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Previous Article | Next Article 
Applied and Environmental Microbiology, July 2008, p. 4059-4069, Vol. 74, No. 13
0099-2240/08/$08.00+0 doi:10.1128/AEM.00190-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Department of Microbiology, University of Georgia, Athens, Georgia,1 Department of Microbiology and Immunology, Loyola University Chicago, Maywood, Illinois2
Received 21 January 2008/ Accepted 7 May 2008
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Traditionally, carbon source utilization patterns have been among the criteria used to distinguish different species within the family Vibrionaceae (6, 18), and this information has also guided the development of semiselective media to enrich for specific species (14). For example, cellobiose utilization is among the variable traits used to describe and distinguish species within the family Vibrionaceae (6), and cellobiose-based media have been used to enrich for certain species, particularly Vibrio vulnificus (11, 33). Molecular markers and DNA sequence analyses are becoming more widespread taxonomic tools with great value, but carbon source utilization phenotypes are still useful discriminators and have been tested through decades of research (6, 18). In the future, once the genetic basis for the utilization of particular carbon sources is better understood, it should be possible to generate molecular DNA-based techniques that draw directly on the wealth of phenotypic information available for identifying Vibrio species.
Understanding the genetic basis for various metabolism patterns among different Vibrio species will also help elucidate the evolutionary history of the Vibrionaceae. Despite interest in both the metabolic variability of this family and the mechanisms underlying its evolution, much remains to be learned about these subjects. For example, it is not known whether cellobiose utilization was an ancestral trait lost by some members of this family or if it was a trait acquired by certain lineages more recently. Bioinformatic analyses of genome sequences in the family Vibrionaceae (12, 21, 32, 46, 52, 64) promise to help answer such questions, but gene and pathway annotations can be ambiguous or incorrect. Therefore, continued experimental determination of metabolic pathways will be necessary to connect genomic and phenotypic variability.
In this study, we describe a gene cluster that is both necessary for cellobiose utilization by V. fischeri and sufficient to confer cellobiose utilization on other Vibrio species. Based on our results and bioinformatic analyses, we propose a model for cellobiose utilization arising from the acquisition of distinct pathways by different lineages within the family Vibrionaceae. We also show that this cellobiose utilization cluster in V. fischeri is responsible for an unexpected cryptic β-galactosidase activity. This observation has immediate practical significance, because the β-galactosidase gene lacZ from Escherichia coli has been used as both a marker (17, 24) and a transcriptional reporter (30, 69, 72, 73) in V. fischeri.
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MATERIALS AND METHODS |
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(20), except conjugative helper plasmid pEVS104 (57), which was maintained in CC118
pir (22); a pCR Blunt-TOPO-derivative, which was maintained in TOP-10 (Invitrogen, Carlsbad, CA); and other plasmids containing the R6K
replication origin, which were maintained in DH5pir (16). E. coli was incubated at 37°C in LB medium (34) or brain heart infusion (BHI) medium (Difco, Sparks, MD). For selection of E. coli, chloramphenicol, kanamycin, and trimethoprim were used at concentrations of 20, 40, and 10 µg ml–1, respectively. For the selection of E. coli with erythromycin (Em), 150 µg ml–1 was added to BHI medium. V. fischeri and all other Vibrionaceae strains were grown at 28°C in LBS medium (55) or by using a specific carbon source, as indicated, added to a minimal salts medium (0.340 mM NaPO4 [pH 7.5], 0.05 M Tris [pH 7.5], 0.3 M NaCl, 0.05 M MgSO4-7H2O, 0.01 M CaCl2-2H2O, 0.01 M NH4Cl, 0.01 M KCl, 0.01 mM FeSO4-7H2O, plus a carbon source). When chloramphenicol, kanamycin, Em, and trimethoprim were added to LBS or minimal medium for selection of V. fischeri or other Vibrionaceae strains, they were used at concentrations of 2, 100, 5, and 10 µg ml–1, respectively. D-Cellobiose (Acros Organics, Geel, Belgium) was added to solid and liquid media at concentrations of 5 mM and 10 mM, respectively. Glucose was added to media at a concentration of 20 mM. Bromocresol purple sodium salt (BCP; Eastman Kodak, Rochester, NY) and 5- bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal; Research Products International, Prospect, IL) were added to LBS medium at concentrations of 0.02 and 100 µg ml–1, respectively. Agar was added to a final concentration of 1.5% for solid media.
