A method for increasing the growth rate of halophilic archaea by mitigating the effects of proviruses within their genomes

Abstract

A method to accelerate the growth rate and enhance the defense mechanisms of halophilic archaea microorganisms by mitigating the effects of proviruses within their genomes. The method involves identifying and deleting integrated proviruses from halophilic archaea cell genomes, exemplified by Haloferax strain 48N, which is chronically infected by HLSV1, a lemon-shaped circular virus. Utilizing a pop-in / pop-out protocol, upstream and downstream flanking regions of the provirus are cloned into a suicide plasmid and transformed into the host cell which is then subjected to selection in growth medium to produce a cell with a deleted provirus. Provirus deletion increases the growth rate and enhances the defense mechanisms of microorganisms by reducing the metabolic strain imposed by such proviruses. The disclosure extends to enhancing the growth rates of other genetically modified microorganisms through provirus mitigation.

Description

A METHOD FOR INCREASING THE GROWTH RATE OF HALOPHILIC ARCHAEA BY MITIGATING THE EFFECTS OF PROVIRUSES WITHIN THEIR GENOMESSTATEMENT REGARDING SPONSORSHIP OF RESEARCH

[0001] The project leading to this application has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement No 787514).STATEMENT OF PRIORITY

[0002] This application claims priority to U.S. Provisional App. No. 63 / 589,848 and for A Method for Increasing The Growth Rate Of Archaea Haloferax Microorganisms by Mitigating The Effects Of Proviruses Within Their Genomes.BACKGROUND

[0003] A major challenge in microbiology is to understand virus-host interactions that involve chronic infections, where cells maintain their viability yet release virions over extended periods without clearing the infection. Such infections are expected to lead to a major loss of fitness for the infected strains, and consequently to their demise, yet in some environments can be paradoxically quite common.

[0004] Archaeal viruses represent a true frontier for virology research, showing an enormous diversity of morphology, egress mechanisms, and molecular biology. Ecologically, the impact of archaeal viruses on their hosts, in some ecosystems such as deep marine sediments, may be greater than that of bacteriophages, yet relatively few of them have been cultivated, compared to bacteriophages or eukaryotic viruses. Chronic infection appears to be common in archaea, and archaeal chronically-infecting viruses are often challenging to isolate since they do not form plaques on soft agar plates.

[0005] A chronically infected organism is one that harbors a persistent, long-term infection caused by a pathogen, such as a virus. This infection is characterized by the pathogen's ability to evade or modify the host's immune response and establish a sustained presence within the organism. Chronic infections can persist for multiplegenerations of the host organism, either through vertical transmission (from parent to offspring) or through repeated exposure to the pathogen.

[0006] Past studies of such viruses have thus relied on metagenomics and single-cell genomics to identify and quantify host-virus interactions in archaea- dominated ecosystems. These approaches have led to major discoveries, including the observation that in some extreme environments >60% of cells are infected with viruses and most of them contain two or more virus types. This has led to the hypothesis that in some natural environments, lysogeny, where a virus is completely dormant or exists as a relatively mild chronic infection, is not just tolerable but may actually be beneficial when it offers protection to cells against more virulent viruses. In other instances, as long as the growth delay is minimal, chronic infection can also be maintained when infected cells produced a toxin that kills off competing non-infected strains, as shown in thermo-acidophilic archaea.

[0007] In haloarchaea, chronic viral infections are common, yet unlike lytic infection, there has not been a good model system to study chronic infection until very recently. The newly established system of Haloferax volcanii pleomorphic virus 1 uses a virus isolated from an environmental sample, whose original host remains unknown, to successfully infect the lab strain of H. volcanii (DS2), the best studied archaeal model organism. This virus has a relatively minor effect on host growth, in line with the “tolerable infection” scenario.

[0008] Many archaea harbor multiple defense systems, including CRISPR-Cas systems, which provide acquired immunity. Chronically infecting viruses must thus avoid triggering such defenses, or subvert them, in order to establish a long-term coexistence with their hosts. In the case of HLSV-1 , lack of CRISPR-mediated acquired immunity by DS2 during exponential growth was attributed to a two-fold down-regulation of the acquisition module gene cas1 , yet no down-regulation was observed during stationary phase, despite sustained virus production.

[0009] The present disclosure broadly relates to genetically modified organisms cured of the chronic viral infections observed in their wildtype counterparts and methods of identifying and making said organisms. More specifically, the present disclosure relatesto the interaction of a chronically infecting lemon-shaped virus and its host the halophilic archaeon Haloferax volcanii 48N. By using a “gene therapy” approach in which the provirus was deleted from the genome, it is possible to cure archaea such as 48N of its virus. As disclosed, the phenotypes and gene expression of the cured strains are greatly improved over infected strains in terms of growth rate and enhanced immune responses. It was observed that under chronic infection, growth is dramatically delayed, anti-viral defense systems are transcriptionally repressed, and the CRISPR-Cas-based acquired immunity is disrupted. Furthermore, the disclosure demonstrates the potential for genetic engineering to mitigate the negative effects of chronic viral infections in archaea, opening up new avenues for research and potential applications in biotechnology.SUMMARY OF THE INVENTION

[0010] In extreme environments, archaea can have chronic viral infections, and such infections are well-tolerated by the hosts and may potentially protect against more lethal infections by lytic viruses. Typically, chronic infection by this virus is never cleared, despite the multiple defense systems of the host, such as CRISPR-Cas, two CBASS systems, and others. The virus subverts host defenses by reducing its transcription and interfering with CRISPR spacer acquisition machinery. The present disclosure shows that the slow growth of many halophilic archaea can be attributed to the effects of chronic infection by proviruses within their genomes which consume resources and alter the gene expression of their hosts.

[0011] According to some embodiments, the present disclosure relates to a natural Haloferax strain, 48N, which is chronically-infected by a lemon-shaped circular virus, named HLSV1 . As disclosed herein, using genetic approaches developed for Haloferax volcanii (H. volcaniifor short 48N was cured of its virus, leading to radical changes in the gene expression profile of 48N and a dramatic improvement in its growth rate. Remarkably, the cured 48N is the fastest-growing halophlic archaeon reported to date, with a generation time of about 1 hour at 45°C compared to the typical 2.5 hours of H. volcanii or its infected isogen.

