PhoB Regulates Motility, Biofilms, and Cyclic di-GMP inVibrio cholerae

Abstract

Signaling through the second messenger cyclic di-GMP (c-di-GMP) is central to the life cycle of Vibrio cholerae. However, relatively little is known about the signaling mechanism, including the specific external stimuli that regulate c-di-GMP concentration. Here, we show that the phosphate responsive regulator PhoB regulates an operon, acgAB, which encodes c-di-GMP metabolic enzymes. We show that induction of acgAB by PhoB positively regulates V. cholerae motility in vitro and that PhoB regulates expression of acgAB at late stages during V. cholerae infection in the infant mouse small intestine. These data support a model whereby PhoB becomes activated at a late stage of infection in preparation for dissemination of V. cholerae to the aquatic environment and suggest that the concentration of exogenous phosphate may become limited at late stages of infection.Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, is a natural inhabitant of temperate aquatic ecosystems throughout the world, including salt, brackish, and some fresh waters (53). The bacterium is known to associate with various aquatic organisms, including cyanobacteria and copepods; additionally, there is evidence that V. cholerae forms biofilms on chitinous surfaces, such as the exoskeletons of zooplankton and phytoplankton (8,19,49,56).Upon entry into a human host via ingestion of contaminated food or water, the bacteria pass through the gastric acid barrier of the stomach and colonize the small intestine. As the bacterium transitions from its natural environment to that of the small intestine, it undergoes a shift from environmental to virulence gene expression (16,25,27,29,50). This transition includes induction of virulence factors, including the toxin coregulated pilus (TCP), required for colonization of the small intestine, and cholera toxin (CT), the A-B toxin responsible for the profuse secretory diarrhea that is characteristic of cholera (20,50). Additionally, the transition from environmental biofilms to the small intestine is accompanied by a decrease in transcription of Vibrio exopolysaccharide synthesis (vps) genes (42).While the overall mechanism controlling this response to changing environments remains unknown, there is evidence to suggest that the bacterial second messenger bis-(3Ј,5Ј)-cyclic di-GMP (c-di-GMP) plays a major role in mediating this adaptive response in V. cholerae (51, 52). The intracellular concentration of c-di-GMP is regulated by the opposing activities of diguanylate cyclase (DGC) and c-di-GMP phosphodiesterase (PDE) enzymes (3, 46). DGCs, containing a GGDEF domain (named for conserved amino acids), catalyze the formation of c-di-GMP from two GTP molecules, while c-di-GMP molecules are hydrolyzed by PDEs, containing an EAL or HD-GYP domain (6,9,33,38,39,41,48). c-di-GMP metabolic enzymes are well conserved throughout the bacterial kingdom. The V. cholerae genome encodes a total of 61 putative c-di-GMP metabolic enzymes: 30 GGDEF, 12 EAL, 9 HD-GYP, and 10 hybrid GGDEF-EAL domain proteins (13,...

Identification and Characterization of a Phosphodiesterase That Inversely Regulates Motility and Biofilm Formation in Vibrio cholerae

“…The shaded region represents the mean level of WT or ⌬cdgJ Ϯ standard deviations. (5,7,33,42). We showed that CdgJ inversely regulates motility and biofilm formation and functions as a PDE.…”

“…Motility and chemotaxis genes are upregulated in the rabbit ileal loop during detachment, a process termed mucosal escape response (37). It also has been shown that some of the genes encoding c-di-GMP-metabolizing enzymes are induced during the late infection stage and help bacteria to become motile and prepare for the aquatic life cycle (42,49,55). A better understanding of the molecular mechanisms involved in c-di-GMP-regulated motility therefore is critical for understanding the pathogenesis and environmental survival of V. cholerae.…”

“…However, relatively little is known about what the players are and how they function mechanistically in each cellular process. In previous studies, nine of the genes encoding c-di-GMP-metabolizing enzymes were shown to have motility-associated phenotypes in V. cholerae (6,7,9,33,42). These studies have revealed that the c-di-GMP regulation of motility is complex.…”

“…In addition, VieA (PDE) positively regulates motility in the V. cholerae classical biotype but not in the El Tor biotype (7). AcgA (PDE) positively regulates motility at low concentrations of inorganic phosphate (42).…”

Integration of Global Regulatory Mechanisms Controlling Vibrio Cholerae Behavior

“…More recently, it was shown that PhoB negatively affects CT and TCP expression by repressing the tcpPH promoter (Pratt et al, 2010). In addition, PhoB negatively regulates biofilm formation in V. cholerae of classical and El Tor biotypes (Pratt et al, 2009;Sultan et al, 2010). A comparison of the levels of expression of known regulators of vps and biofilm between wild-type and phoB V. cholerae of the El Tor biotype revealed that VpsR, is negatively regulated by PhoB (Sultan et al, 2010).…”