Natural transformation of Vibrio fischeri requires tfoX and tfoY

SUMMARY

Recent evidence has indicated that natural genetic transformation occurs in Vibrio cholerae, and that it requires both induction by chitin oligosaccharides, like chitohexaose, and expression of a putative regulatory gene designated tfoX. Using sequence and phylogenetic analyses we have found two tfoX paralogues in all sequenced genomes of the genus Vibrio. Like V. cholerae, when grown in chitohexaose, cells of V. fischeri are able to take up and incorporate exogenous DNA. Chitohexaose-independent transformation by V. fischeri was observed when tfoX was present in multi-copy. The second tfoX paralogue, designated tfoY, is also required for efficient transformation in V. fischeri, but is not functionally identical to tfoX. Natural transformation of V. fischeri facilitates rapid transfer of mutations across strains, and provides a highly useful tool for experimental genetic manipulation in this species. The presence of chitin-induced competence in several vibrios highlights the potential for a conserved mechanism of genetic exchange across this family of environmentally important marine bacteria.

INTRODUCTION

Vibrio fischeri can be isolated from several niches in the marine environment including chitin surfaces, and as a part of a specific, beneficial relationship with the Hawaiian bobtail squid, Euprymna scolopes (Ruby and Lee, 1998). In their role as bioluminescent symbionts, a dense population of up to 10⁹ V. fischeri cells is housed within a specialized light-emitting organ of the squid host (Nyholm and McFall-Ngai, 2004). The association is long-term and dynamic: the squid will maintain the polyclonal symbiont population (Wollenberg and Ruby, 2009) throughout its life, which can last up to a year. An important part of the persistent nature of this relationship is that V. fischeri cells experience a daily cycle of expulsion of the light-organ contents and re-growth of the remaining population (Nyholm and McFall-Ngai, 1998). This periodic behavior, while providing the host with a fresh inoculum of symbionts each day, exerts a continuous selective pressure on the V. fischeri population. These characteristics of the light-organ environment reveal a potential for both genetic exchange and allelic fixation.

Natural competence is a form of bacterial genetic exchange that can be dependent on the presence of inducing conditions in the environment (Chen and Dubnau, 2004). Recently, it has been reported that both Vibrio cholerae and Vibrio vulnificus can be transformed when grown on chitinaceous surfaces such as shrimp shells or crab-shell tiles (Meibom et al., 2005; Udden et al., 2008; Gulig et al., 2009). Thus, chitin-induced competence may be a shared trait of marine vibrios that is related to their capability to utilize this common nutrient (Hunt et al., 2008). The discovery of chitin-induced competence in these two vibrio species, and evidence that chitin is present in the squid-vibrio symbiosis (Wier et al., 2010), led us to ask whether V. fischeri shares this capability.

Disruption of the V. cholerae tfoX gene (tfoX^VC; VC_1153), encoding a putative regulator of competence, abolished detectable transformation (Meibom et al., 2005). BLAST searches of the V. fischeri ES114 genome revealed two putative tfoX^VC homologues, which we have named tfoX^VF (VF_0896) and tfoY^VF (VF_1573) (Fig. 1). In this study we sought to determine whether, like V. cholerae and V. vulnificus, V. fischeri cells grown in the presence of chitin derivatives are genetically competent for transformation. We further investigated whether the V. fischeri homologue of tfoX^VC is required for transformation in culture. Observing the sequence similarity to tfoX^VF, we also asked whether tfoY^VF was required for transformation, and whether the two V. fischeri tfoX homologues were functionally redundant. Finally, given the value of natural transformation as a tool for genetic manipulation, we investigated methods for increasing the frequency and convenience of transformation in V. fischeri.

Figure 1.

Two paralogous tfoX-like sequences exist in all fully-sequenced Vibrionaceae. Phylogenetic reconstruction of tfoX homologues found in 13 Vibrionaceae and two Gram-negative outgroups, rooted with Yersinia pestis KIM. The two shaded clades contain two groups of closely related tfoX-like sequence homologues found in all Vibrionaceae. One clade includes only previously identified tfoX-like sequence homologues (tfoX, light grey) while the other contains another, previously unidentified group of tfoX-like sequence homologues (tfoY, dark grey). Identical nodes obtained from three reconstruction methods (Bayesian, Maximum Likelihood [ML] and Maximum Parsimony [MP]) with support >50% are identified by three numbers: (top left) posterior probabilities from Bayesian analysis, multiplied by 100; (top right) bootstrap percentage from 500 likelihood pseudoreplicates; (bottom) bootstrap percentage from 1000 parsimony pseudoreplicates. The bar indicates 0.1 expected change per site; the asterisk (*) indicates that all methods give identical support values of 100.

