The contribution of mobile genetic elements to the evolution and ecology of Vibrios

Authors


  • Present address: Tracy H. Hazen, Centers for Disease Control and Prevention, Division of Foodborne, Waterborne, and Environmental Diseases, Atlanta, GA 30329, USA.

  • Editor: Ian Head

Correspondence: Patricia A. Sobecky, Department of Biological Sciences, University of Alabama, 250 Hackberry Lane, Tuscaloosa, AL 35401, USA. Tel.: +1 205 348 8330; fax: +1 205 348 1786; e-mail: psobecky@bama.ua.edu

Abstract

An increase in the frequency of seafood-borne gastroenteritis in humans and Vibrio-related disease of fish and invertebrates has generated interest in the ecology of disease-causing Vibrios and the mechanisms driving their evolution. Genome sequencing studies have indicated a substantial contribution of horizontal gene transfer (HGT) to the evolution of Vibrios. Of particular interest is the contribution of HGT to the evolution of Vibrios pathogens and the adaptation of disease-causing Vibrios for survival in diverse environments. In this review, we discuss the diversity and distribution of mobile genetic elements (MGEs) isolated from Vibrios and the contribution of these elements to the expansion of the ecological and pathogenic niches of the host strain. Much of the research on Vibrio MGEs has focused on understanding phages and plasmids and we will primarily discuss the evolution of these elements and also briefly highlight the other diverse elements characterized from Vibrios, which includes genomic islands and conjugative elements.

Introduction

The Vibrionaceae is a diverse family of bacteria that occupy habitats ranging from the deep sea to shallow coastal marine environments to symbionts of invertebrates in marine environments (Reen et al., 2006). Some Vibrios are pathogens that are capable of infecting invertebrates, fish, or humans (Thompson et al., 2004a, b). Vibrios occur as free-living inhabitants in the water column or sediment in coastal marine environments worldwide (Thompson et al., 2004a, b). It has been reported that for some Vibrio pathogens, such as Vibrio parahaemolyticus, known to cause illness in humans, it is frequently difficult to detect isolates directly from the environment that are capable of causing disease (Bej et al., 1999; Nair et al., 2007). One possible explanation is that strains are acquiring virulence traits by horizontal gene transfer (HGT) in the environment. Thus, the contributions of HGT to the emergence of disease-causing Vibrios from environmental populations have become a topic of special interest in recent years. Furthermore, HGT may contribute to niche adaptation as demonstrated for some environmental Vibrios (Hunt et al., 2008), which may eventually lead to speciation.

The number of worldwide Vibrio-related illnesses each year is unknown; however, toxigenic Vibrio cholerae causes frequent infections, with 190 130 cases and 5143 deaths reported to the World Health Organization in 2008 [World Health Organization (WHO), 2009]. The pathogenic Vibrios have been estimated to cause approximately 8000 illnesses annually in the United States; however, it is believed that fewer than 10% of the estimated cases are reported (Mead et al., 1999). Vibrio-related food-borne illnesses in the United States have been increasing since 1998 [Center for Disease Control and Prevention (CDC), 2007]. The number of reported Vibrio-related infections in the United States in 2006 was 78% higher than those reported during 1996–1998 [Center for Disease Control and Prevention (CDC), 2007]. The majority of Vibrio-related human illnesses that occur each year are caused by V. cholerae, Vibrio vulnificus, and V. parahaemolyticus (Thompson et al., 2004a, b).

In this minireview, we discuss current knowledge of the contribution of mobile genetic elements (MGEs) to the evolution and ecology of the Vibrionaceae. We provide specific examples of how MGEs have shaped the genomes and contributed to phenotypes for the survival and adaptation of Vibrios to the natural environment as well as conferring pathogenicity in a host. We conclude by providing examples of how MGEs are likely driving the ongoing evolution of Vibrios by allowing them to expand their environmental niche by acquiring genes conferring novel phenotypes.

Ecology of Vibrios and the mechanisms that drive their evolution

The Vibrionaceae includes organisms from nearly every type of aquatic environment. The family Vibrionaceae consists of 10 genera including the Photococcus, Listonella, Grimontia, Allomonas, Catenococcus, Aliivibrio, Salinivibrio, Enterovibrio, Photobacterium, and Vibrio (Thompson et al., 2005). The majority of research has focused on characterizing Vibrio and Photobacterium spp., and significantly more is known regarding the ecology of these organisms. There are at least 84 Vibrio spp. that are represented by at least one or more gene sequences in GenBank as of March 2010. In addition, a group of related Vibrios discussed in this review comprised of Vibrio salmonicida, Vibrio logei, Vibrio fischeri, and Vibrio wodanis has been redesignated as the genus Aliivibrio (Urbanczyk et al., 2007).

The mechanisms of HGT that shape Vibrio genomes include transduction, conjugation, and natural transformation (Otto et al., 1997; Frost et al., 2005; Meibom et al., 2005). Transduction by diverse phages such as V. parahaemolyticus phages capable of infecting only V. parahaemolyticus (Comeau et al., 2006) or CTXΦ, which is able to move between multiple Vibrio hosts (Faruque et al., 1999; Boyd et al., 2000), could facilitate gene transfer if the phages integrate into the host chromosome and take additional genes following excision. Conjugation of a 67-kb plasmid with the aid of a mobilizable plasmid was shown for the V. vulnificus plasmid pC4602-2 in the presence of the mobilizable pC4602-1 (Lee et al., 2008). Furthermore, predation by the heteroflagellate Cafeteria roenbergensis increased the frequency of conjugal transfer of plasmid DNA among Vibrio strains (Meibom et al., 2005).

