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Novel Locus Required for Expression of High-Level Macrolide-Lincosamide-Streptogramin B Resistance in Staphylococcus aureus. P. K. Martin, 2002.The yycF1(Ts) mutation in Staphylococcus aureus conferred hypersensitivity to macrolide-lincosamide-streptogramin B (MLSB) antibiotics on strains either containing or lacking ermB . The overexpression of the S . aureus Ssa protein restored the yycF1 mutant to wild-type levels of susceptibility . Inactivation of ssa in an unmutagenized strain dramatically reduced ermB-based resistance . Conditional loss of function or expression of ssa in the yycF1 mutant is proposed to result in the observed hypersensitivity to MLSB antibiotics . Transcriptional Interference by a Complex Formed at the Centromere-Like Partition Site of Plasmid P1. James A. Sawitzke, 2002.The partition site, parS, promotes accurate segregation of the replicated P1 plasmid to daughter cells when the P1-encoded ParA and ParB proteins are supplied . The parS site was inserted into the Escherichia coli chromosome between the promoter and the structural gene for ß-galactosidase, lacZ . There was little interference with lacZ expression when ParA and ParB were supplied in trans . However, when a mutant ParA protein, ParAM314I, was supplied along with ParB, expression of lacZ was shut down . ParAM314I, ParB, and parS appear to form a nucleoprotein complex that blocks transcription . Mutations in parA and parB that relieved the parAM314I-dependent block were found . In addition, new mutations which impose the block were selected . Five of the latter mapped to parA and one to parB; all had a propagation-defective phenotype (ParPD) similar to that of parAM314I . Thus, whereas a null par mutant P1 plasmid segregates its DNA randomly, these mutants prevent even random distribution of the plasmid . We propose that ParA protein normally interacts transiently with the ParB-parS complex for partition to proceed but that the mutations block ParA dissociation . This "permanent" ParA-ParB-parS complex acts as a transcription block . Consistent with this hypothesis, we found that three of the seven blocking mutations lie within regions of ParA and ParB that are known to interact with each other . When the transcription block is imposed, regional silencing of nearby genes occurs . However, the requirement for ParA and a mutant parA or parB allele distinguishes the transcription block from the regional ParB-dependent gene silencing previously described . Nucleotide-Dependent Conformational Changes in the  54-Dependent Activator DctD.Ying-Kai Wang, 2003.Activators of 54-RNA polymerase holoenzyme couple ATP hydrolysis to formation of an open promoter complex . DctD 1-142, a truncated and constitutively active form of the
54-dependent activator DctD from Sinorhizobium meliloti, displayed an altered DNase I footprint at its binding site located upstream of the dctA promoter in the presence of ATP . The altered footprint was not observed for a mutant protein with a substitution at or near the putative arginine finger, a conserved arginine residue thought to contact the nucleotide . These data suggest that structural changes in DctD1-142 during ATP hydrolysis can be detected by alterations in the DNase I footprint of the protein and may be communicated by interactions between bound nucleotide and the arginine finger . In addition, kinetic data for changes in fluorescence energy transfer upon binding of 2'(3')-O-(N-methylanthraniloyl)-ATP (Mant-ATP) to DctD1-142 and DctD suggested that these proteins undergo multiple conformational changes following ATP binding .
Characterization of Microbial Communities and Composition in Constructed Dairy Wetland Wastewater Effluent. A. Mark Ibekwe, 2003.Constructed wetlands have been recognized as a removal treatment option for high concentrations of contaminants in agricultural waste before land application . The goal of this study was to characterize microbial composition in two constructed wetlands designed to remove contaminants from dairy washwater . Water samples were collected weekly for 11 months from two wetlands to determine the efficiency of the treatment system in removal of chemical contaminants and total and fecal coliforms . The reduction by the treatment was greatest for biological oxygen demand, suspended solids, chemical oxygen demand, nitrate, and coliforms . There was only moderate removal of total nitrogen and phosphorus . Changes in the total bacterial community and ammonia-oxidizing bacterial composition were examined by using denaturing gradient gel electrophoresis (DGGE) and sequencing of PCR-amplified fragments of the gene carrying the  subunit of the ammonia monooxygenase gene (amoA) recovered from soil samples and DGGE bands . DGGE analysis of wetlands and manure samples revealed that the total bacterial community composition was dominated by bacteria from phylogenetic clusters related to Bacillus, Clostridium, Mycoplasma, Eubacterium, and Proteobacteria originally retrieved from the gastrointestinal tracts of mammals . The population of ammonia-oxidizing bacteria showed a higher percentage of Nitrosospira-like sequences from the wetland samples, while a higher percentage of Nitrosomonas-like sequences from manure, feces, raw washwater, and facultative pond was found . These results show that the wetland system is a natural process dependent upon the development of healthy microbial communities for optimal wastewater treatment .
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