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Construction of mutants and complementation plasmids.
Descriptions of select plasmids and the primers used in their construction are provided in Table 1. Details of plasmid construction are as follows. To generate a 2-bp insertion in celG, we first screened an existing library of XbaI-digested ES114 DNA cloned into pBluescript (Stratagene, La Jolla, CA) to isolate pKV150, which contains the cel gene cluster. pKV150 was digested with BglII, and the cel gene cluster was subcloned into BamHI-digested pEVS79, yielding pKV153. pKV153 was digested with SpeI and self-ligated to roughly center celG within the insert, making pKV156. pKV156 was then digested with ClaI, the overhangs were filled in with Klenow fragment, and the plasmid was recircularized by self-ligation, yielding pKV162, which has a frame-shifting 2-bp insertion in celG. This mutation was placed on the chromosomes of ES114 and DMA420 by allelic exchange, yielding strains KV1319 and DMA401, respectively.
The celI complementation plasmid pDMA171 was generated by first amplifying celI with 
500 bp of upstream sequence, thereby incorporating NheI sites that were engineered near the 5' end of each primer (dma91 and dma92). This PCR product was cleaned and digested with NheI before being cloned directly into AvrII-digested pVSV107. Plasmid pKV151 contains the active cel cluster and was isolated from a library of BglII-digested ES114 DNA cloned into pV08 (2).
To construct pDMA193, the Pcel-gfp reporter, the intergenic DNA upstream of celC was PCR amplified with NheI sites incorporated near the 5' ends of the primers (dma93 and dma94) and cloned into pCR-BluntII-TOPO (Invitrogen, Carlsbad, CA), yielding pDMA181. pDMA181 was digested with SpeI and XhoI, and the promoter fragment was subcloned into pEVS79, digested with the same enzymes, making pDMA184. pDMA184 was digested with NheI, and the promoter was subcloned into AvrII-digested pVSV209, which contains a promoterless gfp gene, completing the construction of pDMA193.
Transposon mutagenesis was performed by conjugating the mini-Tn5 delivery plasmid pEVS170 (N. Lyell and E. Stabb, unpublished results) into wild-type V. fischeri strain ES114. After conjugation proceeded for more than 8 h, the conjugation mixture was diluted and plated onto selective media. The mini-Tn5 mutagenesis was performed in three independent experiments for each screen, encompassing 10,000 colonies per screen. In one screen, mutant colonies were isolated based on their blue color on LBS-X-gal supplemented with Em. In the other screen, mutant colonies were examined for yellowish-white color on LBS medium supplemented with cellobiose, Em, and X-gal. The site of transposon insertion in each mutant strain was determined by cloning the transposon and flanking DNA and then sequencing across the transposon::chromosome junction, using the M13 Forward primer. Insertions were cloned by digesting chromosomal DNA with HhaI, self-ligating the fragments, and recovering the transposon and flanking DNA as a plasmid, taking advantage of the origin of replication and Em resistance gene contained within the transposon.
Carbon utilization assays.
The ability to grow on glucose or on cellobiose as the sole carbon source was tested by adding these sugars to a minimal medium and then streaking single colonies of each strain onto plates, which were incubated at 28°C for 48 h and assessed for growth. To test for acid production by strains in the presence of glucose or cellobiose, single colonies were used to inoculate test tubes containing LBS medium with BCP and either cellobiose, glucose, or no sugar added. Cultures were incubated at 28°C with shaking (200 rpm) for 24 h, and acidification was scored as a change in the BCP from purple to yellow.
cel induction measurements in culture.
