Bacterial plasmid conjugational transfer system identification and structural feature analysis method

Abstract

The invention discloses a bacterial plasmid conjugational transfer system identification and structural feature analysis method, and belongs to the field of microbial bioinformatics analysis. A bacterial plasmid conjugational transfer system identification and structural feature analysis method comprises the following steps: step 1, establishing a database by using a protein sequence of a plasmid to be analyzed, and searching the protein sequence of the plasmid to be analyzed in the database by using the protein sequence of the bacterial plasmid conjugational transfer system as a query sequence; 2, screening and classifying search results; identifying the type of the conjugational transfer system gene contained in the plasmid to be analyzed according to the classification result; 3, sequencing the conjugational transfer system protein sequences contained in the plasmids to be analyzed according to the sequence of the gene loci, and determining the structural characteristics of the conjugational transfer system of the plasmids to be analyzed. The bacterial plasmid conjugational transfer system identification and structural feature analysis method has the beneficial effects that automatic batch processing can be realized, and the method is efficient, accurate and convenient.

Description

Technical Field

[0001] The present application relates to the field of microbial bioinformatics analysis, and in particular, to a method for identifying bacterial plasmid conjugative transfer systems and analyzing structural characteristics. Background Art

[0002] Bacterial plasmids such as IncA, IncC, and IncF have the ability to spread drug resistance genes against carbapenems, third-generation cephalosporins, etc. between bacteria through the conjugative transfer system. The conjugative transfer system of bacterial plasmids includes important components such as relaxase, type IV coupling protein, and type IV secretion system genes.

[0003] Relaxase is a gene that is widely present in all mobile plasmids. According to the differences in the relaxase domains in different plasmids, the conjugative transfer module can be divided into several families: MOB H 、MOB F 、MOB Q 、MOB C 、MOB P and MOB V wait.

[0004] Type IV coupling proteins are present in all conjugative plasmids and transfer substrate DNA or macromolecular proteins from relaxases to secretory channels. Type IV coupling proteins are generally divided into three types based on the domains they contain, namely TrwB / TraD, VirD / TraG, and Other types.

[0005] The type IV secretion system in IncA, IncC, and IncF plasmids is a transmembrane structure that can transfer nucleic acids or macromolecular protein substrates outside the cell. According to the morphology of the conjugative pili representing the plasmid, the type IV secretion system genes of the plasmid are divided into F type, P type, I type, G type, and GP type. In addition, the P type and I type IV secretion systems also include many subtypes, for example, the P type IV secretion system includes Trb, Trw, and VirB subtypes, each of which contains many genes, while the I type IV secretion system includes I_Tra and Dot / Icm subtypes.

[0006] In the past, the analysis of the genome structural characteristics of IncA, IncC, and IncF plasmid conjugative transfer systems usually involves uploading the plasmid sequence to an online website to identify the relaxase, type IV coupling protein, and type IV secretion system gene types in the plasmid. This method is more applicable when the number of plasmids is small, but it is difficult to efficiently and accurately perform the plasmid conjugative transfer system analysis task when faced with large-scale plasmid genome analysis. In addition, previous online analysis tools ran slowly and were inefficient.

[0007] Therefore, there is a need for a method for identifying and analyzing the structural characteristics of bacterial plasmid conjugative transfer systems that can realize automated batch processing and is efficient, accurate, and convenient. Summary of the invention

[0008] The content of this application is used to introduce concepts in a brief form, which will be described in detail in the detailed implementation section below. The content of this application is not intended to identify the key features or essential features of the technical solution claimed for protection, nor is it intended to limit the scope of the technical solution claimed for protection.

[0009] In order to solve the technical problems mentioned in the above background technology section, some embodiments of the present application provide a method for identifying a bacterial plasmid conjugative transfer system and analyzing structural characteristics, comprising the following steps:

[0010] Step 1: Establish a database with the protein sequence of the plasmid to be analyzed, use the protein sequence of the bacterial plasmid conjugative transfer system as the query sequence, and search for the protein sequence of the plasmid to be analyzed in the database;

[0011] Step 2: Screening and classifying the search results; identifying the type of conjugative transfer system gene contained in the plasmid to be analyzed according to the classification results;

[0012] Step 3: Sort the conjugative transfer system protein sequences contained in the plasmid to be analyzed according to the order of the gene loci to determine the structural characteristics of the conjugative transfer system of the plasmid to be analyzed.

