Three New Regulators of Swarming in Vibrio parahaemolyticus
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《细菌学杂志》
Department of Microbiology, The University of Iowa, Iowa City, Iowa 52242
ABSTRACT
Movement on surfaces, or swarming motility, is effectively mediated by the lateral flagellar (laf) system in Vibrio parahaemolyticus. Expression of laf is induced by conditions inhibiting rotation of the polar flagellum, which is used for swimming in liquid. However, not all V. parahaemolyticus isolates swarm proficiently. The organism undergoes phase variation between opaque (OP) and translucent (TR) cell types. The OP cell produces copious capsular polysaccharide and swarms poorly, whereas the TR type produces minimal capsule and swarms readily. OPTR switching is often the result of genetic alterations in the opaR locus. Previously, OpaR, a Vibrio harveyi LuxR homolog, was shown to activate expression of the cpsA locus, encoding capsular polysaccharide biosynthetic genes. Here, we show that OpaR also regulates swarming by repressing laf gene expression. However, in the absence of OpaR, the swarming phenotype remains tightly surface regulated. To further investigate the genetic controls governing swarming, transposon mutagenesis of a TR (opaR1) strain was performed, and SwrT, a TetR-type regulator, was identified. Loss of swrT, a homolog of V. harveyi luxT, created a profound defect in swarming. This defect could be rescued upon isolation of suppressor mutations that restored swarming. One class of suppressors mapped in swrZ, encoding a GntR-type transcriptional regulator. Overexpression of swrZ repressed laf expression. Using reporter fusions and quantitative reverse transcription-PCR, SwrT was demonstrated to repress swrZ transcription. Thus, we have identified the regulatory link that inhibits swarming of OP strains and have begun to elucidate a regulatory circuit that modulates swarming in TR strains.
INTRODUCTION
Vibrio parahaemolyticus is a gram-negative marine bacterium found in coastal and estuarine waters worldwide (11, 31). It possesses two distinct flagellar systems: the polar system produces a single sheathed flagellum designed for swimming motility, whereas the lateral flagellar (laf) system is adapted for movement over moist or viscous surfaces (3, 46). The lateral flagella are expressed only when the cell senses a surface environment (45). Swarmer cells are elongated (5 to 20 times the length of the swimmer cell) and multinucleoid, and they produce a multitude of lateral flagella. Two environmental signals are known to be required for laf expression: iron-limiting conditions and inhibition of polar flagellar rotation (32, 43, 44). The polar flagellum acts as a tactile sensor for the cell. When the cell encounters a surface or sufficiently viscous environment, flagellar rotation is impaired and laf is induced. Thus, the lateral flagellar system is intimately linked to the polar system. However, the molecular mechanism for sensing polar flagellar inhibition and the signal transduction pathway regulating laf expression are not known. It was the goal of this work to begin to elucidate the regulatory circuit controlling laf expression.
Many bacterial species are known to swarm, including some with mixed polar and peritrichous flagellar systems. In species with dual flagellar systems, such as Rhodospirillum centenum, Azospirillum, and Aeromonas species, surface contact appears to be required for induction of the swarming response, but little is known about how these flagellar systems are regulated (33, 46). Enteric swarming species such as Proteus mirabilis, Salmonella enterica serovar Typhimurium, and Serratia liquefaciens use one flagellar system for both swimming and swarming. In these species, flagellar gene expression is controlled by and dependent upon the global regulator FlhDC (1, 16, 54). In P. mirabilis, transcriptional studies have shown that flhDC is upregulated in swarmer cells (6), and many Swarm– mutants that are defective in signaling swarmer cell differentiation appear to downregulate flhDC expression (12, 25). In S. liquefaciens, flhDC is required for swarmer cell differentiation, and overexpression of flhDC induces swarmer cell differentiation and increased flagellar production (21). In contrast, 54 and LafK, a 54-dependent regulator, regulate the lateral flagellar system of V. parahaemolyticus (59). Analysis of the V. parahaemolyticus genome shows that V. parahaemolyticus does not possess flhDC homologs nor does it possess homologs to many of the swarming regulatory factors that have been identified in enteric species with only one flagellar system. Thus, while swarming in V. parahaemolyticus certainly shares many of the same phenotypic characteristics that are found in other swarming species, the genetic and regulatory features appear to be different.
In addition to the swimmer swarmer cell transition, V. parahaemolyticus switches between opaque (OP) and translucent (TR) cell types. OP cells produce a thick capsular polysaccharide (CPS), which promotes surface adhesion and cell-cell adhesion (15, 48). CPS production by OP strains also affects cell packing and colony structure and is the primary determinant of the opaque appearance of colonies (15). Both clinical and environmental isolates undergo reversible OP TR switching at a low frequency. The environmental cues which lead to OPTR switching are not well understood, but salt and nutrient levels are thought to be important (29, 53). Although the environmental signals have not been well defined, the principal genetic factors determining opacity have been identified. OpaR is the central transcriptional regulator. Disruption of opaR converts the OP strain to the TR phenotype, and expression of OpaR confers the opaque phenotype upon a stable TR (opaR1) strain (48). Other TR strains have also been found to contain lesions in the opaR locus and can be repaired to OP by supplying opaR+ on a plasmid (14; L. L. McCarter, unpublished). Transposon mutagenesis studies of the OP strain revealed that mutations in an 11-gene capsular polysaccharide biosynthesis operon (cpsA locus) turned the OP strain to a nearly TR phenotype, demonstrating that CPSA (the CPS product of the cpsA operon) is the primary component of the OP phenotype (23). It was further shown that OpaR activates cpsA expression. Thus, OpaR regulation of the cpsA locus is the fundamental genetic pathway for the OP TR switch (23).
Although it has been observed that OP strains of V. parahaemolyticus swarm poorly relative to TR strains, a genetic link between swarming and the OP/TR state of the cell has never been demonstrated. Two possibilities could explain this result: the increased CPSA production of OP strains might make the cells "sticky" and inhibit swarming or OpaR might directly repress swarming. We discounted the first possibility when cps mutants in the OP background were still repressed for swarming relative to TR cps mutants (Z. T. Guvener, S. Jaques, and L. L. McCarter, unpublished data). Here, we examine the possibility that OpaR plays a more direct role in controlling swarming of OP strains.
We show that OpaR inhibits swarming and does so by repressing laf gene expression. We further examined swarming in the TR (opaR1) background to identify additional regulators. Transposon mutagenesis for swarm-defective mutants identified swrT (VPA0420), a TetR family regulator. swrT mutants are highly impaired for swarming and laf expression. A second transposon mutagenesis was performed on a swrT mutant to look for Swarm+ suppressors. Three mutants contained insertions in swrZ (VP0355), encoding a GntR family regulator. Overexpression of swrZ repressed swarming of the prototype TR strain and repressed laf expression, confirming that SwrZ acts as a repressor of laf. We further show that SwrT regulates laf gene expression by repressing swrZ expression.
MATERIALS AND METHODS
Strains. The strains and plasmids used in this work are described in Table 1. All V. parahaemolyticus strains are derived from BB22 (4). LM5312 (also known as BB22OP) is the prototype OP strain. LM5674 (also known as BB22TR) is the prototype TR strain; it contains an 85-bp deletion in the upstream and N-terminal coding region of opaR (opaR1) (14, 48). Hence, LM5674 and its derivatives are unable to revert to the OP (opaR+) phenotype. A second TR strain (LM5431), which was derived from LM5312, contains a spontaneous 5-bp insertion in the opaR coding region (opaR2) causing a frameshift mutation (14, 59). The lateral flagellar reporter strain, TR flgBL (LM5738), contains a Tn5lux insertion in a lateral flagellar rod gene and was obtained by transposon mutagenesis of LM5431 (opaR2) (59). The OP flgBL strain was obtained by converting the TR flgBL (LM5738) to opaR+ with the opaR+ cosmid (pLM1950, Tetr) and screening for Tets recombinants.
Media and growth conditions. V. parahaemolyticus strains were grown at 30°C in heart infusion (HI) medium containing 25 g heart infusion (Difco) and 20 g NaCl per liter. Swarm-minus (Swarm–) plates contained HI media with 20 g Difco agar per liter, and Swarm-plus (Swarm+) plates contained HI media with 15 g Bacto agar. Motility plates contained 10 g tryptone, 20 g NaCl, and 3.35 g Bacto agar per liter. Antibiotics were used at final concentrations of 50 μg/ml kanamycin, 10 μg/ml chloramphenicol, 25 μg/ml gentamicin, 100 μg/ml streptomycin, and 10 μg/ml tetracycline or other concentrations as indicated. The iron-chelator dipyridyl (2,2'-dipyridyl; Sigma) was used at 100 μM, and phenamil (methanesulfonate salt; Sigma) was used at 25 μM or as indicated. Expression plasmids were induced with 1 mM isopropyl--D-galactopyranoside (IPTG).
Genetic and molecular techniques. General molecular biology methods were followed (55). Chromosomal DNA was prepared as described previously (60). Insertion/deletion mutations were made in Escherichia coli (LLM2657) using a Red recombinase system (9) to introduce Camr, Kanr, or Genr insertion-deletion mutations onto transposon-junction plasmids or cosmids carrying 20-kb insertions of wild-type V. parahaemolyticus DNA. Recombinants were retransformed into DH5, screened for appropriate drug markers, and analyzed by restriction digest. The insertion-deletion constructs were then conjugated into V. parahaemolyticus, and the standard allelic replacement procedure followed (58). All allelic replacements and transposon mutants were confirmed with Southern blot analysis by using Hybond-NX membranes (Amersham) and [32P]dCTP-labeled DNA probes (Pharmacia).