Overnight cultures of V. fischeri carrying pDMA193(Pcel-gfp) were grown in LBS with appropriate antibiotics and diluted 1:500 into 30 ml of antibiotic-free LBS medium, with or without cellobiose or glucose, in 125-ml baffled flasks and were then incubated at 24°C with shaking (200 rpm). The reporter and control plasmids used are derived from a vector that is stable in V. fischeri and does not require selection for maintenance (17). Samples (500-µl) were removed at intervals and the culture optical density at 595 nm (OD595) was determined by using a BioPhotometer unit (Brinkman Instruments, Westbury, NY). Fluorescence was measured using a TD-700 fluorometer (Turner Designs, Sunnyvale, CA), using excitation and emission filters of 486 nm and >510 nm, respectively. The fluorescence reported is the average of measurements taken when the OD595 readings were approximately 2.5. The fluorescence of strains carrying the promoterless gfp construct in pVSV209 was subtracted as background.
To examine the ability of various carbon sources to induce the cel operon, 15 µl of 100 mM stocks of cellobiose, raffinose, sucrose, maltose, lactose, N-acetyl-glucosamine, fructose, mannose, ribose, galactose, xylose, arabinose, and glucose were spotted onto filter disks placed on LBS-X-gal plates and spread-plated with ES114. After 24 h of incubation, plates were examined for the induction of the cel operon, which was scored as rings of blue in the lawn surrounding the sugar-impregnated disk. Parallel plates with the celG mutant KV1319 served as negative controls and did not develop blue color.
Enzyme assays using pNP-conjugated substrates.
The strains tested were grown to an OD595 of 2.0, pelleted, and lysed by freezing at –80°C for 20 min, and the pellets were resuspended in the original volume of a 500 mM sodium phosphate buffer (pH 7.0). One hundred microliters of this lysate was added to 400 µl of a 10 mM p-nitrophenol (pNP)-conjugated substrate dissolved in 50 mM sodium phosphate buffer (pH 7.0). Parallel reaction mixtures were incubated at 28°C and 37°C until a yellow color was observed or for a maximum of 24 h. The assay was stopped by adding 2 ml of 1 M Na2CO3 (final concentration of 800 mM). A 1-ml sample from the reaction mixture was centrifuged for 5 min to pellet cell debris. The absorbance was read at 410 nm (A410) to determine the amount of pNP generated from enzymatic cleavage, and at A550 to determine light scattering from residual cell debris. To calculate pmol of pNP generated min–1 ml–1, the A410 reading from each sample minus the A550 reading for each sample was compared to a linear standard of pNP, and this was divided by the incubation time, and the 0.1 ml of lysate was added to the reaction mixture.
Squid colonization assays.
E. scolopes host animals were maintained in Instant Ocean (Aquarium Systems, Mantor, OH) mixed to 36 ppt. To determine whether a mutant strain had a competitive disadvantage in the symbiosis relative to the wild type, cultures for inoculation were grown as previously described (17), and juvenile squid were exposed to a 1:1 mixture of the wild-type and mutant strains for 14 h and then moved to V. fischeri-free Instant Ocean. Squid were homogenized after 48 h to determine the ratio of wild-type to mutant strains. The relative competitive index (RCI) was determined by dividing the mutant-to-wild type ratio for each individual squid by the ratio for the inoculum. Log-transformed data were used to calculate the average RCI and to determine statistical significance.
Bioinformatic analyses.
Protein sequence comparisons to GenBank entries were generated using BLASTp (3). V. salmonicida LFI1238 sequence was obtained from the Sanger Institute (http://www.sanger.ac.uk/Projects/V_salmonicida/) as a shotgun database, and homologs of specific V. fischeri genes were determined by using Artemis software (48). Genomes with similar regions surrounding the CelC open reading frame (ORF) were found by using the SEED pinned region search (40). The similarities reported between homologs were determined by MatGAT software using the default settings (10). Phylogenetic and molecular evolutionary analyses were conducted with MEGA software version 4.0, using the default settings (60). Using the MEGA program, consensus neighbor-joining phylogenetic trees were constructed by using the amino-Poisson correction. The unweighted-pair group method with arithmetic mean (UPGMA) and minimum evolution trees were also constructed with similar results (data not shown). Bootstrap values for the trees were obtained from a consensus tree based on 1,000 randomly generated trees, using MEGA 4.0 software (60).
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RESULTS |
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celG encodes a β-glucosidase with lower β-galactosidase activity.
