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Mutational Analysis of Xanthomonas Harpin HpaG Identifies a Key Functional Region That Elicits the Hypersensitive Response in Nonhost Plants. Jung-Gun Kim, 2004.HpaG is a type III-secreted elicitor protein of Xanthomonas axonopodis pv . glycines . We have determined the critical amino acid residues important for hypersensitive response [HR] elicitation by random and site-directed mutagenesis of HpaG and its homologXopA . A plasmid clone carrying hpaG was mutagenized by site-directed mutagenesis, hydroxylamine mutagenesis, and error-prone PCR.A total of 52 mutants were obtained, including 51 single missensemutants and 1 double missense mutant . The HR elicitation activitywas abolished in the two missense mutants [HpaG[L50P] and HpaG[L43P/L50P]].Seven single missense mutants showed reduced activity, and theHR elicitation activity of the rest of the mutants was similarto that of wild-type HpaG . Mutational and deletion analysesnarrowed the region essential for elicitor activity to the 23-amino-acidpeptide [H2N-NQGISEKQLDQLLTQLIMALLQQ-COOH] . A synthetic peptideof this sequence possessed HR elicitor activity at the same concentration as the HpaG protein . This region has 78 and 74% homology with 23- and 27-amino-acid regions of the HrpW harpin domains, respectively, from Pseudomonas and Erwinia spp . The secondary structure of the peptide is predicted to be an  -helix,
as is the HrpW region that is homologous to HpaG . The predicted
-helix
of HpaG is probably critical for the elicitation of theHR in tobacco
plants . In addition, mutagenesis of a xopA geneyielded two
gain-of-function mutants: XopA[F48L] and XopA[F48L/M52L].These
results indicate that the 12 amino acid residues betweenL39 and L50
of HpaG have critical roles in HR elicitation intobacco plants.
Increasing Prevalence of Quinolone Resistance in Human Nontyphoid Salmonella enterica Isolates Obtained in Spain from 1981 to 2003. José M. Marimón, 2004.From January 1981 to December 2003, susceptibility to nalidixic acid was tested in 10,504 nontyphoid Salmonella enterica isolates from patients with acute enteric disease in Gipuzkoa, Spain . The prevalence of nalidixic acid resistance steadily increased from less than 0.5% before 1991 to 38.5% in 2003, mainly due to the increase in resistance among isolates of the most prevalent serovar, S . enterica serovar Enteritidis . For nalidixic acid-resistant isolates, the ciprofloxacin MIC was eightfold higher than that for susceptible isolates, and the nalidixic acid-resistant isolates contained a single point mutation in the gyrA gene (at codons for Ser83 or Asp87) . The same mutations were found in a sample of nalidixic acid-resistant nontyphoid Salmonella strains isolated between 1999 and 2003 from retail food for human consumption . In 2003, we identified five S . enterica serovar Typhimurium clinical isolates with high-level fluoroquinolone resistance (ciprofloxacin MIC, 16 µg/ml) with two point mutations in the gyrA gene (coding for Ser83  Phe and Asp87Asn) and one point mutation in the parC gene (coding for Ser80Arg) . Strict sanitary controls are needed to avoid the spread of ciprofloxacin-resistant serovar Typhimurium isolates, and a more efficient veterinary policy must be adopted to decrease the large burden of Salmonella serovar Enteritidis infections in humans in our region .
Replication of the Endosymbiotic Bacterium Blochmannia floridanus Is Correlated with the Developmental and Reproductive Stages of Its Ant Host. Florian Wolschin, 2004.The dynamics of replication of the intracellular endosymbiotic bacterium Blochmannia floridanus was determined during the larval development of its host ant Camponotus floridanus by real-time quantitative PCR . The bacteria were found to proliferate during pupation and immediately after the eclosion of the imagines (adult ants) . In older workers the number of bacteria present in the midgut bacteriocytes decreased significantly . In contrast, the bacterial population in the ovaries was dependent on the reproductive state of the animal . An age-dependent degeneration of the midgut bacteriocytes was also investigated by microscopic techniques in males and female castes of the closely related ant species C . herculeanus and C . sericeiventris, respectively, with similar results and supports the concept of age-dependent degeneration of the midgut bacteriocytes in all castes . Expression of spoIIIJ in the Prespore Is Sufficient for Activation of  G and for Sporulation in Bacillus subtilis.Mónica Serrano, 2003.During sporulation in Bacillus subtilis, the prespore-specific developmental program is initiated soon after asymmetric division of the sporangium by the compartment-specific activation of RNA polymerase sigma factor F .