Transposon mutagenesis of TR strain. The transposon plasmid pRL27 contains a hyperactive transposase gene driven by the tetA promoter (35). An oriR6K gene and a Kanr gene are contained within the inverted repeat ends of the deliverable transposon. The recipient Fla– Laf+ Camr strain (LM4281) was grown overnight, diluted, and grown for approximately 6 h to mid-exponential phase without antibiotic. This culture was mixed with an equal volume of an exponentially growing E. coli strain carrying the transposon donor plasmid pRL27. HI plates with no NaCl were spotted with 10 aliquots of the mating mixture and incubated overnight at 37°C. The mating plates were suspended in 5 ml of 0.3 M sucrose and diluted 10-fold to a final optical density at 600 nm (OD600) of 0.2 to 0.5. The mating suspension (100-μl aliquot) was spread onto HI plates containing chloramphenicol and kanamycin for selection of transposon mutants. Approximately 2,500 colonies were picked onto gridded plates and then transferred onto Swarm and Mot plates for screening. To identify the site of Tn5 insertion of individual mutants, chromosomal DNA was prepared and arbitrarily primed PCR was performed using primers specific to the transposon and a set of arbitrary primers essentially as described previously (52).
swrT2 suppressor mutagenesis. swrT2 suppressor mutagenesis was performed essentially the same as the TR cell mutagenesis, except that the swrT2::lacZ (Camr) strain (LM6820) was the recipient and pools from mutagenesis mating mixtures were plated onto HI Swarm+ plates containing chloramphenicol at 5 μg/ml and kanamycin at 75 μg/ml. Plates were incubated for 3 days, and swarming flares were streaked out for single colonies. Isolated colonies were screened for their swarming phenotype in comparison to the parent and a Swarm+ control strain.
Transposon-junction plasmids. Transposon-junction plasmids were obtained by preparing chromosomal DNA and digested with a restriction enzyme that does not cut within the Tn5 insert, which contains the oriR6K and Kanr markers (35). An aliquot of the digested chromosomal DNA was ligated and transformed into S17-1pir cells. Transformants were selected on LB containing kanamycin and streptomycin. Plasmid from transformants was isolated and examined by restriction digest and then sequenced using transposon-specific primers. To recreate transposon mutants, the transposon-junction plasmid was linearized by digestion with the appropriate restriction enzyme, ligated into the compatibly digested gene replacement vector, pLAFRII, and transformed into DH5 with selection for kanamycin and tetracycline. The transposon-junction-pLAFRII clone could then be used in standard gene replacement procedure (58).
swrT2::lacZ and swrZ::lacZ reporter construction. Several lacZ transcriptional reporters were constructed. SmaI-digested antibiotic resistance cassettes were obtained from p34sCam, pUCGM, or pGM1 (10, 57) and were inserted into the NaeI site in pTL61, which carries a promoterless lacZ gene with an RNase III cleavage site (40) to create a suite of lacZ fusion plasmids. PCR primers were designed and used to amplify the region containing lacZ and the antibiotic cassette (5 kb); the purified PCR product was used to electroporate LLM2657 carrying either the swrT1::Tn5 cosmid (pLM3096) or the swrZ2::Tn5 cosmid (pLM3463). Transformants were selected for the appropriate antibiotic resistance marker and screened for beta-galactosidase activity and Kans. The resultant swrT2::lacZ(Camr) cosmid (pLM3240) and swrZ4::lacZ(Genr) cosmid (pLM3471) were then used for conjugation into V. parahaemolyticus.
Beta-galactosidase assays. Two independent transformants of LM7824 and LM7874 were grown overnight (15 h) in HI broth and as colonies on plates. swrZ-lacZ expression was measured in triplicate from each sample as per Miller (49).
Luminescence measurement. Bioluminescence from plate-grown colonies was examined by exposing overnight colonies in a Fujifilm luminescence image analyzer (LAS-1000) for 1 to 5 min. For time course experiments of plate-grown cells, either overnight cultures (opaR+ experiment) or single colonies (swrT and swrZ+ experiments) of each strain being analyzed were spotted or placed with toothpicks onto a grid array on Swarm+ plates containing antibiotic and 1 mM IPTG, as appropriate, and incubated at 30°C. At each time point, cells were scraped from one or more colonies and suspended in HI, and OD600 and luminescence were measured. Luminescence was quantified by measuring 0.1-ml samples for 30 s in a Turner TD20/22 luminometer (Turner Designs, Sunnyvale, CA). Dilutions were made to keep all measurements within a linear range. Luminescence is reported in specific light units (SLU), which is luminescence min–1 ml–1 OD600–1.
Immunoblots. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed with 12% acrylamide gels according to standard protocols. Samples were normalized to an equal OD600. Immunoblots on polyvinylidene difluoride membranes were probed with pooled antisera raised against either the lateral flagellin protein (LafA antibody no. 127, used at 1:10,000 dilution) or an outer membrane protein (Omp antibody no. 634, used at 1:5,000 dilution). Secondary antibody was peroxidase-conjugated rabbit immunoglobulin G (1:10,000 dilution). The Pierce Supersignal West detection system (Rockford, Ill.) and a Fuji luminescence imager were used for used for signal detection. For examination of LafA from swrZ1 swrT mutants grown in liquid, cultures were inoculated to OD600 of 0.01 in HI plus antibiotic with or without the iron chelator dipyridyl (100 μM) or the polar flagellar inhibitor phenamil (25 μM); cells were harvested at late-logarithmic phase (OD600 = 1.0).
Quantitative RT-PCR. Overday cultures of LM5674 and LM6820 were normalized to an OD600 of 1.0 and diluted 1:1,000, and 25 μl was spread onto Swarm+ plates. Cells were harvested from plates after 15 h of incubation, and RNA was extracted using the QIAGEN RNeasy kit (Valencia, CA). In addition, RNA was treated twice with DNase I. Random hexamers were used to prime total RNA using the Access reverse transcription (RT)-PCR kit (Promega). Primers for quantitative PCR were designed using the Primer Express program (Applied Biosystems) and the RIMD2210633 genome sequence. cDNA was used as a template for quantitative PCR with the SYBR green PCR master mix (Applied Biosystems) and a TaqMan PCR thermocycler (Applied Biosystems) at the University of Iowa DNA Core Facility. Changes in gene expression were calculated by the CT method (User Bulletin no. 2; Applied Biosystems).
RESULTS
OpaR is a repressor of swarming and laf expression. Initial studies of swarming motility in V. parahaemolyticus focused on the TR strain because it was a proficient swarmer when grown on agar plates. By comparison, the OP strain was observed to swarm poorly. Swarming of the prototype OP (LM5312) and TR strains (LM5674, opaR1) is shown in Fig. 1A. The TR colonies have merged together due to their rapid expansion, whereas the OP colonies swarmed minimally. The TR Swarm– and OP Swarm– control strains, which contain Tn5lux insertions in a lateral flagellar rod gene (flgBL), completely failed to swarm.
OpaR activates capsular polysaccharide (CPSA) production, which mediates adhesiveness of cells to each other (15); therefore, it seemed possible that the OP strain was inhibited for swarming simply due to increased CPSA but still made similar amounts of lateral flagella as the TR strain. To test this, we examined lateral flagellin production of cells from actively swarming colonies. Figure 1B shows an immunoblot of cells scraped from the outer edge (2 to 3 mm) of the colonies from the plate in Fig. 1A. The immunoblot was probed with antisera to lateral flagellin (LafA) and an outer membrane protein (Omp), which was used as a loading control. The results showed that the OP strain made significantly less lateral flagellin than did the TR strain.
The OP strain was a poor swarmer and produced much less lateral flagellin, so it was hypothesized that opaR, which is intact in the OP strain, controlled swarming by acting as a negative regulator of laf expression. Lateral flagellar gene expression was examined directly in the OP flgBL::Tn5lux and TR flgBL::Tn5lux luminescence reporter strains, which carry the wild-type opaR (OP flgBL) or the opaR2 allele (TR flgBL), respectively. Figure 1C shows a dark-field photograph of the bottom portion of the plate shown in Fig. 1A. Colonies of the TR flgBL strain produced high levels of luminescence and appear as dark spots in Fig. 1C, whereas colonies of the OP flgBL strain had very low levels of luminescence and did not produce visible spots. This indicated that OpaR was affecting Laf production via repression of laf gene expression. To further test this hypothesis, opaR+ was cloned into an IPTG-inducible expression plasmid and introduced into the TR (opaR2) strain (LM5431) and the laf::lux reporter strain (LM5738), along with the vector control. The swarm-competent strains were tested on Swarm+ plates either lacking or containing 1 mM IPTG. Figure 1D shows that induction of opaR+ repressed swarming and turned the colonies opaque. Next we examined the laf::lux reporter strains to determine whether OpaR repressed laf expression from plate-grown cells. Figure 1E shows that opaR+ repressed laf more than 940-fold at the point of maximal luminescence for the control strain (16,000 versus 17 SLU at 7 h time point) and 180-fold or more from 5 h onward. To control for the possibility that OP strains are unable to transmit light as efficiently as TR strains due to the presence of increased capsular polysaccharide in OP strains, a control mutant containing a Tn5lux fusion in a gene not regulated by OpaR was transformed with the IPTG-inducible opaR+ plasmid and luminescence was examined. In the absence of IPTG, this strain was TR, and in the presence of IPTG, this strain was OP. However, the amount of light produced was equivalent in both growth conditions and, thus, capsular polysaccharide of OP strains does not significantly affect the detection of light (data not shown). Taken together, these data indicate that OpaR is a negative regulator of swarming and acts by repressing laf expression.
Regulation of swarming in a TR strain. Although TR strains are derepressed for swarming due to mutations in the opaR locus, they still require several environmental signals to initiate swarming, namely inhibition of polar flagellar rotation and starvation for iron. Other regulatory factors must be involved in invoking the swarming response in opaR mutant strains. To identify these regulatory factors, a transposon mutagenesis of a TR strain was undertaken and approximately 2,500 transposon mutants were screened for swarming defects. A polar flagellum-defective strain (Fla– Laf+, LM4281) was chosen for mutagenesis because it had a "super" swarming phenotype. It was reasoned that a broader range of swarming defects might be detected in a strain with elevated swarming; furthermore, their effects on motility would be isolated from the polar flagellar system in a Fla– background. One potential regulatory mutant, LM8103, contained a Tn5 insertion in VPA0420, which codes for a TetR family transcriptional regulator. VPA0420 exists as a single open reading frame (ORF), i.e., upstream and downstream genes are transcribed oppositely. BLAST analysis showed that homologs to VPA0420 exist in all the sequenced Vibrio genomes in the public databases; in Vibrio harveyi, the gene has been named luxT. LuxT was shown to affect quorum sensing in V. harveyi by binding to the promoter region of the luxOU operon and repressing luxOU expression (38). We designated VPA0420 as swrT for swarming regulator homolog to luxT of V. harveyi.