Environmental stimuli such as nutrient limitation and the presence of chitin have been identified as signals of HGT by stimulating natural transformation in V. cholerae (Meibom et al., 2005). Vibrio cholerae environmental strains were shown to transform and recombine DNA segments ranging from 7.9 to 44.9 kb (Miller et al., 2007). The natural competence mechanism of V. cholerae was shown to be widely distributed among V. cholerae strains (Miller et al., 2007). Among the mechanisms that prevent HGT are the production of restriction endonucleases that prevent the acquisition of foreign DNA (Frost et al., 2005). A nuclease from V. cholerae was recently shown to reduce the frequency of transformation of extracellular DNA; however, the cells were able to downregulate the production of the nuclease in response to the induction of competence (Blokesch & Schoolnik, 2008).

Types of Vibrio MGEs and their host range

The study of the diversity and distribution of Vibrio MGEs has mostly been limited to disease-causing Vibrios. Recent genome sequencing projects have expanded our knowledge of the types and occurrence of MGEs associated with Vibrios. The frequent types of MGEs characterized from Vibrios are plasmids, phages, insertion sequence (IS) elements, and genomic islands. MGEs have been identified for all of the frequently studied Vibrios, which include not only the disease-causing but also the symbiotic and environmental Vibrios (Fig. 1; Table 1).

Figure 1.

 Phylogenetic analysis of recA nucleotide sequences from select species of the Vibrionaceae. The neighbor-joining phylogeny was constructed in mega v. 4.0 (Tamura et al., 2007) using the Kimura 2-parameter model with 1000 bootstrap replications. Only bootstrap values ≥50 are shown.Scale bar=0.02 nucleotide changes per distance. *Species that have been shown to possess MGEs (plasmids, phages, or conjugative elements). The disease-causing Vibrio spp. shown to have MGEs associated with pathogenicity are indicated in bold. Vibrios that are known to cause illness among humans or marine organisms are indicated in bold.

Table 1. Vibrio plasmids characterized to date
Host organismPlasmidPhenotypeSize (bp)CDSsAccession numbers
V. alginolyticuspVAE259Unknown60755NC_013178
V. choleraepSIO1Unknown49063NC_006860
V. choleraepVCG1.1Unknown44395NC_010897
V. choleraepVCG1.2Unknown23574NC_010899
V. choleraepVCG4.1Unknown21633NC_010910
V. fischeripES213Unknown55019NC_011403
V. fischeripES100Unknown45 84957NC_006842
V. fischeripMJ100Unknown179 459195NC_011185
V. fluvialispBD146Unknown74726NC_011797
V. harveyipVIBHARUnknown89 008120NC_009777
V. nigripulchritudopSFn1Pathogenicity11 23710NC_010733
V. parahaemolyticuspSA19Unknown48394NC_002088
V. parahaemolyticuspZY5Unknown35043NC_012859
V. parahaemolyticusp22702BUnknown28 83726Hazen et al. (unpublished data)
V. shiloniipAK1Unknown13 41510NC_010734
V. campbelliip09022AUnknown31 03632NC_010114
V. fluvialisp0908Unknown81 41395NC_010113
V. mediterraneip23023Unknown52 52764NC_010112
Vibrio sp.pPS41Unknown68868NC_004961
Vibrio sp.pTC68Unknown78472NC_008690
V. tapetispVT1Unknown82 26685NC_010614
V. vulnificuspC4602-1Pathogenicity56 62869NC_009702
V. vulnificuspC4602-2Unknown66 94667NC_009703
V. vulnificuspMP1Unknown76284NC_012758
V. vulnificuspR99Pathogenicity68 44671NC_009701
V. vulnificuspYJ016Unknown48 50869NC_005128
P. damselae piscicidapP9014Unknown55 85161NC_012919
P. damselae piscicidapP91278Unknown131 520161NC_008613
P. damselae piscicidapP99-018Unknown150 157187NC_008612
P. profundumpPBPR1Unknown80 03367NC_005871
V. anguillarumpJM1Unknown65 00957NC_005250
V. anguillarumpLO2Unknown794110NC_009531
A. salmonicidapVAL43Unknown53603NC_011315
A. salmonicidapVSAL320Unknown30 80729NC_011314
A. salmonicidapVSAL43Unknown43273NC_011316
A. salmonicidapVSAL840Unknown83 54072NC_011311

Plasmids

A few studies have characterized the diversity and distribution of plasmids associated with a particular Vibrio spp. primarily in correlation with the pathogenic phenotype of the strain in question. For example, numerous studies have contributed to our knowledge of the diversity of plasmids associated with the fish pathogens Vibrio anguillarum and V. vulnificus (Lee et al., 2008). In addition, recent research has characterized virulence-associated plasmids from the shrimp pathogens Vibrio shilonii and Vibrio nigripulchritudo (Reynaud et al., 2008). Our own research has shown that plasmids isolated from several environmental Vibrios exhibit little genetic similarity (Hazen et al., 2007).

