A novel dnaJ family gene, sflA, encodes an inhibitor of flagellation in marine Vibrio species

Abstract

The marine bacterium Vibrio alginolyticus has a single polar flagellum. Formation of that flagellum is regulated positively and negatively by FlhF and by FlhG, respectively. The ΔflhF mutant makes no flagellum, whereas the ΔflhFG double-deletion mutant usually lacks a flagellum. However, the ΔflhFG mutant occasionally reverts to become motile by forming peritrichous flagella. We have isolated a suppressor pseudorevertant from the ΔflhFG strain (ΔflhFG-sup). The suppressor strain forms peritrichous flagella in the majority of cells. We identified candidate suppressor mutations by comparing the genome sequence of the parental strain, VIO5, with the genome sequences of the suppressor strains. Two mutations were mapped to a gene, named sflA (suppressor of ΔflhFG), at the VEA003730 locus of the Vibrio sp. strain EX25 genome. This gene is specific for Vibrio species and is predicted to encode a transmembrane protein with a DnaJ domain. When the wild-type gene was introduced into the suppressor strain, motility was impaired. Introducing a mutant version of the sflA gene into the ΔflhFG strain conferred the suppressor phenotype. Thus, we conclude that loss of the sflA gene is responsible for the suppressor phenotype and that the wild-type SflA protein plays a role in preventing polar-type flagella from forming on the lateral cell wall.

Fig 1

Schematic diagram summarizing our current knowledge of the flhFG genes and their effects on flagellation. Both overexpression of FlhF and depletion of FlhG lead to an increase in polar flagella. Overexpression of FlhG and depletion of FlhF result in nonflagellated cells, although depletion of FlhF occasionally produces cells with abnormal flagella. flhFG-double-knockout strains usually do not possess flagella; however, a small fraction of this population becomes peritrichously flagellated. A suppressor mutant (ΔflhFG-sup) regains some swarming ability, and most ΔflhFG-sup cells have peritrichous flagella. Abbreviations: Pof, polar flagella; Laf, lateral flagella.

Fig 2

(A) Schematic diagram showing the VEA003729 and sflA genes cloned in pSU21. Four mutations were introduced into the plasmid. BamHI (in the multiple cloning site [MCS] of pSU21)/BglII double digestion removes VEA003729, leaving only sflA (ΔBB); NotI treatment also disables VEA003729 at its N terminus (ΔNot). The digestion with MluI and Eco47III renders sflA nonfunctional (ΔMlu and ΔEco, respectively). (B) Amino acid sequence of the wild-type SflA polypeptide from V. alginolyticus VIO5. Black arrowhead, underlined letters, and gray-shaded sections, predicted signal sequence cleavage site, a transmembrane (TM) domain, and a DnaJ domain, respectively. The suppressor mutations shown by the arrows were found in the sflA gene. The 9 amino acids in parentheses at the end of the sequence were attached to the mutant version of SflA (SflA-Δ9) that was found in the ΔflhFG-sup strain. The substitution shown was found in the ΔflhF-sup strain. (C) Amino acid sequence alignment of the DnaJ domains. When the residues are matched with 100% and 75% identity, the positions are indicated by asterisks and dots, respectively. The sequences aligned were those of DnaJ of E. coli (Ec-DnaJ), CbpA of E. coli (Ec-CbpA), DjlA of E. coli (Ec-DjlA), and SflA of V. alginolyticus (Va-SflA). The numbers in parentheses show the residue numbers of the gene products. The shaded region indicates the highly conserved tripeptide segment of the coil region.

Fig 3

(A) Motility assay showing that SflA expression suppresses the phenotype of the ΔflhFG-sup mutant. Three single colonies of the ΔflhFG and ΔflhFG-sup mutants harboring the plasmid vector pSU21 (vec), plasmid pNT30 with the wild-type (WT) sequence (pSU21::VEA003729 sflA), and pNT40 (pSU21::VEA003729 sflA-Δ9 [Δ9]) were inoculated on VPG soft agar and were incubated for 8 h at 30°C before the pictures were taken. (B) Bacterial strains VIO5, LPN2 (ΔflhFG), LPN2-sup (ΔflhFG-sup), LPN3 (ΔsflA), and LPN3 (ΔflhFG ΔsflA) were grown overnight in VC medium, and 1.5 ml of each culture was inoculated on VPG soft agar plates. The picture was taken after 6.5 h of incubation at 30°C.

Fig 4

Electron micrographs of cells. Cells were negatively stained with potassium phosphotungstate. (A) VIO5 (wild type); (B) LPN3 (ΔsflA); (C) LPN4 (ΔflhFG ΔsflA); (D) LPN2-sup (ΔflhFG-sup). White arrows, positions of the flagellar basal bodies. Bars, 2 μm.

Fig 5

Western blot analysis showing the expression levels of flagellin and FliF for the indicated strains. Midlogarithmically growing cells were harvested by centrifugation, and pelleted samples were subjected to SDS-PAGE and immunoblotting using antibodies against flagellin and FliF, as noted.

Fig 6

Detection of SflA protein. (A) Midlogarithmically growing cells of strains VIO5 (lane 1) and LPN3 (ΔsflA) (lane 2) were harvested and disrupted by sonication. After unbroken cells were removed by low-speed centrifugation, the supernatants were subjected to high-speed centrifugation. The pellets (membrane fraction) were subjected to SDS-PAGE and immunoblotting with the SflA antibody. (B) Midlogarithmically growing cells of the ΔsflA mutant (LPN3) carrying pSU21 (lane 1) or pNT31 (lanes 2 to 5) were fractionated: lane 2, intact cells; lane 3, whole-cell fraction; lane 4, cytoplasmic fraction; lane 5, membrane fraction. The samples were subjected to SDS-PAGE and immunoblotting with the SflA antibody.