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Molecular Analysis of Cytolysin A (ClyA) in Pathogenic Escherichia coli Strains. Albrecht Ludwig, 2004.Cytolysin A (ClyA) of Escherichia coli is a pore-forming hemolytic protein encoded by the clyA (hlyE, sheA) gene that was first identified in E . coli K-12 . In this study we examined various clinical E . coli isolates with regard to the presence and integrity of clyA . PCR and DNA sequence analyses demonstrated that 19 of 23 tested Shiga toxin-producing E . coli (STEC) strains, all 7 tested enteroinvasive E . coli (EIEC) strains, 6 of 8 enteroaggregative E . coli (EAEC) strains, and 4 of 7 tested enterotoxigenic E . coli (ETEC) strains possess a complete clyA gene . The remaining STEC, EAEC, and ETEC strains and 9 of the 17 tested enteropathogenic E . coli (EPEC) strains were shown to harbor mutant clyA derivatives containing 1-bp frameshift mutations that cause premature termination of the coding sequence . The other eight EPEC strains and all tested uropathogenic and new-born meningitis-associated E . coli strains (n = 14 and 3, respectively) carried only nonfunctional clyA fragments due to the deletion of two sequences of 493 bp and 204 or 217 bp at the clyA locus . Expression of clyA from clinical E . coli isolates proved to be positively controlled by the transcriptional regulator SlyA . Several tested E . coli strains harboring a functional clyA gene produced basal amounts of ClyA when grown under standard laboratory conditions, but most of them showed a clyA-dependent hemolytic phenotype only when SlyA was overexpressed . The presented data indicate that cytolysin A can play a role only for some of the pathogenic E . coli strains . Differential Expression Levels of MRP1, MRP4, and MRP5 in Response to Human Immunodeficiency Virus Infection in Human Macrophages. Sylvie Jorajuria, 2004.Multidrug resistance proteins (MRPs) have been reported to be involved in the efflux of some anti-human immunodeficiency virus (HIV) drugs . We show here that MRP1, MRP4, and MRP5 are expressed at the mRNA level in human monocyte-derived macrophages . HIV infection caused increased transcription of these MRPs; however, temporal differences in stimulation are reported . Role of ppGpp in rpoS Stationary-Phase Regulation in Escherichia coli. Matthew Hirsch, 2002.The bacterial sigma factor RpoS is strongly induced under a variety of stress conditions and during growth into stationary phase . Here, we used rpoS-lac fusions in Escherichia coli to investigate control acting at the level of RpoS synthesis, which is especially evident when cells approach stationary phase in rich medium . Previous work has shown that the small molecule ppGpp is required for normal levels of RpoS in stationary phase . Despite the attraction of a model in which the ppGpp level controls stationary-phase induction of RpoS, careful measurement of rpoS-lac expression in a mutant lacking ppGpp showed similar effects during both exponential growth and stationary phase; the main effect of ppGpp was on basal expression . In addition, a modest regulatory defect was associated with the mutant lacking ppGpp, delaying the time at which full expression was achieved by 2 to 3 h . Deletion analysis showed that the defect in basal expression was distributed over several sequence elements, while the regulatory defect mapped to the region upstream of the rpoS ribosome-binding site (RBS) that contains a cis-acting antisense element . A number of other genes that have been suggested as regulators of rpoS were tested, including dksA, dsrA, barA, ppkx, and hfq . With the exception of the dksA mutant, which had a modest defect in Luria-Bertani medium, none of these mutants was defective for rpoS stationary-phase induction . Even a short rpoS segment starting at 24 nucleotides upstream of the AUG initiation codon was sufficient to confer substantial stationary-phase regulation, which was mainly posttranscriptional . The effect of RBS-proximal sequence was independent of all known trans-acting factors, including ppGpp . In Vitro Serial Passage of Staphylococcus aureus: Changes in Physiology, Virulence Factor Production, and agr Nucleotide Sequence. Greg A. Somerville, 2002.Recently, we observed that Staphylococcus aureus strains newly isolated from patients had twofold-higher aconitase activity than a strain passaged extensively in vitro, leading us to hypothesize that aconitase specific activity decreases over time during in vitro passage . To test this hypothesis, a strain recovered from a patient with toxic shock syndrome was serially passaged for 6 weeks, and the aconitase activity was measured . Aconitase specific activity decreased 38% (P < 0.001) by the sixth week in culture . During serial passage, S . aureus existed as a heterogeneous population with two colony types that had pronounced (wild type) or negligible zones of beta-hemolytic activity . The cell density-sensing accessory gene regulatory (agr) system regulates beta-hemolytic activity . Surprisingly, the percentage of colonies with a wild-type beta-hemolytic phenotype correlated strongly with aconitase specific activity (  = 0.96), suggesting a common cause of the decreased aconitase specific activity and the variation in percentage of beta-hemolytic colonies . The loss of the beta-hemolytic phenotype also coincided with the occurrence of mutations in the agrC coding region or the intergenic region between agrC and agrA in the derivative strains . Our results demonstrate that in vitro growth is sufficient to result in mutations within the agr operon . Additionally, our results demonstrate that S . aureus undergoes significant phenotypic and genotypic changes during serial passage and suggest that vigilance should be used when extrapolating data obtained from the study of high-passage strains .
Saprotrophic and Mycoparasitic Components of Aggressiveness of Trichoderma harzianum Groups toward the Commercial Mushroom Agaricus bisporus. Josie Williams, 2003.We examined the mycoparasitic and saprotrophic behavior of isolates representing groups of Trichoderma harzianum to establish a mechanism for the aggressiveness towards Agaricus bisporus in infested commercial compost . Mycoparasitic structures were infrequently observed in interaction zones on various media, including compost, with cryoscanning electron microscopy . T . harzianum grows prolifically in compost in the absence or presence of A . bisporus, and the aggressive European (Th2) and North American (Th4) isolates produced significantly higher biomasses (6.8- and 7.5-fold, respectively) in compost than did nonaggressive, group 1 isolates . All groups secreted depolymerases that could attack the cell walls of A . bisporus and of wheat straw, and some were linked to aggressiveness . Growth on mushroom cell walls in vitro resulted in rapid production of chymoelastase and trypsin-like proteases by only the Th2 and Th4 isolates . These isolates also produced a dominant protease isoform (pI 6.22) and additional chitinase isoforms . On wheat straw, Th4 produced distinct isoforms of cellulase and laminarinase, but there was no consistent association between levels or isoforms of depolymerases and aggressiveness . Th3's distinctive profiles confirmed its reclassification as Trichoderma atroviride. Proteases and glycanases were detected for the first time in sterilized compost colonized by T . harzianum . Xylanase dominated, and some isoforms were unique to compost, as were some laminarinases . We hypothesize that aggressiveness results from competition, antagonism, or parasitism but only as a component of, or following, extensive saprotrophic growth involving degradation of wheat straw cell walls .
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