Bordetella bronchiseptica in a paediatric cystic fibrosis patient: possible transmission from a household cat.

Bordetella bronchiseptica is a zoonotic respiratory pathogen commonly found in domesticated farm and companion animals, including dogs and cats. Here, we report isolation of B. bronchiseptica from a sputum sample of a cystic fibrosis patient recently exposed to a kitten with an acute respiratory illness. Genetic characterization of the isolate and comparison with other isolates of human or feline origin strongly suggest that the kitten was the source of infection.

Diversity in the Bordetella virulence regulon: transcriptional control of a Bvg-intermediate phase gene.

The BvgAS signal transduction system controls the expression of at least three distinct phenotypic phases that lie along a continuum of gene expression states. The Bvg+ phase is characterized by the expression of adhesins and toxins, whereas the Bvg- phase is characterized by motility in Bordetella bronchiseptica and the expression of vrg loci in Bordetella pertussis. The Bvg-intermediate (Bvgi) phase is characterized by the absence of Bvg-repressed phenotypes, the expression of some, but not all, Bvg-activated virulence factors and the presence of a recently discovered set of antigens and phenotypes that are unique to this phase. We report here the transcriptional regulation of bipA, the first-identified Bvgi phase gene. We have mapped the bipA promoter and identified numerous BvgA binding sites in the transcriptional control region. Based on these data, we present a model in which phase-dependent expression of bipA results from the spatial distribution and relative affinities of multiple BvgA binding sites relative to the start site of transcription.

ZntR is an autoregulatory protein and negatively regulates the chromosomal zinc resistance operon znt of Staphylococcus aureus.

A chromosomally encoded znt operon of Staphylococcus aureus consists of two consecutive putative genes designated zntR and zntA. The zntA gene encodes a transmembrane protein that facilitates extrusion of Zn2+ and Co2+, whereas the zntR gene encodes a putative regulatory protein that controls the expression of the znt operon. The zntR gene was amplified using the polymerase chain reaction, cloned into Escherichia coli for overexpression as His-tagged ZntR and purified by Ni2+-affinity column. His-tag-free ZntR was purified to near homogeneity after digestion with enterokinase. Electrophoretic mobility shift assays (EMSAs) indicated that the ZntR bound to a fragment of DNA corresponding to the chromosomal znt promoter region with an affinity of about 8.0 x 10-12 M. The addition of 25 microM Zn2+ or Co2+ in the binding reaction completely or significantly inhibited association of ZntR with the znt promoter. DNase I footprinting assays identified a ZntR binding site encompassing 49 nucleotides in the znt promoter region that contained repeated TGAA sequences. These sequences have been proposed to be the binding sites for SmtB, a metallorepressor protein from the cyanobacterium Synechococcus, to its corresponding operator/promoter. In vitro transcription assays, using S. aureus RNA polymerase, revealed that ZntR represses transcription from the znt promoter in a concentration-dependent fashion. The EMSAs, DNase I footprinting and in vitro transcription assays indicate that ZntR is a trans-acting repressor protein that binds to the znt promoter region and regulates its own transcription together with that of zntA.

Alternative transcription factor sigmaSB of Staphylococcus aureus: characterization and role in transcription of the global regulatory locus sar.

A homolog of the multiple-stress-responsive transcription factor sigmaB of Bacillus subtilis was predicted from the DNA sequence analysis of a region of the Staphylococcus aureus chromosome. A hybrid between the coding sequence of the first 11 amino acids of the gene 10 leader peptide of phage T7 (T7.Tag) and the putative sigB gene of S. aureus was constructed and cloned into Escherichia coli BL21(DE3)pLysS for overexpression from a T7 promoter. A homogeneous preparation of the overproduced protein was obtained by affinity chromatography with a T7.Tag monoclonal antibody coupled to agarose. The amino-terminal amino acid sequence of the first 22 residues of the purified protein matched that deduced from the nucleotide sequence. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein, designated sigmaSB, indicated that it migrated as an approximately 39-kDa polypeptide. Promoter-specific transcription from the B. subtilis sigmaB-dependent PB promoter of the sigB operon was stimulated by sigmaSB in a concentration-dependent fashion when reconstituted with the S. aureus core RNA polymerase (RNAP). Specific transcript from the predicted sigmaB-dependent PB promoter of the sigB operon of S. aureus was obtained by the reconstituted RNAP in a runoff transcription reaction. The sar operon of S. aureus contains three promoter elements (P1, P2, and P3) and is known to partly control the synthesis of a number of extracellular toxins and several cell wall proteins. Our in vitro studies revealed that transcription from the P1 promoter is dependent on the primary sigma factor sigmaSA, while that of the P3 promoter is dependent on sigmaSB. As determined by primer extension studies, the 5' end of the sigmaSB-initiated mRNA synthesized in vitro from the sar P3 promoter is in agreement with the 5' end of the cellular RNA.

Characterization of the primary sigma factor of Staphylococcus aureus.

RNA polymerase (RNAP) isolated from Staphylococcus aureus is deficient in sigma factor and is poorly active in transcription assays. Based on amino acid sequence homology of the Bacillus subtilis vegetative sigma factor sigmaA and the predicted product of the chromosomally located plaC gene of S. aureus, it was hypothesized that plaC could encode the vegetative sigma factor. We cloned plaC under a T7 promoter and overexpressed it in Escherichia coli strain BL21(DE3)pLysE. The overproduced protein, present in inclusion bodies, was solubilized with guanidine hydrochloride, renatured, and purified by DEAE-Sephacel and Sephadex G-75 chromatography. The purified protein, designated sigmaSA, cross-reacted with the B. subtilis anti-sigmaA antibody. E. coli core RNAP, reconstituted with sigmaSA, initiated promoter-specific transcription from the S. aureus promoters hla, sea, and sec and from the E. coli promoters rpoH P1, rpoH P4, and ColE1 RNA-1, which are recognized by the E. coli sigma70. sigmaSA, when added to the purified RNAP from S. aureus, stimulated transcriptional activity of the RNAP up to 72-fold. As determined by primer extension studies, the 5'-ends of the sigmaSA-initiated mRNAs synthesized in vitro from the agr P2 and sea promoters are in general agreement with the 5'-ends of the cellular RNAs. Disruption of the plaC gene on the S. aureus chromosome was lethal. We conclude that plaC encodes the primary sigma factor in S. aureus.

Purification and characterization of DNA dependent RNA polymerase from Staphylococcus aureus.

DNA dependent RNA polymerase from exponentially growing Staphylococcus aureus cells was purified. An SDS-polyacrylamide gel analysis of the most purified preparation revealed that it consists of beta, beta', alpha, and sigma with apparent molecular masses of 151, 147, 42, and 55 kDa, respectively. The sigma subunit cross reacted with a polyclonal antibody against Bacillus subtilis sigma 43. The cross reacting peptide co-migrated with the B. subtilis sigma 43 subunit. The implications of these results are discussed. Promoter specific in vitro run-off transcripts were obtained using the purified enzyme preparation. Specific conditions for the polymerization reaction are defined.