RESULTS

Two paralogous, tfoX-like loci are present in the genomes of all sequenced Vibrionaceae

A BLASTp alignment using the translated protein sequence of tfoX^VC identified two likely matches from the V. fischeri ES114 genome. The TfoX^VC sequence was a close match to a translation of VF_0896 (tfoX^VF), and a more distant match to a translation of VF_1573 (tfoY^VF). Alignment of predicted TfoX^VF and TfoY^VF sequences show 32% identity and 55% similarity. Surprisingly, BLASTn searching using tfoX^VF found two sequence homologues in each Vibrionaceae genome (Table S1). In each genome, one sequence was more similar to tfoX^VF, whereas the other shared more similarity with tfoY^VF. We hypothesized that the two similar, but distinct genes were evidence of a duplication event in the ancestral Vibrionaceae lineage, leading to paralogous sequences in all extant vibrio species. Phylogenetic analyses of the multiple alignment of all tfoX and tfoY homologues from the Vibrionaceae confirmed this hypothesis; two sister groups, corresponding to extant tfoX- and tfoY-like sequences, had strong statistical support in all reconstructions (Fig. 1). Alignment of the tfoX and tfoY gene neighborhoods of V. fischeri, V. cholerae, and V. vulnificus—organisms that exhibit chitin-induced competence—revealed two local sets of ORFs that appear conserved (Fig. 2).

Figure 2.

Loci surrounding the tfoX paralogues of V. fischeri, V. cholerae, and V. vulnificus. Schematic representations of orthologous genomic regions of three Vibrio species. Solid black arrows indicate tfoX or tfoY. Grey arrows indicate ORFs found in the tfoX or tfoY loci of all completed Vibrio genomes. White arrows indicate ORF’s found in all completed Vibrio genomes except V. fischeri strains ES114 and MJ11. Unlined grey arrows indicate ORFs flanking the conserved tfoX or tfoY loci.

Transformation occurs in the presence of chitin oligosaccharides

To determine the extent to which V. fischeri is capable of uptake and incorporation of exogenous DNA, we asked whether a chromosome-encoded chloramphenicol-resistance marker inserted within the ainS gene (VF_1037) could be transformed into wild-type V. fischeri. Cells provided with both DNA and soluble chitin oligosaccharides (in the form of chitohexaose) produced putative transformants with an average efficiency of ~10^-7, a level that is >800-times the limit of detection (Table 1). V. fischeri did not produce detectable transformants when provided with either polymeric chitin (i.e., crab-shell tiles) or the chitin monomer GlcNAc instead of chitohexaose (Table 1). The frequency of spontaneous Cm resistance was below the limit of detection (<1 × 10^-10), as estimated in the same medium, either in the absence of added DNA, or with DNAse-treated DNA.

TABLE 1.

Effect of carbon source on transformation frequency of ES114 by chromosomal DNA^*

Medium†	Plasmid	Mean transformation frequency‡	Range
+ GlcNAc	-	<0.1∥	-
+ Crab shell	-	<0.1∥	-
+ (GlcNAc)6	-	80	2 to 130
+ (GlcNAc)6	pMulTfoX	1,500	400 to 3,500
+ (GlcNAc)6	pLosTfoX	430¶	340 to 520
+ GlcNAc	pMulTfoX	65	2 to 170
+ GlcNAc	pLosTfoX	260	4 to 520
+Glcn	pMulTfoX	360	7 to 710
+Glcn	pLosTfoX	520	100 to 950

^*In the absence of added DNA, transformation frequency was <1 × 10^-10.

^†Grown in minimal media (MM) containing either chitohexaose (GlcNAc)₆, N-acetylglucosamine (GlcNAc), or glucosamine (Glcn)

^‡Average of two experiments (× 10⁹)

^∥Below detection limit, 1 × 10^-10

^¶Antibiotics not added to maintain pLosTfoX.

Putative transformants were selected by Cm resistance, and further analyzed by a PCR screen with primers specific to the ainS∷Cm^R region (Table S2). The appearance of PCR products of the predicted size and sequence indicated transformation had occurred in all 10 of 10 putative transformants (Fig. S1). Because an ainS mutant is non-bioluminescent in culture, recombination of the ainS mutant allele into the chromosome was further confirmed in all of these transformants by the loss of luminescence (Fig. S2).

