris species is understood to be a genetic variant of any L. cremoris species or subspecies. The different Lactococcus subspecies disclosed herein, and in particular the lactis and the cremoris subspecies, may be defined herein based on DNA sequences coding for 16S ribosomal RIMA, or based on other genes of taxonomic relevance.

[0055] DNA acquisition

[0056] Bacteria may naturally acquire exogenous DNA via one of three possible mechanisms: competence (natural transformation), conjugation, or transduction.

[0057] As used herein the term "competence for natural transformation" (sometimes also referred to as "natural competence" or just "competence") refers to a (transient) physiological state of a bacterium that allows natural DNA transformation, including extracellular DNA capture, internalization and integration into the genome of the recipient bacterium. As used herein "transformable through natural transformation" refers to a bacterium displaying competence for natural transformation.

[0058] As used herein the term "natural transformation" refers to the active uptake of exogenous genetic material (e.g. a DNA polynucleotide) by a proteinaceous machinery (i.e. transfor- masome) from the external medium into a bacterium. Since transformation requires that genetic material cross the bacterial cell wall and membrane and the uptake of exogenous genetic material is energetically costly, the process is tightly regulated. Accordingly, bacterial cells may only be transformed under certain conditions. Bacterial cells which are in a transformable state are said to be competent. Transformation may be artificially induced in the laboratory, e.g. by electroporation or exposure to divalent cations (e.g. CaCI2) and heat shock. Alternatively, some species of bacteria express a proteinaceous machinery that provides natural transformation by competence; this system of natural transformation has been widely studied in streptococci.

[0059] Negative regulators of ComX availability

[0060] The present invention relates to global nutritional / stress regulators repressing comX transcription directly, modulating the ComX abundance and / or to proteins involved in degrading ComX to limit its availability.

[0061] These regulators include the adaptor protein MecA, the global transcriptional regulator CodY, the two-component regulatory system CovRS, and the Clp proteins CIpP and CIpC. Furthermore, modulating the production or availability of CcpA encoded by the ccpA gene may also be used to modulate competence for natural transformation.

[0062] As used herein "MecA" refers to the adaptor protein MecA encoded by the mecA gene, and to a homologue thereof, such as a protein with a sequence according to SEQ ID NO:1 or SEQ ID NO:2.

[0063] As used herein "CodY" refers to the transcriptional regulator protein CodY encoded by the codY gene.

[0064] As used herein "CovRS" refers to the two-component regulatory system comprising proteins CovR and CovS, which proteins are encoded by the covR and covS genes, respectively.

[0065] As used herein "Clp proteins CIpP and CIpC" refers to the two proteins CIpP and CIpC encoded by each of the c / pP and the dpC genes.

[0066] As used herein "CcpA" refers to the CcpA protein encoded by the ccpA gene.

[0067] Reference to the protein names MecA (SEQ ID NO: 1 and SEQ ID NO:2), CodY (SEQ ID NO:4), CovR (SEQ ID NO:6), CovS (SEQ ID NO:5), CcpA (SEQ ID NO:3), CIpP (SEQ ID NO:8), CIpC (SEQ ID NO:7), and ComX and their corresponding gene names mecA, codY, covR, covS, ccpA, c / pP, c / pC, and comX are not intended to be limiting, but are intended to encompass respective homologs (i.e., which may be endogenous to a related microbial organism) and polymorphic variants. Homologs and variants can be identified based on sequence identity and / or similar biological (e.g., enzymatic) activity. In certain embodiments, the invention includes a polynucleotide or polypeptide sequence with at least 50%, 60%, 70%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the named gene mecA, codY, covR, covS, ccpA, c / pP, c / pC, and comX or its gene product. "Identity" or "percent identity" in the context of two or more polynucleotide or polypeptide sequences, refers to two or more sequences or sub-sequences that are the same or have a specified percentage of nucleotides or amino acid residues, respectively, that are the same. Percent identity may be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which may also contain gaps to optimize the alignment) for alignment of the two sequences. For example, the sequence can have a percent identity of at least 50%, 60%, 70%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% over a specified region to a reference sequence when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection.

[0068] Alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, 1981, Adv. AppL Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et aL, eds., Current Protocols, John Wiley & Sons, Inc. (1995 Supplement) (Au- subel)).

[0069] The term "variant" is used to mean a naturally occurring polypeptide or nucleotide sequences which differs from a wild-type sequence.

[0070] ComX protein

[0071] As detailed above, the present invention relates to genes encoding global nutri- tional / stress regulators repressing comX transcription directly and proteins involved in degrading ComX to limit its availability. These genes negatively control and modulate ComX abundance. The provision of strains with either an inherently low, reduced or disrupted activity of these negative competence repressors may lead to an increased level or availability of ComX protein further leading to increased natural transformation capacities by competence.

[0072] In some embodiments of the present invention the levels of available and functional endogenous ComX protein in the strain used is directly modulated, either by inducing an increase in the level of endogenous ComX protein, such as by either inducing an increase in the expression of an endogenous comX gene, or by inducing a reduction in ComX protein degradation. In some specific embodiments the strain used according to the present invention has been transformed with a plasmid expressing an exogenous comX gene in order to increase available ComX protein in the strain.

[0073] ComX protein is an alternative sigma factor, also known as ox, which acts as master regulator for the expression of late com genes and is responsible for transcriptional reprogramming of cells including the induction of genes strictly required for DNA transformation (Peterson et al. 2004).

[0074] ComX may bind to a specific target sequence (or box) termed the Com-box (or Cin-box). Com-boxes are located in the vicinity of the promoters of late competence (com) genes / operons responsible for DNA uptake (e.g., comG, comF, and comE operons), DNA protection (e.g. ssb) and DNA recombination (e.g. recA, dprA, coiA), and positively control their expression (Campbell et a!., 1998; Luo and Morrison, 2003).

[0075] The production of the ComX protein in a strain of interest may be increased relatively to an appropriate control strain, i.e., a strain of a lactic acid bacterium, such as of the genus Lactococcus strain in which the production of the ComX protein has not been modulated. ComX protein may be increased if already present or produced if not following modulation as compared to an appropriate control strain, i.e., the lactic acid bacterium, such as the Lactococcus strain in which the ComX protein is not produced. Such modulation of a strain of a lactic acid bacterium may be any genetic modulation of e.g. the endogenous genes of the strain, or it may be a change of various growth conditions of the strain.

[0076] In some embodiments, the production of the ComX protein is constitutive or inducible.

[0077] The production of ComX protein may be monitored using any method known in the art, for example, by western blotting using an antibody specific for the ComX protein. Alternatively, comX gene mRNA transcript levels may be measured by qPCR.

[0078] Alternatively, the ComX protein abundance may be monitored using a reporter construct polynucleotide. The reporter construct polynucleotide may comprise genes encoding one or more reporter proteins, preferably the genes encoding the reporter proteins are operably linked to the comX promoter or a promoter comprising a Com-box sequence. The reporter proteins may be LuxAB, Luc or GFP. Accordingly, ComX production (and activity) may be detected and measured using a luciferase assay (Fontaine et al., 2010).

[0079] In some embodiments of the method of the present invention, the step of modulating the production of a ComX protein is performed by expressing a comX gene in the strain or increasing the expression of a comX gene in the strain. In a particular embodiment, the step of modulating the production of a ComX protein is performed by expressing a comX gene in the strain in some growth conditions, whereas the strain does not express the ComX protein outside of these growth conditions. In a particular embodiment, the step of modulating the production of a ComX protein is performed by increasing the expression of a comX gene in the strain in some growth conditions.

[0080] The comX gene may be an exogenous comX gene. As used herein an "exogenous comX gene" is understood to be a comX gene which is brought into the cytoplasm of a strain of a lactic acid bacterium, such as the genus Lactococcus in order to be expressed. The exogenous comX gene may have the same sequence as the comX gene found in the genome of the lactic acid bacterium, such as the Lactococcus strain or may have a different sequence (i.e., a distinct allele) from the comX gene found in the genome of the lactic acid bacterium, such as the Lactococcus strain. When different, the comX gene may be derived from a strain of a different species, a different subspecies or a different strain of a lactic acid bacterium, such as the Lactococcus.

[0081] The exogenous comX gene may be integrated within the genome of the lactic acid bacterium, such as the Lactococcus strain.

[0082] Alternatively, the exogenous comX gene may be located onto a vector. The vector may be selected from a plasmid, a viral vector (e.g. a phage), a cosmid, or a bacterial artificial chromosome.

[0083] The plasmid used may be transferred into the lactic acid bacterium, such as the Lactococcus strain by conjugation, transformation or transduction. This plasmid may be auto-replicative in the transformed lactic acid bacterium, such as the Lactococcus strain, or not.

[0084] The exogenous comX gene may be operably linked to transcription regulator(s). The exogenous comX gene may be located on a linear or circular polynucleotide.

[0085] Alternatively, in some embodiments of the method of the present invention, the comX gene is the endogenous comX gene of the lactic acid bacterium, such as the Lactococcus strain. As used herein "the endogenous comX gene of the strain" is understood to be a comX gene that is naturally present in the genome of the strain.

[0086] In some embodiments, the comX gene is a Lactococcus comX gene. In an embodiment, the comX gene is a Lactococcus lactis comX gene. In a particular embodiment, the comX gene is a Lactococcus lactis subsp. lactis comX gene. In a particular embodiment, the comX gene is a Lactococcus cremoris subsp. cremoris comX gene.

[0087] The comX gene used in some embodiments according to the present invention may comprise or consist of a nucleotide sequence as specifically disclosed in WO2018114983, which disclosure is hereby incorporated by reference in its entirety.

[0088] As used herein a comX gene is understood to be a gene that encodes a functional ComX protein in the strain where it is expressed. By "functional ComX protein" it is meant a protein which induces or is able to induce the expression of genes regulated by the Com-box, and at least one of the genes selected from comEA, comFA, comGA, dprA, coiA, ssb, radA, radC, recA, and recX.

[0089] As used herein "an inherently low, reduced or disrupted activity" in relation to any one of the adaptor protein MecA, the global transcriptional regulator CodY, the two-component regulatory system CovRS, and Clp proteins CIpP and CIpC, refers to any release of competence repression by any of these proteins as measured by ComX activity, possibly achieved by either affecting gene activity, such as by gene disruption / inactivation, mutations in expression signals, gene mutations affecting protein functionality, or by e.g. specific modifications at the protein level affecting folding and thereby interaction with DNA (e.g. for CodY, CovR), binding co-effectors (e.g. CodY), phosphorylation level (e.g. CovR), phosphatase activity of e.g. CovS, or interaction with ComX or CIpC of e.g. MecA. This competence repression as measured by ComX activity may be an effect directly on comX gene expression, or it may be a post-transcriptional regulation mechanism to control ComX abundance.

[0090] As used herein "integration of the exogenous DNA polynucleotide or parts of it into the genome" refers to the incorporation of a DNA polynucleotide or parts of it from an outside source, i.e. a donor DNA being part of the genome of a lactic acid bacterium, such as a DNA polynucleotide or parts of it not already present in the genome, such as a DNA polynucleotide or parts of it foreign for the recipient lactic acid bacterial strain to be transformed or at least not present in this number of DNA polynucleotide sequences in the recipient lactic acid bacterium. This exogenous DNA polynucleotide or parts of may be a complete gene or it may be a part of a gene, or it may be any part of a DNA polynucleotide that provides some functional effect, such as regulating or disrupting DNA polynucleotide sequences. The donor DNA polynucleotide may be integrated into the genome of the recipient lactic acid bacterial strain in a continuous sequence, or it may be integrated as a mosaic of the donor DNA polynucleotide.

[0091] As used herein the term "full set of endogenous late competence genes" refers to the competence of lactococcal strains with a complete and efficient set of at least 16 late competence genes, in addition to the comX gene, providing a functional natural transformation machinery. The 16 genes include comGA, comGB, comGC, comGD, comGE, comGF, comGG, comC, comFA, comFC, comEA, comEC, ssb, coiA, dprA, and recA (as shown in fig. 1) (Peterson et al., 2004). It is to be understood that some specific strains of a lactic acid bacterium may harbor one or more of these genes in non-functional inefficient genes. Such non-efficient genes may be repaired and used a a suitable strain for the methods of the invention. Accordingly, in some embodiments, any of these genes are repaired genes. The method for transforming a strain of a lactic acid bacterium, such as of the genus Lactococcus, may accordingly comprise a step of repairing any one or more of the 16 late competence genes, such as any one or more of comGA, comGB, comGC, comGD, comGE, comGF, comGG, comC, comFA, comFC, comEA, comEC, ssb, coiA, dprA, and / or recA.

[0092] As used herein "increased levels of available and functional endogenous ComX protein in the strain" refers to levels that are increased over an appropriate control strain, i.e., the Lactococcus strain in which the production or availability of the ComX protein has not been modulated according to the embodiments of this invention. Increased levels of available and functional endogenous ComX protein in the strain may be monitored using any method known in the art. Methods examples have been given here above: western blotting using an antibody specific for the ComX protein, and / or measurement of comX gene mRNA transcript levels by qPCR, and / or using a reporter construct polynucleotide comprising genes encoding one or more reporter proteins operably linked to the comX promoter or a promoter comprising a Com-box sequence. In some embodiments, the increased levels of available and functional endogenous ComX protein referes to levels that are compatible with the obtention of a rate of at least IO-8transformants per pg of DNA. Numbered embodiments of the invention:

[0093] 1. A method for transforming a strain of a lactic acid bacterium, such as of the genus Lactococcus, with an exogenous DNA polynucleotide comprising the steps of:

[0094] (a) providing a strain of the lactic acid bacterium, such as of the genus Lactococcus, which strain is transformable through natural transformation;

[0095] (b) contacting the strain of step (a) with an exogenous DNA polynucleotide in a growth medium and incubating the resulting mixture for integration of the exogenous DNA polynucleotide or parts of it into the genome of the strain; and

[0096] (c) selecting a derivative of said strain which has integrated the exogenous DNA polynucleotide or parts of it into its genome; which strain provided under step a) has any one or more of i) an inherently low, reduced or disrupted activity of the adaptor protein MecA relative to a control strain having a normal activity of the MecA protein; and / or ii) an inherently low, reduced, or disrupted activity of the global transcriptional regulator CodY and / or the two-component regulatory system CovRS relative to a control strain having a normal activity of CodY and / or CovRS; and / or iii) an inherently low, reduced, or disrupted activity of any of Clp proteins CIpP, and / or CIpC.

[0097] 2. The method according to embodiment 1, which strain contains a MecA protein with an inherently low, reduced, or disrupted activity relative to a control strain due to a modification or disruption of the activity of the mecA gene encoding adaptor protein MecA in the strain, such as having an arginine in the protein sequence at a position corresponding to position 125 of SEQ ID NO:2 (R125), such as by having a single amino acid substitution from a leucine to an arginine in the protein sequence at a position corresponding to position 125 of SEQ ID NO:1 (L125R), such as by swapping mecA genes or providing an extra copy of the mecA gene coding for an arginine in the protein sequence at a position corresponding to position 125 of SEQ ID NO:2 (R125), by deletion of the mecA gene, or by disruption of the mecA gene.

