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Lactococcal Phage Genes Involved in Sensitivity to AbiK and Their Relation to Single-Strand Annealing Proteins. Julie D. Bouchard, 2004.Lactococcal phage mutants insensitive to the antiviral abortive infection mechanism AbiK are divided into two classes . One comprises virulent phages that result from DNA exchanges between a virulent phage and the host chromosome . Here, we report the analysis of the second class of phage mutants, which are insensitive to AbiK as a result of a single nucleotide change causing an amino acid substitution . The mutated genes occupy the same position in the various lactococcal phage genomes, but the deduced proteins do not share amino acid sequence similarity . Four nonsimilar proteins involved in the sensitivity to AbiK (Sak) were identified . Two of these Sak proteins are related to Erf and RAD52, single-strand annealing proteins involved in homologous recombination . Adhesion of Enterococcus faecalis in the Nonculturable State to Plankton Is the Main Mechanism Responsible for Persistence of This Bacterium in both Lake and Seawater. Caterina Signoretto, 2004. N4 RNA Polymerase II, a Heterodimeric RNA Polymerase with Homology to the Single-Subunit Family of RNA Polymerases. S. H. Willis, 2002.Bacteriophage N4 middle genes are transcribed by a phage-coded, heterodimeric, rifampin-resistant RNA polymerase, N4 RNA polymerase II (N4 RNAPII) . Sequencing and transcriptional analysis revealed that the genes encoding the two subunits comprising N4 RNAPII are translated from a common transcript initiating at the N4 early promoter Pe3 . These genes code for proteins of 269 and 404 amino acid residues with sequence similarity to the single-subunit, phage-like RNA polymerases . The genes encoding the N4 RNAPII subunits, as well as a synthetic construct encoding a fusion polypeptide, have been cloned and expressed . Both the individually expressed subunits and the fusion polypeptide reconstitute functional enzymes in vivo and in vitro . Trinucleotide GAA Repeats Dictate pMGA Gene Expression in Mycoplasma gallisepticum by Affecting Spacing between Flanking Regions. Li Liu, 2002.The pMGA genes of the avian respiratory pathogen Mycoplasma gallisepticum encode a family of hemagglutinins that are subject to phase variation . A trinucleotide GAA repeat region is located upstream of the pMGA transcription start site . The length of the repeat region varies at a high frequency due to changes in the number of repeat units . Previous studies have shown that pMGA genes are transcribed when 12 GAA repeats are present but are not transcribed when the number of repeats is not 12 . To further analyze the mechanism of gene regulation, the pMGA promoter region was modified either by deleting the nucleotides 5" of the GAA repeats or by inserting linkers of 10 or 12 bp at a position 3" of the repeats . The modified promoter region was fused to a promoterless lacZ gene and transformed into M . gallisepticum by using transposon Tn4001 as a vector . Transformants and successive generations of progeny were analyzed with 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal) to monitor ß-galactosidase activity . For the transformants of M . gallisepticum containing the reporter with deletion of nucleotides 5" of the GAA repeats, GAA-dependent pMGA gene regulation was abolished . For the transformants containing the reporter with an addition of 10- or 12-bp linkers, lacZ was expressed only when eight GAA repeats were present . These data indicate that the nucleotides 5" of the GAA repeats as well as the spacing between the GAA repeats and sequences downstream (3") of the repeats are important for pMGA gene expression . Functional Characterization of Penicillin-Binding Protein 1b from Streptococcus pneumoniae. Anne Marie Di Guilmi, 2003.The widespread use of antibiotics has encouraged the development of drug resistance in pathogenic bacteria . In order to overcome this problem, the modification of existing antibiotics and/or the identification of targets for the design of new antibiotics is currently being undertaken . Bifunctional penicillin-binding proteins (PBPs) are membrane-associated molecules whose transpeptidase (TP) activity is irreversibly inhibited by ß-lactam antibiotics and whose glycosyltransferase (GT) activity represents a potential target in the antibacterial fight . In this work, we describe the expression and the biochemical characterization of the soluble extracellular region of Streptococcus pneumoniae PBP1b (PBP1b*) . The acylation efficiency for benzylpenicillin and cefotaxime was characterized by stopped-flow fluorometry and a 40-kDa stable TP domain was generated after limited proteolysis . In order to analyze the GT activity of PBP1b*, we developed an electrophoretic assay which monitors the fluorescence signal from PBP1b*-bound dansylated lipid II . This binding was inhibited by the antibiotic moenomycin and was specific for the GT domain, since no signal was observed in the presence of the purified functional TP domain . Binding studies performed with truncated forms of PBP1b* demonstrated that the first conserved motif of the GT domain is not required for the recognition of lipid II, whereas the second motif is necessary for such interaction .
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