V. fischeri tfoX is required for normal transformation

To determine whether V. fischeri tfoX plays a role in transformation, we made an internal deletion of 78% of the gene, creating strain AGP200 (Table 2). The deletion was confirmed by PCR (Fig. S3). Analysis of AGP200 indicated that this strain was transformation defective: no transformants were detected under conditions where the wild type was competent (Table 3). The transformation defect of AGP200 could be genetically complemented by supplying tfoX^VF in trans on the plasmid pMulTfoX. The pMulTfoX plasmid carries the tfoX^VF gene on the plasmid vector pVSV104, which is maintained in V. fischeri with ~10 copies per genome (Dunn et al., 2006).

TABLE 2.

Bacterial strains and plasmids used.

Strain or plasmid	Relevant characteristicsa	Source or reference
Bacterial strain
V. fischeri
ES114	Wild-type isolate from E. scolopes light organ	(Ronquist and Huelsenbeck, 2003)
ESR1	Spontaneous RfR derivative of ES114
AGP200	ES114 tfoX gene partially deleted	This study
BF3	ES114 hadA gene inactivated by mini-Tn10 insertion; CmR	(Meibom et al., 2005)
CL21	ES114 ainS gene partially deleted and replaced by CmR marker	(Stabb and Ruby, 2002)
JRM100	ES114 with mini-Tn7 insertion; EmR	(Lyell et al., 2008)
JRM200	ES114 with mini-Tn7 insertion; CmR	(Dunn et al., 2006)
KV618	ESR1 rpoN∷TnluxAB ΔluxA∷erm; RfR	(Boettcher and Ruby, 1990)
NL1	ES114 with tfoY gene inactivated by mini-Tn5 insertion; EmR	(Graf et al., 1994)
Plasmid
pEVS79	V. fischeri suicide cloning vector, CmR, TcR	(Feliciano, 2000)
pEVS104	Conjugative plasmid with tra and trb genes	(Lupp et al., 2003)
pEVS122	R6Kγ oriV, oriTRP4 EmR, lacZα, cosN, loxP, incD	(McCann et al., 2003)
pLosTfoX	995 bp of V. fischeri ES114 DNA containing the tfoXVF ORF cloned into pEVS79	This study
pMU106	Allelic exchange vector carrying partially deleted ainS ORF replaced by CmR marker	(McCann et al., 2003)
pMulTfoX	995 bp of V. fischeri ES114 DNA containing the tfoXVF ORF cloned into pVSV104; KmR	This study
pMulTfoY	930 bp of V. fischeri ES114 DNA containing the tfoYVF ORF cloned into pVSV104; KmR	This study
pUBRtfoX	pEVS122 carrying the tfoX deletion construct.	This study
pVSV104	V. fischeri stable cloning vector, KmR	(Wolfe et al., 2004)

^aRf^R Rifampicin resistance; Cm^R, chloramphenicol resistance; Em^R, erythromycin resistance; Tc^R, tetracycline resistance; Km^R, kanamycin resistance.

TABLE 3.

Transformation of ΔtfoX^VF and tfoY^VF∷Tn5 strains^*

Recipient strain	Plasmid	Mean transformation frequency†	Range
ES114 (wild type)	pVSV104 (vector)	54	2 to 120
	pMulTfoX	12,000	7,700 to 16,000
	pMulTfoY	<0.1‡	-
AGP200 (ΔtfoXVF)	pVSV104 (vector)	<0.1‡	-
	pMulTfoX	4,400	190 to 8,600
	pMulTfoY	<0.1‡	-
NL1 (tfoYVF∷Tn5)	pVSV104 (vector)	<0.1‡	-
	pMulTfoX	21,000	350 to 41,000
	pMulTfoY	1	0.1 to 2

^*Grown in minimal media with chitohexaose (GlcNAc)₆

^†Average of two experiments (× 10⁹)

^‡Below detection limit, 1 × 10^-10

Presence of tfoX^VF in multi-copy confers chitin-independent competence

Carriage of pMulTfoX not only restored competence to a tfoX^VF mutant, but also increased its frequency of transformation by 50- to 80-fold over that of the wild type (Table 3). This transformation enhancement in the presence of multiple copies of tfoX^VF also occurred in wild type. In addition, when V. fischeri carried tfoX^VF on this multi-copy plasmid, transformation became independent of chitin oligosaccharides. That is, growth on two compounds that normally did not induce competence (GlcNAc or Glcn) by wild-type V. fischeri produced transformants when pMulTfoX was present (Table 1), but not when the cells carried the vector plasmid (pVSV104) alone (data not shown).

