# Exception to the Rule: Genomic Characterization of Naturally Occurring UnusualVibrio cholerae Strains with a Single Chromosome

Authors: Gary Xie; Shannon L. Johnson; Karen W. Davenport; Mathumathi Rajavel; Torsten Waldminghaus; John C. Detter; Patrick S. Chain; Shanmuga Sozhamannan
Journal: International Journal of Genomics
 (2017)

---

## Abstract

The genetic make-up of most bacteria is encoded in a single chromosome while about 10% have more than one chromosome. Among these,Vibrio cholerae, with two chromosomes, has served as a model system to study various aspects of chromosome maintenance, mainly replication, and faithful partitioning of multipartite genomes. Here, we describe the genomic characterization of strains that are an exception to the two chromosome rules: naturally occurring single-chromosome V. cholerae. Whole genome sequence analyses of NSCV1 and NSCV2 (natural single-chromosome vibrio) revealed that the Chr1 and Chr2 fusion junctions contain prophages, IS elements, and direct repeats, in addition to large-scale chromosomal rearrangements such as inversions, insertions, and long tandem repeats elsewhere in the chromosome compared to prototypical two chromosome V. cholerae genomes. Many of the known cholera virulence factors are absent. The two origins of replication and associated genes are generally intact with synonymous mutations in some genes, as are recA and mismatch repair (MMR) genes dam, mutH, and mutL; MutS function is probably impaired in NSCV2. These strains are ideal tools for studying mechanistic aspects of maintenance of chromosomes with multiple origins and other rearrangements and the biological, functional, and evolutionary significance of multipartite genome architecture in general.


---

## Body

## 1. Introduction

The genetic endowment of the majority of bacteria (~90%) is inherited in a single chromosome of their respective genomes [1]. An exception to this rule is the presence of two chromosomes in V. cholerae, the causative agent of cholera disease [2, 3]. In fact, all tested genera/species in the family Vibrionaceae possess two chromosomes [4]. The two V. cholerae chromosomes (Chr1 and Chr2) are of unequal sizes (~2.96 Mb and ~1.07 Mb, resp.). Most of our knowledge on the control of chromosome maintenance of multipartite genomes is derived from studies on V. cholerae [5–7]. It was postulated that Chr2 is derived from plasmid based on its plasmid-like origin and evolved into a secondary chromosome by acquiring additional layers of regulation for its replication [3, 8]. Although Chr1 encodes the majority of the housekeeping genes and is considered as the main chromosome, Chr2 also harbors essential genes beside many genes with unknown functions [9–11]. The genes on Chr1 and Chr2 are differentially expressed in specific niches. For example, when the bacterium was grown midexponentially in rabbit ileal loops, it showed expression of many more genes of Chr2 than those expressed in aerobically grown cells in rich medium and harvested at the midexponential phase [12]. Majority of these are probably important niche-specific genes and hence expressed preferentially in ileal loop. The results were similar when the bacteria were collected from stools of cholera patients [13].Chr1 replication follows the traditionalE. coli paradigm in that the replication origin, ori1, contains multiple DnaA boxes where DnaA binds to initiate DNA replication [14–16]. A V. cholerae Chr1 minireplicon can replicate in E. coli without the need for V. cholerae-transacting factors but merely using E. coli proteins such as DnaA. Similarly, V. cholerae ori1 could functionally substitute E. coli replication origin oriC [11, 14, 16].TheV. cholerae ori2 resembles those of low-copy-number plasmids such as P1 and F in that it contains an array of repeats (iterons) where Chr2-specific initiator protein, RctB, binds to unwind the DNA for ori2 firing [14, 17]. In V. cholerae, Chr1 initiates at the onset of the replication period while initiation of Chr2 is delayed and occurs only when 2/3 of the Chr1 replication has already been completed. Because Chr2 is 1/3 the size of Chr1, both chromosomes terminate their replication roughly at the same time [18, 19]. It was found recently that a site, termed crtS (Chr2 replication triggering site), present on Chr1 triggers ori2 initiation when it is replicated [20, 21].The two chromosomes ofV. cholerae are longitudinally arranged in the cell [22]. While Chr1 appears to be spread along the entire longitudinal axis of the cell, Chr2 is restricted to the younger half of the cell. In newborn cells, Chr1 extends from the old pole to the new pole and Chr2 extends from midcell to the new pole [22]. The differential positioning of the chromosomes within the cell is accompanied by a distinct segregation choreography owing to chromosome-specific ParAB/parS-based segregation systems. [22–24]. One of the last steps in chromosome segregation before cell division involves the resolution of dimeric chromosomes that are frequently produced by homologous recombination between sister chromatids following DNA damage [25]. In V. cholerae, dimers of Chr1 and Chr2, are resolved by the action of the same machinery, XerC and XerD site-specific recombinases at the dif sites (dif1 and dif2), located in the ter regions of Chr1 and Chr2, respectively [26]. While the ter regions of both chromosomes replicate at the same time point within the cell cycle, Chr1 sister termini are held together at midcell much longer than Chr2 sister termini [22]. The MatP/matS system was found to impede separation of Chr1 sister termini and restrict movement of Chr2 sister termini to allow processing by FtsK right before cell division [27]. In addition, a nucleoid-associated, FtsZ-binding protein termed SlmA has been shown to be required for blocking septal ring assembly. SlmA is a DNA-associated division inhibitor that is directly involved in preventing Z ring assembly on portions of the membrane surrounding the nucleoid [28].Chr2 is indispensable for viability of the cell since elimination of Chr2 is lethal due to multiple “suicidal” toxin-antitoxin systems encoded by Chr2 [29–31]. V. cholerae with single chromosomes has been created by genetic engineering to fuse the two natural chromosomes [32]. Chromosomal fusions in V. cholerae were also isolated as suppressor mutations for a deletion of the dam (DNA adenine methyl transferase) gene [33]. Dam is essential for replication of Chr2 because the initiator protein RctB binds to the target sites in ori2 only when they are methylated [14, 16, 32]. The only way to replicate Chr2 without a functional ori2, that is, unmethylated ori2, was a Chr1 and Chr2 chromosomal fusion that permits replication of the entire chromosome from ori1 [33]. In dam-suppressor mutants, Chr1 and Chr2, fusions had occurred either by homologous recombination between IS elements or site-specific recombination between dif sites. Interestingly, fusion occurred preferentially in the terminus regions of the chromosomes [33]. Recently, it was found that chromosomal fusion in V. cholerae can also occur as a suppressor of ΔcrtS mutations [20].Until recently, evidence for a naturally occurringVibrio with fused chromosomes was missing. In an attempt to assess the genomic diversity of non-O1/non-O139 V. cholerae, a whole genome mapping strategy was applied on a well-defined, geographically, and temporally diverse strain collection, the Sakazaki serogroup type strains [34]. In that study, the whole genome map data on 91 of the 206 serogroup type strains supported the hypothesis that V. cholerae has an unprecedented genetic and genomic structural diversity with very few clonal complexes. Fortuitously, chromosomal fusions in two unusual strains that possess a single chromosome instead of the two chromosomes usually found in V. cholerae were discovered which was further confirmed by pulse field gel electrophoresis (PFGE) [35].Earlier, we reported the whole genome sequencing and generation of a gapless single-contig sequence of the two unusual single-chromosome strains, 1154-74 (serogroup O49) and 10432-62 (serogroup O27), hereafter referred to as NSCV1 and NSCV2, respectively [36]. In the current paper, we report further genome sequence analyses of NSCV1 and NSCV2. We delineate the Chr1 and Chr2 fusion junctions, other structural anomalies such as indels, inversions and duplications, salient features of their gene content and the origins of replication, and their potential activity. Furthermore, we analyze the genes involved in replication of the multiple origins in the same chromosome. Thus, the primary focus of this paper is to lay the foundation for future studies on functional and mechanistic aspects of chromosome and structural maintenance in these unusual V. cholerae strains.


## 2. Materials and Methods

### 2.1. Bacterial Strains and Growth Conditions

V. cholerae 1154-74 is a strain isolated in India in 1974 from a diarrheal sample, and it belongs to the O49 serogroup. V. cholerae 10432-62 is a strain isolated in the Philippines in 1962 from a diarrheal sample, and it belongs to the O27 serogroup. These strains are referred to as NSCV1 and NSCV2, respectively, in this manuscript. The corresponding Los Alamos National Labs (LANL) Sequencing Center designations are VAAO49 and VABO27, and the old locus tags in the Gen Bank entries carry these designations. The bacterial strains were grown in LB medium at 37°C using normal standard laboratory protocols. Genomic DNAs for sequencing were extracted using Qiagen genomic DNA extraction kits (Qiagen Inc.).


### 2.2. Whole Genome Sequencing Method

The draft genomes ofVibrio cholerae strains NSCV1 (1154-74) and NSCV2 (10432-62) were generated at the LANL Genome Science Group using a combination of Illumina [37] and 454 technologies [38]. For each genome, we constructed and sequenced an Illumina GAiiX shotgun library, a 454 Titanium standard library and a paired end 454 library, and a Pacific Biosciences RS long read library (P4-C2 chemistry). The 454 Titanium standard data and the 454 paired end data were assembled together with Newbler, version 2.3-PreRelease-6/30/2009. The Newbler consensus sequences were computationally shredded into 2 kb overlapping fake reads (shreds). Illumina sequencing data was assembled with VELVET, version 1.0.13 [39], and the consensus sequence was computationally shredded into 1.5 kb overlapping fake reads (shreds). We integrated the 454 Newbler consensus shreds, the Illumina VELVET consensus shreds, the PacBio subreads, and the read pairs in the 454 paired end library using parallel phrap, version SPS - 4.24 (High Performance Software, LLC). Illumina data were used to correct potential base errors and increase consensus quality using the software Polisher developed at Joint Genome Institute (JGI) [40]. Possible misassemblies were corrected using gapResolution [41], or Dupfinisher [42]. Completed genome assemblies were compared to optical maps to ensure consensus. Each final assembly consisted of a single ca 4.1 Mb chromosome.


