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Effect of the CopB Auxiliary Replication Control System on Stability of Maintenance of Par+ Plasmid R1. Jan A. Olsson, 2004.Plasmid R1 is a low-copy-number plasmid that is present at a level of about four or five copies per average cell . The copy number is controlled posttranscriptionally at the level of synthesis of the rate-limiting initiator protein RepA . In addition to this, R1 has an auxiliary system that derepresses a second promoter at low copy numbers, leading to increased repA mRNA synthesis . This promoter is normally switched off by a constitutively synthesized plasmid-encoded repressor protein, CopB; in cells with low copy numbers, the concentration of CopB is low and the promoter is derepressed . Here we show that the rate of loss of a Par+ derivative of the basic replicon of R1 increased about sevenfold when the cells contained a high concentration of the CopB protein formed from a compatible plasmid . Cloning, Nucleotide Sequence, and Overexpression of the L-Rhamnose Isomerase Gene from Pseudomonas stutzeri in Escherichia coli. Khim Leang, 2004.The gene encoding L-rhamnose isomerase (L-RhI) from Pseudomonas stutzeri was cloned into Escherichia coli and sequenced . A sequence analysis of the DNA responsible for the L-RhI gene revealed an open reading frame of 1,290 bp coding for a protein of 430 amino acid residues with a predicted molecular mass of 46,946 Da . A comparison of the deduced amino acid sequence with sequences in relevant databases indicated that no significant homology has previously been identified . An amino acid sequence alignment, however, suggested that the residues involved in the active site of L-RhI from E . coli are conserved in that from P . stutzeri . The L-RhI gene was then overexpressed in E . coli cells under the control of the T5 promoter . The recombinant clone, E . coli JM109, produced significant levels of L-RhI activity, with a specific activity of 140 U/mg and a volumetric yield of 20,000 U of soluble enzyme per liter of medium . This reflected a 20-fold increase in the volumetric yield compared to the value for the intrinsic yield . The recombinant L-RhI protein was purified to apparent homogeneity on the basis of three-step chromatography . The purified recombinant enzyme showed a single band with an estimated molecular weight of 42,000 in a sodium dodecyl sulfate-polyacrylamide gel . The overall enzymatic properties of the purified recombinant L-RhI protein were the same as those of the authentic one, as the optimal activity was measured at 60°C within a broad pH range from 5.0 to 11.0, with an optimum at pH 9.0 . AerR, a Second Aerobic Repressor of Photosynthesis Gene Expression in Rhodobacter capsulatus. Chen Dong, 2002.Open reading frame orf192, which is located immediately upstream of the aerobic repressor gene crtJ, was genetically and biochemically demonstrated to code for a second aerobic repressor (AerR) of photosynthesis gene expression in Rhodobacter capsulatus . Promoter-mapping studies indicate that crtJ has its own promoter but that a significant proportion of crtJ expression is promoted by read-through transcription of orf192 (aerR) transcripts through crtJ . Disruption of aerR resulted in increased photopigment biosynthesis during aerobic growth to a level similar to that of disruption of crtJ . Like that reported for CrtJ, ß-galactosidase assays of reporter gene expression indicated that disruption of aerR resulted in a two- to threefold increase in aerobic expression of the crtI and pucB operons . However, unlike CrtJ, AerR aerobically represses puf operon expression and does not aerobically repress bchC expression . Gel mobility shift analysis with purified AerR indicates that AerR does not bind to a bchC promoter probe but does bind to the crtI, puc, and puf promoter probes . These results indicate that AerR is a DNA-binding protein that targets genes partially overlapping a subset of genes that are also controlled by CrtJ . We also provide evidence for cooperative binding of AerR and CrtJ to the puc promoter region . Differential Recognition of Surface Proteins in Streptococcus pyogenes by Two Sortase Gene Homologs. Timothy C. Barnett, 2002.The interaction of Streptococcus pyogenes (group A streptococcus [GAS]) with its human host requires several surface proteins . In this study, we isolated mutations in a gene required for the surface localization of protein F by transposon mutagenesis of the M6 strain JRS4 . This gene (srtA) encodes a protein homologous to Staphylococcus aureus sortase, which covalently links proteins containing an LPXTG motif to the cell wall . The GAS srtA mutant was defective in anchoring the LPXTG-containing proteins M6, protein F, ScpA, and GRAB to the cell surface . This phenotype was complemented when a wild-type srtA gene was provided in trans . The surface localization of T6, however, was unaffected by the srtA mutation . The M1 genome sequence contains a second open reading frame with a motif characteristic of sortase proteins . Inactivation of this gene (designated srtB) in strain JRS4 affected the surface localization of T6 but not M6, protein F, ScpA, or GRAB . This phenotype was complemented by srtB in trans . An srtA probe hybridized with DNA from all GAS strains tested (M types 1, 3, 4, 5, 6, 18, 22, and 50 and nontypeable strain 64/14) and from streptococcal groups C and G, while srtB hybridized with DNA from only a few GAS strains . We conclude that srtA and srtB encode sortase enzymes required for anchoring different subsets of proteins to the cell wall . It seems likely that the multiple sortase homologs in the genomes of other gram-positive bacteria have a similar substrate-specific role .
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