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 ⌬flhF mutant makes no flagellum, whereas the ⌬flhFG double-deletion mutant usually lacks a flagellum. However, the ⌬flhFG mutant occasionally reverts to become motile by forming peritrichous flagella. We have isolated a suppressor pseudorevertant from the ⌬flhFG strain (⌬flhFG-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 ⌬flhFG), 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 ⌬flhFG 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. Vibrio alginolyticus, ubiquitously found in marine and estuarine environments, is a halophilic Gram-negative bacterium that is occasionally the cause of human infections. Indeed, reports have shown that this bacterium is responsible for a large portion of wound infections (1, 2), ear infections (otitis media and otitis externa) (3), and some instances of gastroenteritis and chronic diarrhea among immunocompromised patients (4).Flagella are the most common way to facilitate motility in bacteria; they are composed of more than 20 proteins and typically exploit 30 other proteins for their precise regulation and assembly (reviewed elsewhere [5-8]). One obvious application of such a complex flagellar apparatus is to propel bacterial cells through liquids (swimming) and on surfaces (swarming) in response to favorable/unfavorable signals and/or to allow the bacteria to successfully compete with other microorganisms. In addition, bacterial flagella are widely associated with adhesion to surfaces, biofilm formation, and the virulent behaviors of a variety of pathogenic bacteria, such as V. cholerae (9-11) and Helicobacter pylori (12)(13)(14). Although many bacteria possess such organelles, mixed patterns of flagellation are observed in different bacterial species. While some bacteria, such as Escherichia coli (15), have peritrichous (lateral) flagella, others have single (e.g., V. cholerae) or multiple (e.g., H. pylori) polar flagella. Several species have been reported to have a dual flagellar system where they express both polar and lateral flagella on the same cell for distinct purposes, i.e., polar flagella for swimming and lateral flagella for swarming (16). Such organisms include V. parahaem...

HubP, a Polar Landmark Protein, Regulates Flagellar Number by Assisting in the Proper Polar Localization of FlhG in Vibrio alginolyticus

“…Deletion of sflA does not confer any phenotypic change affecting motility, but it causes flagellation at random positions in the absence of both FlhF and FlhG. It was suggested that SflA inhibits flagellation in an FlhF/FlhG-independent manner and regulates the number and position of flagella through a different pathway (23,24).…”

“…ATPase activity and/or ATP binding of FlhG is involved in the polar localization of FlhG (17). In Vibrio species, the DnaJ family protein SflA works as a putative negative regulator of flagellar biogenesis (23,24). Deletion of sflA does not confer any phenotypic change affecting motility, but it causes flagellation at random positions in the absence of both FlhF and FlhG.…”

Localization and domain characterization of the SflA regulator of flagellar formation in Vibrio alginolyticus

“…; Kitaoka et al . ). The Δ flhFG Δ sflA cells showed weak but significantly greater motility than Δ flhFG cells because of the formation of multiple peritrichous flagella (Kitaoka et al .…”

“…Whole‐genome sequencing of the Δ flhF ‐sup and Δ flhFG ‐sup strains showed suppressor mutations in a previously uncharacterized gene unlinked to the flagellar gene cluster (Kitaoka et al . ). This gene was named sflA and its translated protein product contains a cleavable, N‐terminal secretion signal sequence followed by a single transmembrane domain and a C‐terminal J‐domain (also referred as the DnaJ‐domain) (Fig.…”

Mycoplasma pneumoniae, an Underutilized Model for Bacterial Cell Biology

“…Again, mutation of cochaperone-associated amino acids is sufficient to achieve this phenotype. Intriguingly, DnaJ is essential for flagellin synthesis in Escherichia coli (49), and a J domain protein in Vibrio alginolyticus is involved in determining the correct location of that organism's flagellum (50). The roles of DnaJ homologs in macromolecular assembly processes may be underappreciated, and M. pneumoniae TopJ provides an excellent example for study of these roles.…”