### 2.3. Genome Annotation

Assembled genomes were annotated using a modified version of the IGS Annotation Engine (released by the University of Maryland, Institute for Genome Sciences at the School of Medicine) on an Ergatis workflow manager. Annotated genomes are available in GenBank under accession numbers NSCV1-1154-74: NZ_CP010811.1 and NSCV2-10432-62: NZ_CP010812.1. Post assembly genome sequence analysis was done using the CLC Genomic Workbench version 6.5.Annotation of the assembled genome sequence was also carried out with genome annotation systems at LANL EDGE serverhttps://bioedge.lanl.gov/ [43] and RAST server [44]. A combined gene prediction strategy was applied by means of the GLIMMER 2.0 system and the CRITICA program suite [45] along with post processing by the RBSfinder tool [46]. tRNA genes were identified with tRNAscan-SE [47]. The deduced proteins were functionally characterized by automated searches in public databases, including SWISS-PROT and TrEMBL [48], Pfam [49], TIGRFAM [50], InterPro [51], and KEGG [52]. Each gene was functionally classified by assigning clusters of orthologous group (COG) number and corresponding COG category [53].


### 2.4. Genomic Comparison

Comparative genome analyses of NSCV1 and NSCV2 to otherV. cholerae strains MS6, O1 biovar El Tor str. N16961, and O139 MO10 were performed by using a set of tools available at LANL and RAST servers. Homology searches were conducted at the nucleotide and amino acid sequence level using BLAST [54]. To obtain a list of orthologs from bacteroidetes genomes, a perl script that determines bidirectional best hits was written. For example, genes g and h are considered orthologs if h is the best BLASTP hit for g and vice versa. E values of 10−15 were acceptable. A gene is considered strain specific if it has no hits with an E value of 10−5 or less. The genome comparisons at the nucleotide level were carried out with genome alignment tools, such as MUMmer2 [55], NUCmer [56], the Artemis Comparison Tool (ACT) [57], and WebAct (http://www.webact.org/WebACT/home) at Imperial College, London.The MUMmer package [58] programs nucmer, repeat_match, and exact_tandems were used for analysis of repeat regions. To identify long inexact genomic repeats, the nucmer program was used with the options –maxmatch and –nosimplify. The resulting dotplot was obtained using mummerplot. The repeat_match and exact_tandems programs were run with default arguments. Tandem repeats finder [59] and inverted repeats finder [60] were run with recommended default arguments to identify tandem and inverted repeats, respectively. Phage islands and putative genomic islands were identified using PHAST (PHAge Search Tool) [61] and Islandviewer [62], respectively. The circular genome diagram (Figure S3 available online at https://doi.org/10.1155/2017/8724304) was drawn using the DNAplotter [63]. Blast Ring Image Generator (BRIG) software was used to generate genome comparison views presented in Figure S2 [64]. Multiple genome alignment shown in Figure S1 was created using progressive MAUVE [65].


## 2.1. Bacterial Strains and Growth Conditions

V. cholerae 1154-74 is a strain isolated in India in 1974 from a diarrheal sample, and it belongs to the O49 serogroup. V. cholerae 10432-62 is a strain isolated in the Philippines in 1962 from a diarrheal sample, and it belongs to the O27 serogroup. These strains are referred to as NSCV1 and NSCV2, respectively, in this manuscript. The corresponding Los Alamos National Labs (LANL) Sequencing Center designations are VAAO49 and VABO27, and the old locus tags in the Gen Bank entries carry these designations. The bacterial strains were grown in LB medium at 37°C using normal standard laboratory protocols. Genomic DNAs for sequencing were extracted using Qiagen genomic DNA extraction kits (Qiagen Inc.).


## 2.2. Whole Genome Sequencing Method

The draft genomes ofVibrio cholerae strains NSCV1 (1154-74) and NSCV2 (10432-62) were generated at the LANL Genome Science Group using a combination of Illumina [37] and 454 technologies [38]. For each genome, we constructed and sequenced an Illumina GAiiX shotgun library, a 454 Titanium standard library and a paired end 454 library, and a Pacific Biosciences RS long read library (P4-C2 chemistry). The 454 Titanium standard data and the 454 paired end data were assembled together with Newbler, version 2.3-PreRelease-6/30/2009. The Newbler consensus sequences were computationally shredded into 2 kb overlapping fake reads (shreds). Illumina sequencing data was assembled with VELVET, version 1.0.13 [39], and the consensus sequence was computationally shredded into 1.5 kb overlapping fake reads (shreds). We integrated the 454 Newbler consensus shreds, the Illumina VELVET consensus shreds, the PacBio subreads, and the read pairs in the 454 paired end library using parallel phrap, version SPS - 4.24 (High Performance Software, LLC). Illumina data were used to correct potential base errors and increase consensus quality using the software Polisher developed at Joint Genome Institute (JGI) [40]. Possible misassemblies were corrected using gapResolution [41], or Dupfinisher [42]. Completed genome assemblies were compared to optical maps to ensure consensus. Each final assembly consisted of a single ca 4.1 Mb chromosome.


## 2.3. Genome Annotation

Assembled genomes were annotated using a modified version of the IGS Annotation Engine (released by the University of Maryland, Institute for Genome Sciences at the School of Medicine) on an Ergatis workflow manager. Annotated genomes are available in GenBank under accession numbers NSCV1-1154-74: NZ_CP010811.1 and NSCV2-10432-62: NZ_CP010812.1. Post assembly genome sequence analysis was done using the CLC Genomic Workbench version 6.5.Annotation of the assembled genome sequence was also carried out with genome annotation systems at LANL EDGE serverhttps://bioedge.lanl.gov/ [43] and RAST server [44]. A combined gene prediction strategy was applied by means of the GLIMMER 2.0 system and the CRITICA program suite [45] along with post processing by the RBSfinder tool [46]. tRNA genes were identified with tRNAscan-SE [47]. The deduced proteins were functionally characterized by automated searches in public databases, including SWISS-PROT and TrEMBL [48], Pfam [49], TIGRFAM [50], InterPro [51], and KEGG [52]. Each gene was functionally classified by assigning clusters of orthologous group (COG) number and corresponding COG category [53].


## 2.4. Genomic Comparison

Comparative genome analyses of NSCV1 and NSCV2 to otherV. cholerae strains MS6, O1 biovar El Tor str. N16961, and O139 MO10 were performed by using a set of tools available at LANL and RAST servers. Homology searches were conducted at the nucleotide and amino acid sequence level using BLAST [54]. To obtain a list of orthologs from bacteroidetes genomes, a perl script that determines bidirectional best hits was written. For example, genes g and h are considered orthologs if h is the best BLASTP hit for g and vice versa. E values of 10−15 were acceptable. A gene is considered strain specific if it has no hits with an E value of 10−5 or less. The genome comparisons at the nucleotide level were carried out with genome alignment tools, such as MUMmer2 [55], NUCmer [56], the Artemis Comparison Tool (ACT) [57], and WebAct (http://www.webact.org/WebACT/home) at Imperial College, London.The MUMmer package [58] programs nucmer, repeat_match, and exact_tandems were used for analysis of repeat regions. To identify long inexact genomic repeats, the nucmer program was used with the options –maxmatch and –nosimplify. The resulting dotplot was obtained using mummerplot. The repeat_match and exact_tandems programs were run with default arguments. Tandem repeats finder [59] and inverted repeats finder [60] were run with recommended default arguments to identify tandem and inverted repeats, respectively. Phage islands and putative genomic islands were identified using PHAST (PHAge Search Tool) [61] and Islandviewer [62], respectively. The circular genome diagram (Figure S3 available online at https://doi.org/10.1155/2017/8724304) was drawn using the DNAplotter [63]. Blast Ring Image Generator (BRIG) software was used to generate genome comparison views presented in Figure S2 [64]. Multiple genome alignment shown in Figure S1 was created using progressive MAUVE [65].