[0012] According to some embodiments, the present disclosure relates to a method for increasing the growth rate of many halophilic archaea by mitigating the effects of proviruses within their genomes that consume resources and alter the gene expression of their hosts. In other aspects, the present disclosure relates to genetically modified microorganisms such as halophilic archaea having increased growth rates and enhanced immune systems through mitigation of proviruses within their genomes.

[0013] Therefore, based on the foregoing and continuing description, the subject invention in its various embodiments may comprise one or more of the following features in any non-mutually-exclusive combination:

[0014] A method for increasing growth rate of a halophilic archaeon, comprising identifying a provirus integrated into a genome of halophilic archaeon;

[0015] A method for increasing growth rate of a halophilic archaeon, comprising deleting said provirus from the genome to create a cured halophilic archaeon;

[0016] A method for increasing growth rate of a halophilic archaeon, comprising deleting a viral integrase gene from the genome of halophilic archaeon;

[0017] A method for increasing growth rate of a halophilic archaeon, wherein the halophilic archaeon is 48N;

[0018] A method for increasing growth rate of a halophilic archaeon, wherein the provirus is HLSV1 ;

[0019] A method for increasing growth rate of a halophilic archaeon, wherein deletion of the provirus is performed via a homologous recombination-based protocol;[0020JA method for increasing growth rate of a halophilic archaeon, wherein deletion of the provirus is performed via a pop-in / pop-out protocol;

[0021] A method for increasing growth rate of a halophilic archaeon wherein the halophilic archaeon is a ApyrE2 mutant;

[0022] A method for increasing growth rate of a halophilic archaeon, wherein an upstream flanking region and a downstream flanking region of a target provirus sequence are cloned into a suicide plasmid;[0023JA method for increasing growth rate of a halophilic archaeon, wherein the suicide plasmid is transformed into the halophilic archaeon;

[0024] A method for increasing growth rate of a halophilic archaeon, wherein the halophilic archaeon is grown in a medium lacking uracil to select for a halophilic archaeon with a chromosomally integrated suicide plasmid;[0025JA method for increasing growth rate of a halophilic archaeon, wherein the halophilic archaeon with the chromosomally integrated suicide plasmid is grown in a medium comprising 5-fluoroorotic acid to select for a cured halophilic archaeon without the provirus;[0026JA method for increasing growth rate of a halophilic archaeon, wherein the suicide plasmid is pTa131 ;

[0027] A method for increasing growth rate of a halophilic archaeon, wherein the halophilic archaeon is Haloferax volcanii;[0028JA method for increasing growth rate of a halophilic archaeon, wherein the genome is substantially free from the provirus;

[0029] A method for increasing growth rate of a halophilic archaeon, wherein the genome is substantially from the viral integrase gene;[0030JA method for increasing growth rate of a halophilic archaeon, wherein the growth rate is increased at least two-fold;[0031JA method for increasing growth rate of a halophilic archaeon, wherein the cured halophilic archaeon possesses a 15 fold increase in mRNA expression levels of archaeal-type H+ ATPase subunits versus wild-type halophilic archaeon;

[0032] A method for increasing growth rate of a halophilic archaeon, wherein expression of cyclases of a CBASS system are upregulated in the cured halophilic archaeon versus wild-type halophilic archaeon;[0033JA genetically modified halophilic archaeon, wherein the halophilic archaeon is genetically modified to be substantially free from at least one provirus;

[0034] A genetically modified halophilic archaeon, wherein the halophilic archaeon is genetically modified to be substantially free from chronic viral infection from HLSV1 ;[0035JA genetically modified halophilic archaeon, wherein the genetically modified archaeon is genetically modified to lack an integrase gene;[0036JA genetically modified halophilic archaeon, wherein a growth rate of the halophilic archaeon is higher than a growth rate of wild-type halophilic archaeon;

[0037] A genetically modified halophilic archaeon, wherein a growth rate of the halophilic archaeon is at least two-fold higher than a growth rate of wild-type halophilic archaeon;[0038JA genetically modified halophilic archaeon, wherein the halophilic archaeon possesses a 15-fold increase in mRNA expression levels of archaeal-type H+ ATPase subunits versus wild-type halophilic archaeon;[0039JA genetically modified halophilic archaeon, wherein expression of levels of cyclases of a CBASS system are higher than expression levels of cyclasses of a CBASS system in wild-type halophilic archaeon;[0040JA genetically modified halophilic archaeon, wherein expression of levels of CRISPR-associated proteins are higher than expression levels of CRISPR-associated proteins in wild-type halophilic archaeon;

[0041] A genetically modified halophilic archaeon, wherein the archaeon exhibits a growth rate that is at least 20% higher than a wild-type archaeon from the genus halophilic;

[0042] A genetically modified halophilic archaeon, wherein the halophilic archaeon exhibits an increase in CRISPR-associated protein CAS 7 gene expression versus wild-type halophilic archaeon;[0043JA genetically modified halophilic archaeon, wherein the halophilic archaeon exhibits an increase in CRISPR-associated protein CAS 5 gene expression versus wild-type halophilic archaeon;

[0044] A genetically modified halophilic archaeon, wherein the halophilic archaeon exhibits an increase in CRISPR-associated protein CAS 4 gene expression versus wild-type halophilic archaeon;[0045JA genetically modified halophilic archaeon, wherein the halophilic archaeon exhibits an increase in CRISPR-associated protein CAS 2 gene expression versus wild-type halophilic archaeon;[0046JA genetically modified halophilic archaeon, wherein the halophilic archaeon exhibits an increase in CBASS 1 Cyclase gene expression versus wild-type halophilic archaeon;

[0047] A genetically modified halophilic archaeon, wherein the halophilic archaeon exhibits an increase in CBASS 2 Cyclase gene expression versus wild-type halophilic archaeon;[0048JA genetically modified halophilic archaeon, wherein the halophilic archaeon exhibits an increase in CBASS 1 Effector gene expression versus wild-type halophilic archaeon;

[0049] A genetically modified halophilic archaeon, wherein the halophilic archaeon exhibits an increase in DUF1837 domain-containing protein / Hachiman A1 gene expression versus wild-type halophilic archaeon;[0050JA genetically modified halophilic archaeon, wherein the halophilic archaeon exhibits an increase in DEAD / DEAH box helicase I Hachiman B1 gene expression versus wild-type halophilic archaeon;

