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Journal of Bacteriology, August 2003, p. 4508-4518, Vol. 185, No. 15
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.15.4508-4518.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Lateral Flagellar Gene System of Vibrio parahaemolyticus

Bonnie J. Stewart and Linda L. McCarter*

Department of Microbiology, The University of Iowa, Iowa City, Iowa 52242

Received 28 January 2003/ Accepted 1 May 2003


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Vibrio parahaemolyticus possesses dual flagellar systems adapted for movement under different circumstances. A single polar flagellum propels the bacterium in liquid (i.e., swimming) with a motor that is powered by the sodium motive force. Multiple proton-driven lateral flagella enable translocation over surfaces (i.e., swarming). The polar flagellum is produced continuously, while production of lateral flagella is induced when the organism is grown on surfaces. This work describes the isolation of mutants with insertions in the structural and regulatory laf genes. A Tn5-based lux transcriptional reporter transposon was constructed and used for mutagenesis and subsequent transcriptional analysis of the laf regulon. Twenty-nine independent insertions were distributed within 16 laf genes. DNA sequence analysis identified 38 laf genes in two loci. Among the mutants isolated, 11 contained surface-induced lux fusions. A hierarchy of laf gene expression was established following characterization of the laf::lux transcriptional fusion strains and by mutational and primer extension analyses of the laf regulon. The laf system is like many enteric systems in that it is a proton-driven, peritrichous flagellar system; however, laf regulation was different from the Salmonella-Escherichia coli paradigm. There is no apparent flhDC counterpart that encodes master regulators known to control flagellar biosynthesis and swarming in many enteric bacteria. A potential {sigma}54-dependent regulator, LafK, was demonstrated to control expression of early genes, and a lateral-specific {sigma}28 factor controls late flagellar gene expression. Another notable feature was the discovery of a gene encoding a MotY-like product, which previously had been associated only with the architecture of sodium-type polar flagellar motors.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Vibrio parahaemolyticus can differentiate into a specialized cell type, called the swarmer cell, which is adapted for rapid movement on surfaces. Upon differentiation, numerous new organelles—lateral flagella—are peritrichously elaborated on the cell surface (4). Lateral flagella are encoded by one of the two distinct flagellar gene sets in V. parahaemolyticus. Each set contains more than 35 structural and regulatory genes. The polar flagellum, which is sheathed by an extension of the cell outer membrane, is constitutively expressed (48). It is a highly effective propulsive organelle in liquid environments. Rotation speed of the polar flagellum, which acts as a propeller, has been measured to average 1,100 revolutions per s for closely related V. alginolyticus (45, 46). The sodium motive force powers this rotation, and swimming speeds achieve ~60 µm per s (9). However, the polar organelle becomes less effective as viscosity increases, and lateral flagella are induced (11, 47). The proton motive force powers the peritrichous lateral flagella, similar to the peritrichous flagella of Escherichia coli (9). Production of numerous lateral flagella enables many marine Vibrio species to move through viscous environments or over surfaces (10). Some other bacteria possess both peritrichous and polar flagellation, including Aeromonas and Azospirillum species and Rhodospirillum centenum; however, there appears to be some functional overlap of the two kinds of flagella (2, 35, 55, 57, 74). Other swarming bacteria typically regulate gene expression of a single peritrichous system to accommodate the need for different numbers of flagella (28, 29). For example, swarming in Proteus mirabilis and Serratia liquefaciens involves upregulation of flagellar biosynthesis through the master regulatory genes flhD and flhC (22, 24).

For V. parahaemolyticus, the two flagellar systems appear distinct, although central chemotaxis elements are shared (73). All mutants that have been isolated with defects in the polar flagellar system (fla) show no defect in swarming (13, 34, 49). Although the polar gene system and its regulation have been studied in V. parahaemolyticus, the lateral flagellar system (laf) has not been well characterized. This work employs transposon mutagenesis by a Tn5 derivative in combination with the sensitive reporter function of a promoterless luxCDABE operon to analyze the laf gene system of V. parahaemolyticus. Until now, the genetics of dual flagellar systems in a single organism have not been dissected.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains and growth conditions. The strains and plasmids used in this work are described in Table 1. V. parahaemolyticus strains were grown at either 30°C or room temperature (~27°C) in heart infusion medium (HI broth) containing 25 g of heart infusion (Difco) and 20 g of NaCl per liter. Swarm plates were prepared by adding 15 g of Bacto agar (Difco) to HI broth. Swimming medium was made with Vogel-Bonner salts medium (84) supplemented with 20 g of NaCl, 3.25 g of agar per liter, and 0.4% galactose. Translucent V. parahaemolyticus strains were used in this study. V. parahaemolyticus undergoes phase variation between opaque and translucent colony types (51). Translucent strains produce less capsular polysaccharide and are more swarming proficient than opaque strains (20). Strain LM5431 was used as the target strain for transposon mutagenesis. This strain, a derivative of the translucent swarming-proficient strain LM5395, was made spontaneously resistant to phosphonomycin (100 µg/ml) according to the method of Alper and Ames (5). Strain LM5431 is translucent, swarm and swim competent, and a prototroph. Antibiotics were used at final concentrations of 50 µg of kanamycin per ml, 10 µg of chloramphenicol per ml, 60 µg of phosphonomycin per ml, 30 µg of gentamicin per ml, and 10 µg of tetracycline per ml. E. coli strains were maintained with the same antibiotic concentrations as just described, except that 15 µg of gentamicin per ml was used. Overexpression plasmids were induced with 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG).


