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Proc Natl Acad Sci U S A. Feb 22, 2005; 102(8): 3004–3009.
Published online Feb 9, 2005. doi:  10.1073/pnas.0409900102
PMCID: PMC549501

Complete genome sequence of Vibrio fischeri: A symbiotic bacterium with pathogenic congeners


Vibrio fischeri belongs to the Vibrionaceae, a large family of marine γ-proteobacteria that includes several dozen species known to engage in a diversity of beneficial or pathogenic interactions with animal tissue. Among the small number of pathogenic Vibrio species that cause human diseases are Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus, the only members of the Vibrionaceae that have had their genome sequences reported. Nonpathogenic members of the genus Vibrio, including a number of beneficial symbionts, make up the majority of the Vibrionaceae, but none of these species has been similarly examined. Here we report the genome sequence of V. fischeri ES114, which enters into a mutualistic symbiosis in the light organ of the bobtail squid, Euprymna scolopes. Analysis of this sequence has revealed surprising parallels with V. cholerae and other pathogens.

Keywords: genomics, pili, symbiosis, toxins, toxin-coregulated pilus

The marine bacterium Vibrio fischeri is best known as the specific symbiont in the light-emitting organs of certain squids and fishes (1), where it produces luminescence by expressing the lux operon, a small cluster of genes found in several of the Vibrionaceae. Luminescence is controlled by acyl-homoserine lactone quorum sensing, which was discovered in V. fischeri but is a common feature of host-associated bacteria in a number of genera (2). The Vibrionaceae comprise several dozen species that are often found associated with animal tissue (3). Among the small number of pathogenic Vibrio species that cause human diseases are Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus, the only members of the Vibrionaceae that have had their genome sequences reported (46). To date, no representative of the much more numerous benign and beneficial Vibrionaceae has been examined at the genome level.

Unlike many symbiotic bacteria, V. fischeri has been studied at the physiological and genetic levels for decades, has a well described environmental biology, and is easily examined by using molecular genetics (7). Thus, this particular nonpathogenic member of the Vibrionaceae is an ideal candidate for comparative genome analyses with pathogenic vibrios. The best understood of the V. fischeri symbiotic associations are those with sepiolid squids. These symbioses involve monospecific populations of V. fischeri cultured extracellularly, but within epithelium-lined crypts, in a specialized host organ. The squid associations have been extensively studied because of the ease of initiating the association and of observing developmental changes in both partners (8). Nevertheless, important questions remain concerning the genetic and metabolic mechanisms by which V. fischeri and other symbiotic bacteria adjust to the special environment of host tissues. To better understand these symbiotic activities in V. fischeri, and to begin to identify features common to beneficial and pathogenic bacteria, we sequenced the genome of strain ES114, the model light-organ symbiont of the squid Euprymna scolopes (9).

Materials and Methods

V. fischeri genomic DNA was mechanically sheared, and 2- to 3-kb fragments were isolated. The ends of the fragments were filled by using Klenow fragment and ligated into SmaI-digested pGEM3 to produce a high-copy-number, small-insert library (10). Higher molecular mass genomic DNA was partially digested with Sau3A to construct a cosmid library containing 30- to 35-kb inserts in Lorist6 (11). Undigested, unsheared DNA was used as template for PCR amplification of chromosomal regions not represented in the plasmid or cosmid libraries. Whole-genome shotgun sequencing was performed on ≈35,000 plasmids and 400 cosmids, as well as gap-spanning PCR products. The average phrap consensus quality was 82. Automated contig-assembly algorithms were ineffective in determining the number and orientations of the highly homologous, tandemly repeated, ribosomal RNA operons; thus, these regions were assembled manually from sequenced PCR products. The genome, with an average coverage of 10×, was assembled into three contigs. Potential ORFs were identified by using both critica (12) and a program developed at Integrated Genomics (Chicago). The finished genome underwent a round of initial automated annotation, followed by a manual gene-by-gene curation. The results of this analysis can be found at www.ergo-light.com.

Results and Discussion

General Features. As in each of the three sequenced pathogenic Vibrio species (46), V. fischeri has two chromosomes (Fig. 1). Genes encoding representatives of 25 classes of cellular function can be found on each of these replicons (Fig. 2). Strain ES114 is also characterized by the presence of a 45.8-kbp plasmid designated pES100. Carriage of a plasmid that is homologous to pES100 is common among other symbiotic strains of V. fischeri, but it is not required for host association (13). The sequence of pES100 suggests that, like a similarly sized replicon in V. vulnificus YJ016 (4), it is a conjugative plasmid, much of whose sequence encodes a putative type IV secretion system. Thus, such plasmids are common to both beneficial and pathogenic Vibrio species.

Fig. 1.
Genome maps of Chr I (2.9 Mbp) and Chr II (1.3 Mbp) of V. fischeri ES114. (From the inside) Ring 1, rrn operons (red); tRNAs (green). Ring 2, “foreign” elements (black). Ring 3, type IV pilus loci (red). Circle 1, G+C content over a 200-kb ...
Fig. 2.
V. fischeri ES114 ORFs on the + and - strands, color-coded to functionality (see key).