[0051] A genetically modified Haloferax archaeon, wherein the Haloferax archaeon exhibits an approximately 3.81 fold increase in CRISPR-associated protein CAS 7 gene expression versus wild-type Haloferax archaeon;

[0052] A genetically modified Haloferax archaeon, wherein the Haloferax archaeon exhibits an approximately 5.79 fold increase in CRISPR-associated protein CAS 5 gene expression versus wild-type Haloferax archaeon;

[0053] A genetically modified Haloferax archaeon, wherein the Haloferax archaeon exhibits an approximately 4.68 fold increase in CRISPR-associated protein CAS 4 gene expression versus wild-type Haloferax archaeon;

[0054] A genetically modified Haloferax archaeon, wherein the Haloferax archaeon exhibits an approximately 17.19 fold increase in CRISPR-associated protein CAS 2 gene expression versus wild-type Haloferax archaeon;

[0055] A genetically modified Haloferax archaeon, wherein the Haloferax archaeon exhibits an approximately 8.29 fold increase in CBASS 1 Cyclase gene expression versus wild-type Haloferax archaeon;

[0056] A genetically modified Haloferax archaeon, wherein the Haloferax archaeon exhibits an approximately 14.58 fold increase in CBASS 2 Cyclase gene expression versus wild-type Haloferax archaeon;

[0057] A genetically modified Haloferax archaeon, wherein the Haloferax archaeon exhibits an approximately 2.79 fold increase in CBASS 1 Effector gene expression versus wild-type Haloferax archaeon;

[0058] A genetically modified halophilic archaeon, wherein the halophilic archaeon is Haloferax; and

[0059] A genetically modified halophilic archaeon, wherein the halophilic archaeon has a generation time of approximately 1 hour when grown at 45°C.DESCRIPTION OF THE DRAWINGS

[0060] Figure 1. Genomics-based evidence for the existence of a virus in 48N. A. CRISPR spacer acquisition against the 48N chromosome by H. volcanii during mating on filters is almost exclusively against one locus in the genome. B. Coverage plot of Illumina™ sequence reads that map to the 48N chromosome. DNA was extracted from cells grown to high cell density over 3 days (stationary culture).

[0061] Figure 2. Viruses infecting Haloferax Atlit 48N. A-C. Transmission electron micrographs of lemon-shaped viruses released to the supernatant. D. Apleomorphic virus present in the same supernatant preparation. E. Lemon-shaped viruses outside (black triangles) and within cells. Dashed arrows mark fibrillar structures that some of the viruses adhered to.

[0062] Figure 3. Effects of virus curing on gene expression and growth of the host. A. growth curves of 48N strains and the H. volcanii lab strain. Samples were grown over-night in YPC rich medium at 45°C to the mid-log phase and then diluted with a fresh medium to OD600nm of 0.1 . The growth curves were carried out in microplate reader at 45°C with continuous shaking while measurements were taken every 30 minutes at a wavelength of 595nm. B. A volcano plot showing differentially abundant transcripts in cultures of, based on RNA-Seq. C. Gene categories affected by infection with LSV-48N.

[0063] Figure 4. Evidence for both replicating and integrated forms of LSV-48N in cells. (A) LSV-48N shown in integrated and circular form. (B) Agarose gel electrophoresis of PGR amplicons obtained from liquid cultures of 48N cells grown under different conditions. The respective wells contain: (1 ) the circular form of LSV- 48N; (2) the integrated form of LSV-48N (up); (3) the integrated form of LSV-48N (down); (4) Amplicon of a control gene (polB).

[0064] Figure 5. Digital droplet PGR quantification of LSV-48N genome copies along the growth curve (increasing OD) taken from a culture of 48N grown on rich (YPC) medium.DETAILED DESCRIPTION

[0065] Haloferax volcanii DS2 acquires CRISPR spacers against a region within Haloferax Atlit 48N genome that encodes a provirus

[0066] Haloferax Atlit 48N (henceforth 48N) is a strain originally isolated from the tidal pools of Atlit. A mating experiment was performed between a uracil auxotroph derivative of 48N (ApyrE2) which carries the pWL102 plasmid conferring resistance to mevinolin and a tryptophan auxotroph derivative of Haloferax volcanii DS2 (AtrpA, pyrE::HVO_0894) known as UG469. This assay, designed to obtain mostly colonies ofUG469 that received pWL102 by mating, was conducted by applying stationary cultures of the two strains onto a filter on a rich medium and then transferring them to selectable plates containing mevinolin and lacking uracil.

[0067] Then, the spacer acquisition profile of the DS2 CRISPR-Cas system was tested in the mating products and observed that spacer acquisition was highly skewed: the vast majority of spacers matched just one region in the 48N genome (Fig. 1 A). A closer examination of that genome region showed that it contained ORFs encoding two homologs of bacteriophage-associated integrases that are typical to temperate phages. The same region also had matches in CRISPR arrays of two Haloferax strains isolated from the same site as 48N: 24N and 47N (Table 1 ).

[0068] Most notably, in both of these strains, the leader proximal spacer, which in type I systems is the one most recently acquired, matched the suspected provirus (Table 1 ). The pattern of acquisition resembled that observed in bacteria following infection with dsDNA phages, i.e., a strong enrichment for a specific part of the viral genome, potentially representing the free end of the injected DNA (Fig. 1 A). Taken together these observations led to the hypothesis that the spacers were acquired from a virus that chronically infected 48N and was able to deliver its DNA into strain UG469, and probably some environmental strains of Haloferax.

[0069] To test whether the region corresponding to the vast majority of newly acquired CRISPR spacers indeed originated from a replicating mobile element, a fully assembled genome of 48N main chromosome was first obtained along with its four natural plasmids by combining the Illumina™ reads with Pacific BioScience™ SMRT sequencing data (see Methods). The raw Illumina reads were then mapped to the genome to obtain coverage scores of different areas of the genome. This analysis showed that coverage of the suspected provirus locus was over 20-fold higher than the genome average (Figure 1 B). Notably, examination of the assembly graph showed that the pro-virus element exists both as an integrated provirus and in a circular form.