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  		TABLE 1. Bacterial strains and plasmids

 
Genetic and molecular techniques. General molecular biology methods were adapted from Sambrook et al. (72). Chromosomal DNA was prepared according to the protocol of Woo et al. (87). Some insertion and deletion mutations were made with a {lambda} Red recombinase system (17) to introduce Camr cassettes onto cosmids carrying 40-kb insertions of wild-type V. parahaemolyticus DNA (53) in E. coli. Conjugation and gene replacement methods for V. parahaemolyticus have been described elsewhere (77). All gene replacements were confirmed by Southern blot analysis performed using Hybond-NX membranes (Amersham) and 32P-dCTP-labeled DNA probes (Pharmacia). RNA isolation for surface- and liquid-grown cells was performed using trizol reagent (Gibco BRL) according to the manufacturer's suggestions. Reverse transcription-PCR (RT-PCR) was performed as specified using Access RT-PCR (Promega).

Primer extension experiments were conducted as previously described (34) using RNA (~15 µg/reaction) prepared from plate- and liquid-grown LM5674. The primer extension reactions were repeated at least two times with independently isolated RNA preparations. For all laf genes examined, there were primer extension products for plate-grown but not for liquid-grown cells. As a control to normalize the RNA preparations, polar flagellin transcription was analyzed. For the polar flagellin gene flaA, there were primer extension products for both plate- and liquid-grown cells. The LafA primer extension product was so abundant that the reaction was diluted 1:25 prior to loading on the sequencing gel.

Mutagenesis. Plasmid pRL27 contains a hyperactive Tn5-transposase gene expressed from the tetA promoter (41) and a Tn5-derived transposon containing the oriR6K replication origin and a gene encoding kanamycin resistance. To make the transposon a luminescence reporter, a promoterless luxCDABE operon (19) from Vibrio fischeri was inserted into the transposon on pRL27. A 7-kb {Delta}Plux cassette was cloned from pLM2683 with BamHI into vector pBSL86, thus making pLM2818. The cassette was excised from pLM2818 by using flanking KpnI sites and inserted into KpnI-digested pRL27 to yield pLM2819. Transposon mutagenesis was performed on the phosphonomycin-resistant strain LM5431. The recipient was grown overnight in medium containing phosphonomycin and diluted to an optical density at 600 nm (OD600) of 0.01 followed by 6 h of growth without antibiotic. This culture was mixed with an equal volume of an exponentially growing E. coli strain carrying the transposon delivery plasmid pLM2819. HI plates with no NaCl were spotted with 10 25-µl drops of the mating mixture and grown overnight at 37°C. Cells from each mating plate were suspended in 5 ml of 0.3 M sucrose and diluted 10-fold to a final OD600 value of 0.2 to 0.5. A volume of 100 µl of this mating suspension was spread onto HI medium that contained the phosphonomycin for counterselection of the E. coli donor and kanamycin for selection of V. parahaemolyticus transposon mutants. Approximately 2,000 colonies were picked from each mating and maintained on HI medium containing kanamycin. Southern blot analysis on representative mutants confirmed the randomness of transposition and absence of multiple insertions.

Lateral flagellar mutant identification. Gridded arrays of mutant colonies were transferred to the appropriate media to examine swarming and swimming phenotypes. Colony immunoblots of the swarm- swim+ mutants were used to assess lateral flagellin (LafA) production. Activity of the lux reporter gene was measured by examining light production of the mutants in a luminescence imager (LAS-1000; Fujifilm). Chromosomal DNA was isolated from selected mutants and arbitrarily primed PCR was performed by using primers designed to a transposon end and arbitrary primers ARB6 and ARB2 as described by O'Toole and Kolter (63). The transposon-specific primers used were KANARB1 (5'-GGCGATTCAGGCCTGGTATGAG-3'), KANARB2 (5'-GCATGCAAGCTTCAGGGTTGAG-3'), LUXARB1 (5'-GTGTTCTCTTCGGCGGCGCTGG-3'), and LUXARB2 (5'-GATCCTCGCCGTACTGCCCGC-3'). PCR products were sequenced to identify the interrupted gene and were also radiolabeled to probe a V. parahaemolyticus cosmid library. Subsequent DNA sequence analysis of the region of interest was performed on the retrieved cosmid.