SwrT regulates swarming and laf expression in V. parahaemolyticus. The original swrT mutant was found to be swarm defective but not completely blocked for swarming. To confirm this, the swrT::Tn5 mutation was recreated in the parent strain, LM4281 (Fla–Laf+). Figure 2A shows that the recreated swrT::Tn5 mutant was also defective for swarming, demonstrating that the Tn5 insertion was responsible for the phenotype. Similar to the original mutant, swarming was not completely abolished. A lafA mutant strain (LM7805), which contains a Tn5 insertion in the gene for lateral flagellin in the LM4281 background, failed to produce lateral flagella and is shown as a Swarm– control.
To observe the phenotype of a swrT mutation in a Fla+ Laf+ strain and to ensure that the swrT::Tn5 mutation did not generate any partial protein products, a second allele that deleted the majority of the swrT coding region was constructed. The swrT mutation was moved into the prototype TR strain (LM5674) to generate strain LM6820, and its swarming phenotype was analyzed (Fig. 2B). Similar to the swrT::Tn5 mutant, the swrT mutant was severely swarm defective, but swarming was not completely eliminated (compared to the flgBL Swarm– control strain). Thus, compared to their respective parental strains, the two swrT mutations appeared to have similar relative effects on swarming in both the Fla– and Fla+ strains.
Immunoblots to lateral flagellin were performed to determine if the swrT mutation affected swarming due to a disruption of the lateral flagellar system or if swrT affected some other system important for swarming but did not directly affect lateral flagellin production and/or function. Immunoblots were performed on cells harvested from the Swarm+ plates shown in Fig. 2A and B (outer 2 to 3 mm of colonies) and probed with anti-LafA antiserum. Figure 2C shows that both swrT mutants produced significantly less lateral flagellin than their respective parent strains. The swrT::Tn5 allele may cause a more profound defect on LafA production in the Fla– Laf+ background (LM4281) than the swrT allele effect in the Fla+ Laf+ background (LM5674). Whether this difference is due to allele specificity or strain background is not known. Nevertheless, both swrT mutant alleles affected swarming and caused significantly decreased LafA production.
Since SwrT is a member of the TetR family of transcriptional regulators, it seemed likely that swrT mutations affected laf gene expression. To examine this, the swrT::Tn5 mutation was introduced into the chromosome of the laf::lux reporter strain, and laf expression (i.e., luminescence) was measured from plate-grown cells. Figure 2D shows that the parent strain expressed high levels of laf, whereas expression from the swrT mutant was greatly reduced. For example, at the point of maximal laf expression (11 h time point), the parent strain produced 400,000 SLU and the swrT mutant produced 4,400 SLU, a 90-fold decrease. Quantitative RT-PCR experiments using the deletion allele corroborated the reporter results obtained with the Tn5 allele. Comparing RNA harvested from plate-grown LM6820 (swrT) and parental LM5674 revealed a 40- to 50-fold decrease in lafA expression in the swrT mutant strain (data not shown) Thus, swrT mutants were defective for induction of laf gene expression.
A swrT+ clone restores swarming but overexpression is toxic. Since swrT mutants are defective for swarming, it was hypothesized that SwrT could be an activator of swarming. To test this, swrT+ was cloned into an IPTG-inducible plasmid and transferred into the swrT::Tn5 mutant (LM6507) and the prototype TR strain (LM5674). The resultant strains were examined for swarming. In the absence of IPTG, the basal level of swrT+ expression partially restored swarming to the swrT mutant, although induction with IPTG severely inhibited growth (Fig. 3, bottom two rows). In the swrT+ parent strain (LM5674), the basal level of swrT+ expression appeared to impair cells for growth and/or swarming. Colonies were slightly smaller, and swarming "fingers" were apparent rather than the uniform radial expansion of the vector control colonies (Fig. 3, compare top two rows in left panel). In the presence of IPTG, the TR strain carrying swrT+ grew very poorly. Growth experiments in liquid media showed that in the absence of IPTG, the swrT+ clone was slightly inhibitory to growth and was severely inhibitory with increasing concentrations of IPTG; 1 mM IPTG almost completely inhibited growth (data not shown). As a result, we were unable to fully examine the effect of swrT+ expression on swarming.
Transposon mutagenesis to look for suppressors of swrT. Although we were unable to test SwrT's role as an activator, we considered an alternative hypothesis. Rather than act as an activator of swarming, SwrT could repress a downstream repressor of swarming such that mutation of swrT allowed expression of the secondary repressor, and this resulted in the decreased swarming phenotype of the swrT mutant. This idea seemed reasonable since SwrT is a member of the TetR family of regulators, many of which act as repressors (22). Furthermore, in V. harveyi, LuxT was shown to act as a transcriptional repressor (38). To test this hypothesis and to identify the secondary repressor(s), we decided to look for suppressor mutants. The swarm-defective swrT strain (LM6820) was mutagenized with a Tn5 transposon, and Swarm+ suppressors were selected on Swarm+ plates. These Tn5 mutants were subsequently identified by arbitrary PCR and Southern blot analysis.
Identification of swrZ suppressor mutants. Transposon mutagenesis identified a number of suppressors that conferred a Swarm+ phenotype to the swrT parent; however, we were primarily interested in finding transcriptional regulators of the laf system. Of 100 suppressor mutants that were isolated, three independent mutants contained Tn5 insertions in a single ORF, VP0355, a putative GntR family transcriptional regulator. The Swarm+ phenotype for two of the VP0355 mutants is shown in Fig. 4 (all three mutants had similar phenotypes). Disruption of VP0355 restored the swrT mutant strain to a level of swarming equivalent to that of the TR parent. The VP0355 ORF appears to be monocistronic; 158 bp separate it from the stop codon of the upstream gene (VP0356, pyruvate kinase), and the downstream gene (VP0354) is transcribed in the opposite orientation. Thus, the transposon insertions found in VP0355 should not have any polar effects. BLAST searches indicated that VP0355 has homologs in the three other Vibrio species whose genomes are publicly available (V. cholerae, V. vulnificus, and V. fischeri). No function has been identified for any of these homologs. However, one report describes a homolog, luxZ, in Photobacterium leiognathi; that study showed that transcription of luxZ was independent of the upstream gene (also pyruvate kinase in P. leiognathi) but did not describe a function for LuxZ (37). In keeping with this report and the phenotype found in V. parahaemolyticus, we have designated VP0355 swrZ for swarming regulator homolog to luxZ of P. leiognathi.
swrZ mutants still require iron depletion and polar flagellar inhibition for laf expression. Since swrZ mutants were derepressed for swarming on plates (Fig. 4), we wondered if they might be derepressed for laf expression in liquid, a condition where the prototype TR strain LM5674 does not produce lateral flagella. V. parahaemolyticus requires two conditions before laf expression is derepressed: inhibition of polar flagellar rotation and starvation for iron. These two conditions are met when V. parahaemolyticus is grown on Swarm+ plates; however, they can also be mimicked in liquid media with the use of the drug phenamil, which specifically inhibits the Na+-driven polar flagellar motor (2, 51), and iron-chelating agents (44). The swrT swrZ suppressor mutant and the swrT parent strain were transformed with either the swrT+ plasmid or a vector control to yield a set of strains that are genotypically wild-type, swrT, swrZ, and swrT swrZ. To test whether any of the mutants still required both signals for lateral flagellin production, each strain was grown in liquid media containing no addition, phenamil, the iron chelator dipyridyl, or both phenamil and dipyridyl. Figure 5 shows that strains containing the swrZ mutation by itself or in combination with the swrT mutation still required polar flagellar inhibition and iron starvation for Laf production. As expected for its swarm-defective phenotype, the swrT strain did not express detectable levels of LafA in the presence of phenamil and dipyridyl. As a control, the prototype TR strain, LM5674, was also grown under the same conditions, demonstrating that both signals were required for LafA production in the parent TR background. In addition, the swrZ2::Tn5 mutation was moved into the laf::lux reporter strain (LM5738) and examined for luminescence in liquid throughout growth. Only when both phenamil and dipyridyl were present was laf expression induced (data not shown). Thus, the swrZ mutation does not alleviate the need for either iron starvation or polar flagellar inhibition to signal Laf expression.
SwrZ is a repressor of swarming and laf expression. If SwrZ is in fact a repressor, then expression of swrZ+ from a plasmid should repress swarming. To test this, swrZ+ was cloned into an IPTG-inducible vector. It and the vector control were introduced into the TR strain, LM5674, and swarming was examined on plates lacking or containing 1 mM IPTG. Uninduced, the swrZ+ plasmid had little or no effect, but induction with IPTG showed strong repression of swarming (Fig. 6A). To assess whether SwrZ repressed swarming by affecting laf gene expression, the swrZ+ clone and vector control were introduced into the laf::lux reporter strain (LM5738), and laf expression was monitored from plate-grown cells. Uninduced, the swrZ+ plasmid had little or no effect on laf gene expression, but when induced with IPTG, laf expression was repressed almost completely (Fig. 6B). At the point of maximal laf expression, the vector control strain produced 220,000 SLU, while the swrZ+ expression strain produced only 300 SLU, approximately 700-fold repression; repression was 200-fold or more for all time points. Thus, swrZ prevents swarming via regulation of the laf system.
Expression of swrZ is repressed by SwrT. To further test the hypothesis that SwrT affects swarming by repressing a downstream repressor of swarming, a low-copy swrZ::lacZ reporter plasmid was constructed and introduced into the swrT+ TR strain and the swrT::Tn5 mutant. LacZ activity was measured from liquid-grown and plate-grown cells to determine if SwrT regulated swrZ expression differently under conditions where laf genes are expressed (plate) from those where they are not expressed (liquid). Figure 7 shows that swrZ expression in the swrT background was 15 to 17 times the level of the swrT+ TR strain. This difference was observed in both liquid-grown and plate-grown cells; however, the level of swrZ expression in liquid was approximately two times the level in plate-grown cells. To confirm these results, quantitative RT-PCR was performed on the TR and swrT mutant strains. RNA was harvested from plate-grown cells of the prototype TR strain (LM5674) and the swrT mutant (LM6820) after overnight growth. PCR amplification signals for the two strains were normalized to gylceraldehyde-3-phosphate dehydrogenase (gapA) levels. In two independent experiments (cells grown on different days), swrZ expression from the swrT strain was 4 to 5 times the level of the TR strain. Taken together, these data demonstrate that SwrZ is a repressor of swarming and laf expression, and that SwrT represses swrZ expression.
