Mutations in the extracellular protein secretion pathway genes (eps) interfere with rugose polysaccharide production in and motility of Vibrio cholerae

Abstract

Vibrio cholerae is the causal organism of the diarrheal disease cholera. The rugose variant of V. cholerae is associated with the secretion of an exopolysaccharide. The rugose polysaccharide has been shown to confer increased resistance to a variety of agents, such as chlorine, bioacids, and oxidative and osmotic stresses. It also promotes biofilm formation, thereby increasing the survival of the bacteria in the aquatic environments. Here we show that the extracellular protein secretion system (gene designated eps) is involved directly or indirectly in the production of rugose polysaccharide. A TnphoA insertion in epsD gene of the eps operon abolished the production of rugose polysaccharide, reduced the secretion of cholera toxin and hemolysin, and resulted in a nonmotile phenotype. We have constructed defined mutations of the epsD and epsE genes that affected these phenotypes and complemented these defects by plasmid clones of the respective wild-type genes. These results suggest a major role for the eps system in pathogenesis and environmental survival of V. cholerae.

FIG. 1

The eps operon of V. cholerae. The eps cluster contains the genes epsC to epsN, and the entire region is 12,077 bp in length. The transcriptional orientation of the eps cluster is indicated by the arrow below the genes. The site of the TnphoA insertion in epsD is indicated by a filled circle, and those of the in-frame Kan^r cassettes are indicated by the filled diamonds. A 146-bp deletion in the epsE gene at the site of Kan^r insertion is indicated by the broken box.

FIG. 2

(A) Smooth and rugose colony morphologies of the wild-type and mutant strains. The various strains were grown to different times, as indicated, in LB agar plates with or without the respective antibiotics. (B) Strain AA1 (epsE::Kan^r) and the complemented strain (epsE::Kan^r/pAA42) after overnight growth.

FIG. 3

Swarming behavior of wild-type and mutant V. cholerae strains on motility agar plates. 1, N16961 R (wild type); 2, NS1 (epsD::TnphoA); 3, AA10 (epsD::Kan^r); 4, AA10/pDSK-2 (epsD⁺); 5, AA1 (epsE::Kan^r); 6, AA1/pAA42 Tet^r (epsE⁺)