Architecture of the superintegron in Vibrio cholerae: identification of core and unique genes

Background: Vibrio cholerae, the etiologic agent of cholera, is indigenous to aquatic environments. The V. cholerae genome consists of two chromosomes; the smallest of these harbors a large gene capture and excision system called the superintegron (SI), of ~120 kbp. The flexible nature of the SI that results from gene cassette capture, deletion and rearrangement is thought to make it a hotspot of V. cholerae diversity, but beyond the basic structure it is not clear if there is a core genome in the SI and if so how it is structured. The aim of this study was to explore the core genome structure and the differences in gene content among strains of V. cholerae. Methods: From the complete genomes of seven V. cholerae and one Vibrio mimicus representative strains, we recovered the SI sequences based on the locations of the structural gene IntI4 and the V. cholerae repeats. Analysis of the pangenome, including cluster analysis of functional genes, pangenome profile analysis, genetic variation analysis of functional genes, strain evolution analysis and function enrichment analysis of gene clusters, was performed using a pangenome analysis pipeline in addition to the R scripts, splitsTree4 and genoPlotR. Results and conclusions: Here, we reveal the genetic architecture of the V. cholerae SI. It contains eight core genes when V. mimicus is included and 21 core genes when only V. cholerae strains are considered; many of them are present in several copies. The V. cholerae SI has an open pangenome, which means that V. cholerae may be able to import new gene cassettes to SI. The set of dispensable SI genes is influenced by the niche and type species. The core genes are distributed along the SI, apparently without a position effect.

V. cholerae the smallest of these harbors a large gene capture and excision system called the superintegron (SI), of ~120 kbp. The flexible nature of the SI that results from gene cassette capture, deletion and rearrangement is thought to make it a hotspot of diversity, but beyond the basic structure it is not clear if V. cholerae there is a core genome in the SI and if so how it is structured. The aim of this study was to explore the core genome structure and the differences in gene content among strains of . V. cholerae From the complete genomes of seven and one Methods: V. cholerae Vibrio representative strains, we recovered the SI sequences based on the mimicus locations of the structural gene and the repeats. Analysis of IntI4 V. cholerae the pangenome, including cluster analysis of functional genes, pangenome profile analysis, genetic variation analysis of functional genes, strain evolution analysis and function enrichment analysis of gene clusters, was performed using a pangenome analysis pipeline in addition to the R scripts, splitsTree4 and genoPlotR.
Here, we reveal the genetic architecture of the Results and conclusions: V. SI. It contains eight core genes when is included and 21 cholerae V. mimicus core genes when only strains are considered; many of them are V. cholerae present in several copies. The SI has an open pangenome, which V. cholerae means that may be able to import new gene cassettes to SI. The V. cholerae set of dispensable SI genes is influenced by the niche and type species. The core genes are distributed along the SI, apparently without a position effect. Vibrio cholerae is a diverse, environmental, gram-negative bacterial species that can be pathogenic and can cause cholera, a severe diarrheal disease that occurs most frequently in epidemic form 1,2 . The V. cholerae genome consists of two chromosomes. The largest chromosome of 2.96 Mbp encodes most essential genes. The 1.07 Mbp small chromosome contains few essential genes and the superintegron (SI), a large gene capture and excision system of ~120 kbp 2 ( Figure 1). The SI is characterized by a site-specific integrase gene (IntI4) closely associated with a cognate recombination site attI and a promoter Pc followed by a large array of gene cassettes. The functions of the majority of the SI genes are unknown; however, a few genes have been characterized and it has been suggested that they are involved in adaptive functions such as toxin-antitoxin (TA) loci. TA loci consist of two genes in an operon encoding a 'toxin' and an 'antitoxin'. The expression of the toxins reduces cell growth and prevents colony formation, thus exerting a bacteriostatic rather than bacteriocidal condition. However, cell viability can be rescued by later overproduction of the cognate antitoxins 6 .
The pangenome describes the complete repertoire of genes in a bacterial species, which includes the "core genome" containing genes present in all strains, a "dispensable genome" containing genes present in two or more strains, and "unique genes" specific to single strains 7 . Previous phylogeographic analysis, considering V. cholerae strains and its sister species Vibrio metecus 8 , showed that, in contrast to the core genome, the SI displays strong geographical differentiation, and cassettes from the V. cholerae group cluster with those of V. metecus from the same place rather than with cassettes from geographically distinct V. cholerae. It suggested that SI structure is influenced by geographic boundaries and in response to environmental conditions. The flexible nature of the SI that results from gene cassette capture, deletion and rearrangement is thought to make it a hotspot of V. cholerae diversity, but beyond the basic structure it is not clear if there is a core genome in the SI and if so how it is structured. The aim of this work was to explore the core genome structure and the differential gene content among strains of V. cholerae.