[0098] 3. The method according to embodiments 1 or 2, which strain has an inherently low, reduced or disrupted activity of the of the global transcriptional regulator CodY and / or the two-com- ponent regulatory system CovRS due to a modification or disruption of the activity of the codY gene and / or any of the covR and / or covS genes in the strain, such as by deleting or mutating the codY, the covR and / or the covS genes in the strain. The method according to any one of embodiments 1-3, which strain provided under step a) contains a gene encoding Carbon Catabolite Control Protein A (CcpA). The method according to any one of embodiments 1-4, which strain has an inherently low, reduced, or disrupted activity of any of Clp proteins CIpP, and / or CIpC due to a modification or disruption of the activity of any of the c / pP and / or the dpC gene. The method according to any one of embodiments 1-5, which strain performs with increased levels of natural transformation induced by increased levels of available and functional endogenous ComX protein in the strain relative to a control strain, which control strain does not have any of the inherently low, reduced or disrupted activities listed under i)-iii), such as by either induced or increased expression of an endogenous comX gene relative to the control strain, by a reduced ComX protein degradation, or by increased availability or abundance. A method for transforming a strain of a lactic acid bacterium, such as of the genus Lactococcus, with an exogenous DNA polynucleotide comprising the steps of:

[0099] (a) providing a strain of the lactic acid bacterium, such as of the genus Lactococcus, which strain is transformable through natural transformation;

[0100] (b) increasing the competence of the strain for natural transformation;

[0101] (c) contacting the strain of step (b) with an exogenous DNA polynucleotide in a growth medium and incubating the resulting mixture for integration of the exogenous DNA polynucleotide or part of it into the genome of the strain; and

[0102] (d) selecting a derivative of said strain which has integrated the exogenous DNA polynucleotide or part of it into its genome; wherein this increase in the ability of the strain to perform natural transformation under step (b) is induced by either of i) growing the strain in a growth medium to induce a diauxic shift in the growth of the strain; and / or ii) growing the strain in a growth medium without any one or more of glutamate, aspartate, xanthine, adenine, thymine, uracil, cytosine and guanine; and / or iii) transforming through natural transformation under step b) and c) at a pH lower than about 8.5, such as in the pH range of 5.0-8.5. The method according to embodiment 7, which method comprises a step of transforming through natural transformation under step b) and c) at a pH lower than about 8.0, such as lower than about 7.9, such as lower than about 7.8, such as lower than about 7.7, such as lower than about 7.6, such as lower than about 7.5, such as lower than about 7.4, such as lower than about 7.3, such as lower than about 7.2, such as lower than about 7.1, such as lower than about 7.0, such as lower than about 6.9, such as lower than about 6.8, such as lower than about 6.7, such as lower than about 6.6, such as lower than about 6.5, such as lower than about 6.4, such as lower than about 6.3, such as lower than about 6.2, such as lower than about 6.1, such as lower than about 6.0, such as lower than about 5.9, such as lower than about 5.8, such as lower than about 5.7, such as lower than about 5.6, such as lower than about 5.5, such as lower than about 5.4, such as lower than about 5.3, such as in the pH range of 5.0-8.0, such as in the pH range of 5.0-7.9, such as in the pH range of 5.0-7.8, such as in the pH range of 5.0-7.7, such as in the pH range of 5.0-7.6, such as in the pH range of 5.0-7.5, such as in the pH range of 5.0-7.4, such as in the pH range of 5.0- 7.3, such as in the pH range of 5.0-7.2, such as in the pH range of 5.0-7.1, such as in the pH range of 5.0-7.0, such as in the pH range of 5.0-6.9, such as in the pH range of 5.0-6.8, such as in the pH range of 5.1-8.5, such as in the pH range of 5.2-8.5, such as in the pH range of 5.3-8.5, such as in the pH range of 5.4-8.5, such as in the pH range of 5.5-8.5, such as in the pH range of 5.6-8.5, such as in the pH range of 5.7-8.5, such as in the pH range of 5.8-8.5, such as in the pH range of 5.9-8.5, such as in the pH range of 6.0-8.5, such as in the pH range of 6.1-8.5, such as in the pH range of 6.2-8.5, such as in the pH range of 6.3-8.5, such as in the pH range of 6.4-8.5, such as in the pH range of 6.5-8.5, such as in the pH range of 6.6-8.5, such as in the pH range of 6.7-8.5, such as in the pH range of 6.8-8.5, such as in the pH range of 6.9-8.5, such as in the pH range of 7.0-8.5. The method according to embodiments 7 or 8, which increased ability of the strain to perform natural transformation is induced by increasing the level of available and functional endogenous ComX protein in the strain relative to a control strain, which control strain has not been induced to increase the level of endogenous ComX protein, such as by either inducing an increase in the expression of an endogenous comX gene relative to the control strain, or by inducing a reduction in ComX protein degradation. The method according to any one of embodiments 1-9, which selected strain provides for a rate of at least lxlO-8transformants per g of exogenous DNA polynucleotide. The method according to any one of embodiments 7-10, wherein the diauxic shift under i) is induced by growth of the strain in a medium with a) limited amount of glucose, such as with less than 0.40% (w / v), such as less than 0.35% (w / v), such as less than 0.30% (w / v), such as less than 0.25% (w / v), such as less than 0.23% (w / v), such as less than 0.20% (w / v) glucose; and b) at least 0.5% (w / v), such as at least 0.6% (w / v), such as at least 0.7% (w / v), such as at least 0.8% (w / v), such as at least 0.9% (w / v), such as at least 1.0% (w / v) of one or more secondary sugar. The method according to embodiment 10, wherein the one or more secondary sugar is selected from maltose, xylose, cellobiose, galactose, arabinose and trehalose. The method according to any one of embodiments 7-12, which strain provided under step a) is as defined in any one of embodiments 2-6. The method according to any one of embodiments 1-13, where the lactic acid bacterium is of the genus Lactococcus, such as a strain selected from the species Lactococcus lactis, Lactococcus cremoris, Lactococcus laudensis, Lactococcus carnosus, Lactococcus formosensis, Lactococcus fujiensis, Lactococcus hircilactis, Lactococcus kimchii, Lactococcus paracarno- sus, Lactococcus plantarum, Lactococcus raffinolactis, and Lactococcus taiwanensis. The method according to any one of embodiments 1-14, which strain provided under step a) has one or more endogenous late competence genes repaired to provide a functional gene, such as a functional comGA, comGB, comGC, comGD, comGE, comGF, comGG, comC, comFA, comFC, comEA, comEC, ssb, coiA, dprA, and / or recA gene. The method according to any one of embodiments 1-15, which strain provided under step a) has the full set of endogenous late competence genes in addition to the comX gene. The method according to any one of embodiments 1-16, which strain transformable through natural transformation provided under step (a) has been transformed with a plasmid expressing an exogenous comX gene having at least 90%, such as 95%, or 100% identity to the endogenous comX gene of the strain. A strain of a lactic acid bacterium, such as of the genus Lactococcus, obtained or obtainable by the method of any one of embodiments 1 to 17. Use of a strain of a lactic acid bacterium, such as of the genus Lactococcus having either of i) an inherently low, reduced or disrupted activity of the adaptor protein MecA relative to a control strain having a normal activity of the MecA protein; and / or ii) an inherently low, reduced, or disrupted activity of the global transcriptional regulator CodY and / or the two-component regulatory system CovRS relative to a control strain having a normal activity of CodY and / or CovRS; and / or iii) an inherently low, reduced, or disrupted activity of any of Clp proteins CIpP, and / or CIpC; for transforming the strain through natural transformation. The use according to embodiment 18, wherein the strain is as defined in any one of embodiments 2-6. A method for increasing the rate of natural transformation of a strain of a lactic acid bacterium, such as of the genus Lactococcus, comprising the steps of

[0103] (a) providing a strain of the lactic acid bacterium, such as of the genus Lactococcus, which strain is transformable through natural transformation;

[0104] (b) performing any one or more of i) reducing or disrupting the activity of the adaptor protein MecA relative to a control strain having a normal activity of the MecA protein; and / or ii) reducing or disrupting the activity of the global transcriptional regulator CodY and / or the two-component regulatory system CovRS relative to a control strain having a normal activity of CodY and / or CovRS; and / or iii) reducing or disrupting the activity of any of Clp proteins CIpP, and / or CIpC; which strain has an increased rate of natural transformation by competence relative to a control strain not having reduced or disrupted activity under any of i) to iii). The method according to embodiment 20, which strain has reduced or disrupted activities due to a modification or disruption as defined in any one of embodiments 2-6. A method for identifying a strain of a lactic acid bacterium, such as of the genus Lactococcus which is transformable through natural transformation comprising the steps of:

[0105] (a) providing a strain of a lactic acid bacterium, such as of the genus Lactococcus;

[0106] (b) measuring the activity of any one or more of i) the adaptor protein MecA relative to a control strain having a normal activity of the MecA protein; and / or ii) the global transcriptional regulator CodY and / or the two-component regulatory system CovRS relative to a control strain having a normal activity of CodY and / or CovRS; and / or iii) the activity of any of Clp proteins CIpP, and / or CIpC;

[0107] (c) select a strain with a reduced or disrupted activity of any one of these proteins defined in (b) i)-iii);

[0108] (d) contacting the strain obtained in step (c) with an exogenous DNA polynucleotide encoding a marker gene in a growth medium and incubating the resulting mixture for integration of the exogenous DNA polynucleotide into the genome of the strain; and

[0109] (e) determining the rate of integration events; wherein a rate of at least 1 x IO-8transformants per g of DNA is indicative of a strain which is transformable through natural transformation. The method according to embodiment 22, which strain has reduced or disrupted activities due to a modification or disruption as defined in any one of embodiments 2-6. SEQUENCES

[0110] SEQ ID NO: 1

[0111] MKYEDINENTIKITLSFDDLTDYDIKLSDFFGNQEVIEQFFYELVDELGLENRFGNVGMLTFQIQPFPQGVHMIV HEEAMLGEGGEIPDDPEEFEELMTGFYNKLNEIGADMARERGITDFKPGLGLPGTKKDEAEQEPDFIYYSIRYE DIMSVLTGIKNVKFADEESEFYRYDGNFYLVVLDNQKEKGKMHVESTRSRMMEYGEATKMSREFLQEYGECLI ATRALDVLRKI

[0112] SEQ ID N0:2 (amino acid sequence of the negative regulator of genetic competence MecA protein in Lactococcus lactis with an arginine in the protein sequence at a position corresponding to position 125 of SEQ ID NO: 1 (125R))

[0113] MKYEDINENTIKITLSFDDLTDYDIKLSDFFGNQEVIEQFFYELVDELGLENRFGNVGMLTFQIQPFPQGVHMIV HEEAMLGEGGEIPDDPEEFEELMTGFYNKLNEIGADMARERGITDFKPGRGLPGTKKDEAEQEPDFIYYSIRYE DIMSVLTGIKNVKFADEESEFYRYDGNFYLVVLDNQKEKGKMHVESTRSRMMEYGEATKMSREFLQEYGECLI ATRALDVLRKI

[0114] SEQ ID NO:3 CcpA

[0115] MVESTTTIYDVARVAGVSMATVSRVVNGNANVKEKTRQKVLEAIAELDYRPNAVARGLASKRTTTVGVILPTIT STYFAAITRGVDDIASMYKYNMILANSDNDVEKEEKVLETFLSKQVDGIVYMGSSLDEKIRTSLKNSRTPVVLV GTIDGDKEIPSVNIDYHLAAYQSTKKLIDSGNKKIAYIMGSLKDVENTERMVGYQEALLEANIEFDENLVFEGN YSYEQGKALAERLLERGATSAVVSHDTVAVGLLSAMMDKGVKVPEDFEIISGANSPITQYTYPTLTSVNQPLYD LGAVAMRLLTKLMLKEDVEQNQLVLDHEIFSRRSTK

[0116] SEQ ID NO:4 CodY

[0117] VATLLEKTRKITAILQDGVTDLQQELPYNSMTERLANVIDCNACVINTKGELLGYSLPYNTNNDRVDQFFYDRK LPDEYVRAAVRIYDTMANVPVDRPLAIFPEESLGDFPKGVTTLAPIYGSGMRLGTFIMWREDGEFTDDDLVLVE LATTVIGVQLSNLKLEQMEENIRKDTMATMAVNTLSYSEMKAVKAIIEELDGEEGHVIASVIADKIGITRSVIVN ALRKLESAG VIESRSLG M KGTYLKVLNTG LFDKLAG RN F

[0118] SEQ ID NO:5 CovS

[0119] MKKLFTFNKKKETVEESSAKRSIMLRWAFANTVFCFITFTLFATLTYQLTISSFIKEEQQLLTRSMDSVEEVLEK ADAPLNSSNLNTYIEATSKIQNGESEGMSLGSIIGTRKAFYIYDLNHKLLYSTNRHTFGFQNQANNEMKEIRGE NPGYLVQRKIISKSTGQVVGYLQAFYDTTTYHRISNLLLIVLLILEIVALIVAQLIGYFMANYFMKPLEKLYQGMQ EMANDPTNDFEPIEIQSGDEIEELAHVYNDMMLKMKAYLEQQNRFVSDVSHELRTPLAVLDGHINLLNRWGK NDPEVLDESLQASLDEVDRMKKMLEEMLALARLENVDLSSEELDCDVGKVCNRALKNFQLLHDDFEIVLDNRL

[0120] IYPTHARISENHFEQGLRILLDNAAKYSPDDRKEIVITVSEDEQFVITSVSDKGIGISEEDINHLFERFFRADKAR NREIGGTGLGLSILARLAENYQGEIEVNSELGLGSTFTLKFPKIK

[0121] SEQ ID NO:6 CovR

[0122] MTSKKILIIEDEKNLARFVSLELEHEGYATEIKDNGRSGLEEATSKDYDLILLDLMLPELDGFEVARRLRKEKDT PIIMMTARDSTMDRVAGLDIGADDYITKPFAIEELLARVRAFFRREEHGHAVERAENTSFRDLVIDKTNRTVHR GKKVIDLTRREYDLLLTLMQNVGDVVTREHLVSQVWGYEEGTETNVVDVYIRYLRNKIDVEGQDSYIQTVRGL GYVMRERK

[0123] SEQ ID NO:7 CIpC

[0124] MKFENIKYTPTLDRILEKAEEYAHQYQYGTIESAHLLAAMATTSGSIAYSLLAGMNVDSSDLLIDLEDLSSHVK

[0125] VKRSTLRFSPRAEEVMTAASFLAIHNNSEAVGTEHLLYALLQVEDGFGLQLLKLQKINIVSLRKELEKRTGLKVP ESKKAVTPMSKRKMAKGVAENSTTPTLDSVSSDLTEEARLGKLDPMIGREAEIDRLIHILSRRTKNNPVLVGEP GVGKSAIIEGLAQRIVNGQVPIGLMNSRIMALNMATVVAGTKFRGEFEDRLTAIVEEVSSDPDVIIFIDELHTII GAGGGMDSVNDAANILKPALARGDFQMVGATTYHEYQKYIEKDEALERRLARINVDEPSPDEAIAILQGLREK

[0126] FEDYHQVKFTDQAIKSAVMLSVRYMTSRKLPDKAIDLLDEAAAAVKISVKNQQTKRLDLEKELAEAQEELSEA

[0127] VIKLDIKASRTKEKAVEKIADKIYKFSVKEDKRQEVTDQAVVAVASTLTGVPITQMTKSESDRLINLEKELHKR

[0128] VVGQEEAISAVSRAIRRARSGVADSRRPMGSFMFLGPTGVGKTELAKALADSVFGSEDNMIRVDMSEFMEKH

[0129] STSRLIGAPPGYVGYDEGGQLTERVRNKPYSVVLLDEVEKAHPDVFNIMLQILDDGFVTDTKGRKVDFRNTIII MTSNLGATALRDDKTVGFGAKNITADYSAMKSRILEELKRHYRPEFLNRIDENIVFHSLESQEIEQIVKIMSKSL IKRLAEQDIHVKLTPSAVKLIAEVGFDPEYGARPLRKALQKEVEDLLSEQLLSGEIKAGNHVSIGASNKKIKIAQ IV

[0130] SEQ ID N0:8 CIpP

[0131] MGYLVPTVIEQSSRGERAYDIYSRLLKDRIIMLTGPVEDGMANSIIAQLLFLDAQDNTKDIYLYVNTPGGSVSA

[0132] GLAIVDTMNFIKSDVQTIVMGMAASMGTIIASSGTKGKRFMLPNAEYLIHQPMGGTGQGTQQTDMAIVAEQL LKTRKRLEQILADNSNRSLEQIHKDAERDHWMDAKETLEYGFIDEIMENNSLK