Surface and liquid luminescence assays. Bioluminescence on plates was monitored by exposing overnight colonies in a Fujifilm luminescent image analyzer (LAS-1000) for 30 s to 5 min. For time course experiments on plate-grown cells, strains were grown on plates and suspended to an OD600 of 0.05 and 50 µl was spread onto multiple fresh HI swarm plates with antibiotics when appropriate. Plates were suspended at specified times in 5 ml of HI broth, and OD and relative light unit (RLU) measurements were recorded. Several plates were harvested for early time points to ensure an adequate cell number. For time courses in liquid, overnight cultures were diluted to OD600 values of 0.10 and 30 µl was inoculated into 250-ml flasks containing 15 ml of HI broth. These cultures were incubated with aeration at room temperature with periodic sampling for OD and light readings. Luminescence was quantified by measuring 0.1-ml samples for 30 s in a TD20/22 luminometer (Turner Designs). Dilutions were made to keep all measurements (RLU) within a linear range. Luminescence is reported here as specific light units (SLU), which describe RLU per minute per milliliter per OD600. Each experiment was performed at least two times, with light readings taken in triplicate.

Immunoblots. Whole-cell preparations were made by suspending 16-h plate-grown cells to an OD600 of 2.0 in 0.5 ml of Laemmli sample buffer (40). Samples (5 µl) were run on 12% acrylamide gels and blotted as previously described (13). Immunoblot probes consisted of a pool of polyclonal antibodies raised to the lateral flagellin protein LafA (antibody no. 127) at a concentration of 1:10,000 (54) and to a ~40 kDa V. parahaemolyticus outer membrane protein (antibody no. 634) at a concentration of 1:5,000. Colony immunoblots were performed essentially as described by Meyer et al. (56). Anti-LafA was used at a concentration of 1:1,000 overnight for colony blots.

DNA sequence analysis. DNA sequence determination was performed by the DNA Core Facility of the University of Iowa. Oligonucleotides were manufactured by Integrated DNA Technologies, Inc. (Coralville, Iowa). The KANARB2 and LUXARB2 primers were used to sequence outward from the ends of the transposon. Sequence assembly was performed by using the Genetics Computer Group (GCG) software package. Searches for homology were carried out by the National Center for Biotechnology Information using the BLAST network service (6). Sequence alignments were performed by using the CLUSTAL W program (found at http://www.ebi.ac.uk/clustalw) (83). Only the sequence for which we obtained double-stranded information was deposited in GenBank.

V. parahaemolyticus lateral flagellar GenBank accession numbers are U52957 (lafA-motBL); U52597 (flgNL-flgEL); and AY22518 (fliML-fliJL).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mutagenesis and identification of lateral flagellar (laf) mutants. The V. parahaemolyticus strain LM5431, which is a phosphonomycin-resistant derivative of the translucent strain LM5395, was mutagenized with a Tn5-derived transposon carrying a reporter luxCDABE operon. Approximately 12,000 mutants were screened for swarming defects on 1.5% agar plates. Mutants defective in swarming motility—but still capable of swimming motility—were assayed for the production of lateral flagellin by probing colony immunoblots with an antibody specific to the lateral flagellin protein, LafA. Laf- insertion mutants were examined for laf-dependent lux gene expression by assaying surface growth-dependent light emission. DNA sequence analysis using either arbitrarily primed PCR fragments or plasmids carrying the kanamycin-resistant end of the Tn5 lux and a portion of flanking chromosomal DNA was used to identify transposon-interrupted laf genes. Twenty-nine transposon insertions were identified in 16 lateral flagellar genes (Table 2). All but two of these lateral flagellar genes contained at least one transcriptional fusion, which produced surface-induced light. An example of surface-induced light production for one of the fusion strains is shown in Fig. 1. Light expression of flgHL, encoding a lateral flagellar L-ring component, was increased by at least 1,000-fold when the fusion strain was grown on a surface. All of the mutants with defects in these genes failed to produce lateral flagellin, except strains with insertions in the lateral flagellar motor gene, motAL. The LafA production profiles of a subset of these mutants are shown in the immunoblot in Fig. 2, lanes 2 to 4.


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  		TABLE 2. Lateral flagellar gene system of V. parahaemolyticus

 


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  		FIG. 1. Expression of the lateral flagellar fusion flgHL::lux is induced by growth on a surface. Luminescence of LM5834 was measured in a luminometer during growth in HI agar ({blacksquare}) and HI broth (•).

 


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  		FIG. 2. Lateral flagellin production in V. parahaemolyticus wild-type and mutant strains. HI plate-grown cells were harvested after 16 h. Lanes: 1, LM5674 (wild type); 2, LM5743 (fliDL::Tn5 lux); 3, LM5840 (motYL::Tn5 lux); 4, LM5741 (motBL::Tn5 lux); 5, LM5674 (wild type); 6, LM6210 (fliAL::Camr); 7, LM6092 (lafK::lacZ-Genr); and 8, LM6209 (rpoN::Camr). The immunoblot was probed with polyclonal antisera to lateral flagellin (LafA) at a concentration of 1:10,000 and an outer membrane protein (Omp) at 1:1,000 as a loading control. LafA and Omp are indicated with arrows.

