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Type IV-Like Pili Formed by the Type II Secreton: Specificity, Composition, Bundling, Polar Localization, and Surface Presentation of Peptides. Guillaume Vignon, 2003.The secreton or type II secretion machinery of gram-negative bacteria includes several type IV pilin-like proteins (the pseudopilins) that are absolutely required for secretion . We previously reported the presence of a bundled pilus composed of the pseudopilin PulG on the surface of agar-grown Escherichia coli K-12 cells expressing the Klebsiella oxytoca pullulanase (Pul) secreton genes at high levels (N . Sauvonnet, G . Vignon, A . P . Pugsley, and P . Gounon, EMBO J . 19:2221-2228, 2000) . We show here that PulG is the only pseudopilin in purified pili and that the phenomenon is not restricted to the Pul secreton reconstituted in E . coli or to PulG . For example, high-level expression of the endogenous E. coli gsp secreton genes caused production of bundled pili composed of the pseudopilin GspG, and the Pul secreton was able to form pili composed of PulG-like proteins from secreton systems of other bacteria . PulG derivatives in which the C terminus was extended by the addition of eight different peptides were also assembled into pili and functioned in secretion . Three of the C-terminal peptides were shown to be exposed along the entire length of the assembled pili . Hence, the C terminus of PulG may represent a permissive site for the insertion of immunogenic epitopes or other peptide sequences . One of these PulG variants, with a six-histidine tag at its C terminus, formed nonpolar, nonbundled pili, suggesting that bundle formation and polar localization are not correlated with the ability of PulG to function in secretion . We propose that the PulG pilus is an artifactual manifestation of a periplasmic "pseudopilus" and that cycles of pseudopilus extension and retraction within the periplasm propel pullulanase through secretin channels in the outer membrane . Abnormally long pili that extend beyond the outer membrane are produced only when pilus length control and retraction are deregulated by overproduction of the major pseudopilus subunit (PulG) . Differential Protein Expression during Growth of Acidithiobacillus ferrooxidans on Ferrous Iron, Sulfur Compounds, or Metal Sulfides. Pablo Ramírez, 2004.A set of proteins that changed their levels of synthesis during growth of Acidithiobacillus ferrooxidans ATCC 19859 on metal sulfides, thiosulfate, elemental sulfur, and ferrous iron was characterized by using two-dimensional polyacrylamide gel electrophoresis . N-terminal amino acid sequencing and mass spectrometry analysis of these proteins allowed their identification and the localization of the corresponding genes in the available genomic sequence of A . ferrooxidans ATCC 23270 . The genomic context around several of these genes suggests their involvement in the energetic metabolism of A . ferrooxidans . Two groups of proteins could be distinguished . The first consisted of proteins highly upregulated by growth on sulfur compounds (and downregulated by growth on ferrous iron): a 44-kDa outer membrane protein, an exported 21-kDa putative thiosulfate sulfur transferase protein, a 33-kDa putative thiosulfate/sulfate binding protein, a 45-kDa putative capsule polysaccharide export protein, and a putative 16-kDa protein of unknown function . The second group of proteins comprised those downregulated by growth on sulfur (and upregulated by growth on ferrous iron): rusticyanin, a cytochrome c552, a putative phosphate binding protein (PstS), the small and large subunits of ribulose biphosphate carboxylase, and a 30-kDa putative CbbQ protein, among others . The results suggest in general a separation of the iron and sulfur utilization pathways . Rusticyanin, in addition to being highly expressed on ferrous iron, was also newly synthesized, as determined by metabolic labeling, although at lower levels, during growth on sulfur compounds and iron-free metal sulfides . During growth on metal sulfides containing iron, such as pyrite and chalcopyrite, both proteins upregulated on ferrous iron and those upregulated on sulfur compounds were synthesized, indicating that the two energy-generating pathways are induced simultaneously depending on the kind and concentration of oxidizable substrates available . In Situ Exposure to Low Herbicide Concentrations Affects Microbial Population Composition and Catabolic Gene Frequency in an Aerobic Shallow Aquifer. Julia R. de Lipthay, 2003.The aim of this study was to evaluate how the in situ exposure of a Danish subsurface aquifer to phenoxy acid herbicides at low concentrations (<40 µg l-1) changes the microbial community composition . Sediment and groundwater samples were collected inside and outside the herbicide-exposed area and were analyzed for the presence of general microbial populations, Pseudomonas bacteria, and specific phenoxy acid degraders . Both culture-dependent and culture-independent methods were applied . The abundance of microbial phenoxy acid degraders (100 to 104 g-1 sediment) was determined by most probable number assays, and their presence was only detected in herbicide-exposed sediments . Similarly, PCR analysis showed that the 2,4-dichlorophenoxyacetic acid degradation pathway genes tfdA and tfdB (102 to 103 gene copies g-1 sediment) were only detected in sediments from contaminated areas of the aquifer . PCR-restriction fragment length polymorphism measurements demonstrated the presence of different populations of tfd genes, suggesting that the in situ herbicide degradation was caused by the activity of a heterogeneous population of phenoxy acid degraders . The number of Pseudomonas bacteria measured by either PCR or plating on selective agar media was higher in sediments subjected to high levels of phenoxy acid . Furthermore, high numbers of CFU compared to direct counting of 4',6-diamidino-2-phenylindole-stained cells in the microscope suggested an increased culturability of the indigenous microbial communities from acclimated sediments . The findings of this study demonstrate that continuous exposure to low herbicide concentrations can markedly change the bacterial community composition of a subsurface aquifer . External pH and Nitrogen Source Affect Secretion of Pectate Lyase by Colletotrichum gloeosporioides. N. Drori, 2003.Accumulation of ammonia and associated tissue alkalinization predispose fruit to attack by Colletotrichumgloeosporioides. As the external pH increases from 4.0 to 6.0, pectate lyase (PL) and other extracellular proteins are secreted and accumulate . At pH 4.0 neither pelB (encoding PL) transcription nor PL secretion were detected; however, they were detected as the pH increased . Nitrogen assimilation also was required for PL secretion at pH 6.0 . Both inorganic and organic nitrogen sources enhanced PL secretion at pH 6.0, but neither was sufficient for PL secretion at pH 4.0 . Sequence analysis of the 5' upstream region of the pelB promoter revealed nine putative consensus binding sites for the Aspergillus transcription factor PacC . Consistent with this result, the transcript levels of pac1 (the C . gloeosporioides pacC homologue) and pelB increased in parallel as a function of pH . Our results suggest that the ambient pH and the nitrogen source are independent regulatory factors for processes linked to PL secretion and virulence of C . gloeosporioides .
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