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Journal of Bacteriology, June 2004, p. 4014-4018, Vol. 186, No. 12
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.12.4014-4018.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Cross-Regulation in Vibrio parahaemolyticus: Compensatory Activation of Polar Flagellar Genes by the Lateral Flagellar Regulator LafK

Yun-Kyeong Kim and Linda L. McCarter^*

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

Received 26 November 2003/ Accepted 17 March 2004

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Gene organization and hierarchical regulation of the polar flagellar genes of Vibrio parahaemolyticus, Vibrio cholerae, and Pseudomonas aeruginosa appear highly similar, with one puzzling difference. Two ⁵⁴-dependent regulators are required to direct different classes of intermediate flagellar gene expression in V. cholerae and P. aeruginosa, whereas the V. parahaemolyticus homolog of one of these regulators, FlaK, appears dispensable. Here we demonstrate that there is compensatory activation of polar flagellar genes by the lateral flagellar regulator LafK.

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Acting as propellers driven by rotary motors, flagella are highly complex and efficient molecular machines. Flagellar assembly, which occurs via a flagellum-specific type three secretion system, initiates with the formation of a ring structure in the membrane. It proceeds to formation of a basal body hook and culminates with the polymerization of the extracytoplasmic propeller-like filament, subunits of which are secreted through the basal body hook structure (reviewed in reference 9). The assembly of this complex organelle requires the products of more than 35 genes. Flagellar gene expression is carefully regulated by a number of mechanisms such that there are sequential classes of gene expression coupled to morphogenesis of the organelle (reviewed in reference 1).

Although the classes of temporally expressed flagellar genes are generally comparable (with a few variations), the specific regulators and sigma factors controlling early, middle, and late gene expression differ considerably. Flagellar gene organization and the specific transcriptional regulators of polar flagellar genes appear similar in many Vibrio and Pseudomonas species (reviewed in reference 14). In these organisms, ⁵⁴-dependent transcription factors regulate expression of intermediate genes, which encode components of the flagellar export-assembly complex and basal body hook structure. An alternate sigma factor (²⁸) directs late polar gene expression, including transcription of propeller-encoding genes (e.g., flagellin genes). In Vibrio cholerae and Pseudomonas aeruginosa, experiments suggest that there are four classes in the temporal hierarchy of expression, with two subclasses of intermediate genes requiring distinct ⁵⁴-dependent regulators (5, 16). Loss of function of either regulatory gene blocks the flagellar gene expression pathway (2, 8, 16, 17). These ⁵⁴-type transcriptional regulators are also found in Vibrio parahaemolyticus and other members of the family Vibrionaceae. In all of these organisms, the regulators are encoded by two linked operons: flaK and flaLM in V. parahaemolyticus, flrA and flrBC in V. cholerae and V. fischeri, and fleQ and fleSR in P. aeruginosa (Fig. 1). FlrA and FleQ are essential for flagellar formation and activate expression of certain intermediate genes, including those encoding export and assembly components (5, 16). FlrA and FleQ are also required for transcription of the downstream flrBC and fleSR operons, which encode two-component regulatory components (2, 16). The sensor kinase component is presumably autophosphorylated in response to as-yet-undefined signals and acts as the phosphodonor to activate the ⁵⁴-dependent response regulator, which is necessary for expression of other intermediate flagellar genes such as those encoding the hook basal body (4). This regulatory scheme is also pertinent to Vibrio fischeri. Mutation of the V. fischeri flrA renders the organism entirely nonmotile (15).