 
Organization of the laf loci. Nucleotide sequence and PCR analysis were used to establish the gene order between insertion mutations and to identify additional potential laf genes. Table 2 lists the predicted genes and gene products in the laf system. A total of 38 open reading frames (ORFs) were found in two distinct regions. DNA sequence analysis revealed little or no intergenic spacing among large groups of ORFs and overlap of potential coding regions. RT PCR was performed by using RNA from surface-grown cells to confirm transcriptional linkage between some ORFs. A PCR product contingent on the presence of both reverse transcriptase and RNA, and similar to that amplified from chromosomal DNA, signified transcriptional coupling between fliRL and flhBL as well as lafKL and fliEL (data not shown). Based on these observations (as well as mutant analysis), at least seven operons within two loci are predicted (shown in Table 2).

A laf-specific regulator: LafK. No counterpart to the master flagellar regulatory operon flhDC of enteric bacteria was found in the laf-encoding regions. Moreover, BLAST analysis of the recently completed V. parahaemolyticus genome (http://genome.gen-info.osaka-u.ac.jp/bacteria/vibrio) does not yield homologous flhD or flhC genes. However, one of the laf-encoding regions contains the coding sequence for a potential regulatory protein, named LafK. The 445-amino acid (aa) predicted protein product contains an N-terminal CheY-like response regulator domain (smart00448; E = 7e-07), a central {sigma}54 interaction domain (pfam00158; E = 4e-87), and a conserved HTH motif (pfam02954; E = 1e-04) at its C terminus. This domain architecture matches that of the AtoC protein family (COG2204; E = 4e-117) and is also similar to that of the polar flagellar regulators FlaK and FlaM of V. parahaemolyticus (34, 79), FlrA and FlrC in Vibrio cholerae (36), and FleQ and FleR in Pseudomonas aeruginosa (7, 71). Amino acid alignment of LafK, other {sigma}54-interacting flagellar proteins, and NtrC from E. coli shows that the four highly conserved residues commonly associated with response regulator receiver domain phosphorylation, corresponding to NtrC residues Asp-11, Asp-12, Asp-54, and Lys-104, are not present in LafK (58, 81). The {sigma}54 interaction domain in LafK extends from aa 126 to 344 and contains two regions involved in ATP hydrolysis. The helix-turn-helix domain spans from aa 390 to 429. No transposon insertions were identified in the lafK coding region; therefore, the mutant strain LM6092 was constructed bearing the lafK::lacZ(Genr) mutation. LM6092 failed to swarm or produce lateral flagellin (Fig. 2, lane 7).

LafK induces lateral flagellar gene expression. The cosmid containing the lafK locus, pLM2910, was transferred to laf::lux reporter strains LM5735 (fliHL) and LM5838 (motYL) for complementation analysis. For both strains, swarming was restored; movement over surfaces was observed upon provision of the lafK+ cosmid pLM2910 but not of an unrelated control cosmid (Fig. 3A). In addition, light on a plate appeared brighter than that for strains carrying a control cosmid (Fig. 3B and C). The lafK+ cosmid also increased luminescence in fusion strains with laf lesions that were not in the locus represented by pLM2910, specifically LM5738 (flgBL), LM5736 (flgLL), and LM5743 (fliDL). For example, light production on a plate was increased on the provision of pLM2910 to LM5738, carrying a fusion to flgBL (Fig. 3B and C), although swarming was not restored (Fig. 3A). These data suggest that restoration of lateral flagellar gene transcription was not simply the result of the restoration of organelle morphogenesis on complementation but rather that a gene product of pLM2910 was activating laf gene expression in trans.



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  		FIG. 3. The lafK+ cosmid induces laf gene expression in trans. Photos are of representative colonies depicting (A) swarming motility after overnight growth on HI medium containing 1.5% agar and tetracycline and (B) light production from 1-min exposures in a luminescence imager after overnight colony growth on HI plates containing 2.0% agar and tetracycline. (C) Light from colonies shown in B was measured in a luminometer in triplicate. Strains: LM6064 (fliHL::lux strain carrying cosmid pLM2910) and LM6065 (fliHL::lux strain carrying control cosmid pLM2102); LM6066 (motYL::lux strain carrying pLM2910) and LM6067 (motYL::lux strain carrying pLM2102); LM6062 (flgBL::lux strain carrying pLM2910) and LM6063 (flgBL::lux strain carrying pLM2102).