This enhanced transformation suggested that providing tfoX^VF in multi-copy to V. fischeri would enable the exploitation of natural transformation as a genetic tool for V. fischeri researchers. To further refine this method, the tfoX^VF complementation fragment was inserted into the vector backbone of plasmid pEVS79, which contains an origin that can be mobilized into V. fischeri cells, but that is not stably maintained without selection (Stabb and Ruby, 2002). The resulting plasmid, pLosTfoX, when introduced into V. fischeri ES114, also conferred an increased transformation frequency that was chitoligosaccharide-independent; however, the plasmid had to be maintained by antibiotic selection, at least until donor DNA was added (Table 1). Conveniently, after overnight recovery in non-selective medium, 19% of these transformants had lost the pLosTfoX plasmid as judged by the absence of both antibiotic resistance to chloramphenicol, and a PCR product when using primers targeting the oriT region of the plasmid (data not shown).

V. fischeri cells can be transformed by chromosomal, plasmid, or linear forms of DNA

Smaller fragments of DNA, such as plasmids or PCR products, are conveniently obtained and contain, by weight, more copies of a given genetic marker than chromosomal DNA. We asked whether such fragments of DNA carrying a Cm-resistance cassette (Cm^R) would transform more efficiently than chromosomal DNA when competent cells were provided with an equivalent weight of DNA in one of these three forms: (i) chromosomal DNA isolated from V. fischeri strain CL21 (containing a chromosomal replacement of ainS with the Cm^R); (ii) plasmid pMU106 (Table 2), originally used to make the CL21 replacement mutant, and isolated from an E. coli host; or, (iii) PCR DNA that was produced by amplification of the region surrounding the ainS mutation, using CL21 chromosomal DNA as the template. To control for the possible presence of residual chromosomal DNA in the PCR-DNA condition, a no-polymerase PCR reaction was run in parallel. No transformants were observed when cells were provided with the products of the no-polymerase PCR control reaction. While transformants were observed with each of the three forms of donor DNA, transformation with either chromosomal or plasmid DNA gave the highest frequency. Similarly, PCR-generated DNA was ~40-fold less effective when compared on the basis of the number of transformants/copy of Cm^R marker added (Table 4). This lower frequency may result from either the shorter stretch of homologous flanking DNA (<10³ base-pairs) present in the PCR product or, perhaps, the absence of methylation or other modification of the PCR product.

TABLE 4.

Influence of the form of extracellular DNA on transformation frequency^*

DNA form	Estimated extent of flanking homology (bp)	Mean transformation frequency†	Range
chromosomal	>104	23,000	12,000 to 33,000
plasmid	103	22,000	16,000 to 28,000
PCR product	102 to 103	530	460 to 610

^*V. fischeri strain ES114 carrying pMulTfoX was grown in minimal media (MM) with (GlcNAc)₆.

^†Transformation frequency when equal weights of DNA were added; average of two experiments (× 10⁹).

Natural transformation can be used to transfer mutations to new strain backgrounds

Existing mutations at several sites in the genome can be experimentally transferred into new V. fischeri strain backgrounds by transformation (Table 5). As anticipated, chromosomal DNA isolated from various mutant strains transformed at similar frequencies. For instance, there was no detectable difference in transformation frequency at the same site on the chromosome, regardless of the antibiotic-resistance cassette being transferred. While most of these transfers of marked DNA into wild-type cells simply re-created existing strains, we also performed transformations that created new mutant derivatives. Specifically, we transformed the Cm^R cassette from CL21 into Em-resistant JRM100 (Table s1), creating a novel strain that carried antibiotic-resistance cassettes at two separate sites. Similarly, transformation using donor DNA from the rpoN∷TnluxAB(Cm^R) ΔluxA∷erm strain, KV618 (Table S1), into wild type V. fischeri, and selection for Cm-resistance, resulted in the transfer of the rpoN mutation from a non-isogenic V. fischeri strain background (ESR1), into a wild-type strain (ES114). In the latter case, putative transformants were immotile on soft agar plates, consistent with the phenotype of the rpoN mutation; however, unlike the donor KV618, the transformants were still luminescent, like the recipient ES114 background (data not shown).

TABLE 5.

Transformation frequency of chromosomal different markers between V. fischeri strains^*

Donor strain	Recipient strain	Transformation frequency†
CL21	ES114	490
CL21	JRM100	900
JRM100	ES114	150
JRM200	ES114	170
BF3	ES114	80
KV618	ES114	100

^*Recipient strains (Table 2) were grown in minimal medium containing (GlcNAc)₆.

