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Substrate Specificity of the Escherichia coli Outer Membrane Protease OmpT. John D. McCarter, 2004.OmpT is a surface protease of gram-negative bacteria that has been shown to cleave antimicrobial peptides, activate human plasminogen, and degrade some recombinant heterologous proteins . We have analyzed the substrate specificity of OmpT by two complementary substrate filamentous phage display methods: (i) in situ cleavage of phage that display protease-susceptible peptides by Escherichia coli expressing OmpT and (ii) in vitro cleavage of phage-displayed peptides using purified enzyme . Consistent with previous reports, OmpT was found to exhibit a virtual requirement for Arg in the P1 position and a slightly less stringent preference for this residue in the P1' position (P1 and P1' are the residues immediately prior to and following the scissile bond) . Lys, Gly, and Val were also found in the P1' position . The most common residues in the P2' position were Val or Ala, and the P3 and P4 positions exhibited a preference for Trp or Arg . Synthetic peptides based upon sequences selected by bacteriophage display were cleaved very efficiently, with kcat/Km values up to 7.3 x 106 M–1 s–1 . In contrast, a peptide corresponding to the cleavage site of human plasminogen was hydrolyzed with a kcat/Km almost 106-fold lower . Overall, the results presented in this work indicate that in addition to the P1 and P1' positions, additional amino acids within a six-residue window (between P4 and P2') contribute to the binding of substrate polypeptides to the OmpT binding site . Involvement of the SppA1 Peptidase in Acclimation to Saturating Light Intensities in Synechocystis sp . Strain PCC 6803. E. Pojidaeva, 2004.The sll1703 gene, encoding an Arabidopsis homologue of the thylakoid membrane-associated SppA peptidase, was inactivated by interposon mutagenesis in Synechocystis sp . strain PCC 6803 . Upon acclimation from a light intensity of 50 to 150 µE m–2 s–1, the mutant preserved most of its phycobilisome content, whereas the wild-type strain developed a bleaching phenotype due to the loss of about 40% of its phycobiliproteins . Using in vivo and in vitro experiments, we demonstrate that the  sppA1
strain does not undergo the cleavage of the LR33
and LCM99 linker proteins that develops in the
wild type exposed to increasing light intensities . We conclude that a
major contribution to light acclimation under a moderate light regime
in cyanobacteria originates from an SppA1-mediated cleavage of
phycobilisome linker proteins . Together with changes in gene
expression of the major phycobiliproteins, it contributes an
additional mechanism aimed at reducing the content in phycobilisome
antennae upon acclimation to a higher light intensity .
Bacterial Community Associated with Black Band Disease in Corals. Jorge Frias-Lopez, 2004.Black band disease (BBD) is a virulent polymicrobial disease primarily affecting massive-framework-building species of scleractinian corals . While it has been well established that the BBD bacterial mat is dominated by a cyanobacterium, the quantitative composition of the BBD bacterial mat community has not described previously . Terminal-restriction fragment length polymorphism (T-RFLP) analysis was used to characterize the infectious bacterial community of the bacterial mat causing BBD . These analyses revealed that the bacterial composition of the BBD mat does not vary between different coral species but does vary when different species of cyanobacteria are dominant within the mat . On the basis of the results of a new method developed to identify organisms detected by T-RFLP analysis, our data show that besides the cyanobacterium, five species of the division Firmicutes, two species of the Cytophaga-Flexibacter-Bacteroides (CFB) group, and one species of  -proteobacteria are also consistently abundant within the infectious mat . Of these dominant taxa, six were consistently detected in healthy corals . However, four of the six were found in much higher numbers in BBD mats than in healthy corals . One species of the CFB group and one species of Firmicutes were not always associated with the bacterial communities present in healthy corals . Of the eight dominant bacteria identified, two species were previously found in clone libraries obtained from BBD samples; however, these were not previously recognized as important . Furthermore, despite having been described as an important component of the pathogenetic mat, a Beggiatoa species was not detected in any of the samples analyzed . These results will permit the dominant BBD bacteria to be targeted for isolation and culturing experiments aimed at deciphering the disease etiology .
NADPH-Dependent L-Sorbose Reductase Is Responsible for L-Sorbose Assimilation in Gluconobacter suboxydans IFO 3291. Masako Shinjoh, 2002.The NADPH-dependent L-sorbose reductase (SR) of L-sorbose-producing Gluconobacter suboxydans IFO 3291 contributes to intracellular L-sorbose assimilation . The gene disruptant showed no SR activity and did not assimilate the once-produced L-sorbose, indicating that the SR functions mainly as an L-sorbose-reducing enzyme in vivo and not as a D-sorbitol-oxidizing enzyme . Dependence of Helicobacter pylori Urease Activity on the Nickel-Sequestering Ability of the UreE Accessory Protein. Stéphane Benoit, 2003.The Helicobacter pylori ureE gene product was previously shown to be required for urease expression, but its characteristics and role have not been determined . The UreE protein has now been overexpressed in Escherichia coli, purified, and characterized, and three altered versions were expressed to address a nickel-sequestering role of UreE . Purified UreE formed a dimer in solution and was capable of binding one nickel ion per dimer . Introduction of an extra copy of ureE into the chromosome of mutants carrying mutations in the Ni maturation proteins HypA and HypB resulted in partial restoration of urease activity (up to 24% of the wild-type levels) . Fusion proteins of UreE with increased ability to bind nickel were constructed by adding histidine-rich sequences (His-6 or His-10 to the C terminus and His-10 as a sandwich fusion) to the UreE protein . Each fusion protein was overexpressed in E . coli and purified, and its nickel-binding capacity and affinity were determined . Each construct was also expressed in wild-type H . pylori and in hypA and hypB mutant strains for determining in vivo urease activities . The urease activity was increased by introduction of all the engineered versions, with the greatest Ni-sequestering version (the His-6 version) also conferring the greatest urease activity on both the hypA and hypB mutants . The differences in urease activities were not due to differences in the amounts of urease peptides . Addition of His-6 to another expressed protein (triose phosphate isomerase) did not result in stimulation of urease, so urease activation is not related to the level of nonspecific protein-bound nickel . The results indicate a correlation between H . pylori urease activity and the nickel-sequestering ability of the UreE accessory protein . Branching of Escherichia coli Cells Arises from Multiple Sites of Inert Peptidoglycan. Miguel A. de Pedro, 2003.Some strains of Escherichia coli defective for dacA, the gene coding for penicillin-binding protein 5, exhibit a strong branching phenotype when cell division is blocked . Since such branch formation implies a differentiation of polar caps at ectopic locations in the cell envelope, we analyzed murein segregation and observed a strong correlation between areas of inert murein and these morphological anomalies . In particular, the tips of branches exhibited the same properties as those described for polar caps of wild-type cells, i.e., the synthesis and turnover of murein were inhibited . Also, the mobility of cell envelope proteins was apparently constrained in areas with morphological defects . Polar regions of branching cells and sacculi had aberrant morphologies with a very high frequency . Of special interest was that areas of inert murein at polar caps were often split by areas of active synthesis, a situation unlike that observed in wild-type cells . These observations suggest that in dacA mutants, branches and other morphological anomalies may arise from split polar caps or by de novo generation of new poles built around inert peptidoglycan patches in the side walls of the cell .
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