DISCUSSION
This work identifies three regulators, OpaR, SwrT, and SwrZ, that control swarming in V. parahaemolyticus; a model showing their regulatory activity is shown in Fig. 8. In addition to its role as an activator of cpsA, we show that OpaR represses swarming and laf expression. This result offers new insight to the dichotomy of the swarming phenotype observed between the OP and TR strains. The reduced swarming phenotype of the OP strain is not simply due to enhanced stickiness from increased CPSA production found in OP cells. In addition to upregulating cps, OpaR also prevents transcription of laf genes. Although OpaR appears to be a potent regulator of swarming, TR strains still exhibit a complex surface-dependent control of swarming. In the TR background, mutation of swrT results in a swarm-defective phenotype and does so by decreasing laf expression. SwrT appears to regulate swarming by repressing transcription of the downstream repressor, swrZ. Importantly, the swrZ mutation does not release swarming from surface dependence; i.e., the swrT swrZ mutant and the swrT swrZ/swrT+ strain do not produce LafA in liquid unless both conditions of iron starvation and polar flagellar inhibition are met. This suggests that SwrZ is not responsible for transmitting either the iron starvation or the polar flagellar inhibition signal. It is not known whether SwrZ represses the laf system directly or works through an intermediary. Also, it remains to be determined how swrT and swrZ affect swarming in an OP (opaR+) background and to what environmental cues or molecular signals they respond before exerting their effect on swarming.
OpaR plays a central role in regulating the OP TR switch and the swarming phenotype of the cell, so understanding the regulation and expression of opaR will be critical to revealing the control of opacity and swarming. Since OpaR is 96% identical to LuxR of V. harveyi and expression of LuxR is regulated by the quorum-sensing system in that organism (36), it is reasonable to think that OpaR might also be regulated by a quorum-sensing system. Sequence gazing supports such an idea. The genome of V. parahaemolyticus possesses homologs to all the known components of the quorum-sensing system in V. harveyi, including the three autoinducer systems (AI-1, AI-2, and CqsAS) as well as the central regulatory genes and sRNAs. We are investigating the potential role of quorum sensing in regulating swarming in V. parahaemolyticus, and preliminary evidence indicates that V. parahaemolyticus may not follow the V. harveyi paradigm. In each Vibrio species, quorum-sensing systems seem to exhibit unique variations in their regulatory schemes despite sharing numerous quorum-sensing homologs (7, 27, 42, 50). Thus, we suspect that the V. parahaemolyticus system will present its own variation on the quorum-sensing regulatory theme.
In non-Vibrio species, quorum sensing has been shown to regulate swarming; for example, in S. liquefaciens, quorum sensing has been shown to regulate production of the surfactant serrawettin, which is required for swarming (39). A quorum-sensing mutant (swrI) is unable to swarm but can be restored to wild-type swarming if exogenous surfactant is supplied (13). However, swrI mutants are not defective for flagellar expression or swarmer cell differentiation (21). This differs from our results, which show that OpaR regulates flagellar gene expression and where no specific surfactant has been identified. Although quorum-sensing systems seem ideally suited to coordinate group activities such as swarming, this is not necessarily the case. In P. mirabilis, a luxS quorum-sensing mutant was examined and found not to have a detectable defect in swarming or virulence in a mouse infection model (56). It seems that considerable diversity exists in whether and how different species use quorum-sensing systems to regulate swarming.
In other bacteria, quorum-sensing systems have been shown to control a number of factors important for colonization, virulence, and biofilm formation. An early study in V. cholerae showed that HapR (the homolog to V. harveyi LuxR) was required for expression of hemagglutinin protease (30). It has since been shown that the quorum-sensing system in V. cholerae regulates extracellular polysaccharide and virulence gene expression through HapR (24, 34, 50, 61). HapR represses extracellular polysaccharide expression and biofilm formation in V. cholerae, whereas OpaR activates CPSA and enhances the kinetics of biofilm formation in V. parahaemolyticus (14, 23, 24). Virulence of V. parahaemolyticus also seems to be regulated by OpaR. A recent report showed that a TR (opaR) strain expressed a type three secretion effector protein, and this was prevented in the OP strain (26). We suspect that OpaR may regulate a number of characteristics important for life on a surface, including virulence factors, and that OP and TR (opaR) strains have significantly different genetic programs for control of these characteristics. In V. cholerae, phase variation between hapR+ and hapR– strains has been observed and shown to result in very different phenotypic profiles and virulence properties (24, 34, 61). Similar to V. cholerae, the regulatory differences between OP/TR phase variants in V. parahaemolyticus may be adaptations to specific environmental niches, where they provide a competitive advantage.
Although quorum sensing may be an important factor in the regulation of swarming, other regulatory controls are clearly required. The TR strain still requires two environmental conditions, iron starvation and inhibition of polar flagellar rotation, for laf expression. Thus, we undertook a transposon mutagenesis of a TR strain to identify new components in these signal transduction pathways. SwrT was identified as a potential regulatory mutant. The partial Swarm+ phenotype of the swrT mutant indicates that SwrT has a modulating effect on laf expression but is not the primary regulator of laf. We do not necessarily expect regulatory mutants to be completely abrogated for swarming, since many signals probably converge to regulate laf. Other regulatory signals may override the absence of one signal under the appropriate conditions (e.g., decreasing iron, changes in nutrient availability, cell density, and the state of polar flagellar rotation). Swarmer cell development and laf expression, which represent a large energy commitment to the cell, probably only occur within a narrow window of operating conditions but are otherwise repressed.
SwrT is 94% identical to LuxT of V. harveyi; both are members of the TetR family of transcriptional regulators. This family of proteins has a DNA binding domain and an effector binding/oligomerization domain; TetR regulators typically act as repressors until an effector molecule is bound, whereupon repression is relieved (e.g., TetR represses tetA expression until a tetracycline molecule is bound) (28). In V. harveyi, LuxT has been shown to bind and repress luxOU expression 2-fold, but no effector molecule has been identified (38). Given its effect on swrZ expression, it seems likely that SwrT also acts as a repressor. In V. harveyi, LuxO and LuxU act as the signal integration center for the three autoinducer systems in that organism (17, 18, 27). Autoinducers influence the level of phosphorylated LuxO, which ultimately controls expression of the luxR gene. LuxR is the central output transcriptional regulator that is homologous to OpaR. A luxT mutant has decreased and delayed luminescence early in growth, presumably due to elevated levels of LuxO and LuxU, but is able to achieve full levels of luminescence after reaching stationary phase (38). The quorum-sensing system of V. parahaemolyticus must be characterized before we can determine if SwrT is involved in quorum-sensing regulation. However, based on LuxT's role in the quorum-sensing system in V. harveyi, one would predict that SwrT would regulate the luxOU homologs in V. parahaemolyticus, which would in turn regulate opaR and hence laf expression. Thus, although we have demonstrated that SwrT has a significant role in a strain that lacks the central quorum-sensing output regulator OpaR, it seems likely that SwrT will have additional, or alternate, regulatory effects on swarming in an opaR+ background.
Homologs to swrT are found in the genomes of all the Vibrionaceae species that have been sequenced to date (V. cholerae, V. fischeri, V. vulnificus, and Photobacterium profundum); however, none of these species possesses a second flagellar system or is known to swarm. Beyond the Vibrio spp., SwrT homologs are only found in Shewanella species, which are also predominantly marine organisms. The primary cellular role of SwrT is probably not restricted to swarming but may be related to life in the marine environment where Vibrio and Shewanella species are typically found. In V. harveyi, the luxT mutant was observed to have increased resistance to high salt concentration (4% NaCl) when grown on plates (38). A transcriptome analysis of Shewanella oneidensis in low salt (1% NaCl) versus high salt (5% NaCl) showed that expression of the S. oneidensis swrT decreased 10-fold under the high-salt conditions (41). Although the V. parahaemolyticus swrT mutant did not show enhanced resistance to high salt concentrations, the wild-type parental strains (OP and TR) are highly resistant to salt. LM5312 and LM5674 strains grew well on salt concentrations as high as 6% NaCl (data not shown). We are currently investigating whether SwrT plays a role in the Na+ cycle of V. parahaemolyticus.
Flagellar systems represent a significant expenditure in cellular resources with respect to number of genes, protein synthesis, and organelle operation. Thus, it is not surprising that flagellar systems are highly regulated. Flagellum-mediated swarming represents an even more significant investment due to the elaboration and operation of the many additional flagella and changes in cellular architecture due to filamentation of the swarmer cell. Furthermore, differentiation to a swarmer cell is transient; the swarming edge of a colony goes through cycles of differentiation and dedifferentiation, which may be regulated by temporal and/or spatial factors. Thus, it is expected that the decision to swarm involves many layers of regulation. In swarming bacteria with one flagellar system, a number of genes have been discovered that regulate the flhDC master flagellar operon, including sdiA, umoABCD, and rcsAB, for which no homologs exist in V. parahaemolyticus (8, 16, 54). Conversely, the Enterobacteriaceae do not have homologs to opaR, swrT, or swrZ. Thus, regulation of swarming in the dual flagellar system of V. parahaemolyticus appears to differ significantly from the monoflagellate system of the enterics.
The homology of OpaR, SwrT, and SwrZ to quorum-sensing components in other Vibrionaceae species suggests that these proteins either function directly in the quorum-sensing system of V. parahaemolyticus or that they modulate its activity in some manner. What is perhaps most interesting is that SwrT and SwrZ function in the opaR mutant genetic background. Although quorum-sensing may be important for regulating swarming in V. parahaemolyticus, the fact that both OP and TR strains exhibit differing capacities to swarm suggests that the cell may have alternative regulatory strategies for swarming in these two cell types. It may be that SwrT and SwrZ are part of a mechanism that allows the cell to adapt its swarming pattern according to its OP or TR phase. If so, swarming in V. parahaemolyticus may present a good model system for examining the regulatory adaptations necessary to manage information from sensory systems such as quorum sensing and accommodate the stochastic mutations that typify phase variation.
ACKNOWLEDGMENTS
We thank Yun-Kyeong Kim for excellent molecular biology support and Bonnie Stewart for critical discussion. We are also indebted to Rachel Larsen and Bill Metcalf for providing pRL27 and Barry Wanner for the Red recombination system.