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FIG. 1. Polar and lateral flagellar regulators. (A) Physical map of the locus encoding the V. parahaemolyticus polar flagellar regulators. A potential rho-independent transcriptional terminator (closed square) occurs between the coding regions for flaK and flaL. Homologous gene clusters are found in other bacteria such as V. cholerae (flrA flrBC) and P. aeruginosa (fleQ fleSR). The percent identities obtained in a pairwise BLAST analysis (22) with the V. parahaemolyticus homolog are provided in parentheses. FlaK, FlaM, and their homologs appear to be transcription factors with potential 54-interacting domains. FlaL and its homologs are potential sensor kinases. FlaM and its homologs, in addition to containing 54-interacting domains, are potential response regulators. The positions of insertions introduced into the flaK coding region are indicated: a chloramphenicol resistance cassette was inserted into the NsiI site at position bp 200 (to make flaK1) and the HpaI site at bp 640 (to make flaK2). (B) The lateral flagellar regulatorygene lafK also encodes a potential 54-interacting transcription factor. LafK is essential for swarming but is not required for swimming motility. The position of the insertion used to disrupt lafK is indicated: a gentamicin resistance lacZ cassette was introduced into the SacI site at bp 154 of the coding region. The percent identities obtained in a BLAST analysis of LafK with its polar flagellar homolog from each organism are shown. (C) Clustal W alignments of FlaK and LafK (http://www.ebi.ac.uk/clustalw). The alignment symbols denoting degree of conservation are as follows: *, residues are identical; :, conserved substitutions; and ., semiconserved substitutions. The top alignment compares FlaK (488 amino acids) in the top line and LafK (444 amino acids) in the lower line. The centrally located, conserved 54-interacting domains (pfam00158) are underlined for both regulatory proteins. Also underlined are N terminally located conserved HTH_8 domains (pfam02954) common to bacterial regulatory proteins in the FIS family. The bottom alignment shows the comparison of the HTH_8 domains.

There has been a puzzling discrepancy between observations of the phenotype of mutants of V. parahaemolyticus that is not in accordance with the regulatory scheme deciphered for other polarly flagellated Vibrio and Pseudomonas species. The flaK gene was discovered as an open reading frame encoding a potential ⁵⁴-dependent regulator located downstream of the flagellin chaperone-encoding gene fliS (formerly named flaJ). The gene was disrupted, but disappointingly it was not found to be important for swimming motility (21). Bacteria with flaK lesions have only slight swimming motility defects, an observation that is not consistent with FlaK being required for intermediate flagellar gene expression. Therefore, another regulatory scenario must be postulated for V. parahaemolyticus. In this work, we reexamined the phenotype of flaK mutants and elucidated a key distinction for polar flagellar regulation in V. parahaemolyticus.

The polar flagellar regulator FlaK is homologous to P. aeruginosa FleQ and V. cholerae FlrA, but loss of flaK is dispensable whereas FleQ and FlrA are essential for swimming motility. In semisolid motility plates, both polar and lateral flagellar systems contribute to motility (3, 13). Moreover, mutations disabling the polar system lead to induction of the lateral system so that impairment of polar flagellar function cannot easily be examined in a swarming-competent strain (12). As a result, defects in swimming motility can best be studied in a strain inactivated for swarming motility. Motility for strain LM1017 (Fla⁺Laf^–) is solely the result of swimming, with no contribution from swarming because of a lesion in the lateral flagellar hook gene (3). In prior work, a chloramphenicol resistance cassette was used to disrupt the flaK-coding region in strain LM1017 (21). Insertions at two restriction sites were used to create the flaK disruptions (Fig. 1). Mutants with flaK lesions showed radial expansion in motility plates (Fig. 2A) and were motile when viewed in the light microscope. To further probe the mutant phenotype, immunoblot analysis of liquid-grown cultures demonstrated that the strains produced polar flagellins (Fig. 2B). Swimming motility and flagellin production were only slightly decreased from the parental strain. For comparison, results for the nonmotile, nonflagellated fliS mutant are shown. The fliS strain (also derived from LM1017) contains a lesion in a polar flagellin chaperone-encoding gene (21).

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FIG. 2. Introduction of mutations in flaK does not abolish swimming motility. (A) Swimming motility in semisolid tryptone motility agar (1% tryptone, 2% NaCl, 0.32% agar) after overnight incubation at room temperature. Three single colonies of each strain were inoculated. (B) Immunoblot of polar flagellin (Fla) profiles with samples prepared from strains grown in heart infusion broth. Immunoblotting conditions have been described (3) except that blots were incubated with primary antiserum for 14 h. The longer incubation period allowed detection of a cross-reacting cellular protein that serves as a loading control. All strains were derived from strain LM1017, which contains a defect in the lateral flagellar hook gene; therefore there is no contribution to motility from the lateral flagella (Fla+ Laf–). Strains: lane 1, LM1017 (flgE313L); lane 2, LM4348 (fliS1::Camr in LM1017); lane 3, LM4347 (flaK1::Camr in LM1017); and lane 4, LM4349 (flaK2::Camr in LM1017). The nonmotile control strain LM4348 contains a defect in the polar flagellar chaperone fliS (formerly named flaJ) (21).