One of the striking characteristics of the V. fischeri genome is the G+C content of the DNA (Table 1), which is the lowest of the 27 species of Vibrionaceae (14). Despite this very low G+C content, V. fischeri is more closely related to the higher G+C pathogenic Vibrio species than to any other sequenced bacterium (www.ergo-light.com). The genome-wide value of 38.3% G+C (Table 1) is similar to the G+C values of each of the three individual replicons, suggesting they have all had a long history in this species. The distribution of 16S ribosomal RNA (rrn) operons in V. fischeri is similar to that reported for V. parahaemolyticus and V. vulnificus. Specifically, there is one rrn on the smaller chromosome (Chr II) of V. fischeri, whereas the 11 remaining operons are on the larger chromosome (Chr I) (Fig. 1). Thus, the absence of an rrn operon on the small chromosome in V. cholerae (5) appears atypical in this genus. In V. fischeri, most of the 11 rrn operons are clustered in three loci (Fig. 1). Such extended series of sequential rrn operons, creating patches of higher G+C content, have not been observed in the other Vibrio species. It is also of interest to note that the chromosomal density of apparent ORFs is almost 10% greater in V. cholerae compared to V. fischeri (Table 1).

Table 1.
Comparison of general features of the V. fischeri ES114 and V. cholerae N16961 genomes

An analysis of the two chromosomes of V. cholerae, the first sequenced Vibrio genome, revealed that ORFs located on Chr II were twice as likely to be unique to this organism than those on Chr I (5). At that time, it was not clear whether this result reflected a dearth of genes reported from other Vibrionaceae or indicated that the smaller replicon was a repository for genes unique to V. cholerae. A similar analysis of the two chromosomes of V. fischeri indicates that, even with the genomes of four members of the genus Vibrio in the database, there is an ≈4-fold greater percentage of unique genes on Chr II of this species (Table 1). Thus, the smaller chromosomes characteristic of this genus may turn out to be a rich source of genes that define the unique potential of individual Vibrio species, and perhaps their specific lifestyles.

Mobile Elements. As in most bacteria, V. fischeri carries evidence of mobile genetic elements on its chromosomes. Although little is known about the importance of these elements in V. fischeri, in other bacteria they play a role in obtaining genes encoding virulence factors or resistance to environmental stresses (15, 16). Evidence of a retron, an integron, and three phage-like loci is found in the genome (Fig. 1). The most intriguing of these foreign elements is a cholera toxin (CTX) phage-like gene cluster on Chr II (Fig. 3). This element is composed of eight ORFs, including four homologs of CTX-phage genes (17): cep, orfU, ace, and zot. The V. fischeri locus differs from the well studied V. cholerae El Tor CTX phage in two important ways: it is missing the downstream RS2 cluster believed to be required for phage excision and multiplication (18) and, in place of the CTX genes (ctxAB), there are two apparently truncated ORFs. Interestingly, the V. fischeri locus shares greater sequence similarity to the V. cholerae CTX phage than the severely diminished homologous locus present in V. parahaemolyticus RIMD 2210633.

Fig. 3.
Comparison of the V. cholerae CTX phage locus (A) with a homologous locus in V. fischeri ES114 (B). ORFs encoding the CTX genes ctxA and ctxB, and the second RS2 element downstream of the locus, are missing in V. fischeri. Identifiable attL and attR sites ...

Pilus Gene Clusters. Extracellular pilus structures are common among bacteria, and have been implicated in diverse colonization functions (19). Several kinds of pili have been described in Vibrio species, some of which are required for pathogenesis (20, 21). The V. fischeri genome contains 10 separate pilus gene clusters, including eight type-IV pilus loci (Table 2). Four of these clusters are found on Chr I of all of the sequenced Vibrio species. They include a mannose-sensitive hemagglutinin (MshA), a homolog of the PilT pilus involved in twitching motility (22), and two Vibrio PilA homologs (21, 23). Two of the remaining four pilus gene clusters are paralogs (one on each V. fischeri chromosome) that are homologous to the Flp1 type IV-B tight adhesion (tad) pilus family. Members of this evolutionarily diverse family are found in many bacteria (24), including V. parahaemolyticus and V. vulnificus, but not the sequenced V. cholerae strain. The last two type-IV pilus loci of V. fischeri have homologs only in V. cholerae: one encodes a highly diverged PilA-like gene (7), and the other is a toxin-coregulated pilus (TCP)-encoding locus (25) (Fig. 4). The two non-type-IV pilus clusters are not found in any other sequenced Vibrio species, and include homologs of the curli-encoding genes of enteric bacteria (26) and a putative conjugative pilin encoded on pES100.

Fig. 4.
The TCP gene cluster in V. fischeri ES114 (green) and V. cholerae N16961 (red). V. fischeri has no identifiable homologs of aldA, tagA, toxT,or tcpJ, and the V. fischeri ORF labeled “hyp.” has no detectable similarity to V. cholerae tcpR ...
Table 2.
Putative pilin loci in V. fischeri

Mutations in only two pilin genes, both on Chr II (Table 2), have been analyzed for their effects on symbiosis. The first is the Flp1 paralog, which is required for achieving normal colonization levels in the light organ.‡‡ The second is the PilA2 pilus, which facilitates efficient light organ colonization by bacteria (7). The presence of multiple pilus gene clusters in the V. fischeri genome suggests that different pili may be expressed to aid this bacterium either in the diverse environments it inhabits or during the multiple stages of its development as a symbiont (27).