To investigate whether the putative 6-phospho-β-glucosidase encoded by celG was responsible for the cleavage of cellobiose and X-gal, we generated mutant KV1319, which contains a 2-bp insertion in celG. KV1319 was unable to cleave X-gal and unable to utilize cellobiose as a sole carbon source and did not acidify cellobiose-containing media in BCP assays. This provides further evidence that CelG is responsible for the cleavage of both cellobiose and X-gal, a supposition that is also supported by the enzymatic assays described below. To test the prediction that the celI mutant colony's blue phenotype on LBS-X-gal was due to the loss of CelI-mediated repression of celG, we incorporated the 2-bp frameshifting mutation in celG into DMA420, a celI transposon mutant. As predicted, the resulting strain (DMA401 celG; mutant, celI::mini-Tn5-Em) frameshift was yellowish-white in contrast to the blue color of the celI mutant on LBS-X-gal plates (data not shown).
To further test the substrates targeted by CelG, we assayed enzymatic activity in cell lysates by using sugar substrates para-linked to a nitrophenol group. We examined lysates of the celI mutant DMA420, which should enhance CelG activity by allowing the derepression of celG. To determine whether the utilization of a particular substrate was specific to CelG and not to some other enzyme in the whole-cell lysate, we compared the activity of celI mutant lysates (Fig. 2, dark gray bars) to that of lysates of the celI celG double mutant, DMA401 (Fig. 2, light gray bars). Thus, Fig. 2 shows both CelG-dependent activity, which is the difference between the activities in strains DMA420 and DMA401, and CelG-independent activity, which is the activity in strain DMA401.
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Although each of the other substrates cleaved by CelG contains a β-1,4 linkage, the promiscuity of the enzyme was evident in its activity as both a β-glucosidase and a β-galactosidase. The latter may reflect a coincidental and physiologically irrelevant activity. Although CelG is apparently able to cleave pNP-β-D-lactopyranoside (Fig. 2, lac), V. fischeri is unable to utilize lactose as a carbon source. Moreover, the data below indicate that CelG is induced by the presence of cellobiose but not by lactose (Fig. 3). Given the ability of CelG to direct X-gal cleavage, the β-galactosidase activity attributed to CelG in this assay was expected; however, it is worth noting that this activity was 50-fold lower than the β-glucosidase activity (Fig. 2).
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-D-xylopyranoside (Fig. 2, xylo). We saw no enzymatic activity toward pNP-β-D-maltoside (Fig. 2, malt).
celI-cellobiose- and glucose-mediated control of cel expression.
To determine when the cel genes are induced, we developed a transcriptional reporter plasmid, pDMA193, containing the region immediately upstream of the cel gene cluster, driving the expression of gfp. Without cellobiose, the reporter's fluorescence in ES114 was slightly elevated above that of the background, but with growth in cellobiose, there was an increase of almost 5-fold in fluorescence (Fig. 3). In the celI::mini-Tn5-Em mutant DMA420, the reporter was expressed with and without added cellobiose, further supporting our prediction that CelI represses the cel gene cluster when cellobiose is not present.
Interestingly, in the presence of cellobiose, fluorescence of the reporter in the celI mutant DMA420 decreases compared to when it is growing without cellobiose. We hypothesized that the generation of glucose due to the cleavage of cellobiose might cause catabolite repression of the gene cluster, resulting in this inhibitory effect of cellobiose on cel expression. To examine this, both the wild type ES114 and the celI mutant DMA420 containing the reporter plasmid were grown in the presence of glucose. In both strains, fluorescence from the reporter plasmid decreased substantially when cells were grown in LBS medium supplemented with glucose (Fig. 3). Furthermore, we moved the reporter plasmid into DMA401, the celI celG double mutant, to assess whether the loss of CelG, which should reduce the breakdown of cellobiose to glucose, would allow induction of the gene cluster in the presence of cellobiose. In media with glucose, reporter expression in DMA401 was reduced, as it was in the other strains (Fig. 3). However, supplementation with cellobiose did not affect reporter expression in DMA401 (Fig. 3). Thus, it appears that cel expression is repressed by glucose that is either added exogenously or generated by CelG-dependent cleavage of cellobiose.
