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41.
Vanja Klepac‐Ceraj Katherine P. Lemon Thomas R. Martin Martin Allgaier Steven W. Kembel Alixandra A. Knapp Stephen Lory Eoin L. Brodie Susan V. Lynch Brendan J. M. Bohannan Jessica L. Green Brian A. Maurer Roberto Kolter 《Environmental microbiology》2010,12(5):1293-1303
Polymicrobial bronchopulmonary infections in cystic fibrosis (CF) cause progressive lung damage and death. Although the arrival of Pseudomonas aeruginosa often heralds a more rapid rate of pulmonary decline, there is significant inter‐individual variation in the rate of decline, the causes of which remain poorly understood. By coupling culture‐independent methods with ecological analyses, we discovered correlations between bacterial community profiles and clinical disease markers in respiratory tracts of 45 children with CF. Bacterial community complexity was inversely correlated with patient age, presence of P. aeruginosa and antibiotic exposure, and was related to CF genotype. Strikingly, bacterial communities lacking P. aeruginosa were much more similar to each other than were those containing P. aeruginosa, regardless of antibiotic exposure. This suggests that community composition might be a better predictor of disease progression than the presence of P. aeruginosa alone and deserves further study. 相似文献
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Truncated K+ channel DNA sequences specifically suppress lymphocyte K+ channel gene expression. 总被引:4,自引:1,他引:3 下载免费PDF全文
We have constructed a series of deletion mutants of Kv1.3, a Shaker-like, voltage-gated K+ channel, and examined the ability of these truncated mutants to form channels and to specifically suppress full-length Kv1.3 currents. These constructs were expressed heterologously in both Xenopus oocytes and a mouse cytotoxic T cell line. Our results show that a truncated mutant Kv1.3 must contain both the amino terminus and the first transmembrane-spanning segment, S1, to suppress full-length Kv1.3 currents. Amino-terminal-truncated DNA sequences from one subfamily suppress K+ channel expression of members of only the same subfamily. The first 141 amino acids of the amino-terminal of Kv1.3 are not necessary for channel formation. Deletion of these amino acids yields a current identical to that of full-length Kv1.3, except that it cannot be suppressed by a truncated Kv1.3 containing the amino terminus and S1. To test the ability of truncated Kv1.3 to suppress endogenous K+ currents, we constructed a plasmid that contained both truncated Kv1.3 and a selection marker gene (mouse CD4). Although constitutively expressed K+ currents in Jurkat (a human T cell leukemia line) and GH3 (an anterior pituitary cell line) cells cannot be suppressed by this double-gene plasmid, stimulated (up-regulated) Shaker-like K+ currents in GH3 cells can be suppressed. 相似文献
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Kinetics and sequence specificity of processing of prepilin by PilD, the type IV leader peptidase of Pseudomonas aeruginosa. 下载免费PDF全文
PilD, originally isolated as an essential component for the biogenesis of the type IV pili of Pseudomonas aeruginosa, is a unique endopeptidase responsible for processing the precursors of the P. aeruginosa pilin subunits. It is also required for the cleavage of the leader peptides from the Pdd proteins, which are essential components of an extracellular secretion pathway specific for the export of a number of P. aeruginosa hydrolytic enzymes and toxins. Substrates for PilD are initially synthesized with short, i.e., 6- to 8-amino-acid-long, leader peptides with a net basic charge and share a high degree of amino acid homology through the first 16 to 30 residues at the amino terminus. In addition, they all have a phenylalanine residue at the +1 site relative to the cleavage site, which is N methylated prior to assembly into the oligomeric structures. In this study, the kinetics of leader peptide cleavage from the precursor of the P. aeruginosa pilin subunit by PilD was determined in vitro. The rates of cleavage were compared for purified enzyme and substrate as well as for enzyme and substrate contained within total membranes extracted from P. aeruginosa strains overexpressing the cloned pilD or pilA genes. Optimal conditions were obtained only when both PilD