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The HWE Histidine Kinases, a New Family of Bacterial Two-Component Sensor Kinases with Potentially Diverse Roles in Environmental Signaling. Baruch Karniol, 2004.Two-component signal transduction pathways play a major role in the response of bacteria to external cues . These pathways are initiated by large collection of histidine kinases (HKs) containing a sensor domain that perceives the environmental signal followed by an HK domain that triggers a histidine-aspartate phosphorelay . Previous phylogenetic analyses identified 11 major families of two-component HKs by comparing signature motifs within the HK domain . Here we describe a new family with homology to Agrobacterium tumefaciens BphP2, an HK first discovered by the presence of a phytochrome sensor domain involved in light perception . Members of this sensor HK family differ from most others by the absence of a recognizable F box and the presence of several uniquely conserved residues, including a histidine in the N box and a tryptophan-X-glutamic acid sequence in the G1 box, which we have used to define the family (HWE) . At least 81 members were identified in a variety of  -
and
 -proteobacteria,
with a significant enrichment in the Rhizobiaceae family . Several
representatives were shown to have HK activity in vitro, supporting
their proposed participation in phosphorelays . One or more domains
related to signal transduction were evident N-terminal to the
HK domain, including chemotactic methyltransferase domains,
suggesting that this family has multiple roles in environmental
signaling . The discovery of the HWE family further extends the
diversity within the HK superfamily and expands the importance of
two-component signaling in bacteria .
Application of Real-Time Quantitative PCR to Molecular Analysis of Candida albicans Strains Exhibiting Reduced Susceptibility to Azoles. Andrew S. Chau, 2004.Real-time quantitative PCR was used to measure expression levels of genes encoding efflux pumps, ERG11 and two control genes, ACT1 and PMA1, in a collection of 14 fluconazole-susceptible Candida albicans isolates . For each gene, average expression levels and variations within the population were determined . These values were then used as reference points to make predictions about the molecular basis of resistance in 38 clinical isolates (the majority of which were resistant to fluconazole) obtained from 18 patients treated with posaconazole for refractory oropharyngeal candidiasis . For each of the 38 isolates, the expression levels of genes encoding efflux pumps, ERG11 and the control genes, were measured as above . Comparison of the two data sets revealed that expression of ACT1 and PMA1 did not vary significantly between the two sets of isolates . In contrast, MDR1, ERG11, CDR1, and CDR2 were overexpressed in 3, 4, 14, and 35, respectively, of the isolates from patients treated with azoles . In addition to these changes, the patient isolates all had at least one and often multiple missense mutations in ERG11 . Select ERG11 alleles were expressed in Saccharomyces cerevisiae; all of the alleles tested conferred reduced susceptibility to fluconazole . Despite both the increases in pump expression and the ERG11 mutations, only one of the patient isolates exhibited a large decrease in posaconazole susceptibility . Control of Enzyme IIscr and Sucrose-6-Phosphate Hydrolase Activities in Streptococcus mutans by Transcriptional Repressor ScrR Binding to the cis-Active Determinants of the scr Regulon. Bing Wang, 2003.In Streptococcus mutans, enzyme IIscr and sucrose-6-phosphate hydrolase are two important enzymes in the transport and metabolism of dietary sucrose . The scr regulon of S . mutans is composed of three genes, scrA and scrB, which code for enzyme IIscr and sucrose-6-phosphate hydrolase, respectively, and scrR, which codes for a GalR-LacI-type transcription regulator . It was previously shown that expression of both scrA and scrB is similarly induced by sucrose . Mutation in the scrR gene resulted in increased expression of scrB relative to that in the wild-type strain . In this study, we employed DNA mobility shift and DNase I protection assays with a purified ScrR-histidine tag fusion protein to examine the DNA binding properties of ScrR to the promoter regions of the scrA and scrB genes . The results showed that ScrR bound specifically to the promoter regions of both scrA and scrB . Two regions with high affinity for ScrR in the promoter sequences of the scrA and scrB genes were identified by DNase I protection assays . One, OC, which includes a 20-bp imperfect inverted-repeat sequence, is located between the two promoters, and the other, OB, is located within the scrB promoter region containing a 37-bp imperfect direct-repeat sequence . Mutations of OB and OC resulted in constitutive transcription and expression of both the scrA and scrB genes . Our results indicated that S . mutans coordinates the activities of enzyme IIscr and sucrose-6-phosphate hydrolase by transcriptional repressor ScrR binding to the promoter regions of the scr regulon .
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