^†Representative experiment, values are the number of transformants per 10⁹ cells.

tfoX^VF and tfoY^VF contribute to competence, but are not identical in function

To determine whether the paralogue tfoY^VF also influences DNA uptake and incorporation, a transposon-insertion mutant of tfoY^VF, NL1 (Table 2), was used as the recipient strain in the transformation assay. Like the tfoX^VF mutant, NL1 yielded no detectable transformants, even in the presence of chitohexaose (Table 3). The transformation defect of NL1 was partially complemented by supplying tfoY^VF in trans on pMulTfoY. These findings suggest that both tfoX^VF and tfoY^VF are required for producing detectable levels of transformation in culture, and, while they may be functionally redundant, they are unable to fully compensate for the loss of the other. If the functions of the two genes were identical, we would predict that the tfoX^VF mutant could be complemented by additional copies of tfoY^VF supplied in trans, and vice versa. In fact, while complementation of NL1 (tfoY^VF∷Tn5) with pMulTfoX restored transformation, complementation of AGP200 (ΔtfoX^VF) with pMulTfoY did not. Furthermore, carriage of pMulTfoY in a wild-type background did not result in the chitoligosaccharide-independent transformation conferred by pMulTfoX, and instead appeared to decrease the frequency observed in wild-type cells. Thus, the influences of tfoX^VF and tfoY^VF on transformation competency are distinct.

DISCUSSION

V. fischeri cells appear capable of at least three forms of genetic transfer. First, they can mobilize and maintain DNA using a plasmid-borne conjugation system (Dunn et al., 2005); this ability to exchange both small and large plasmids may contribute to the large number and diversity of extrachromosomal elements they can carry (Boettcher, 1994). In addition, an inspection of the completed genomes of two strains of V. fischeri provides evidence of several phage-incorporation events (Ruby 2005; Mandel 2008), suggesting transduction as another potential mechanism for genetic exchange. Transduction is believed to be a central element in the ecology and virulence of V. cholerae (Jensen et al., 2006), and lytic phage have been shown to increase the frequency of V. cholerae transformation by releasing host cell DNA into the environment (Udden et al., 2008). However, there is little information about the role of phage and transduction in V. fischeri. In the work presented here, we show evidence of a third mechanism of genetic exchange in V. fischeri: transformation resulting from genetic competence.

Natural competence can be mediated by environmental signals, and is facilitated by the induction of specific structural and enzymatic proteins (Solomon and Grossman, 1996). When these factors are present, bacterial proteins bind extracellular DNA and transport it into the cell. These nucleic acids can then be either catabolized to serve as nutrients (Palchevskiy and Finkel, 2006) or, if the DNA is sufficiently similar to its own, incorporated into the cell’s genome by homologous recombination (Hamilton and Dillard, 2006). While the function(s) of competence and/or transformation in ecologically relevant settings remains poorly understood for most bacteria, the machinery by which extracellular DNA is mobilized into another genome appears to be relatively conserved among Gram-negative bacteria (Chen and Dubnau, 2004). Nevertheless, the regulation of this process is often tailored to a given species’ unique lifestyle.

Transformation was first demonstrated in the genus Vibrio in 2005, when chitin-induced competence and chromosomal incorporation of extracellular DNA was reported in V. cholerae (Meibom et al., 2005). This role for chitin suggested that chitinous structures are important sites not only for growth, but also for horizontal gene transfer. The report also showed that a regulatory protein encoded by tfoX was required for efficient transformation by V. cholerae. Soon after this discovery, expression studies of cultured V. fischeri cells grown on acetylchitobiose, the soluble dimeric subunit of chitin, indicated that this nutrient caused an upregulation of not only the genes of the chitin-utilization program but also several putative competence genes, such as tfoX and comE (A. Schaefer and E. Ruby, unpubl. data.). We show here that V. fischeri becomes genetically competent in a tfoX-dependent manner when grown in the presence of chitin oligosaccharides (Tables 1 and 3). As in V. cholerae, this induction was specific and did not occur when other carbon sources, including the chitin monomer N-acetylglucosamine (GlcNAc), were provided (Meibom et al., 2005). However, unlike V. cholerae, transformation by V. fischeri cells was not detectable induced by the presence of chitin in the form of crab shell tiles (Table 1), perhaps due to their relatively poor attachment and growth on this insoluble substrate. More recently, chitin-dependent induction of competence has also been demonstrated in V. vulnificus, although the contribution of this bacterium’s tfoX gene to this process was not examined (Gulig et al., 2009). Taken together, these results make a strong argument that chitin is an important factor in the ecological genetics of the Vibrionaceae.

