Sequence Analysis of Tn phoA Insertion Sites in Vibrio cholerae Mutants Defective in Rugose Polysaccharide Production

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Abstract
Vibrio cholerae can switch from a smooth to a wrinkled or rugose colony phenotype characterized by the secretion of a polysaccharide that enables the bacteria to survive harsh environmental conditions. In order to understand the genetic basis of rugosity, we isolated Tn phoA -induced stable, smooth mutants of two O1 El Tor rugose strains and mapped the insertion sites in several of the mutants using a modified Y-adapter PCR technique. One of the Tn phoA insertions was mapped to the first gene of the vps region that was previously shown to encode the rugose polysaccharide biosynthesis cluster. Three insertions were mapped to a previously unknown hlyA -like gene, also in the vps region. Five other insertions were found in loci unlinked to the vps region: (i) in the epsD gene (encodes the “secretin” of the extracellular protein secretion apparatus), (ii) in a hydG -like gene (encodes a ς 54 -dependent transcriptional activator similar to HydG involved in labile hydrogenase production in Escherichia coli , (iii) in a gene encoding malic acid transport protein upstream of a gene similar to yeiE of E. coli (encodes a protein with similarities to LysR-type transcriptional activators), (iv) in dxr (encodes 1-deoxy- d -xylulose 5-phosphate reductoisomerase), and (v) in the intergenic region of lpd and odp (encode enzymes involved in the pyruvate dehydrogenase complex formation). These data suggest the involvement of a complex regulatory network in rugose polysaccharide production and highlight the general utility of the Y-adapter PCR technique described here for rapid mapping of transposon insertion sites.