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Analysis of the Conjugal Transfer System of the Pheromone- Independent Highly Transferable Enterococcus Plasmid pMG1: Identification of a tra Gene (traA) Up-Regulated during Conjugation. Koichi Tanimoto, 2002.pMG1 (65.1 kbp) is a pheromone-independent Enterococcus faecium conjugative plasmid conferring gentamicin resistance . pMG1 is able to transfer to enterococci at a high frequency in broth mating experiments, and it does not respond to the sex pheromones which are involved in the high-frequency transfer system of some conjugative plasmids in Enterococcus faecalis . To analyze regulation of tra gene expression in pMG1, transcripts of pMG1 were examined during conjugation . RNA samples were prepared from mating mixtures 20, 40, 80, and 160 min after initiation of mating and were subsequently analyzed by Northern hybridization by using a variety of pMG1 DNA fragments as probes . One transcript of gene 71ORF2 increased to the maximal level 20 min after the start of mating . The level of this transcript decreased after 40 min of mating to the same level as the level in the control donor culture . The increase was not observed in cultures of the donor cells or recipient cells . These findings suggested that the increase was a conjugation-specific event . The 71ORF2 gene of pMG1 was disrupted by using the suicide vector pMG226 carrying an erythromycin resistance gene that is expressed in enterococci . The transfer frequencies of mutant plasmid pMG229, which had a disrupted 71ORF2 gene in pMG1, and the parent plasmid pMG1 were 1.6 x 10-7 and 1.1 x 10-3 per donor cell, respectively, in broth mating experiments, and the transfer frequencies of pMG229 and pMG1 were 2.7 x 10-3 and 8.5 x 10-2 per donor cell, respectively, in filter mating experiments . This indicated that the transfer frequency of plasmid pMG229 was reduced during broth mating and was not altered during filter mating . 71ORF2, which is designated traA, is a gene involved in the tra gene system for conjugation, and the product of this gene is associated with the formation or stabilization of mating aggregates during broth mating . Reduced Water Availability Influences the Dynamics, Development, and Ultrastructural Properties of Pseudomonas putida Biofilms. Woo-Suk Chang, 2003.Pseudomonas putida strain mt-2 unsaturated biofilm formation proceeds through three distinct developmental phases, culminating in the formation of a microcolony . The form and severity of reduced water availability alter cell morphology, which influences microcolony size and ultrastructure . The dehydration (matric stress) treatments resulted in biofilms comprised of smaller cells, but they were taller and more porous and had a thicker extracellular polysaccharide layer at the air interface . In the solute stress treatments, cell filamentation occurred more frequently in the presence of high concentrations of ionic (but not nonionic) solutes, and these filamented cells drastically altered the biofilm architecture . Propionyl Coenzyme A Is a Common Intermediate in the 1,2-Propanediol and Propionate Catabolic Pathways Needed for Expression of the prpBCDE Operon during Growth of Salmonella enterica on 1,2-Propanediol. Sergio Palacios, 2003.The studies reported here identify propionyl coenzyme A (propionyl-CoA) as the common intermediate in the 1,2-propanediol and propionate catabolic pathways of Salmonella enterica serovar Typhimurium LT2 . Growth on 1,2-propanediol as a carbon and energy source led to the formation and excretion of propionate, whose activation to propionyl-CoA relied on the activities of the propionate kinase (PduW)/phosphotransacetylase (Pta) enzyme system and the CobB sirtuin-controlled acetyl-CoA and propionyl-CoA (Acs, PrpE) synthetases . The different affinities of these systems for propionate ensure sufficient synthesis of propionyl-CoA to support wild-type growth of S . enterica under low or high concentrations of propionate in the environment . These redundant systems of propionyl-CoA synthesis are needed because the prpE gene encoding the propionyl-CoA synthetase enzyme is part of the prpBCDE operon under the control of the PrpR regulatory protein, which needs 2-methylcitrate as a coactivator . Because the synthesis of 2-methylcitrate by PrpC (i.e., the 2-methylcitrate synthase enzyme) requires propionyl-CoA as a substrate, the level of propionyl-CoA needs to be raised by the Acs or PduW-Pta system before 2-methylcitrate can be synthesized and prpBCDE transcription can be activated . Increased Exopolysaccharide Production in Lactococcus lactis due to Increased Levels of Expression of the NIZO B40 eps Gene Cluster. Ingeborg C. Boels, 2003.Exopolysaccharides (EPS) play an important role in the rheology and texture of fermented food products . This is the first report demonstrating that homologous overexpression of a complete eps gene cluster in Lactococcus lactis leads to increased EPS production levels . A ninefold-elevated EPS plasmid copy number led to an almost threefold increase in the eps expression level, resulting in an almost fourfold increase in the NIZO B40 EPS production level . It was previously reported that increased EPS precursor levels did not influence NIZO B40 EPS production levels . However, the present results indicate that the maximal NIZO B40 EPS production level is limited by the activity level of the expression products of the eps gene cluster rather than by the level of EPS precursors .
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