The role of TfoX in transformation has been studied not only in V. cholerae, but also in Haemophilus influenzae, Aggregatibacter (Actinobacillus) actinomycetemcomitans and Escherichia coli (Williams et al., 1994; Zulty and Barcak, 1995; Bhattacharjee et al., 2007; Sinha et al., 2009). In each case, TfoX (or the homologous Sxy) is thought to promote transcription of competence-related genes by assisting the cyclic-AMP receptor protein (CRP) in binding to Sxy-dependent CRP (CRP-S) sites within their promoter region (Sinha et al., 2009). In addition, the presence of the tfoX/sxy gene in multi-copy, either over-expressed or under the control of its native promoter, results in increased, constitutive or, in the case of V. cholerae, chitin-independent competence (Williams et al., 1994; Meibom et al., 2005; Bhattacharjee et al., 2007).

The rate of transformation we report for V. fischeri in culture is ~100-fold lower than that observed in V. cholerae (Meibom et al., 2005). These data suggest that V. fischeri has either a more stringent competence regulation, or a higher level of extracellular DNAse activity (Blokesch and Schoolnik, 2008). This lower transformation frequency of cells grown with soluble chitin oligosaccharides could explain the absence of observed transformation of V. fischeri grown on crab shell tiles. Nevertheless, the increased transformation frequency conferred by the carriage of multiple copies of tfoX^VF provides a new technique for rapidly introducing mutations into V. fischeri (Table 4), probably through recA-mediated homologous recombination (Dunn et al., 2005). This approach not only adds a new method to the genetic repertoire of researchers working on V. fischeri, but also suggests that, given the widespread distribution of tfoX genes (Fig. 1), the introduction of multiple copies of tfoX homologues into other Vibrionaceae may facilitate the discovery of natural competence in these species as well.

We also report that a novel gene and putative paralogue of tfoX^VF, designated tfoY^VF, is required for normal levels of transformation in culture. These loci, tfoX^VF and tfoY^VF, resemble each other in sequence and appear to converge in their influence on transformation under similar experimental conditions. However, they are unable to compensate fully for the loss of the other and may have distinct influences on transformation frequency (Table 3). Transcriptional studies in V. fischeri have also shown different regulation patterns for the two genes. For instance, in the light organ both genes are differentially regulated over the course of the day, and their transcription appears sequential: tfoX^VF peaks first, with tfoY^VF continuing an upward trajectory of expression (Wier et al., 2010). Like its V. cholerae orthologue, the tfoY^VF gene of V. fischeri contains a putative riboswitch motif upstream of the start site (Weinberg et al., 2007; Sudarsan et al., 2008), while tfoX^VF does not. This difference further suggests that tfoX^VF and tfoY^VF have distinct cellular inputs for their activation.

The presence of tfoX homologues in all sequenced Vibrionaceae (Fig. 1), combined with a common capability to utilize chitin as a nutrient (Hunt et al., 2008), indicates that chitin-induced transformation is likely a shared-derived trait of this family of marine bacteria. Given the different ecologies of V. cholerae, V. vulnificus, and V. fischeri, it is intriguing that they each respond to the presence of chitin oligosaccharides by inducing competence. Perhaps this common response to chitin reflects a shared life stage in seawater, or the common presence of chitin in association with a host. One of the best understood natural environments of Vibrio species is the light-organ symbiosis of V. fischeri (Nyholm and McFall-Ngai, 2004). Whether the uptake of DNA results in a fitness advantage for competent V. fischeri in the light organ, by providing an additional nutrient source and/or as a means of generating further diversity within the polyclonal population (Wollenberg and Ruby, 2009), is a direction for future studies. To date, we have observed no colonization defect for either a tfoX^VF or a tfoY^VF mutant within the first 48 h of symbiosis (data not shown). However, recent evidence indicates that rscS, a gene that is essential for colonization of the squid light organ, may have been acquired horizontally (Mandel et al., 2009), perhaps by transformation during the evolution of V. fischeri. Thus, over evolutionary time scales, there may be a direct connection between the capacity for genetic competence and symbiotic fitness. Further investigation may reveal how this mechanism of genetic exchange has been tailored to the individual lifestyles of diverse vibrios, from pathogens to beneficial symbionts.