## 3. Results and Discussion

### 3.1. Whole Genome Sequencing ofV. cholerae Strains NSCV1-(1154-74/VAA_O49 and NSCV2-(10432-62/VAB_O27)

Whole genome mapping (WGM) also known as optical mapping and PFGE data provided evidence of Chr1 and Chr2 fusions inV. cholerae strains NSCV1 (1154-74) and NSCV2 (10432-62) [35]. We performed whole genome sequencing of the two V. cholerae strains in order to understand the molecular mechanisms that might have led to Chr1 and Chr2 fusions in these strains [36]. We generated Roche 454 (21× and 29×), Illumina (2178× and 507×), and Pac Bio RS (350× and 190×) sequence data at the indicated coverages for NSCV1 and NSCV2, respectively, and used a hybrid assembly approach that resulted in a single-contig, gapless genome (Table S1). Further resolution of the ambiguous regions was carried out using Sanger-based primer walking according to Las Alamos National Labs (LANL) genome finishing pipeline yielding a final, finished, and closed single-contig sequence [66]. Consistent with the WGM results and in contrast to the previous genomic reports of V. cholerae and its close neighbors [2–4], these two sequences assembled into genomes consisting of a single chromosome of approximately the same size as the two expected chromosomes added together. All other genomic statistics for NSCV1 and NSCV2 appear to be highly similar to other V. cholerae genomes currently available in GenBank (Table S1). The genomes of NSCV1 and NSCV2 are comprised of a single circular chromosome of 3,928,357 and 4,074,462 bp in length with an overall G + C content of 47.73% and 47.66%, respectively (Figure 1). Following the sequencing convention, the nucleotide position 1 was placed upstream of the dnaA gene (VAA_049_1 and VAB_027_1) encoding the chromosomal replication initiator protein. Chr2 portion of NSCV1 and NSCV2 spans from genome positions 1,720,246–2,772,395 (1,052,150 bps) and 297,221–1,375,947 (1,078,727 bps), respectively. All other NSCV1 and NSCV2 genome statistics are presented in Table S1.Figure 1
Circular genome maps of NSCV1 (1154-74_VAAO49) and NSCV2 (10432-62_VABO27). Fusion of Chr1 (dark grey) to Chr2 (blue) is shown in the circle at the respective locations. Various unique features such as prophages and the origins of replication- and replication-associated genes are indicated around the circles.


### 3.2. Whole Genome Sequence Comparison of NSCV1 and NSCV2 to OtherV. cholerae

DNA sequence ofV. cholerae strain MS6 is the closest match to NSCV1 and NSCV2 chromosomes in public genome databases based on whole genome sequence homology [67]. MS6 is a V. cholerae O1 strain with a novel genetic background designated in Thailand–Myanmar and isolated from a stool sample of a diarrheal patient [68]. NSCV1 and NSCV2 genome sequences exhibit a high degree of synteny with MS6, N16961, MO10, and TSY216 as visualized in the Mauve alignment despite genetic rearrangements such as insertions and inversions across the chromosome (Figure S1) [69]. A pairwise Megablast comparison of NSCV1 and NSCV2 genomes to four pathogenic V. cholerae (N16961, MO10, MS6, and TSY 216) was performed. At 85%, identity > 88–94% is shared between NSCV1 and NSCV2 compared to N16961, MS6, or MO10 genomes and 74% and 80% is shared between NSCV1 and NSCV2 compared to TSY216, respectively. The unique regions varied from 5 to 10% (Table S2).To further delineate the differences between NSCV1 and NSCV2 against all other genomes, the locations and gene content of the unique regions were determined (Table S3) and displayed as BRIG view (Figure S2). In the whole genome comparison of NSCV1 and NSCV2 to V. cholerae strains MS6, N16961, and TSY216, large regions (~10 kb or more) missing in comparator strains are highlighted in the outer circle of the BRIG view (Figure S2). As expected, many features (e.g., prophages) that are unique to NSCV1 and NSCV2 are absent in the pathogenic strains and similarly, many of the known virulence regions (e.g., CTX and VPI) that are present in pathogenic V. cholerae such as N16961 are absent in NSCV1 and NSCV2. In Chr2 segment, the super integron region is present in NSCV1 and NSCV2 with many subtle indels/variations.


### 3.3. Comparison of Genomic Content of NSCV1 and NSCV2 to PathogenicV. cholerae

Further analysis of the genomic content of NSCV1 and NSCV2 was performed by comparing the genome annotations. We were interested to see if there are any differences in the genetic content that might explain the mechanism of genomic fusion. Overall, the number of CDS encoded by NSCV1 and NSCV2 is very similar to otherV. cholerae (Table S1). NSCV1 and NSCV2 have 2759 CDS in common with all other genomes compared here. Comparison of the genomes of NSCV1 and NSCV2 with MS6, N16961, and MO10 (an epidemic V. cholerae strain belonging to the O139 serogroup) revealed that the four organisms have approximately 3188/3259 genes in common, depending on whether NSCV1 or NSCV2 is used as the query, respectively (Figure 2). There are 173/304 CDSs unique to NSCV1 and NSCV2, respectively.Figure 2
Comparison of the genomic content of NSCV1 (a), NSCV2 (b), and NSCV1 and NSCV2 (c) to various other genomes. Venn diagram showing the number of NSCV1 or NSCV2 predicted CDS with significant homology (1e−5) with the predicted products of the near neighbors: V. cholerae MS6, N16961 (serogroup O1 biovar El Tor), and O139 MO10 (serogroup O139). The number outside the circles (173 or 304) represents the number of NSCV1 or NSCV2 CDS that does not have significant homologs in the three strains compared. Conserved genes among them were defined by whole-genome pairwise sequence comparisons using the sequence-based comparison tool in RAST.
(a)
(b)
(c)


### 3.4. Genomic Islands, Prophages, and Other Mobile Genetic Elements

In the NSCV1 and NSCV2 chromosomes, 49 and 20 genomic islands amounting to 677, 625 bps and 205, 247 bps or 16.32% and 4.94% of the entire genome, respectively, were detected. Clustered regularly, interspaced short palindromic repeat (CRISPR) element is a widely found defense mechanism of prokaryotes against entry of foreign DNA including plasmids and phages [70]. In the NSCV1 and NSCV2 chromosomes, one CRISPR element was located at 3,606,088–3,606,203 bps and 3,572,464–3,572,579 bps in NSCV1 and NSCV2 genomes, respectively. The NSCV1 and NSCV2 genomes contained 58 and 55 mobile genetic elements, respectively, that encode phage integrases, transposases, and site-specific recombinase (Figure S3), compared to 46 mobile element genes in V. cholerae MS6. NSCV1 has 5 prophages located at (1) 685,729–702,622; (2) 1,458,739–1,476,484; (3) 1,756,187–1,818,403; (4) 1,879,405–1,883,642; and (5) 2,643,793–2,650,802. Prophages 3 and 5 are at the immediate boundary region and inside of the Chr2 insertion locus. Only prophage 3 at the 5′ end of Chr2 insertion appears to be intact and encodes 56 CDS. NSCV2 has 5 prophages located at (1) 3,113,341–3,130,219; (2) 3,416,918–3,428,588; (3) 299,327–349,041; (4) 1,359,430–1,369,981; and (5) 1,676,903 to 1,714,350. Only prophage 5 is intact whereas the other four are defective or incomplete. Prophages 3 and 4 are at the immediate flanking region of the Chr2 insertion junction (Figure 1).


### 3.5. Virulence and Antimicrobial Resistance Genes

V. cholerae strains NSCV1 and NSCV2 were isolated from patients exhibiting atypical cholera symptoms [34]. It is of interest to see if these strains carry any of the usual V. cholerae virulence factors and if not, what other virulence factors or toxins might be present that would explain the diarrheal symptoms. The major virulence factor of V. cholerae, the cholera toxin encoded by ctxAB genes, is not found in NSCV1; however, other toxin genes ace and zot and RTX toxin cluster can be found, in addition to toxRS genes. The toxin genes are absent in NSCV2. NSCV1 and NSCV2 each has 57 genes encoding putative resistance to antibiotics and toxic compounds such as colicin V, bacteriocin, cobalt-zinc-cadmium, copper homeostasis, fluoroquinolones, and multidrug resistance efflux pumps.


### 3.6. O-Antigen Regions

NSCV1 and NSCV2 belong to serogroups O49 and O27, respectively, and as seen in other serogroups ofV. cholerae, the O-antigen biosynthesis genes are encoded in a cluster (wb∗) on Chr1 part of the respective genomes from 3,777,887–3,815,081 (CDS VAAO49_3428-VAAO49_3463) and 3,888,708–3,920,841 (CDS VABO27_3582-VABO27_3611) of NSCV1 and NSCV2, respectively. Also, as seen in other wb∗ regions, the boundary regions of this cluster are highly conserved whereas the region between the conserved genes is highly divergent. More specifically, in the NSCV1 wb∗ region, 2 segments of 2974 bps and 6212 bps at the left end and another segment of 5733 bps at the right end, respectively, are 96% and 94% identical to those in the wbe region (O1 serogroup). A blast analysis of the wb∗ region of NSCV2 revealed segments that are similar to V. vulnificus (GenBank Accession # CP009261) gene cluster (around 42 kb) with 4 segments that are >65–85% identical, ranging in length from ~1.0 kb to 8.831 kb to the NSCV2 wb∗ cluster. In addition, identities to V. mimicus at 83–92% in segments of 4176 bps, 2789 bps, and 1410 bps and to Plesiomonas shigelloides and Shewanella baltica at ~80% (3281 bps), and to NSCV1 at 94–96% (4325, and 3007 bps) identities were also found as depicted in Figure 3.Figure 3
Genetic maps of O-antigen regions (wb∗) of NSCV1 and NSCV2. The various genes and their orientations are indicated by the arrows. Homologous regions to other serogroups are indicated by the red arrows. The V. vulnificus wb∗ cluster that has extensive homology to NSCV2 wb∗ cluster is shown in (c).
(a)
(b)
(c)