The lateral flagellar regulator is also homologous to FlaK. What is distinctive about V. parahaemolyticus? One clear difference between V. parahaemolyticus and the other polarly flagellated bacteria is that V. parahaemolyticus possesses dual flagellar systems: the polar system is utilized for swimming in dilute liquid environments, and the lateral (or peritrichous) flagella are employed for swarming, i.e., movement over surfaces or through viscous conditions (reviewed in reference 11). The two flagellar gene sets are distinct, and in fact they are found on different chromosomes (10). Approximately 50 polar and 40 lateral flagellar genes have been identified (7, 13, 20). Flagellar gene mutants that have a cell defective for swimming motility are not affected in swarming motility and vice versa. We have recently elucidated the flagellar hierarchy for the lateral system (20). To our surprise, regulation appeared different from the FlhDC-mediated regulation of peritrichous flagella observed for many swarming bacteria including Escherichia coli, Salmonella enterica serovar Typhimurium, and Proteus mirabilis (6). Like the polar system, the lateral flagellar system requires rpoN (encoding ⁵⁴) and a ⁵⁴-dependent transcriptional activator. The lateral transcriptional regulator LafK is homologous to the polar flagellar regulators (Fig. 1). In a pairwise BLAST comparison (22), LafK showed 43% identities and 66% positives with FlaK (E value = 2e-60). A sequence alignment for FlaK and LafK is shown in Fig. 1C; not only is there high similarity observed within the central ⁵⁴interaction domains but there is also considerable alignment in the C-terminal helix-turn-helix domains. This observed similarity between polar and lateral flagellar regulators suggested the hypothesis that perhaps LafK was responsible for compensating for the loss of FlaK.

The lafK gene can substitute for loss of flaK function. To test the idea that the lateral flagellar regulator was responsible for polar flagellar gene expression, a mutant with defects in both flaK and lafK was constructed. The lafK1::Gen^r allele and methods for introduction onto the chromosome via allelic exchange have been described (19, 20). Introduction of lafK1::Gen^r into a swarming-competent strain abolishes swarming motility (20) and production of lateral flagella (Fig. 3A, compare the mutant in lane 2 with the parent in lane 1). These lanes in the immunoblot also show no effect by lafK1 on levels of polar flagellin in the flaK⁺ strain. Introduction of the lafK1 allele into the swimming-only strain LM1017 (strain 3) to make LM7010 had little effect on swimming motility (Fig. 3B, strain 4) or polar flagellin production (Fig. 3A, lane 4). In contrast, introduction of the lafK1 allele into the flaK-defective strain LM4347 (Fig. 3, strain 5) to make the double mutant strain LM6856 (Fig. 3, strain 6) decreased polar flagellin production and swimming motility. These results indicate that the lateral flagellar regulator LafK can compensate for loss of FlaK and direct transcription of polar flagellar genes.

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FIG. 3. flaK or lafK mutants swim, but flaK lafK mutants are impaired for swimming motility. (A) Polar (Fla) and lateral (Laf) flagellin profiles in immunoblots with samples prepared from strains grown overnight on HI plates. Strains: lane 1, LM5674 (wild type, Fla+ Laf+); lane 2, LM7011 (lafK1::Genr in LM5674); lane 3, LM1017 (flgE313L); lane 4, LM7010 (lafK1::Genr in LM1017); lane 5, LM4347 (flaK1::Camr in LM1017); and lane 6, LM6856 (lafK1::Genr flaK1::Camr in LM1017). (B) Swimming motility in semisolid tryptone motility agar. Plates were inoculated with three single colonies of each strain and incubated at room temperature overnight. Strains are numbered as for panel A.