Researchers have demonstrated that V. anguillarum requires plasmid-encoded genes in order to cause disease in fish (Di Lorenzo et al., 2003; Wu et al., 2004). Plasmid pJM1 encodes proteins involved in siderophore production that contribute to the V. anguillarum pathogenic mechanism (Di Lorenzo et al., 2003). Furthermore, plasmids from V. anguillarum strains that were isolated from as far away as Japan and the United States shared similar restriction patterns that indicated that these plasmids were likely genetically conserved (Di Lorenzo et al., 2003). In addition, V. vulnificus biotype 2 strains that cause disease in eels require the presence of a 56–68-kb plasmid that is required for pathogenicity (Lee et al., 2005, 2008). The plasmid-encoded rtx genes that are known virulence-associated genes and the loss of the plasmids resulted in a loss of resistance to eel serum and virulence (Lee et al., 2008). A survey of the plasmid content of V. vulnificus revealed that there were 28 different plasmid profiles; however, there was a specific plasmid type that was detected among all biotype 2 and biotype 3 strains (Roig & Amaro, 2009). In addition, there was a plasmid of a size similar to the V. vulnificus plasmid pYJ016 that was detected in nearly half of biotype 1 V. vulnificus strains (Roig & Amaro, 2009). Many strains contained small plasmids that remain uncharacterized and may be prophages as demonstrated for other Vibrios (Chang et al., 2002).

An 11.2-kb plasmid, pSFn1, isolated from V. nigripulchritudo, was associated with virulence toward shrimp (Reynaud et al., 2008). In addition, a similar-size plasmid was detected among other highly virulent strains of V. nigripulchritudo (Reynaud et al., 2008). The coral pathogen V. shilonii possessed a similar-size plasmid (13.4 kb) that exhibited significant genetic conservation to pSFn1 of V. nigripulchritudo (Reynaud et al., 2008). The similarity of the virulence-associated plasmid from a shrimp pathogen to the plasmid of a coral pathogen indicated that plasmids may play a crucial role in the HGT of genes required by Vibrios to infect marine invertebrates.

Few studies have examined the sequence diversity of plasmids associated with environmental Vibrios. In a previous study, we demonstrated the considerable sequence diversity of three plasmids isolated from Vibrio fluvialis, Vibrio mediterranei, and Vibrio campbellii from a coastal environment (Hazen et al., 2007). Several plasmids have been characterized from the mutualistic symbiotic bacterium V. fischeri, which produces light that helps camouflage the bobtail squid Euprymna scolopes (Ruby et al., 2005). Genome sequencing of the squid symbiont V. fischeri ES114 revealed the presence of plasmid pES100 (45.8 kb) (Ruby et al., 2005), while sequencing of the fish symbiont V. fischeri MJ11 revealed the plasmid pMJ100 (179 kb) (Mandel et al., 2009). For both of these plasmids, the majority of the predicted protein-encoding regions encoded hypothetical proteins and it is unclear whether the plasmids contribute to host phenotypes. In addition, the small mobilizable plasmid pES213 (5.5 kb) was characterized and used to develop molecular tools for the study of V. fischeri (Dunn et al., 2005).

Several small plasmids (3.5–4.8 kb) have been characterized from V. parahaemolyticus (Table 1); however, these plasmids encode only hypothetical proteins and their contribution to host phenotypes and their role in HGT are not known. Sequence analysis of a 28.8-kb plasmid isolated from a V. parahaemolyticus environmental strain revealed that the plasmid had a gene with 98% nucleotide identity to a hypothetical protein of the V. parahaemolyticus genomic island VPaI-6 that is associated with pandemic strains (P.A. Sobecky & T.H. Hazen, unpublished data). This hypothetical protein is also similar to a protein encoded on the Vibrio harveyi ATCC BAA-1116 genome, indicating that plasmids may have contributed to interspecies gene exchange of genomic island genes that lead to the emergence of the V. parahaemolyticus pandemic clone. In addition, p22702B had a replication protein-encoding gene (rep) with 85% identity to the rep of the V. campbellii plasmid p09022 that was identified in a previous study (Hazen et al., 2007). Further investigation of the distribution of the conserved rep type revealed a plasmid family present in V. parahaemolyticus, V. harveyi, and V. campbellii strains isolated from environmental samples (Hazen et al., unpublished data). These plasmids include p09022, which we characterized in a previous study (Hazen et al., 2007), and pVIBHAR, which was sequenced recently as part of the V. harveyi ATCC BAA-1116 genome (Fig. 2). Additional large MGEs (80–90 kb) isolated from V. parahaemolyticus clinical and environmental strains that did not have the conserved rep type instead exhibited a conserved genetic structure indicated by restriction endonuclease digestion (data not shown; Hazen et al., unpublished data).

Figure 2.

 Phylogenetic analysis of the predicted amino acid sequence of replication protein-encoding genes, rep, encoded by plasmids isolated from Vibrio spp. The neighbor-joining phylogeny was constructed in mega v. 4.0 (Tamura et al., 2007) using the p-distance estimation model and 1000 bootstrap replications. Only bootstrap values ≥50 are shown. Scale bar=0.1 amino acid changes per distance. A ‘g’ prefix on the strain name indicates Rep sequences obtained from genomic data and an individual replicon had not been described, while a ‘p’ prefix to the plasmid name indicates sequences associated with known replicons. The host organisms of the plasmids are indicated.