Taking advantage of the promiscuity of CelG and its ability to degrade X-gal, we next determined whether carbon sources other than cellobiose could induce the expression of the cel gene cluster, using a disc assay with ES114 or the celG mutant KV1319 plated on LBS-X-gal plates. We tested glucose, galactose, lactose, chitin-hexamers, cellulose, N-acetyl-glucosamine, and cellobiose and found that only cellobiose was able to induce the expression of the cel gene cluster, resulting in CelG-dependent cleavage of X-gal and development of blue color in the growth of ES114 cells around the disc (data not shown). Thus, even if CelG is able to cleave substrates other than cellobiose (as described above), the substrates above do not induce celG and are therefore unlikely to be physiologically relevant targets for the cel gene cluster.
The celCBGKAI gene cluster on pKV151 confers cellobiose utilization on six cellobiose-negative Vibrio strains.
To determine whether this cellobiose-utilizing gene cluster in V. fischeri was sufficient to confer cellobiose utilization to other Vibrio strains, pKV151 (Fig. 1) was moved into six different Vibrio or Photobacterium strains that are negative for cellobiose utilization (Table 2). Transconjugants were tested for growth on solid medium with cellobiose as the sole carbon source and were also tested for growth in the presence of glucose (the breakdown product of cellobiose) as a control. Both ES114 and the celG frameshift mutant were also included as positive and negative controls, respectively. All of the strains grew regardless of the plasmid when glucose was the sole carbon source (data not shown); however, of the strains carrying the insertless vector pVO8, only ES114 grew on cellobiose (Table 2). Thus, for each of the other Vibrio or Photobacterium strains, the V. fischeri cel gene cluster on pKV151 conferred the ability to grow on cellobiose (Table 2).
Substrate utilization is often tested indirectly based on the production of fermentation acids in the presence of a particular sugar, resulting in a pH shift that can be detected by the colorimetric change in the dye BCP. We therefore also tested strains carrying pKV151 in BCP assays. All strains with either the control vector or pKV151 acidified glucose-containing LBS medium (Table 2). For Vibrio cholerae VC4103, V. mimicus, and V. parahaemolyticus, pKV151 conferred not only the ability to grow on cellobiose but also the production of acid in LBS supplemented with cellobiose. For the other strains, the ability to grow on cellobiose did not correlate with acid production in the presence of cellobiose. Photobacterium leiognathi and V. hollisae were unable to acidify the cellobiose-containing medium regardless of whether they carried the control vector or pKV151, whereas V. cholerae VC4056 acidified the medium regardless of whether it contained pKV151 or the control vector. Thus, although acid production is often used as an indirect indicator of sugar catabolism by Vibrio species (6, 18), direct testing for growth on cellobiose was a more reliable measure of this metabolic capability.
Bioinformatic analyses of the cel gene cluster.
Using a combination of bioinformatic programs and databases (see Materials and Methods), we sought to determine whether celCBGKAI was an ancestral locus present in all cellobiose-utilizing members of the family Vibrionaceae and, if this was not the case, to determine the likely origin(s) of these genes. Comparisons of nucleotide or encoded protein sequences yielded similar results, and for the most part, only the latter are reported here, with nucleotide sequences used for reporting gene arrangement. We found that V. salmonicida strain LFI1238, which utilizes cellobiose (N.-P. Willassen, personal communication), has a homologous celCBGKAI cluster. The cel gene order is conserved in V. salmonicida, the encoded proteins were >90% similar to the respective homologs in V. fischeri, and the V. salmonicida cel gene cluster included both the genes for cellobiose utilization (Fig. 4A) and the regulator celI (not shown).