### 3.7. Identification of Large Tandem Repeats and Inversion

A closer analysis of the WGMs (whole genome maps) of NSCV1 and NSCV2 strains revealed a large duplication in the general region of 1240 and 1506 kb on a reference genome (M66–2) coordinates based on optical restriction maps [35]. Experimental WGM data is further supported by in silico WGM generated from whole genome sequence data of NSCV1 and NSCV2 (Figure S4). Sequence read mapping results showed that there is a large duplication of 200 kbs and 70 kbs regions in NSCV1 and NSCV2, respectively, as evidenced by >1×, but <2×, depth of average coverage of the genome at 1,503,484 to 1,718,861 bps in NSCV1 and 2,221,105 to 2,300,924 bps in NSCV2 compared to that in the rest of the genome (Figure S5). Manual validation indicated that the 200 kbs and 70 kbs regions indeed are fragmental duplications, existing among subpopulation of NSCV1 and NSCV2 cells, respectively. Both 1 copy and 2 copy versions of genome assembly have enough evidence supporting variations among population. These duplicated sequences spanned from positions 1,503,484 to 1,718,861 (unit 1) and from 1,718,862 to 1,934,269 bp (unit 2) in NSCV1 and from positions 2,221,105 to 2,300,924 (unit 1) and from 2,300,925 to 2,380,745 (unit 2) in NSCV2 in genome sequences with repeats included (Figure S6). The duplicated sequences span a total length of 430,785 bp and 159,640 bp on Chr1 region and encode 177 and 59 duplicated genes in NSCV1 and NSCV2, respectively. It should be noted that the two copies of repeat units are not identical. There is a 31 bp indel between the two copies in NSCV1, which resides at the locus of VAA_049_1509, encoding exonuclease family protein. There are 5 single bp indels between the two copies in NSCV2. Although tandem repeats are present at different locations on NSCV1 and NSCV2, there is a 40 kb DNA segment that overlapped at the 5′ end of NSCV1 and NSCV2 repeats with 98% identities (Figure S7). However, the tandem duplications could not be verified by PCR of the junctions since the orientation of the two copies cannot be ascertained in the assembly. Hence, we have decided to keep the genome sequence with a single copy as the ref seq (GenBank submission) for all the analyses. This long duplication anomaly could not be unequivocally resolved with the existing sequence data. In addition, there is a large inversion from 1,477,189 to 3,484,983 bps in NSCV2 compared to MS6 chromosome 1 region from 612,067 to 2,541,958 bps (Figure S1b).


### 3.8. Mapping the Fusion Junctions and Possible Mechanism of Genome Fusion

Chromosomal fusions were observed previously in suppressor mutants resulting from a genetic screen [33]. These fusions were analyzed in detail and found to occur via two different mechanisms, either by homologous recombination at IS elements or by site-specific recombination at dif sites [32] or as transient chromosomal fusions in ΔcrtS suppressor mutants [20]. In this study, we analyzed the fusion sites of NSCV1 and NSCV2 in detail to see if any one of these mechanisms also led to chromosomal fusion in the natural single-chromosome isolates. The circular single chromosomes of NSCV1 and NSCV2 are shown in Figure 1, and along with N16961, Chr1- and Chr2-concatenated circular maps are shown in Figure S2, and the genome sequence statistics are provided in Table S1. The insertion sites on Chr1 backbone (as an accepter) and Chr2 backbone (as a donor) are not the same for NSCV1 and NSCV2 (see below). Artemis Comparison Tool views (zoomed in view) of the fusion junctions are presented in Figures S8 and S9. Further description of the hypothetical events leading to the fusions is provided below.


### 3.9. Chr1 and Chr2 Fusion Junctions inV. cholerae Strain NSCV1

A closer examination of the Chr1-Chr2 insertion junction in NSCV1 to Chr1 and Chr2 sequences of MS6 indicates that multiple events probably occurred before the final NSCV1 was derived. In an NSCV1-like intermediate, on Chr1 between VAA049_1545 and VAA049_2500, there probably was an insertion of MS6_A0562- and MS6_A0272-like CDS (Chr2 CDS) (Figure4 (top panel)). On Chr2, there was an insertion of a prophage to the right of MS6_A0272 (Figure 4 (top panel)). In the next step, Chr2 with the new prophage was inserted into Chr1 via the homologies between the two CDS (VAA049_1594 versus MS6_A0272 and VAA049_2432 versus MS6_A0562), and upon the resolution of the cointegrate, one copy each of the two homologous CDS along with intervening sequences was deleted (Figure 4 (bottom panel)). Thus, Chr2 in NSCV1 spans from 1,714,319 to 2,772,395 bps (including its flanking unique region, Figure 4 (bottom panel)) or from 1,757,711 to 2,722,145 bps (including boundary of Chr2 homologous region) (Figure 4 (bottom panel)) spanning NSCV1 CDS VAAO49_1594–2432. This recombination event likely occurred between MS6_1029 and MS6_1028 or between 1,168,144 and 1,168,021 bp in MS6-like Chr1 background and inserted from the breakpoint between MS6_A0272 and MS6_A0562 or between 303,999 and 446,488 bp on MS6-like Chr2 background (Figure 4 (bottom panel)). There are 2 prophages (3 and 5) closer to the boundaries of Chr2 insertion locus. Flanking the Chr2 insertion sites, there are unique regions of 37,425 bps and 50,250 bps in length at 5′ end and 3′ end, respectively. The role of any of these unique regions in the recombination event or the precise mechanism of recombination cannot be ascertained with available whole genome sequence data in public databases.Figure 4
Putative recombination event that resulted in Chr1 and Chr2 fusions in NSCV1. Top panel: MS6 Chr1 CDS at the fusion junction and the same location in a putative NSCV1-like intermediate and the MS6 Chr2 circle and the codons at the fusion junction are depicted. The cross-over region between Chr1 and Chr2 is indicated by the long X. A prophage insertion event to the right fusion junction prior or post to Chr 2 fusion is depicted by a green line. Bottom panel: The recombination products after fusion event are shown with NSCV 1 in the middle and an excision product that contains one copy of the cross-over genes with the intervening sequences at the bottom.


### 3.10. Chr1 and Chr2 Fusion Junctions inV. cholerae Strain NSCV2

A closer examination of the insertion junctions indicates that an NSCV2-like intermediate probably possessed MS6_A0925- and MS6_A0924-like Chr2 CDS on Chr1 flanked by 2 prophages (Figure5 (top panel)). In that intermediate strain, Chr2 was inserted via recombination of the homologous segments (VAB027_307 versus MS6_A0924 and VAB027_1228 versus MS6_A0925). As a result, Chr2 spans from 337,822 bps to 1,351,337 bps (without its flanking prophage regions between CDS VABO27_307-VABO27_1228) or from 297,221 bps to 1,375,947 bps (including prophage regions between CDS VABO27_275-VABO27_1225). This recombination event likely occurred between 2,643,219 and 2,643,208 bp or between MS6_2336 and MS6_2335 on MS6 Chr1 backbone and inserted between MS6_A0924 and MS6_A0925 break points (852,904–852,973) on MS6 Chr2 backbone (Figure 5 (bottom panel)). The insertion boundary is flanked by 245 bp repeats located at 337,552 to 337,796 bp and 1,351,093 to 1,351,337 bps with 96% identities, then flanked by 2 prophages. In the flanking regions of 2 prophages, there are 12 bp identical repeats (caccgcagggtg) (297,210–297,221 bps and 1,375,947–1,375,958 bps). The 245 bps repeat also overlaps 155 bps with VABO27_1228 that encodes L-threonine 3-dehydrogenase.Figure 5
Putative recombination event that resulted in Chr1 and Chr2 fusions in NSCV2. Top panel: MS6 Chr1 CDS at the fusion junction and the same location in a putative NSCV2-like intermediate and the MS6 Chr2 circle and the codons at the fusion junction are depicted. The cross-over region between Chr1 and Chr2 is indicated by the long X. Bottom panel: The recombination products after fusion event are shown with NSCV2 in the middle and an excision product that contains one copy of the cross-over genes with the intervening sequences at the bottom.As mentioned above for NSCV1, the role of any of these unique regions in the recombination event or the mechanism of recombination that resulted in chromosomal fusion cannot be ascertained with availableV. cholerae whole genome sequence data. Since the immediate predecessor or recombination intermediate strains are not known, it is difficult to decipher the exact events that led to the NSCV1 and NSCV2. For example, the exact mechanism of recombination (site specific versus generalized recombination) or the precise recombination cross-over points cannot be predicted. It also appears that there is more than one event before the final NSCV1 and NSCV2 arose. The presence of prophages at the insertion junctions leads us to speculate that the Chr2 insertion events in NSCV1 and NSCV2 appear to be mediated by generalized homologous recombination via homologies provided by prophages or parts/genes homologous to prophage genes and mobile gene elements present in both Chr1 and Chr2 in the precursor strain.


