Identification of the mutation responsible for the temperature-sensitive lipopolysaccharide O-antigen defect in the Pseudomonas aeruginosa cystic fibrosis isolate 2192.
Pseudomonas aeruginosa in the lungs of cystic fibrosis (CF) patients is characterized by a series of genotypic and phenotypic changes that reflect the transition from acute to chronic infection. These include the overproduction of the exopolysaccharide alginate and the loss of complete lipopolysaccharide (LPS). LPS is a major component of the Gram-negative outer membrane and is composed of lipid A, core oligosaccharide, and O antigen. In this report, we show that the LPS defect of the P. aeruginosa chronic infection isolate 2192 is temperature sensitive. When grown at 25°C, 2192 expresses serotype O1 LPS with a moderate chain length and in reduced amounts relative to those of a wild-type serotype O1 laboratory strain (stO1). In contrast, 2192 expresses no LPS O antigen when grown at 37°C. This is the first time that a temperature-sensitive defect in O-antigen production has been reported. Using complementation analyses with a constructed wbpM deletion mutant of stO1, we demonstrate that the temperature-sensitive O-antigen production defect in 2192 is due to a mutation in wbpM, which encodes a UDP-4,6-GlcNAc dehydratase involved in O-antigen synthesis. The mutation, a deletion of a single amino acid (V636) from the extreme C terminus of WbpM, renders the protein less stable than its wild-type counterpart. This residue of WbpM, which is critical for stability and function, is located outside of the recognized domains of the protein and may provide insight into the structure-function relationship of this enzyme, which is found in all 20 serotypes of P. aeruginosa. We also identify a promoter of wbpM, map a transcriptional start site of wbpM, and show that mucoidy plays a role in the loss of expression of high-molecular-weight LPS in this CF isolate.
Department of Microbiology, Immunology, and Cancer Biology, University of Virginia Health Sciences Center, Charlottesville, VA, USA.
Michael R
MR
Davis
Artur
A
Muszynski
Ivonne V
IV
Lollett
Christopher L
CL
Pritchett
Russell W
RW
Carlson
Joanna B
JB
Goldberg
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D053898:Biosynthetic Pathways / Q000235:genetics*; D003550:Cystic Fibrosis / Q000150:complications; D005816:Genetic Complementation Test; D006801:Humans; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics*; D019081:O Antigens / Q000096:biosynthesis* / Q000235:genetics; D011401:Promoter Regions, Genetic; D055550:Protein Stability; D011552:Pseudomonas Infections / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D017384:Sequence Deletion; D013696:Temperature; D024363:Transcription Initiation Site
D001426:Bacterial Proteins; D019081:O Antigens; C095976:O antigen, Pseudomonas; D006836:Hydro-Lyases; C441972:wbpM protein, Pseudomonas aeruginosa