Cross-regulation in Vibrio parahaemolyticus: compensatory activation of polar flagellar genes by the lateral flagellar regulator LafK

Abstract

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 sigma(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.

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 σ⁵⁴-interacting domains. FlaL and its homologs are potential sensor kinases. FlaM and its homologs, in addition to containing σ54-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 σ⁵⁴-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 σ⁵⁴-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.

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 (flgE313_L); lane 2, LM4348 (fliS1::Cam^r in LM1017); lane 3, LM4347 (flaK1::Cam^r in LM1017); and lane 4, LM4349 (flaK2::Cam^r in LM1017). The nonmotile control strain LM4348 contains a defect in the polar flagellar chaperone fliS (formerly named flaJ) (21).

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::Gen^r in LM5674); lane 3, LM1017 (flgE313_L); lane 4, LM7010 (lafK1::Gen^r in LM1017); lane 5, LM4347 (flaK1::Cam^r in LM1017); and lane 6, LM6856 (lafK1::Gen^r flaK1::Cam^r 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.

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::Gen^r), LM4471 (flaK1::Cam^r), LM7305 (flaK1::Cam^r lafK1::Gen^r), LM4472 (fliS1::Cam^r), and LM7395 (fliS1::Cam^r lafK1::Gen^r).