[0133] SEQ ID N0:9 Rpskt Lactococcus lactis

[0134] MPTINQLVRKPRRAQVTKSKSPAMNVGYNSRKKVQTKLASPQKRGVATRVGTMTPKKPNSALRKFARVRLSN

[0135] LMEVTAYIPGIGHNLQEHSVVLLRGGRVKDLPGVRYHIVRGALDTAGVADRKQSRSKYGAKKPKA

[0136] SEQ ID NO: 10 RpsLu* Lactococcus lactis

[0137] MPTINQLVRKPRRAQVTKSKSPAMNVGYNSRKKVQTKLASPQKRGVATRVGTMTPRKPNSALRKFARVRLSN

[0138] LMEVTAYIPGIGHNLQEHSVVLLRGGRVKDLPGVRYHIVRGALDTAGVADRKQSRSKYGAKKPKA

[0139] SEQ ID NO: 11 RpsQc Lactococcus cremoris

[0140] MPTINQLVRKPRRAQVTKSKSPAMNVGYNSRKKVQTKLASPQKRGVATRVGTMTPKKPNSALRKFARVRLSN

[0141] LMEVTAYIPGIGHNLQEHSVVLLRGGRVKDLPGVRYHIVRGALDTAGVADRKQSRSKYGAKKPKA

[0142] SEQ ID NO: 12 RpsQc* Lactococcus cremoris

[0143] MPTINQLVRKPRRAQVTKSKSPAMNVGYNSRKKVQTKLASPQKRGVATRVGTMTPIKPNSALRKFARVRLSN

[0144] LMEVTAYIPGIGHNLQEHSVVLLRGGRVKDLPGVRYHIVRGALDTAGVADRKQSRSKYGAKKPKA

[0145] SEQ ID NO: 283 DprA*

[0146] MGQVAQLKSIPNFIEQYKNQDVSKLREEYKIYPSFSLLDEIYPERLKEIYNPPVLLFYQGDIRLLKTPKLAFVGSR

[0147] QASSQGIKAVQKIVTELNQNFTIVSGLAKGIDTASHLSAIKNKIPTIAVIGTGLDIFYPLENRKIQEYLAKNQLIL SEY

[0148] SEQ ID NO: 284 DprA

[0149] MITNFDLYRWKKAGMTNLGVNKLLKFFHRYGKRISLRQMGQVAQLKSIPNFIEQYKNQDVSKLREEYKKYPSF

[0150] SLLDEIYPERLKEIYNPPVLLFYQGDIRLLKTPKLAFVGSRQASSQGIKAVQKIVTELNQNFTIVSGLAKGIDTAS HLSAIKNKIPTIAVIGTGLDIFYPLENRKIQEYLAKNQLILSEYSVGEKPLRYHFPERNRIIAGLSHGVVVIEAKLR SGSLITCERALEEGRDIFAVPGNIADGFSDGCNHLIQQGAKLIYQAQDILEEYLYD

[0151] SEQ ID NO: 285 ComX*

[0152] MKLMKQIRIRTWKIEDYLQEGMIILHLLLEEQNDGQKLHTKFKVKYHQRLIDELRRSYAKKRSHDHFIGLDVYE

[0153] CSDWINSGDTSPDNEVVFNHLLAEVYEGLSAHYQDLLLRQMRGEELTRMQRYRLREKIKAILFSEDEE

[0154] SEQ ID NO: 286 ComX

[0155] MTYYLEEEDFENLFLEMKPIVMKLMKQIRIRTWKIEDYLQEGMIILHLLLEEQSDGQKLHTKFKVKYHQRLIDEL

[0156] RRSYAKKRSHDHFIGLDVYECSDWINSGDTSPDNEVVFNHLLAEVYEGLSAHYQDLLLRQMRGEELTRMQRY RLREKIKAILFSEDEE

[0157] SEQ ID NO: 287 ComFA* MSTNQEKLFGRLLLKNDILQLIKNTDKISVSKIFSNFLLETKVNPILGMTSISSNKIKCNRCGTVHIKNSVKLPIG VFYCPSCIQLGRVRSDEFLYFLPQKNFPKKSYINWSGKLTENQKSISNALCQEINSHQQIIVQAVTGAGKTAMI YQVIEQILESGGVVGLASPRIDVCLELHQRLSRDFSCKIPLLYHDGDSYFRAPLIIMTSHQLLRFKEAFDLLIIDE VDAFPFRDNEMLYVAAEKARKIEGNLIYLTATSTDKLEKDIKKQKLYPLFLPRRFHNFPLVVPKFFWKNKFDKKL IEQRNSGFPLLIFAAEIEFGQEFAKQLQLKFPKEKIASVASTTKDRLEIVKAFRNKEITILIATSILERGVTFPNVD VFVINSEHPNFTKSALIQMAGRVGRSPERPTGLVSFFITENPKQCVRQLEKSKNEPTGRFFMIKCLLCSRNIAQ NFLFSELFLLKSPQNLLCSKCQKNFEKIPEAHCPCCCKPNVHEICTDCQNWEKRGYKIRHQAIFHYNQAMKDY FSQYKFVGDYRLYQIFEPYFKNISQKSPLVPIPISPKRLEERGFNQVTAFLQQDNFIELLEKENSVKQSSLNRKE

[0158] RLESPNPFRLKKGLKVPTKVILIDDIYTTGTTLYHASQILKEAGVHEIRSFSLCC

[0159] SEQ ID NO: 288 ComFA

[0160] MSTNQEKLFGRLLLKNDILQLIKSTDKISVSKIFSNFLLEAKVNPILGMTSISSNKIKCNRCGTVHIKNSVKLPIG VFYCPSCIQLGRVRSDEFLYFLPQKNFPKKSYINWSGKLTENQKSISNALCQEINSHQQIIVQAVTGAGKTEMI YQVIEQILESGGVVGLASPRIDVCLELHQRLSRDFSCKIPLLYHDGDSYFRAPLIIMTSHQLLRFKEAFDLLIIDE VDAFPFRDNEMLYFAAEKARKIEGNLIYLTATSTDKLEKDIKKQKLYPLFLPRRFHNFPLVVPKFFWKNKFDKKL IEQRNSGFPLLIFAAEIESGQEFAKQLQLKFPKEKITFVASTTKDRLEIVKAFRNKEITILIATSILERGVTFPNVD VFVINSEHPNFTKSALIQMAGRVGRSPERPTGLVSFFHYGKSKAMCQAVREIKKMNQLGGFS

[0161] SEQ ID NO: 289 ComEC* and correspond to the 3 existing stop codons in the corresponding DNA sequence, TAG, TAA, TGA, respectively)

[0162] MRFLLIKFTLIFLTYLVVLLYFCIFTYNLPLLILFICSILLALFRKYYLMIPILIVFAVFFLLINSLSIRDNENQPKNLSK IELIPDTIQVNGDLLSFQGRMNGQKYQVYDTLKSLKEKEFYQNLSQNCQLSFTGNLEIPEIQRNFNGFDDQKYL ASQNIYRQITIQQITNVEIKKNFDLHTLRRKAIVWTQNHFPKPMSDYMTGLLFGFLSKDFNQMSDIYSSLGIIH LFALSGMQVNFFIDWFRKILLRMGLTRERVNLVQIPFSIFYAFMTGLSVSVLRALFQKNIRLTSLDNIGVTTLVL MIISPKFLLTTGGQLTLFYAFVISMLNHSFSNLNGLKKC*QNHQSFHFVFFRYSSLISIFFSLFQYY#QLYLVFFLI LCCSRCFYAVFLYHFWDFRSI#ILFFKF+NG + FIKLIYPYITLLFWGNLVHSN#LFYLC#LVYS+IHFLIKKGDFF #LDLYFYFFSSLKIQ*EQALQRLISDKAIVFFYKISLIKKTF*LILGDNLLFLKKIGKKRRRNQMLTKRLFLI + NPW

[0163] GWLILTNLF*LIQMLTMLVISYP+LIK#K#EKSGSVREN#PTVVLWKN#KKQKFRFMLVKLGIKFRFLIVFFKF YQMVTLKKVIIMIP+*LMETFTIQNFFLQEIWNKKEKKSFYKIIPNYR+MC#KWGIMAVKQVQVRNSSKRYIL NLH#SRLEKLIAMVIPIKKL+KP+PNIR#RHSELTKKGQ#N#QK#ISNGKSKQLD

[0164] SEQ ID NO: 290 ComEC

[0165] MRFLLIKFPLIFLTYLVVLLYFCIFTYNLPLLILFICSILLALFRKYYLMIPILIVFAVFFLLINSLSIRDNENQPKTLSKI ELISDTIQVNGDLLSFQGR.MNGQNYQVYDTLKSLKEKKFYQNLSQNCQLSFTGNLQIPETQR.NFNGFDDQKY LASQNIYRQITIQQITNVEIKKNFDLHTLRRKAIVWTQNHFPKPMSDYMTGLLFGFLSKDFNQMSDIYSSLGII HLFALSGMQVNFFIDWFRKILLRMGLTREIVNLVQIPFSIFYAFMTGLSVSVLRALFQKNIRLTSLDNIGVTTLVL MLISPKFLLTTGGQLTLFYAFVISMLNHPFSNLNGLKKVLTESAVLSLCVLPLLIFDFHIFQPLSILLTIIFGFLFDF MLLPLLLCCFLISFLGFSLNLNFIFQILEWLVHKVDLPLHYPFVLGKPSSLELIILFVLIGLFVDSFSHKKRRFFLVG FIFLFFFITKNPMRASITAVDIGQGDSIFLQDKFNKENILIDTGGRLALPQKNWQKAQTQSNADKTLIPYLESM GVAHIDQLILTHTDADHVGDFLSLADKIKIREIWVSPGELTNSSFVEKLKKAKIPIHVSKVGDKIPIFDSFLQVL

[0166] SNGYTKKGDNNDSIVTYGNFYHTKFLFTGDLEQEGEKKLLQNYPKLQVDVLKVGHHGSKTSSSPEFIKEIHPK LALISVGKANRYGHPNQETLETLAKYQVKTLRTDQKGAIKLTEVNQQWQIKTVR

[0167] SEQ ID NO: 291 CoiA* (Sign "+" correspond to the stop codon in the corresponding DNA sequence (TAG)

[0168] MLTAIDENNKIINLLELDRKELTGKFYCPSCHSELLIKNGQIKVLHFAHKSLKSCNLWLENESDQHLGLKKILY+ WFKKTDKVEIERYLPELNQRPDLLVNDKIAIEIQCSHLSIKRLKERTENYKAHGFKVLWLMGKDLWLAEQVTEL QKNLVYFSENRGFFYWELDFQRKKLRLKSLIHEDLRGRIICLQEEIPFGKGRLIAHLRLPYLAQKLVKIPTFKDSK LSSFIRQQLYYQSPKWMKIQEKYYQKGENLLTKKFEGPYIAPLGLNLLENFTDEMTITTFTQIDQNVKLYYENFL INFQRNSLEMLYPPRFYAIMGKQKKEK

[0169] SEQ ID NO: 292 CoiA

[0170] MRTAIDENNKIINLLELDRKELTGKFYCPSCHSELLIKNGQIKVLHFAHKSLKSCNLWLENESEQHLGLKKILYQ

[0171] WFKRTDKVEIERYLPELNQRPDLLVNDKIAIEIQCSHLSIKRLKERTENYKTHGFKVLWLMGKDLWLAEQVTEL QKNLVYFSENRGFFYWELDFQRKKLRLKSLIHEDLRGRIICLQEEIPFGKGRLIAHLRLPYLAQKLVKIPTFKDSK LSSFIRQQLYYQSPKWMKIQEKYYQKGENLLTKKFEGPYIAPLGLNLLENFTDEMTITTFTQIDQNVKLYYENFL INFQRNSLEMLYPPRSYAIMGKQKKEK

[0172] SEQ ID NO: 13 rpsLu_ [IL1403_L0389] Lactococcus lactis

[0173] ATGCCTACAATTAACCAATTGGTACGCAAACCACGTCGTGCTCAAGTGACTAAATCTAAATCACCAGCAAT GAACGTTGGCTACAACAGCCGTAAAAAAGTACAAACTAAACTTGCAAGCCCACAAAAACGTGGAGTAGCA ACTCGTGTTGGTACTATGACTCCTAAAAAACCTAACTCAGCGCTTCGTAAATTCGCGCGTGTACGTCTTTCA AACCTTATGGAAGTAACAGCGTACATCCCAGGTATCGGACACAACCTCCAAGAACACAGTGTTGTACTTCT

[0174] TCGTGGTGGACGTGTAAAAGACCTTCCAGGGGTACGTTACCATATCGTTCGTGGTGCACTTGATACAGCA GGTGTCGCTGACCGTAAACAAAGCCGTTCTAAATACGGTGCTAAAAAACCAAAAGCTTAA

[0175] SEQ ID NO: 14 rpsLu.* Lactococcus lactis

[0176] ATGCCTACAATTAACCAATTGGTACGCAAACCACGTCGTGCTCAAGTGACTAAATCTAAATCACCAGCAAT GAACGTTGGCTACAACAGCCGTAAAAAAGTACAAACTAAACTTGCAAGCCCACAAAAACGTGGAGTAGCA ACTCGTGTTGGTACTATGACTCCTAGAAAACCTAACTCAGCGCTTCGTAAATTCGCGCGTGTACGTCTTTC AAACCTTATGGAAGTAACAGCGTACATCCCAGGTATCGGACACAACCTCCAAGAACACAGTGTTGTACTTC

[0177] TTCGTGGTGGACGTGTAAAAGACCTTCCAGGGGTACGTTACCATATCGTTCGGGGTGCACTTGATACAGC AGGTGTCGCTGACCGTAAACAAAGCCGTTCTAAATACGGTGCTAAAAAACCAAAAGCTTAA

[0178] SEQ ID NO: 15 rpsLcc [llmg_2558] Lactococcus cremoris

[0179] ATGCCTACAATTAACCAATTGGTACGCAAACCTCGTCGTGCTCAAGTGACTAAATCTAAATCACCAGCAAT GAACGTTGGCTACAACAGCCGTAAAAAAGTACAAACTAAACTTGCAAGCCCACAAAAACGTGGAGTAGCA ACTCGTGTTGGTACAATGACTCCTAAAAAACCTAACTCAGCGCTTCGTAAATTCGCGCGTGTACGTCTTTC AAACCTTATGGAAGTAACAGCGTACATCCCAGGTATCGGACACAACCTCCAAGAACACAGTGTTGTACTTC

[0180] TTCGTGGTGGACGTGTAAAAGACCTTCCAGGGGTACGTTACCATATCGTTCGTGGTGCACTTGATACAGCA GGTGTCGCTGACCGTAAACAAAGCCGTTCTAAATACGGTGCTAAAAAACCAAAAGCTTAA

[0181] SEQ ID NO: 16 rpsLLc* Lactococcus cremoris

[0182] ATGCCTACAATTAACCAATTGGTACGCAAACCTCGTCGGGCTCAAGTGACTAAATCTAAATCACCAGCAAT GAACGTTGGCTACAACAGCCGTAAAAAAGTACAAACTAAACTTGCAAGCCCACAAAAACGTGGAGTAGCA ACTCGTGTTGGTACAATGACTCCTATAAAACCTAACTCAGCGCTTCGTAAATTCGCGCGTGTACGTCTTTCA AACCTTATGGAAGTAACAGCGTACATCCCAGGTATCGGACACAACCTCCAAGAACACAGTGTTGTACTTCT

[0183] TCGTGGTGGACGTGTAAAAGACCTTCCAGGGGTACGTTACCATATCGTTCGTGGTGCACTTGATACAGCA GGTGTCGCTGACCGTAAACAAAGCCGTTCTAAATACGGTGCTAAAAAACCAAAAGCTTAA

[0184] SEQ ID NO: 293 dprA* [IL1403_L200051]

[0185] ATGGGACAAGTAGCACAACTTAAATCAATTCCCAACTTTATTGAACAATATAAAAATCAAGACGTAAGTAAA CTGAGAGAAGAGTATAAAATATACCCATCATTTTCTCTTCTTGATGAAATTTATCCCGAAAGGCTCAAGGAA ATTTACAATCCACCGGTTCTATTATTTTATCAAGGAGATATAAGACTCTTAAAAACTCCTAAATTAGCTTTTG TTGGTAGTCGACAAGCAAGCTCACAAGGAATTAAAGCCGTTCAAAAAATTGTGACAGAGTTGAACCAAAAT

[0186] TTTACGATTGTCAGCGGACTCGCTAAAGGAATTGATACAGCAAGTCATCTCTCTGCCATTAAAAATAAAATC CCTACAATTGCAGTTATCGGTACAGGCTTAGATATCTTTTATCCTTTAGAAAATCGAAAAATTCAAGAATAT CTTGCTAAAAACCAACTCATACTATCAGAATATTGA

[0187] SEQ ID NO: 294 dprA [DGCC12653_02130]

[0188] ATGATAACAAATTTTGATTTATATCGTTGGAAAAAAGCTGGCATGACCAATTTAGGAGTAAATAAACTTTTA AAATTTTTTCATAGATATGGTAAAAGAATAAGTTTAAGACAAATGGGACAAGTAGCACAACTTAAATCAATT CCCAACTTTATTGAACAATATAAAAATCAAGACGTCAGTAAACTGAGAGAAGAGTATAAAAAATACCCATCA TTTTCTCTTCTTGATGAAATTTATCCCGAAAGGCTCAAGGAAATTTACAATCCACCGGTTCTATTATTTTATC