[0013] Furthermore, the conjugative transfer system includes a relaxase gene, a type IV coupling protein gene and a type IV secretion system gene.

[0014] Furthermore, the plasmid to be analyzed includes a plasmid to be detected and a reference plasmid of the plasmid to be detected; the reference plasmid is obtained by literature retrieval.

[0015] Furthermore, in step 1, the protein sequence of the bacterial plasmid conjugative transfer system is collected and obtained through public literature and public databases.

[0016] Furthermore, in step 1, the search method is: running a local Blastp tool in a computer system, using the protein sequences of the relaxase gene, the type IV coupling protein gene and the type IV secretion system gene as query sequences, and performing a Blastp search on the protein sequence of the plasmid to be analyzed.

[0017] Furthermore, in step 2, the screening method is: screening the search results according to the conventional standards for homologous sequence comparison.

[0018] Furthermore, in step 3, the method of sorting is: starting from the relaxase gene of the plasmid to be analyzed, and arranging in the direction of the type IV coupling protein gene.

[0019] Furthermore, in step 3, the structural features of the plasmid conjugative transfer system to be analyzed are determined by comparing the sorted plasmids to be detected with the reference plasmids to determine the structural features of each plasmid to be detected compared with the reference plasmid.

[0020] Furthermore, in step 3, the structural features include gene deletion features, gene insertion features and fragment inversion features.

[0021] The beneficial effect of the present application is that it provides a method for identifying and analyzing the structural characteristics of bacterial plasmid conjugation transfer systems that can realize automated batch processing, and is efficient, accurate, and convenient. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings constituting a part of this application are used to provide a further understanding of this application, so that other features, purposes and advantages of this application become more obvious. The illustrative embodiment drawings and their descriptions of this application are used to explain this application and do not constitute an improper limitation on this application.

[0023] In addition, throughout the drawings, the same or similar reference numerals represent the same or similar elements. It should be understood that the drawings are schematic and that the components and elements are not necessarily drawn to scale.

[0024] In the attached picture:

[0025] Figure 1 It is a schematic diagram of the overall process according to an embodiment of the present application. DETAILED DESCRIPTION

[0026] Embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although certain embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided to provide a more thorough and complete understanding of the present disclosure. It should be understood that the drawings and embodiments of the present disclosure are only for exemplary purposes and are not intended to limit the scope of protection of the present disclosure.

[0027] It should also be noted that, for ease of description, only the parts related to the invention are shown in the drawings. In the absence of conflict, the embodiments and features in the embodiments of the present disclosure can be combined with each other.

[0028] It should be noted that the concepts such as "first" and "second" mentioned in the present disclosure are only used to distinguish different devices, modules or units, and are not used to limit the order or interdependence of the functions performed by these devices, modules or units.

[0029] It should be noted that the modifications of "one" and "plurality" mentioned in the present disclosure are illustrative rather than restrictive, and those skilled in the art should understand that unless otherwise clearly indicated in the context, it should be understood as "one or more".

[0030] The present disclosure will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments.

[0031] Embodiment, a bacterial plasmid conjugative transfer system identification and structural feature analysis method, referring to Figure 1 , including the following steps:

[0032] Step 1: Establish a database with the protein sequence of the plasmid to be analyzed, use the protein sequence of the bacterial plasmid conjugative transfer system as the query sequence, and search the protein sequence of the plasmid to be analyzed in the database. The protein sequence of the bacterial plasmid conjugative transfer system is collected and obtained through public literature and public databases. The plasmid to be analyzed includes a plasmid to be detected and a reference plasmid of the plasmid to be detected; the reference plasmid is obtained through literature retrieval. The conjugative transfer system includes a relaxase gene, a type IV coupling protein gene, and a type IV secretion system gene. The search method is: run the local Blastp tool in the computer system, use the protein sequences of the relaxase gene, the type IV coupling protein gene, and the type IV secretion system gene as query sequences, and perform a Blastp search on the protein sequence of the plasmid to be analyzed. The specific process is as follows:

[0033] Create a working folder Conjugation_analysis and download the protein sequences of the plasmids to be tested with Accession numbers NZ_LT985225 and NZ_MT559989 from the public database. Since both plasmids to be tested are IncC type 2 plasmids, select NC_016976 as the reference plasmid and download its protein sequence. Merge the above three sequences into one file, name it Sequences.fasta, and store it in the Conjugation_analysis folder.