Phages

In contrast to the limited knowledge of Vibrio plasmids, numerous studies have examined the diversity and distribution of Vibrio phages. The phages listed in Table 2 are those that have been characterized and deposited in GenBank as of March 2010. Some of the replication proteins of the genome-associated phages are included in a phylogenetic analysis of replication proteins of phages isolated from Vibrios (Fig. 3). In particular, phages related to the filamentous phages based on the replication protein-encoding gene are present in nearly every Vibrio genome sequenced to date including V. fischeri, Vibrio mimicus, V. shilonii, Vibrio splendidus, and V. vulnificus (Fig. 3). Following the characterization of the filamentous phage CTX that encodes the cholera toxin genes ctxAB (Waldor & Mekalanos, 1996), significant research was conducted on the diversity of filamentous vibriophages (Chang et al., 1998, 2002; Nasu et al., 2000; Comeau et al., 2005, 2006; Comeau & Suttle, 2007). The filamentous vibriophage f237 was characterized from V. parahaemolyticus strains of the O3:K6 pandemic group (Nasu et al., 2000). The filamentous phages isolated from V. parahaemolyticus have been shown to share significant genetic similarity (Chang et al., 1998, 2002; Nasu et al., 2000). Phylogenetic analysis of the predicted amino acid sequences of replication-associated protein-encoding genes showed the degree of relatedness of the replication proteins from selected filamentous vibriophages (Fig. 3). For example, the replication protein of the prophage (pO3K6) of the V. parahaemolyticus pandemic phage f237 was nearly identical to the replication protein of phage VfO4K68, which is the deleted form of the pandemic phage that is missing the gene ORF8 (Chang et al., 2002). Vibriophages capable of infecting V. parahaemolyticus are abundant in sediment and water (Baross et al., 1978; Comeau et al., 2006). In addition, phages specific to V. vulnificus were abundant in the water and sediment of the Gulf of Mexico (DePaola et al., 1997, 1998). The V. vulnificus phages isolated were morphologically diverse and exhibited different patterns of host infectivity (Pelon et al., 1995; DePaola et al., 1998). CTXΦ has been shown to move from V. cholerae to closely related species such as V. mimicus, leading to the emergence of toxigenic V. mimicus (Boyd et al., 2000).

Table 2. Vibrio phages characterized to date
Host
organism
PhagePhenotypeSize
(bp)
CDSsAccession
number
V. choleraeCTXPathogenicity700010ACF98534
V. choleraepTLCUnknown47195NC_004982
V. choleraeK139Unknown33 10644NC_003313
V. choleraeKSF-1phiUnknown710712NC_006294
V. parahaemolyticusKVP40Unknown244 834381NC_005083
V. choleraeVEJphiUnknown684211NC_012757
V. choleraeVGJphiUnknown754213NC_004736
V. harveyiVHMLUnknown43 19857NC_004456
V. choleraeVP2Unknown39 85347NC_005879
V. choleraeVP5Unknown39 78648NC_005891
V. parahaemolyticusVP882Unknown38 19771NC_009016
V. parahaemolyticusVP93Unknown43 93144NC_012662
V. choleraeVSKUnknown688214NC_003327
V. parahaemolyticuspO3K6Unknown878410NC_002473
V. parahaemolyticusVf12Unknown79657NC_005949
V. parahaemolyticusVf33Unknown79657NC_005948
V. parahaemolyticusVfO3K6Unknown878410NC_002362
V. parahaemolyticusVfO4K68Unknown68918NC_002363
V. choleraefs1Unknown634015NC_004306
V. choleraefs2Unknown86519NC_001956
V. choleraekappaUnknown33 13445NC_010275
UnknownVP4Unknown39 50331NC_007149
V. parahaemolyticusVpV262Unknown46 01267NC_003907
Figure 3.

 Phylogenetic analysis of the predicted amino acid sequences of replication protein-encoding genes from phages isolated from Vibrios. The neighbor-joining tree was constructed in mega v. 4.0 (Tamura et al., 2007) using the p-distance estimation model and 1000 bootstrap replications. Only bootstrap values ≥50 are shown. Scale bar=0.1 amino acid changes per distance. The host organisms of the phage are indicated when known. A ‘g’ prefix on the strain name indicates Rep sequences obtained from genomic data and an individual phage had not been described.

The majority of the vibriophages characterized have been small (≤10 kb) (Chang et al., 1998, 2002; Nasu et al., 2000). Several large phages have been characterized that are capable of infecting V. parahaemolyticus (Seguritan et al., 2003). Among the large phages is the 244.8 kb T4-like phage KVP40 (Miller et al., 2003) and the V. parahaemolyticus phages VP16C (47.5 kb) and VP16T (49.5 kb) (Seguritan et al., 2003). KVP40 encoded numerous T4 phage proteins and a putative NAD salvage pathway that may aid host metabolism (Miller et al., 2003). Both of the V. parahaemolyticus phages and KVP40 encoded numerous hypothetical proteins with no known contribution to functions of the bacterial host. The V. parahaemolyticus phage VpV262 (46 kb) was characterized as an ancestor to the T7-like phages (Hardies et al., 2003).