 
To test the hypothesis that the potential regulator, LafK, encoded in the flagellar gene cluster on pLM2910 was responsible for the laf induction caused by pLM2910, light production was examined in flgBL, flgLL, motYL, and fliHL lux reporter strains carrying pLM2910 and pLM2910 containing an insertion in lafK (pLM3034). In all cases, the mutated cosmid failed to promote laf::lux gene induction on plates (data not shown). Thus, lafK or a gene in the lafK operon appeared capable of activating laf gene expression. The lafK gene was subsequently subcloned into an expression vector and transferred into flgBL and fliDL reporter strains. Figure 4A shows that in cells harvested from plates, gene expression of flgBL::Tn5 lux was increased when lafK was overexpressed. Light was also induced by lafK overexpression in strain LM5743, containing fliDL::Tn5 lux (data not shown). Moreover, overexpression of lafK resulted in the release of laf genes from their normal surface dependence. As shown in Fig. 4B, induction of flgBL::lux expression occurred in broth-grown cells when lafK expression was driven by the Ptac promoter (LM6108 grown in the presence of IPTG) or by the chloramphenicol resistance gene promoter (LM6107). These data suggest that LafK is a transcriptional regulator of laf gene expression.



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  		FIG. 4. Overexpression of lafK is sufficient to activate lateral flagellar gene expression on plates and in liquid. (A) Cells were harvested from HI plates containing chloramphenicol and 1 mM IPTG: {blacksquare}, LM6108 (flgBL::Tn5 lux carrying lafK+ plasmid pLM3109); {blacktriangleup}, LM6179 (flgBL::Tn5 lux carrying vector pLM1835). (B) Cells were harvested from broth containing chloramphenicol (and IPTG when indicated): {blacksquare}, LM6108 (flgBL::Tn5 lux carrying lafK+ plasmid pLM3109 grown with IPTG); •, LM6108 (lgBL::Tn5 lux carrying lafK+ plasmid pLM3109); {blacktriangleup}, LM6179 (flgBL::Tn5 lux carrying vector pLM1835 grown with IPTG); and {diamondsuit}, LM6109 (flgBL::Tn5 lux carrying pLM3108). Plasmid pLM3109 contains the lafK-coding region aligned for transcription with the IPTG-inducible Ptac promoter. Plasmid pLM3108 contains the lafK-coding region inserted in the opposite direction and aligned for transcription with the chloramphenicol-resistance promoter.

 
Swarming and laf gene expression are {sigma}54 dependent. Regulation by LafK homologs in V. cholerae and P. aeruginosa has been demonstrated to be dependent on interaction with the alternative sigma factor {sigma}54 (36, 71). To determine whether {sigma}54 plays a role in swarming, a small deletion in rpoN was introduced into translucent strain LM5674. Swarming in LM6209 ({Delta}rpoN::Camr) was abolished, as was swimming motility. Moreover, no LafA was produced (Fig. 2, lane 8). To assess the {sigma}54 dependency of lateral flagellar gene expression, rpoN mutations were introduced into LM5735, LM5743, LM5738, and LM5736 with lux reporter fusions in fliHL, fliDL, flgBL, and flgLL, respectively. In all four fusion strains, luminescence was greatly reduced in the absence of RpoN (Fig. 5).



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  		FIG. 5. Lateral flagellar gene expression is dependent on {sigma}54, {sigma}28, and the lafK locus. Luminescence (reported as SLU) of each lux reporter strain was monitored during growth on 1.5% agar HI plates. The following mutations were introduced into each reporter strain: {blacksquare}, no mutation; •, rpoN; {blacktriangleup}, lafK; and {blacklozenge}, fliAL. (A) Reporter strain LM5735 (fliHL::Tn5 lux); (B) reporter strain LM5743 (fliDL::Tn5 lux); (C) reporter strain LM5736 (flgLL::Tn5 lux); (D) reporter strain LM5738 (flgBL::Tn5 lux). SLU light values are the average of three measurements, and standard deviation is less than 5%.

 
{sigma}28-dependent laf gene expression. Within the fliD operon is a gene, fliAL, which encodes a potential sigma factor homologous to FliA ({sigma}28) of E. coli. This gene was previously shown to be required for expression of the V. parahaemolyticus lateral flagellin gene lafA in E. coli (54). In order to determine which of the laf genes are dependent on this {sigma}28-type factor, fliAL deletion and insertion mutations were introduced into the wild-type and laf::Tn5 lux reporter strains. The fliAL gene was confirmed to be required for LafA production as shown by immunoblot analysis of surface-grown cells (Fig. 2, lane 6). Light production of four laf::lux strains with and without the fliAL::Camr mutation was compared (Fig. 5). Expression of fliHL and flgBL was not dependent on {sigma}28 (Fig. 5A and D), whereas expression of fliDL and flgLL was FliAL dependent (Fig. 5B and C). Light production of flgML::lux strain LM5739 was also greatly reduced on the introduction of the fliAL lesion (data not shown).