[0034] Run the local Blastp tool in the Linux system, build a library with the Sequences.fasta sequence, name it Plasmid_sequences, and perform Blastp searches with the collected relaxase genes, type IV coupling protein genes, and type IV secretion system protein sequences as query sequences;

[0035] Among them, the instruction to establish the database is:

[0036] "makeblastdb-in Sequences.fasta-dbtype prot-parse_seqids-hash_index-out Database\Plasmid_sequences";

[0037] Among them, the command for Blastp search is:

[0038] "blastp-query Relaxase.fasta\T4cp.fasta\T4ss.fasta-db Database\Plasmid_sequences-outfmt"6qseqid sseqid pident length qcovs qcovhsp qcovusmismatch gapopen qstart qend sstart send evalue bitscore"-out Relaxase_result.txt\T4cp_result.txt\T4ss_result.txt".

[0039] Step 2: Screen and classify the search results using conventional standards for homologous sequence alignment; identify the type of conjugative transfer system gene contained in the plasmid to be analyzed based on the classification results.

[0040] The conventional standards for homologous sequence alignment are e-value < 0.001, identity > = 50% and coverage > = 50%; the search results of the protein sequences of the relaxase gene, type IV coupling protein gene and type IV secretion system gene of the plasmid to be analyzed are screened in turn according to these standards. According to the collected protein sequence classification information of the bacterial plasmid relaxase gene, type IV coupling protein gene and type IV secretion system gene, the results obtained by the Blastp search are annotated by type. In the screening process, for a protein sequence corresponding to multiple genes of different types, only the result with the largest identity is retained; when the identities are the same, the results with a larger coverage are retained; when both the identity and coverage are the same, only the results with a smaller e-value are retained.

[0041] According to the search and screening results, the conjugative transfer system gene of the plasmid NZ_LT985225 to be tested is: F_TraI (MOB H), F_TraD (TrwB / TraD), F_TraL, F_TraE, F_TraK, F_TraB, F_TraV, F_TraA, F_TraC, F_TraW, F_TraU, F_TraN, P_TrbB, F_TraF, F_TraH, F_TraG, F_Orf169, P_TraG; the conjugative transfer system genes of the plasmid NZ_MT559989 to be tested are: F_TraI (MOB H ), F_TraD (TrwB / TraD), F_TraL, F_TraE, F_TraK, F_TraB, F_TraV, F_TraA, F_TraC, F_TraW, F_TraU, F_TraN, F_Orf169, F_TraG, F_TraH, F_TraF; the conjugative transfer system genes of the reference plasmid NC_016976 are: F_TraI (MOB H ), F_TraD(TrwB / TraD), F_TraL, F_TraE, F_TraK, F_TraB, F_TraV, F_TraA, F_TraC, F_TraW, F_TraU, F_TraN, P_TrbB, F_TraF, F_TraH, F_TraG, F_Orf169.

[0042] Step 3: Sort the conjugative transfer system protein sequences contained in the plasmid to be analyzed according to the order of the gene loci, and determine the structural characteristics of the conjugative transfer system of the plasmid to be analyzed. The method of the sorting is: starting from the relaxase gene of the plasmid to be analyzed, arranging in the direction of the type IV coupling protein gene; if the plasmid to be analyzed lacks the relaxase gene, starting from the type IV coupling protein gene, arranging in the direction of the type IV secretion system gene protein. The structural characteristics of the conjugative transfer system of the plasmid to be analyzed are: comparing the sorted plasmid to be detected with the reference plasmid, and determining the structural characteristics of each plasmid to be detected compared with the reference plasmid. The structural characteristics include gene deletion characteristics, gene insertion characteristics and fragment inversion characteristics.

[0043] Among them, the method for confirming the inversion feature is: compare the arranged conjugative transfer system gene cluster of the plasmid to be tested with the conjugative transfer system gene cluster of the reference plasmid. If the arrangement order of 3 or more consecutive conjugative transfer system protein fragments is opposite to the arrangement order of these protein fragments in the reference plasmid, and the arrangement order of the conjugative transfer system protein fragments on both sides of the fragment is consistent with the reference plasmid, it is considered that the conjugative transfer system genome of the fragment has an inversion feature.