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Despite further analyses, the ancestry of the celCBGKAI genes in V. fischeri and V. salmonicida remains uncertain, and these genes may have multiple origins. It seems likely that celI, which encodes the LacI family regulator, originated within the family Vibrionaceae, as it shares high similarity with many regulators in this bacterial family (data not shown). Interestingly, however, CelG and CelK clustered most closely with ORFs found in the phylum Firmicutes, particularly in the Clostridium, Bacillus, and Listeria species. This is illustrated both in the comparisons of similar gene clusters (Fig. 4) and in the neighbor-joining trees that compare the specific proteins encoded by these gene clusters (Fig. 5). Gene clusters in Clostridium acetobutylicum and Listeria monocytogenes were more similar to the V. fischeri cel cluster than were the gene clusters from other Vibrio species (Fig. 4A). The closest homolog of CelG was found in C. acetobutylicum (Fig. 4A and 5D), while close homologs to CelK were found in L. monocytogenes and Yersinia intermedia (Fig. 4A and 5C). Although Y. intermedia is a gammaproteobacterium, it appeared to be exceptional in this group in that the proteins encoded by the Y. intermedia cluster group more closely with homologs in Listeria than with proteins in other proteobacteria. The gene cluster in L. monocytogenes is also similar to the V. fischeri cel cluster, and the gene order is conserved with that in V. fischeri, except that the PTS IIC and glucosidase genes are switched (Fig. 4A). Moreover, codon usage by Listeria is so similar to that in V. fischeri that genes transferred between the two cannot be recognized as foreign by using this criterion (data not shown). The origin of the celA, celB, and celC genes is especially difficult to infer, as the PTS IIC component tends to group with homologs in the phylum Firmicutes (Fig. 5B), whereas the PTS IIB and IIA components tend to group more closely with homologs within the family Vibrionaceae (Fig. 5A and data not shown). The PTS IIB and IIA components must interface with other endogenous proteins, and the respective genes might be expected to face greater selective pressure to adapt to a new host and therefore appear less foreign. Overall, it seems plausible that at least some of the genes for cellobiose uptake and catabolism may have been transferred horizontally from a marine Firmicutes organism to an ancestor of the V. fischeri/V. salmonicida clade, although other scenarios remain possible.
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Mutants with insertions in each of the cel genes were checked for their ability to compete with the wild type for colonization in E. scolopes. Mutants with an insertion in celI or in the noncoding region upstream of celI were consistently outcompeted by 2.2-fold as indicated by RCI values of 0.45 (Table 3). In contrast, strains with mutations in genes responsible for the transport and degradation of cellobiose, celA, celB, celC, celG, and celK, had no significant competitive defect relative to that of the wild type (Table 3). We competed DMA401 (celG mutant, celI::mini-Tn5-Em) to see if the competitive defect of a mutation in celI was dependent on the overexpression of a functional cel gene cluster and cellobioase activity. This competition yielded an RCI that was essentially the same as the single celI mutant alone, indicating that the negative effect of knocking out celI on symbiotic colonization is independent of cellobioase activity. Instead, this attenuation of competitiveness may simply be from the overexpression of the Cel proteins. Not surprisingly, the ptsI mutant DMA424 was outcompeted by the wild type; however, this mutant's slower growth in culture indicates that its defect in colonizing the host cannot be considered symbiosis specific.
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Cellobiose is a disaccharide-breakdown product of cellulose, but V. fischeri apparently lacks cellulose-degrading capacity, and the importance of its cel gene cluster is unclear. There is no evidence that cellobiose is released by E. scolopes to symbiotic V. fischeri, and it seems unlikely that this predatory invertebrate would produce or accumulate cellulose or cellobiose. Moreover, losing the ability to utilize cellobiose did not result in symbiotic attenuation (Table 3). V. fischeri has been isolated from the water column, even in areas where symbiotic hosts are not found (27, 45), and it has also been isolated from the bacterial consortium in the guts of herbivorous marine fishes, where it can survive and persist (42, 59). These observations suggest a niche where cellobioase activity may be important for V. fischeri. Many herbivorous fish partially digest cellulose by acid hydrolysis in their stomachs (74), and it is believed that bacterial cellulases contribute to the digestion of the cellulose in the gut, as with termites and ruminants (47, 70). Although V. fischeri does not contain a cellulase, it is possible that either the acidity of the fish's stomach or other bacteria in a fish's intestine could break down cellulose into cellobiose, allowing V. fischeri to consume it.