The genes flgBL and flgLL are located in the same locus but clearly exhibited different regulation by {sigma}28. This suggested that flgKL and flgLL, which encode the two hook-associated proteins, constitute a separate {sigma}28-dependent operon that is distinct from the operon encoding hook basal body components (flgBCDEFGHIJL), which was subsequently confirmed with epistasis experiments (Fig. 6). Upon the introduction of an flgBL deletion-insertion mutation into the flgLL::Tn5 lux reporter strain (to make LM5847), expression of the reporter was greatly reduced. Luminescence was restored when flgBL was provided to LM5847 on a cosmid (to make LM6034), demonstrating that flgBL::Camr was not polar on expression of flgLL::lux in the double-mutant strain. Rather, the data suggest that the flgBL mutation caused a block in the laf hierarchy such that subsequent flagellar gene expression was prevented. Provision of flgBL in trans allowed completion of the hook basal body structure and rescued transcriptional regulation of flgKLL::Tn5 lux.



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  		FIG. 6. Expression of flgBL and flgLL are at different levels in the lateral flagellar hierarchy. (A) Luminescence of colonies of the flgLL reporter strain after overnight growth on HI plates. Plates were exposed in a luminescence imager for 1 min. (B) Luminescence (reported as SLU) of the same colonies was measured in a luminometer in triplicate. Strains: 1, LM5736 (flgLL::Tn5 lux); 2, LM5847 (flgLL::Tn5 lux flgBL::Camr); 3, LM6035 (flgLL::Tn5 lux flgBL::Camr carrying control cosmid pLM2102); and 4, LM6034 (flgLL::Tn5 lux flgBL::Camr carrying flgBL+ cosmid pLM1776). Strains 1 and 2 were grown on HI plates containing kanamycin, and strains 3 and 4 were grown on HI plates containing chloramphenicol and tetracycline.

 
Identification of transcriptional initiation sites. Primer extension analysis using RNA prepared from plate-grown cells was used to identify promoter regions (Fig. 7). The results are summarized in Table 3, which shows the nucleotide sequence upstream of the start point of transcription. Some promoter regions appeared complex, as multiple primer extension products were observed.



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  		FIG. 7. Primer extension analysis of lateral flagellar gene transcription. RNA was prepared from exponentially growing cultures of strain LM5674 that were grown in liquid HI (L) or on HI plates (P). Arrows indicate the primer extension product. Lanes g, a, t, and c correspond to the dideoxy nucleotide used in the DNA sequencing reactions. DNA sequence and primer extension products were generated with the following primers: LafA, 5'-GTGCGTAGTTAGTGTGCATTG-3'; FliDL, 5'-CTATTGAACTCACGAGCTTACTC-3'; FlgBL, 5'-GCTAGGTTACTGGCGAGGAC-3'; FlgML, 5'-GTCGATTTTCATGCTGTGGTC-3'; MotYL, 5'-GCCACTGAAGCTCTATAACTGGCAT-3'; FlgKL, 5'-CGCCGTCACGTCAAGTGCAACGCGG-3'; FlgAL, 5'-GCACCCACAAATCGTACTCCAG-3'; and FliML, 5'-CTCGAATGATATGAATAGGCTTGCC-3'.

 

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  		TABLE 3. Lateral flagellar promoter regionsa

 
Identification of a motY-like gene. Most components of the well-studied Escherichia coli and Salmonella enterica serovar Typhimurium flagellar systems are represented in the lateral system of V. parahaemolyticus, but one ORF particular to the lateral flagellar system was observed. Several mutations were found in a 1-kb ORF divergently transcribed from fliML. The ORF encodes a predicted 340-aa protein with a single transmembrane domain near its N terminus and a C-terminal OmpA domain (COG2885; E = 8e-25). This domain contains an NRRV amino acid sequence that is highly conserved among outer membrane proteins and peptidoglycan-associated proteins. Members of the OmpA domain family include membrane-bound flagellar motor elements MotB and MotY (18, 50). Twelve of the 16 residues marking a potential peptidoglycan interaction domain described in the polar-specific MotY of V. parahaemolyticus (50) are conserved in the carboxyl terminus of this predicted protein, and overall, it shares 25% identity and 42% positives (E = 2 e-15) in a BLAST analysis with MotYP. We have named this ORF motYL and predict that its protein product plays a functional role in lateral flagella. It is clearly not the lateral flagellar motB homolog, which is encoded by motBL and is linked to motAL. The motBL and motAL genes have been shown to complement motility in an E. coli {Delta}motAB mutant (30).