Methodology
Based on the complete genomes of seven V. cholerae and one V. mimicus representative strains (Table 1), we searched repeats above 10 nucleotides and used one VCR sequence (AAC AAA CGC CTC AAG AGG GAC TGT CAA CGC GTG GCG TTT CCA GTC CCA TTG AGC CGT GGT GGT TTC GGT TGT TGT GTT TGA GTT TAG TGT TAT GCG TTG TCA GCC CCT TAG GCG GGC G) to search for sequences with more of 45% nucleotide identity. The SI sequences were recovered using the locations of the structural gene IntI4 and VCRs identified with the UGENE software 9 . Cluster analysis of functional genes was performed using the pangenome analysis pipeline 10 , which searches for homologs or orthologs among multiple genomes using the MultiParanoid (MP) method (based on a 90% nucleotide identity threshold). For each pair of genes in the same cluster, the local matched region is no less than 25% of the longer protein coding sequence and the global matched region is no less than 50% of the longer protein coding sequence. The minimum score value and E-value in BLAST are 50 and 1e-8 10 . The gene content was converted to a presence/ absence (0/1) matrix and then the core, dispensable and unique genes were identified by in-house R scripts. The phylogenetic tree based in gene content and split network for gene content were constructed with SplitsTree4 11 using the GeneContentDistance method 12 . The SI structure and comparison of seven V. cholerae and, their sister species, V. mimicus were performed using genoPlotR 13 .

Results and discussion
SI regions were extracted from the seven V. cholerae and one V. mimicus genomes ( Table 1). The 1285 genes recovered were clustered and a total of 408 clusters were detected ( Figure 2A; Table S1). The pangenome of the SI of Vibrio strains evaluated was 408 genes, of which eight correspond to core genes, 196 are   distributed or dispensable genes and 204 are unique genes. Six of the eight core genes are present in many copies ( Table 2). The pangenome profile analysis shows that the cluster numbers of core genome are almost the same, when the SI considered reaches nine, while the pangenome is still increasing (Figure 2A). We infer that the V. cholerae SI has an open pangenome, which means that V. cholerae may have the ability to import new SI gene cassettes, which affect its plasticity and diversity. On the other hand, the set of SIs, from clinical and environmental lineages, used in this study are apparently representative of this species because allowed to establish that the core genome is close to being completed.
Function enrichment analysis of gene clusters were performed according to description of gene annotation (File S1) supplied to the pangenome analysis pipeline 10 . From the 408 clusters, 329 were unclassified by the function enrichment analysis. Following the categorization of Cluster of Orthologous Groups (COG), the characterized clusters were rich in the following categories: translation, ribosomal structure and biogenesis, transcription, replication, recombination and repair, cell cycle control, cell division, chromosome partitioning, defense mechanisms, cell wall/membrane/ envelope biogenesis and posttranslational modification, protein turnover, chaperones, amino acid transport and metabolism, nucleotide transport and metabolism, lipid transport and metabolism, secondary metabolites biosynthesis, transport and catabolism ( Figure 2B).
In the SI, random excisions occur throughout the cassette array to form nonreplicative circular intermediates containing one or several cassettes; integration events preferentially occur at the attI site 5 and are subjected to selection. It is expected that SI core genes would be arranged and stay together; however, we found the core genes are distributed along the SI (Figure 3), apparently without any position effect.
We identified 204 unique genes, 94 belonging to V. mimicus MB451, nine to LMA3984, 45 to O395, nine to 2010EL1786, 14 to MJ1236, seven to IEC224, 20 to M66, and six to N16961 (Figure 3; Table S1). Considering only the V. cholerae SI, there are 21 core genes, most of them present in many copies and rich in the transcription, replication, recombination and repair, translation, ribosomal structure and biogenesis categories.
Pandey and Gerdes 14 identified 13 TA loci within the SI of the N16961 strain. Here we identified six TA genes as part of core SI genes (Table 2), of which the relB genes (VCA0349 and VCA0504) were present in all V. cholerae strains (including V. mimicus) SIs.
The previous authors 14 also identified higBA-1 TA loci (VCA0392 and VCA0391); in our results, these two TA loci are present in all clinical V. cholerae strains (Table S1). These results suggest that V. cholerae TA loci function as essential stress response elements that help cells survive 6 , as well as act to stabilize the massive arrays of SI cassettes, as reported previously 15 .
A previous study suggested that SI structure is influenced by geographic boundaries in response to environmental conditions 8 . Here, we found that the clinical nature of the V. cholerae and V. mimicus strains evaluated were not grouped together by the analyses  performed. Therefore, the ability of V. cholerae to cause disease must be explained by other virulence factors found outside the SI region.
There are 199 clusters involved with indel or mutation events (Table S2). As for the non-synonymous/synonymous substitution (dN/dS) ratio, we found that 30 clusters were suffering positive selection pressure (dN/dS > 1). At the same time, we could also select those variable clusters as the markers for different strains. Based on pangenome profiles and single nucleotide polymorphism (SNP) information, gene content and phylogenetic trees were constructed (Figure 4). The SNP information from SI was useful for separating V. cholerae from V. mimicus, but nevertheless lacked the resolution to distinguish between the different lineages of V. cholerae. However, using gene content information (Figure 4), a good resolution was reached that was coherent with the evolution of the species and the environmental or clinical nature of the strains. These results indicate that the evolution of V. cholerae into different lineages is reflected in the diversity of the SI, which would be also influenced by horizontal gene transfer in these region, as proposed elsewhere 8,16,17 .

