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Production of Heterologous and Chimeric Scaffoldins by Clostridium acetobutylicum ATCC 824. S. Perret, 2004.Clostridium acetobutylicum ATCC 824 converts sugars and various polysaccharides into acids and solvents . This bacterium, however, is unable to utilize cellulosic substrates, since it is able to secrete very small amounts of cellulosomes . To promote the utilization of crystalline cellulose, the strategy we chose aims at producing heterologous minicellulosomes, containing two different cellulases bound to a miniscaffoldin, in C . acetobutylicum . A first step toward this goal describes the production of miniCipC1, a truncated form of CipC from Clostridium cellulolyticum, and the hybrid scaffoldin Scaf 3, which bears an additional cohesin domain derived from CipA from Clostridium thermocellum . Both proteins were correctly matured and secreted in the medium, and their various domains were found to be functional . Sustained-Release Formulation of Doxycycline Hyclate for Prophylaxis of Tick Bite Infection in a Murine Model of Lyme Borreliosis. N. S. Zeidner, 2004.The prophylactic potential of a single injection of sustained-release doxycycline hyclate (Atridox) was compared to that of a single oral dose of doxycycline hyclate in a murine model of Lyme borreliosis . Prophylaxis, as measured by the lack of cultivable spirochetes and demonstrable pathology, was noted for 43% of orally treated mice; in contrast, the sustained-release doxycycline hyclate completely protected mice from infection and resultant pathology . Conserved Response Regulator CtrA and IHF Binding Sites in the  -Proteobacteria Caulobacter crescentus and Rickettsia prowazekii Chromosomal Replication Origins.Ann Karen C. Brassinga, 2002.CzcR is the Rickettsia prowazekii homolog of the Caulobacter crescentus global response regulator CtrA . CzcR expression partially compensates for developmental defects in ctrA mutant C . crescentus cells, and CzcR binds to all five CtrA binding sites in the C . crescentus replication origin . Conversely, CtrA binds to five similar sites in the putative R . prowazekii replication origin (oriRp) . Also, Escherichia coli IHF protein binds over a central CtrA binding site in oriRp . Therefore, CtrA and IHF regulatory proteins have similar binding patterns in both replication origins, and we propose that CzcR is a global cell cycle regulator in R . prowazekii . Escherichia coli phnN, Encoding Ribose 1,5-Bisphosphokinase Activity (Phosphoribosyl Diphosphate Forming): Dual Role in Phosphonate Degradation and NAD Biosynthesis Pathways. Bjarne Hove-Jensen, 2003.An enzymatic pathway for synthesis of 5-phospho-D-ribosyl  -1-diphosphate (PRPP) without the participation of PRPP synthase was analyzed in Escherichia coli . This pathway was revealed by selection for suppression of the NAD requirement of strains with a deletion of the prs gene, the gene encoding PRPP synthase (B . Hove-Jensen, J . Bacteriol . 178:714-722, 1996) . The new pathway requires three enzymes: phosphopentomutase, ribose 1-phosphokinase, and ribose 1,5-bisphosphokinase . The latter activity is encoded by phnN; the product of this gene is required for phosphonate degradation, but its enzymatic activity has not been determined previously . The reaction sequence is ribose 5-phosphate
 ribose 1-phosphate
ribose 1,5-bisphosphate
PRPP . Alternatively, the synthesis of ribose 1-phosphate in the first step, catalyzed by phosphopentomutase, can proceed via phosphorolysis of a nucleoside, as follows: guanosine + Pi
guanine + ribose 1-phosphate . The ribose 1,5-bisphosphokinase-catalyzed phosphorylation of ribose 1,5-bisphosphate is a novel reaction and represents the first assignment of a specific chemical reaction to a polypeptide required for cleavage of a carbon-phosphorus (C—P) bond by a C-P lyase . The phnN gene was manipulated in vitro to encode a variant of ribose 1,5-bisphosphokinase with a tail consisting of six histidine residues at the carboxy-terminal end . PhnN was purified almost to homogeneity and characterized . The enzyme accepted ATP but not GTP as a phosphoryl donor, and it used ribose 1,5-bisphosphate but not ribose, ribose 1-phosphate, or ribose 5-phosphate as a phosphoryl acceptor . The identity of the reaction product as PRPP was confirmed by coupling the ribose 1,5-bisphosphokinase activity to the activity of xanthine phosphoribosyltransferase in the presence of xanthine, which resulted in the formation of 5'-XMP, and by cochromatography of the reaction product with authentic PRPP .
Functional Replacement of the Escherichia coli D-(-)-Lactate Dehydrogenase Gene (ldhA) with the L-(+)-Lactate Dehydrogenase Gene (ldhL) from Pediococcus acidilactici. Shengde Zhou, 2003.The microbial production of L-(+)-lactic acid is rapidly expanding to allow increased production of polylactic acid (PLA), a renewable, biodegradable plastic . The physical properties of PLA can be tailored for specific applications by controlling the ratio of L-(+) and D-(-) isomers . For most uses of PLA, the L-(+) isomer is more abundant . As an approach to reduce costs associated with biocatalysis (complex nutrients, antibiotics, aeration, product purification, and waste disposal), a recombinant derivative of Escherichia coli W3110 was developed that contains five chromosomal deletions (focA-pflB frdBC adhE ackA ldhA) . This strain was constructed from a D-(-)-lactic acid-producing strain, SZ63 (focA-pflB frdBC adhE ackA), by replacing part of the chromosomal ldhA coding region with Pediococcus acidilactici ldhL encoding an L-lactate dehydrogenase . Although the initial strain (SZ79) grew and fermented poorly, a mutant (SZ85) was readily isolated by selecting for improved growth . SZ85 exhibited a 30-fold increase in L-lactate dehydrogenase activity in comparison to SZ79, functionally replacing the native D-lactate dehydrogenase activity . Sequencing revealed mutations in the upstream, coding, and terminator regions of ldhL in SZ85, which are presumed to be responsible for increased L-lactate dehydrogenase activity . SZ85 produced L-lactic acid in M9 mineral salts medium containing glucose or xylose with a yield of 93 to 95%, a purity of 98% (based on total fermentation products), and an optical purity greater than 99% . Unlike other recombinant biocatalysts for L-lactic acid, SZ85 remained prototrophic and is devoid of plasmids and antibiotic resistance genes .
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