Genomic islands and conjugative elements

Integrons, transposons, integrative conjugative elements (ICEs), and genomic islands of Vibrios have been characterized primarily as a result of genome sequencing projects (Waldor et al., 1996; Heidelberg et al., 2000; Chen et al., 2003; Faruque & Mekalanos, 2003; Hurley et al., 2006; Hjerde et al., 2008). Among the largest of the integrative elements are the genomic islands, which can be up to several hundred kilobases. Although genomic islands do not encode genes for self-mobilization, they do encode genes for self-excision from the genome and may then be subject to HGT by natural transformation. A recent article established that genomic islands are evolutionarily distinct integrative elements (Boyd et al., 2009). Genomic islands may facilitate the transfer of virulence-associated genes such as a type III secretion system (T3SS) of non-O1/non-O139 V. cholerae strains, which is similar to a T3SS encoded on a genomic island of V. parahaemolyticus strains of the O3:K6 pandemic group (Park et al., 2004; Dziejman et al., 2005).

In addition, superintegrons such as the V. cholerae superintegron have been identified that are approximately 100 kb and are present in the genomes of almost every Vibrio sequenced to date (Mazel et al., 1998; Biskri et al., 2005). ICEs such as the multidrug resistance element, SXT, of V. cholerae increase the frequency of HGT under stress (Beaber & Waldor, 2004). Much remains unknown about the diversity and evolution of Vibrio integrative elements. A comprehensive assessment of these elements is beyond the scope of the current review. Each additional Vibrio genome that is sequenced contributes more to our knowledge of the diversity and distribution of integrative elements and their potential role in HGT and the evolution of Vibrio genomes. In the following sections of this minireview, we will focus on the role of plasmids and phages in the evolution of Vibrios.

Contributions of MGEs to Vibrio evolution

Phylogenetic analysis of replication proteins from plasmids isolated from diverse Vibrios indicated that several main modes of replication (rep types) were present among Vibrio plasmids (Fig. 2). We chose to assess the relatedness of Vibrio plasmids by analyzing their predicted replication proteins because these are often the most conserved protein-encoding regions of a plasmid (Sobecky et al., 1997, 1998). A prominent rep group present in the replication phylogeny consists of putative replication proteins encoded on plasmids from V. campbellii, V. parahaemolyticus, V. harveyi, Vibrio furnissii, and V. cholerae. We have recently characterized the sequence of a 28.8-kb plasmid, p22702B, from a V. parahaemolyticus environmental strain that exhibits this same conserved rep type (Hazen et al., unpublished data). While these plasmids encoded a replication protein with a conserved rep domain, additional plasmids had unique replication proteins with no conserved domains. For example, four plasmids (48–68 kb) isolated from three different strains of V. vulnificus that formed a closely related group exhibited a unique rep type (Fig. 2). This unique rep type was detected on plasmids from V. vulnificus biotype 2 strains that cause disease in eels and a strain that caused human illness. The most closely related rep is from plasmid pVT1 of Vibrio tapetis, suggesting that this rep type may have come from a Vibrio ancestor and the V. vulnificus and V. tapetis strains have stably maintained the plasmids encoding this rep type. This rep type may have been maintained by select V. vulnificus strains if the plasmid that encoded the Rep protein also possessed addiction systems or encoded genes that conferred a beneficial phenotype to the host. The V. vulnificus plasmids, pC4602-2 and pR99, of biotype 2 strains that encode this rep type also have a protein involved in the virulence of these strains toward eels (Lee et al., 2008). The most distantly related Rep in this group was encoded by plasmid pC4602-1, which was present in the same host strain with pC4602-2 requiring some evolved divergence in the replication proteins of these two plasmids in order for them to co-occur. In addition, Rep proteins of plasmids from Photobacterium profundum, Aliivibrio salmonicida, and V. fischeri (recently renamed Aliivibrio fischeri) exhibited similarity. As with the Rep proteins of V. vulnificus, these Rep proteins were detected among related species, suggesting that the Rep proteins originated from a plasmid encoded by an ancestor of these species. The plasmids with this rep type may have been maintained because they encoded proteins that conferred a beneficial phenotype to the host strain; however, the function of proteins encoded by these plasmids is largely unknown (Ruby et al., 2005; Hjerde et al., 2008).

To survey the relatedness of Vibrio phages that have been characterized to date by sequencing, we constructed a neighbor-joining phylogeny of amino acid sequences of the replication proteins from all phages present in GenBank as of March 2010 (Fig. 3). Several prominent groups of phages are identifiable in the phylogenetic analysis of the predicted replication proteins (Fig. 3). The filamentous vibriophages, which are among the most abundant phages detected among Vibrios, were present in diverse disease-causing species. The V. parahaemolyticus filamentous phages exhibited significant amino acid identity and were most related to two V. harveyi phages present in the genomes of two different V. harveyi strains that were sequenced recently (Fig. 3). Interestingly, related phage replication proteins were detected in the genomes of the fish pathogen A. salmonicida and the human pathogen V. vulnificus YJ016. A second group of related replication proteins were comprised of the independently isolated and characterized phages fs1, VEJΦ, VGJΦ, KSF-1Φ, and VSK (Fig. 3). These phages have a predicted rolling circle type of replication. This group of phages likewise had small genome sequences and the divergence in the amino acid sequences may indicate that these phages have a different mode of replication.

