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Expression of a Functional Secreted YopN-TyeA Hybrid Protein in Yersinia pestis Is the Result of a +1 Translational Frameshift Event. Franco Ferracci, 2004.YopN is a secreted protein that prior to secretion directly interacts with the cytosolic SycN/YscB chaperone complex and TyeA . This study identifies a secreted YopN-TyeA hybrid protein that is expressed by Yersinia pestis, but not by Yersinia enterocolitica . DNA sequence analysis and site-directed mutagenesis studies demonstrate that the hybrid protein is the result of a +1 translational frameshift event . Diversity and Evolution of the Class A Chromosomal Beta-Lactamase Gene in Klebsiella pneumoniae. S. Hæggman, 2004.We investigated the diversity of the chromosomal class A beta-lactamase gene in Klebsiella pneumoniae in order to study the evolution of the gene . A 789-bp portion was sequenced in a panel of 28 strains, representative of three phylogenetic groups, KpI, KpII, and KpIII, recently identified in K . pneumoniae and of different chromosomal beta-lactamase variants previously identified . Three groups of sequences were found, two of them corresponding to the families SHV (pI 7.6) and LEN (pI 7.1), respectively, and one, more heterogeneous, corresponding to a new family that we named OKP (for other K . pneumoniae beta-lactamase) . Levels of susceptibility to ampicillin, cefuroxime, cefotaxime, ceftazidime, and aztreonam and inhibition by clavulanic acid were similar in the three groups . One new SHV variant, seven new LEN variants, and four OKP variants were identified . The OKP variants formed two subgroups based on nucleotide sequences, one with pIs of 7.8 and 8.1 and the other with pIs of 6.5 and 7.0 . The nucleotide sequences of the housekeeping genes gyrA, coding for subunit A of gyrase, and mdh, coding for malate dehydrogenase, were also determined . Phylogenetic analysis of the three genes studied revealed parallel evolution, with the SHV, OKP, and LEN beta-lactamase families corresponding to the phylogenetic groups KpI, KpII, and KpIII, respectively . This correspondence was fully confirmed for 34 additional strains in PCR assays specific for the three beta-lactamase families . We estimated the time since divergence of the phylogenetic groups KpI and KpIII at between 6 and 28 million years, confirming the ancient presence of the beta-lactamase gene in the genome of K . pneumoniae . Molecular Cloning and Expression of Mn2+-Dependent Sphingomyelinase/Hemolysin of an Aquatic Bacterium, Pseudomonas sp . Strain TK4. Noriyuki Sueyoshi, 2002.We report here the molecular cloning and expression of a hemolytic sphingomyelinase from an aquatic bacterium, Pseudomonas sp . strain TK4 . The sphingomyelinase gene was found to consist of 1,548 nucleotides encoding 516 amino acid residues . The recombinant 57.7-kDa enzyme hydrolyzed sphingomyelin but not phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, or phosphatidylethanolamine, indicating that the enzyme is a sphingomyelin-specific sphingomyelinase C . The hydrolysis of sphingomyelin by the enzyme was found to be most efficient at pH 8.0 and activated by Mn2+ . The enzyme shows quite a broad specificity, i.e., it hydrolyzed 4-nitrobenz-2-oxa-1,3-diazole (NBD)-sphingomyelin with short-chain fatty acids and NBD-sphingosylphosphorylcholine, the latter being completely resistant to hydrolysis by any sphingomyelinase reported so far . Significant sequence similarities were found in sphingomyelinases from Bacillus cereus, Staphylococcus aureus, Listeria ivanovii, and Leptospira interrogans, as well as a hypothetical protein encoded in Chromobacterium violaceum, although the first three lacked one-third of the sequence corresponding to that from the C terminus of the TK4 enzyme . Interestingly, the deletion mutant of strain TK4 lacking 186 amino acids at the C-terminal end hydrolyzed sphingomyelin, whereas it lost all hemolytic activity, indicating that the C-terminal region of the TK4 enzyme is indispensable for the hemolytic activity . Signal Detection and Target Gene Induction by the CpxRA Two-Component System. Patricia A. DiGiuseppe, 2003.The Cpx pathway is a two-component signal transduction system that senses a variety of envelope stresses, including misfolded proteins, and responds by upregulating periplasmic folding and trafficking factors . CpxA resides in the inner membrane and has both kinase and phosphatase activities . CpxR, the response regulator, mediates a response by activating transcription of stress-combative genes . Signal transduction is subject to feedback inhibition via regulon member CpxP and autoamplification . Recently, it was shown that the Cpx pathway is also upregulated when cells adhere to hydrophobic surfaces and that this response is dependent on the outer membrane lipoprotein NlpE . Here we show that while NlpE is required for induction of the Cpx pathway by adhesion, induction by envelope stress and during growth is NlpE independent . We show that while all of the envelope stresses tested induce the Cpx pathway in a manner that is dependent on the periplasmic domain of CpxA, induction during growth is independent of CpxA . Therefore, we propose that the Cpx pathway can sense inducing cues that enter the signaling pathway at three distinct points . Although CpxP is not required for induction of the Cpx pathway, we show that its activity as a negative regulator of CpxA is inactivated by envelope stress . Moreover, the cpxP promoter is more inducible than any other regulon member tested . Consistent with these results, we suggest that CpxP performs a second function, most likely that of a chaperone . Finally, we show that two Cpx-regulated genes are differentially upregulated in response to different envelope stresses, suggesting the existence of three stress-responsive systems .
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