[0189] AAGGAGATATAAGACTCTTAAAAACTCCTAAATTAGCTTTTGTTGGTAGTCGACAAGCAAGCTCACAAGGA ATTAAAGCCGTTCAAAAAATTGTGACAGAGTTGAACCAAAATTTTACGATTGTCAGCGGACTCGCTAAAGG AATTGATACAGCAAGTCATCTTTCTGCCATTAAAAATAAAATCCCTACAATTGCAGTTATCGGTACAGGCTT AGATATCTTTTATCCTTTAGAAAATCGAAAAATTCAAGAGTATCTTGCTAAAAACCAACTCATACTATCAGAA TATTCAGTCGGAGAAAAGCCACTTCGTTATCATTTTCCTGAACGCAATCGTATCATTGCGGGACTTTCACAT GGTGTAGTAGTGATAGAAGCAAAGTTACGAAGTGGTAGTTTAATTACTTGTGAGCGCGCTCTGGAAGAAG

[0190] GAAGAGATATTTTCGCTGTACCAGGAAACATAGCAGATGGATTTTCAGATGGCTGTAATCATCTAATACAA CAAGGAGCAAAATTAATCTATCAAGCCCAAGATATCTTAGAAGAATATTTGTATGATTAA

[0191] SEQ ID NO: 295 comX* [IL1403_L6973]

[0192] ATGAAATTAATGAAACAAATTCGCATTAGAACATGGAAAATAGAGGATTATCTTCAAGAGGGGATGATTAT TTTACATCTTCTATTAGAAGAGCAGAACGATGGTCAAAAGCTGCATACAAAATTTAAGGTAAAGTATCATCA AAGATTAATAGATGAATTAAGACGAAGTTATGCAAAGAAACGAAGCCATGACCATTTTATAGGTTTAGATG TTTATGAATGCTCAGACTGGATAAATTCAGGTGATACTAGTCCAGATAATGAAGTGGTCTTCAATCATTTGC

[0193] TGGCAGAAGTATATGAAGGTTTGAGCGCACATTATCAAGACTTACTACTTCGACAAATGCGAGGAGAAGA ACTAACTCGCATGCAACGGTATCGCCTTCGTGAAAAAATAAAGGCCATCTTATTTTCAGAAGACGAAGAGT GA

[0194] SEQ ID NO: 296 comX [DGCC12653_13390]

[0195] ATGACATATTACTTGGAAGAAGAGGATTTTGAAAATCTGTTTTTAGAAATGAAACCTATAGTTATGAAATTA ATGAAACAAATTCGCATTAGAACATGGAAAATAGAGGATTATCTTCAAGAGGGGATGATTATTTTACATCTT CTATTAGAAGAGCAGAGCGATGGTCAAAAGCTGCATACAAAATTTAAGGTAAAGTATCATCAAAGATTAAT AGATGAATTAAGACGAAGTTATGCAAAGAAACGAAGCCATGACCATTTTATAGGTTTAGATGTTTATGAAT

[0196] GCTCAGACTGGATAAATTCAGGTGATACTAGTCCAGATAATGAAGTGGTCTTCAATCATTTGCTGGCAGAA GTATATGAAGGTTTGAGCGCACATTATCAAGACTTACTACTTCGACAAATGCGAGGAGAAGAACTAACTCG CATGCAACGGTATCGCCTTCGTGAAAAAATAAAGGCCATCTTATTTTCAGAAGACGAAGAGTGA

[0197] SEQ ID NO: 297 comFA* [1AA59_ RS06700]

[0198] ATGAGCACTAATCAAGAAAAGTTATTTGGCCGTTTATTATTAAAAAATGATATTTTGCAACTTATAAAAAATA

[0199] CTGACAAGATTTCTGTCAGTAAAATTTTTAGTAATTTTTTGTTAGAAACGAAGGTGAATCCAATTTTGGGAA TGACTTCAATTTCTTCCAATAAAATAAAATGCAACCGTTGTGGGACTGTTCATATAAAAAATTCTGTCAAAC TTCCAATTGGTGTATTTTACTGTCCAAGTTGTATTCAATTAGGTCGAGTCCGCTCCGATGAATTCTTGTACT TTCTGCCACAAAAGAATTTCCCAAAGAAATCATATATAAACTGGTCTGGAAAACTGACAGAGAATCAAAAAT

[0200] CAATTTCAAATGCCCTTTGTCAGGAAATTAATTCTCATCAGCAAATAATTGTCCAAGCTGTGACTGGAGCTG GAAAAACTGCAATGATTTATCAAGTCATTGAGCAAATTTTAGAAAGCGGTGGGGTTGTTGGTCTAGCTAGT CCAAGAATTGATGTTTGTCTTGAACTTCATCAGCGATTATCACGTGATTTTTCCTGTAAGATTCCACTCTTAT ATCATGATGGCGACAGCTATTTCCGAGCTCCATTAATAATAATGACCAGTCATCAGCTTTTACGTTTCAAGG

[0201] AAGCTTTTGATTTGCTGATTATTGATGAAGTTGATGCCTTTCCCTTTAGAGATAATGAAATGCTTTATGTTG CGGCAGAAAAAGCAAGAAAAATAGAAGGAAATTTAATATATTTGACCGCAACTTCTACTGACAAACTTGAA AAAGATATAAAAAAGCAAAAACTCTATCCTTTGTTTCTCCCGCGTCGTTTTCACAATTTCCCTTTAGTGGTG CCTAAATTTTTTTGGAAAAATAAATTTGATAAGAAATTAATTGAGCAAAGAAATAGTGGCTTTCCTCTTCTTA

[0202] TTTTTGCTGCTGAAATTGAATTTGGACAAGAATTTGCAAAACAACTACAATTGAAATTTCCTAAAGAAAAAA TTGCTTCCGTTGCTTCAACAACAAAAGATAGATTGGAAATTGTTAAAGCTTTTAGAAATAAAGAAATTACTA TTTTAATAGCGACATCAATTCTTGAACGGGGAGTCACTTTTCCAAATGTTGATGTTTTTGTCATCAACAGTG AGCACCCAAACTTCACTAAATCCGCACTGATACAAATGGCTGGACGTGTGGGTCGTAGTCCTGAACGTCC

[0203] AACAGGCTTAGTTAGTTTTTTCATTACGGAAAATCCAAAGCAATGTGTCAGGCAGTTAGAGAAATCAAAAA ATGAACCAACTGGGAGGTTTTTCATGATAAAGTGTTTGCTTTGTTCAAGAAATATTGCTCAAAACTTTCTAT TTAGTGAACTTTTCCTACTCAAAAGTCCTCAAAATCTTTTATGTTCTAAATGTCAAAAAAATTTTGAAAAGAT CCCTGAAGCTCACTGTCCTTGCTGCTGTAAGCCAAATGTCCATGAGATCTGTACTGATTGTCAAAATTGGG

[0204] AAAAAAGAGGATACAAAATCAGACATCAGGCAATTTTTCACTACAATCAAGCAATGAAAGATTATTTTAGCC AGTATAAATTTGTTGGAGATTATCGATTATACCAAATTTTTGAGCCCTATTTTAAAAATATTTCCCAAAAATC TCCTTTGGTTCCAATTCCTATTTCACCAAAAAGATTAGAGGAACGTGGTTTCAATCAAGTGACTGCTTTTCT TCAACAAGATAATTTTATTGAACTTCTTGAAAAAGAGAATTCGGTCAAACAATCTAGCCTTAATCGAAAAGA

[0205] ACGATTGGAAAGTCCAAATCCTTTTCGATTAAAAAAAGGGCTAAAAGTCCCTACAAAAGTTATATTAATTGA CGATATTTATACAACAGGTACCACCTTATATCACGCATCACAAATTCTAAAAGAAGCTGGTGTTCATGAAAT AAGGAGCTTTTCTCTCTGCTGTTAA SEQ ID NO: 298 comFA [DGCC12653_02995]

[0206] ATGAGCACTAATCAAGAAAAGTTATTTGGCCGTTTATTATTAAAAAATGATATTTTACAACTTATAAAAAGTA

[0207] CTGACAAGATTTCTGTCAGTAAAATTTTTAGTAATTTTTTGTTAGAAGCGAAGGTGAATCCAATTTTGGGAA

[0208] TGACTTCAATTTCTTCCAATAAAATAAAATGTAACCGTTGTGGGACTGTTCATATAAAAAATTCTGTCAAACT

[0209] TCCAATTGGTGTATTTTACTGTCCAAGTTGTATTCAATTAGGTCGAGTCCGCTCCGATGAATTCTTGTACTT

[0210] TCTGCCACAAAAGAATTTCCCAAAGAAATCATATATAAACTGGTCGGGAAAACTGACAGAGAATCAAAAAT

[0211] CAATTTCAAATGCCCTCTGTCAGGAAATTAATTCTCATCAGCAAATAATTGTCCAAGCTGTGACTGGAGCTG

[0212] GAAAAACTGAAATGATTTATCAAGTCATTGAGCAAATTTTAGAAAGCGGTGGAGTTGTTGGTCTAGCTAGT

[0213] CCAAGAATTGATGTTTGTCTTGAACTTCATCAGCGATTATCACGTGATTTTTCCTGTAAGATTCCACTCTTAT

[0214] ATCATGATGGCGACAGCTATTTCCGAGCTCCATTAATAATAATGACCAGTCATCAGCTTTTACGTTTCAAGG

[0215] AAGCTTTTGATTTGCTGATTATTGATGAAGTTGATGCCTTTCCCTTTAGAGATAATGAAATGCTTTATTTTGC

[0216] GGCAGAAAAAGCAAGAAAAATAGAAGGAAATTTAATATATTTGACCGCAACTTCTACTGACAAACTTGAAA

[0217] AAGATATAAAAAAGCAAAAACTCTATCCTTTGTTTCTCCCGCGTCGTTTTCACAATTTCCCTTTAGTGGTGC

[0218] CTAAATTTTTTTGGAAAAATAAATTTGATAAGAAATTAATTGAGCAAAGAAATAGTGGCTTTCCTCTTCTTAT

[0219] TTTTGCTGCTGAAATTGAATCTGGACAAGAATTTGCAAAACAACTACAATTGAAATTTCCTAAAGAAAAAAT

[0220] TACTTTCGTTGCTTCAACAACAAAAGATAGATTGGAAATTGTTAAAGCTTTTAGAAATAAAGAAATTACTATT

[0221] TTAATAGCGACATCAATTCTTGAACGGGGAGTCACTTTTCCAAATGTTGATGTTTTTGTCATCAACAGTGAG

[0222] CACCCAAACTTCACTAAATCTGCACTGATACAAATGGCTGGACGTGTAGGTCGTAGTCCTGAGCGTCCAAC AGGCTTAGTTAGTTTTTTTCATTACGGAAAATCCAAAGCAATGTGTCAGGCAGTTAGAGAAATCAAAAAAAT GAACCAACTGGGAGG I I I I I CATGA

[0223] SEQ ID NO: 299 comEC* [MG1363_RS09775]

[0224] ATGAGATTTCTATTGATTAAATTTACCCTGATTTTTCTAACTTATCTTGTTGTGCTACTTTATTTTTGTATCTT

[0225] TACTTACAATTTACCCTTGCTTATTTTATTTATTTGCTCAATTTTACTAGCTTTATTCAGAAAATATTATCTAA

[0226] TGATTCCGATATTAATTGTTTTTGCTGTATTTTTTCTGTTAATCAATAGTCTTAGTATTAGGGATAATGAAAA

[0227] TCAACCAAAAAATCTCTCAAAAATTGAATTAATTCCAGATACAATCCAAGTCAATGGTGATTTATTGAGTTTT

[0228] CAGGGGAGAATGAATGGACAAAAATATCAAGTCTATGACACATTGAAAAGTCTAAAAGAGAAAGAGTTTTA

[0229] TCAGAACTTAAGTCAAAATTGTCAGTTAAGTTTCACAGGAAACCTTGAAATACCAGAAATTCAGCGAAATTT

[0230] TAATGGTTTTGATGACCAAAAATATTTAGCTAGTCAAAATATCTATCGACAAATTACCATCCAGCAAATTAC

[0231] AAATGTGGAAATAAAAAAGAATTTTGACCTTCATACTTTAAGGCGAAAGGCAATTGTTTGGACACAAAATC

[0232] ATTTTCCAAAACCGATGAGTGACTATATGACAGGTTTGCTTTTTGGCTTTTTAAGTAAGGATTTTAATCAGA

[0233] TGAGCGATATTTACAGCTCTTTAGGAATTATTCATCTTTTTGCTCTATCAGGAATGCAAGTAAATTTTTTTAT

[0234] TGACTGGTTTCGTAAAATACTACTTCGCATGGGACTGACGAGAGAAAGAGTGAATCTTGTTCAAATTCCAT

[0235] TTTCGATTTTTTATGCTTTTATGACGGGATTATCGGTTTCAGTTTTAAGAGCCCTTTTTCAAAAAAATATTCG

[0236] TCTGACTTCTTTGGATAATATCGGAGTGACGACATTAGTTTTGATGATCATAAGTCCAAAATTTTTACTGAC

[0237] GACTGGCGGTCAGCTTACCTTGTTTTATGCTTTTGTCATTTCGATGCTTAACCATTCTTTTTCTAATTTAAAT

[0238] GGATTGAAAAAGTGCTGACAGAATCATCAGTCCTTTCATTTTGTGTTCTTCCGTTACTCATCTTTGATTTCC

[0239] ATATTTTTCAGCCTCTTTCAATATTATTAACAATTATATTTGGTTTTCTTTTTGATTTTATGCTGCTCCCGCTG

[0240] CTTTTATGCTGTTTTCTTATATCATTTTTGGGATTTTCGCTCAATTTAAATTTTATTTTTCAAATTCTAGAATG

[0241] GCTAGTTCATAAAGTTGATTTACCCTTACATTACCCTTTTGTTTTGGGGAAACCTAGTTCACTCGAATTAATT

[0242] ATTCTATTTGTGTTAATTGGTTTATTCGTAGATTCATTTTCTCATAAAAAAAGGAGATTTTTTTTAATTGGATT

[0243] TATATTTTTATTTTTTTTCATCACTAAAAATCCAATGAGAGCAAGCATTACAGCGGTTGATATCGGACAAGG

[0244] CGATAGTATTTTTCTACAAGATAAGTTTAATAAAGAAAACATTCTGATTGATACTGGGGGACAACTTGCTCT

[0245] TCCTCAAAAAAATTGGCAAAAAGCGCAGACGCAATCAAATGCTGACAAAACGCTTATTCCTTATTTAGAATC

[0246] CATGGGGGTGGCTCATATTGACCAACTTATTCTGACTCATACAGATGCTGACCATGTTGGTGATTTCTTATC

[0247] CTTAGCTGATAAAATAAAAATAAGAGAAATCTGGGTCAGTCCGGGAGAATTAACCAACAGTAGTTTTGTGG

[0248] AAAAATTAAAAAAAGCAAAAATTCCGATTCATGTTAGTAAAGTTGGGGATAAAATTCCGATTTTTGATAGTT

[0249] TTCTTCAAGTTTTATCAAATGGTTACACTAAAAAAGGTGATAATAATGATTCCATAGTGACTTATGGAAACT

[0250] TTTACCATACAAAATTTCTTTTTACAGGAGATTTGGAACAAGAAGGAGAAAAAAAGCTTCTACAAAATTATC

[0251] CCAAATTACAGGTAGATGTGTTAAAAGTGGGGCATCATGGCAGTAAAACAAGTTCAAGTCCGGAATTCATC

[0252] AAAGAGATACATCCTAAACTTGCACTAATCTCGGTTGGAAAAGCTAATCGCTATGGTCATCCCAATCAAGA AACTTTAGAAACCTTAGCCAAATATCAGGTAAAGACACTCAGAACTGACCAAAAAGGGGCAATAAAATTAA CAGAAGTAAATCAGCAATGGCAAATCAAAACAGTTAGATAA

