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Characterization of the Xanthomonas axonopodis pv . glycines Hrp Pathogenicity Island. Jung-Gun Kim, 2003.We sequenced an approximately 29-kb region from Xanthomonas axonopodis pv . glycines that contained the Hrp type III secretion system, and we characterized the genes in this region by Tn3-gus mutagenesis and gene expression analyses . From the region, hrp (hypersensitive response and pathogenicity) and hrc (hrp and conserved) genes, which encode type III secretion systems, and hpa (hrp-associated) genes were identified . The characteristics of the region, such as the presence of many virulence genes, low G+C content, and bordering tRNA genes, satisfied the criteria for a pathogenicity island (PAI) in a bacterium . The PAI was composed of nine hrp, nine hrc, and eight hpa genes with seven plant-inducible promoter boxes . The hrp and hrc mutants failed to elicit hypersensitive responses in pepper plants but induced hypersensitive responses in all tomato plants tested . The Hrp PAI of X . axonopodis pv . glycines resembled the Hrp PAIs of other Xanthomonas species, and the Hrp PAI core region was highly conserved . However, in contrast to the PAI of Pseudomonas syringae, the regions upstream and downstream from the Hrp PAI core region showed variability in the xanthomonads . In addition, we demonstrate that HpaG, which is located in the Hrp PAI region of X . axonopodis pv . glycines, is a response elicitor . Purified HpaG elicited hypersensitive responses at a concentration of 1.0 µM in pepper, tobacco, and Arabidopsis thaliana ecotype Cvi-0 by acting as a type III secreted effector protein . However, HpaG failed to elicit hypersensitive responses in tomato, Chinese cabbage, and A . thaliana ecotypes Col-0 and Ler . This is the first report to show that the harpin-like effector protein of Xanthomonas species exhibits elicitor activity . In Vitro Activities of the ß-Lactamase Inhibitors Clavulanic Acid, Sulbactam, and Tazobactam Alone or in Combination with ß-Lactams against Epidemiologically Characterized Multidrug-Resistant Acinetobacter baumannii Strains. Paul G. Higgins, 2004.Acinetobacter baumannii is an important nosocomial pathogen usually in the context of serious underlying disease . Multidrug resistance in these organisms is frequent . The ß-lactamase inhibitors clavulanic acid, sulbactam, and tazobactam have intrinsic activity against Acinetobacter strains . To evaluate their potential therapeutic usefulness, we determined the in vitro activity of ampicillin, sulbactam, ampicillin-sulbactam, cefoperazone, cefoperazone-sulbactam, piperacillin, piperacillin-sulbactam, tazobactam, piperacillin-tazobactam, amoxicillin, clavulanic acid, amoxicillin-clavulanic acid, ticarcillin, and ticarcillin-clavulanic acid against multidrug-resistant A . baumannii . All isolates were epidemiologically characterized by RAPD [random(ly) amplified polymorphic DNA] analysis and/or pulsed-field gel electrophoresis and represented different strain types, including sporadic strains, as well as outbreak-related strains . The MICs were determined by agar dilution on Mueller-Hinton agar (using fixed concentrations, as well as fixed ratios for ß-lactamase inhibitors) and the E-test . The majority of E-test results were within two dilutions of those recorded by agar dilution, with the exception of piperacillin-tazobactam . Sulbactam was superior to clavulanic acid and tazobactam and may represent an alternative treatment option for infections due to multiresistant A . baumannii strains . ß-Lactamase inhibitors have intrinsic activity but do not enhance activity of ß-lactams against A . baumannii . Testing with the inhibitor added at a fixed concentration as recommended for piperacillin-tazobactam and ticarcillin-clavulanic acid by the National Committee for Clinical Laboratory Standards may falsely suggest high activity or gives uninterpretable results due to trailing . If combinations are used for testing, fixed ratios may give more useful results . Discrimination of Modes of Action of Antifungal Substances by Use of Metabolic Footprinting. Jess Allen, 2004.Diploid cells of Saccharomyces cerevisiae were grown under controlled conditions with a Bioscreen instrument, which permitted the essentially continuous registration of their growth via optical density measurements . Some cultures were exposed to concentrations of a number of antifungal substances with different targets or modes of action (sterol biosynthesis, respiratory chain, amino acid synthesis, and the uncoupler) . Culture supernatants were taken and analyzed for their "metabolic footprints" by using direct-injection mass spectrometry . Discriminant function analysis and hierarchical cluster analysis allowed these antifungal compounds to be distinguished and classified according to their modes of action . Genetic programming, a rule-evolving machine learning strategy, allowed respiratory inhibitors to be discriminated from others by using just two masses . Metabolic footprinting thus represents a rapid, convenient, and information-rich method for classifying the modes of action of antifungal substances . Organization and Regulation of Pentachlorophenol-Degrading Genes in Sphingobium chlorophenolicum ATCC 39723. Mian Cai, 2002.The first three enzymes of the pentachlorophenol (PCP) degradation pathway in Sphingobium chlorophenolicum (formerly Sphingomonas chlorophenolica) ATCC 39723 have been characterized, and the corresponding genes, pcpA, pcpB, and pcpC, have been individually cloned and sequenced . To search for new genes involved in PCP degradation and map the physical locations of the pcp genes, a 24-kb fragment containing pcpA and pcpC was completely sequenced . A putative LysR-type transcriptional regulator gene, pcpM, and a maleylacetate reductase gene, pcpE, were identified upstream of pcpA . pcpE was found to play a role in PCP degradation . pcpB was not found on the 24-kb fragment . The four gene products PcpB, PcpC, PcpA, and PcpE were responsible for the metabolism of PCP to 3-oxoadipate in ATCC 39723, and inactivational mutation of each gene disrupted the degradation pathway . The organization of the pcp genes is unusual because the four PCP-degrading genes, pcpA, pcpB, pcpC, and pcpE, were found to be located at four discrete locations . Two hypothetical LysR-type regulator genes, pcpM and pcpR, have been identified; pcpM was not required, but pcpR was essential for the induction of pcpB, pcpA, and pcpE . The coinducers of PcpR were PCP and other polychlorinated phenols . The expression of pcpC was constitutive . Thus, the organization and regulation of the genes involved in PCP degradation to 3-oxoadipate were documented . Escherichia coli Cells Bearing a Ribosomal Ambiguity Mutation in rpsD Have a Mutator Phenotype That Correlates with Increased Mistranslation. Sergey Balashov, 2003.Escherichia coli cells bearing certain mutations in rpsD (coding for the 30S ribosomal protein S4) show a ribosomal ambiguity (Ram) phenotype characterized by increased translational error rates . Here we show that spontaneous mutagenesis increases in Ram cells bearing the rpsD14 allele, suggesting that the recently described translational stress-induced mutagenesis pathway is activated in Ram cells . Characterization and Molecular Cloning of a Novel Enzyme, Inorganic Polyphosphate/ATP-Glucomannokinase, of Arthrobacter sp . Strain KM. Takako Mukai, 2003.A bacterium exhibiting activities of several inorganic polyphosphate [poly(P)]- and ATP-dependent kinases, including glucokinase, NAD kinase, mannokinase, and fructokinase, was isolated, determined to belong to the genus Arthrobacter, and designated Arthrobacter sp . strain KM . Among the kinases, a novel enzyme responsible for the poly(P)- and ATP-dependent mannokinase activities was purified 2,200-fold to homogeneity from a cell extract of the bacterium . The purified enzyme was a monomer with a molecular mass of 30 kDa . This enzyme phosphorylated glucose and mannose with a high affinity for glucose, utilizing poly(P) as well as ATP, and was designated poly(P)/ATP-glucomannokinase . The Km values of the enzyme for glucose, mannose, ATP, and hexametaphosphate were determined to be 0.50, 15, 0.20, and 0.02 mM, respectively . The catalytic sites for poly(P)-dependent phosphorylation and ATP-dependent phosphorylation of the enzyme were found to be shared, and the poly(P)-utilizing mechanism of the enzyme was shown to be nonprocessive . The gene encoding the poly(P)/ATP-glucomannokinase was cloned from Arthrobacter sp . strain KM, and its nucleotide sequence was determined . This gene contained an open reading frame consisting of 804 bp coding for a putative polypeptide with a calculated molecular mass of 29,480 Da . The deduced amino acid sequence of the polypeptide exhibited homology to the amino acid sequences of the poly(P)/ATP-glucokinase of Mycobacterium tuberculosis H37Rv (level of homology, 45%), ATP-dependent glucokinases of Corynebacterium glutamicum (45%), Renibacterium salmoninarum (45%), and Bacillus subtilis (35%), and proteins of bacteria belonging to the order Actinomyces whose functions are not known . Alignment of these homologous proteins revealed seven conserved regions . The mannose and poly(P) binding sites of poly(P)/ATP-glucomannokinase are discussed .
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