The TCP Genes in V. fischeri. In V. cholerae, the TCP is an important virulence factor (28). The genes encoding its synthesis are located in a single large cluster (Fig. 4) that is believed to have entered the genome as a Vibrio pathogenicity island (VPI) (29, 30). Homologs of most of the TCP genes are present in the genome of V. fischeri. A majority of the genes in the tcp cluster exhibit synteny between the two species (Fig. 2); however, eight of the homologs are located elsewhere in the V. fischeri genome. Although the reasons for the different gene arrangements in V. cholerae and V. fischeri are unknown, the G+C content and apparent absence of flanking insertion elements in the V. fischeri cluster suggest that it was not horizontally acquired in the recent past (Fig. 5). In contrast, the presence of insertion elements, as well as the G+C content of the V. cholerae VPI, support the idea that this region is foreign, and originated in an unusually low G+C genome like that of V. fischeri.

Fig. 5.
G+C content of ORFs in and around the TCP gene clusters of V. fischeri ES114 (green) and V. cholerae N16961 (red) plotted against their relative linear position on the chromosome. The average G+C content of the two species' genomes are indicated by the ...

Homologs of Toxin-Encoding Genes. Although V. fischeri is not known to be pathogenic, and strain ES114 is a beneficial symbiont, its genome carries homologs of Vibrio genes that may have toxin activity (Table 3, which is published as supporting information on the PNAS web site). As mentioned earlier, these include two CTX phage-encoded genes, zot (zona occludins toxin) and ace (accessory cholera enterotoxin), the latter of which has been found only in the V. cholerae and V. fischeri genome sequences. The proteins coded for by these genes have been shown to contribute to the structure of CTX phage (17), and their possible roles as toxins remain controversial (31, 32). At this time, it is not known whether the V. fischeri homologs of these two genes are expressed in this species, or whether they might play a role in this bacterium's symbiotic associations.

All sequenced Vibrio species carry genes encoding another putative toxin called RTX (repeats in structural toxins). RTX activity in V. cholerae appears to affect regulators of host-cell actin polymerization, causing dramatic changes in cytoskeletal structure, leading to the loss of tight-junction integrity (31, 33). The expression or potential activity of a V. fischeri RTX protein has not yet been investigated, but it is intriguing that symbiotic infection of the squid host results in a set of controlled changes in actin deployment in epithelial cells surrounding the bacteria in the light organ (34, 35). Because these changes are a part of the host's normal developmental program, the RTX protein may provide an important signal in symbiosis. Similarly, it has been discovered recently that V. fischeri secretes a degradation product of peptidoglycan that is responsible for inducing normal tissue development in the nascent light organ (36). This extracellular product is identical in structure to tracheal cytotoxin (TCT), produced by Bordetella pertussis and Neisseria meningitides, indicating that bacterial virulence factors not only are context specific, but also may be required symbiotic signals. The protein believed to be responsible for TCT production may be encoded by a distinct transglycosylase gene that is present in these two pathogens and V. fischeri, but is not found in the other sequenced Vibrio species (A. Schaefer, unpublished data). The carriage by V. fischeri of genes encoding RTX and TCT suggests that the activities these effectors encode may result in either a beneficial or a pathogenic outcome, depending on the host species or tissue location colonized.

Conclusions. Countless bacterial species interact with animals and plants in persistent associations that are often essential to their host's existence. Such symbiotic associations may share similarly derived colonization factors with pathogens. If we are to understand the unifying themes underlying these contrasting bacteria–host interactions, we must begin to use comparative genomic approaches with closely related pathogenic and beneficial microbial species. Such studies within the genus Vibrio may help reveal not only the evolutionary origins of host-targeted virulence factors, but also those mechanisms by which pathogens commonly associate with marine invertebrate reservoirs as benign or even beneficial symbionts.

Supplementary Material

Supporting Table:


We thank the following members of Integrated Genomics, Inc., for their work on the project: A. Lapidus and L. Chu for sequencing; E. Goltsman, N. Larson, and G. Pusch for assembly, and I. Anderson, V. Kapatral, A. Bhattacharyya, G. Reznik, M. D'Souza, T. Walunas, Y. Grechkin, and N. Kyrpides for initial annotation. This sequencing project was supported by a grant from the W. M. Keck Foundation.


Abbreviations: Chr, chromosome; CTX, cholera toxin; TCP, toxin-coregulated pilus.

Data deposition: The sequences reported in this paper have been deposited in the GenBank database [accession nos. CP000020 (large chromosome), CP000021 (small chromosome), and CP000022 (plasmid)].


‡‡Feliciano, B. & Ruby, E. G. (1999) Abstr. Gen. Meeting Am. Soc. Microbiol. 99, 462 (abstr.).


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