[0253] SEQ ID NO: 300 comEC [KW2_1750] ATGAGATTTCTATTGATTAAATTTCCCCTGATTTTTCTAACTTATCTTGTTGTGCTCCTTTATTTTTGTATCTT

[0254] TACTTACAATTTACCCTTGCTTATTTTATTTATTTGCTCAATTTTACTAGCTTTATTCAGAAAATATTATCTAA

[0255] TGATTCCGATATTAATTGTTTTTGCTGTATTTTTTCTGTTAATCAATAGTCTTAGTATTAGGGATAATGAAAA

[0256] TCAACCAAAAACTCTCTCAAAAATTGAATTAATTTCAGATACAATTCAAGTCAATGGTGATTTATTGAGTTTT

[0257] CAGGGGAGAATGAACGGACAAAACTATCAAGTCTATGACACATTGAAAAGTCTAAAAGAGAAAAAATTTTA

[0258] TCAGAACTTAAGTCAAAATTGTCAGTTAAGTTTCACAGGAAACCTTCAAATACCAGAAACTCAGAGAAATTT

[0259] TAATGGTTTTGATGACCAAAAATATTTAGCTAGTCAAAATATCTATCGACAGATTACCATCCAACAAATTAC

[0260] AAATGTGGAAATAAAAAAGAATTTTGACCTTCATACTTTAAGGCGAAAGGCAATTGTTTGGACACAAAATC

[0261] ATTTTCCAAAACCGATGAGTGACTATATGACAGGCTTACTTTTTGGCTTTTTAAGCAAGGATTTTAATCAGA

[0262] TGAGCGATATTTACAGCTCTTTAGGAATTATTCATCTTTTTGCTCTATCAGGAATGCAAGTAAATTTTTTTAT

[0263] TGATTGGTTTCGTAAAATACTTCTTCGCATGGGACTGACGAGAGAAATAGTGAATCTTGTTCAAATTCCATT

[0264] TTCTATTTTTTATGCTTTTATGACGGGATTATCAGTTTCAGTTTTAAGAGCCCTTTTTCAAAAAAATATTCGT

[0265] CTGACTTCTTTGGATAATATCGGAGTGACGACATTAGTTTTGATGCTCATAAGTCCAAAATTTTTACTGACG

[0266] ACTGGCGGTCAGCTTACCTTGTTTTATGCTTTTGTCATTTCGATGCTTAACCATCCTTTTTCTAATTTAAATG

[0267] GATTGAAAAAAGTGCTGACAGAATCAGCAGTCCTTTCACTTTGTGTTCTTCCGTTACTCATCTTTGATTTCC

[0268] ATATTTTTCAGCCTCTTTCAATATTATTAACAATTATATTTGGTTTTCTTTTTGATTTTATGCTGCTCCCGCTG

[0269] CTTTTATGCTGTTTTCTTATATCATTTTTGGGATTTTCGCTCAATTTAAATTTTATTTTTCAAATTCTAGAATG

[0270] GCTAGTTCATAAAGTTGATTTACCCTTACATTACCCCTTTGTTTTGGGGAAACCTAGTTCACTCGAATTAATT

[0271] ATTCTATTTGTGTTAATTGGTTTATTCGTAGATTCATTTTCTCATAAAAAAAGGAGATTTTTTTTAGTTGGAT

[0272] TTATATTTTTATTTTTTTTCATCACTAAAAATCCAATGAGAGCAAGCATTACAGCGGTTGATATCGGACAAG

[0273] GCGATAGTATTTTTCTACAAGATAAGTTTAATAAAGAAAATATTCTGATTGATACAGGGGGGCGACTTGCT

[0274] CTTCCTCAAAAAAATTGGCAAAAAGCGCAGACGCAATCAAATGCTGACAAAACGCTTATTCCTTATTTAGAA

[0275] TCCATGGGGGTGGCTCATATTGACCAACTTATTCTGACTCATACAGATGCTGACCATGTTGGCGATTTCTT

[0276] GTCCTTAGCTGATAAAATAAAAATAAGAGAAATCTGGGTCAGTCCGGGAGAATTAACCAACAGTAGTTTTG

[0277] TGGAAAAATTAAAAAAAGCAAAAATTCCGATTCATGTTAGTAAAGTTGGGGATAAAATTCCGATTTTTGATA

[0278] GTTTTCTTCAAGTTTTATCAAATGGTTACACTAAAAAAGGGGATAATAATGATTCCATAGTGACTTATGGAA

[0279] ACTTTTACCATACAAAATTTCTTTTTACAGGAGATTTGGAACAAGAAGGAGAAAAAAAGCTTCTACAAAATT

[0280] ATCCCAAATTACAGGTAGATGTGTTAAAAGTGGGGCATCATGGCAGTAAAACAAGTTCAAGTCCGGAATTC

[0281] ATCAAAGAGATACATCCTAAACTTGCACTAATCTCGGTTGGAAAAGCTAATCGCTATGGTCATCCCAATCA

[0282] AGAAACTTTAGAAACCTTAGCCAAATATCAGGTAAAGACACTCAGAACTGACCAAAAAGGGGCAATAAAAT

[0283] TAACAGAAGTAAATCAGCAATGGCAAATCAAAACAGTTAGATAA

[0284] SEQ ID NO: 301 co / A* [MG1363_RS09560]

[0285] ATGCTGACAGCAATTGATGAAAATAATAAAATAATAAACTTACTGGAATTAGACCGCAAAGAATTGACCGG

[0286] AAAATTTTATTGTCCTTCTTGTCATTCAGAGCTACTTATTAAAAATGGGCAGATAAAAGTTCTTCATTTTGCC

[0287] CATAAATCTCTAAAGAGCTGCAATTTATGGTTGGAAAATGAGTCAGACCAGCATTTAGGTTTGAAAAAAAT

[0288] ATTATATTAGTGGTTTAAAAAGACTGACAAAGTCGAAATTGAACGCTATTTGCCCGAACTTAACCAAAGACC

[0289] AGATTTATTAGTAAATGACAAAATCGCTATTGAGATACAATGCTCTCATTTATCTATTAAGCGTCTCAAAGA

[0290] ACGTACAGAAAATTATAAGGCGCATGGCTTTAAAGTGCTTTGGTTGATGGGAAAGGATTTATGGCTGGCTG

[0291] AACAGGTGACAGAACTTCAAAAAAATTTAGTTTATTTCTCGGAAAATAGAGGATTCTTTTATTGGGAGTTAG

[0292] ATTTTCAGAGGAAAAAATTAAGATTAAAATCTCTTATTCATGAGGATTTACGTGGCAGAATCATTTGCTTAC

[0293] AAGAGGAAATTCCTTTTGGAAAAGGACGACTTATTGCGCATCTACGCTTACCTTATTTGGCTCAAAAACTG

[0294] GTGAAAATTCCTACTTTTAAAGATTCTAAACTTTCTAGTTTTATTCGTCAGCAATTATACTATCAATCACCAA

[0295] AATGGATGAAAATTCAAGAAAAATATTATCAAAAAGGAGAAAATTTACTGACGAAAAAATTTGAAGGTCCAT

[0296] ACATTGCTCCACTAGGTTTAAATTTACTAGAGAATTTTACTGACGAAATGACAATCACAACTTTTACTCAGA

[0297] TTGATCAAAATGTTAAGCTTTACTATGAAAATTTTTTAATAAATTTCCAAAGAAATAGCCTTGAAATGCTCTA

[0298] TCCTCCCCGTTTCTATGCTATAATGGGAAAGCAGAAGAAGGAAAAATAA

[0299] SEQ ID NO: 302 coiA [KW2_1705]

[0300] ATGCGGACAGCAATTGATGAAAATAATAAAATAATAAACTTACTGGAATTAGACCGCAAAGAATTGACCGG

[0301] AAAATTTTATTGTCCTTCTTGTCATTCAGAGCTACTTATTAAAAATGGGCAGATAAAAGTTCTTCATTTTGCC

[0302] CATAAATCTCTAAAGAGCTGCAATTTATGGTTGGAAAATGAGTCAGAACAGCATTTAGGTTTGAAAAAAAT

[0303] ATTATATCAGTGGTTTAAAAGAACTGACAAAGTCGAAATTGAACGCTATTTGCCCGAACTTAACCAAAGAC

[0304] CAGATTTATTAGTAAATGACAAAATCGCTATTGAGATACAATGCTCTCATTTATCTATTAAGCGTCTCAAAG

[0305] AACGTACAGAAAATTATAAGACGCATGGCTTTAAAGTGCTTTGGTTGATGGGAAAGGATTTATGGCTGGCT GAACAGGTGACAGAACTTCAAAAAAATTTAGTTTATTTCTCGGAAAATAGAGGATTCTTTTATTGGGAGTTA GATTTTCAGAGGAAAAAATTAAGATTAAAATCTCTTATTCATGAGGATTTACGTGGCAGAATCATTTGCTTA CAAGAGGAAATTCCTTTTGGAAAAGGACGACTTATTGCGCATCTACGCTTACCTTATTTGGCTCAAAAACT GGTGAAAATTCCTACTTTTAAAGATTCTAAACTTTCTAGTTTTATTCGTCAGCAATTATACTATCAATCACCA AAATGGATGAAAATTCAAGAAAAATATTATCAAAAAGGAGAAAATTTACTGACGAAAAAATTTGAGGGTCC ATACATTGCTCCACTAGGTTTAAATTTACTAGAGAATTTTACTGACGAAATGACAATCACAACTTTTACTCA GATTGATCAAAATGTTAAGCTTTACTATGAAAATTTTTTAATAAATTTCCAAAGAAATAGCCTTGAAATGCTC TATCCTCCCCGTTCCTATGCTATAATGGGAAAGCAGAAGAAGGAAAAATAA

[0306] EXAMPLES

[0307] Bacterial strains, plasmids, and oligonucleotides. Bacterial strains, plasmids, and oligonucleotides used in this work are listed below.

[0308] Growth conditions. L. lactis strains were cultivated in M17 (Difco Laboratories, Detroit, MI) or CDM (adapted from Sissler M. et al, 1999) supplemented with 0.5% (w / v) glucose (M17G and CDMG, respectively), or supplemented with 0.21% (w / v) glucose and 1.1% (w / v) secondary sugar (CDM*) when required, at 30°C without agitation. The composition of the CDM is as follows: NH4CI 0.5 g I-1, xanthine 0.01 g I-1, adenine 0.01 g I-1, uracil 0.01 g I-1, guanine 0.01 g I-1, K2HPO4 7.5 g I1, MOPS 8.3 g I1, NaCI 2 g I1, MgSO40.08 g I1, FeSO40.004 g I1, MnCI20.0012 g I-1, glutamine 0.1 g I-1, asparagine 0.1 g I-1, riboflavin 0.001 g I-1, pantothenate calcium 0.001 g I’1, nicotinic acid 0.001 g I-1, pyridoxine 0.001 g I-1, thiamine HCI 0.001 g I-1, biotin 0.001 g I-1, tyrosine 0.05 g I-1, cysteine 0.1 g I-1, isoleucine 0.1 g I-1, leucine 0.1 g I-1, methionine 0.1 g I-1, tryptophan 0.1 g I-1, valine 0.1 g I-1, histidine 0.1 g I-1, arginine 0.1 g I-1, glycine 0.1 g I-1, lysine 0.1 g I-1, phenylalanine 0.2 g I-1, threonine 0.2 g I-1, alanine 0.3 g I-1, proline 0.3 g I-1, serine 0.3 g I-1, aspartate 0.2 g I-1, glutamate 0.2 g I-1. Glutamate, aspartate, xanthine, adenine, uracil, and guanine were omitted in the competence-optimized CDM (CDM*-DEB). Escherichia coli ToplO was cultivated at 37°C in LB (Lysogeny Broth) medium with agitation. Solid agar plates were prepared by adding 2% (w / v) agar to the medium. When required, 5 pg ml1of erythromycin, 1 mg ml1of streptomycin, 0.5 mg ml1of spectinomycin, and / or 10 pg ml1of chloramphenicol were added to the medium for L. lactis; and 250 pg ml1of erythromycin, 250 pg ml1of ampicillin, and 10 pg ml1of chloramphenicol for E. coli.

[0309] DNA techniques and electrotransformation. Electrotransformations of L. lactis (Holo and Nes, 1989) and E. coli (Dower et a / ., 1988) were performed as previously described. Chromosomal DNA of / ., lactis used as template for PCR was extracted as previously described (Ferain et al., 1996). PCRs were performed using the Q5 DNA polymerase (NEB) in a GeneAmp PCR system 2400 (Applied Biosystems). Isolation of a rpsL* mutant conferring resistance to streptomycin in L. lactis. Spontaneous streptomycin-resistant IL1403 mutants were isolated on 1 mg ml1streptomycin-containing plates. After the sequencing of the rpsL gene with primers BID_RpsLUnivUp / BID_RpsLUnivDown, one spontaneous mutant containing the mutation K56R into the ribosomal protein S12 (SEQID 10), that was previously shown to confer resistance to streptomycin, was retained (David et al. 2017). A 3.7-kb fragment containing the rpsL mutated gene strA2 allele, SEQ ID 14, named rpsLLL*) was amplified by PCR with primers BID_LLcfusARpsL / BID_LLIdacARpsL and cloned into the pGEM®-T Easy vector (Promega), yielding plasmid pGEM-rps!.*. This plasmid was used as template to generate the 3.7-kb PCR product (named rpsL*) with primers BID_LLcfusARpsL / BID_LLIdacARpsL that was used as donor DNA in natural transformation assays.

[0310] Isolation of a rpsL* mutant conferring resistance to streptomycin in L. cremoris. Spontaneous streptomycin-resistant MG1363 clones were isolated on 1 mg ml1streptomycin containing plates. After the sequencing of the rpsL gene with primers RpsLUnivlIP / RpsLUnivDN, one spontaneous mutant resulting in a mutation (K56I) into the ribosomal protein S12 (SEQ ID 12) that was previously shown to confer resistance to streptomycin was selected. A 3.7-kb fragment containing the rpsL mutated gene (strAl allele, SEQ ID 16, named rpsLcc*) was amplified by PCR with primers BID_LLcdacARpsL / BID_LLIcfusARpsL and cloned into the pGEM®-T easy vector (Promega), yielding plasmid pGEM-rpsf.MG*. This plasmid was used as template to generate the 3.7-kb PCR product (named rpsL c*) with primers BID_LLcdacARpsL / BID_LLIcfusARpsL that was used as donor DNA in natural transformation assays of strain KW2.

[0311] Xylose-induced comX expression and natural transformation. The L. lactis strains containing plasmid pGhPx / / T-comX (Px / / r and comX from L. lactis IO-1) were grown overnight at 30°C. Cells were washed twice in distilled water, and OD6oo was adjusted to 0.1 in M17 supplemented with 0.1% (w / v) glucose and 1% (w / v) xylose and erythromycin. Typically, 2 pg of DNA was added to 100 pl of inoculated transformation medium, and the culture was further incubated during 24 hours at 30°C. Cells were then spread on M17G agar plates supplemented with appropriate antibiotics, and CFUs were counted after 48 hours of incubation at 30°C. The transformation rate was calculated as the number of antibiotic-resistant CFU ml1divided by the total number of viable CFU ml-1.

[0312] Spontaneous transformation assays. After an overnight preculture in M17G at 30°C of L. lactis DGCC12653 (and other strains or mutants), 100 p\ of the preculture was diluted in 900 p\ of fresh M17G to restart the culture. After 2 hours of growth, cells were washed twice in distilled water and inoculated to a final OD6oo of 0.1 in competence-activating medium (CDM* or CDM*- DEB). Typically, 1.5-2.0 pg of donor DNA was added to 100 pl of transformation medium, and the culture was further incubated for 24 hours at 30°C. Cells were then spread on M17G agar plates supplemented or not with appropriate antibiotics and CFUs were counted after 48 hours of incubation. The transformation rate was calculated as reported above. The transfer of the mutation conferring streptomycin resistance was confirmed by DNA sequencing of the rpsL gene after its amplification by PCR using primers BID_RpsLUnivUp / BID_RpsLUnivDown.

[0313] Detection of absorbance and luminescence. Overnight precultures in M17G were washed with sterile water and inoculated in the adequate medium to a final OD6oo of 0.1. After inoculation in 300 pl-culture volumes and before luminescence analysis, nonyl-aldehyde was diluted 100 times in mineral oil, and 50 pl of this preparation was disposed between the wells of a white 96- well plate with a transparent bottom (Greiner, Alphen a / d Rijn, The Netherlands). Growth (OD6oo) and luciferase activity (Lux) were monitored at 5-minute intervals in a Hidex Sense microplate reader (Hidex, Lemminkaisenkatu, Finland) for a maximum of 24 hours. The luciferase activity is expressed in relative light units (RLU) and the specific luciferase activity in RLU ODeoo1.