### 3.11. Sequence Analysis of the Origins of Replication and Associated Genes

Theori1 and ori2 origins of replications in NSCV1 and NSCV2 chromosomes were identified by a homology to the Chr1 and Chr2 origins of MS6. The ori1 is colocalized with the genes (rpmH, dnaA, dnaN, recF, and gyrA) often found near the oriC in prokaryotic genomes, and origin of the location corresponded with GC nucleotide skew (G − C/G + C) analysis as illustrated in Figure S2 and Figure S3. Based on these data, we assigned base-pair 1 in an intergenic region located in the putative ori1. Similarly, ori2 was located based on sequence homology to the ori2 in other prototypical V. cholerae genomes. The genome positions of the ori, their orientation, and genes within the ori are indicated in Table 1. A list of putative dif sites (chromosome dimer resolution sites) and their locations are provided in Table S4. A previous study had found chromosome fusions in V. cholerae as suppressors of impaired Chr2 replication [33]. As mentioned above, some of these fusions had occurred by site-specific recombination of the two dif sites of Chr1 and Chr2, respectively. The positions of the intact dif sites on the fused chromosomes in NSCV1 and NSCV2 support the notion that this was not the mechanism how fusion occurred in these strains (Table S4). It remains to be seen which of the two dif sites on NSCV1 and NSCV2 chromosomes is active and used for chromosome dimer resolution. This might depend on the molecular mechanism involved in positioning the respective XerCD recombinase at the dif site.Table 1
Genome locations of origins of replication and replication-associated genes.
Ori1 and Ori2 and the genes within Ori
Locus ID
Gene/Locus
N16961_O1
Locus ID
1154-74_O49
Locus ID
10432-62_O27
Start
End
Size
Strand
Orientation
Start
End
Size
Strand
Orientation
Start
End
Size
Strand
Orientation
Rep_ori_Chr_1
2956820
2961149/1
806
5136
Clockwise
Rep_ori_Chr_1
3916660
3921794
5135
Clockwise
Rep_ori_Chr_1
4039130
4044265
5136
Clockwise
VC2772
parB
2956823
2957704
882
Complement
VAA049_RS18130
3916663
3917544
882
Complement
VAB027_RS18795
4039133
4040014
882
Complement
VC2773
parA
2957731
2958504
774
Complement
VAA049_RS18135
3917571
3918344
774
Complement
VAB027_RS18800
4040041
4040814
774
Complement
VC2774
gidB
2958519
2959151
633
Complement
VAA049_RS18140
3918359
3918991
633
Complement
VAB027_RS18805
4040829
4041461
633
Complement
VC2775
gidA
2959151
2961046
1896
Complement
VAA049_RS18145
3918991
3920886
1896
Complement
VAB027_RS18810
4041461
4043356
1896
Complement
Ori_1
2961047
2961149
371
474
Ori_1
3920887
3921359
473
Ori_I
4043357
4043830
474
VC0001
hypo
235
402
168
Complement
VAA049_RS18150
3921224
3921390
167
Complement
VAB027_RS18815
4043694
4043861
168
Complement
VC0002
mioC
372
806
435
Complement
VAA049_RS18155
3921360
3921794
435
Complement
VAB027_RS18800
2269384
2269818
435
Complement
Rep_ori_Chr_2
1069696
1072315/1
3191
5811
Clockwise
Rep_ori_Chr_2
2096979
2102790
5812
Counterclockwise
Rep_ori_Chr_2
1115023
1120832
5810
Counterclockwise
VCA1113
1068927
1069958
1032
VAA049_RS09630
2102528
2103559
1032
Complement
VAB027_RS05240
1120570
1121601
1032
Complement
VCA1114
parB
1070018
1070989
972
Complement
VAA049_RS09625
2101496
2102476
972
VAB027_RS05235
1119539
1120510
972
VCA1115
parA
1070997
1072220
1224
Complement
VAA049_RS09620
2100271
2101488
1218
VAB027_RS05230
1118314
1119531
1218
VCA0001
rctA
112
246
135
Complement
rctA
2099924
2100058
135
rctA
1117968
1118102
135
Ori_2
247
1133
887
Ori_2
2099037
2099923
887
Ori_II
1117081
1117967
887
VCA0002
rctB
1134
3110
1977
VAA049_RS09615
2097060
2099036
1977
Complement
VAB027_RS05225
1115104
1117080
1977
Complement
Replication-associated genes
Locus ID
Gene
N16961_O1
Locus ID
1154-74_O49
Locus ID
10432-62_O27
Start
End
Size
Orientation
Start
End
Size
Strand
Start
End
Size
Orientation
VC2626
dam
2796912
2797745
834
Complement
VAA049_RS01410
295053
295886
834
VAB027_RS01350
282403
283236
834
VC0543
recA
574696
575760
1065
VAA049_RS016045
3495035
3496099
1065
Complement
VAB027_RS16670
3602,557
3603621
1065
Complement
VC0668
mutH
715847
716512
666
Complement
VAA049_RS015395
3352580
3353245
666
VAB027_RS16030
3461932
3462597
666
VC0345
mutL
367072
369033
1962
VAA049_RS016985
3680484
3682445
1962
Complement
VAB027_RS17585
3784248
3786209
1962
Complement
VC0535
mutS
565832
568420
2589
Complement
VAA049_RS16080
3502248
3504836
2589
VAB027_RS016705
3609897
3612475
2580
VC0128
xerC
122005
122940
936
VAA049_RS00590
114667
115602
936
VAB027_RS00575
114596
115531
936
VC2419
xerD
2593375
2594283
909
Complement
VAA049_RS02440
498541
499449
909
VAB027_RS15355
3316085
3316993
909
Complement
VC0012
dnaA
7397
8815
1419
VAA049_RS00005
38
1441
1404
VAB027_RS00005
38
1441
1404
dif-1
1564104
1564131
28
Complement
IGR of VAA049_RS06840-VAA049_RS06845
1476590
1476617
28
IGR of VAB027_RS10790-VAB027_RS10795
2301152
2301179
28
Complement
dif-2
507983
508010
28
VAA049_RS12025
2643791
2643818
28
Complement
IGR of VAB027_RS03005-VAB027_RS03010
664645
664672
28
ComplementThere is extensive genetic conservation in theori1 and ori2 of NSCV1 (Figure 6) and NSCV2 (Figure 7) compared to a prototypical reference genome such as N16961. A closer look at the sequences of the origin regions including the genes within the origin of replication indicated that there is no significant indels between the respective origins compared to a reference genome such as N16961. However, a number of SNPs were found in the origins of replication. The nucleotide and amino acid changes at the origins of NSCV1 and NSCV2 are presented in Table S5. It remains to be analyzed experimentally if both origins on the fused chromosomes are active in replication.Figure 6
Genetic organization ofori1 of NSCV1 and NSCV2 in comparison to the respective ori in N16961. The old locus tags with known gene designations have been used to indicate the ORFs. The physical ori and unannotated ORFs are indicated by red arrows.Figure 7
Genetic organization ofori2 of NSCV1 and NSCV2 in comparison to the respective ori in N16961. The old locus tags with known gene designations have been used to indicate the ORFs. The physical ori is indicated by a red arrow.


### 3.12. Sequence Analyses of Replication Associated and Mismatch Repair Genes That May Be Potentially Involved in Single-Chromosome Maintenance

Earlier studies have indicated that thedam gene is essential for the viability of V. cholerae, and depletion of DNA adenine methyl transferase (Dam protein) leads to successful spontaneous fusion of the two chromosomes [33]. Hence, we examined the genetic status of DNA adenine methylase gene (dam) in natural single-chromosome V. cholerae, NSCV1, and NSCV2 and found the dam gene to be intact. We also inspected the status of RecA and the MMR genes since they have been implicated in maintenance of chromosomal rearrangements such as large tandem repeats, inversion, and fusion. The nucleotide and amino acid changes in various replication and MMR genes in NSCV1 and NSCV2 are presented in Table S6. RecA-mediated homologous recombination was probably involved in the fusion event, and the fact that the resolution of the single chromosome into 2 chromosomes has not been observed during normal growth conditions indicates that the RecA in NSCV1 and NSCV2 is nonfunctional or altered due to the presence of multiple SNPs, of which one leads to single-amino acid change in RecA protein (Y305C). Preliminary data indicate that NSCV1 and NSCV2 are probably recombination deficient since construction of recombinants via natural transformation has been unsuccessful. The MMR genes are generally intact except for mutS. The mutS gene is impaired, that is, deletion of amino acid residues 132–150, in NSCV2. Among the other mismatch repair system genes, mutH and mutL have single-nucleotide polymorphisms that lead to amino acid changes in the respective proteins. Other replication-associated proteins such as DnaA, ParAB, and XerC have amino acid changes whereas XerD is intact (Table S6). Preliminary data indicate that the ori1 is active in both strains suggesting a functional DnaA protein. However, the effect of these protein alterations in ParAB, XerC, RecA, and MMR proteins on recombination, replication, and maintenance of chromosomal fusions and other large-scale genome rearrangements described above awaits more stringent functional studies including cloning and intra-/interspecific complementation and these studies are underway.