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ABSTRACT
Movement on surfaces, or swarming motility, is effectively mediated by the lateral flagellar (laf) system in Vibrio parahaemolyticus. Expression of laf is induced by conditions inhibiting rotation of the polar flagellum, which is used for swimming in liquid. However, not all V. parahaemolyticus isolates swarm proficiently. The organism undergoes phase variation between opaque (OP) and translucent (TR) cell types. The OP cell produces copious capsular polysaccharide and swarms poorly, whereas the TR type produces minimal capsule and swarms readily. OPTR switching is often the result of genetic alterations in the opaR locus. Previously, OpaR, a Vibrio harveyi LuxR homolog, was shown to activate expression of the cpsA locus, encoding capsular polysaccharide biosynthetic genes. Here, we show that OpaR also regulates swarming by repressing laf gene expression. However, in the absence of OpaR, the swarming phenotype remains tightly surface regulated. To further investigate the genetic controls governing swarming, transposon mutagenesis of a TR (opaR1) strain was performed, and SwrT, a TetR-type regulator, was identified. Loss of swrT, a homolog of V. harveyi luxT, created a profound defect in swarming. This defect could be rescued upon isolation of suppressor mutations that restored swarming. One class of suppressors mapped in swrZ, encoding a GntR-type transcriptional regulator. Overexpression of swrZ repressed laf expression. Using reporter fusions and quantitative reverse transcription-PCR, SwrT was demonstrated to repress swrZ transcription. Thus, we have identified the regulatory link that inhibits swarming of OP strains and have begun to elucidate a regulatory circuit that modulates swarming in TR strains.
INTRODUCTION
Vibrio parahaemolyticus is a gram-negative marine bacterium found in coastal and estuarine waters worldwide (11, 31). It possesses two distinct flagellar systems: the polar system produces a single sheathed flagellum designed for swimming motility, whereas the lateral flagellar (laf) system is adapted for movement over moist or viscous surfaces (3, 46). The lateral flagella are expressed only when the cell senses a surface environment (45). Swarmer cells are elongated (5 to 20 times the length of the swimmer cell) and multinucleoid, and they produce a multitude of lateral flagella. Two environmental signals are known to be required for laf expression: iron-limiting conditions and inhibition of polar flagellar rotation (32, 43, 44). The polar flagellum acts as a tactile sensor for the cell. When the cell encounters a surface or sufficiently viscous environment, flagellar rotation is impaired and laf is induced. Thus, the lateral flagellar system is intimately linked to the polar system. However, the molecular mechanism for sensing polar flagellar inhibition and the signal transduction pathway regulating laf expression are not known. It was the goal of this work to begin to elucidate the regulatory circuit controlling laf expression.
Many bacterial species are known to swarm, including some with mixed polar and peritrichous flagellar systems. In species with dual flagellar systems, such as Rhodospirillum centenum, Azospirillum, and Aeromonas species, surface contact appears to be required for induction of the swarming response, but little is known about how these flagellar systems are regulated (33, 46). Enteric swarming species such as Proteus mirabilis, Salmonella enterica serovar Typhimurium, and Serratia liquefaciens use one flagellar system for both swimming and swarming. In these species, flagellar gene expression is controlled by and dependent upon the global regulator FlhDC (1, 16, 54). In P. mirabilis, transcriptional studies have shown that flhDC is upregulated in swarmer cells (6), and many Swarm– mutants that are defective in signaling swarmer cell differentiation appear to downregulate flhDC expression (12, 25). In S. liquefaciens, flhDC is required for swarmer cell differentiation, and overexpression of flhDC induces swarmer cell differentiation and increased flagellar production (21). In contrast, 54 and LafK, a 54-dependent regulator, regulate the lateral flagellar system of V. parahaemolyticus (59). Analysis of the V. parahaemolyticus genome shows that V. parahaemolyticus does not possess flhDC homologs nor does it possess homologs to many of the swarming regulatory factors that have been identified in enteric species with only one flagellar system. Thus, while swarming in V. parahaemolyticus certainly shares many of the same phenotypic characteristics that are found in other swarming species, the genetic and regulatory features appear to be different.
In addition to the swimmer swarmer cell transition, V. parahaemolyticus switches between opaque (OP) and translucent (TR) cell types. OP cells produce a thick capsular polysaccharide (CPS), which promotes surface adhesion and cell-cell adhesion (15, 48). CPS production by OP strains also affects cell packing and colony structure and is the primary determinant of the opaque appearance of colonies (15). Both clinical and environmental isolates undergo reversible OP TR switching at a low frequency. The environmental cues which lead to OPTR switching are not well understood, but salt and nutrient levels are thought to be important (29, 53). Although the environmental signals have not been well defined, the principal genetic factors determining opacity have been identified. OpaR is the central transcriptional regulator. Disruption of opaR converts the OP strain to the TR phenotype, and expression of OpaR confers the opaque phenotype upon a stable TR (opaR1) strain (48). Other TR strains have also been found to contain lesions in the opaR locus and can be repaired to OP by supplying opaR+ on a plasmid (14; L. L. McCarter, unpublished). Transposon mutagenesis studies of the OP strain revealed that mutations in an 11-gene capsular polysaccharide biosynthesis operon (cpsA locus) turned the OP strain to a nearly TR phenotype, demonstrating that CPSA (the CPS product of the cpsA operon) is the primary component of the OP phenotype (23). It was further shown that OpaR activates cpsA expression. Thus, OpaR regulation of the cpsA locus is the fundamental genetic pathway for the OP TR switch (23).
Although it has been observed that OP strains of V. parahaemolyticus swarm poorly relative to TR strains, a genetic link between swarming and the OP/TR state of the cell has never been demonstrated. Two possibilities could explain this result: the increased CPSA production of OP strains might make the cells "sticky" and inhibit swarming or OpaR might directly repress swarming. We discounted the first possibility when cps mutants in the OP background were still repressed for swarming relative to TR cps mutants (Z. T. Guvener, S. Jaques, and L. L. McCarter, unpublished data). Here, we examine the possibility that OpaR plays a more direct role in controlling swarming of OP strains.
We show that OpaR inhibits swarming and does so by repressing laf gene expression. We further examined swarming in the TR (opaR1) background to identify additional regulators. Transposon mutagenesis for swarm-defective mutants identified swrT (VPA0420), a TetR family regulator. swrT mutants are highly impaired for swarming and laf expression. A second transposon mutagenesis was performed on a swrT mutant to look for Swarm+ suppressors. Three mutants contained insertions in swrZ (VP0355), encoding a GntR family regulator. Overexpression of swrZ repressed swarming of the prototype TR strain and repressed laf expression, confirming that SwrZ acts as a repressor of laf. We further show that SwrT regulates laf gene expression by repressing swrZ expression.
MATERIALS AND METHODS
Strains. The strains and plasmids used in this work are described in Table 1. All V. parahaemolyticus strains are derived from BB22 (4). LM5312 (also known as BB22OP) is the prototype OP strain. LM5674 (also known as BB22TR) is the prototype TR strain; it contains an 85-bp deletion in the upstream and N-terminal coding region of opaR (opaR1) (14, 48). Hence, LM5674 and its derivatives are unable to revert to the OP (opaR+) phenotype. A second TR strain (LM5431), which was derived from LM5312, contains a spontaneous 5-bp insertion in the opaR coding region (opaR2) causing a frameshift mutation (14, 59). The lateral flagellar reporter strain, TR flgBL (LM5738), contains a Tn5lux insertion in a lateral flagellar rod gene and was obtained by transposon mutagenesis of LM5431 (opaR2) (59). The OP flgBL strain was obtained by converting the TR flgBL (LM5738) to opaR+ with the opaR+ cosmid (pLM1950, Tetr) and screening for Tets recombinants.
Media and growth conditions. V. parahaemolyticus strains were grown at 30°C in heart infusion (HI) medium containing 25 g heart infusion (Difco) and 20 g NaCl per liter. Swarm-minus (Swarm–) plates contained HI media with 20 g Difco agar per liter, and Swarm-plus (Swarm+) plates contained HI media with 15 g Bacto agar. Motility plates contained 10 g tryptone, 20 g NaCl, and 3.35 g Bacto agar per liter. Antibiotics were used at final concentrations of 50 μg/ml kanamycin, 10 μg/ml chloramphenicol, 25 μg/ml gentamicin, 100 μg/ml streptomycin, and 10 μg/ml tetracycline or other concentrations as indicated. The iron-chelator dipyridyl (2,2'-dipyridyl; Sigma) was used at 100 μM, and phenamil (methanesulfonate salt; Sigma) was used at 25 μM or as indicated. Expression plasmids were induced with 1 mM isopropyl--D-galactopyranoside (IPTG).
Genetic and molecular techniques. General molecular biology methods were followed (55). Chromosomal DNA was prepared as described previously (60). Insertion/deletion mutations were made in Escherichia coli (LLM2657) using a Red recombinase system (9) to introduce Camr, Kanr, or Genr insertion-deletion mutations onto transposon-junction plasmids or cosmids carrying 20-kb insertions of wild-type V. parahaemolyticus DNA. Recombinants were retransformed into DH5, screened for appropriate drug markers, and analyzed by restriction digest. The insertion-deletion constructs were then conjugated into V. parahaemolyticus, and the standard allelic replacement procedure followed (58). All allelic replacements and transposon mutants were confirmed with Southern blot analysis by using Hybond-NX membranes (Amersham) and [32P]dCTP-labeled DNA probes (Pharmacia).