[0070] In addition to the CRISPR-targeted proviral region, three other genomic regions within the 48N main chromosome had sequence coverage of about 2.5-3.5 foldhigher than the mean coverage, indicating that additional replicating elements are present in the 48N culture (Figure 1 B).

[0071] 48N encodes a chronically infecting lemon-shaped virus.

[0072] To test whether the replicating element identified is indeed a virus, the supernatant of a 48N culture was concentrated and subjected to transmission electron microscopy (TEM). TEM images indicated the presence of predominantly lemon-shaped virus-like particles about 65-90 nm long and 35-50 nm wide (Figure 2 A-C), similar to in shape to lemon / spindle viruses previously observed in other archaeal species such as Haloarcula and Sulfolobus, despite the lack of sequence similarity of any of the ORFs encoded by the provirus to any known capsid protein. Additional spherical virus-like particles of about 75-90 nm in diameter (Figure 2D) were also observed. Both intracellular viruses and a fraction of the secreted ones appeared attached to fibrillar structures (dashed arrows, Figure 2C and 2E).

[0073] When DNA was extracted from the supernatant and submitted to Illumina sequencing and de-novo genome assembly (see Methods), the following was observed: numerous small (>5kbp) contigs of relatively low coverage depth (averaging between 0.95 and 4.82 fold coverage) mapping to the host chromosome and plasmids, and two prominent contigs, 24kbp and 16kbp long, of higher coverage depth (7403.05 and 17.29 average fold, respectively (Figure 4), the 24kbp contig corresponded to a circular replicon. The 16kbp contig encoded two putative proteins which show similarity to proteins conserved in haloarchaeal pleomorphic viruses, one matching ORF7 of Halogeometricum sp. pleomorphic virus 1 , and the other ORF6 of that virus with a closer match to Haloferax volcanii pleomorphic virus 1. Additionally, the 16kbp contig encoded a tyrosine recombinase / integrase typical to archaeal proviruses and temperate bacteriophages. This implies that the 16kbp contig likely belongs to a pleomorphic virus, while the 24kbp genome corresponds to a lemon-shaped virus (henceforth LSV-48N). LSV-48N virions were also observed within 48N cells (Figure 2E).

[0074] LSV-48N is non-lethal and continuously shed during growth

[0075] PCR-based analysis of DNA extracted from 48N cultures, using outwardfacing primers that either amplify a circularized genome, or the integrated form, indicated that both forms are present under both standard, lower-salt and low- phosphate conditions (Figure 4).

[0076] In order to determine at what stages during 48N growth curve are viruses produced and secreted, we used digital droplet PCR (ddPCR) to quantify viral genome copies in DNA extracted from the supernatant as well as from the cells at different time points when growing 48N in liquid culture (see Methods). Results showed that during early exponential growth (OD of 0.1 ) there is maximal accumulation of viral genome copies within cells, yet the maximal release of viruses to the medium occurs during late exponential growth-early stationary phase (OD of 0.7-1 ). (Figure 5). When the viability of the cells was tested using fluorescent microscopy and the LIVE / DEAD™ BacLight™ kit, there were very few dead cells present (roughly 1 .5%). Furthermore, 48N consistently grew faster than H. volcanii lab strain (Figure 3), indicating that chronic infection with LSV-48N is well-tolerated by the host.

[0077] LSV-48N profoundly affects the growth and physiology of its host

[0078] Even when grown for 400 generations in rich medium, it was not possible to obtain colonies that spontaneously lost LSV-48N (tested by virus-specific colony PCR). Therefore, the provirus was deleted from the genome using a two-step strategy, beginning from a 48N derivative in which the pyrE gene was deleted (making it a uracil auxotroph, see Methods, henceforth 48NApyrE2). First, the gene for the putative phage-like integrase from 48NApyrE2 was deleted using the pop-in / pop-out approach. Then, the remaining part of the viral genome was deleted using pop-in / pop-out and confirmed the clean deletion via whole genome sequencing. As expected, TEM images showed lemon-shaped viruses in the infected 48NApyrE2 strain but not the virus-cured strain (cured 48N).

[0079] Chronic viral infection often results in a considerable growth burden and a major shift of the host gene expression. Indeed, when the growth of the wild-type infected 48N was compared to its cured 48N, a major growth improvement wasobserved, with a growth rate over two-fold higher, the fastest ever observed for a haloarchaeon at 45% (Figure3A). Then RNA was extracted and compared the transcriptome of 48N and cured 48N using RNA-seq. As can be expected from such striking differences in growth, RNA-seq analysis revealed an immense change in the transcription pattern of 48N with almost 60% of the genes in cured 48N exhibiting a significant difference in their expression (Figure 3B) that spanned all functional categories (Figure 3C). The large increase in generation time in the virus-infected 48N indicates that the virus is consuming much of the cellular resources and potentially modulating host metabolism.

[0080] Indeed, the RNA-seq results showed that not only were viral genes encoding membrane proteins at very highly transcribed levels, but the expression of many host genes which are associated with different functions, including metabolism, were unexpectedly altered. Thus, the two cytochrome bd-type quinol oxidase subunits that play a critical role in respiration in haloarchaea were more than 10-fold higher in the infected strain, implying that respiration may be increased during infection. In contrast, many host metabolic genes were strongly downregulated, such as the archaeal-type H+ ATPase subunits, nearly all of which had mRNA levels decrease of more than 15 fold.

[0081] LSV downregulates the defense systems of its host

[0082] Based on genomic analysis using PADLOC (https: / / doi.org / 10.1093 / nar / gkac400), the 48N genome has multiple potential anti-viral defenses encoded on its natural plasmids. Among those, CRISPR-Cas and the two CBASS systems should be able to prevent chronic viral infection: CRISPR-Cas can generally acquire spacers against replicating selfish elements and then target their DNA for degradation, while CBASS systems cause abortive-infection by recognizing viral infection and killing the host. However, a comparison of the gene expression levels of defense genes in 48N and cured-48N showed that these defense systems, and additional ones, such as Hachiman, were strongly downregulated in the infected strain (Table 2). Notably, the genes encoding the cyclases of the CBASS systems, required for their activation in the presence of viral infection had much lower levels in 48N compared to cured 48N (8.29 and 14.6 fold lower in CBASS type 1 and 2, respectively),as were the cas2 and cas4, genes required for new spacer acquisition (17.19 and 4.68 lower, respectively). Thus, LSV-48N infection downregulates cellular defenses, either directly or indirectly at the level of transcription, sufficiently to allow chronic and sustained infection.