## 3.1. Whole Genome Sequencing ofV. cholerae Strains NSCV1-(1154-74/VAA_O49 and NSCV2-(10432-62/VAB_O27)

Whole genome mapping (WGM) also known as optical mapping and PFGE data provided evidence of Chr1 and Chr2 fusions inV. cholerae strains NSCV1 (1154-74) and NSCV2 (10432-62) [35]. We performed whole genome sequencing of the two V. cholerae strains in order to understand the molecular mechanisms that might have led to Chr1 and Chr2 fusions in these strains [36]. We generated Roche 454 (21× and 29×), Illumina (2178× and 507×), and Pac Bio RS (350× and 190×) sequence data at the indicated coverages for NSCV1 and NSCV2, respectively, and used a hybrid assembly approach that resulted in a single-contig, gapless genome (Table S1). Further resolution of the ambiguous regions was carried out using Sanger-based primer walking according to Las Alamos National Labs (LANL) genome finishing pipeline yielding a final, finished, and closed single-contig sequence [66]. Consistent with the WGM results and in contrast to the previous genomic reports of V. cholerae and its close neighbors [2–4], these two sequences assembled into genomes consisting of a single chromosome of approximately the same size as the two expected chromosomes added together. All other genomic statistics for NSCV1 and NSCV2 appear to be highly similar to other V. cholerae genomes currently available in GenBank (Table S1). The genomes of NSCV1 and NSCV2 are comprised of a single circular chromosome of 3,928,357 and 4,074,462 bp in length with an overall G + C content of 47.73% and 47.66%, respectively (Figure 1). Following the sequencing convention, the nucleotide position 1 was placed upstream of the dnaA gene (VAA_049_1 and VAB_027_1) encoding the chromosomal replication initiator protein. Chr2 portion of NSCV1 and NSCV2 spans from genome positions 1,720,246–2,772,395 (1,052,150 bps) and 297,221–1,375,947 (1,078,727 bps), respectively. All other NSCV1 and NSCV2 genome statistics are presented in Table S1.Figure 1
Circular genome maps of NSCV1 (1154-74_VAAO49) and NSCV2 (10432-62_VABO27). Fusion of Chr1 (dark grey) to Chr2 (blue) is shown in the circle at the respective locations. Various unique features such as prophages and the origins of replication- and replication-associated genes are indicated around the circles.


## 3.2. Whole Genome Sequence Comparison of NSCV1 and NSCV2 to OtherV. cholerae

DNA sequence ofV. cholerae strain MS6 is the closest match to NSCV1 and NSCV2 chromosomes in public genome databases based on whole genome sequence homology [67]. MS6 is a V. cholerae O1 strain with a novel genetic background designated in Thailand–Myanmar and isolated from a stool sample of a diarrheal patient [68]. NSCV1 and NSCV2 genome sequences exhibit a high degree of synteny with MS6, N16961, MO10, and TSY216 as visualized in the Mauve alignment despite genetic rearrangements such as insertions and inversions across the chromosome (Figure S1) [69]. A pairwise Megablast comparison of NSCV1 and NSCV2 genomes to four pathogenic V. cholerae (N16961, MO10, MS6, and TSY 216) was performed. At 85%, identity > 88–94% is shared between NSCV1 and NSCV2 compared to N16961, MS6, or MO10 genomes and 74% and 80% is shared between NSCV1 and NSCV2 compared to TSY216, respectively. The unique regions varied from 5 to 10% (Table S2).To further delineate the differences between NSCV1 and NSCV2 against all other genomes, the locations and gene content of the unique regions were determined (Table S3) and displayed as BRIG view (Figure S2). In the whole genome comparison of NSCV1 and NSCV2 to V. cholerae strains MS6, N16961, and TSY216, large regions (~10 kb or more) missing in comparator strains are highlighted in the outer circle of the BRIG view (Figure S2). As expected, many features (e.g., prophages) that are unique to NSCV1 and NSCV2 are absent in the pathogenic strains and similarly, many of the known virulence regions (e.g., CTX and VPI) that are present in pathogenic V. cholerae such as N16961 are absent in NSCV1 and NSCV2. In Chr2 segment, the super integron region is present in NSCV1 and NSCV2 with many subtle indels/variations.


## 3.3. Comparison of Genomic Content of NSCV1 and NSCV2 to PathogenicV. cholerae

Further analysis of the genomic content of NSCV1 and NSCV2 was performed by comparing the genome annotations. We were interested to see if there are any differences in the genetic content that might explain the mechanism of genomic fusion. Overall, the number of CDS encoded by NSCV1 and NSCV2 is very similar to otherV. cholerae (Table S1). NSCV1 and NSCV2 have 2759 CDS in common with all other genomes compared here. Comparison of the genomes of NSCV1 and NSCV2 with MS6, N16961, and MO10 (an epidemic V. cholerae strain belonging to the O139 serogroup) revealed that the four organisms have approximately 3188/3259 genes in common, depending on whether NSCV1 or NSCV2 is used as the query, respectively (Figure 2). There are 173/304 CDSs unique to NSCV1 and NSCV2, respectively.Figure 2
Comparison of the genomic content of NSCV1 (a), NSCV2 (b), and NSCV1 and NSCV2 (c) to various other genomes. Venn diagram showing the number of NSCV1 or NSCV2 predicted CDS with significant homology (1e−5) with the predicted products of the near neighbors: V. cholerae MS6, N16961 (serogroup O1 biovar El Tor), and O139 MO10 (serogroup O139). The number outside the circles (173 or 304) represents the number of NSCV1 or NSCV2 CDS that does not have significant homologs in the three strains compared. Conserved genes among them were defined by whole-genome pairwise sequence comparisons using the sequence-based comparison tool in RAST.
(a)
(b)
(c)


## 3.4. Genomic Islands, Prophages, and Other Mobile Genetic Elements

In the NSCV1 and NSCV2 chromosomes, 49 and 20 genomic islands amounting to 677, 625 bps and 205, 247 bps or 16.32% and 4.94% of the entire genome, respectively, were detected. Clustered regularly, interspaced short palindromic repeat (CRISPR) element is a widely found defense mechanism of prokaryotes against entry of foreign DNA including plasmids and phages [70]. In the NSCV1 and NSCV2 chromosomes, one CRISPR element was located at 3,606,088–3,606,203 bps and 3,572,464–3,572,579 bps in NSCV1 and NSCV2 genomes, respectively. The NSCV1 and NSCV2 genomes contained 58 and 55 mobile genetic elements, respectively, that encode phage integrases, transposases, and site-specific recombinase (Figure S3), compared to 46 mobile element genes in V. cholerae MS6. NSCV1 has 5 prophages located at (1) 685,729–702,622; (2) 1,458,739–1,476,484; (3) 1,756,187–1,818,403; (4) 1,879,405–1,883,642; and (5) 2,643,793–2,650,802. Prophages 3 and 5 are at the immediate boundary region and inside of the Chr2 insertion locus. Only prophage 3 at the 5′ end of Chr2 insertion appears to be intact and encodes 56 CDS. NSCV2 has 5 prophages located at (1) 3,113,341–3,130,219; (2) 3,416,918–3,428,588; (3) 299,327–349,041; (4) 1,359,430–1,369,981; and (5) 1,676,903 to 1,714,350. Only prophage 5 is intact whereas the other four are defective or incomplete. Prophages 3 and 4 are at the immediate flanking region of the Chr2 insertion junction (Figure 1).


## 3.5. Virulence and Antimicrobial Resistance Genes

V. cholerae strains NSCV1 and NSCV2 were isolated from patients exhibiting atypical cholera symptoms [34]. It is of interest to see if these strains carry any of the usual V. cholerae virulence factors and if not, what other virulence factors or toxins might be present that would explain the diarrheal symptoms. The major virulence factor of V. cholerae, the cholera toxin encoded by ctxAB genes, is not found in NSCV1; however, other toxin genes ace and zot and RTX toxin cluster can be found, in addition to toxRS genes. The toxin genes are absent in NSCV2. NSCV1 and NSCV2 each has 57 genes encoding putative resistance to antibiotics and toxic compounds such as colicin V, bacteriocin, cobalt-zinc-cadmium, copper homeostasis, fluoroquinolones, and multidrug resistance efflux pumps.


## 3.6. O-Antigen Regions

NSCV1 and NSCV2 belong to serogroups O49 and O27, respectively, and as seen in other serogroups ofV. cholerae, the O-antigen biosynthesis genes are encoded in a cluster (wb∗) on Chr1 part of the respective genomes from 3,777,887–3,815,081 (CDS VAAO49_3428-VAAO49_3463) and 3,888,708–3,920,841 (CDS VABO27_3582-VABO27_3611) of NSCV1 and NSCV2, respectively. Also, as seen in other wb∗ regions, the boundary regions of this cluster are highly conserved whereas the region between the conserved genes is highly divergent. More specifically, in the NSCV1 wb∗ region, 2 segments of 2974 bps and 6212 bps at the left end and another segment of 5733 bps at the right end, respectively, are 96% and 94% identical to those in the wbe region (O1 serogroup). A blast analysis of the wb∗ region of NSCV2 revealed segments that are similar to V. vulnificus (GenBank Accession # CP009261) gene cluster (around 42 kb) with 4 segments that are >65–85% identical, ranging in length from ~1.0 kb to 8.831 kb to the NSCV2 wb∗ cluster. In addition, identities to V. mimicus at 83–92% in segments of 4176 bps, 2789 bps, and 1410 bps and to Plesiomonas shigelloides and Shewanella baltica at ~80% (3281 bps), and to NSCV1 at 94–96% (4325, and 3007 bps) identities were also found as depicted in Figure 3.Figure 3
Genetic maps of O-antigen regions (wb∗) of NSCV1 and NSCV2. The various genes and their orientations are indicated by the arrows. Homologous regions to other serogroups are indicated by the red arrows. The V. vulnificus wb∗ cluster that has extensive homology to NSCV2 wb∗ cluster is shown in (c).
(a)
(b)
(c)


