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Reconstitution of a Staphylococcal Plasmid-Protein Relaxation Complex In Vitro. Jamie A. Caryl, 2004.The isolation of plasmid-protein relaxation complexes from bacteria is indicative of the plasmid nicking-closing equilibrium in vivo that serves to ready the plasmids for conjugal transfer . In pC221 and pC223, the components required for in vivo site- and strand-specific nicking at oriT are MobC and MobA . In order to investigate the minimal requirements for nicking in the absence of host-encoded factors, the reactions were reconstituted in vitro . Purified MobA and MobC, in the presence of Mg2+ or Mn2+, were found to nick at oriT with a concomitant phosphorylation-resistant modification at the 5' end of nic . The position of nic is consistent with that determined in vivo . MobA, MobC, and Mg2+ or Mn2+ therefore represent the minimal requirements for nicking activity . Cross-complementation analyses showed that the MobC proteins possess binding specificity for oriT DNA of either plasmid and are able to complement each other in the nicking reaction . Conversely, nicking by the MobA proteins is plasmid specific . This suggests the MobA proteins may encode the nicking specificity determinant . Mechanisms of Azole Resistance in Petite Mutants of Candida glabrata. Sophie Brun, 2004.We previously showed that resistant colonies of Candida glabrata inside the azole inhibition zones had respiratory deficiency due to mutations in mitochondrial DNA . Here, we analyzed the mechanisms of azole resistance in petite mutants of C . glabrata obtained by exposure to fluconazole or induced by ethidium bromide . The respiratory deficiency of these mutants was confirmed by oxygraphy and flow cytometric analysis with rhodamine 123, and its mitochondrial origin was demonstrated by transmission electron microscopy and restriction endonuclease analysis of the mitochondrial DNA . Flow cytometry with rhodamine 6G suggested an increased drug efflux in mutant cells, which was further supported by Northern blot analysis of the expression of the C . glabrata CDR1 (CgCDR1) and CgCDR2 genes, encoding efflux pumps . Conversely, the expression of CgERG11, which encodes the azole target, was not affected by petite mutations, and no differences were seen in the sequence of this gene between parent isolates and mutants . Moreover, sterol analysis showed similar overall amount of sterols in parent and mutant cells, but quantitative modifications were observed in the mutants, with almost undetectable biosynthesis intermediates . Further analysis performed after separation of free sterols from steryl esters revealed a defect in sterol esterification in mutant cells, with free ergosterol representing 92% of the overall sterol content . Thus, resistance or decreased susceptibility to azoles in petite mutants of C . glabrata is associated with increased expression of CgCDR1 and, to a lesser extent, of CgCDR2 . In addition, the marked increase in free ergosterol content would explain their increased susceptibility to polyenes . Effect of Forage or Grain Diets with or without Monensin on Ruminal Persistence and Fecal Escherichia coli O157:H7 in Cattle. M. J. Van Baale, 2004.Twelve ruminally cannulated cattle, adapted to forage or grain diet with or without monensin, were used to investigate the effects of diet and monensin on concentration and duration of ruminal persistence and fecal shedding of E . coli O157:H7 . Cattle were ruminally inoculated with a strain of E . coli O157:H7 (1010 CFU/animal) made resistant to nalidixic acid (Nalr) . Ruminal and fecal samples were collected for 11 weeks, and then cattle were euthanized and necropsied and digesta from different gut locations were collected . Samples were cultured for detection and enumeration of Nalr E . coli O157:H7 . Cattle fed forage diets were culture positive for E . coli O157:H7 in the feces for longer duration (P < 0.05) than cattle fed a grain diet . In forage-fed cattle, the duration they remained culture positive for E . coli O157:H7 was shorter (P < 0.05) when the diet included monensin . Generally, ruminal persistence of Nalr E . coli O157:H7 was not affected by diet or monensin . At necropsy, E . coli O157:H7 was detected in cecal and colonic digesta but not from the rumen . Our study showed that cattle fed a forage diet were culture positive longer and with higher numbers than cattle on a grain diet . Monensin supplementation decreased the duration of shedding with forage diet, and the cecum and colon were culture positive for E . coli O157:H7 more often than the rumen of cattle . The metD D-Methionine Transporter Locus of Escherichia coli Is an ABC Transporter Gene Cluster. József Gál, 2002.The metD D-methionine transporter locus of Escherichia coli was identified as the abc-yaeE-yaeC cluster (now renamed metNIQ genes) . The abc open reading frame is preceded by tandem MET boxes bracketed by the -10 and -35 boxes of a promoter . The expression driven by this promoter is controlled by the MetJ repressor and the level of methionine . Production of 6-Phenylacetylene Picolinic Acid from Diphenylacetylene by a Toluene-Degrading Acinetobacter Strain. Jim C. Spain, 2003.Several strategies for using enzymes to catalyze reactions leading to the synthesis of relatively simple substituted picolinic acids have been described . The goal of the work described here was to synthesize a more complex molecule, 6-phenylacetylene picolinic acid [6-(2-phenylethynyl)pyridine-2-carboxylic acid], for use as a potential endcapping agent for aerospace polymers . We screened 139 toluene-degrading strains that use a variety of catabolic pathways for the ability to catalyze oxidative transformation of diphenylacetylene . Acinetobacter sp . strain F4 catalyzed the overall conversion of diphenylacetylene to a yellow metabolite, which was identified as a putative meta ring fission product (2-hydroxy-8-phenyl-6-oxoocta-2,4-dien-7-ynoic acid [RFP]) . The activity could be sustained by addition of toluene at a flow rate determined empirically so that the transformations were sustained in spite of the fact that toluene is a competitive inhibitor of the enzymes . The overall rate of transformation was limited by the instability of RFP . The RFP was chemically converted to 6-phenylacetylene picolinic acid by treatment with ammonium hydroxide . The results show the potential for using the normal growth substrate to provide energy and to maintain induction of the enzymes involved in biotransformation during preliminary stages of biocatalyst development .
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