Conclusions
In this study, we have revealed the genetic architecture of the V. cholerae SI, which contains eight core genes, many of them present in many copies. The V. cholerae SI has an open pangenome, which means that V. cholerae may have the ability to import new gene cassettes into the SI. The set of the dispensable SI gene cassettes is influenced by the niche and type species. The core genes are distributed along the SI, apparently without a position effect.
Author contributions MAM and ACPV designed the study. MAM and ACPV analyzed the data. MAM and ACPV wrote the article. All authors have approved the final manuscript for publication.

Competing interests
No relevant competing interests were disclosed.

Gran t information
This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) doctoral fellowship to MAM.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Supplementary tables Table S1. Orthologs clusters identification among SIs from V. cholerae and V. mimicus genomes. These clusters were identified using the pangenome analysis pipeline 10 , strains without genes in the cluster are marked with "-". Table S2. Clusters involved with indel or mutation events. The 1 st column is the Cluster ID, which is consistent with the ID in Table S1. The 2 nd column is the cluster conservation of current cluster. The 3 rd column is the variation position, which counts according to the alignment result of protein sequences in this cluster. For indel events, the position is an integer. For synonymous mutation and non synonymous mutation, the position is a floating number, in which the integer part marks the position of the amino acid in the alignment result of protein sequences, while the decimal part mark the position of codon. The 4 th column shows the amino acid types on current position. The 5 th column shows the nucleotide types on current position, indel is marked with "-". The 6 th column shows all gene nucleotide profile in current position (for indel, amino acid will be listed). The 7 th column shows the variation type (indel, synonymous and non synonymous). The CDS.variation.analysis spreadsheet shows the summary result for CDS.variation.

Supplementary files
File S1. Gene data of SI region from seven V. cholerae and one V. mimicus genomes used in this study.
File S2. R scripts used in this study.

Current Referee Status:
Referee The core genes estimates for SI (21 and 8, among and respectively) V. cholerae V. cholerae-V. mimicus reported in this study may not be very meaningful as the core genome might diminish or at least reduced further if additional genomes from distinct lineages are included. I strongly recommend inclusion of additional genomes at least from the major phylogenetic lineages of O1.

V. cholerae
The function of TA loci as an essential stress response element needs to be supported by some experimental data.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
No competing interests were disclosed. The article, although it has a very narrow focus, investigates an interesting question about integron regions in that scientists have so far mostly applied to whole genome sequences: what is their V. cholerae pan-genome and core genome? The scope of the question is quite narrow, as it focuses on a single species ( ), but is certainly novel.

V. cholerae
The methods and approach used are sound and the results generally well presented. I have two major issues with the analysis: Why use only seven genomes when >200 are available? This is quite puzzling to me, as no data should be excluded if it is available. Some of the >200 genomes available are relatively V. cholerae redundant (they all belong to the 7 pandemic group) and present little gene content/SNP th 1.

2.
redundant (they all belong to the 7 pandemic group) and present little gene content/SNP diversity, but the variations they present are crucial in understanding small scale variability. Many available environmental genomes were also not included and should have been.

V. cholerae
The article is mostly descriptive and needs to make more informative statements about the results. What is the significance of such a small core genome? How does the pan-genome of the integron overlap with the rest of the genome? What is the meaning of the specific subset of functions found in the integron? How are sites under positive selection distributed in the integron? What genes are under positive/ negative selection? Much more can be said about integrons from the analyses performed.
Basically, the manuscript is interesting but could really benefit from a broader (more genomes) analysis and a more in-depth look at the results to infer hypotheses about integrons in . V. cholerae I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
No competing interests were disclosed.

Competing Interests:
Thandavarayan Ramamurthy National Institute of Cholera and Enteric Diseases, Kolkata, India O1 and genetic relatedness of SIs at the biotype level. cholerae Comments: Abstract: Results and Conclusions: replace the word 'reveal' with "describe" Introduction: first paragraph: replace the word 'standing' with "standby" Page 6. The role of TA gene should be validated with non-toxigenic strains of (e.g., V. cholerae sequence comparison with non-O1, non-O139 strains of V. cholerae I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
No competing interests were disclosed. Competing Interests: th