Identification of an immunoreactive Brucella abortus HtrA stress response protein homolog

An 11-kb fragment of Brucella abortus genomic DNA cloned into the BamHI site of pUC9 expressed a 60-kDa protein in Escherichia coli DH5-alpha. Antibodies reactive with this 60-kDa protein were detected by Western blot (immunoblot) analysis in sera from mice, cattle, and goats experimentally infected...

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Bibliographic Details
Published in:	Infection and Immunity Vol. 62; no. 3; pp. 1000 - 1007
Main Authors:	Roop, R.M. II, Fletcher, T.W, Sriranganathan, N.M, Boyle, S.M, Schurig, G.G
Format:	Journal Article
Language:	English
Published:	Washington, DC American Society for Microbiology 01-03-1994
Subjects:	Amino Acid Sequence Animals BACTERIA Bacterial Proteins - immunology Bacteriology Base Sequence Biological and medical sciences BRUCELLA ABORTUS Brucella abortus - chemistry Brucella abortus - immunology CLONACION CLONAGE ESTRES TERMICO EXPRESION GENICA EXPRESSION DES GENES Fundamental and applied biological sciences. Psychology GENE GENES GENETICA Genetics GENETIQUE Heat-Shock Proteins - immunology Mice Mice, Inbred BALB C Microbiology Molecular Sequence Data Periplasmic Proteins PROTEINAS PROTEINE SECUENCIA NUCLEICA SEQUENCE NUCLEIQUE Serine Endopeptidases - chemistry Serine Endopeptidases - genetics Serine Endopeptidases - immunology STRESS THERMIQUE Brucellaceae Antigenicity Nucleotide sequence Gene product Heat shock protein Restriction map Bacteria Brucella abortus Stress Aminoacid sequence
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Summary:	An 11-kb fragment of Brucella abortus genomic DNA cloned into the BamHI site of pUC9 expressed a 60-kDa protein in Escherichia coli DH5-alpha. Antibodies reactive with this 60-kDa protein were detected by Western blot (immunoblot) analysis in sera from mice, cattle, and goats experimentally infected with B. abortus, in sera from mice experimentally infected with Brucella melitensis, and in serum from a dog experimentally infected with Brucella canis. Similar results were seen with sera obtained from cattle and dogs with naturally acquired brucellosis. The gene encoding the 60-kDa Brucella protein was localized to a 2-kb EcoRI fragment which was also reactive in Southern blots with genomic DNA from other strains of B. abortus as well as with genomic DNA from B. melitensis and B. canis. Nucleotide sequence analysis of the cloned EcoRI fragment revealed an open reading frame encoding a protein with a predicted molecular mass of 51,847 Da and an isoelectric point of 5.15. Comparison of the deduced amino acid sequence of the immunoreactive Brucella protein with the SWISS-PROT protein sequence data base revealed that it shares 40% amino acid sequence identity with the E. coli and Salmonella typhimurium HtrA stress response proteins. Computer-assisted analysis of this amino acid sequence also predicted that the putative Brucella HtrA homolog contains an export signal sequence and a serine protease active site, two structural features characteristic of previously described HtrA proteins. A potential sigma(E) type heat shock promoter sequence was detected upstream of the cloned Brucella htrA gene, and Northern (RNA) blot analysis demonstrated that exposure of B. abortus 2308 to heat shock conditions resulted in a transient elevation of htrA transcription. These results strongly suggest that the immunoreactive 60-kDa Brucella protein is a member of the HtrA class of stress response proteins
Bibliography:	L73 9509383 ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2
ISSN:	0019-9567 1098-5522
DOI:	10.1128/iai.62.3.1000-1007.1994