## 3.7. Identification of Large Tandem Repeats and Inversion

A closer analysis of the WGMs (whole genome maps) of NSCV1 and NSCV2 strains revealed a large duplication in the general region of 1240 and 1506 kb on a reference genome (M66–2) coordinates based on optical restriction maps [35]. Experimental WGM data is further supported by in silico WGM generated from whole genome sequence data of NSCV1 and NSCV2 (Figure S4). Sequence read mapping results showed that there is a large duplication of 200 kbs and 70 kbs regions in NSCV1 and NSCV2, respectively, as evidenced by >1×, but <2×, depth of average coverage of the genome at 1,503,484 to 1,718,861 bps in NSCV1 and 2,221,105 to 2,300,924 bps in NSCV2 compared to that in the rest of the genome (Figure S5). Manual validation indicated that the 200 kbs and 70 kbs regions indeed are fragmental duplications, existing among subpopulation of NSCV1 and NSCV2 cells, respectively. Both 1 copy and 2 copy versions of genome assembly have enough evidence supporting variations among population. These duplicated sequences spanned from positions 1,503,484 to 1,718,861 (unit 1) and from 1,718,862 to 1,934,269 bp (unit 2) in NSCV1 and from positions 2,221,105 to 2,300,924 (unit 1) and from 2,300,925 to 2,380,745 (unit 2) in NSCV2 in genome sequences with repeats included (Figure S6). The duplicated sequences span a total length of 430,785 bp and 159,640 bp on Chr1 region and encode 177 and 59 duplicated genes in NSCV1 and NSCV2, respectively. It should be noted that the two copies of repeat units are not identical. There is a 31 bp indel between the two copies in NSCV1, which resides at the locus of VAA_049_1509, encoding exonuclease family protein. There are 5 single bp indels between the two copies in NSCV2. Although tandem repeats are present at different locations on NSCV1 and NSCV2, there is a 40 kb DNA segment that overlapped at the 5′ end of NSCV1 and NSCV2 repeats with 98% identities (Figure S7). However, the tandem duplications could not be verified by PCR of the junctions since the orientation of the two copies cannot be ascertained in the assembly. Hence, we have decided to keep the genome sequence with a single copy as the ref seq (GenBank submission) for all the analyses. This long duplication anomaly could not be unequivocally resolved with the existing sequence data. In addition, there is a large inversion from 1,477,189 to 3,484,983 bps in NSCV2 compared to MS6 chromosome 1 region from 612,067 to 2,541,958 bps (Figure S1b).


## 3.8. Mapping the Fusion Junctions and Possible Mechanism of Genome Fusion

Chromosomal fusions were observed previously in suppressor mutants resulting from a genetic screen [33]. These fusions were analyzed in detail and found to occur via two different mechanisms, either by homologous recombination at IS elements or by site-specific recombination at dif sites [32] or as transient chromosomal fusions in ΔcrtS suppressor mutants [20]. In this study, we analyzed the fusion sites of NSCV1 and NSCV2 in detail to see if any one of these mechanisms also led to chromosomal fusion in the natural single-chromosome isolates. The circular single chromosomes of NSCV1 and NSCV2 are shown in Figure 1, and along with N16961, Chr1- and Chr2-concatenated circular maps are shown in Figure S2, and the genome sequence statistics are provided in Table S1. The insertion sites on Chr1 backbone (as an accepter) and Chr2 backbone (as a donor) are not the same for NSCV1 and NSCV2 (see below). Artemis Comparison Tool views (zoomed in view) of the fusion junctions are presented in Figures S8 and S9. Further description of the hypothetical events leading to the fusions is provided below.


## 3.9. Chr1 and Chr2 Fusion Junctions inV. cholerae Strain NSCV1

A closer examination of the Chr1-Chr2 insertion junction in NSCV1 to Chr1 and Chr2 sequences of MS6 indicates that multiple events probably occurred before the final NSCV1 was derived. In an NSCV1-like intermediate, on Chr1 between VAA049_1545 and VAA049_2500, there probably was an insertion of MS6_A0562- and MS6_A0272-like CDS (Chr2 CDS) (Figure4 (top panel)). On Chr2, there was an insertion of a prophage to the right of MS6_A0272 (Figure 4 (top panel)). In the next step, Chr2 with the new prophage was inserted into Chr1 via the homologies between the two CDS (VAA049_1594 versus MS6_A0272 and VAA049_2432 versus MS6_A0562), and upon the resolution of the cointegrate, one copy each of the two homologous CDS along with intervening sequences was deleted (Figure 4 (bottom panel)). Thus, Chr2 in NSCV1 spans from 1,714,319 to 2,772,395 bps (including its flanking unique region, Figure 4 (bottom panel)) or from 1,757,711 to 2,722,145 bps (including boundary of Chr2 homologous region) (Figure 4 (bottom panel)) spanning NSCV1 CDS VAAO49_1594–2432. This recombination event likely occurred between MS6_1029 and MS6_1028 or between 1,168,144 and 1,168,021 bp in MS6-like Chr1 background and inserted from the breakpoint between MS6_A0272 and MS6_A0562 or between 303,999 and 446,488 bp on MS6-like Chr2 background (Figure 4 (bottom panel)). There are 2 prophages (3 and 5) closer to the boundaries of Chr2 insertion locus. Flanking the Chr2 insertion sites, there are unique regions of 37,425 bps and 50,250 bps in length at 5′ end and 3′ end, respectively. The role of any of these unique regions in the recombination event or the precise mechanism of recombination cannot be ascertained with available whole genome sequence data in public databases.Figure 4
Putative recombination event that resulted in Chr1 and Chr2 fusions in NSCV1. Top panel: MS6 Chr1 CDS at the fusion junction and the same location in a putative NSCV1-like intermediate and the MS6 Chr2 circle and the codons at the fusion junction are depicted. The cross-over region between Chr1 and Chr2 is indicated by the long X. A prophage insertion event to the right fusion junction prior or post to Chr 2 fusion is depicted by a green line. Bottom panel: The recombination products after fusion event are shown with NSCV 1 in the middle and an excision product that contains one copy of the cross-over genes with the intervening sequences at the bottom.


## 3.10. Chr1 and Chr2 Fusion Junctions inV. cholerae Strain NSCV2

A closer examination of the insertion junctions indicates that an NSCV2-like intermediate probably possessed MS6_A0925- and MS6_A0924-like Chr2 CDS on Chr1 flanked by 2 prophages (Figure5 (top panel)). In that intermediate strain, Chr2 was inserted via recombination of the homologous segments (VAB027_307 versus MS6_A0924 and VAB027_1228 versus MS6_A0925). As a result, Chr2 spans from 337,822 bps to 1,351,337 bps (without its flanking prophage regions between CDS VABO27_307-VABO27_1228) or from 297,221 bps to 1,375,947 bps (including prophage regions between CDS VABO27_275-VABO27_1225). This recombination event likely occurred between 2,643,219 and 2,643,208 bp or between MS6_2336 and MS6_2335 on MS6 Chr1 backbone and inserted between MS6_A0924 and MS6_A0925 break points (852,904–852,973) on MS6 Chr2 backbone (Figure 5 (bottom panel)). The insertion boundary is flanked by 245 bp repeats located at 337,552 to 337,796 bp and 1,351,093 to 1,351,337 bps with 96% identities, then flanked by 2 prophages. In the flanking regions of 2 prophages, there are 12 bp identical repeats (caccgcagggtg) (297,210–297,221 bps and 1,375,947–1,375,958 bps). The 245 bps repeat also overlaps 155 bps with VABO27_1228 that encodes L-threonine 3-dehydrogenase.Figure 5
Putative recombination event that resulted in Chr1 and Chr2 fusions in NSCV2. Top panel: MS6 Chr1 CDS at the fusion junction and the same location in a putative NSCV2-like intermediate and the MS6 Chr2 circle and the codons at the fusion junction are depicted. The cross-over region between Chr1 and Chr2 is indicated by the long X. Bottom panel: The recombination products after fusion event are shown with NSCV2 in the middle and an excision product that contains one copy of the cross-over genes with the intervening sequences at the bottom.As mentioned above for NSCV1, the role of any of these unique regions in the recombination event or the mechanism of recombination that resulted in chromosomal fusion cannot be ascertained with availableV. cholerae whole genome sequence data. Since the immediate predecessor or recombination intermediate strains are not known, it is difficult to decipher the exact events that led to the NSCV1 and NSCV2. For example, the exact mechanism of recombination (site specific versus generalized recombination) or the precise recombination cross-over points cannot be predicted. It also appears that there is more than one event before the final NSCV1 and NSCV2 arose. The presence of prophages at the insertion junctions leads us to speculate that the Chr2 insertion events in NSCV1 and NSCV2 appear to be mediated by generalized homologous recombination via homologies provided by prophages or parts/genes homologous to prophage genes and mobile gene elements present in both Chr1 and Chr2 in the precursor strain.


