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New Protein-Protein Interactions Identified for the Regulatory and Structural Components and Substrates of the Type III Secretion System of the Phytopathogen Xanthomonas axonopodis Pathovar citri. Marcos C. Alegria, 2004.We have initiated a project to identify protein-protein interactions involved in the pathogenicity of the bacterial plant pathogen Xanthomonas axonopodis pv . citri . Using a yeast two-hybrid system based on Gal4 DNA-binding and activation domains, we have focusedon identifying interactions involving subunits, regulators,and substrates of the type III secretion system coded by thehrp [for hypersensitive response and pathogenicity], hrc [for hrp conserved], and hpa [for hrp associated] genes . We have identified several previously uncharacterized interactions involving [i] HrpG, a two-component system response regulator responsible for the expression of X . axonopodis pv . citri hrp operons, and XAC0095, a previously uncharacterized protein encountered only in Xanthomonas spp.; [ii] HpaA, a protein secreted by the type III secretion system, HpaB, and the C-terminal domain of HrcV;[iii] HrpB1, HrpD6, and HrpW; and [iv] HrpB2 and HrcU . Homotropic interactions were also identified for the ATPase HrcN . Thesenewly identified protein-protein interactions increase our understandingof the functional integration of phytopathogen-specific typeIII secretion system components and suggest new hypotheses regardingthe molecular mechanisms underlying Xanthomonas pathogenicity. Cell Wall Composition and Decreased Autolytic Activity and Lysostaphin Susceptibility of Glycopeptide-Intermediate Staphylococcus aureus. Jennifer L. Koehl, 2004.The cell wall composition and autolytic properties of passage-selected glycopeptide-intermediate Staphylococcus aureus (GISA) isolates and their parent strains were studied in order to investigate the mechanism of decreased vancomycin susceptibility . GISA had relatively modest changes in peptidoglycan composition involving peptidoglycan interpeptide bridges and somewhat decreased cross-linking compared to that of parent strains . The cell wall phosphorus content of GISA strains was lower than that of susceptible parent strains, indicating somewhat lower wall teichoic acid levels in the GISA strains . Similar to whole cells, isolated crude cell walls retaining autolytic activity of GISA had drastically reduced autolytic activity compared to that of parent strains, and this arose early in the development of the GISA phenotype . This was due to an alteration in the autolytic enzymes of GISA as revealed by normal susceptibility of GISA-purified cell walls to parental strain autolysin extract and lower activity and altered peptidoglycan hydrolase activity profiles in GISA autolysin extracts compared to those of parent strains . Northern blot analysis indicated that expression of atl, the major autolysin gene, was significantly downregulated in a GISA strain compared to that of its parent strain . In contrast to whole cells, which showed decreased lysostaphin susceptibility, purified cell walls of GISA showed increased susceptibility to lysostaphin . We suggest that in our GISA strains, decreased autolytic activity is involved in the tolerance of vancomycin and the activities of endogenous autolysins are important in conferring sensitivity to lysostaphin on whole cells . Quorum Sensing Is Not Required for Twitching Motility in Pseudomonas aeruginosa. Scott A. Beatson, 2002.It has been reported that mutations in the quorum-sensing genes lasI and rhlI in Pseudomonas aeruginosa result in, among many other things, loss of twitching motility (A . Glessner, R . S . Smith, B . H . Iglewski, and J . B . Robinson, J . Bacteriol . 181:1623-1629, 1999) . We constructed knockouts of lasI and rhlI and the corresponding regulatory genes lasR and rhlR and found no effect on twitching motility . However, twitching-defective variants accumulated during culturing of lasI and rhlI mutants . Further analysis showed that the stable twitching-defective variants of lasI and rhlI mutants had arisen as a consequence of secondary mutations in vfr and algR, respectively, both of which encode key regulators affecting a variety of phenotypes, including twitching motility . In addition, when grown in shaking broth culture, lasI and rhlI mutants, but not the wild-type parent, also accumulated unstable variants that lacked both twitching motility and swimming motility and appeared to be identical in phenotype to the S1 and S2 variants that were recently reported to occur at high frequencies in P . aeruginosa strains grown as a biofilm or in static broth culture (E . Deziel, Y . Comeau, and R . Villemur, J . Bacteriol . 183:1195-1204, 2001) . These results indicate that mutations in one regulatory system may create distortions that select during subsequent culturing for compensatory mutations in other regulatory genes within the cellular network . This problem may have compromised some past studies of regulatory hierarchies controlled by quorum sensing and of bacterial regulatory systems in general . Identification of Pyrene-Induced Proteins in Mycobacterium sp . Strain 6PY1: Evidence for Two Ring-Hydroxylating Dioxygenases. Serge Krivobok, 2003.In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation were investigated in the pyrene-degrading Mycobacterium sp . strain 6PY1 . [14C]pyrene mineralization experiments showed that bacteria grown with either pyrene or phenanthrene produced high levels of pyrene-catabolic activity but that acetate-grown cells had no activity . As a means of