[0314] Construction of competence reporter plasmids. The Pcomx-luxAB reporter plasmid for luminescence assays was constructed as follows. The comX promoter was amplified by PCR from chromosomal DNA of L. lactis DGCC12653 with primers FT886_PcomX_fw / FT887_PcomX_rec_rv. The plasmid containing the luxAB genes pGhPComGA[MG]luxAB (David et al., 2017) was amplified by PCR using primers FT884_pGhPcomXlux_fw / FT925_PcomXlux_rec_rv. Both PCR fragments were joined by the Gibson assembly method (Gibson et al., 2009) and the resulting plasmid was named pGhPcomx- / t / AB. The PcomGA-luxAB reporter plasmid for luminescence assays was constructed as follows. The comGA promoter was amplified by PCR from chromosomal DNA of f., lactis IO-1 using primers LuxcIOFl_XhoI / LuxIORl. The plasmid pGhPcomGA[MG]luxAB was amplified by PCR using primers LuxcIOR2_XhoI / LuxIOF2. Both PCR fragments were joined by overlap PCR, digested by Xhol, and self-ligated using T4 DNA ligase. The resulting plasmid was named pGhP-

[0315] Construction of deletion mutants in L. lactis strains. The comX, comEC, ccpA, codY, covRS, and mecA genes as well as Rgg and TCSs encoding genes were similarly inactivated by the exchange of their coding sequences with either P32-cat or spc resistance cassette (conferring chloramphenicol or spectinomycin resistance, respectively) using double crossing-over events. For this purpose, overlapping PCR products containing the resistance cassette flanked by two recombination arms of ~1.2 kb (upstream and downstream homologous regions) were generated as previously reported (David et al. 2017). Briefly, upstream, downstream, and resistance-cassette fragments were separately amplified by PCR, purified, mixed in equimolecular amount, and assembled by overlapping PCR by using the forward and reverse primers of the upstream and downstream fragments, respectively (see Primers Table). Typically, 5 pg of the obtained overlapping PCR product was used as donor DNA for xylose-induced natural transformation of the L. lactis strains harboring plasmid pGhPx / / r-comX. The correct insertion of the resistance cassette in each targeted locus of the transformants was validated by PCR using primers in the resistance cassette and in the chromosome outside of the construct, (see Primers Table). To obtain the final mutant strains, the curing of the thermosensitive vector pGhPx / / r-com ' was performed by growing the cells overnight at 37°C without erythromycin. The cultures were subsequently diluted and plated on M17G agar without erythromycin at 30°C. The resulting colonies were streaked in parallel on M17G plates with and without erythromycin. The absence of plasmid pGhPx / / r-com ' in erythromycin-sensitive clones was validated by PCR.

[0316] Swapping of mecA genes. The mecA mutants (EmecA: mat) of strains DGCC12653 and DGCC12671 harboring plasmid pGhPx / / T-comX were used for this exchange as they display high rates of natural transformation. Both mecA genes from strains DGCC12653 and DGCC12671 were amplified by PCR with primers FT1319 / FT1320, generating ~5-kb fragments used as donor DNA. Replacement of the EmecA::cat deletion by the counterpart mecA gene was performed by xylose-induced natural transformation as described above. Five hundred isolated colonies were streaked in parallel on M17G-agar plates containing or not chloramphenicol, searching for antibiotic-sensitive transformants. Then, the swapping of mecA genes from sensitive clones was validated by PCR and sequencing. Finally, the curing of the pGhPx / / T-comX plasmid was performed as described above.

[0317] Construction of strain DGCC12653 nisRK for nisin-induced overexpression. The nisRK genes from strain IO-1 were inserted between DGCC12653_06785 and DGCC12653_06790 genes in DGCC12653 using double crossing-over events. For this purpose, an overlapping PCR product containing nisRK genes associated with the spc cassette in opposite orientation and flanked by two recombination arms of ~1.2 kb was generated as reported above (see Primers Table). The obtained overlapping PCR product (5 g) was used as donor DNA for natural transformation of strain DGCC12653 harboring plasmid pGhPx / / T-comX. The correct insertion of nisRK genes in the targeted locus of the transformants was validated by PCR. To obtain the final mutant strain, the curing of the thermosensitive vector pGhPx / / T-comX was performed as described above.

[0318] Purification of transcriptional regulators. The overexpression plasmids were constructed as follows. The ccpA, codY, and covR genes flanked by a sequence encoding an N-terminal 6-his tag were amplified by PCR from strain DGCC12653. For regulator overexpression, pBAD (for ccpA) or pNZ8048 (for codY and covR) were amplified by PCR and joined to the regulator-encoding genes by the Gibson assembly method (see Primers Table) (Gibson et al, 2009). The final constructs, pBAD-6his-ccpA, pNZ8048-6his-coc / Y, and pNZ8048-6his-cov / ? were validated by sequencing. The 6His-CcpA (rCcpA) protein was overexpressed in E. coli ToplO. The 6His-CodY (rCodY) and 6His-CovR (rCovR) were overexpressed in L. lactis DGCC12653 nisRK (nisin induction with 1 ng ml1). The recombinant proteins were purified using Ni-NTA resin according to the manufacturer's instructions (ProBond Resin, Novex).

[0319] Electromobility shift assays (EMSA). The whole promoter region of comX (PCOmx, intergenic region) from DGCC12653 was amplified by PCR using fluorescent-labeled primers (FT851_Cy3_PcomX_up / FT853_Cy5_PcomX_dw). In each assay, the purified proteins were incubated in 2-fold serial dilutions with a constant quantity of labeled probe (2.5 ng l-1). As a negative control, a 150-bp DNA sequence located in the coding region (CDS) of the dnaE gene was amplified by PCR using primers AK350 / AK303 (Knoops et al, 2022). CodY-binding assays were performed in CodY buffer (Tris-HCI 20 mM pH 7.5, NaCI 150 mM, BSA 1 mg ml-1, EDTA 1 mM, glycerol 10% [w / v]) at 30°C for 20 minutes. CcpA-binding assays were performed in CodY buffer supplemented with glucose-6-phosphate 25 mg ml1and poly-dldC 2 g ml1as reported above. CovR-binding assays were performed in Buffer-CovR (NaPO450 mM pH 6.5, NaCI 50 mM, MgCI21 mM, CaCI21 mM, DTT 1 mM, poly-dldC 2 pg ml-1, glycerol 10% [w / v]) at 30°C for 15 minutes. Prior to incubation with the DNA probe, CovR proteins were incubated in CovR buffer supplemented with 50 mM acetyl phosphate for 30 minutes at room temperature. Samples were migrated in native poly-acrylamide gels in non-denaturing MOPS buffer, and revealed by the Amer- sham Typhoon device.

[0320] RNA sequencing of L. lactis mutants. RNA extraction and sequencing were performed on the wild type, EcodY mutant, and EcovRS mutant of strain DGCC12653. Each mutant and the wildtype control were grown in their respective efficient competence-activating medium (CDM*-DEB and CDM* for EcodY and EcovRS, respectively) and harvested at their respective diauxic shift. RNA was extracted using the RNeasy plus bacteria kit (Qiagen) according to manufacturer indications. Total RNA was checked for integrity with an RNA Nano chip (Agilent Technologies) and sent for Illumina sequencing (GeneWiz). Raw data were processed on the Galaxy server (use.galaxy.org) using Bowtie2 algorithm to yield BAM files containing the read coordinates and Seqmonk to count the number of reads per coding sequence (CDS). The dataset was exported into an Excel file for further analyses. First, the dataset was standardized to CDS-mapped reads per ~10 million overall reads. Then, we estimated the ratio of CDS-mapped reads in mutants vs. WT.

[0321] Construction of competence inducer plasmids. The PmtiA-comX inducer plasmid for natural transformation was constructed as follows. The mtlA promoter (PmtM) was amplified by PCR from chromosomal DNA of L. cremoris MG1363 with primers FT3A_PmanT_rec_up / FT5A_PmanT_rev. The pGhPx / / r-com ' plasmid (David et al., 2017) was amplified by PCR using primers FT7A_comXMG_rec_fw / FT25L_pGh_fw. Both PCR fragments were joined by the Gibson assembly method (Gibson et al., 2009) and the resulting plasmid was named pGhPmtM-cornX. The ComRS induced plasmid for natural transformation was constructed as follows. The empty pG+host9 plasmid was amplified by PCR using primers FT25L_pGh_fw / FT26L_pGh_rv. The construct containing PcomR-cornR from Streptococcus thermophilus, the terminator tLDH from Lactobacillus plantarum, and shp0064 promoter (Ps / ,poos4) from S. thermophilus, was amplified from the pGIMCD107 plasmid (Duchene et al., 2019) with primers FT17A_Pshp0064_rec_rv / FT19A_pGh_Pshp0064_rec_fw. The gene comX was amplified from chromosomal DNA of f., cremoris KW2 with primers FT32A_comXkw2_rec_rv / FT458_XLS_UPF. These three PCR fragments were joined by the Gibson assembly method (Gibson et al., 2009) and the resulting plasmid was named pGhcomRS-comX.

[0322] Mannitol-induced comX expression and natural transformation. The L. cremoris MG 1363 strains containing plasmid pGhPmtM-comX were grown overnight at 30°C. Cells were washed twice in distilled water, and OD6oo was adjusted to 0.1 in M17 supplemented with 0.1% (w / v) glucose and 1% (w / v) mannitol and erythromycin. Typically, 2 g of donor DNA was added to 100 l of inoculated transformation medium, and the culture was further incubated during 24 hours at 30°C. Cells were then spread on M17G agar plates supplemented with appropriate antibiotics, and CFUs were counted after 48 hours of incubation at 30°C. The transformation rate was calculated as the number of antibiotic-resistant CFU ml1divided by the total number of viable CFU ml T

[0323] XIP-induced comX expression and natural transformation. The L. cremoris MG1363 strains containing plasmid pGhcomRS-comX were grown overnight at 30°C. Cells were washed twice in distilled water, and OD6oo was adjusted to 0.1 in M17G supplemented or not with XIP 10 pM and erythromycin. Typically, 2 pg of donor DNA was added to 100 pl of inoculated transformation medium, and the culture was further incubated during 24 hours at 30°C. Cells were then spread on M17G agar plates supplemented with appropriate antibiotics, and CFUs were counted after 48 hours of incubation at 30°C. The transformation rate was calculated as the number of antibioticresistant CFU ml1divided by the total number of viable CFU ml T

[0324] Construction of trans-repair plasmids. The Pxyn-comX-comFAC overexpression plasmid for trans-repair of 1AA59 L. lactis strain was constructed as follows. The locus containing the comFA-comFC operon and its promoter PcomFwas amplified by PCR from chromosomal DNA of L. lactis DGCC12653 with primers LPll_comFAC_rec_fw / LP12_comFAC_rec_rv. The plasmid containing the comX-overexpression construct pGhPx / / r-com ' was amplified by PCR using primers LPl_pGh_rev / LP2_pGh_fw. Both PCR fragments were joined by the Gibson assembly method (Gibson et al., 2009) and the resulting plasmid was named pGhPxyiT-comX-comFAC. The Pxyn- comX-dprA overexpression plasmid for trans-repair of IL1403 L. lactis strain was constructed as follows. The locus containing the dprA gene was amplified by PCR from chromosomal DNA of L. lactis DGCC12653 with primers LP3_dprA_op_rec_fw / LP4_dprA_op_rec_rv. The plasmid containing the comX-overexpression construct pGhPx / / r-comX was amplified by PCR using primers LPl_pGh_rev / LP2_pGh_fw. Both PCR fragments were joined by the Gibson assembly method (Gibson et a / ., 2009) and the resulting plasmid was named pGhPxyiT-comX-dprA.

[0325] Construction of c / s-repair plasmids. The pGhcomEA-rep plasmid for chromosomal repair of 1AA59 L. lactis strain by double-crossover homologous integration was constructed as follows. The locus containing the comFA gene was amplified by PCR, generating a ~2.800-bp DNA fragment centered around the comFA mutation. This PCR fragment was amplified from chromosomal DNA of L. lactis DGCC12653 with primers FT41L_PcomFAC2_rec_fw / FT42L_PcomFAC2_rec_rv. The empty pG+host9 plasmid was amplified by PCR using primers FT25L_pGh_fw / FT26L_pGh_rv. Both PCR fragments were joined by the Gibson assembly method (Gibson et al., 2009) and the resulting plasmid was named pGhcomFAC-rep. The pGhcomEC-rep plasmid for chromosomal repair of MG1363 L. cremoris strain by double-crossover homologous integration was constructed as follows. The locus containing the comEC gene was amplified by PCR, generating a ~5.200-bp DNA fragment centered around the comEC mutation. This PCR fragment was amplified from chromosomal DNA of f., cremoris KW2 with primers FT56L_comEC2_rec_fw / FT57L_comEC2_rec_rv. The empty pG+host9 plasmid was amplified by PCR using primers FT25L_pGh_fw / FT26L_pGh_rv. Both PCR fragments were joined by the Gibson assembly method (Gibson et al., 2009) and the resulting plasmid was named pGhcomEC-rep.

[0326] The pGhco / A-rep plasmid for chromosomal repair of MG1363 L. cremoris strain by double-crossover homologous integration was constructed as follows. The locus containing the coiA gene was amplified by PCR, generating a ~4.000-bp DNA fragment centered around the coiA mutation. This PCR fragment was amplified from chromosomal DNA of f., cremoris KW2 with primers FT48L_coiA_rec_fw / FT49L_coiA_rec_rv. The empty pG+host9 plasmid was amplified by PCR using primers FT25L_pGh_fw / FT26L_pGh_rv. Both PCR fragments were joined by the Gibson assembly method (Gibson et al., 2009) and the resulting plasmid was named pGhco / A-rep.

[0327] C / s-repair of mutated genes by double-crossover homologous integration. The mutated comFA, comEC and coiA genes were similarly repaired by exchange of their coding sequences with a functional version of it, by double-crossover homologous integration (Biswas et al., 1993). For this purpose, c / s-repair plasmids (pGhcomFAC-rep, pGhcomEC-rep and pGhco / A-rep) were electroporated in the appropriate strains. Cells were cultured in M17G supplemented with erythromycin overnight at 37°C to favor plasmid integration. After being plated as isolated colonies on M17G agar containing erythromycin, and grown overnight at 37°C, a series of colonies were picked and cultured overnight in M17G at 30°C to favor plasmid excision. The correct repair of mutated genes was validated by PCR and sequencing. To obtain the final repaired strains, the curing of the thermosensitive vectors was performed by growing the cells overnight at 37°C without erythromycin. The cultures were subsequently diluted and plated on M17G agar without erythromycin at 30°C. The resulting colonies were streaked in parallel on M17G plates with and without erythromycin. The absence of plasmids in erythromycin-sensitive clones was validated by PCR.

[0328] C / s-repair of mutated genes by natural transformation. The mecA mutant (AmecA.-.-cat) of strain IL1403 dprA* comX* harboring plasmid pG PxyiT-comX-dprA was used for the exchange of dprA gene as it displays high rates of natural transformation. The dprA gene from DGCC12653 was amplified by PCR using primers FT35L_dprA_locus_up / FT36L_dprA_locus_dw, generating a ~4-kb fragment used as donor DNA. Repair of dprA* by the functional dprA gene was performed by xylose-induced natural transformation, supplemented with rpsLLi* [dprA] [rpsL*] - 10: 1). This allowed selection of transformed cells on streptomycin. Twenty isolated colonies were validated for their dprA gene repair by PCR and sequencing. Three IL1403 dprA comX* mecA' rspL* clones harboring plasmid pG Pxyi—comX-dprA were subsequently transformed by natural transformation using 2 g of rpsLLi_ as donor DNA. Five hundred isolated colonies were streaked in parallel on M17G-agar plates containing or not streptomycin, searching for streptomycin-sensitive transformants, reaching strain IL1403 dprA comX* mecA'. The comX* gene was repaired exactly as for dprA, but comX gene from DGCC12653 was amplified by PCR using primers FT708_PxylcomX_fw / FT1412_comX_locus_dw, generating a ~3.5-kb fragment used as donor DNA. Finally, replacement of the EmecA::cat deletion by the original mecA gene was performed by xylose-induced natural transformation as described above. Five hundred isolated colonies were streaked in parallel on M17G-agar plates containing or not chloramphenicol, searching for chloramphenicol-sensitive transformants.

