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Infection and Immunity, December 2000, p. 6857-6864, Vol. 68, No. 12
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Afsar Ali,1,2 Zahid Hayat Mahmud,1,2 J. Glenn Morris Jr.,1,2 Shanmuga Sozhamannan,1,2,* and Judith A. Johnson2,3

Departments of Epidemiology and Preventive Medicine1 and Pathology,3 University of Maryland School of Medicine, and Veterans Affairs Maryland Health Care System,2 Baltimore, Maryland 21201

Received 10 July 2000/Returned for modification 11 August 2000/Accepted 6 September 2000

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 TnphoA-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 TnphoA 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 sigma 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.

* Corresponding author. Mailing address: Department of Epidemiology and Preventive Medicine, University of Maryland School of Medicine, 934-MSTF, 10 S. Pine St., Baltimore, MD 21201. Phone: (410) 706-5157. Fax: (410) 706-4581. E-mail: ssozhama{at}medicine.umaryland.edu.

Infection and Immunity, December 2000, p. 6857-6864, Vol. 68, No. 12
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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