identifying specific catabolic enzymes, protein extracts from bacteria grown on pyrene or on other carbon sources were analyzed by two-dimensional gel electrophoresis . Pyrene-induced proteins were tentatively identified by peptide sequence analysis . Half of them resembled enzymes known to be involved in phenanthrene degradation, with closest similarity to the corresponding enzymes from Nocardioides sp . strain KP7 . The genes encoding the terminal components of two distinct ring-hydroxylating dioxygenases were cloned . Sequence analysis revealed that the two enzymes, designated Pdo1 and Pdo2, belong to a subfamily of dioxygenases found exclusively in gram-positive bacteria . When overproduced in Escherichia coli, Pdo1 and Pdo2 showed distinctive selectivities towards PAH substrates, with the former enzyme catalyzing the dihydroxylation of both pyrene and phenanthrene and the latter preferentially oxidizing phenanthrene . The catalytic activity of the Pdo2 enzyme was dramatically enhanced when electron carrier proteins of the phenanthrene dioxygenase from strain KP7 were coexpressed in recombinant cells . The Pdo2 enzyme was purified as a brown protein consisting of two types of subunits with Mrs of about 52,000 and 20,000 . Immunoblot analysis of cell extracts from strain 6PY1 revealed that Pdo1 was present in cells grown on benzoate, phenanthrene, or pyrene and absent in acetate-grown cells . In contrast, Pdo2 could be detected only in PAH-grown cells . These results indicated that the two enzymes were differentially regulated depending on the carbon source used for growth . Chemolithoorganotrophic Growth of Nitrosomonas europaea on Fructose. Norman G. Hommes, 2003.The nitrifying bacterium Nitrosomonas europaea can obtain all its carbon for growth from CO2 and all its energy and reductant for growth from the oxidation of NH3 and is considered an obligate chemolithoautotroph . Previous studies have shown that N . europaea can utilize limited amounts of certain organic compounds, including amino acids, pyruvate, and acetate, although no organic compound has been reported to support the growth of N . europaea . The recently completed genomic sequence of N . europaea revealed a potential permease for fructose . With this in mind, we tested if N . europaea could utilize fructose and other compounds as carbon sources to support growth . Cultures were incubated in the presence of fructose or other organic compounds in sealed bottles purged of CO2 . In these cultures, addition of either fructose or pyruvate as the sole carbon source resulted in a two- to threefold increase in optical density and protein content in 3 to 4 days . Studies with [14C]fructose showed that >90% of the carbon incorporated by the cells during growth was derived from fructose . Cultures containing mannose, glucose, glycerol, mannitol, citrate, or acetate showed little or no growth . N . europaea was not able to grow with fructose as an energy source, although the presence of fructose did provide an energy benefit to the cells . These results show that N . europaea can be grown in CO2-free medium by using fructose and pyruvate as carbon sources and may now be considered a facultative chemolithoorganotroph . Recovery and Enumeration of Cryptosporidium parvum from Animal Fecal Matrices. Cheryl M. Davies, 2003.Accurate quantification of Cryptosporidium parvum oocysts in animal fecal deposits on land is an essential starting point for estimating watershed C . parvum loads . Due to the general poor performance and variable recovery efficiency of existing enumeration methods, protocols were devised based on initial dispersion of oocysts from feces by vortexing in 2 mM tetrasodium pyrophosphate, followed by immunomagnetic separation . The protocols were validated by using an internal control seed preparation to determine the levels of oocyst recovery for a range of fecal types . The levels of recovery of 102 oocysts from cattle feces (0.5 g of processed feces) ranged from 31 to 46%, and the levels of recovery from sheep feces (0.25 g of processed feces) ranged from 21% to 35% . The within-sample coefficients of variation for the percentages of recovery from five replicates ranged from 10 to 50% . The ranges for levels of recovery of oocysts from cattle, kangaroo, pig, and sheep feces (juveniles and adults) collected in a subsequent watershed animal fecal survey were far wider than the ranges predicted by the validation data . Based on the use of an internal control added to each fecal sample, the levels of recovery ranged from 0 to 83% for cattle, from 4 to 62% for sheep, from 1 to 42% for pigs, and from 40 to 73% for kangaroos . Given the variation in the levels of recovery of oocysts from different fecal matrices, it is recommended that an internal control be added to at least one replicate of every fecal sample analyzed to determine the percentage of recovery . Depending on the animal type and based on the lowest approximate percentages of recovery, between 10 and 100 oocysts g of feces-1 must be present to be detected . Unusual Water Flux in the Extracellular Polysaccharide of the Cyanobacterium Nostoc commune. Eric Shaw, 2003.The speed of water uptake by desiccated Nostoc commune was found to depend upon the duration of desiccation . The rehydration of desiccated colonies led to marked, time-dependent changes in structure and ultrastructure and fluctuations in the composition of the transcriptome . Physical evaporative water loss is an active process that was influenced by inhibitors of transcription and translation .
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