Immediately downstream of motYL is lafK and the genes encoding some of the components required for export and assembly (fliHIJL), a switch component (fliGL), and early basal body formation (fliEFL). Transcription of lafK was linked with fliHL by RT PCR. Mutants with a transposon insertion in motYL fail to synthesize lateral flagellin (Fig. 2, lane 3), but this appears to be due to a polar effect because the mutation could not be complemented by motY on plasmid pLM3019. Introduction of the lafK expression clone (pLM3109) also failed to resurrect swarming in the motYL mutant; however, lateral flagellin production was restored (data not shown). Thus, evidence suggests that MotYL plays role in flagellar rotation; it is required for motility but not for the production of flagella.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The best-understood systems of flagellar regulation are those of E. coli and S. enterica serovar Typhimurium (reviewed in references 1 and 44). Flagellar genes are transcribed in a temporal process in which the timing of gene expression is linked to assembly of the flagellum. At the top of the cascade is the flhDC operon, which encodes a heteromultimeric complex that directs the {sigma}70-dependent activation of class II flagellar genes. Class II genes encode components of the hook basal body, export and assembly proteins, and a flagellar-specific {sigma}28. This sigma factor then directs initiation of transcription at class III flagellar promoters. Morphogenesis of the flagellar organelle is linked to gene expression, as transcription of class III genes—which encode components of the motor, propeller, and navigation system (i.e., chemotaxis)—is prohibited until an anti-{sigma}28 factor (FlgM) can be exported through a completed hook basal body structure. The master flagellar operon flhDC also directs transcription of many nonflagellar genes (65). It has been implicated in the regulation of cell division (66, 67), and overproduction is known to induce swarming in many enteric bacteria (22). Although the V. parahaemolyticus lateral gene system encodes proton-powered peritrichous flagella that are upregulated in response to growth on a surface, as are the flagella of E. coli and S. enterica, we have discovered that the hierarchy of the laf system is unlike the system of these enteric bacteria. There are no flhDC counterparts, and the V. parahaemolyticus lateral flagellar system possesses classes of {sigma}54-dependent as well as {sigma}28-dependent genes.

The hierarchical regulation of gene expression and assembly of the polar flagellum of Caulobacter crescentus has also been carefully studied (31, 32, 88). In contrast to E. coli, none of the flagellar genes requires a specialized {sigma}28-type factor for expression, and {sigma}54-dependent transcriptional regulators activate some of the genes. The third known permutation of the flagellar cascade of gene expression is found with the polar flagellar systems of Vibrio species and P. aeruginosa (33, 52, 64). In these organisms, flagellar genes are transcribed sequentially from {sigma}54-dependent and then {sigma}28-dependent promoters.

Although prior work has demonstrated that the gene encoding the lateral flagellin required a specialized cognate {sigma}28 for expression in E. coli (in contrast to polar flagellin genes, which could be expressed by using E. coli {sigma}28) (54), little else was known about the lateral flagellar gene system and its regulation. To gain this information, we performed a search for mutations in both structural and regulatory flagellar genes of the swarming V. parahaemolyticus strain LM5431. This work describes the first use of the transposon Tn5 in V. parahaemolyticus. A promoterless luxCDABE operon was engineered as the reporter, and approximately 75% of the insertions in lateral flagellar genes resulted in transcriptional fusions, which were then used to probe the hierarchy of regulation. Twenty-nine independent (i.e., nonidentical) insertions were identified within 16 laf genes in two separate loci. The propensity for insertions in some genes and not others suggests some bias in Tn5 transposition in V. parahaemolyticus.

Mapping the genes containing the insertions and further sequencing resulted in identification of a lateral flagellar system containing 38 genes, outlined in Table 2. Region 1 (~14 kb) encodes many of the structural proteins that are assembled to make the hook basal body structure. Region 2 (~25 kb) encodes switch, motor, export-assembly, and flagellin genes. The switch and export-assembly coding regions are divided among two divergently transcribed sets of genes. One predicted operon contains fliMNPQRL and flhBAL; the second operon contains motYL and fliFEGHIJL. Neither of the two laf regions contains a counterpart for fliOL, which is typically found in other flagellar systems. The role of FliO is poorly understood, even in S. enterica serovar Typhimurium and E. coli (75). It has been postulated that FliO is involved in flagellar export, although it shows no homology to other Type III secretion proteins (59). The lateral flagellar gene motYL is a new element of the machinery of the peritrichous, proton-driven flagella. The MotYL predicted protein sequence shows similarities to many peptidoglycan-binding proteins and in particular to the Vibrio polar flagellar protein MotYP. MotYP is thought to function as part of the sodium-type motor. Recent work suggests that MotY localizes to the outer membrane, in contrast to the cytoplasmic location of MotB (60). Until now, the function of motY-like genes has been assigned only to sodium-driven polar motility systems (25, 50, 61, 62). This work reveals that a MotY homolog can play a role in proton-driven peritrichous flagella.

