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Mechanism of Transcription Activation at the comG Promoter by the Competence Transcription Factor ComK of Bacillus subtilis. K. A. Susanna, 2004.The development of genetic competence in Bacillus subtilis is regulated by a complex signal transduction cascade, which resultsin the synthesis of the competence transcription factor, encodedby comK . ComK is required for the transcription of the late competence genes that encode the DNA binding and uptake machineryand of genes required for homologous recombination . In vivoand in vitro experiments have shown that ComK is responsiblefor transcription activation at the comG promoter . In this study,we investigated the mechanism of this transcription activation.The intrinsic binding characteristics of RNA polymerase withand without ComK at the comG promoter were determined, demonstratingthat ComK stabilizes the binding of RNA polymerase to the comG promoter . This stabilization probably occurs through interactions with the upstream DNA, since a deletion of the upstream DNAresulted in an almost complete abolishment of stabilizationof RNA polymerase binding . Furthermore, a strong requirementfor the presence of an extra AT box in addition to the commonComK-binding site was shown . In vitro transcription with B.subtilis RNA polymerase reconstituted with wild-type α-subunits and with C-terminal deletion mutants of the α-subunits was performed,demonstrating that these deletions do not abolish transcriptionactivation by ComK . This indicates that ComK is not a type Iactivator . We also show that ComK is not required for open complexformation . A possible mechanism for transcription activation is proposed, implying that the major stimulatory effect of ComK is on binding of RNA polymerase. Microscale and Molecular Assessment of Impacts of Nickel, Nutrients, and Oxygen Level on Structure and Function of River Biofilm Communities. J. R. Lawrence, 2004.Studies were carried out to assess the influence of nutrients, dissolved oxygen (DO) concentration, and nickel (Ni) on river biofilm development, structure, function, and community composition . Biofilms were cultivated in rotating annular reactors with river water at a DO concentration of 0.5 or 7.5 mg liter–1, with or without a combination of carbon, nitrogen, and phosphorus (CNP) and with or without Ni at 0.5 mg liter–1 . The effects of Ni were apparent in the elimination of cyanobacterial populations and reduced photosynthetic biomass in the biofilm . Application of lectin-binding analyses indicated changes in exopolymer abundance and a shift in the glycoconjugate makeup of the biofilms, as well as in the response to all treatments . Application of the fluorescent live-dead staining (BacLight Live-Dead staining kit; Molecular Probes, Eugene, Oreg.) indicated an increase in the ratio of live to dead cells under low-oxygen conditions . Nickel treatments had 50 to 75% fewer ‘live’ cells than their corresponding controls . Nickel at 0.5 mg liter–1 corresponding to the industrial release rate concentration for nickel resulted in reductions in carbon utilization spectra relative to control and CNP treatments without nickel . In these cases, the presence of nickel eliminated the positive influence of nutrients on the biofilm . Other culture-dependent analyses (plate counts and most probable number) revealed no significant treatment effect on the biofilm communities . In the presence of CNP and at both DO levels, Ni negatively affected denitrification but had no effect on hexadecane mineralization or sulfate reduction . Analysis of total community DNA indicated abundant eubacterial 16S ribosomal DNA (rDNA), whereas Archaea were not detected . Amplification of the alkB gene indicated a positive effect of CNP and a negative effect of Ni . The nirS gene was not detected in samples treated with Ni at 0.5 mg liter–1, indicating a negative effect on specific populations of bacteria, such as denitrifiers, resulting in a reduction in diversity . Denaturing gradient gel electrophoresis revealed that CNP had a beneficial impact on biofilm bacterial diversity at high DO concentrations, but none at low DO concentrations, and that the negative effect of Ni on diversity was similar at both DO concentrations . Notably, Ni resulted in the appearance of unique bands in 16S rDNA from Ni, DO, and CNP treatments . Sequencing results confirmed that the bands belonged to bacteria originating from freshwater and marine environments or from agricultural soils and industrial effluents . The observations indicate that significant interactions occur between Ni, oxygen, and nutrients and that Ni at 0.5 mg liter–1 may have significant impacts on river microbial community diversity and function . Regulation of the Hydrogenase-4 Operon of Escherichia coli by the  54-Dependent Transcriptional Activators FhlA and HyfR.David A. G. Skibinski, 2002. Alterations of Cell Wall Structure and Metabolism Accompany Reduced Susceptibility to Vancomycin in an Isogenic Series of Clinical Isolates of Staphylococcus aureus. K. Sieradzki, 2003.A series of isogenic methicillin-resistant Staphylococcus aureus isolates recovered from a bacteremic patient were shown to acquire gradually increasing levels of resistance to vancomycin during chemotherapy with the drug (K . Sieradzki, T . Leski, L . Borio, J . Dick, and A . Tomasz, J . Clin . Microbiol . 41:1687-1693, 2003) . We compared properties of the earliest (parental) vancomycin-susceptible isolate, JH1 (MIC, 1 µg/ml), to two late (progeny) isolates, JH9 and JH14 (vancomycin MIC, 8 µg/ml) . The resistant isolates produced abnormally thick cell walls and poorly separated cells when grown in antibiotic-free medium . Chemical analysis of the resistant isolates showed decreased cross-linkage of the peptidoglycan and drastically reduced levels of PBP4 as determined by the fluorographic assay . Resistant isolates showed reduced rates of cell wall turnover and autolysis . In vitro hydrolysis of resistant cell walls by autolytic extracts prepared from either susceptible or resistant strains was also slow, and this abnormality could be traced to a quantitative (or qualitative) change in the wall teichoic acid component of resistant isolates . Some change in the structure and/or metabolism of teichoic acids appears to be an important component of the mechanism of decreased susceptibility to vancomycin in S. aureus .
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