Transposon mutagenesis of TR strain. The transposon plasmid pRL27 contains a hyperactive transposase gene driven by the tetA promoter (35). An oriR6K gene and a Kanr gene are contained within the inverted repeat ends of the deliverable transposon. The recipient Fla– Laf+ Camr strain (LM4281) was grown overnight, diluted, and grown for approximately 6 h to mid-exponential phase without antibiotic. This culture was mixed with an equal volume of an exponentially growing E. coli strain carrying the transposon donor plasmid pRL27. HI plates with no NaCl were spotted with 10 aliquots of the mating mixture and incubated overnight at 37°C. The mating plates were suspended in 5 ml of 0.3 M sucrose and diluted 10-fold to a final optical density at 600 nm (OD600) of 0.2 to 0.5. The mating suspension (100-μl aliquot) was spread onto HI plates containing chloramphenicol and kanamycin for selection of transposon mutants. Approximately 2,500 colonies were picked onto gridded plates and then transferred onto Swarm and Mot plates for screening. To identify the site of Tn5 insertion of individual mutants, chromosomal DNA was prepared and arbitrarily primed PCR was performed using primers specific to the transposon and a set of arbitrary primers essentially as described previously (52).
swrT2 suppressor mutagenesis. swrT2 suppressor mutagenesis was performed essentially the same as the TR cell mutagenesis, except that the swrT2::lacZ (Camr) strain (LM6820) was the recipient and pools from mutagenesis mating mixtures were plated onto HI Swarm+ plates containing chloramphenicol at 5 μg/ml and kanamycin at 75 μg/ml. Plates were incubated for 3 days, and swarming flares were streaked out for single colonies. Isolated colonies were screened for their swarming phenotype in comparison to the parent and a Swarm+ control strain.
Transposon-junction plasmids. Transposon-junction plasmids were obtained by preparing chromosomal DNA and digested with a restriction enzyme that does not cut within the Tn5 insert, which contains the oriR6K and Kanr markers (35). An aliquot of the digested chromosomal DNA was ligated and transformed into S17-1pir cells. Transformants were selected on LB containing kanamycin and streptomycin. Plasmid from transformants was isolated and examined by restriction digest and then sequenced using transposon-specific primers. To recreate transposon mutants, the transposon-junction plasmid was linearized by digestion with the appropriate restriction enzyme, ligated into the compatibly digested gene replacement vector, pLAFRII, and transformed into DH5 with selection for kanamycin and tetracycline. The transposon-junction-pLAFRII clone could then be used in standard gene replacement procedure (58).
swrT2::lacZ and swrZ::lacZ reporter construction. Several lacZ transcriptional reporters were constructed. SmaI-digested antibiotic resistance cassettes were obtained from p34sCam, pUCGM, or pGM1 (10, 57) and were inserted into the NaeI site in pTL61, which carries a promoterless lacZ gene with an RNase III cleavage site (40) to create a suite of lacZ fusion plasmids. PCR primers were designed and used to amplify the region containing lacZ and the antibiotic cassette (5 kb); the purified PCR product was used to electroporate LLM2657 carrying either the swrT1::Tn5 cosmid (pLM3096) or the swrZ2::Tn5 cosmid (pLM3463). Transformants were selected for the appropriate antibiotic resistance marker and screened for beta-galactosidase activity and Kans. The resultant swrT2::lacZ(Camr) cosmid (pLM3240) and swrZ4::lacZ(Genr) cosmid (pLM3471) were then used for conjugation into V. parahaemolyticus.
Beta-galactosidase assays. Two independent transformants of LM7824 and LM7874 were grown overnight (15 h) in HI broth and as colonies on plates. swrZ-lacZ expression was measured in triplicate from each sample as per Miller (49).
Luminescence measurement. Bioluminescence from plate-grown colonies was examined by exposing overnight colonies in a Fujifilm luminescence image analyzer (LAS-1000) for 1 to 5 min. For time course experiments of plate-grown cells, either overnight cultures (opaR+ experiment) or single colonies (swrT and swrZ+ experiments) of each strain being analyzed were spotted or placed with toothpicks onto a grid array on Swarm+ plates containing antibiotic and 1 mM IPTG, as appropriate, and incubated at 30°C. At each time point, cells were scraped from one or more colonies and suspended in HI, and OD600 and luminescence were measured. Luminescence was quantified by measuring 0.1-ml samples for 30 s in a Turner TD20/22 luminometer (Turner Designs, Sunnyvale, CA). Dilutions were made to keep all measurements within a linear range. Luminescence is reported in specific light units (SLU), which is luminescence min–1 ml–1 OD600–1.
Immunoblots. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed with 12% acrylamide gels according to standard protocols. Samples were normalized to an equal OD600. Immunoblots on polyvinylidene difluoride membranes were probed with pooled antisera raised against either the lateral flagellin protein (LafA antibody no. 127, used at 1:10,000 dilution) or an outer membrane protein (Omp antibody no. 634, used at 1:5,000 dilution). Secondary antibody was peroxidase-conjugated rabbit immunoglobulin G (1:10,000 dilution). The Pierce Supersignal West detection system (Rockford, Ill.) and a Fuji luminescence imager were used for used for signal detection. For examination of LafA from swrZ1 swrT mutants grown in liquid, cultures were inoculated to OD600 of 0.01 in HI plus antibiotic with or without the iron chelator dipyridyl (100 μM) or the polar flagellar inhibitor phenamil (25 μM); cells were harvested at late-logarithmic phase (OD600 = 1.0).
Quantitative RT-PCR. Overday cultures of LM5674 and LM6820 were normalized to an OD600 of 1.0 and diluted 1:1,000, and 25 μl was spread onto Swarm+ plates. Cells were harvested from plates after 15 h of incubation, and RNA was extracted using the QIAGEN RNeasy kit (Valencia, CA). In addition, RNA was treated twice with DNase I. Random hexamers were used to prime total RNA using the Access reverse transcription (RT)-PCR kit (Promega). Primers for quantitative PCR were designed using the Primer Express program (Applied Biosystems) and the RIMD2210633 genome sequence. cDNA was used as a template for quantitative PCR with the SYBR green PCR master mix (Applied Biosystems) and a TaqMan PCR thermocycler (Applied Biosystems) at the University of Iowa DNA Core Facility. Changes in gene expression were calculated by the CT method (User Bulletin no. 2; Applied Biosystems).
RESULTS
OpaR is a repressor of swarming and laf expression. Initial studies of swarming motility in V. parahaemolyticus focused on the TR strain because it was a proficient swarmer when grown on agar plates. By comparison, the OP strain was observed to swarm poorly. Swarming of the prototype OP (LM5312) and TR strains (LM5674, opaR1) is shown in Fig. 1A. The TR colonies have merged together due to their rapid expansion, whereas the OP colonies swarmed minimally. The TR Swarm– and OP Swarm– control strains, which contain Tn5lux insertions in a lateral flagellar rod gene (flgBL), completely failed to swarm.
OpaR activates capsular polysaccharide (CPSA) production, which mediates adhesiveness of cells to each other (15); therefore, it seemed possible that the OP strain was inhibited for swarming simply due to increased CPSA but still made similar amounts of lateral flagella as the TR strain. To test this, we examined lateral flagellin production of cells from actively swarming colonies. Figure 1B shows an immunoblot of cells scraped from the outer edge (2 to 3 mm) of the colonies from the plate in Fig. 1A. The immunoblot was probed with antisera to lateral flagellin (LafA) and an outer membrane protein (Omp), which was used as a loading control. The results showed that the OP strain made significantly less lateral flagellin than did the TR strain.
The OP strain was a poor swarmer and produced much less lateral flagellin, so it was hypothesized that opaR, which is intact in the OP strain, controlled swarming by acting as a negative regulator of laf expression. Lateral flagellar gene expression was examined directly in the OP flgBL::Tn5lux and TR flgBL::Tn5lux luminescence reporter strains, which carry the wild-type opaR (OP flgBL) or the opaR2 allele (TR flgBL), respectively. Figure 1C shows a dark-field photograph of the bottom portion of the plate shown in Fig. 1A. Colonies of the TR flgBL strain produced high levels of luminescence and appear as dark spots in Fig. 1C, whereas colonies of the OP flgBL strain had very low levels of luminescence and did not produce visible spots. This indicated that OpaR was affecting Laf production via repression of laf gene expression. To further test this hypothesis, opaR+ was cloned into an IPTG-inducible expression plasmid and introduced into the TR (opaR2) strain (LM5431) and the laf::lux reporter strain (LM5738), along with the vector control. The swarm-competent strains were tested on Swarm+ plates either lacking or containing 1 mM IPTG. Figure 1D shows that induction of opaR+ repressed swarming and turned the colonies opaque. Next we examined the laf::lux reporter strains to determine whether OpaR repressed laf expression from plate-grown cells. Figure 1E shows that opaR+ repressed laf more than 940-fold at the point of maximal luminescence for the control strain (16,000 versus 17 SLU at 7 h time point) and 180-fold or more from 5 h onward. To control for the possibility that OP strains are unable to transmit light as efficiently as TR strains due to the presence of increased capsular polysaccharide in OP strains, a control mutant containing a Tn5lux fusion in a gene not regulated by OpaR was transformed with the IPTG-inducible opaR+ plasmid and luminescence was examined. In the absence of IPTG, this strain was TR, and in the presence of IPTG, this strain was OP. However, the amount of light produced was equivalent in both growth conditions and, thus, capsular polysaccharide of OP strains does not significantly affect the detection of light (data not shown). Taken together, these data indicate that OpaR is a negative regulator of swarming and acts by repressing laf expression.
Regulation of swarming in a TR strain. Although TR strains are derepressed for swarming due to mutations in the opaR locus, they still require several environmental signals to initiate swarming, namely inhibition of polar flagellar rotation and starvation for iron. Other regulatory factors must be involved in invoking the swarming response in opaR mutant strains. To identify these regulatory factors, a transposon mutagenesis of a TR strain was undertaken and approximately 2,500 transposon mutants were screened for swarming defects. A polar flagellum-defective strain (Fla– Laf+, LM4281) was chosen for mutagenesis because it had a "super" swarming phenotype. It was reasoned that a broader range of swarming defects might be detected in a strain with elevated swarming; furthermore, their effects on motility would be isolated from the polar flagellar system in a Fla– background. One potential regulatory mutant, LM8103, contained a Tn5 insertion in VPA0420, which codes for a TetR family transcriptional regulator. VPA0420 exists as a single open reading frame (ORF), i.e., upstream and downstream genes are transcribed oppositely. BLAST analysis showed that homologs to VPA0420 exist in all the sequenced Vibrio genomes in the public databases; in Vibrio harveyi, the gene has been named luxT. LuxT was shown to affect quorum sensing in V. harveyi by binding to the promoter region of the luxOU operon and repressing luxOU expression (38). We designated VPA0420 as swrT for swarming regulator homolog to luxT of V. harveyi.