Another related group in the phage replication protein phylogeny was that of the V. cholerae filamentous phage CTXΦ and related phages present in the genomes of additional V. cholerae strains and the recently sequenced V. mimicus V573. The remaining phage replication proteins in the phylogeny likely represent phages that have distinct modes of replication from the filamentous phages. Molecular studies have demonstrated that HGT events between diverse Vibrios such as V. fischeri and V. cholerae were facilitated by MGEs. For example, remnants of the filamentous phage CTXΦ were identified in the V. fischeri genome (Ruby et al., 2005). Furthermore, CTXΦ has been shown to transfer the cholera-toxin-encoding virulence genes from V. cholerae to V. mimicus (Boyd et al., 2000).

Overall, in both the plasmid and the phage replication protein phylogenies, there is substantial conservation relative to the host organism from which the MGEs were isolated. For example, the medium-size plasmids of V. parahaemolyticus are most related to similar-size plasmids from closely related hosts including other V. parahaemolyticus strains, V. campbellii, and V. harveyi. This conservation of the plasmid replication type to a narrow range of hosts indicates that these elements may have been acquired from a common ancestor to V. parahaemolyticus, V. harveyi, and V. campbellii. Conversely, these plasmids may move frequently between these different host species and have maintained their ability to replicate in these different hosts. The ability of these plasmids to move among different Vibrios would facilitate gene exchange and help maintain their relatedness and ability to occupy similar environmental niches. Similarly, analysis of rep proteins associated with plasmids from several of the Aliivibrios (V. fischeri and A. salmonicida) suggested that these replicons may be restricted to a few closely related host species (Fig. 2). In contrast, a phage replication protein was detected in the genome of V. fischeri that was most similar to the CTX phages of V. cholerae (Ruby et al., 2005). This suggests that the plasmids may have a narrower host range, while the filamentous Vibrio phages may frequently move genes among unrelated Vibrios.

MGEs facilitate the expansion of the Vibrio ecological niche

A number of studies have examined the diversity of vibriophages associated with Vibrios isolated from coastal environments (Chang et al., 1998, 2002; Nasu et al., 2000; Miller et al., 2003; Seguritan et al., 2003), and a few studies have determined the diversity and distribution of plasmids among environmental Vibrios (Shehane & Sizemore, 2002; Roig & Amaro, 2009). In a previous study, we characterized three plasmids isolated from diverse environmental Vibrios including V. mediterranei, V. campbellii, and V. fluvialis (Hazen et al., 2007). Research investigating the presence of bacteriocinogenic Vibrios in environmental samples has demonstrated that V. parahaemolyticus strains have a high frequency of plasmid DNA (Shehane & Sizemore, 2002).

Vibrios undergo phenotypic variation in response to changing environmental conditions such as fluctuating nutrients, temperature, and aeration (Hilton et al., 2006). Phenotypic switching is the change in colony phenotypes that has been observed for V. vulnificus environmental strains in response to increased temperature and static growth conditions (Hilton et al., 2006). Similarly, V. parahaemolyticus exhibits phenotypic switching, resulting in changes in capsular polysaccharide production and in biofilm structure in response to changing environmental conditions (McCarter, 1998; Enos-Berlage et al., 2005). Vibrios in the environment can frequently possess plasmids and phages (DePaola et al., 1998; Hardies et al., 2003; Comeau et al., 2006; Comeau & Suttle, 2007); however, it is unclear how these MGEs may contribute to survival under changing environmental conditions (Hazen et al., 2007). We demonstrated in a previous study that a V. parahaemolyticus environmental strain isolated from Spartina-dominated salt marsh sediment had horizontally acquired an nifH homolog and the ability to fix atmospheric nitrogen at low levels (Criminger et al., 2007). Vibrio natriegens, Vibrio diazotrophicus, and Vibrio cincinnatiensis have previously been shown to fix atmospheric nitrogen; however, until recently, V. parahaemolyticus was not known to possess nitrogen-fixation genes (Urdaci et al., 2004). It is unclear whether the nifH homolog was horizontally acquired from an MGE such as a plasmid; however, in a previous study, we demonstrated that nif homologs occur on plasmids (Beeson et al., 2002).

The V. parahaemolyticus pandemic strains have enhanced swarming compared with nonpandemic strains (Yeung et al., 2002), which potentially results from proteins encoded by the VPaI-1 genomic island. Swarming motility is the movement of cells across a surface and involves the production of multiple lateral flagella (Jacques & McCarter, 2006). Multiple swarming regulators have been identified for V. parahaemolyticus (Park et al., 2005; Jacques & McCarter, 2006). Vibrio parahaemolyticus is able to produce either a single polar flagellum or multiple lateral flagella in response to environmental signals (Jacques & McCarter, 2006). Iron limitation and the expression of lateral flagella have been shown to regulate swarming motility (Jacques & McCarter, 2006). In addition, a horizontally acquired H-NS-like protein, vpaH, was shown to regulate the production of lateral flagella in trh+V. parahaemolyticus strains and increase swarming motility (Park et al., 2005). Furthermore, the V. parahaemolyticus O3:K6 pandemic clone has been shown to exhibit enhanced swarming motility compared with nonpandemic strains (Yeung et al., 2002). Surprisingly, we found that nearly all environmental strains isolated from a North Carolina sample site exhibited enhanced swarming motility while <40% of strains from other environmental sample locations swarmed (Hazen et al., unpublished data). These findings suggested that swarming motility may be beneficial to strains occupying certain pathogenic or ecological niches. The enhanced swarming of the O3:K6 pandemic clone may be caused by the presence of several genomic islands unique to the pandemic clone (Hurley et al., 2006; Boyd et al., 2008); however, it is not known whether the V. parahaemolyticus environmental strains that swarmed had these genes.