A regulatory gene, lafK, was found immediately downstream of motY. Overexpression of lafK increased gene expression of all laf::lux fusion strains examined. Furthermore, overexpression released gene expression from its normal surface dependence, and fusion strains produced light in liquid. Containing response regulator receiver, {sigma}54-interacting, and DNA-binding motifs, LafK domain architecture suggests that the protein is a response regulator and bears resemblance to the polar flagellar {sigma}54-type regulators FlaK and FlaM (and their homologs in other bacteria). The polar {sigma}54-dependent flagellar regulator FlaM and its homologs FlrCV. cholerae and FleRP. aeruginosa possess the highly conserved residues of typical response regulators. They also have cognate sensor proteins (FlaL, FlrB, and FleS, respectively). In addition, phosphorylation has been demonstrated to be necessary for FlrC-mediated regulation in V. cholerae (15). In contrast, LafK does not possess a cognate sensory protein, nor does it contain the signature highly conserved residues found in the receiver domain of response regulators. In particular, it lacks the site of phosphorylation corresponding to Asp54 of NtrC. In these respects, LafK is most similar to the polar flagellar regulators FlaKV. parahaemolyticus, FlrAV. cholerae, and FleQP. aeruginosa, which also possess no known sensor partners and are not believed to be phosphorylated. Evidence suggests that the activity of nonphosphorylated FleQ is modified posttranslationally by the binding of the antiactivator protein FleN in P. aeruginosa (16). How lafK is regulated, and/or LafK activity modulated, remains to be determined.

Swarming and swimming motility were entirely dependent on {sigma}54, whereas FliAL (lateral {sigma}28) and LafK appear to be swarming-specific regulators, as only swarming was abolished, not swimming, when mutations in the genes were introduced into the wild-type strain. To dissect the regulatory cascade, lux reporter fusions were employed in combination with mutations in rpoN, fliAL, and lafK. Upon the introduction of an rpoN lesion, luminescence was greatly reduced in all fusion strains examined. Expression of fliHL, motYL, and flgBL was not affected by a mutation in fliAL. Expression of fliDL, flgML, and flgLL showed significant FliAL dependence. Primer extension results, which identified the start points of transcription and localized the promoter regions, were generally consistent with the fusion analysis. The regions preceding the transcription initiation sites for lafA, fliDL, flgML, and flgKL contained conserved nucleotides that could represent a {sigma}28-dependent consensus promoter. The promoter region for fliDL appeared to have two transcriptional starts, a major promoter (i.e., strong primer extension band) containing sequences resembling the {sigma}28-dependent consensus, and a weaker promoter containing sequences that might constitute a {sigma}54-type promoter. Such an arrangement would be consistent with the need to have some middle gene expression of the fliDL operon because it encodes {sigma}28 (FliAL). No Tn5 lux fusions were isolated in the potential operon defined by fliMNPQRflhBAL, and for this reason the placement of the operon in the hierarchy is uncertain. Primer extension analysis identified one major and one minor product originating upstream of fliML; however, the upstream sequences do not clearly resemble other promoter regions. This was also true for the major product of motYL. Taken together, these results suggest the backbone of the potential hierarchy diagrammed in Fig. 8. However, it should be emphasized that future work is required to confirm and establish the details of the circuitry.



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  		FIG. 8. Model for the lateral flagellar hierarchy of lateral flagellar gene expression. This scheme, based on lux reporter fusion and primer extension analyses, represents the simplest model for temporal regulation of laf genes. Details of the regulatory cascade remain to be elucidated. No lux fusions were obtained in the fliM operon, so it has not been placed in the hierarchy.

 
Many bacteria have invested heavily in flagellar systems with respect to numbers of genes and amount of protein and energy expended for organelle biogenesis and propulsion. As a result, flagellar systems are carefully regulated, both transcriptionally and posttranscriptionally, by a number of environmental conditions and global regulators (1, 8, 27, 37, 38, 68, 69, 76, 82). Many of these global regulators act to modulate the level of expression of the master flagellar operon flhDC, e.g., CAP, OmpR, H-NS, CsrA, and SdiA (12, 39, 42, 78, 85, 86). V. parahaemolyticus is remarkable in that it possesses two distinct flagellar systems and at times, both flagella are simultaneously assembled. On contact with surfaces, the polarly flagellated swimmer cell differentiates to an elongated swarmer cell and produces hundreds of lateral flagella. The molecular mechanism for these cells' surface recognition and gene expression reprogramming is not known, although surface sensing is linked to polar flagellar performance and iron starvation (48). Recent work has identified a GGDEF-EAL motif containing protein that modulates laf gene expression (14). Since the V. parahaemolyticus lateral system does not possess the FlhDC regulators, how the global coordination of gene expression converges to regulate the lateral flagellar hierarchy will be most interesting to dissect.


   ACKNOWLEDGMENTS
 
We thank Yun-Kyeong Kim and Debbie Noack for their excellent molecular biology support and Sandford Jaques for critical discussion. We are indebted to Rachel Larsen and Bill Metcalf for their kind provision of pRL27 and Barry Wanner for {lambda} Red.

This work was supported by the National Science Foundation research grant MCB0077327 to L.L.M. and the National Institutes of Health training grant T32 AI07511 to B.J.S.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, The University of Iowa, Iowa City, IA 52242. Phone: (319) 335-9721. Fax: (319) 335-7679. E-mail: linda-mccarter{at}uiowa.edu. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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