SwrT regulates swarming and laf expression in V. parahaemolyticus. The original swrT mutant was found to be swarm defective but not completely blocked for swarming. To confirm this, the swrT::Tn5 mutation was recreated in the parent strain, LM4281 (Fla–Laf+). Figure 2A shows that the recreated swrT::Tn5 mutant was also defective for swarming, demonstrating that the Tn5 insertion was responsible for the phenotype. Similar to the original mutant, swarming was not completely abolished. A lafA mutant strain (LM7805), which contains a Tn5 insertion in the gene for lateral flagellin in the LM4281 background, failed to produce lateral flagella and is shown as a Swarm– control.
To observe the phenotype of a swrT mutation in a Fla+ Laf+ strain and to ensure that the swrT::Tn5 mutation did not generate any partial protein products, a second allele that deleted the majority of the swrT coding region was constructed. The swrT mutation was moved into the prototype TR strain (LM5674) to generate strain LM6820, and its swarming phenotype was analyzed (Fig. 2B). Similar to the swrT::Tn5 mutant, the swrT mutant was severely swarm defective, but swarming was not completely eliminated (compared to the flgBL Swarm– control strain). Thus, compared to their respective parental strains, the two swrT mutations appeared to have similar relative effects on swarming in both the Fla– and Fla+ strains.
Immunoblots to lateral flagellin were performed to determine if the swrT mutation affected swarming due to a disruption of the lateral flagellar system or if swrT affected some other system important for swarming but did not directly affect lateral flagellin production and/or function. Immunoblots were performed on cells harvested from the Swarm+ plates shown in Fig. 2A and B (outer 2 to 3 mm of colonies) and probed with anti-LafA antiserum. Figure 2C shows that both swrT mutants produced significantly less lateral flagellin than their respective parent strains. The swrT::Tn5 allele may cause a more profound defect on LafA production in the Fla– Laf+ background (LM4281) than the swrT allele effect in the Fla+ Laf+ background (LM5674). Whether this difference is due to allele specificity or strain background is not known. Nevertheless, both swrT mutant alleles affected swarming and caused significantly decreased LafA production.
Since SwrT is a member of the TetR family of transcriptional regulators, it seemed likely that swrT mutations affected laf gene expression. To examine this, the swrT::Tn5 mutation was introduced into the chromosome of the laf::lux reporter strain, and laf expression (i.e., luminescence) was measured from plate-grown cells. Figure 2D shows that the parent strain expressed high levels of laf, whereas expression from the swrT mutant was greatly reduced. For example, at the point of maximal laf expression (11 h time point), the parent strain produced 400,000 SLU and the swrT mutant produced 4,400 SLU, a 90-fold decrease. Quantitative RT-PCR experiments using the deletion allele corroborated the reporter results obtained with the Tn5 allele. Comparing RNA harvested from plate-grown LM6820 (swrT) and parental LM5674 revealed a 40- to 50-fold decrease in lafA expression in the swrT mutant strain (data not shown) Thus, swrT mutants were defective for induction of laf gene expression.
A swrT+ clone restores swarming but overexpression is toxic. Since swrT mutants are defective for swarming, it was hypothesized that SwrT could be an activator of swarming. To test this, swrT+ was cloned into an IPTG-inducible plasmid and transferred into the swrT::Tn5 mutant (LM6507) and the prototype TR strain (LM5674). The resultant strains were examined for swarming. In the absence of IPTG, the basal level of swrT+ expression partially restored swarming to the swrT mutant, although induction with IPTG severely inhibited growth (Fig. 3, bottom two rows). In the swrT+ parent strain (LM5674), the basal level of swrT+ expression appeared to impair cells for growth and/or swarming. Colonies were slightly smaller, and swarming "fingers" were apparent rather than the uniform radial expansion of the vector control colonies (Fig. 3, compare top two rows in left panel). In the presence of IPTG, the TR strain carrying swrT+ grew very poorly. Growth experiments in liquid media showed that in the absence of IPTG, the swrT+ clone was slightly inhibitory to growth and was severely inhibitory with increasing concentrations of IPTG; 1 mM IPTG almost completely inhibited growth (data not shown). As a result, we were unable to fully examine the effect of swrT+ expression on swarming.
Transposon mutagenesis to look for suppressors of swrT. Although we were unable to test SwrT's role as an activator, we considered an alternative hypothesis. Rather than act as an activator of swarming, SwrT could repress a downstream repressor of swarming such that mutation of swrT allowed expression of the secondary repressor, and this resulted in the decreased swarming phenotype of the swrT mutant. This idea seemed reasonable since SwrT is a member of the TetR family of regulators, many of which act as repressors (22). Furthermore, in V. harveyi, LuxT was shown to act as a transcriptional repressor (38). To test this hypothesis and to identify the secondary repressor(s), we decided to look for suppressor mutants. The swarm-defective swrT strain (LM6820) was mutagenized with a Tn5 transposon, and Swarm+ suppressors were selected on Swarm+ plates. These Tn5 mutants were subsequently identified by arbitrary PCR and Southern blot analysis.
Identification of swrZ suppressor mutants. Transposon mutagenesis identified a number of suppressors that conferred a Swarm+ phenotype to the swrT parent; however, we were primarily interested in finding transcriptional regulators of the laf system. Of 100 suppressor mutants that were isolated, three independent mutants contained Tn5 insertions in a single ORF, VP0355, a putative GntR family transcriptional regulator. The Swarm+ phenotype for two of the VP0355 mutants is shown in Fig. 4 (all three mutants had similar phenotypes). Disruption of VP0355 restored the swrT mutant strain to a level of swarming equivalent to that of the TR parent. The VP0355 ORF appears to be monocistronic; 158 bp separate it from the stop codon of the upstream gene (VP0356, pyruvate kinase), and the downstream gene (VP0354) is transcribed in the opposite orientation. Thus, the transposon insertions found in VP0355 should not have any polar effects. BLAST searches indicated that VP0355 has homologs in the three other Vibrio species whose genomes are publicly available (V. cholerae, V. vulnificus, and V. fischeri). No function has been identified for any of these homologs. However, one report describes a homolog, luxZ, in Photobacterium leiognathi; that study showed that transcription of luxZ was independent of the upstream gene (also pyruvate kinase in P. leiognathi) but did not describe a function for LuxZ (37). In keeping with this report and the phenotype found in V. parahaemolyticus, we have designated VP0355 swrZ for swarming regulator homolog to luxZ of P. leiognathi.
swrZ mutants still require iron depletion and polar flagellar inhibition for laf expression. Since swrZ mutants were derepressed for swarming on plates (Fig. 4), we wondered if they might be derepressed for laf expression in liquid, a condition where the prototype TR strain LM5674 does not produce lateral flagella. V. parahaemolyticus requires two conditions before laf expression is derepressed: inhibition of polar flagellar rotation and starvation for iron. These two conditions are met when V. parahaemolyticus is grown on Swarm+ plates; however, they can also be mimicked in liquid media with the use of the drug phenamil, which specifically inhibits the Na+-driven polar flagellar motor (2, 51), and iron-chelating agents (44). The swrT swrZ suppressor mutant and the swrT parent strain were transformed with either the swrT+ plasmid or a vector control to yield a set of strains that are genotypically wild-type, swrT, swrZ, and swrT swrZ. To test whether any of the mutants still required both signals for lateral flagellin production, each strain was grown in liquid media containing no addition, phenamil, the iron chelator dipyridyl, or both phenamil and dipyridyl. Figure 5 shows that strains containing the swrZ mutation by itself or in combination with the swrT mutation still required polar flagellar inhibition and iron starvation for Laf production. As expected for its swarm-defective phenotype, the swrT strain did not express detectable levels of LafA in the presence of phenamil and dipyridyl. As a control, the prototype TR strain, LM5674, was also grown under the same conditions, demonstrating that both signals were required for LafA production in the parent TR background. In addition, the swrZ2::Tn5 mutation was moved into the laf::lux reporter strain (LM5738) and examined for luminescence in liquid throughout growth. Only when both phenamil and dipyridyl were present was laf expression induced (data not shown). Thus, the swrZ mutation does not alleviate the need for either iron starvation or polar flagellar inhibition to signal Laf expression.
SwrZ is a repressor of swarming and laf expression. If SwrZ is in fact a repressor, then expression of swrZ+ from a plasmid should repress swarming. To test this, swrZ+ was cloned into an IPTG-inducible vector. It and the vector control were introduced into the TR strain, LM5674, and swarming was examined on plates lacking or containing 1 mM IPTG. Uninduced, the swrZ+ plasmid had little or no effect, but induction with IPTG showed strong repression of swarming (Fig. 6A). To assess whether SwrZ repressed swarming by affecting laf gene expression, the swrZ+ clone and vector control were introduced into the laf::lux reporter strain (LM5738), and laf expression was monitored from plate-grown cells. Uninduced, the swrZ+ plasmid had little or no effect on laf gene expression, but when induced with IPTG, laf expression was repressed almost completely (Fig. 6B). At the point of maximal laf expression, the vector control strain produced 220,000 SLU, while the swrZ+ expression strain produced only 300 SLU, approximately 700-fold repression; repression was 200-fold or more for all time points. Thus, swrZ prevents swarming via regulation of the laf system.
Expression of swrZ is repressed by SwrT. To further test the hypothesis that SwrT affects swarming by repressing a downstream repressor of swarming, a low-copy swrZ::lacZ reporter plasmid was constructed and introduced into the swrT+ TR strain and the swrT::Tn5 mutant. LacZ activity was measured from liquid-grown and plate-grown cells to determine if SwrT regulated swrZ expression differently under conditions where laf genes are expressed (plate) from those where they are not expressed (liquid). Figure 7 shows that swrZ expression in the swrT background was 15 to 17 times the level of the swrT+ TR strain. This difference was observed in both liquid-grown and plate-grown cells; however, the level of swrZ expression in liquid was approximately two times the level in plate-grown cells. To confirm these results, quantitative RT-PCR was performed on the TR and swrT mutant strains. RNA was harvested from plate-grown cells of the prototype TR strain (LM5674) and the swrT mutant (LM6820) after overnight growth. PCR amplification signals for the two strains were normalized to gylceraldehyde-3-phosphate dehydrogenase (gapA) levels. In two independent experiments (cells grown on different days), swrZ expression from the swrT strain was 4 to 5 times the level of the TR strain. Taken together, these data demonstrate that SwrZ is a repressor of swarming and laf expression, and that SwrT represses swrZ expression.
