Cloning, characterization and construction of htrA and htrA-like mutants of Brucella abortus and their survival in BALB/c mice.
A genomic library of Brucella abortus S2308 was screened for expression of recombinant proteins recognized by sera from mice and from cattle infected with B. abortus. A positive clone, BA1, expressing a 50 kDa peptide was recognized by both sera. Plasmid pBA1, isolated from BA1, was shown by restriction enzyme digestion to possess a 1.9 kb insert. The nucleotide sequence of the pBA1 insert revealed an open reading frame with of 1539 bases with a coding capacity of 513 amino acids and a predicted molecular weight of 50,992. The predicted amino acid sequence showed 37% identity to E. coli HtrA, a temperature inducible serine protease. A second B. abortus htrA gene, designated htrA-like, was identified on a different cloned fragment that also encoded B. abortus recA. The nucleotide sequence of the htrA-like gene revealed an open reading frame of 1422 nucleotides with a coding capacity of 474 amino acids and a predicted molecular weight of 50,155. The deduced amino acid sequence of the htrA-like gene showed 42% and 36% identity with B. abortus and E. coli HtrAs respectively. Western blotting of E. coli lysate containing the htrA-like gene was not recognized by sera from B. abortus-infected cattle or mice. B. abortus htrA but not htrA-like relieved the temperature sensitive phenotype and permitted growth of an E. coli htrA mutant at 42 degrees C. B. abortus htrA and htrA-like mutants were constructed and their survival and growth in BALB/c mice was compared to the parental strain S2308.(ABSTRACT TRUNCATED AT 250 WORDS)
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U.S. Department of Agriculture, Agriculture Research Service, Ames, IA 50010.
Cheville
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Morfitt
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D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D001426:Bacterial Proteins / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D002003:Brucella abortus / Q000235:genetics* / Q000502:physiology; D002006:Brucellosis / Q000382:microbiology; D003001:Cloning, Molecular; D015698:Genomic Library; D006360:Heat-Shock Proteins; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D033903:Periplasmic Proteins; D012697:Serine Endopeptidases / Q000235:genetics* / Q000502:physiology; D014169:Transformation, Bacterial
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; D033903:Periplasmic Proteins; C061682:DegP protease; D012697:Serine Endopeptidases