Another phenotype associated with environmental adaptation that has been transferred by MGEs is bacteriocin production. Among the Vibrios that produce a bacteriocin-like substance is V. harveyi (McCall & Sizemore, 1979; Hoyt & Sizemore, 1982). A V. harveyi strain was shown to produce a bacteriocin-like substance that was associated with the presence of plasmid DNA (McCall & Sizemore, 1979), indicating that there may be bacteriocinogenic-like plasmids among Vibrios. The V. harveyi strain producing the bacteriocin-like substance was able to outcompete an isogenic plasmid-free strain that no longer produced the bacteriocin (Hoyt & Sizemore, 1982). Vibrio vulnificus was also shown to produce a bacteriocin-like substance that inhibited the growth of diverse Vibrios including V. parahaemolyticus (Shehane & Sizemore, 2002). Likewise, a V. mediterranei strain was identified that inhibited the growth of V. parahaemolyticus (Carraturo et al., 2006). We have characterized the sequence of a plasmid isolated from a V. parahaemolyticus environmental strain with a gene that had 98% amino acid identity to hypothetical protein-encoding genes of the VPaI-6 genomic island (Hazen et al., unpublished data). This hypothetical protein-encoding gene had >90% amino acid identity to a predicted bacteriocinogenic protein that is encoded in the V. harveyi genome and may represent a novel class of bacteriocins present in Vibrios.

While it is yet to be shown that the small, but abundant filamentous vibriophages contribute significantly to gene exchange that drives niche expansion, researchers have demonstrated that these smaller MGEs directly influence Vibrio ecology by regulating population dynamics. For example, phages capable of infecting disease-causing V. cholerae were detected in water samples when V. cholerae was absent, and the phages were absent when V. cholerae was present (Faruque et al., 2005). The inverse relationship of the presence of phages and V. cholerae indicated that the phages were reducing the population of V. cholerae. In addition, as infected V. cholerae lyse, they release genetic material available for natural transformation by other V. cholerae (Meibom et al., 2005; Miller et al., 2007) or possibly by other diverse Vibrios. Vibrio parahaemolyticus strains possessing a telomeric phage were more sensitive to UV irradiation, which triggered phage induction (Zabala et al., 2009). This phage was similar to the phage VHML of V. harveyi, which suggested that these phages may belong to a family that can transfer between V. parahaemolyticus and V. harveyi, which may facilitate the transfer of genes that would contribute to pathogen emergence.

Insights from genomics and metagenomics into the contributions of MGEs to Vibrio evolution

Comparative analysis of genomes from six Vibrios demonstrated large regions of homology between the disease-causing genomes analyzed that may correspond with genomic islands (Reen et al., 2006). Furthermore, a large number of inversions between species such as V. vulnificus and V. parahaemolyticus suggested that rearrangements occurred as these species diverged, and these events may have been mediated by mobile elements. A comparison of the genomic dissimilarity (δ*), which measured the dinucleotide relative abundance values of the two Vibrio chromosomes among more recently sequenced Vibrios, suggested that chromosome II may have been acquired by HGT (Thompson et al., 2009). As of March 2010, there have been approximately 58 Vibrio genomes sequenced and deposited in GenBank. Numerous additional Vibrios genome projects are currently underway including those for the less frequent human pathogenic species V. fluvialis, Vibrio alginolyticus, Vibrio metschnikovii, and Vibrio coralliilyticus. The completion of these additional genomes likely will considerably expand our knowledge of the types and diversity of MGEs associated with Vibrios.

Among the genomes that have been completed are those of the Vibrio pathogens V. cholerae (Heidelberg et al., 2000), V. parahaemolyticus (Makino et al., 2003), and V. vulnificus (Chen et al., 2003; Kim et al., 2003). In addition, the genome of the fish pathogen V. splendidus was completed recently (Le Roux et al., 2009) and two genomes of the squid light organ symbiotic bacterium V. fischeri have been sequenced (Ruby et al., 2005; Mandel et al., 2009). Horizontal transfer of virulence-associated genes has been shown to occur between different Vibrio spp. including the acquisition of the V. parahaemolyticus thermostable direct hemolysin trh by a V. alginolyticus strain (González-Escalona et al., 2006). Comparison of the V. parahaemolyticus and V. cholerae chromosomes revealed rearrangements between the genomes as well as the acquisition of additional genes on chromosome II of V. parahaemolyticus (Makino et al., 2003). The T3SS located on chromosome II of V. parahaemolyticus is most related to a T3SS of non-O1/non-O139 V. cholerae strains (Dziejman et al., 2005; Okada et al., 2009). Other genes involved in the pathogenic mechanism of V. parahaemolyticus that may have been horizontally acquired are a histone-like protein vpaH that is involved in swarming motility (Park et al., 2005). The effect of HGT on the evolution of the genome of V. parahaemolyticus has been demonstrated in numerous studies (Chang et al., 1998; Park et al., 2005; Hurley et al., 2006; Boyd et al., 2008).