[0044] Compared with the reference plasmid NC_016976, the tested plasmid NZ_MT559989 lacks the P_TrbB gene; the tested plasmid NZ_LT985225 does not have the conjugative transfer system gene deletion;

[0045] Compared with the reference plasmid NC_016976, the tested plasmid NZ_LT985225 contained the P_TraG gene; the tested plasmid NZ_MT559989 did not have the conjugative transfer system gene inserted;

[0046] Compared with the reference plasmid NC_016976, the tested plasmid NZ_MT559989 had inversion of the F_Orf169, F_TraG, F_TraH, and F_TraF gene segments, while no inversion of the genome segments was found in the tested plasmid NZ_LT985225.

[0047] The above descriptions are only some preferred embodiments of the present disclosure and an explanation of the technical principles used. Those skilled in the art should understand that the scope of the invention involved in the embodiments of the present disclosure is not limited to the technical solutions formed by a specific combination of the above-mentioned technical features, but should also cover other technical solutions formed by any combination of the above-mentioned technical features or their equivalent features without departing from the above-mentioned inventive concept. For example, the above-mentioned features are replaced with the technical features with similar functions disclosed in the embodiments of the present disclosure (but not limited to) and the technical solutions formed.

[0048] In summary, the method of the present application can improve the accuracy and convenience of the analysis of the genome structural characteristics of the bacterial plasmid conjugative transfer system. Based on the data downloaded from public literature and public databases, it can realize automated and batch processing, and realize efficient analysis of the genome structural characteristics of the bacterial plasmid conjugative transfer system.

Claims

1. A method for identifying bacterial plasmid conjugative transfer systems and analyzing structural characteristics, characterized in that: The following steps are involved: Step 1: Establish a database with the protein sequence of the plasmid to be analyzed, use the protein sequence of the bacterial plasmid conjugative transfer system as the query sequence, and search for the protein sequence of the plasmid to be analyzed in the database; Step 2: Screening and classifying the search results; identifying the type of conjugative transfer system gene contained in the plasmid to be analyzed according to the classification results; Step 3: Sort the conjugative transfer system protein sequences contained in the plasmid to be analyzed according to the order of the gene loci to determine the structural characteristics of the conjugative transfer system of the plasmid to be analyzed.

2. The method for identifying bacterial plasmid conjugative transfer system and analyzing structural characteristics according to claim 1, characterized in that: The conjugative transfer system includes a relaxase gene, a type IV coupling protein gene, and a type IV secretion system gene.

3. The method for identifying bacterial plasmid conjugative transfer system and analyzing structural characteristics according to claim 2, characterized in that: The plasmid to be analyzed includes a plasmid to be detected and a reference plasmid of the plasmid to be detected; the reference plasmid is obtained by literature retrieval.

4. The method for identifying bacterial plasmid conjugative transfer system and analyzing structural characteristics according to claim 3, characterized in that: In step 1, the protein sequence of the bacterial plasmid conjugative transfer system is collected and obtained through public literature and public databases.

5. The method for identifying bacterial plasmid conjugative transfer system and analyzing structural characteristics according to claim 4, characterized in that: In step 1, the search method is: running a local Blastp tool in a computer system, using the protein sequences of relaxase gene, type IV coupling protein gene and type IV secretion system gene as query sequences, and performing a Blastp search on the protein sequence of the plasmid to be analyzed.

6. The method for identifying bacterial plasmid conjugative transfer system and analyzing structural characteristics according to claim 5, characterized in that: In step 2, the screening method is: screening the search results according to the conventional standards for homologous sequence comparison.

7. The method for identifying bacterial plasmid conjugative transfer system and analyzing structural characteristics according to claim 6, characterized in that: In step 3, the method of sorting is: starting from the relaxase gene of the plasmid to be analyzed, and arranging in the direction of the type IV coupling protein gene.

8. The method for identifying bacterial plasmid conjugative transfer system and analyzing structural characteristics according to claim 7, characterized in that: In step 3, the structural features of the plasmid conjugative transfer system to be analyzed are determined by comparing the sorted plasmids to be detected with the reference plasmids to determine the structural features of each plasmid to be detected compared with the reference plasmid.

9. The method for identifying bacterial plasmid conjugative transfer system and analyzing structural characteristics according to claim 8, characterized in that: In step 3, the structural features include gene deletion features, gene insertion features and fragment inversion features.

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