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Biosynthesis Pathway of ADP-L-glycero-ß-D-manno-Heptose in Escherichia coli. Bernd Kneidinger, 2002.The steps involved in the biosynthesis of the ADP-L-glycero-ß-D-manno-heptose (ADP-L-ß-D-heptose) precursor of the inner core lipopolysaccharide (LPS) have not been completely elucidated . In this work, we have purified the enzymes involved in catalyzing the intermediate steps leading to the synthesis of ADP-D-ß-D-heptose and have biochemically characterized the reaction products by high-performance anion-exchange chromatography . We have also constructed a deletion in a novel gene, gmhB (formerly yaeD), which results in the formation of an altered LPS core . This mutation confirms that the GmhB protein is required for the formation of ADP-D-ß-D-heptose . Our results demonstrate that the synthesis of ADP-D-ß-D-heptose in Escherichia coli requires three proteins, GmhA (sedoheptulose 7-phosphate isomerase), HldE (bifunctional D-ß-D-heptose 7-phosphate kinase/D-ß-D-heptose 1-phosphate adenylyltransferase), and GmhB (D,D-heptose 1,7-bisphosphate phosphatase), as well as ATP and the ketose phosphate precursor sedoheptulose 7-phosphate . A previously characterized epimerase, formerly named WaaD (RfaD) and now renamed HldD, completes the pathway to form the ADP-L-ß-D-heptose precursor utilized in the assembly of inner core LPS . Isolation and Characterization of cLV25, a Bacteroides fragilis Chromosomal Transfer Factor Resembling Multiple Bacteroides sp . Mobilizable Transposons. Kathleen A. Bass, 2002.Horizontal DNA transfer contributes significantly to the dissemination of antibiotic resistance genes in Bacteroides fragilis . To further our understanding of DNA transfer in B . fragilis, we isolated and characterized a new transfer factor, cLV25 . cLV25 was isolated from B . fragilis LV25 by its capture on the nonmobilizable Escherichia coli-Bacteroides shuttle vector pGAT400  BglII . Similar to other Bacteroides sp . transfer factors, cLV25 was mobilized in E . coli by the conjugative plasmid R751 . Using Tn1000 mutagenesis and deletion analysis of cLV25, two mobilization genes, bmgA and bmgB, were identified, whose predicted proteins have similarity to DNA relaxases and mobilization proteins, respectively . In particular, BmgA and BmgB were homologous to MocA and MocB, respectively, the two mobilization proteins of the B . fragilis mobilizable transposon Tn4399 . A cis-acting origin of transfer (oriT) was localized to a 353-bp region that included nearly all of the intergenic region between bmgB and orf22 and overlapped with the 3' end of orf22 . This oriT contained a putative nic site sequence but showed no significant similarity to the oriT regions of other transfer factors, including Tn4399 . Despite the lack of sequence similarity between the oriTs of cLV25 and Tn4399, a mutation in the cLV25 putative DNA relaxase, bmgA, was partially complemented by Tn4399 . In addition to the functional cross-reaction with Tn4399, a second distinguishing feature of cLV25 is that predicted proteins have similarity to proteins encoded not only by Tn4399 but by several Bacteroides sp . transfer factors, including NBU1, NBU2, CTnDOT, Tn4555, and Tn5520 .
Involvement of the Multidomain Regulatory Protein XynR in Positive Control of Xylanase Gene Expression in the Ruminal Anaerobe Prevotella bryantii B14. Kohji Miyazaki, 2003.The xylanase gene cluster from the rumen anaerobe Prevotella bryantii B14 was found to include a gene (xynR) that encodes a multidomain regulatory protein and is downstream from the xylanase and ß-xylosidase genes xynA and xynB. Additional genes identified upstream of xynA and xynB include xynD, which encodes an integral membrane protein that has homology with Na:solute symporters; xynE, which is related to the genes encoding acylhydrolases and arylesterases; and xynF, which has homology with the genes encoding  -glucuronidases . XynR includes, in a single 833-amino-acid polypeptide, a putative input domain unrelated to other database sequences, a likely transmembrane domain, histidine kinase motifs, response regulator sequences, and a C-terminal AraC-type helix-turn-helix DNA binding domain . Two transcripts (3.7 and 5.8 kb) were detected with a xynA probe, and the start site of the 3.7-kb transcript encoding xynABD was mapped to a position upstream of xynD . The DNA binding domain of XynR was purified after amplification and overexpression in Escherichia coli and was found to bind to a 141-bp DNA fragment from the region immediately upstream of xynD. In vitro transcription assays demonstrated that XynR stimulates transcription of the 3.7-kb transcript . We concluded that XynR acts as a positive regulator that activates expression of xynABD in P . bryantii B14 . This is the first regulatory protein that demonstrates significant homology with the two-component regulatory protein superfamily and has been shown to be involved in the regulation of polysaccharidase gene expression .
Quorum-Sensing System and Stationary-Phase Sigma Factor (rpoS) of the Onion Pathogen Burkholderia cepacia Genomovar I Type Strain, ATCC 25416. Claudio Aguilar, 2003.Bacterial strains belonging to Burkholderia cepacia can be human opportunistic pathogens, plant pathogens, and plant growth promoting and have remarkable catabolic activity . B . cepacia consists of several genomovars comprising what is now known as the B . cepacia complex . Here we report the quorum-sensing system of a genomovar I onion rot type strain ATCC 25416 . Quorum sensing is a cell-density-dependent regulatory response which involves the production of N-acyl homoserine lactone (HSL) signal molecules . The cep locus has been inactivated in the chromosome, and it has been shown that CepI is responsible for the biosynthesis of an N-hexanoyl HSL (C6-HSL) and an N-octanoyl HSL (C8-HSL) and that the cep locus regulates protease production as well as onion pathogenicity via the expression of a secreted polygalacturonase . A cep-lacZ-based sensor plasmid has been constructed and used to demonstrate that CepR responded to C6-HSL with only 15% of the molar efficiency of C8-HSL, that a cepR knockout mutant synthesized 70% less HSLs, and that CepR responded best towards long-chain HSLs . In addition, we also report the cloning and characterization of the stationary-phase sigma factor gene rpoS of B . cepacia ATCC 25416 . It was established that quorum sensing in B . cepacia has a negative effect on rpoS expression as determined by using an rpoS-lacZ transcriptional fusion; on the other hand, rpoS-null mutants displayed no difference in the accumulation of HSL signal molecules .
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