F directs transcription of spoIIIG, encoding the late forespore-specific regulator
G . Following synthesis,
G is initially kept in an inactive form, presumably because it is bound to the SpoIIAB anti-sigma factor . Activation of
G occurs only after the complete engulfment of the prespore by the mother cell . Mutations in spoIIIJ arrest sporulation soon after conclusion of the engulfment process and prevent activation of
G . Here we show that
G accumulates but is mostly inactive in a spoIIIJ mutant . We also show that expression of the spoIIIGE155K allele, encoding a form of
G that is not efficiently bound by SpoIIAB in vitro, restores
G-directed gene expression to a spoIIIJ mutant . Expression of spoIIIJ occurs during vegetative growth . However, we show that expression of spoIIIJ in the prespore is sufficient for
G activation and for sporulation . Mutations in the mother cell-specific spoIIIA locus are known to arrest sporulation just after completion of the engulfment process . Previous work has also shown that
G accumulates in an inactive form in spoIIIA mutants and that the need for spoIIIA expression for
G activation can be circumvented by the spoIIIGE155K allele . However, in contrast to the case for spoIIIJ, we show that expression of spoIIIA in the prespore does not support efficient sporulation . The results suggest that the activation of
G at the end of the engulfment process involves the action of spoIIIA from the mother cell and of spoIIIJ from the prespore .
pH-Dependent Modulation of Cyclic AMP Levels and GadW-Dependent Repression of RpoS Affect Synthesis of the GadX Regulator and Escherichia coli Acid Resistance. Zhuo Ma, 2003.Extreme acid resistance is a remarkable property of virulent and avirulent Escherichia coli . The ability to resist environments in which the pH is 2.5 and below is predicted to contribute significantly to the survival of E . coli during passage through the gastric acid barrier . One acid resistance system imports glutamate from acidic environments and uses it as a proton sink during an intracellular decarboxylation reaction . Transcription of the genes encoding the glutamate decarboxylases and the substrate-product antiporter required for this system is induced under a variety of conditions, including the stationary phase and a low pH . Acid induction during log-phase growth in minimal medium appears to occur through multiple pathways . We recently demonstrated that GadE, the essential activator of the genes, was itself acid induced . In this report we present evidence that there is a regulatory loop involving cross-repression of two AraC-like regulators, GadX and GadW, that can either assist or interfere with GadE activation of the gad decarboxylase and antiporter genes, depending on the culture conditions . Balancing cross-repression appears to be dependent on cAMP and the cAMP regulator protein (CRP) . The control loop involves the GadX protein repressing the expression of gadW and the GadW protein repressing or inhibiting RpoS, which is the alternative sigma factor that drives transcription of gadX . CRP and cAMP appear to influence GadX-GadW cross-repression from outside the loop by inhibiting production of RpoS . We found that GadW represses the decarboxylase genes in minimal medium and that growth under acidic conditions lowers the intracellular cAMP levels . These results indicate that CRP and cAMP can mediate pH control over gadX expression and, indirectly, expression of the decarboxylase genes . Mutational or physiological lowering of cAMP levels increases the level of RpoS and thereby increases the production of GadX . Higher GadX levels, in turn, repress gadW and contribute to induction of the gad decarboxylase genes . The presence of multiple pH control pathways governing expression of this acid resistance system is thought to reflect different environmental routes to a low pH . Development and Application of Different Methods for the Detection of Toxoplasma gondii in Water. Christina Kourenti, 2003.Two methods, centrifugation and flocculation, were evaluated to determine their efficiencies of recovery of Toxoplasma gondii oocysts from contaminated water samples . Demineralized and tap water replicates were inoculated with high numbers of sporulated or unsporulated T . gondii oocysts (1 x 105 and 1 x 104 oocysts) . The strain, age, and concentration of the seeded oocysts were recorded . Oocysts were recovered either by centrifugation of the contaminated samples at various g values or by flocculation with two coagulants, Fe2(SO4)3 and Al2(SO4)3 . The recovery rates were determined with the final pellets by phase-contrast microscopy . Sporulated oocysts were recovered more effectively by flocculation with Al2(SO4)3 (96.5% ± 21.7%) than by flocculation with Fe2(SO4)3 (93.1% ± 8.1%) or by centrifugation at 2,073 x g (82.5% ± 6.8%) . For the unsporulated oocysts, flocculation with Fe2(SO4)3 was more successful (100.3% ± 26.9%) than flocculation with Al2(SO4)3 (90.4% ± 19.1%) or centrifugation at 2,565 x g (97.2% ± 12.5%) . The infectivity of the sporulated oocysts recovered by centrifugation was confirmed by seroconversion of all inoculated mice 77 days postinfection . These data suggest that sporulated Toxoplasma oocysts purified by methods commonly used for waterborne pathogens retain their infectivity after mechanical treatment and are able to induce infections in mammals . This is the first step in developing a systematic approach for the detection of Toxoplasma oocysts in water . Survival of F-Specific RNA Coliphage, Feline Calicivirus, and Escherichia coli in Water: a Comparative Study. Paul B. Allwood, 2003.The relationship between the survival of enteric viral pathogens and their indicators (coliform bacteria and coliphages) is not well understood . We compared the survival rates of feline calicivirus (FCV), Escherichia coli, and a male-specific RNA coliphage MS2 at 4, 25, and 37°C for up to 28 days in dechlorinated water . The survival rates of E . coli and FCV, a surrogate of noroviruses (NV), had a high degree of correlation at 4 and 25°C, while MS2 phage survived significantly longer (P < 0.05) at these two temperatures . At 37°C, the survival rates for all three organisms were highly correlated . Decimal reduction values indicating the number of days needed for 90% reduction in titer (D values) decreased for all three organisms as storage temperatures increased . FCV had the shortest D value among all three organisms at all temperatures investigated . These findings indicate that F-specific RNA phages may be useful indicators of NV in the environment .
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