EXPERIMENTAL PROCEDURES

Bacteria and culturing techniques

The bacterial strains and plasmids used in this study are described in Table S1. V. fischeri srains were grown at 28° C in either a defined seawater minimal medium (MM) (0.33 mM β-glycerophosphate, 300 mM NaCl, 50 mM MgSO₄, 10 mM CaCl₂, 10 mM KCl, 0.01 mM FeSO₄, and 100 mM Tris-HCl [pH 7.5]), Luria-Bertani salt medium (LBS) (Dunlap, 1989), or a seawater-based tryptone (SWT) medium (5 g Bacto-tryptone [Difco Inc.], 3 g yeast extract, 3 mL glycerol, 700 mL filtered Instant Ocean [Aquarium Systems, Inc, Mentor, OH], and 300 mL distilled water) (Boettcher and Ruby, 1990). Escherichia coli strains used in construction of plasmids were grown at 37° C in either Luria-Bertani medium (LB) (Miller, 1992) or brain-heart infusion medium (BHI; BD, Sparks, MD). Media were solidified with 1.5% agar as needed. When added to LBS, chloramphenicol (Cm), erythromycin (Em), and kanamycin (Km) were used at concentrations of 5 μg/mL, 5 μg/mL, and 100 μg/mL respectively. When added to LB, Cm and Km were used at concentrations of 25 μg/mL and 50 μg/mL respectively. When added to BHI, Em was used at a concentration of 150 μg/mL.

Sequence collection

The amino acid sequence for the tfoX-like ORF in V. fischeri ES114 (VF_0896) was used to query GenBank using the BLASTp algorithm (Altschul et al., 1997). The results of this search revealed that all large chromosomes of fully-assembled Vibrionaceae genomes encode two protein sequences with similarity to the query sequence. The nucleotide sequences for both loci in V. fischeri ES114 (VF_0896 and VF_1573) were used to query GenBank using the BLASTn algorithm; from these searches, a collection of tfoX-like sequences in the 13 fully-assembled Vibrionaceae (and three outgroup) species were tabulated (Table S1).

Phylogenetic analyses

Nucleotide sequences were aligned using the default pairwise and multiple alignment parameters in ClustalX 2.0.11. Phylogenetic reconstructions were completed using three methods: maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference. MP reconstructions were performed by treating gaps as missing, searching heuristically using simple addition, tree-bisection reconnection for swaps, and swapping on best only with 1000 repetitions. For ML and Bayesian reconstructions, likelihood scores of 56 potential evolutionary models were tested using the Akaike Information Criteria (AIC) as implemented in Modeltest 3.7 (Larkin et al., 2007); based on AIC, a general time-reversible model with a gamma-shape parameter (GTR+Γ) was used. ML reconstructions were performed by treating gaps as missing, searching heuristically using simple addition, subtree pruning and regrafting for swaps, and swapping on best only with 1000 repetitions as implemented by PAUP*4.0b10 (Posada and Buckley, 2004). Bayesian inference was performed by invoking rates=gamma and nst=6 settings (GTR+Γ model) and temp=0.15 (to insure an appropriate amount of chain swapping) in the software package MrBayes3.1.2. Confidence in the topology of the ML and MP reconstructions was statistically assessed using either 100 bootstrap pseudoreplicates with the above search parameters. To test the Bayesian inference, an appropriately stationary posterior-probability distribution was sampled every 200 generations; a stationary distribution was defined as having an average standard deviation of split frequencies between two chains in a Metropolis-coupled Markov chain Monte Carlo (MCMCMC) run <0.01 for ~70-90% of samples (~500,000-2,000,000 total generations; 2501 of 10001 trees discarded as ‘burn-in’). Consensus trees drawn from the sample distribution generated by MCMCMC were used for the assessment of the posterior probabilities of all clades. Y. pestis strain KIM (Table S1) was used to root all reconstructions.

DNA

Chromosomal DNA was purified using the MasterPure DNA Purification kit (Epicentre, Madison, WI) and served as a template both for amplifying the regions that flanked genes targeted for deletion, and for direct transfer of chromosomal markers between strains. Plasmid DNA was isolated using a QIAprep spin miniprep kit (Qiagen, Valencia, CA). PCR primers (Table S2) were purchased from either IDT (Coralville, IA) or the UW Biotechnology Center (Madison, WI). PCR fragments used for transformation were amplified with Pfx50 DNA polymerase (Invitrogen, Carlsbad, CA) and purified with Wizard^® SV Gel and PCR Clean-Up System (Promega, Madison, WI).

