Document Detail


Identification of rfbA, involved in B-band lipopolysaccharide biosynthesis in Pseudomonas aeruginosa serotype O5.
MedLine Citation:
PMID:  7537247     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Previous work from this laboratory has shown that a 26-kb insert in cosmid clone pFV100, isolated from a Pseudomonas aeruginosa gene library, contained genes that could restore serotype-specific B-band lipopolysaccharide (LPS) expression in rough mutant ge6. In this study, subclones from pFV100 were made to identify genes responsible for B-band LPS synthesis. Transformation of Escherichia coli HB101 with cosmid clone pFV100 resulted in expression of P. aeruginosa serotype O5 B-band LPS, indicating the presence of an rfb cluster in pFV100. Expression of P. aeruginosa LPS could not be achieved in E. coli HB101 transformed with any of the subclones. Complementation studies of well-characterized rough mutants of P. aeruginosa PAO1 deficient in B-band LPS biosynthesis were performed with the various subclones. Subclone pFV110, containing a 1.4-kb XbaI-HindIII insert, restored B-band LPS biosynthesis in mutant AK44 (A+B-; complete core). Probing chromosomal DNA from the 20 International Antigenic Typing Scheme serotypes with the 1.4-kb insert from pFV110 in Southern hybridizations revealed a positive reaction to restriction fragments in serotypes O2, O5, O16, O20, and O18. LPS of serotypes O2, O5, O16, and O20 were shown earlier to have a similar backbone structure in their O antigen. The insert in pFV110 was sequenced, and the deduced amino acid sequence was compared with sequences of protein databases. No significant homology could be detected with any sequences in the database. Open reading frame analysis identified one region, ORF303, which could encode a 33-kDa protein. Using E. coli maxicells for protein expression, orf303 mediated the expression of a unique polypeptide with an apparent molecular mass of 32.5 kDa. The deficiency in the synthesis of B-band LPS biosynthesis in mutant AK44 is apparently complemented by the 33-kDa protein encoded by orf303. We have designated this ORF rfbA. This investigation is the first report on cloning and sequencing of an rfb gene involved specifically in O-antigen biosynthesis in P. aeruginosa PAO1.
Authors:
T Dasgupta; J S Lam
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Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Infection and immunity     Volume:  63     ISSN:  0019-9567     ISO Abbreviation:  Infect. Immun.     Publication Date:  1995 May 
Date Detail:
Created Date:  1995-06-01     Completed Date:  1995-06-01     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  0246127     Medline TA:  Infect Immun     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  1674-80     Citation Subset:  IM    
Affiliation:
Canadian Bacterial Diseases Network, University of Guelph, Ontario, Canada.
Data Bank Information
Bank Name/Acc. No.:
GENBANK/U17293;  Z47767
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Base Sequence
Blotting, Southern
Chromosomes, Bacterial / genetics
Cloning, Molecular
Escherichia coli / genetics
Genes, Bacterial / genetics*
Genetic Complementation Test
Lipopolysaccharides / biosynthesis*
Mannose-6-Phosphate Isomerase*
Molecular Sequence Data
Mutagenesis, Insertional
Nucleotidyltransferases / biosynthesis,  genetics*,  metabolism
O Antigens
Polysaccharides, Bacterial / biosynthesis*
Protein Conformation
Pseudomonas aeruginosa / classification,  genetics*,  metabolism
Recombinant Proteins / biosynthesis
Sequence Analysis, DNA
Serotyping
Chemical
Reg. No./Substance:
0/Lipopolysaccharides; 0/O Antigens; 0/Polysaccharides, Bacterial; 0/Recombinant Proteins; EC 2.7.7.-/Nucleotidyltransferases; EC 2.7.7.-/dTDP-D-glucose synthase; EC 5.3.1.8/Mannose-6-Phosphate Isomerase
Comments/Corrections
Comment In:
Infect Immun. 1995 Dec;63(12):4964-5   [PMID:  7591168 ]

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