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Bacteriophage-Based Genetic System for Selection of Nonsplicing Inteins. Isaac K. O. Cann, 2004.A genetic selection system that detects splicing and nonsplicing activities of inteins was developed based on the ability to rescue a T4 phage strain with a conditionally inactive DNA polymerase . This phage defect can be complemented by expression of plasmid-encoded phage RB69 DNA polymerase . Insertion of an intein gene into the active site of the RB69 DNA polymerase gene renders polymerase activity and phage viability dependent on protein splicing . The effectiveness of the system was tested by screening for thermosensitive splicing mutants . Development of genetic systems with the potential of identifying protein splicing inhibitors is a first step towards controlling proliferation of pathogenic microbes harboring inteins in essential proteins . Regulation of the metC-cysK Operon, Involved in Sulfur Metabolism in Lactococcus lactis. María Fernández, 2002.Sulfur metabolism in gram-positive bacteria is poorly characterized . Information on the molecular mechanisms of regulation of genes involved in sulfur metabolism is limited, and no regulator genes have been identified . Here we describe the regulation of the lactococcal metC-cysK operon, encoding a cystathionine ß-lyase (metC) and cysteine synthase (cysK) . Its expression was shown to be negatively affected by high concentrations of cysteine, methionine, and glutathione in the culture medium, while sulfur limitation resulted in a high level of expression . Other sulfur sources tested showed no significant effect on metC-cysK gene expression . In addition we found that O-acetyl-l-serine, the substrate of cysteine synthase, was an inducer of the metC-cysK operon . Using a random mutagenesis approach, we identified two genes, cmbR and cmbT, involved in regulation of metC-cysK expression . The cmbT gene is predicted to encode a transport protein, but its precise role in regulation remains unclear . Disruption of cmbT resulted in a two- to threefold reduction of metC-cysK transcription . A 5.7-kb region containing the cmbR gene was cloned and sequenced . The encoded CmbR protein is homologous to the LysR family of regulator proteins and is an activator of the metC-cysK operon . In analogy to CysB from Escherichia coli, we propose that CmbR requires acetylserine to be able to bind the activation sites and subsequently activate transcription of the metC-cysK operon . Characterization of the Bacillus subtilis ywtD Gene, Whose Product Is Involved in  -Polyglutamic Acid Degradation.Takao Suzuki, 2003.The ywtD gene, which codes for an enzyme that degrades -polyglutamic acid (PGA), was cloned from Bacillus subtilis IFO16449 . The gene is located immediately downstream of ywsC and ywtABC, a PGA operon involved in PGA biosynthesis, and it showed partial similarity to genes coding for DL-endopeptidase, a peptidoglycan-degrading enzyme . The ywtD gene, from which signal sequence is excised, was inserted into pET15b, and the recombinant plasmid was then transformed into Escherichia coli . Histidine-tagged YwtD was purified from sonicated cells of the transformant . The purified YwtD degraded PGA to yield two hydrolyzed products, a high-molecular-mass product (490 kDa with nearly 100% L-glutamic acid) and an 11-kDa product (with D-glutamic acid and L-glutamic acid in an 80:20 ratio) . This finding and results of enzymatic analysis of the two products with carboxypeptidase G suggest that YwtD is a novel enzyme cleaving the
-glutamyl bond only between D- and L-glutamic acids of PGA, and it may be designated
-DL-glutamyl hydrolase .
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