Luminescence in culture

To determine the luminescence characteristics of V. fischeri wild-type and mutant strains, 2 mL of SWT were inoculated to an optical density at 600 nm (OD) of about 0.05 with cells that had been pre-grown overnight in LBS. Cultures were maintained at 28° C with shaking, and sampled at various times during growth to measure both luminescence and OD. Maximum specific luminescence (luminescence/OD) values were averaged for measurements made between OD 0.2 and 3.0. Growth rates were also determined during these experiments by observing the change in OD as a function of time.

Transformation of V. fischeri

To determine the frequency of transformation in liquid culture, cells were grown to mid-log phase in MM supplemented with different sources of carbon. These sources included: soluble chitin oligosaccharides, provided in the form of chitohexaose ([GlcNAc]₆; 2 μM) (Associates of Cape Cod, East Falmouth; MA); or N-acetylglucosamine (GlcNAc; 10 μM) or glucosamine (Glcn; 10 μM) (Sigma, St. Louis, MO). Approximately 48 μg of DNA was added per mL of culture, which was incubated at 25° C for 30 min, and the cells were then diluted into LBS for a recovery period of between 4 and 14 h. These transformation reactions were plated onto LBS with the appropriate antibiotic selection. Antibiotic-resistant colonies that arose were considered putative transformants. Serial dilutions plated in parallel without antibiotic were used to determine the total number of cells placed on the selective plates. The frequency of transformation was calculated as the proportion of transformants arising from the total cell population. Because there was no difference in the growth rates of the wild type and any of the transformants made in this study (data not shown), the length of recovery is unlikely to change the calculation of transformation efficiency. In any given experiment, the frequency of transformation could vary by as much as an order of magnitude; therefore, we report both the mean and range of frequencies observed. Transformation on crab-shell tiles was performed as previously described (Meibom et al., 2005). Briefly, V. fischeri cells were grown statically for 24 h in MM containing a crab-shell tile. The supernatant was then removed, and the tiles were resuspended in MM containing chromosomal DNA, and incubated for an additional 24 h. The tiles were removed and placed in LBS overnight for recovery before plating on selective and non-selective media as described above.

Verification of recombination in putative transformants

To confirm the introduction of a mutant copy of the ainS gene (VF_1037) into the recipient, chromosomal DNA isolated from putative transformants was used as the template in PCR reactions. Primer CamRCheckRev1 (Table S2) targeted the transformed Cm^R cassette, while primer ainSCheckRev1 targeted the region outside the ainS gene on the recipient’s chromosome. To further confirm the identity of the putative transformants, we took advantage of the ainS defect in luminescence. The luminescence of candidate transformants grown in SWT was measured as a function of cell density; the presence of the wild-type ainS allele resulted in detectable luminescence, whereas a mutant allele did not.

Construction of tfoX^VF and tfoY^VF mutants

To generate the ΔtfoX mutant allele, approximately 2.0 kb of DNA upstream, and including a portion, of the tfoX^VF gene (VF_0896) was PCR-amplified using primers tfoXUSFor and tfoXUSRev (Table S2). An approximately 1.6-kb DNA fragment containing part of the tfoX^VF gene, as well as sequence downstream of the stop codon, was PCR-amplified using tfoXDSFor and tfoXDSRev (Table S2). These upstream and downstream fragments were fused by an engineered restriction site, resulting in a 492-bp internal deletion. The deletion leaves a 50-bp N-terminal sequence and an 82-bp C-terminal sequence intact, carried on the pUBRtfoX plasmid (Table S1). The mutant construct was introduced into ES114 via tri-parental mating, and incorporation of the deletion was confirmed by amplification with PCR primers flanking the tfoX^VF gene (tfoXconfirmFor4 and tfoXconfirmRev; Table S2). The tfoY^VF mutant NL1 contains a mini-Tn5 transposon insertion in VF_1573, and was obtained from a previously described transposon-mutant library (Lyell et al., 2008).

Construction of tfoX^VF and tfoY^VF complementation plasmids

To supply the tfoX^VF gene in trans, approximately 979 bp of DNA containing the tfoX^VF gene and 150 bp of upstream sequence were PCR amplified using primers tfoXcompFor and tfoXcompRev (Table S2). The resulting product was cloned as a KpnI/SacI fragment into the complementation vector pVSV104 (Table S1), the origin of which is stably maintained in V. fischeri without selection. To supply the tfoY^VF gene in trans, approximately 930 bp of DNA containing the tfoY^VF gene and 233 bp of upstream sequence was PCR amplified using primers tfoYcompFor and tfoYcompRev. The resulting product was similarly cloned into pVSV104.