Lateral Flagellar Gene System of Vibrio parahaemolyticus

Abstract

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 54 -dependent regulator, LafK, was demonstrated to control expression of early genes, and a lateral-specific 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.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 ϳ60 m 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)....

Plasticity of the domain structure in FlgJ, a bacterial protein involved in flagellar rod formation

“…1). The former gene belongs to the gene set responsible for the polar flagellar system (McCarter, 2001), whereas the latter gene belongs to that responsible for the lateral flagellar system (Stewart and McCarter, 2003). Y. pestis also possesses both dual-and single-domain FlgJ homologs, the latter of which is encoded by the flagellar gene cluster homologous to the lateral flagellar gene set of V. parahaemolyticus (Parkhill et al, 2003).…”

“…1). These bacteria have two different sets of flagellar genes (Parkhill et al, 2001;Kaneko et al, 2002;Makino et al, 2003;Stewart and McCarter, 2003), each of which includes either one of the two FlgJ homologs.…”

Genetic determinants of biofilm development of opaque and translucent Vibrio parahaemolyticus

“…OP conversion was observed by streaking 1 month old liquid cultures for single colonies). LM5432 and LM5431 are phosphonomycin-resistant derivatives of LM5312 and LM5395 respectively (Stewart and McCarter, 2003). Phosphonomycin-resistance served as the counterselection for the mutagenesis with pRL27 and its derivatives.…”

“…It appeared that strong promoters were required to confer sufficient levels of chloramphenicol resistance. Subsequently, methods for using Larsen and Metcalf's Tn 5 -RL27 transposon (Larsen et al ., 2002) in V. parahaemolyticus were developed (Stewart and McCarter, 2003) and used to isolate pellicle mutants in both OP (bank II) and TR backgrounds (bank III).…”

“…Three derivatives of Tn5 were employed for transposon mutagenesis: Tn5cat-TS (this work), Tn5-RL27 (Larsen et al, 2002) and Tn5lux-RL27 (Stewart and McCarter, 2003). Tn5cat-TS contains a promoterless reporter gene encoding chloramphenicol resistance, a kanamycin-resistance gene, and an ori6K origin of replication; it was constructed by introducing a promoterless chloramphenicol acetyl transferase gene (derived from pUCM; Schweizer, 1990) into the KpnI site of pTnMod-RKm' (Dennis and Zylstra, 1998) to make pLM2274.…”