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Genomic Analysis and Initial Characterization of the Chitinolytic System of Microbulbifer degradans Strain 2-40. Michael B. Howard, 2003.The marine bacterium Microbulbifer degradans strain 2-40 produces at least 10 enzyme systems for degrading insoluble complex polysaccharides (ICP) . The draft sequence of the 2-40 genome allowed a genome-wide analysis of the chitinolytic system of strain 2-40 . The chitinolytic system includes three secreted chitin depolymerases (ChiA, ChiB, and ChiC), a secreted chitin-binding protein (CbpA), periplasmic chitooligosaccharide-modifying enzymes, putative sugar transporters, and a cluster of genes encoding cytoplasmic proteins involved in N-acetyl-D-glucosamine (GlcNAc) metabolism . Each chitin depolymerase was detected in culture supernatants of chitin-grown strain 2-40 and was active against chitin and glycol chitin . The chitin depolymerases also had a specific pattern of activity toward the chitin analogs 4-methylumbelliferyl-ß-D-N,N'-diacetylchitobioside (MUF-diNAG) and 4-methylumbelliferyl-ß-D-N,N',N"-triacetylchitotrioside (MUF-triNAG) . The depolymerases were modular in nature and contained glycosyl hydrolase family 18 domains, chitin-binding domains, and polycystic kidney disease domains . ChiA and ChiB each possessed polyserine linkers of up to 32 consecutive serine residues . In addition, ChiB and CbpA contained glutamic acid-rich domains . At 1,271 amino acids, ChiB is the largest bacterial chitinase reported to date . A chitodextrinase (CdxA) with activity against chitooligosaccharides (degree of polymerization of 5 to 7) was identified . The activities of two apparent periplasmic (HexA and HexB) N-acetyl-ß-D-glucosaminidases and one cytoplasmic (HexC) N-acetyl-ß-D-glucosaminidase were demonstrated . Genes involved in GlcNAc metabolism, similar to those of the Escherichia coli K-12 NAG utilization operon, were identified . NagA from strain 2-40, a GlcNAc deacetylase, was shown to complement a nagA mutation in E . coli K-12 . Except for the GlcNAc utilization cluster, genes for all other components of the chitinolytic system were dispersed throughout the genome . Further examination of this system may provide additional insight into the mechanisms by which marine bacteria degrade chitin and provide a basis for future research on the ICP-degrading systems of strain 2-40 . The Vibrio cholerae vieSAB Locus Encodes a Pathway Contributing to Cholera Toxin Production. Anna D. Tischler, 2002.The genes encoding cholera toxin (CT), ctxAB, are coregulated with those for other Vibrio cholerae virulence factors by a cascade of transcriptional activators, including ToxR, TcpP, and ToxT . Additional regulators that modulate expression of ctxAB during infection were recently identified in a genetic selection . A transposon insertion in vieS, the sensor kinase of the VieSAB three-component signal transduction system, resulted in failure to induce expression of a ctxA-recombinase fusion during murine infection . To determine which components of the VieSAB system are essential for CT regulation, ctxAB transcript levels were assessed by RNase protection assay in various vieSAB in-frame deletion mutants after growth in vitro under virulence gene inducing conditions . A threefold reduction in ctxAB transcript levels was observed for the  vieSAB strain; consistent with this, the
vieSAB strain produced twofold less CT protein than the wild type, and this defect was complementable in trans . These results suggest that the VieSAB three-component system is required for full activation of the ctxAB operon during in vitro growth as well as during infection . The VieSAB system may regulate ctxAB expression indirectly by affecting production of ToxT, because decreased toxT transcript levels were observed in the
vieSAB strain .
Genes for Chlorogenate and Hydroxycinnamate Catabolism (hca) Are Linked to Functionally Related Genes in the dca-pca-qui-pob-hca Chromosomal Cluster of Acinetobacter sp . Strain ADP1. Michael A. Smith, 2003.Hydroxycinnamates are ubiquitous in the environment because of their contributions to the structure and defense mechanisms of plants . Additional plant products, many of which are formed in response to stress, support the growth of Acinetobacter sp . strain ADP1 through pathways encoded by genes in the dca-pca-qui-pob chromosomal cluster . In an appropriate genetic background, it was possible to select for an Acinetobacter strain that had lost the ability to grow with caffeate, a commonly occurring hydroxycinnamate . The newly identified mutation was shown to be a deletion in a gene designated hcaC and encoding a ligase required for conversion of commonly occurring hydroxycinnamates (caffeate, ferulate, coumarate, and 3,4-dihydroxyphenylpropionate) to thioesters . Linkage analysis showed that hcaC is linked to pobA . Downstream from hcaC and transcribed in the direction opposite the direction of pobA transcription are open reading frames designated hcaDEFG . Functions of these genes were inferred from sequence comparisons and from the properties of knockout mutants . HcaD corresponded to an acyl coenzyme A (acyl-CoA) dehydrogenase required for conversion of 3,4-dihydroxyphenylpropionyl-CoA to caffeoyl-CoA . HcaE appears to encode a member of a family of outer membrane proteins known as porins . Knockout mutations in hcaF confer no discernible phenotype . Knockout mutations in hcaG indicate that this gene encodes a membrane-associated esterase that hydrolyzes chlorogenate to quinate, which is metabolized in the periplasm, and caffeate, which is metabolized by intracellular enzymes . The chromosomal location of hcaG, between hcaC (required for growth with caffeate) and quiA (required for growth with quinate), provided the essential clue that led to the genetic test of HcaG as the esterase that produces caffeate and quinate from chlorogenate . Thus, in this study, organization within what is now established as the dca-pca-qui-pob-hca chromosomal cluster provided essential information about the function of genes in the environment .
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