Bioinformatics analysis indicated that the second type III secretion system (T3SS2) located on the second chromosome and two copies of tdh compose a genomic island flanked by Tn7-like transposase genes (Boyd et al., 2008; Sugiyama et al., 2008). The putative pathogenicity island encoding tdh and T3SS2 has been demonstrated by sequencing and comparative genome hybridization to be associated with V. parahaemolyticus clonal pandemic strains (Boyd et al., 2008; Izutsu et al., 2008). A recent study has demonstrated that the enterotoxicity of pathogenic strains possessing only trh and lacking T3SS2 genes can be attributed to the presence of an additional T3SS2 encoded within the region surrounding trh (Okada et al., 2009). This newly identified T3SS2 is designated T3SS2β and is located within a 100-kb genomic island present on chromosome II of V. parahaemolyticus strains that possess trh and not tdh (Okada et al., 2009). This demonstrated the convergent evolution of similar pathogenic mechanisms of V. parahaemolyticus. Interestingly, both of the T3SS2s are located in genomic islands flanked by IS elements (Hurley et al., 2006; Okada et al., 2009).

The recent completion of an A. salmonicida genome revealed the extent to which MGEs can shape a Vibrio genome (Hjerde et al., 2008). The strain characterized possessed four plasmids as was seen for numerous other A. salmonicida strains that had been analyzed previously (Sørum et al., 1990; Hjerde et al., 2008). The plasmids ranged in size from 4.3 to 83.5 kb and encoded genes for mobility as well as acyltransferases that may aid in preventing a host organism from recognizing the A. salmonicida that has established an infection (Hjerde et al., 2008). In addition, the genome contained 20 different types of IS elements, nine phage-like elements, and 16 genomic islands (Hjerde et al., 2008). Of the 288 IS elements identified in the genome, there were many that had inserted into genes, likely rendering them nonfunctional. Therefore, this analysis demonstrated that MGEs not only expand the environmental niche of a Vibrio but also limit it by reducing the number of functioning phenotypes encoded within the genome.

A recent metagenome study of an induced viral sample revealed abundant Vibrio-like integrase genes from phages (McDaniel et al., 2008). Detection of the Vibrio-like integrases from total microbial DNA using real-time PCR estimated that there were 1.9 × 107 copies L−1 of the integrases. This indicated a large presence of phages capable of infecting Vibrios. In addition, they detected the Vibrio-like integrases in over half of the global ocean survey sample sites (Yooseph et al., 2007) and four sites from which marine viral metagenomes were prepared (Angly et al., 2006).

The role of MGEs and HGT in the emergence of Vibrio pathogens

Recent studies demonstrating the sequence conservation of MGEs present in different Vibrio spp. have indicated that MGEs likely play a significant role in the ongoing evolution of Vibrios. MGEs also likely blur the species definition of Vibrios by distributing what were considered to be species-specific genes. The sequence characterization of a plasmid isolated from V. parahaemolyticus demonstrated that plasmids contribute to interspecific gene exchange between V. parahaemolyticus and V. harveyi.

Furthermore, Vibrio MGEs likely play a significant role in the emergence of disease-causing strains from environmental populations. We recently characterized a V. parahaemolyticus environmental strain isolated from sediment of a Florida brackish estuary that closely resembled the pandemic clones based on multilocus sequence analysis (Hazen et al., unpublished data). Although this environmental strain was phylogenetically similar to the O3:K6 pandemic clone based on MLSA, it lacked the known virulence-associated genes and genomic islands that are characteristic of the pandemic clone (Hazen et al., unpublished data). The identification of this strain as well as other environmental strains that were phylogenetically similar to clinical strains isolated from disease-associated samples indicated that HGT may play a significant role in the rapid emergence of disease-causing strains from nonpathogenic environmental populations. Following the acquisition of the genomic islands of the pandemic clone, these strains may cause human illness under permissive conditions.

Summary

Research findings to date have indicated a clear contribution of MGEs toward the ongoing evolution of Vibrio genomes. Genetic elements such as phages and plasmids have contributed to the distribution of disease-associated genes within and between species. In addition, recent evidence has suggested a significant role of MGEs in the expansion of the environmental niche of pathogenic and environmental Vibrios. While recent research has demonstrated the diversity and distribution of MGEs within Vibrio spp., little is known regarding the distribution of MGEs among diverse Vibrios. Further studies are required to understand the extent of MGE-mediated HGT between diverse Vibrios and the contribution of this gene exchange toward the development of novel pathogenic phenotypes and the ability of Vibrios to expand their ecological niche.

Acknowledgements

We would like to thank two anonymous reviewers for their comments, which improved this manuscript. Previous research support has been provided by an Office of Naval Research Grant N00014-021-0228 to P.A.S., NSF LExEn Grant, and a GT/CDC seed grant no. 1241326 to P.A.S. and C. Bopp. T.H.H. is currently supported by an ASM/CCID postdoctoral research fellowship.

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