Interestingly, other cellobiose-utilizing Vibrio species, notably V. vulnificus, lack the celCBGKAI gene cluster, and we speculate that cellobiose catabolism may have arisen in members of the family Vibrionaceae by multiple distinct events. Based on our bioinformatic data, we speculate that a Firmicutes species horizontally transferred the cellobiose degradation glucokinase and glucosidase genes, and possibly the PTS genes, to an ancestor of the V. fischeri and V. salmonicida lineages. Firmicutes species have been found in the guts of marine fishes (23, 36) and in the marine environment (9, 44), so it seems plausible for the ancestor of V. fischeri to have acquired the gene cluster from a member of the phylum Firmicutes by horizontal gene transfer. Moreover, codon usage patterns of V. fischeri and Listeria species isolated from marine environments are not distinguishably different, suggesting that the expression of genes transferred between these species may be readily possible. As genome sequences become available for additional marine species, particularly those of Firmicutes, the origin of the V. fischeri gene cluster may become more apparent.
Our laboratory and those of others use the β-galactosidase gene lacZ as a transcriptional reporter (30, 69, 72, 73) in V. fischeri, and the discovery of a cryptic β-galactosidase activity in V. fischeri strikes a cautionary note for such applications. For example, when lacZ is used as a transcriptional reporter and transposon mutants are screened to find regulators of these lacZ fusions, knockouts of celI will also result in blue colonies on media containing X-gal. It may be useful in such situations to use the celG mutant allele in KV1319 in the reporter strain to prevent celG expression from confounding screens for lacZ activity. Alternatively, celI could be introduced on the pDMA171 plasmid into strains with apparent increases in lacZ activity to eliminate the possibility that a celI mutation and concomitant celG expression are responsible for β-galactosidase activity. Whatever the experimental setup, appropriate controls and careful interpretations are warranted whenever lacZ is used in a celG+ background.
lacZ has also been used as a marker in V. fischeri, so that the ratio of two strains in a mixed inoculum or infection can be determined by blue/white screen plating on media with X-gal. Determining strain ratios underlies competition experiments, which enable researchers to detect even subtle differences in symbiotic fitness (8, 24, 26, 29, 31, 35, 39, 56, 65, 66, 71, 72). Recently, V. fischeri strains have been marked for competition assays with the introduction of a stable plasmid containing lacZ (1, 17, 24). However, the lacZ-carrying plasmids can be lost, albeit at a low rate, and their use is inconsistent with other plasmids (e.g., for complementation). Our data suggest a fresh approach that does not rely on a plasmid-borne lacZ gene but retains the convenience of blue/white scoring to determine strain ratios. In this approach, one strain could be marked with the celG mutant allele present in KV1319. We have shown that this mutation has no effect on colonization competitiveness (Table 3), yet it results in the loss of blue color when it is plated on medium containing cellobiose and X-gal.
The family Vibrionaceae is an important and diverse family of bacteria in which species are continually being discovered (5, 25, 28, 41, 43, 50, 62) and with an apparent capacity for rapid evolution, given the periodic emergence of new pathogenic biotypes (13, 19, 49, 51). Traditionally, phenotypic markers such as strains' catabolic capacities have been used to help define Vibrio species. However, as more genomes are sequenced for important Vibrio species, molecular probes and DNA-based techniques will likely play an ever-larger role in identifying and defining important species or emergent biotypes. Our bioinformatic and phenotypic analyses suggest, not surprisingly, that caution is warranted when automated genome annotations are viewed. For example, an automated annotation of celA indicated that it directed "diacetylchitobiose-specific" transport, which seems, clearly, not to be the case given its importance in cellobiose catabolism. Similarly, PTS gene clusters were annotated as cellobiose transport systems in V. parahaemolyticus and V. cholerae, two species that are cellobiose negative. Experimental studies linking genes with taxonomically useful phenotypes, such as our dissection of the cel gene cluster reported here, will be useful in the future to improve Vibrio genome annotations and to connect molecular and phenotypic identification techniques.
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ACKNOWLEDGMENTS |
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This work was supported by NIH grant GM59690 to K.L.V., National Science Foundation CAREER grant MCB-0347317 to E.V.S., and NIH grant A150661 to M. McFall-Ngai.
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FOOTNOTES |
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Published ahead of print on 16 May 2008.
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