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Identification of psl, a Locus Encoding a Potential Exopolysaccharide That Is Essential for Pseudomonas aeruginosa PAO1 Biofilm Formation. Kara D. Jackson, 2004.Bacteria inhabiting biofilms usually produce one or more polysaccharides that provide a hydrated scaffolding to stabilize and reinforce the structure of the biofilm, mediate cell-cell and cell-surface interactions, and provide protection from biocides and antimicrobial agents . Historically, alginate has been considered the major exopolysaccharide of the Pseudomonas aeruginosa biofilm matrix, with minimal regard to the different functions polysaccharides execute . Recent chemical and genetic studies have demonstrated that alginate is not involved in the initiation of biofilm formation in P . aeruginosa strains PAO1 and PA14 . We hypothesized that there is at least one other polysaccharide gene cluster involved in biofilm development . Two separate clusters of genes with homology to exopolysaccharide biosynthetic functions were identified from the annotated PAO1 genome . Reverse genetics was employed to generate mutations in genes from these clusters . We discovered that one group of genes, designated psl, are important for biofilm initiation . A PAO1 strain with a disruption of the first two genes of the psl cluster (PA2231 and PA2232) was severely compromised in biofilm initiation, as confirmed by static microtiter and continuous culture flow cell and tubing biofilm assays . This impaired biofilm phenotype could be complemented with the wild-type psl sequences and was not due to defects in motility or lipopolysaccharide biosynthesis . These results implicate an as yet unknown exopolysaccharide as being required for the formation of the biofilm matrix . Understanding psl-encoded exopolysaccharide expression and protection in biofilms will provide insight into the pathogenesis of P . aeruginosa in cystic fibrosis and other infections involving biofilms . Glycerol-3-Phosphate-Induced Catabolite Repression in Escherichia coli. Tanja Eppler, 2002.The formation of glycerol-3-phosphate (G3P) in cells growing on TB causes catabolite repression, as shown by the reduction in malT expression . For this repression to occur, the general proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), in particular EIIAGlc, as well as the adenylate cyclase and the cyclic AMP-catabolite activator protein system, have to be present . We followed the level of EIIAGlc phosphorylation after the addition of glycerol or G3P . In contrast to glucose, which causes a dramatic shift to the dephosphorylated form, glycerol or G3P only slightly increased the amount of dephosphorylated EIIAGlc . Isopropyl-ß-D-thiogalactopyranoside-induced overexpression of EIIAGlc did not prevent repression by G3P, excluding the possibility that G3P-mediated catabolite repression is due to the formation of unphosphorylated EIIAGlc . A mutant carrying a C-terminally truncated adenylate cyclase was no longer subject to G3P-mediated repression . We conclude that the stimulation of adenylate cyclase by phosphorylated EIIAGlc is controlled by G3P and other phosphorylated sugars such as D-glucose-6-phosphate and is the basis for catabolite repression by non-PTS compounds . Further metabolism of these compounds is not necessary for repression . Two-dimensional polyacrylamide gel electrophoresis was used to obtain an overview of proteins that are subject to catabolite repression by glycerol . Some of the prominently repressed proteins were identified by peptide mass fingerprinting . Among these were periplasmic binding proteins (glutamine and oligopeptide binding protein, for example), enzymes of the tricarboxylic acid cycle, aldehyde dehydrogenase, Dps (a stress-induced DNA binding protein), and D-tagatose-1,6-bisphosphate aldolase . Secretion of  -Amylase from Pseudoalteromonas haloplanktis TAB23: Two Different Pathways in Different Hosts.Maria Luisa Tutino, 2002.Secretion of cold-adapted -amylase from Pseudoalteromonas haloplanktis TAB23 was studied in three Antarctic bacteria . We demonstrated that the enzyme is specifically secreted in the psychrophilic hosts even in the absence of a protein domain that has been previously reported to be necessary for
-amylase secretion in Escherichia coli . The occurrence of two different secretion pathways in different hosts is proposed .
Identification of [2Fe-2S] Clusters in Microbial Ferrochelatases. Tamara A. Dailey, 2002.The terminal enzyme of heme biosynthesis, ferrochelatase (EC 4.99.1.1), catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme . Prior to the present work, [2Fe-2S] clusters have been identified and characterized in animal ferrochelatases but not in plant or prokaryotic ferrochelatases . Herein we present evidence that ferrochelatases from the bacteria Caulobacter crescentus and Mycobacterium tuberculosis possess [2Fe-2S] clusters . The enzyme from C . crescentus is a homodimeric, membrane-associated protein while the enzyme from M . tuberculosis is monomeric and soluble . The clusters of the C . crescentus and M . tuberculosis ferrochelatases are ligated by four cysteines but possess ligand spacings that are unlike those of any previously characterized [2Fe-2S] cluster-containing protein, including the ferrochelatase of the yeast Schizosaccharomyces pombe . Thus, the microbial ferrochelatases represent a new group of [2Fe-2S] cluster-containing proteins .
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