[0329] Swapping of mecA genes in IL1403. The mecA mutant (AmecA.-.-cat) of strain IL1403 harboring plasmid pGhPxyrrComX-dprA was used for this exchange as it displays high rates of natural transformation. The mecA gene from strain DGCC12653 was amplified by PCR with primers FT1319 / FT1320, generating ~5-kb fragments used as donor DNA. Replacement of the AmecA.-.-cat deletion by the counterpart mecA gene was performed by xylose-induced natural transformation as described above. Five hundred isolated colonies were streaked in parallel on M17G-agar plates containing or not chloramphenicol, searching for antibiotic-sensitive transformants. Then, the swapping of mecA genes from sensitive clones was validated by PCR and sequencing. Finally, the curing of the pGhPxyn-comX-dprA plasmid was performed as described above.

[0330] Table 1. Strains used and generated in these examples

[0331]

[0332] Table 2. Plasmids used and generated in these examples

[0333]

[0334] Apr, Emr, CnT, and Spcr: ampicillin, erythromycin, chloramphenicol, and spectinomycin resistance, respectively

[0335] Ts: thermosensitive

[0336] Table 3. Oligonucleotides used in these examples

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344] Example 1: Natural DNA transformation by comX overexpression in multiple L. lactis strains

[0345] The ability of ComX overproduction to achieve natural DNA transformation was tested in 15 Lactococcus lactis strains harboring the intact and full set of late competence (com) genes (16 genes), in addition to comX (Fig. 1A). For this purpose, an inducible comX expression plasmid (pGhPx / mo-co Xio) was generated by cloning the comX gene under the control of the xylose-inducible PxyiT promoter (both from L. lactis strain IO-1) in the thermosensitive plasmid pG+host9. The latter was introduced in 15 L. lactis strains by electroporation (Fig. IB). Transformation assays were performed in M17 supplemented with glucose 0.1% (w / v) and xylose 1% (w / v) with rpsL* as donor DNA (20 g / ml). Donor DNA rpsL* consists of a 3.7-kb fragment containing the rpsL mutated gene (strA2 allele, K56R), conferring resistance to streptomycin. After a 24-hours incubation, cells were spread on M17G (M17 with glucose 0.5%) agar plates supplemented or not with streptomycin and CFUs were counted after 48 hours of incubation. The transformation efficiency was calculated as the number of antibiotic-resistant CFU ml1divided by the total number of viable CFU ml1. All 15 strains harboring plasmid pGhPx / mo-cornXiOwere able to transform exogenous DNA, with rates of natural transformation from 1.7 x IO-7(DGCC12696 [pGhPx / mo- co Xio]) to 6.2 x IO-2(DGCC12671 [pGhPx / / no-co 'io]) (Fig. IB). These results show that natural DNA transformation can be achieved by comX overexpression in multiple L. lactis strains harboring the complete set of late com gene.

[0346] Example 2: Spontaneous DNA transformation in multiple L. lactis strains

[0347] The ability of spontaneous natural transformation (i.e, competence) was tested in 18 L. lactis strains harboring the intact full set of late com genes (16 genes), in addition to comX. For this purpose, these 18 L. lactis strains were cultured in M17G with donor DNA. Transformation assays were performed in M17 supplemented with glucose 0.5% (w / v) and rpsL* as donor DNA (20 g / ml) (Fig. 2A). After a 24-hours incubation, cells were spread on M17G agar plates supplemented or not with streptomycin and CFUs were counted after 48 hours of incubation. The transformation efficiency was calculated as described in example 1. The strain DGCC12653 showed spontaneous natural transformation in M17G with a rate of natural transformation of ~2 x IO-6(Fig. 2A). We validated that natural transformation took place through the transformation machinery since a deficiency in ComX or in the DNA channel ComEC abolished competence (Fig. 2B). Moreover, a time-lapse experiment showed that transformation occurred at the entry of the stationary growth phase (Fig. 2C), contrasting with competence development in streptococci that takes place during the early logarithmic phase. As L. lactis is believed to originate from plants, several plant sugars were tested as an alternative to glucose to potentially improve rates of natural transformation in DGCC12653. Transformation assays with DGCC12653 in M17 supplemented with maltose 1% (w / v) instead of glucose rpsL* as donor DNA) allowed increased transformation capacities up to ~10-5(Fig. 2D). These results show that the plant sugar maltose has a positive effect on competence efficiency in L. lactis.

[0348] In order to have a better control on various nutrients that could impact spontaneous natural transformation, transformation assays were performed in chemically-defined medium (CDM) with different mixtures of glucose and maltose, as carbon sources. These results showed that DGCC12653 is unable to achieve natural transformation in CDM supplemented with glucose alone, and at a low level with an alternative plant sugar as sole carbon source (Fig. 2E). However, mixing a small amount of glucose with an alternative sugar in excess, such as maltose, increased transformation efficiency above 5 x IO-6in the optimized proportions (glucose 0.21% [w / v] and maltose 1.1% [w / v]) (Fig. 2E). This competence-inducing medium was named CDM*. The effect of amino acid and nitrogen base (purines and pyrimidines) omissions was also investigated on spontaneous transformation. By removing aspartate, glutamate and nitrogen bases ( / .e., adenine, guanine, uracil, cytosine, and xanthine) from the CDM medium (named CDM*- DEB), the rate of natural transformation was ~10-fold increased, reaching ~4.5 x IO-5(Fig. 2F). In this optimized medium, various sugar combinations between glucose (0.21% [w / v]) and maltose, xylose, galactose, cellobiose, arabinose, or trehalose (all at 1.1% [w / v]) were also tested. The results showed spontaneous natural transformation with all sugar mixtures, the glucosemaltose mixture showing the highest performance (Fig. 2G). Finally, transformation assays in this optimized medium with rpsL* as donor DNA were performed with 8 L. lactis strains harboring the complete set of late com genes. In those conditions, spontaneous transformation was observed with a rate of natural transformation ranging from ~10-8to ~10-5. Among these strains, DGCC12653 remained the most efficient spontaneous transformer (Fig. 2H).

[0349] Together, these results highlight that a sugar diauxic shift improves the rate of natural transformation of a range of L. lactis strains, showing that growth on alternative carbon sources to glucose is a key physiological trait to activate spontaneous transformation in this species. Example 3. Involvement of CcpA in competence repression

[0350] To further investigate the role of the glucose-maltose diauxic shift on spontaneous natural transformation of strain DGCC12653, the expression of the comX gene was monitored. For this purpose, a luminescence reporter system was designed by cloning the luciferase genes luxAB under the control of the comX promoter (PCOmx) from DGCC12653 in pG+host9 plasmid (pGhPcomx- luxAB). Luminescence assays showed that comX expression was boosted after 7 hours of culture, which corresponds to the diauxic shift between glucose starvation and a second growth consuming maltose (Fig. 3A). Parallelly, transformation assays were performed by adding rpsL* DNA at the beginning of the culture and the rate of natural transformation was monitored every hour after DNA addition. While no transformation was observed before 7 hours of culture, transformation events occurred at the diauxic shift and increased until 10 hours post DNA addition to reach a maximum rate of natural transformation of ~6 x IO-5(Fig. 3A). These data show that the diauxic shift stimulates ComX production at a sufficient level to allow competence activation.

[0351] As a stimulating effect of the diauxic shift on competence development was observed, the role of the global carbon regulator CcpA (carbon catabolite control protein A) was investigated. A ccpA- deleted mutant was constructed (see Examples) and then transformed with the pGhPramx- / <JxAB reporter system. An increased expression of comX was observed in the early growth phase (~2- fold) along with a major growth defect for the ccpA mutant compared to the wild-type in optimized CDM*-DEB conditions (Fig. 3B). To test whether CcpA is directly involved in competence regulation by binding the promoter of comX (Pcomx), electrophoretic mobility shift assays (EMSAs) were performed with purified CcpA of strain DGCC12653 (see Examples). The purified protein was incubated with fluorescently-labelled PCOmx (Cy3Pcomx) as a DNA probe, which corresponds to the intergenic region located upstream of the comX gene. The results showed that CcpA significantly interacts with PCOmx compared to the control probe (CDS of dnaE) (Fig. 3C).

[0352] Together, these results show that a sugar diauxic shift activates comX expression at the transcriptional level. They also show that ccpA disruption increases comX transcription on glucose and that CcpA binds to the comX promoter.

[0353] Example 4. Involvement of CodY in competence repression

[0354] As starvation in puric and pyrimidic bases and amino acids impacts transformability, the potential role of the global nitrogen regulator CodY on competence control was investigated. For this purpose, a coc / Y-deleted mutant was constructed (see Examples) and transformed with the pGhP- comx-luxAB reporter system. CodY inactivation resulted in a nearly 10-fold increase in comX expression at the diauxic shift (Fig. 4A) and a drastic improvement of the rate of natural transformation (maximum of ~ IO-3) compared to the wild type in CDM*-DEB (Fig. 4B). In addition, a RNAseq analysis was performed to compare the wild type and the codY mutant using total RNAs extracted during the diauxic shift. This analysis confirmed the higher expression of comX along with the complete late com regulon in the CodY-deficient strain (Fig. 4C). These results show that CodY downregulates the expression of comX and late com genes. To question the possible direct interaction between CodY and PCOmx, CodY protein of strain DGCC12653 was purified for binding assays (see Examples). EMSAs performed withCy3Pcomx confirmed the direct binding of CodY to Pcomx (Fig. 4D). Subsequent analyses revealed that CodY binding was favored by the presence of branched-chain amino acids with the strongest effect observed with isoleucine (5 mM) (Fig. 4D).

[0355] Altogether, these results show that CodY represses competence development by downregulating comX expression through a direct binding on Pcomx in L. lactis.

[0356] Example 5. Involvement of CovRS in competence repression

[0357] In order to determine if competence in L. lactis is controlled by a cell-to-cell communication system or stress sensor(s), the ten members of the Rgg family and the eight complete two-compo- nent systems (TCS) were systematically inactivated in strain DGCC12653 (see Examples). The analyses of the mutants revealed that only CovRS inactivation allowed both an increase in comX expression (up to 15-fold) (Fig. 5A) and rate of natural transformation (up to ~ IO-3) (Fig. 5B). A RNAseq analysis was also performed to compare the transcriptomes of the covRS mutant with the wild type. Total RNAs were extracted from cells collected during the diauxic shift, when spontaneous transformation was shown to take place. This experiment confirmed the increase of comX expression, as well as the complete set of late com genes, in the covRS mutant compared to the wild type (Fig. 5C). To question the possibility that CovR directly regulates competence by binding PCOmx, the CovR protein from strain DGCC12653 was purified (see Examples) and EMSAs were performed withCy3PCOmx as a probe. EMSAs showed that CovR was able to bind PCOmx, thus acting as a direct repressor of comX expression in L. lactis (Fig. 5D).

[0358] Altogether, these results show that the general stress sensor system CovRS directly represses comX expression and thus transformation efficiency in L. lactis.

[0359] Example 6. Impact of initial pH on competence repression

[0360] As CovRS was previously shown to respond to pH in streptococci, the rates of natural transformation were monitored in a range of initial pH values (pH 5.5 to 8.0) (Fig. 6). Transformation assays were performed in a series of CDM*-DEB cultures individually adjusted for their initial pH, with rpsL* as donor DNA (20 g / ml). After a 24-hours incubation, cells were spread on M17G agar plates supplemented or not with streptomycin and CFUs were counted after 48 hours of incubation. The transformation efficiency was calculated as described in example 1. While spontaneous transformation in the wild-type strain was abolished below pH 6.3, the covRS mutant remained highly transformable in a much larger range of low pH values (Fig. 6).

[0361] Example 7. Involvement of MecA in competence repression

[0362] Variations of spontaneous natural transformation between strain DGCC12653 and other / ., lactis strains (such as DGCC12651, DGCC12671 and DGCC12678) in optimized conditions suggest an additional layer of regulation at the post-transcriptional level between ComX production and transcriptional activation of the late com genes. As it was previously reported that the MecA- CIpCP machinery known to degrade ComX negatively affects artificially-induced natural transformation of L. cremoris (David et al., 2017), the protein sequences of MecA, CIpC, and CIpP of strains DGCC12651, DGCC12671 and DGCC12678 were compared with those from strain DGCC12653. While alignments of CIpC and CIpP proteins showed no specific variation, the adaptor protein MecA displayed a single amino-acid change (L125R) that is only present in strain DGCC12653 (Fig. 7A). To test whether the L125R substitution could be responsible for the spontaneous transformability of DGCC12653, mecA genes were reciprocally swapped between strains DGCC12653 (R.125; variant) and DGCC12671 (L125; wild type) (Fig. 7B). We also generated mecA deletion mutants in both genetic backgrounds. Notably, we observed that DGCC12653 that produces the native version of MecA (L125) nearly lost its capacity to be transformed by exogenous DNA. Conversely, the production of the MecA variant (R.125) in a non-spontaneously transformable strain (DGCC12671) activated natural transformation at a rate comparable to a MecA- deficient strain (Fig. 7C).

[0363] In addition, the mecA gene was inactivated in 8 L. lactis strains harboring the complete set of late com genes (see Examples). Natural transformation assays performed in four different competence-inducing media rpsL* as donor DNA) showed that mecA deletion allowed transformation events to different extents in these 8 strains (Fig. 7D). Transformation assays were also performed in a mecA mutant of strain KW2 from L. cremoris in the same competence-inducing conditions (Fig. 7E). For these transformation assays, a 3.7-kb DNA fragment containing a mutated version of rpsL (rps / .MG*) isolated from L. cremoris MG1363 was used at the same concentration. The results showed an increased transformability of the mecA mutant compared to the wild-type (Fig. 7E). These results highlight that L. cremoris and L. lactis share a similar post- transcriptional regulation mechanism to control ComX abundance.

[0364] Together, these results showed that MecA plays a crucial role in competence repression of lactococci and that the single variation MecA-L125R explains the unique transformation behavior of strain DGCC12653. Comparison of competence regulation at the global level between Lactococcus, Streptococcus, and Bacillus

[0365] Together, these data depict a complete rewiring of the regulatory network controlling competence in L. lactis when compared to closely related streptococci (Fig. 8). Although ComX is the common central regulator of competence in both, the proximal regulation of its activation does not appear to be controlled in lactococci by a cell-to-cell communication module like in streptococci, but rather by global regulators sensing cellular nutritional status (CcpA and CodY) or stress conditions (CovRS). By comparison, global regulators are distantly controlling ComX by modulating the communication module in streptococci. Moreover, the post-transcriptional layer of regulation through ComX degradation by the MecA-CIpCP machinery plays a predominant role in L. lactis, while it is a secondary locking device in streptococci. In addition, the kinetics of competence activation during growth on a single carbon source are totally different: stationary phase in L. lactis and transitory during early logarithmic growth for streptococci, controlled by the on / off switch of the communication module. Notably, the timing of competence activation and the network hierarchy share some similarities with competence regulation in bacilli, where the central transcriptional regulator ComK is proximally modulated by global regulators and primarily controlled through degradation, which may or may not be regulated by a cell-to-cell signaling system.

[0366] Example 8: Natural DNA transformation by comX overexpression in a repaired version of L. lactis 1AA59.

[0367] The L. lactis strain 1AA59 harbors a single nucleotide deletion in the comFA gene, leading to a shift in the open reading frame and to a translational fusion with comFC comFA*), beside the 15 supposedly intact late com genes, and in addition to comX. The ability to achieve natural transformation by ComX production was tested in the presence of intact copies of comFA-comFC genes, either on a plasmid or in the chromosome (trans- and cis-repair, respectively) (Fig. 9A). For trans-repair, an inducible comX-comFAC-expression plasmid (pGhPxyrrio-comXio-comFAC) was generated by cloning the Pcomr-comFA-cornFC operon from L. lactis DGCC12653 downstream of the comX gene in the comX-expression plasmid (pGhPy / mO-comX / O). The comX-comFAC-expres- sion plasmid was introduced in L. lactis 1AA59 by electroporation. For c / s-repair, a ~2.800 bp region centered around the comFA single nucleotide deletion was amplified from L. lactis DGCC12653 and cloned in the thermosensitive plasmid pG+host9 (pGhcomFAC-rep). The latter was introduced in 1AA59 L. lactis strain by electroporation, and the allelic exchange was performed by double cross-over homologous integration (Biswas et al., 1993), resulting in the mutant 1AA59 comFA. This mutant was electroporated with the comX-expression plasmid (pGhPx / mO-comXro). Transformation assays were performed in M17 supplemented with glucose 0.25% (w / v) and xylose 1% (w / v) with rpsLLi* as donor DNA (20 g / ml) (Fig. 9A). After a 48- hour incubation, cells were spread on M17G-agar plates supplemented or not with streptomycin, and CFUs were counted after 48 hours of incubation. The transformation efficiency was calculated as the number of antibiotic-resistant CFU ml1divided by the total number of viable CFU ml- T Harboring a functional allele of comFA-comFC genes in the chromosome (c / s-repair) allowed a significant transformation of exogenous DNA, with rates of natural transformation of ~1 x IO-5(pGhPx / / TO-com ro), while trans-repair was much less efficient with only a few transformants per assay. These results show that natural transformation can be achieved by comX overexpression in a strain repaired for its com regulon.