[0083] To further investigate why 48N does not acquire CRISPR immunity against LSV-48N, a spacer acquisition assay was performed on the 48N CRISPR arrays using amplicon sequencing. Extremely low spacer acquisition levels were observed and no spacers that originated from the genome of LSV-48N. Furthermore, when spacers that had partial matches to the 48N genome were examined, it was noticed that a number of them did not have matches in any 48N replicon. Thus, it appears that either LSV-48N or another selfish genetic element within the 48N genome encodes a factor that interferes with the spacer acquisition process of the 48N CRISPR- Cas.

[0084] Conclusions

[0085] Extreme environments that are often dominated by archaea tend to have lower diversity and lower cell density than bacteria-dominated hyper-diverse ecosystems such as soil or the mammalian gut. Consequently, host-virus interactions of extremophilic archaea may differ greatly from phage-bacteria interactions, and it has been suggested that when chronic infection protects against lethal infection, it can actually be beneficial. The viruses in such environments, in turn, may benefit from a chronic lifestyle when the host cell density is low, making long-term exploitation of the host and its progeny much more desirable.

[0086] Here, the physiology and gene expression of a chronically infected extremophile strain was examined. Importantly, because it was isolated directly from an evaporation pond where other strains of the same species abound, it can be assumed to be relatively fit, despite being infected. Furthermore, it grows faster than DS2 in the lab when grown on standard media, and quickly outcompetes it in head-to-head growth experiments.

[0087] Nonetheless, the seemingly mild burden of chronic infection is in reality not mild at all. Curing the virus dramatically increases growth rate as well as expression of anti-viral defense genes. While it may seem paradoxical that such a burden is sustained in a natural habitat, one should keep in mind that in many ecosystems that are continuously limited for resources, such as available carbon and nitrogen, and are not subject to feast or famine fluctuations, the maximal growth rate may not be the key trait determining ecological fitness.

[0088] Furthermore, the two strains which were isolated in the same sampling round with 48N (24N and 47N) that were not infected by LSV-48N and had CRISPR spacers matching its genome had similar growth rates to that of 48N (generation times on rich medium of 2.08 and 2.96 h. for 24N and 47N, respectively, vs. 2.18h for 48N). Thus, as observed previously for other extreme environments, hypersaline systems may allow chronic infection with viruses to be tolerated.

[0089] How LSV-48N can efficiently downregulate different defense systems at the level of transcript abundance is unclear. One possibility is that one or more of the three putative DNA-binding proteins encoded by the LSV-48N is a master regulator able to suppress promoters of multiple defense genes. Alternatively, it could act indirectly, for example through one or more protein-protein interactions with the host transcriptional regulation machinery.

[0090] Materials and Methods

[0091] Culture conditions

[0092] Haloferax strains were routinely grown aerobically at 45°C in either Hv- YPC (rich medium) or in Hv-Ca / Hv-Ca+ (minimal medium). When required, uracil was added at a concentration of 50 pg / ml. For pop-out selection medium, 5-fluoroorotic acid (5-FOA) was added to a concentration of 50 pg / ml.

[0093] Droplet Digital PCR (ddPCR) analysis on circular LSV-48N

[0094] The number of viral particles in 1 ml of 48N culture was inferred from ddPCR experiments. 1 ml from cultures at different growth stages (OD600nm: 0.1 , 0.2, 0.3, 0.5, 0.7, 1 .0, 1 .5) were analyzed. In order to measure the amount of secreted viralparticles in the media, DNA was extracted from 200pl of the supernatant using Quick- DNA_viral_kit following centrifugation at 12,000 rpm. For "in cells" viral particles quantification, 1 ml from the cell culture from each growth stage was vortexed and washed twice with Hv-Ca broth to remove cell adhesed viruses. The cells were then diluted and lysed (suspended pellet cells from 1 ml with 100 pl ST buffer [1 M NaCI, 20 mM Tris.HCI] and 100 pl lysis buffer [100 mM EDTA pH 8.0, 0.2% SDS]) before viral DNA was extracted from lysed cells (Quick-DNA viral kit). Viral DNA extractions were diluted to final concentration of 0.2 pg / pl before added to the ddPCR reaction mix. Primers used are: Is24, Is25 for the circular form of the virus and: ls660 Is661 for control housekeeping gene. 48N cells were grown to OD600nm of: 0.1 , 0.2, 0.3, 0.5, 0.7, 1 .0, 1 .5. in YPC. CFU counts was were made for each culture in order to deduce the average number of archaeal cells in 1 ml.

[0095] LSV-48N particles imaging

[0096] HLSV1 particles were visualized using a negative staining approach: Samples were adsorbed on formvar / carbon coated grids and stained with 2% aqueous uranyl acetate. Samples were examined using a transmission electron microscope.

[0097] Growth curves

[0098] To compare the growth of the 48N strains, each sample was grown overnight in appropriate media at 45°C to the mid-log phase and then diluted to a fresh medium to OD600nm of 0.1 . The growth curves were carried out in 96-well plates at 45°C with continuous shaking, using a microplate reader. Optical density was measured every 30 minutes at a wavelength of 595nm.

[0099] LSV-48N circular I integrated form detection by PCR

[0100] Integrated and circulated form of LSV-48N inferred using PCR analysis with specific PCR oligonucleotides. The integrated form was inferred when a PCR product of 1000 bp was visible after gel electrophoresis using IT556 and IT557 for upstream integration site and IT558 and IT559 for downstream integration site. One primer corresponds to the genome and the other one to the virus in the integration site. The circular form was obtained using IT560 and IT561 The primers matched the ends ofthe virus and the 1000 bp product is only obtained if the virus closes in a circle. PCR was performed using KAPA 2G ready mix by Roche®.

