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Identification of Genes Controlled by the Essential YycG/YycF Two-Component System of Staphylococcus aureus. Sarah Dubrac, 2004.The YycG/YycF essential two-component system [TCS], originally identified in Bacillus subtilis, is very highly conserved and appears to be specific to low-G+C gram-positive bacteria, including several pathogens such as Staphylococcus aureus . By studying growth of S . aureus cells where the yyc operon is controlled by an isopropyl-ß-D-thiogalactopyranoside [IPTG]-induciblepromoter, we have shown that this system is essential in S.aureus during growth at 37°C and that starvation for theYycG/YycF regulatory system leads to cell death . During a previousstudy of the YycG/YycF TCS of B . subtilis, we defined a potentialYycF consensus recognition sequence, consisting of two hexanucleotidedirect repeats, separated by five nucleotides [5'-TGT[A/T]A[A/T/C]-N5-TGT[A/T]A[A/T/C]-3']. A detailed DNA motif analysis of the S . aureus genome indicates that there are potentially 12 genes preceded by this sequence, 5 of which are involved in virulence . An in vitro approach was undertaken to determine which of these genes are controlledby YycF . The YycG and YycF proteins of S . aureus were overproducedin Escherichia coli and purified . Autophosphorylation of theYycG kinase and phosphotransfer to YycF were shown in vitro.Gel mobility shift and DNase I footprinting assays were usedto show direct binding in vitro of purified YycF to the promoterregion of the ssaA gene, encoding a major antigen and previouslysuggested to be controlled by YycF . YycF was also shown to bindspecifically to the promoter regions of two genes, encodingthe IsaA antigen and the LytM peptidoglycan hydrolase, in agreementwith the proposed role of this system in controlling virulenceand cell wall metabolism. Transcriptional Response of Pasteurella multocida to Defined Iron Sources. Michael L. Paustian, 2002.Pasteurella multocida was grown in iron-free chemically defined medium supplemented with hemoglobin, transferrin, ferritin, and ferric citrate as iron sources . Whole-genome DNA microarrays were used to monitor global gene expression over seven time points after the addition of the defined iron source to the medium . This resulted in a set of data containing over 338,000 gene expression observations . On average, 12% of P . multocida genes were differentially expressed under any single condition . A majority of these genes encoded P . multocida proteins that were involved in either transport and binding or were annotated as hypothetical proteins . Several trends are evident when the data from different iron sources are compared . In general, only two genes (ptsN and sapD) were expressed at elevated levels under all of the conditions tested . The results also show that genes with increased expression in the presence of hemoglobin did not respond to transferrin or ferritin as an iron source . Correspondingly, genes with increased expression in the transferrin and ferritin experiments were expressed at reduced levels when hemoglobin was supplied as the sole iron source . Finally, the data show that genes that were most responsive to the presence of ferric citrate did not follow a trend similar to that of the other iron sources, suggesting that different pathways respond to inorganic or organic sources of iron in P . multocida . Taken together, our results demonstrate that unique subsets of P . multocida genes are expressed in response to different iron sources and that many of these genes have yet to be functionally characterized . Structural Analysis of the Domain Interface in DrrB, a Response Regulator of the OmpR/PhoB Subfamily. Victoria L. Robinson, 2003.The N-terminal regulatory domains of bacterial response regulator proteins catalyze phosphoryl transfer and function as phosphorylation-dependent regulatory switches to control the output activities of C-terminal effector domains . Structures of numerous isolated regulatory and effector domains have been determined . However, a detailed understanding of regulatory interactions among these domains has been limited by the relative paucity of structural data for intact multidomain response regulator proteins . The first multidomain structures determined, those of transcription factor NarL and methylesterase CheB, both revealed extensive interdomain interfaces . The regulatory domains obstruct access to the functional sites of the effector domains, indicating a regulatory mechanism based on inhibition . In contrast, the recently determined structure of the OmpR/PhoB homologue DrrD revealed no significant interdomain interface, suggesting that the domains are tethered by a flexible linker and lack a fixed orientation relative to each other . To address the generality of this feature, we have determined the 1.8-Å resolution crystal structure of Thermotoga maritima DrrB, providing a second structure of a multidomain response regulator of the OmpR/PhoB subfamily . The structure reveals an extensive domain interface of 751 Å2 and therefore differs greatly from that observed in DrrD . Residues that are crucial players in defining the activation state of the regulatory domain contribute to this interface, implying that conformational changes associated with phosphorylation will influence these intramolecular contacts . The DrrB and DrrD structures are suggestive of different signaling mechanisms, with intramolecular communication between N- and C-terminal domains making substantially different contributions to effector domain regulation in individual members of the OmpR/PhoB family .
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