and substrate were contained within total membranes. PilD catalysis of P. aeruginosa prepilin followed normal Michaelis-Menten kinetics, with a measured apparent Km of approximately 650 microM, and a kcat of 180 min-1. The kinetics of PilD processing of another type IV pilin precursor, that from Neisseria gonorrhoeae with a 7-amino-acid-long leader peptide, were essentially the same as that measured for wild-type P. aeruginosa prepilin. Quite different results were obtained for a number of prepilin substrates containing substitutions at the conserved phenylalanine at the +1 position relative to the cleavage site, which were previously shown to be well tolerated in vivo. Substitutions of methionine, serine, and cysteine for phenylalanine show that Km values remain close to that measured for wild-type substrate, while kcat and kcat/Km values were significantly decreased. This indicates that while the affinity of enzyme for substrate is relatively unaffected by the substitutions, the maximum rate of catalysis favors a phenylalanine at this position. Interesting, PilD cleavage of one mutated pillin (asparagine) resulted in a lower Km value of 52.5 microM, which indicates a higher affinity for the enzyme, as well as a lower kcat value of 6.1 min m(-1). This suggests that it may be feasible to design peptide inhibitors of PilD. 相似文献
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Cristina Landeta Laura McPartland Ngoc Q. Tran Brian M. Meehan Yifan Zhang Zaidi Tanweer Shoko Wakabayashi Jeremy Rock Taehyun Kim Deepak Balasubramanian Rebecca Audette Melody Toosky Jessica Pinkham Eric J. Rubin Stephen Lory Gerald Pier Dana Boyd Jon Beckwith 《Molecular microbiology》2019,111(4):918-937
In bacteria, disulfide bonds confer stability on many proteins exported to the cell envelope or beyond, including bacterial virulence factors. Thus, proteins involved in disulfide bond formation represent good targets for the development of inhibitors that can act as antibiotics or anti‐virulence agents, resulting in the simultaneous inactivation of several types of virulence factors. Here, we present evidence that the disulfide bond forming enzymes, DsbB and VKOR, are required for Pseudomonas aeruginosa pathogenicity and Mycobacterium tuberculosis survival respectively. We also report the results of a HTS of 216,767 compounds tested against P. aeruginosa DsbB1 and M. tuberculosis VKOR using Escherichia coli cells. Since both P. aeruginosa DsbB1 and M. tuberculosis VKOR complement an E. coli dsbB knockout, we screened simultaneously for inhibitors of each complemented E. coli strain expressing a disulfide‐bond sensitive β ‐galactosidase reported previously. The properties of several inhibitors obtained from these screens suggest they are a starting point for chemical modifications with potential for future antibacterial development. 相似文献
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Brück TB Brück WM Santiago-Vázquez LZ McCarthy PJ Kerr RG 《Marine biotechnology (New York, N.Y.)》2007,9(5):561-576
This study examined the microbiota associated with the marine azooxanthellate octocorals Leptogorgia minimata, Swiftia exertia, and Iciligorgia schrammi collected from moderate depths (45 m). Traditional aerobic plate culture, fluorescence in situ hybridization (FISH), and
molecular identification of the 16S rDNA region were used for this purpose. In general, cultures were found to be selective
for Gammaproteobacteria, Alphaproteobacteria, and Firmicutes. Interestingly, FISH counts for Firmicutes in the whole coral
(holobiont) were near the detection limit of this assay, representing less than 6% of the total detectable microbiota in all
counts. Proteobacteria, especially Alpha- and Gammaproteobacteria, made up the majority of the total microbiota in the holobionts.
In addition, the absence of zooxanthellae in these three corals was confirmed by the use of polymerase chain reaction (PCR)
and dinoflagellate-specific primers, and spectrophotometric chlorophyll pigment measurements. No evidence of zooxanthellae
could be found in any of the corals by either of these techniques. This is the first study examining the microbiota marine
octocorals, which grow at moderate depth (40 to 100 m) in the absence of direct sunlight.
Thomas B. Brück and Wolfram M. Brück have made equal contributions to this publication. 相似文献