[0101] Mating experiments

[0102] The mating experiments were conducted using an uracil auxotroph derivative of 48N (ApyrE2) carrying the pWL102 plasmid conferring resistance to mevinolin and a tryptophan auxotroph derivative of Haloferax volcanii DS2 (AtrpA, pyrE::HVO_0894) known as UG469). Liquid cultures of both parental strains were grown to an G.D600 of ~1 .8. The parental strains were then mixed in 1 :1 ratio and applied to a nitrocellulose 0.45pm filters using a Swinnex 25mm filter holder. The filter with the mating products was transferred to a rich medium plate (Hv-YPC) for 24 hours for phenotypic expression. The cells were then re-suspended and washed in Hv-Ca broth before plated on Hv-Ca media containing mevinolin and lacking uracil. After 14 days in 45°C incubator, we were able to detect colonies that grew on the selectable plates. Around 200 colonies were picked and extracted their DNA using the DNA spooling protocol and continued to the next step of new CRISPR spacers detection.

[0103] Nutritional competence experiment

[0104] H. volcanii lab strain H729 cells were grown for a week in YPC-HV broth containing extracted DNA from 48N strain (50ng / pl). DNA from that culture was then extracted and used for detection of new spacers in arrays C and D of H. volcanii as described below.

[0105] New CRISPR spacers detection

[0106] Extracted DNA was used as PCR template for either H. volcanii or 48N CRISPR arrays using specific primers amplifying the region between the leader and the third spacer for each. When analyzed by agarose gel electrophoresis, longer PCR products indicate new spacer-repeat acquisitions. To obtain visible acquisition bands in the agarose gel, we extracted the approximated elongated length region from the gel, isolated DNA and amplified the fragment through another cycle of PCR. New acquisition events could then be detected via the presence of a higher band. PCR products were then sent for processing and Illumina amplicon sequencing (240,000-290,000 reads perarray per biological repeat) at the Center for Genomic Research, University of Illinois, USA.

[0107] Briefly, the elongated PCR product was enriched using Ampure™ beads size-selection; sample specific barcodes and Illumina adaptors were added by PCR; and the resulting products were purified, pooled, and paired-ends sequenced on a MiSeq Illumina platform. Notably, even after these consecutive steps of size selection many reads still represented amplicons derived from no acquisition amplifications.

[0108] Initial data processing

[0109] Paired-end raw Illumina reads were quality-filtered (Q>20) and merged using PEAR (paired-end read merger, yielding, for most samples, 240000-290000 high- quality sequences; samples which yielded over 300000 seqs per sample were subsampled randomly, using VSEARCH, to 280000 seqs / sample. Biological replicates within each array were then converted to a single fasta file using QIIME's multiple_split_libraries_fastq.py script 13, followed by de-replication, abundance sorting and clustering (99% threshold) using VSEARCH 14. Pairwise identity at the clustering step was defined as [matching columns] / [alignment length] (set in VSERACH as -iddef 1 ), which ensures terminal gaps are not ignored and sequences of different length do not cluster together. The length distribution of the clustered sequences is similar to that of the raw sequences; the arrays show a peak at 250 bp (size of the original fragment, containing 3 repeats), with progressively smaller peaks at 320 bp (corresponding to one new acquisition) and 390 bp (2 new acquisitions). The raw reads were then mapped back to the clusters, again with a 99% identity threshold, to create a table presenting the abundance of each cluster in each biological repeat.

[0110] Spacer extraction

[0111] The centroid sequence of each cluster was used for extraction of acquired new spacer sequences. To identify true acquisition events while excluding DNA rearrangement events (which may also result in an extended PCR product), a custom-made R script based on the 'Biostrings' and 'tools' R packages was used to count the number of repeats in each centroid sequence, allowing up to 2 mismatchesper repeat. Fragments containing more than 3 repeats were tagged as putative acquisitions, and the sequence between the 2 repeats closest to the leader end was extracted. “False positives”, which are the result of rearrangement of spacers within or between arrays rather than canonical acquisition, were eliminated by screening the extracted putative spacers against all original CRISPR arrays from both species, allowing up to 5 mismatches per spacer; spacers with matches in existing CRISPR arrays were excluded from further analysis. To facilitate comparison of CRISPR activity between arrays, we also counted the number of sequences containing the original number of repeats.

[0112] Mapping new spacers to genomic location

[0113] In order to establish the protospacer location for each new acquisition, we used the blastn-short program 15 at an E-value of .0001 against a file containing genome sequences of either H. volcanii or 48N including their natural plasmids. Blast results were refined using Custom made R scripts based on the 'stringr' package. In brief, no more than 3 mismatches between the spacer and the protospacer were allowed; in cases of multiple matches, the location with the highest score was preserved (while conserving abundance information); and spacers with equally high scores across multiple locations were excluded. Furthermore, spacers that were aligned only partially were removed when the alignment length was shorter than the total spacer length by five or more bases.

[0114] Quantification of spacer acquisition events

[0115] Using the mapping approach described above we quantified total acquisition events for each spacer and the mean number of events across biological replicates for each spacer was calculated. 4 spacers that showed a highly aberrant pattern when comparing the 3 biological replicates (abundance in one sample more than 1000-fold higher than in the other 2 samples) were corrected for by replacing the aberrantly high value with the average counts of that spacer in the other 2 biological replicates.

[0116] Generating ApyrE2 48N strain

[0117] Strain 48N was verified for its sensitivity to 5F0A (150 pg / ml) in YPC medium and its ability to grow on CA medium. Knock out of the pyrE2 gene was done with pGB68 by pop-in and then pop-out. Pop-in transformants were selected on YPC plates containing Novobiocin (0.3pg / ml) and individual colonies were then transferred into liquid YPC medium with no novobiocin and shaken in 45°C for 48 hours after which they were streaked on YPC plates supplemented with 5FOA. Resistant colonies were picked and streaked on CA plates and YPC plates. Notably, half of the colonies retained their ability to grow on CA while' resistant to 5FOA. To obtain true knockout colonies, 3 cycles of 5FOA selection were performed. The genotype of the pop-in colonies, as well as knockout colonies was confirmed by PCR using the primers:V-PyrE2-F, V-PyrE2-R.