FlaK cannot substitute for LafK. To confirm and extend the above observations, flaK and lafK mutations were recombined singly and in combination into the wild-type chromosome. Our initial studies were performed in a swimming-only strain background that was disabled for swarming motility. This background is most suitable for directly assessing effects on the polar system as contributions from the lateral system mask motility defects in semisolid "swim" plates; however, such a background is not suitable for determining effects on the lateral system. Figure 4 shows that mutations in the polar flagellar genes flaK or fliS do not affect the capacity of strains to swarm. The figure also illustrates the complication of dual flagellar systems and the compensatory action of lateral flagella in swim plates. The rates of radial expansion of flaK or fliS strains in swim plates are equivalent and indistinguishable from those of the wild-type parental strain, even though the fliS strain is completely blocked for polar flagellin synthesis and nonmotile in the absence of lateral flagella while the flaK strain synthesizes almost normal amounts of polar flagellin and can swim in the absence of lateral flagella (Fig. 2 and similar immunoblot data not shown for wild-type backgrounds). Nevertheless, movement on swim and swarm plates is effectively stopped by the introduction of the lafK1 allele into flaK or fliS strains, whereas the lafK1 allele abolished swarming in the wild-type background but had no effect on swimming motility (Fig. 4, top row). We interpret these results to indicate that (i) LafK is not necessary for swimming motility, (ii) FlaK cannot compensate for the loss of LafK by acting on the lateral system, and (iii) LafK is essential for swarming. In summary, the results obtained with the Fla⁺ Laf⁺ background are consistent with our findings obtained with the Fla⁺Laf^– background, specifically that both flaK and lafK must be mutated to cause severe loss of movement in swimming motility plates. Furthermore, the data extend our findings to demonstrate that that loss of LafK is sufficient to eliminate swarming motility, suggesting the FlaK has no significant compensatory role in the lateral system.

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FIG. 4. In the wild-type background, the flaK mutation has a phenotype only in the context of a lafK mutation. Two single colonies of each strain were inoculated on a swarm plate (2.5% heart infusion, 2% NaCl, 1.5% agar [DIFCO]) and into a swim plate (semisolid tryptone motility agar; 0.325% agar [Difco]). Plates were incubated at room temperature overnight. Strains: LM5674 (wild type), LM7011 (lafK1::Genr), LM4471 (flaK1::Camr), LM7305 (flaK1::Camr lafK1::Genr), LM4472 (fliS1::Camr), and LM7395 (fliS1::Camr lafK1::Genr).

Although the introduction of the lafK defect caused a profound loss of motility and polar flagellin production in flaK strains, there was slight residual motility observed in swim plates and some low-level polar flagellin synthesis. In the light microscope, the majority of the cells (>99%) were nonmotile; however an occasional, highly motile bacterium could be observed. The phenotype was observed in both the wild-type and LM1017 backgrounds and with two flaK alleles (data not shown). It is not clear why these strains were not completely defective for polar motility; however, it is probably not due to reversion or suppression. Residual motility was observed in the presence of growth in gentamicin and chloramphenicol to maintain selection for both alleles; it was observed as slow radial expansion in a swim plate and was not due to flares of motility; and if one retested the periphery of the swim zone, the rate of swimming was slow and equivalent to that of the starting strain. Similar residual motility (or rather, lack of a completely nonmotile phenotype) has been reported for flrA mutants of V. cholerae but was not observed in V. fischeri (8, 15).

Thus, the mystery and apparent discrepancy between polar flagellar regulation of V. parahaemolyticus and regulation in other Vibrio or Pseudomonas species can be in part accounted for by the unique, compensatory ability of the second flagellar system of V. parahaemolyticus. LafK was absolutely required for expression of the lateral flagellar genes and swarming motility. In contrast, FlaK was not absolutely required for expression of the polar flagellar genes and swimming motility. Mutants with flaK defects can still swim (albeit with a slightly slower expansion rate) and produce polar flagellins (albeit with a slight decrease in the amount of protein). Introduction of the lafK defect into flaK strains caused a significant decrease in swimming motility and production of polar flagellins. The overlapping activity of the lateral and flagellar regulators is not reciprocal, as flaK cannot compensate for lafK. Thus, this shared regulation occurs in only one direction. It will be interesting to explore what this regulation means in terms of flagellar gene expression and motile behavior. Although distinct gene sets encode each flagellar system, the constituents of the navigation system (i.e., chemotaxis signal transduction) are shared (18). There is only one set of central cytoplasmic chemotaxis phosphorelay-encoding genes, and these are found in association with polar flagellar genes (10). Future work will examine the effect of these mutations on chemotaxis gene expression. Perhaps this cross-regulation of polar flagellar genes by the lateral flagellar regulator is a mechanism to ensure expression or upregulation of the chemotaxis genes when the bacterium is growing on a surface and locomotion is driven by the numerous lateral flagella.

 
This work was supported by National Science Foundation award number MCB-0315617.

 
* 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.

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Journal of Bacteriology, June 2004, p. 4014-4018, Vol. 186, No. 12
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.12.4014-4018.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kim, Y.-K. Articles by McCarter, L. L. Search for Related Content PubMed PubMed Citation Articles by Kim, Y.-K. Articles by McCarter, L. L.