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Role of the Pilot Protein YscW in the Biogenesis of the YscC Secretin in Yersinia enterocolitica. Peter Burghout, 2004.The YscC secretin is a major component of the type III protein secretion system of Yersinia enterocolitica and forms an oligomeric structure in the outer membrane . In a mutant lacking the outer membrane lipoprotein YscW, secretion is strongly reduced, and it has been proposed that YscW plays a role in the biogenesis of the secretin . To study the interaction between the secretin and this putative pilot protein, YscC and YscW were produced in trans in a Y . enterocolitica strain lacking all other components of the secretion machinery . YscW expression increased the yield of oligomeric YscC and was required for its outer membrane localization, confirming the function of YscW as a pilot protein . Whereas the pilot-binding site of other members of the secretin family has been identified in the C terminus, a truncated YscC derivative lacking the C-terminal 96 amino acid residues was functional and stabilized by YscW . Pulse-chase experiments revealed that  30
min were required before YscC oligomerization was completed . In the
absence of YscW, oligomerization was delayed and the yield of YscC
oligomers was strongly reduced . An unlipidated form of the YscW
protein was not functional, although it still interacted with the
secretin and caused mislocalization of YscC even in the presence of
wild-type YscW . Hence, YscW interacts with the unassembled YscC
protein and facilitates efficient oligomerization, likely at the
outer membrane .
Diversity of Toxic Shock Syndrome Toxin 1-Positive Staphylococcus aureus Isolates. John E. Warner, 2004. Clearance of Human-Pathogenic Viruses from Sludge: Study of Four Stabilization Processes by Real-Time Reverse Transcription-PCR and Cell Culture. S. Monpoeho, 2004.Sludges derived from wastewater treatment are foul-smelling, biologically unstable substances . As well as containing numerous pathogenic microorganisms, they also consist of organic matter that can be used as agricultural fertilizer . Legislation nevertheless requires sludges to be virologically tested prior to spreading by the counting of infectious enterovirus particles . This method, based on culture of enterovirus on BGM cells, is lengthy and not very sensitive . The aim of this study was to propose an alternative method of genome quantification for all enteroviruses that is applicable to verifying the elimination of viruses in complex samples such as sludges . Our complete protocol was compared to the official method, consisting of enterovirus enumeration with the most probable number of cythopathic unit (MPNCU) assay through the study of four stabilization procedures: liming, composting, heat treatment, and mesophile anaerobic digestion . Enterovirus quantities at the start of the stabilization procedures were between 37 and 288 MPNCU/g on the one scale and between 4 and 5 log genome copies/g on the other . It was shown that all procedures except mesophile anaerobic digestion were highly effective in the elimination of enterovirus particles and genomes in wastewater sludges . Reduction of viruses by mesophile anaerobic digestion was by only 1 log (infectious particles and genomes) . In conclusion, stabilization processes can indeed be checked by virological quality control of sludges with gene amplification . However, the infectivity of genomes needs to be confirmed with cell culture or a correlation model if the virological risk inherent in the agricultural use of such sludges is to be fully addressed . Temperature- and H-NS-Dependent Regulation of a Plasmid-Encoded Virulence Operon Expressing Escherichia coli Hemolysin. Cristina Madrid, 2002.Proteins H-NS and Hha form a nucleoprotein complex that modulates expression of the thermoregulated hly operon of Escherichia coli. We have been able to identify two H-NS binding sites in the hly regulatory region . One of them partially overlaps the promoter region (site II), and the other is located about 2 kbp upstream (site I) . In contrast, Hha protein did not show any preference for specific sequences . In vitro, temperature influences the affinity of H-NS for a DNA fragment containing both binding sites and H-NS-mediated repression of hly operon transcription . Deletion analysis of the hly regulatory region confirms the relevance of site I for thermoregulation of this operon . We present a model to explain the temperature-modulated repression of the hly operon, based on the experiments reported here and other, preexisting data . Disruption of lolCDE, Encoding an ATP-Binding Cassette Transporter, Is Lethal for Escherichia coli and Prevents Release of Lipoproteins from the Inner Membrane. Shin-ichiro Narita, 2002.ATP-binding cassette transporter LolCDE was previously identified, by using reconstituted proteoliposomes, as an apparatus catalyzing the release of outer membrane-specific lipoproteins from the inner membrane of Escherichia coli . Mutations resulting in defective LolD were previously shown to be lethal for E . coli . The amino acid sequences of LolC and LolE are similar to each other, but the necessity of both proteins for lipoprotein release has not been proved . Moreover, previous reconstitution experiments did not clarify whether or not LolCDE is the sole apparatus for lipoprotein release . To address these issues, a chromosomal lolC-lolD-lolE null mutant harboring a helper plasmid that carries the lolCDE genes and a temperature-sensitive replicon was constructed . The mutant failed to grow at a nonpermissive temperature because of the depletion of LolCDE . In addition to functional LolD, both LolC and LolE were required for growth . At a nonpermissive temperature, the outer membrane lipoproteins were mislocalized in the inner membrane since LolCDE depletion inhibited the release of lipoproteins from the inner membrane . Furthermore, both LolC and LolE were essential for the release of lipoproteins . On the other hand, LolCDE depletion did not affect the translocation of a lipoprotein precursor across the inner membrane and subsequent processing to the mature lipoprotein . From these results, we conclude that the LolCDE complex is an essential ABC transporter for E . coli and the sole apparatus mediating the release of outer membrane lipoproteins from the inner membrane .
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