DISCUSSION
This work identifies three regulators, OpaR, SwrT, and SwrZ, that control swarming in V. parahaemolyticus; a model showing their regulatory activity is shown in Fig. 8. In addition to its role as an activator of cpsA, we show that OpaR represses swarming and laf expression. This result offers new insight to the dichotomy of the swarming phenotype observed between the OP and TR strains. The reduced swarming phenotype of the OP strain is not simply due to enhanced stickiness from increased CPSA production found in OP cells. In addition to upregulating cps, OpaR also prevents transcription of laf genes. Although OpaR appears to be a potent regulator of swarming, TR strains still exhibit a complex surface-dependent control of swarming. In the TR background, mutation of swrT results in a swarm-defective phenotype and does so by decreasing laf expression. SwrT appears to regulate swarming by repressing transcription of the downstream repressor, swrZ. Importantly, the swrZ mutation does not release swarming from surface dependence; i.e., the swrT swrZ mutant and the swrT swrZ/swrT+ strain do not produce LafA in liquid unless both conditions of iron starvation and polar flagellar inhibition are met. This suggests that SwrZ is not responsible for transmitting either the iron starvation or the polar flagellar inhibition signal. It is not known whether SwrZ represses the laf system directly or works through an intermediary. Also, it remains to be determined how swrT and swrZ affect swarming in an OP (opaR+) background and to what environmental cues or molecular signals they respond before exerting their effect on swarming.
OpaR plays a central role in regulating the OP TR switch and the swarming phenotype of the cell, so understanding the regulation and expression of opaR will be critical to revealing the control of opacity and swarming. Since OpaR is 96% identical to LuxR of V. harveyi and expression of LuxR is regulated by the quorum-sensing system in that organism (36), it is reasonable to think that OpaR might also be regulated by a quorum-sensing system. Sequence gazing supports such an idea. The genome of V. parahaemolyticus possesses homologs to all the known components of the quorum-sensing system in V. harveyi, including the three autoinducer systems (AI-1, AI-2, and CqsAS) as well as the central regulatory genes and sRNAs. We are investigating the potential role of quorum sensing in regulating swarming in V. parahaemolyticus, and preliminary evidence indicates that V. parahaemolyticus may not follow the V. harveyi paradigm. In each Vibrio species, quorum-sensing systems seem to exhibit unique variations in their regulatory schemes despite sharing numerous quorum-sensing homologs (7, 27, 42, 50). Thus, we suspect that the V. parahaemolyticus system will present its own variation on the quorum-sensing regulatory theme.
In non-Vibrio species, quorum sensing has been shown to regulate swarming; for example, in S. liquefaciens, quorum sensing has been shown to regulate production of the surfactant serrawettin, which is required for swarming (39). A quorum-sensing mutant (swrI) is unable to swarm but can be restored to wild-type swarming if exogenous surfactant is supplied (13). However, swrI mutants are not defective for flagellar expression or swarmer cell differentiation (21). This differs from our results, which show that OpaR regulates flagellar gene expression and where no specific surfactant has been identified. Although quorum-sensing systems seem ideally suited to coordinate group activities such as swarming, this is not necessarily the case. In P. mirabilis, a luxS quorum-sensing mutant was examined and found not to have a detectable defect in swarming or virulence in a mouse infection model (56). It seems that considerable diversity exists in whether and how different species use quorum-sensing systems to regulate swarming.
In other bacteria, quorum-sensing systems have been shown to control a number of factors important for colonization, virulence, and biofilm formation. An early study in V. cholerae showed that HapR (the homolog to V. harveyi LuxR) was required for expression of hemagglutinin protease (30). It has since been shown that the quorum-sensing system in V. cholerae regulates extracellular polysaccharide and virulence gene expression through HapR (24, 34, 50, 61). HapR represses extracellular polysaccharide expression and biofilm formation in V. cholerae, whereas OpaR activates CPSA and enhances the kinetics of biofilm formation in V. parahaemolyticus (14, 23, 24). Virulence of V. parahaemolyticus also seems to be regulated by OpaR. A recent report showed that a TR (opaR) strain expressed a type three secretion effector protein, and this was prevented in the OP strain (26). We suspect that OpaR may regulate a number of characteristics important for life on a surface, including virulence factors, and that OP and TR (opaR) strains have significantly different genetic programs for control of these characteristics. In V. cholerae, phase variation between hapR+ and hapR– strains has been observed and shown to result in very different phenotypic profiles and virulence properties (24, 34, 61). Similar to V. cholerae, the regulatory differences between OP/TR phase variants in V. parahaemolyticus may be adaptations to specific environmental niches, where they provide a competitive advantage.
Although quorum sensing may be an important factor in the regulation of swarming, other regulatory controls are clearly required. The TR strain still requires two environmental conditions, iron starvation and inhibition of polar flagellar rotation, for laf expression. Thus, we undertook a transposon mutagenesis of a TR strain to identify new components in these signal transduction pathways. SwrT was identified as a potential regulatory mutant. The partial Swarm+ phenotype of the swrT mutant indicates that SwrT has a modulating effect on laf expression but is not the primary regulator of laf. We do not necessarily expect regulatory mutants to be completely abrogated for swarming, since many signals probably converge to regulate laf. Other regulatory signals may override the absence of one signal under the appropriate conditions (e.g., decreasing iron, changes in nutrient availability, cell density, and the state of polar flagellar rotation). Swarmer cell development and laf expression, which represent a large energy commitment to the cell, probably only occur within a narrow window of operating conditions but are otherwise repressed.
SwrT is 94% identical to LuxT of V. harveyi; both are members of the TetR family of transcriptional regulators. This family of proteins has a DNA binding domain and an effector binding/oligomerization domain; TetR regulators typically act as repressors until an effector molecule is bound, whereupon repression is relieved (e.g., TetR represses tetA expression until a tetracycline molecule is bound) (28). In V. harveyi, LuxT has been shown to bind and repress luxOU expression 2-fold, but no effector molecule has been identified (38). Given its effect on swrZ expression, it seems likely that SwrT also acts as a repressor. In V. harveyi, LuxO and LuxU act as the signal integration center for the three autoinducer systems in that organism (17, 18, 27). Autoinducers influence the level of phosphorylated LuxO, which ultimately controls expression of the luxR gene. LuxR is the central output transcriptional regulator that is homologous to OpaR. A luxT mutant has decreased and delayed luminescence early in growth, presumably due to elevated levels of LuxO and LuxU, but is able to achieve full levels of luminescence after reaching stationary phase (38). The quorum-sensing system of V. parahaemolyticus must be characterized before we can determine if SwrT is involved in quorum-sensing regulation. However, based on LuxT's role in the quorum-sensing system in V. harveyi, one would predict that SwrT would regulate the luxOU homologs in V. parahaemolyticus, which would in turn regulate opaR and hence laf expression. Thus, although we have demonstrated that SwrT has a significant role in a strain that lacks the central quorum-sensing output regulator OpaR, it seems likely that SwrT will have additional, or alternate, regulatory effects on swarming in an opaR+ background.
Homologs to swrT are found in the genomes of all the Vibrionaceae species that have been sequenced to date (V. cholerae, V. fischeri, V. vulnificus, and Photobacterium profundum); however, none of these species possesses a second flagellar system or is known to swarm. Beyond the Vibrio spp., SwrT homologs are only found in Shewanella species, which are also predominantly marine organisms. The primary cellular role of SwrT is probably not restricted to swarming but may be related to life in the marine environment where Vibrio and Shewanella species are typically found. In V. harveyi, the luxT mutant was observed to have increased resistance to high salt concentration (4% NaCl) when grown on plates (38). A transcriptome analysis of Shewanella oneidensis in low salt (1% NaCl) versus high salt (5% NaCl) showed that expression of the S. oneidensis swrT decreased 10-fold under the high-salt conditions (41). Although the V. parahaemolyticus swrT mutant did not show enhanced resistance to high salt concentrations, the wild-type parental strains (OP and TR) are highly resistant to salt. LM5312 and LM5674 strains grew well on salt concentrations as high as 6% NaCl (data not shown). We are currently investigating whether SwrT plays a role in the Na+ cycle of V. parahaemolyticus.
Flagellar systems represent a significant expenditure in cellular resources with respect to number of genes, protein synthesis, and organelle operation. Thus, it is not surprising that flagellar systems are highly regulated. Flagellum-mediated swarming represents an even more significant investment due to the elaboration and operation of the many additional flagella and changes in cellular architecture due to filamentation of the swarmer cell. Furthermore, differentiation to a swarmer cell is transient; the swarming edge of a colony goes through cycles of differentiation and dedifferentiation, which may be regulated by temporal and/or spatial factors. Thus, it is expected that the decision to swarm involves many layers of regulation. In swarming bacteria with one flagellar system, a number of genes have been discovered that regulate the flhDC master flagellar operon, including sdiA, umoABCD, and rcsAB, for which no homologs exist in V. parahaemolyticus (8, 16, 54). Conversely, the Enterobacteriaceae do not have homologs to opaR, swrT, or swrZ. Thus, regulation of swarming in the dual flagellar system of V. parahaemolyticus appears to differ significantly from the monoflagellate system of the enterics.
The homology of OpaR, SwrT, and SwrZ to quorum-sensing components in other Vibrionaceae species suggests that these proteins either function directly in the quorum-sensing system of V. parahaemolyticus or that they modulate its activity in some manner. What is perhaps most interesting is that SwrT and SwrZ function in the opaR mutant genetic background. Although quorum-sensing may be important for regulating swarming in V. parahaemolyticus, the fact that both OP and TR strains exhibit differing capacities to swarm suggests that the cell may have alternative regulatory strategies for swarming in these two cell types. It may be that SwrT and SwrZ are part of a mechanism that allows the cell to adapt its swarming pattern according to its OP or TR phase. If so, swarming in V. parahaemolyticus may present a good model system for examining the regulatory adaptations necessary to manage information from sensory systems such as quorum sensing and accommodate the stochastic mutations that typify phase variation.
ACKNOWLEDGMENTS
We thank Yun-Kyeong Kim for excellent molecular biology support and Bonnie Stewart for critical discussion. We are also indebted to Rachel Larsen and Bill Metcalf for providing pRL27 and Barry Wanner for the Red recombination system.
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