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Global Gene Expression in Staphylococcus aureus Biofilms. Karen E. Beenken, 2004.We previously demonstrated that mutation of the staphylococcal accessory regulator (sarA) in a clinical isolate of Staphylococcus aureus (UAMS-1) results in an impaired capacity to form a biofilm in vitro (K . E . Beenken, J . S . Blevins, and M . S . Smeltzer, Infect . Immun . 71:4206-4211, 2003) . In this report, we used a murine model of catheter-based biofilm formation to demonstrate that a UAMS-1 sarA mutant also has a reduced capacity to form a biofilm in vivo . Surprisingly, mutation of the UAMS-1 ica locus had little impact on biofilm formation in vitro or in vivo . In an effort to identify additional loci that might be relevant to biofilm formation and/or the adaptive response required for persistence of S . aureus within a biofilm, we isolated total cellular RNA from UAMS-1 harvested from a biofilm grown in a flow cell and compared the transcriptional profile of this RNA to RNA isolated from both exponential- and stationary-phase planktonic cultures . Comparisons were done using a custom-made Affymetrix GeneChip representing the genomic complement of six strains of S . aureus (COL, N315, Mu50, NCTC 8325, EMRSA-16 [strain 252], and MSSA-476) . The results confirm that the sessile lifestyle associated with persistence within a biofilm is distinct by comparison to the lifestyles of both the exponential and postexponential phases of planktonic culture . Indeed, we identified 48 genes in which expression was induced at least twofold in biofilms over expression under both planktonic conditions . Similarly, we identified 84 genes in which expression was repressed by a factor of at least 2 compared to expression under both planktonic conditions . A primary theme that emerged from the analysis of these genes is that persistence within a biofilm requires an adaptive response that limits the deleterious effects of the reduced pH associated with anaerobic growth conditions . Establishment of a Real-Time PCR-Based Approach for Accurate Quantification of Bacterial RNA Targets in Water, Using Salmonella as a Model Organism. Axel Fey, 2004.Quantitative PCR (Q-PCR) is a fast and efficient tool to quantify target genes . In eukaryotic cells, quantitative reverse transcription-PCR (Q-RT-PCR) is also used to quantify gene expression, with stably expressed housekeeping genes as standards . In bacteria, such stable expression of housekeeping genes does not occur, and the use of DNA standards leads to a broad underestimation . Therefore, an accurate quantification of RNA is feasible only by using appropriate RNA standards . We established and validated a Q-PCR method which enables the quantification of not only the number of copies of target genes (i.e., the number of bacterial cells) but also the number of RNA copies . The genes coding for InvA and the 16S rRNA of Salmonella enterica serovar Typhimurium were selected for the evaluation of the method . As DNA standards, amplified fragments of the target genes were used, whereas the same DNA standards were transcribed in vitro for the development of appropriate RNA standards . Salmonella cultures and environmental water samples inoculated with bacteria were then employed for the final testing . Both experimental approaches led to a sensitive, accurate, and reproducible quantification of the selected target genes and RNA molecules by Q-PCR and Q-RT-PCR . It is the first time that RNA standards have been successfully used for a precise quantification of the number of RNA molecules in prokaryotes . This demonstrates the potential of this approach for determining the presence and metabolic activity of pathogenic bacteria in environmental samples . Transcription Activation by FNR: Evidence for a Functional Activating Region 2. Timo Blake, 2002.The FNR protein of Escherichia coli controls the transcription of target genes in response to anoxia via the assembly-disassembly of oxygen-labile iron-sulfur clusters . Previous work identified patches of surface-exposed amino acids (designated activating regions 1 and 3 [AR1 and AR3, respectively]) of FNR which allow it to communicate with RNA polymerase (RNAP) and thereby activate transcription . Previously it was thought that FNR lacks a functional activating region 2 (AR2), although selecting for mutations that compensate for defective AR1 or a miscoordinated iron-sulfur cluster can reactivate AR2 . Here we show that the substitution of two surface-exposed lysine residues (Lys49 and Lys50) of FNR impaired transcription from class II (FNR box centered at -41.5) but not class I (FNR box centered at -71.5) FNR-dependent promoters . The degree of impairment was greater when a negatively charged residue (Glu) replaced either Lys49 or Lys50 than when uncharged amino acid Ala was substituted . Oriented heterodimers were used to show that only the downstream subunit of the FNR dimer was affected by the Lys  Ala substitutions at a class II promoter . Site-directed mutagenesis of a negatively charged patch (162EEDE165) within the N-terminal domain of the RNAP
 subunit that interacts with the positively charged AR2 of the cyclic AMP receptor protein suggested that Lys49 and Lys50 of FNR interact with this region of the
subunit of RNAP . Thus, it was suggested that Lys49 and Lys50 form part of a functional AR2 in FNR .
Characterization of the Interaction of Bacillus subtilis PyrR with pyr mRNA by Site-Directed Mutagenesis of the Protein. Heather K. Savacool, 2002.The Bacillus subtilis PyrR protein regulates transcriptional attenuation of the pyrimidine nucleotide (pyr) operon by binding in a uridine nucleotide-dependent manner to specific sites on pyr mRNA and stabilizing a secondary structure of the downstream RNA that favors termination of transcription . The high-resolution structure of unliganded PyrR was used to guide site-directed mutagenesis of 12 amino acid residues that were thought likely to be involved in the binding of RNA . Missense mutations were constructed and evaluated for their effects on regulation of pyr genes in vivo and their uracil phosphoribosyltransferase activity, which is catalyzed by wild-type PyrR . A substantial fraction of the mutant PyrR proteins did not have native structures, but eight PyrR mutants were purified and characterized physically, for their uracil phosphoribosyltransferase activity and for their ability to bind pyr RNA in vitro . On the basis of these studies Thr-18, His-22, Arg-141, and Arg-146 were implicated in RNA binding . Arg-27 and Lys-152 were also likely to be involved in RNA binding, but Gln substitution mutations in these residues may have altered their subunit-subunit interactions slightly . Arg-19 was implicated in pyr regulation, but a specific role in RNA binding could not be demonstrated because the R19Q mutant protein could not be purified in native form . The results confirm a role in RNA binding of a positively charged face of PyrR previously identified from the crystallographic structure . The RNA binding residues lie in two sequence segments that are conserved in PyrR proteins from many species .
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