## 3.11. Sequence Analysis of the Origins of Replication and Associated Genes

Theori1 and ori2 origins of replications in NSCV1 and NSCV2 chromosomes were identified by a homology to the Chr1 and Chr2 origins of MS6. The ori1 is colocalized with the genes (rpmH, dnaA, dnaN, recF, and gyrA) often found near the oriC in prokaryotic genomes, and origin of the location corresponded with GC nucleotide skew (G − C/G + C) analysis as illustrated in Figure S2 and Figure S3. Based on these data, we assigned base-pair 1 in an intergenic region located in the putative ori1. Similarly, ori2 was located based on sequence homology to the ori2 in other prototypical V. cholerae genomes. The genome positions of the ori, their orientation, and genes within the ori are indicated in Table 1. A list of putative dif sites (chromosome dimer resolution sites) and their locations are provided in Table S4. A previous study had found chromosome fusions in V. cholerae as suppressors of impaired Chr2 replication [33]. As mentioned above, some of these fusions had occurred by site-specific recombination of the two dif sites of Chr1 and Chr2, respectively. The positions of the intact dif sites on the fused chromosomes in NSCV1 and NSCV2 support the notion that this was not the mechanism how fusion occurred in these strains (Table S4). It remains to be seen which of the two dif sites on NSCV1 and NSCV2 chromosomes is active and used for chromosome dimer resolution. This might depend on the molecular mechanism involved in positioning the respective XerCD recombinase at the dif site.Table 1
Genome locations of origins of replication and replication-associated genes.
Ori1 and Ori2 and the genes within Ori
Locus ID
Gene/Locus
N16961_O1
Locus ID
1154-74_O49
Locus ID
10432-62_O27
Start
End
Size
Strand
Orientation
Start
End
Size
Strand
Orientation
Start
End
Size
Strand
Orientation
Rep_ori_Chr_1
2956820
2961149/1
806
5136
Clockwise
Rep_ori_Chr_1
3916660
3921794
5135
Clockwise
Rep_ori_Chr_1
4039130
4044265
5136
Clockwise
VC2772
parB
2956823
2957704
882
Complement
VAA049_RS18130
3916663
3917544
882
Complement
VAB027_RS18795
4039133
4040014
882
Complement
VC2773
parA
2957731
2958504
774
Complement
VAA049_RS18135
3917571
3918344
774
Complement
VAB027_RS18800
4040041
4040814
774
Complement
VC2774
gidB
2958519
2959151
633
Complement
VAA049_RS18140
3918359
3918991
633
Complement
VAB027_RS18805
4040829
4041461
633
Complement
VC2775
gidA
2959151
2961046
1896
Complement
VAA049_RS18145
3918991
3920886
1896
Complement
VAB027_RS18810
4041461
4043356
1896
Complement
Ori_1
2961047
2961149
371
474
Ori_1
3920887
3921359
473
Ori_I
4043357
4043830
474
VC0001
hypo
235
402
168
Complement
VAA049_RS18150
3921224
3921390
167
Complement
VAB027_RS18815
4043694
4043861
168
Complement
VC0002
mioC
372
806
435
Complement
VAA049_RS18155
3921360
3921794
435
Complement
VAB027_RS18800
2269384
2269818
435
Complement
Rep_ori_Chr_2
1069696
1072315/1
3191
5811
Clockwise
Rep_ori_Chr_2
2096979
2102790
5812
Counterclockwise
Rep_ori_Chr_2
1115023
1120832
5810
Counterclockwise
VCA1113
1068927
1069958
1032
VAA049_RS09630
2102528
2103559
1032
Complement
VAB027_RS05240
1120570
1121601
1032
Complement
VCA1114
parB
1070018
1070989
972
Complement
VAA049_RS09625
2101496
2102476
972
VAB027_RS05235
1119539
1120510
972
VCA1115
parA
1070997
1072220
1224
Complement
VAA049_RS09620
2100271
2101488
1218
VAB027_RS05230
1118314
1119531
1218
VCA0001
rctA
112
246
135
Complement
rctA
2099924
2100058
135
rctA
1117968
1118102
135
Ori_2
247
1133
887
Ori_2
2099037
2099923
887
Ori_II
1117081
1117967
887
VCA0002
rctB
1134
3110
1977
VAA049_RS09615
2097060
2099036
1977
Complement
VAB027_RS05225
1115104
1117080
1977
Complement
Replication-associated genes
Locus ID
Gene
N16961_O1
Locus ID
1154-74_O49
Locus ID
10432-62_O27
Start
End
Size
Orientation
Start
End
Size
Strand
Start
End
Size
Orientation
VC2626
dam
2796912
2797745
834
Complement
VAA049_RS01410
295053
295886
834
VAB027_RS01350
282403
283236
834
VC0543
recA
574696
575760
1065
VAA049_RS016045
3495035
3496099
1065
Complement
VAB027_RS16670
3602,557
3603621
1065
Complement
VC0668
mutH
715847
716512
666
Complement
VAA049_RS015395
3352580
3353245
666
VAB027_RS16030
3461932
3462597
666
VC0345
mutL
367072
369033
1962
VAA049_RS016985
3680484
3682445
1962
Complement
VAB027_RS17585
3784248
3786209
1962
Complement
VC0535
mutS
565832
568420
2589
Complement
VAA049_RS16080
3502248
3504836
2589
VAB027_RS016705
3609897
3612475
2580
VC0128
xerC
122005
122940
936
VAA049_RS00590
114667
115602
936
VAB027_RS00575
114596
115531
936
VC2419
xerD
2593375
2594283
909
Complement
VAA049_RS02440
498541
499449
909
VAB027_RS15355
3316085
3316993
909
Complement
VC0012
dnaA
7397
8815
1419
VAA049_RS00005
38
1441
1404
VAB027_RS00005
38
1441
1404
dif-1
1564104
1564131
28
Complement
IGR of VAA049_RS06840-VAA049_RS06845
1476590
1476617
28
IGR of VAB027_RS10790-VAB027_RS10795
2301152
2301179
28
Complement
dif-2
507983
508010
28
VAA049_RS12025
2643791
2643818
28
Complement
IGR of VAB027_RS03005-VAB027_RS03010
664645
664672
28
ComplementThere is extensive genetic conservation in theori1 and ori2 of NSCV1 (Figure 6) and NSCV2 (Figure 7) compared to a prototypical reference genome such as N16961. A closer look at the sequences of the origin regions including the genes within the origin of replication indicated that there is no significant indels between the respective origins compared to a reference genome such as N16961. However, a number of SNPs were found in the origins of replication. The nucleotide and amino acid changes at the origins of NSCV1 and NSCV2 are presented in Table S5. It remains to be analyzed experimentally if both origins on the fused chromosomes are active in replication.Figure 6
Genetic organization ofori1 of NSCV1 and NSCV2 in comparison to the respective ori in N16961. The old locus tags with known gene designations have been used to indicate the ORFs. The physical ori and unannotated ORFs are indicated by red arrows.Figure 7
Genetic organization ofori2 of NSCV1 and NSCV2 in comparison to the respective ori in N16961. The old locus tags with known gene designations have been used to indicate the ORFs. The physical ori is indicated by a red arrow.


## 3.12. Sequence Analyses of Replication Associated and Mismatch Repair Genes That May Be Potentially Involved in Single-Chromosome Maintenance

Earlier studies have indicated that thedam gene is essential for the viability of V. cholerae, and depletion of DNA adenine methyl transferase (Dam protein) leads to successful spontaneous fusion of the two chromosomes [33]. Hence, we examined the genetic status of DNA adenine methylase gene (dam) in natural single-chromosome V. cholerae, NSCV1, and NSCV2 and found the dam gene to be intact. We also inspected the status of RecA and the MMR genes since they have been implicated in maintenance of chromosomal rearrangements such as large tandem repeats, inversion, and fusion. The nucleotide and amino acid changes in various replication and MMR genes in NSCV1 and NSCV2 are presented in Table S6. RecA-mediated homologous recombination was probably involved in the fusion event, and the fact that the resolution of the single chromosome into 2 chromosomes has not been observed during normal growth conditions indicates that the RecA in NSCV1 and NSCV2 is nonfunctional or altered due to the presence of multiple SNPs, of which one leads to single-amino acid change in RecA protein (Y305C). Preliminary data indicate that NSCV1 and NSCV2 are probably recombination deficient since construction of recombinants via natural transformation has been unsuccessful. The MMR genes are generally intact except for mutS. The mutS gene is impaired, that is, deletion of amino acid residues 132–150, in NSCV2. Among the other mismatch repair system genes, mutH and mutL have single-nucleotide polymorphisms that lead to amino acid changes in the respective proteins. Other replication-associated proteins such as DnaA, ParAB, and XerC have amino acid changes whereas XerD is intact (Table S6). Preliminary data indicate that the ori1 is active in both strains suggesting a functional DnaA protein. However, the effect of these protein alterations in ParAB, XerC, RecA, and MMR proteins on recombination, replication, and maintenance of chromosomal fusions and other large-scale genome rearrangements described above awaits more stringent functional studies including cloning and intra-/interspecific complementation and these studies are underway.


## 4. Conclusions

All species of the genus Vibrio known to date harbor two chromosomes. Here, we present an exception to this rule by describing the genomic architecture of two naturalV. cholerae isolates with one fused chromosome. For many years, the quest to understand why and how Vibrios evolved their bipartite genomes remains enigmatic. The strains described here appeared to have taken an evolutionary path backwards and might be instrumental in future unraveling of long-standing questions on chromosome biology in Vibrios. One fascinating question to address is whether the replication of the fused chromosome is dominated by one of the subchromosomes or if they share the two replicons. If the latter is true then this would be the first example of a bacterial chromosome with two active replication origins. A tantalizing idea that has been proposed is that Chr2 is actually a “parasitic replicon.” The concept of selfish replication origins has been established recently [71]. It could be that the V. cholerae strains described here are the result of a selfish replicon taking over the “host.” Currently, studies are underway to begin to address these questions to unravel the mystery of a single chromosome with multiple replication origins. A second question that is worth exploring pertains to how the chromosomal fusions are maintained and if and how or under what conditions they ever revert into two separate chromosomes. Many of the genes involved in these functions have suffered changes. It remains to be seen if NSCV1 and NSCV2 strains are defective or functionally altered in any of these genes in order to maintain the chromosomal fusions and other structural alterations.



---

*Source: 8724304-2017-08-29.xml*