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The sufR Gene (sll0088 in Synechocystis sp. Strain PCC 6803) Functions as a Repressor of the sufBCDS Operon in Iron-Sulfur Cluster Biogenesis in Cyanobacteria. Tao Wang, 2004.The suf operon is composed of four genes (sufB, sufC, sufD, and sufS) and is highly conserved in the genomes of cyanobacteria . Open reading frame sll0088 in Synechocystis sp . strain PCC 6803 is located near the 5' end of the suf operon but is transcribed in the direction opposite that of the suf operon . We previously reported the isolation of two independent suppressor strains of C14SPsaC that mapped to sll0088 and restored photoautotrophic growth . The protein encoded by sll0088 has two significant features: (i) a DNA-binding domain near the N terminus and (ii) four highly conserved cysteine residues near the C terminus . The protein has high sequence similarity to transcription regulatory proteins with a conserved DNA-binding domain and can be classified in the DeoR family of helix-loop-helix proteins . The protein falls into a further subclass that contains a C-X12-C-X13-C-X14-C motif near the C terminus, which may represent a metal-binding site . The expressed Sll0088 protein harbored an iron-sulfur cluster as shown by optical and electron paramagnetic resonance spectroscopy . Compared to the wild type, expression levels of the sufBCDS genes were elevated when cells were grown under conditions of oxidative and iron stress and were even higher in a null mutant of Synechococcus sp . strain PCC 7002 in which the sll0088 homolog was insertionally inactivated . In agreement with the proposed role of the sufBCDS genes in iron metabolism, the growth rate of the null mutant was significantly higher than that of the wild type under iron-limiting conditions . We propose that the protein encoded by sll0088 is a transcriptional repressor of the suf operon, and we name the gene sufR . Enterococcus faecalis 3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase, an Enzyme of Isopentenyl Diphosphate Biosynthesis. Autumn Sutherlin, 2002.Biosynthesis of the isoprenoid precursor isopentenyl diphosphate (IPP) proceeds via two distinct pathways . Sequence comparisons and microbiological data suggest that multidrug-resistant strains of gram-positive cocci employ exclusively the mevalonate pathway for IPP biosynthesis . Bacterial mevalonate pathway enzymes therefore offer potential targets for development of active site-directed inhibitors for use as antibiotics . We used the PCR and Enterococcus faecalis genomic DNA to isolate the mvaS gene that encodes 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, the second enzyme of the mevalonate pathway . mvaS was expressed in Escherichia coli from a pET28 vector with an attached N-terminal histidine tag . The expressed enzyme was purified by affinity chromatography on Ni2+-agarose to apparent homogeneity and a specific activity of 10 µmol/min/mg . Analytical ultracentrifugation showed that the enzyme is a dimer (mass, 83.9 kDa; s20,w, 5.3) . Optimal activity occurred in 2.0 mM MgCl2 at 37oC . The  Ha was 6,000 cal . The pH activity profile, optimum activity at pH 9.8, yielded a pKa of 8.8 for a dissociating group, presumably Glu78 . The stoichiometry per monomer of acetyl-CoA binding was 1.2 ± 0.2 and that of covalent acetylation was 0.60 ± 0.02 . The Km for the hydrolysis of acetyl-CoA was 10 µM . Coupled conversion of acetyl-CoA to mevalonate was demonstrated by using HMG-CoA synthase and acetoacetyl-CoA thiolase/HMG-CoA reductase from E . faecalis .
Molecular Characterization and Expression in Escherichia coli of Three ß-1,3-Glucanase Genes from Lysobacter enzymogenes Strain N4-7. Jeffrey D. Palumbo, 2003.Lysobacter enzymogenes strain N4-7 produces multiple biochemically distinct extracellular ß-1,3-glucanase activities . The gluA, gluB, and gluC genes, encoding enzymes with ß-1,3-glucanase activity, were identified by a reverse-genetics approach following internal amino acid sequence determination of ß-1,3-glucanase-active proteins partially purified from culture filtrates of strain N4-7 . Analysis of gluA and gluC gene products indicates that they are members of family 16 glycoside hydrolases that have significant sequence identity to each other throughout the catalytic domain but that differ structurally by the presence of a family 6 carbohydrate-binding domain within the gluC product . Analysis of the gluB gene product indicates that it is a member of family 64 glycoside hydrolases . Expression of each gene in Escherichia coli resulted in the production of proteins with ß-1,3-glucanase activity . Biochemical analyses of the recombinant enzymes indicate that GluA and GluC exhibit maximal activity at pH 4.5 and 45°C and that GluB is most active between pH 4.5 and 5.0 at 41°C . Activity of recombinant proteins against various ß-1,3 glucan substrates indicates that GluA and GluC are most active against linear ß-1,3 glucans, while GluB is most active against the insoluble ß-1,3 glucan substrate zymosan A . These data suggest that the contribution of ß-1,3-glucanases to the biocontrol activity of L . enzymogenes may be due to complementary activities of these enzymes in the hydrolysis of ß-1,3 glucans from fungal cell walls . Rhamnolipid Surfactant Production Affects Biofilm Architecture in Pseudomonas aeruginosa PAO1. Mary E. Davey, 2003.In response to certain environmental signals, bacteria will differentiate from an independent free-living mode of growth and take up an interdependent surface-attached existence . These surface-attached microbial communities are known as biofilms . In flowing systems where nutrients are available, biofilms can develop into elaborate three-dimensional structures . The development of biofilm architecture, particularly the spatial arrangement of colonies within the matrix and the open areas surrounding the colonies, is thought to be fundamental to the function of these complex communities . Here we report a new role for rhamnolipid surfactants produced by the opportunistic pathogen Pseudomonas aeruginosa in the maintenance of biofilm architecture . Biofilms produced by mutants deficient in rhamnolipid synthesis do not maintain the noncolonized channels surrounding macrocolonies . We provide evidence that surfactants may be able to maintain open channels by affecting cell-cell interactions and the attachment of bacterial cells to surfaces . The induced synthesis of rhamnolipids during the later stages of biofilm development (when cell density is high) implies an active mechanism whereby the bacteria exploit intercellular interaction and communication to actively maintain these channels . We propose that the maintenance of biofilm architecture represents a previously unrecognized step in the development of these microbial communities .
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