[0118] 48N cured strain construction

[0119] Construction of the 48 cured strain was performed according to the pop-in / pop- out protocol. In this method, the upstream and downstream flanking regions of the sequence to be deleted are amplified by PCR and cloned together into the ‘suicide plasmid’ pTA131 , which cannot replicate autonomously and carries the pyrE2 selectable genetic marker. The plasmids are then transformed (Transformation of 48N was carried out using the polyethylene glycol 600 method into the 48N ApyrE2 mutant, and the transformants, in which the plasmids have been integrated into the chromosome, are selected for on plates that lack uracil ('pop-in'). Upon counter-selection on plates containing uracil and 5-fluoroorotic acid (5-FOA), the only cells that survive are those in which the integrated plasmids have been excised by spontaneous intra-chromosomal homologues recombination ('pop-out'), either restoring the WT gene or resulting in allele exchange. The generation of the 48N HLSV1 cured strain was performed in two steps. First, the viral integrase gene was deleted so that viral genomes present in the circular form will not be able to re-integrate after the deletion has occurred. The A-integrase strain UG595 was used and inserted with ZZZ plasmid to delete the entire virus from the chromosome. The 'pop-out' strains were screened using pairs of primers IT1 and IT4 located upstream and downstream to the LSV-48N integration site and the sequence was validated by sequencing.

[0120] Preprocessing of Illumina and PacBio sequencing data

[0121] The Illumina raw reads used for the previous, publicly available, assembly of Haloferax volcanii spp. 48N were fetched. Reads mapping to PhiX174 (a library preparation spike-in) were identified using bowtie2. Subsequently, unmapped reads were filtered using Trimmomatic™ using the command 'adapters / NexteraPE- PE.fa:2:30:10 LEADINGS TRAILINGS SLIDINGWINDOW:4:15 MINLEN:36'. PacificBio Science (“long”) reads were filtered using bbduk.sh from the bbtools suite with the following flags: minlen=100 maq=8, i.e. reads were retained only if they were of a minimal length of 100 nucleotides and had a minimal average quality of 8.

[0122] Assembly of 48N and LSV-48N genomes

[0123] LSV-48N processed reads were assembled using SPAdes™ (version 3.1 1.0) (cite 10.1101 / gr.213959.116). Manual examination of the assembly graph, together with PGR amplification, were used to verify the genome terminals and the circular topology.

[0124] The hybrid assembly of Haloferax volcanii spp. 48N was performed using Unicycler (version 0.4.4) with default options, supplying pilon for genome polishing. The assembly graph was examined and the topology of the virus form was observed. Mapping of the raw reads and manual examination of the coverage suggested both integrated and circular forms of the virus were present in the original culture. For simplicity, the resolved form used for coverage (depth) calculation was set as the circular form (i.e. as a standalone replicon as a separate contig) in order to report the virus coverage separately from that of the host chromosome.

[0125] Coverage reports (including estimated depth) where generated using bbmap.sh from the bbtools suite, via the 'covstats' output. For these estimation, only the Illumina reads were provided as a query and the resolved 48N genome as reference.

[0126] Finally, the hybrid assembly of Haloferax volcanii spp. 48N completeness was estimated at 99.57% with contamination of 0.13% (CheckM version 1 .0.18).

[0127] Genome structural and functional annotations

[0128] For gene prediction, the assembled genomes were analyzed using genemark. Predicted protein sequences were then searched (using hmmsearch), mmseqs and psi-BLAST against several public protein domain databases (Pfam, PDB, CDD and arCOGs. The functional / descriptive label of profile matches with good alignment statistics was cloned onto the query protein. Predicted proteins without sufficiently reliable match were manually examined further using web-hhpred and CollabFold2.

[0129] Tables

[0130] Table 1 . Spacers matching the provirus locus from 48N in genomes of natural isolates obtained from the same site and season (leader proximal spacer locations are highlighted in bold type).

[0131] Table 2. Anti-viral immunity genes whose expression is significantly higher in the cured strain vs. its infected isogen.

[0132] In some embodiments, the disclosure is related to archaea of the genus Haloferax-, however, the present disclosure is not limited to the genus Haloferax. The present disclosure is applicable to all halophilic archaea, and the methods and techniques disclosed herein with respect to specific archaeons are equally applicable to all halophilic archaea. Halophilic archaea are a diverse group of microorganisms that thrive in high-salt environments. By applying the methods described herein, it is possible to identify and eliminate proviruses harbored therein, leading to improved growth rates and altered gene expression profiles in various halophilic archaeal species. In some embodiments, it is contemplated that the methods disclosed herein, such as the pop-in / pop-out protocol and CRISPR-Cas-based gene editing, can be adapted to target and remove proviruses from all halophilic archaea.

[0133] In some embodiments, the present disclosure relates to a method for increasing the growth rate of a Haloferax archaeon. This involves identifying and deleting a provirus integrated into the archaeon's genome. The provirus can be HLSV1 , and the deletion can be achieved through techniques like homologous recombination or the pop-in / pop-out protocol.

[0134] In some embodiments, the pop-in / pop-out protocol typically involves using a suicide plasmid, such as pTA131 , and selecting for transformed archaeons that have lost the plasmid. This can result in the deletion of the provirus and associated genes like the viral integrase gene.

[0135] The cured archaeon often exhibits increased expression of genes involved in energy production and defense systems, leading to faster growth. For instance, the cured archaeon may possess a generation time of approximately 1 hour when grown at 45°C, compared to the typical 2.5 hours of wild-type Haloferax volcanii. Additionally, the cured archaeon may exhibit upregulated expression of genes associated with CRISPR- Cas systems and CBASS systems, indicating improved defense mechanisms against viral infections.

[0136] Furthermore, the disclosure relates to genetically modified halophilic archaea that are genetically modified to be substantially free from at least one provirus. In some embodiments, the halophile archaea is Haloferax volcanii. These halophilic archaeons can be generated through techniques that target and eliminate specific proviruses or viral genes. The resulting archaeons often exhibit increased growth rates, altered gene expression patterns, and improved resistance to viral infections.

Claims

CLAIMS1 . A method for increasing growth rate of a halophilic archaeon, comprising: identifying a provirus integrated into a genome of a halophilic archaeon; and deleting said provirus from the genome to create a cured halophilic archaeon.

2. The method of claim 1 , further comprising the step of deleting a viral integrase gene from the genome of a halophilic archaeon.

