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In Vivo Expression of the Mannose-Resistant Fimbriae of Photorhabdus temperata K122 during Insect Infection. L. M. Meslet-Cladiere, 2004.Photorhabdus temperata K122 is an entomopathogenic bacterium symbiotically associated with nematodes of the family Heterorhabditidae . Surface fimbriae are important for the colonization of many pathogenic bacteria, and here we report the nucleotide sequence and analysis of the expression of a 12-kbp fragment encoding the mannose-resistant fimbriae of P . temperata (mrf) . The mrf gene cluster contains 11 genes with an organization similar to that of the mrp locus from Proteus mirabilis . mrfI (encoding a putative recombinase) and mrfA (encoding pilin), the first gene in an apparent operon of nine other genes, are expressed from divergent promoters . The mrfI-mrfA intergenic region contains inverted repeats flanking the mrfA promoter . This region was shown to be capable of inversion, consistent with an ON/OFF regulation of the operon . In in vitro liquid cultures, both orientations were detected . Nevertheless, when we analyzed the expression of all of the genes in the mrf locus by semiquantitative reverse transcription-PCR during infection of Galleria mellonella (greater wax moth) larvae, expression of mrfA was not detected until 25 h postinfection, preceding the death of the larvae at 32 h . In contrast, mrfJ (a putative inhibitor of flagellar synthesis) was expressed throughout infection . Expression of mrfI was also detected only late in infection (25 to 30 h), indicating a possible increase in inversion frequency at this stage . In both in vitro liquid cultures and in vivo larval infections, the distal genes of the operon were expressed at substantially lower levels than mrfA . These results indicate the complex regulation of the mrf cluster during infection . Biosynthesis of the Cyanobacterial Light-Harvesting Polypeptide Phycoerythrocyanin Holo-  Subunit in a Heterologous Host.Aaron J. Tooley, 2002.The entire pathway for the biosynthesis of the phycobiliviolin-bearing His-tagged holo- subunit of the cyanobacterial photosynthetic accessory protein phycoerythrocyanin was reconstituted in Escherichia coli. Cyanobacterial genes encoding enzymes required for the conversion of heme to 3Z-phycocyanobilin, a precursor of phycobiliviolin (namely, heme oxygenase 1 and 3Z-phycocyanobilin:ferredoxin oxidoreductase), were expressed from a plasmid under the control of the hybrid trp-lac (trc) promoter . Genes for the apo-phycoerythrocyanin
subunit (pecA) and the heterodimeric lyase/isomerase (pecE and pecF), which catalyzes both the covalent attachment of phycocyanobilin and its concurrent isomerization to phycobiliviolin, were expressed from the trc promoter on a second plasmid . Upon induction, recombinant E . coli used endogenous heme to produce holo-PecA with absorbance and fluorescence properties similar to those of the same protein produced in cyanobacteria . About two-thirds of the apo-PecA was converted to holo-PecA . No significant bilin addition took place in a similarly engineered E . coli strain that lacks pecE and pecF. By using immobilized metal affinity chromatography, both apo-PecA and holo-PecA were isolated as ternary complexes with PecE and PecF . The identities of all three components in the ternary complexes were established unambiguously by protein and tryptic peptide analyses performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry .
Role of Two Novel Two-Component Regulatory Systems in Development and Phosphatase Expression in Myxococcus xanthus. Aurelio Moraleda-Muñoz, 2003.We have cloned a two-component regulatory system (phoR2-phoP2) of Myxococcus xanthus while searching for genes that encode proteins with phosphatase activity, where phoR2 encodes the histidine kinase and phoP2 encodes the response regulator . A second system, phoR3-phoP3, was identified and isolated by using phoP2 as a probe . These two systems are quite similar, sharing identities along the full-length proteins of 52% on the histidine kinases and 64% on the response regulators . The predicted structures of both kinases suggest that they are anchored to the membrane, with the sensor domains being located in the periplasmic space and the kinase domains in the cytoplasm . The response regulators (PhoP2 and PhoP3) exhibit a helix-loop-helix motif typical of DNA-binding proteins in the effector domains located in the C-terminal region . Studies on two single-deletion mutants and one double-deletion mutant have revealed that these systems are involved in development . Mutant fruiting bodies are not well packed, originating loose and flat aggregates where some myxospores do not reshape properly, and they remain as elongated cells . These systems are also involved in the expression of Mg-independent acid and neutral phosphatases, which are expressed during development . The neutral phosphatase gene is especially dependent on PhoP3 . Neither PhoP2 nor PhoP3 regulates the expression of alkaline phosphatases and the pph1 gene . Identification and Characterization of Lactobacillus helveticus PepO2, an Endopeptidase with Post-Proline Specificity. Yo-Shen Chen, 2003.A post-proline endopeptidase (PepO2) was detected in cell extracts from a genomic library of Lactobacillus helveticus CNRZ32 by using the synthetic substrate N-acetyl-ß-casein-(f203-209)-  -nitroanilide in a coupled reaction with aminopeptidase N . Isolates with activity for this substrate contained plasmids with visually indistinguishable restriction profiles . Nucleotide sequence analysis revealed a 1,947-bp open reading frame, designated pepO2, encoding a putative 71.4-kDa protein . Analysis of the predicted peptide sequence revealed that L . helveticus PepO2 contained the zinc-dependent metalloprotease motif HEXXH and exhibited levels of amino acid sequence similarity of 72, 61, 59, and 53% to L . helveticus PepO, Lactococcus lactis PepO2, L . lactis PepO, and Lactobacillus rhamnosus PepO, respectively . Northern hybridization results indicated that the transcript containing pepO2 was monocistronic . Despite the high degrees of amino acid similarity to PepO proteins from other lactic acid bacteria, the specificity of the L . helveticus PepO2 for post-proline bonds distinguishes it from other PepO-type endopeptidases characterized to date . The specificity for post-proline bonds also suggests that this enzyme may play a central role in the hydrolysis of casein-derived bitter peptides, such as ß-casein(f193-209) .
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