[0368] As proportions of glucose and xylose could impact transformation efficiency, transformation assays were performed on the 1AA59 comFA (pGhPxy / no-com ro) mutant in M17 with different glucose concentrations and xylose 1% (w / v) (Fig. 9B). These results showed that natural transformation occurs with glucose concentrations ranging from 0.20% to 0.35% (w / v), reaching a maximum efficiency around glucose 0.25% (w / v) supplemented with xylose 1% (w / v).

[0369] Example 9: repairing dprA and comX genes unleashed spontaneous natural DNA transformation in L. lactis IL1403.

[0370] The L. lactis strain IL1403 harbors a single nucleotide deletion in the dprA gene (dprA*), along with a nucleotide substitution in the comX-start codon comX*), leading to an early stop codon and a late start codon, respectively. The ability of IL1403 dprA* comX* to perform natural DNA transformation was tested in presence (pGhPXyiTo-comXio-dprA trans -repair) and absence (pGhPxy / no-com ro) of a functional copy of the dprA gene (from strain DGCC12653) cloned in op- eron with comX on the expression plasmid (Fig. 10A). The same test was performed with the chromosomal repaired version of dprA gene in IL1403, also harboring either pGhPxymo-comX / o or pGhPXyiTo-comXio-dprA plasmids (Fig 10A). In order to repair the IL1403 strain in the chromosome, the dprA* mutated gene was replaced with a functional copy of dprA, initially amplified from L. lactis DGCC12653 strain. The allelic exchange was performed using natural DNA transformation (pGhPxyiTio-comXio-dprA) with a ~4.000-bp PCR fragment amplified around the dprA gene from L. lactis strain DGCC12653. Gene repair was validated by PCR and sequencing, and the partially repaired IL1403 dprA comX* mutant was obtained. Transformation assays were performed in M17 supplemented with glucose 0.1% (w / v) and xylose 1% (w / v). Two different donor DNA were tested : rpsLLi* and 07535: -.spec (10 g / ml). While rpsLLi* consists of an all-along high homology fragment, donor DNA 07535: :spec is composed by a spectinomycin-resistance cassette (specR: 1.032 bp) surrounded by ~1.3-kb up- and down-recombination arms corresponding to the environment of the yhgC (L162840) gene in IL1403 (DGCC12653_07535 in DGCC12653), in order to knock it out. After a 24-hour incubation, cells were spread on M17G agar plates supplemented or not with the adequate antibiotics, and CFUs were counted after 48 hours of incubation. The transformation efficiency was calculated as described in example 8. These results show that trans-repairing IL1403 with a functional copy of the dprA gene improves and even unleashes natural transformation with rpsLu* and 07535: -.spec as donor DNA, respectively. Additionally, bringing back a functional version of dprA gene in the chromosome still improved transformation rates. Finally, IL1403 harboring both a cis- and trans-repaired version of dprA displays the highest transformation rates, reaching 6.5xl0-2and 3xl0-3with rpsLLi* and 07535: -.spec, respectively.

[0371] In order to fully repair IL1403 strain in the chromosome, the comX* mutated gene was also replaced with a functional copy of comX, amplified from L. lactis DGCC12653 strain. The allelic exchange was performed as above using natural DNA transformation with a ~3.500-bp PCR fragment amplified around the comX gene from L. lactis strain DGCC12653. Gene repair was validated by PCR and sequencing, and the repaired IL1403 dprA comX mutant was obtained. This mutant was tested for its ability to transform rpsLLi* donor DNA by spontaneous natural transformation in the optimized medium CDM*-DEB supplemented with rpsLu* donor DNA and showed transformation capabilities, while no transformants were observed when cultured in CDM* (Fig 10B). The effect of MecA on spontaneous natural transformation was also investigated by generating MecAR125 (in which the original mecA allele [MecAL125: SEQ ID NO:1] has been replaced by the one from DGCC12653 [MecAR125: SEQ ID NO:2]) and MecA- (in which the mecA gene has been deleted) strains. These two mutants were constructed as described before, using the pGhPXyiTio-comXio-dprA overexpression system. As expected, the mecA' deletion mutant displayed a higher transformation rate than the native version (~50x) while the mutant MecAR125 showed an intermediate response (~13x) (Fig 10B).

[0372] In order to get rid of the sugar-controlled Pxyrr system, a heterologous comX-overexpression system (ComRS) was developed in the thermosensitive plasmid pG+host9 (Duchene et al., 2019). In this case, comXio was cloned under the control of the shp0064 gene promoter (Ps / ,poos4), along with the comR gene and its native promoter (PcomR-com / ?), all PCR-amplified from Streptococcus thermophilus (pGhcomRS-comXio). Thanks to the constitutive expression of comR, comX expression is triggered by adding XIP (ComX-Inducing Peptide) directly into the transformation medium, activating the ComRS-response mechanism (Fontaine et al. 2010).

[0373] This plasmid was also electroporated in the repaired strain IL1403 dprA comX. Transformation assays were performed in various media (M17 supplemented with glucose 0.5% [w / v], CDM*, CDM*-DEB and M17*), supplemented or not with XIP 10 M, and with rpsL * as donor DNA (20 g / ml) (Fig. 10C). After a 24-hour incubation, cells were spread on M17G agar plates supplemented or not with streptomycin, and CFUs were counted after 48 hours of incubation. These results show that the ComRS-based comX-overexpression system is functional in L. lactis using various growth media, and that CDM*-DEB seems to provide the best transformation environment. Example 10. Natural DNA transformation by comX overexpression in a repaired version of L. cremoris MG1363.

[0374] The L. cremoris strain MG1363 harbors a single nucleotide deletion in the comEC gene (comEC*), along with a nucleotide substitution in the coiA gene (co / A*), leading both to early stop codons. Both genes are sequentially repaired in MG1363 chromosome. To do so, regions centered around mutated genes {comEC and co / A) are amplified from L. cremoris KW2 and cloned in the thermosensitive plasmid pG+host9 (pGhcomEC-rep and pGhco / A-rep). These two plasmids are introduced in the L. cremoris MG1363 strain by electroporation and the allelic exchanges are performed by double cross-over homologous integration, reaching first the MG1363 coiA comEC* mutant and then the MG1363 coiA comEC mutant.

[0375] As MG1363 is unable to metabolize xylose, two new comX-overexpression plasmids are generated. The first one is based on the ComRS system from S. thermophilus, as described in example 9. However, in this case, comX is amplified from the L. cremoris strain KW2 and then cloned under the control of the shp0064 gene promoter (Ps / ,poos4), along with the comR gene and its native promoter (PramP-comR), all amplified from Streptococcus thermophilus pGhcomRSSt-comXKw2')- The second one is based on mannitol catabolism. To this end, comX is amplified from the L. cremoris strain KW2 and cloned under the control of the mtlA gene promoter (PmtM) from MG1363 (pGhPmtwMG-cornX / z^). These two plasmids are electroporated in the repaired and nonrepaired MG1363 strains. Transformation assays are performed differently for each comX-over- expression plasmid (Fig. 11). In strains harboring the pGhcomRSSt-comXKw2 plasmid, transformation assays are performed in M17 supplemented with glucose 0.5% (w / v) and XIP 10 pM with rpsLMG* used as donor DNA (20 g / ml). In strains harboring the pGhPmtiAMG-comXKW2 plasmid, transformation assays are performed in M17 supplemented with glucose 0.1% (w / v) and mannitol 1% (w / v) with rpsLMG* used as donor DNA (20 g / ml). After a 24-hour incubation, cells are spread on M17G agar plates supplemented or not with streptomycin, and CFUs are counted after 48 hours of incubation. The transformation efficiency is calculated as described in example 8. These results show that the repaired MG1363 strain is able to perform natural DNA transformation under ComX overproduction, while the strains harboring mutations in coiA and / or comEC are not.

[0376] REFERENCES

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Claims

CLAIMS1. A method for transforming a strain of a lactic acid bacterium, such as of the genus Lactococcus, with an exogenous DNA polynucleotide comprising the steps of:(a) providing a strain of the lactic acid bacterium, such as of the genus Lactococcus, which strain is transformable through natural transformation;(b) contacting the strain of step (a) with an exogenous DNA polynucleotide in a growth medium and incubating the resulting mixture for integration of the exogenous DNA polynucleotide or parts of it into the genome of the strain; and(c) selecting a derivative of said strain which has integrated the exogenous DNA polynucleotide or parts of it into its genome; which strain provided under step a) has any one or more of i) an inherently low, reduced or disrupted activity of the adaptor protein MecA relative to a control strain having a normal activity of the MecA protein; and / or ii) an inherently low, reduced, or disrupted activity of the global transcriptional regulator CodY and / or the two-component regulatory system CovRS relative to a control strain having a normal activity of CodY and / or CovRS; and / or iii) an inherently low, reduced, or disrupted activity of any of Clp proteins CIpP, and / or CIpC.

2. The method according to claim 1, which strain contains a MecA protein with an inherently low, reduced, or disrupted activity relative to a control strain due to a modification or disruption of the activity of the mecA gene encoding adaptor protein MecA in the strain, such as having an arginine in the protein sequence at a position corresponding to position 125 of SEQ ID NO:2 (R125), such as by having a single amino acid substitution from a leucine to an arginine in the protein sequence at a position corresponding to position 125 of SEQ ID NO: 1 (L125R), such as by swapping mecA genes or providing an extra copy of the mecA gene coding for an arginine in the protein sequence at a position corresponding to position 125 of SEQ ID NO:2 (R125), by deletion of the mecA gene, or by disruption of the mecA gene.

3. The method according to claims 1 or 2, which strain has an inherently low, reduced or disrupted activity of the of the global transcriptional regulator CodY and / or the two-component regulatory system CovRS due to a modification or disruption of the activity of the codY gene and / or any of the covR and / or covS genes in the strain, such as by deleting or mutating the codY, the covR and / or the covS genes in the strain.

4. The method according to any one of claims 1-3, which strain provided under step a) contain a gene encoding Carbon Catabolite Control Protein A (CcpA).

5. The method according to any one of claims 1-4, which strain has an inherently low, reduced, or disrupted activity of any of Clp proteins CIpP, and / or CIpC due to a modification or disruption of the activity of any of the c / pP and / or the dpC gene.

6. The method according to any one of claims 1-5, which strain performs with increased levels of natural transformation induced by increased levels of available and functional endogenous ComX protein in the strain relative to a control strain, which control strain does not have any of the inherently low, reduced or disrupted activities listed under i)-iii), such as by either induced or increased expression of an endogenous comX gene relative to the control strain, by a reduced ComX protein degradation, or by increased availability or abundance.

7. A method for transforming a strain of a lactic acid bacterium, such as of the genus Lactococcus, with an exogenous DNA polynucleotide comprising the steps of:(a) providing a strain of the lactic acid bacterium, such as of the genus Lactococcus, which strain is transformable through natural transformation;(b) increasing the competence of the strain for natural transformation;(c) contacting the strain of step (b) with an exogenous DNA polynucleotide in a growth medium and incubating the resulting mixture for integration of the exogenous DNA polynucleotide or part of it into the genome of the strain; and(d) selecting a derivative of said strain which has integrated the exogenous DNA polynucleotide or part of it into its genome; wherein this increase in the ability of the strain to perform natural transformation under step (b) is induced by either of i) growing the strain in a growth medium to induce a diauxic shift in the growth of the strain; and / or ii) growing the strain in a growth medium without any one or more of glutamate, aspartate, xanthine, adenine, thymine, uracil, cytosine and guanine; and / or iii) transforming through natural transformation under step b) and c) at a pH lower than about 8.5, such as in the pH range of 5.0-8.5.

8. The method according to claim 7, which increased ability of the strain to perform natural transformation is induced by increasing the level of available and functional endogenous ComX protein in the strain relative to a control strain, which control strain has not been induced to increase the level of endogenous ComX protein, such as by either inducing an increase in the expression of an endogenous comX gene relative to the control strain, or by inducing a reduction in ComX protein degradation.

9. The method according to any one of claims 1-8, which selected strain provides for a rate of at least lxlO-8transformants per g of exogenous DNA polynucleotide.

10. The method according to any one of claims 7-9, wherein the diauxic shift under i) is induced by growth of the strain in a medium with a) limited amount of glucose, such as with less than 0.40% (w / v), such as less than 0.35% (w / v), such as less than 0.30% (w / v), such as less than 0.25% (w / v), such as less than 0.23% (w / v), such as less than 0.20% (w / v) glucose; and b) at least 0.5% (w / v), such as at least 0.6% (w / v), such as at least 0.7% (w / v), such as at least 0.8% (w / v), such as at least 0.9% (w / v), such as at least 1.0% (w / v) of one or more secondary sugar, such as one or more secondary sugar selected from maltose, xylose, cellobiose, galactose, arabinose and trehalose.

11. The method according to any one of claims 1-10, where the lactic acid bacterium is of the genus Lactococcus, such as a strain selected from the species Lactococcus lactis, Lactococcus cremoris, Lactococcus laudensis, Lactococcus carnosus, Lactococcus formosensis, Lactococcus fujiensis, Lactococcus hircilactis, Lactococcus kimchii, Lactococcus paracarnosus, Lactococcus plantarum, Lactococcus raffinolactis, and Lactococcus taiwanensis.

12. The method according to any one of claims 1-11, which strain transformable through natural transformation provided under step (a) has been transformed with a plasmid expressing an exogenous comX gene having at least 90%, such as 95%, or 100% identity to the endogenous comX gene of the strain.

13. Use of a strain of a lactic acid bacterium, such as of the genus Lactococcus having either of i) an inherently low, reduced or disrupted activity of the adaptor protein MecA relative to a control strain having a normal activity of the MecA protein; and / or ii) an inherently low, reduced, or disrupted activity of the global transcriptional regulator CodY and / or the two-component regulatory system CovRS relative to a control strain having a normal activity of CodY and / or CovRS; and / or iii) an inherently low, reduced, or disrupted activity of any of Clp proteins CIpP, and / or CIpC; for transforming the strain through natural transformation.

14. A method for increasing the rate of natural transformation by competence of a strain of a lactic acid bacterium, such as of the genus Lactococcus, comprising the steps of(a) providing a strain of the lactic acid bacterium, such as of the genus Lactococcus, which strain is transformable through natural transformation;(b) performing any one or more of i) reducing or disrupting the activity of the adaptor protein MecA relative to a control strain having a normal activity of the MecA protein; and / or ii) reducing or disrupting the activity of the global transcriptional regulator CodYand / or the two-component regulatory system CovRS relative to a control strain having a normal activity of CodY and / or CovRS; and / or iii) reducing or disrupting the activity of any of Clp proteins CIpP, and / or CIpC; which strain has an increased rate of natural transformation by competence relative to a control strain not having reduced or disrupted activity under any of i) to iii).

15. A method for identifying a strain of a lactic acid bacterium, such as of the genus Lactococcus which is transformable through natural transformation comprising the steps of:(a) providing a strain of a lactic acid bacterium, such as of the genus Lactococcus;(b) measuring the activity of any one or more of i) the adaptor protein MecA relative to a control strain having a normal activity of the MecA protein; and / or ii) the global transcriptional regulator CodY and / or the two-component regulatory system CovRS relative to a control strain having a normal activity of CodY and / or CovRS; and / or iii) the activity of any of Clp proteins CIpP, and / or CIpC;(c) select a strain with a reduced or disrupted activity of any one of these proteins defined in (b) i)-iii);(d) contacting the strain obtained in step (c) with an exogenous DNA polynucleotide encoding a marker gene in a growth medium and incubating the resulting mixture for integration of the exogenous DNA polynucleotide into the genome of the strain; and(e) determining the rate of integration events; wherein a rate of at least 1 x IO-8transformants per g of DNA is indicative of a strain which is transformable through natural transformation.