3. The method of claim 1 , wherein the halophilic archaeon is 48N.

4. The method of claim 1 , wherein the provirus is HLSV1 .

5. The method of claim 1 , wherein deletion of the provirus is performed via a homologous recombination-based protocol.

6. The method of claim 1 , wherein deletion of the provirus is performed via a pop- in / pop-out protocol.

7. The method of claim 6, wherein: the halophilic archaeon is a ApyrE2 mutant; an upstream flanking region and a downstream flanking region of a target provirus sequence are cloned into a suicide plasmid; the suicide plasmid is transformed into the halophilic archaeon; the halophilic archaeon is grown in a medium lacking uracil to select for a halophilic archaeon with a chromosomally integrated suicide plasmid; and the halophilic archaeon with the chromosomally integrated suicide plasmid is grown in a medium comprising 5-fluoroorotic acid to select for a cured halophilic archaeon without the provirus.

8. The method of claim 7, wherein the suicide plasmid is pTa131 .

9. The method of claim 1 , wherein the halophilic archaeon is a Haloferax archaeon.

10. The method of claim 9, wherein the Haloferax archaeon is Haloferax volcanii.11 . The method of claim 1 , wherein the genome is substantially free from the provirus.

12. The method of claim 2, wherein the genome is substantially from the viral integrase gene.

13. The method of claim 1 , wherein the growth rate is increased at least two-fold.

14. The method of claim 1 , wherein the cured halophilic archaeon possesses a 15- fold increase in mRNA expression levels of archaeal-type H+ ATPase subunits versus wild-type Haloferax archaeon.

15. The method of claim 1 , wherein expression of cyclases of a CBASS system are upregulated in the cured halophilic archaeon versus wild-type halophilic archaeon.

16. A genetically modified halophilic archaeon, wherein the halophilic archaeon is genetically modified to be substantially free from at least one provirus.

17. The genetically modified archaeon of claim 16, wherein the halophilic archaeon is genetically modified to be substantially free from chronic viral infection from HLSV1.

18. The genetically modified archaeon of claim 16, wherein the genetically modified archaeon is genetically modified to lack an integrase gene.

19. The genetically modified archaeon of claim 16, wherein a growth rate of the halophilic archaeon is higher than a growth rate of wild-type halophilic archaeon.

20. The genetically modified archaeon of claim 16, wherein a growth rate of the halophilic archaeon is at least two-fold higher than a growth rate of wild-type halophilic archaeon.21 . The genetically modified archaeon of claim 16, wherein the halophilic archaeon possesses a 15-fold increase in mRNA expression levels of archaeal-type H+ ATPase subunits versus wild-type halophilic archaeon.

22. The genetically modified archaeon of claim 16, wherein expression of levels of cyclases of a CBASS system are higher than expression levels of cyclases of a CBASS system in wild-type halophilic archaeon.

23. The genetically modified archaeon of claim 16, wherein expression of levels of CRISPR-associated proteins are higher than expression levels of CRISPR- associated proteins in the wild-type halophilic archaeon.

24. The genetically modified archaeon of claim 16, wherein the halophilic archaeon exhibits a growth rate that is at least 20% higher than a wild-type halophilic archaeon.

25. The genetically modified archaeon of claim 16, wherein the halophilic archaeon exhibits an increase in CRISPR-associated protein CAS 7 gene expression versus wild-type halophilic archaeon.

26. The genetically modified archaeon of claim 16, wherein the halophilic archaeon exhibits an increase in CRISPR-associated protein CAS 5 gene expression versus wild-type halophilic archaeon.

27. The genetically modified archaeon of claim 16, wherein the halophilic archaeon exhibits an increase in CRISPR-associated protein CAS 4 gene expression versus wild-type halophilic archaeon.

28. The genetically modified archaeon of claim 16, wherein the halophilic archaeon exhibits an increase in CRISPR-associated protein CAS 2 gene expression versus wild-type halophilic archaeon.

29. The genetically modified archaeon of claim 16, wherein the halophilic archaeon exhibits an increase in CBASS 1 Cyclase gene expression versus wild-type halophilic archaeon.

30. The genetically modified archaeon of claim 16, wherein the halophilic archaeon exhibits an increase in DUF1837 domain-containing protein / Hachiman A1 gene expression versus wild-type halophilic archaeon.31 . The genetically modified archaeon of claim 16, wherein the halophilic archaeon exhibits an increase in DEAD / DEAH box helicase I Hachiman B1 gene expression versus wild-type halophilic archaeon.

32. The genetically modified archaeon of claim 16, wherein the halophilic archaeon is a member of the genus Haloferax.

33. The genetically modified archaeon of claim 32, wherein the member of the genus Haloferax exhibits an approximately 3.81 fold increase in CRISPR-associated protein CAS 7 gene expression versus wild-type member of the genus Haloferax.

34. The genetically modified archaeon of claim 32, wherein the member of the genus Haloferax exhibits an approximately 5.79 fold increase in CRISPR-associated protein CAS 5 gene expression versus wild-type member of the genus Haloferax.

35. The genetically modified archaeon of claim 32, wherein the member of the genus Haloferax exhibits an approximately 4.68 fold increase in CRISPR-associated protein CAS 4 gene expression versus wild-type member of the genus Haloferax.

36. The genetically modified archaeon of claim 32, wherein the member of the genus Haloferax exhibits an approximately 17.19 fold increase in CRISPR-associated protein CAS 2 gene expression versus the wild-type member of the genus Haloferax.

37. The genetically modified archaeon of claim 32, wherein the member of the genus Haloferax exhibits an approximately 8.29 fold increase in CBASS 1 Cyclase gene expression versus the wild-type member of the genus Haloferax.

38. The genetically modified archaeon of claim 32, wherein the member of the genus Haloferax exhibits an approximately 14.58 fold increase in CBASS 2 Cyclase gene expression versus the wild-type member of the genus Haloferax.

39. The genetically modified archaeon of claim 32, wherein the member of the genus Haloferax exhibits an approximately 2.79 fold increase in CBASS 1 Effector gene expression versus the wild-type member of the genus Haloferax.

40. The genetically modified archaeon of claim 32, wherein the member of the genusHaloferax has a generation time of approximately 1 hour when grown at 45°C.

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