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Role of RepA and DnaA Proteins in the Opening of the Origin of DNA Replication of an IncB Plasmid. T. Betteridge, 2004.The replication initiator protein RepA of the IncB plasmid pMU720 was shown to induce localized unwinding of its cognate origin of replication in vitro . DnaA, the initiator protein of Escherichia coli, was unable to induce localized unwinding of this origin of replication on its own but enhanced the opening generated by RepA . The opened region lies immediately downstream of the last of the three binding sites for RepA (RepA boxes) and covers one turn of DNA helix . A 6-mer sequence, 5'-TCTTAA-3', which lies within the opened region, was essential for the localized unwinding of the origin in vitro and origin activity in vivo . In addition, efficient unwinding of the origin of replication of pMU720 in vitro required the native positioning of the binding sites for the initiator proteins . Interestingly, binding of RepA to RepA box 1, which is essential for origin activity, was not required for the localized opening of the origin in vitro . Novel Approach to Characterization of Combined Pharmacodynamic Effects of Antimicrobial Agents. Vincent H. Tam, 2004.There is considerable need for new modeling approaches in the study of combined antimicrobial effects . Current methods based on the Loewe additivity and Bliss independence models are associated with implicit assumptions about the interacting system . To circumvent these limitations, we propose an alternative approach to the quantification of pharmacodynamic drug interaction (PDI) . Pilot time-kill studies were performed with 108 CFU of Pseudomonas aeruginosa/ml at baseline with meropenem or tobramycin alone . The studies were repeated with 25 concentration combinations of meropenem (0 to 64 mg/liter) and tobramycin (0 to 32 mg/liter) in a five-by-five array . The data were modeled with a three-dimensional response surface using effect summation as the basis of null interaction . The interaction index (Ii) is defined as the ratio of the volumes under the planes (VUP) of the observed and expected surfaces: VUPobserved/VUPexpected . Synergy and antagonism are defined as Ii values of <1 and >1, respectively . In all combinations, an enhanced killing effect was seen compared to that of either drug at the same concentration . The most significant synergism was observed between 1 and 5 mg/liter of meropenem and between 1 and 4 mg/liter of tobramycin; seven out of nine combinations had a >2-log drop compared to the more potent agent . The Ii was found to be 0.76 (95% confidence interval, 0.65 to 0.91) for the concentration ranges of the agents . The results corroborate previous data indicating that meropenem is synergistic with an aminoglycoside when used in combination against P . aeruginosa . Our parametric approach to quantifying PDI appears robust and warrants further investigations . Identification of an Na+-Dependent Transporter Associated with Saxitoxin-Producing Strains of the Cyanobacterium Anabaena circinalis. Francesco Pomati, 2004.Blooms of the freshwater cyanobacterium Anabaena circinalis are recognized as an important health risk worldwide due to the production of a range of toxins such as saxitoxin (STX) and its derivatives . In this study we used HIP1 octameric-palindrome repeated-sequence PCR to compare the genomic structure of phylogenetically similar Australian isolates of A . circinalis . STX-producing and nontoxic cyanobacterial strains showed different HIP1 (highly iterated octameric palindrome 1) DNA patterns, and characteristic interrepeat amplicons for each group were identified . Suppression subtractive hybridization (SSH) was performed using HIP1 PCR-generated libraries to further identify toxic-strain-specific genes . An STX-producing strain and a nontoxic strain of A . circinalis were chosen as testers in two distinct experiments . The two categories of SSH putative tester-specific sequences were characterized by different families of encoded proteins that may be representative of the differences in metabolism between STX-producing and nontoxic A . circinalis strains . DNA-microarray hybridization and genomic screening revealed a toxic-strain-specific HIP1 fragment coding for a putative Na+-dependent transporter . Analysis of this gene demonstrated analogy to the mrpF gene of Bacillus subtilis, whose encoded protein is involved in Na+-specific pH homeostasis . The application of this gene as a molecular probe in laboratory and environmental screening for STX-producing A . circinalis strains was demonstrated . The possible role of this putative Na+-dependent transporter in the toxic cyanobacterial phenotype is also discussed, in light of recent physiological studies of STX-producing cyanobacteria . Effects of Deletion of Genes Encoding Fe-Only Hydrogenase of Desulfovibrio vulgaris Hildenborough on Hydrogen and Lactate Metabolism. Brant K. J. Pohorelic, 2002.The physiological properties of a hyd mutant of Desulfovibrio vulgaris Hildenborough, lacking periplasmic Fe-only hydrogenase, have been compared with those of the wild-type strain . Fe-only hydrogenase is the main hydrogenase of D . vulgaris Hildenborough, which also has periplasmic NiFe- and NiFeSe-hydrogenases . The hyd mutant grew less well than the wild-type strain in media with sulfate as the electron acceptor and H2 as the sole electron donor, especially at a high sulfate concentration . Although the hyd mutation had little effect on growth with lactate as the electron donor for sulfate reduction when H2 was also present, growth in lactate- and sulfate-containing media lacking H2 was less efficient . The hyd mutant produced, transiently, significant amounts of H2 under these conditions, which were eventually all used for sulfate reduction . The results do not confirm the essential role proposed elsewhere for Fe-only hydrogenase as a hydrogen-producing enzyme in lactate metabolism (W . A . M . van den Berg, W . M . A . M . van Dongen, and C . Veeger, J . Bacteriol . 173:3688–3694, 1991) . This role is more likely played by a membrane-bound, cytoplasmic Ech-hydrogenase homolog, which is indicated by the D . vulgaris genome sequence . The physiological role of periplasmic Fe-only hydrogenase is hydrogen uptake, both when hydrogen is and when lactate is the electron donor for sulfate reduction . Comparison of Campylobacter Isolates from Poultry and Humans: Association between In Vitro Virulence Properties, Biotypes, and Pulsed-Field Gel Electrophoresis Clusters. Eacute;ric Nadeau, 2003.The in vitro virulence properties of 197 temporally and geographically related Campylobacter isolates from chicken broilers and humans were compared . Comparisons of the virulence properties associated with genotypes and biotypes were made . All isolates adhered to, and 63% invaded, INT-407 cells, whereas 13% were cytotoxic for CHO cells . CHO cell-cytotoxic extracts were also cytotoxic for INT-407 cells, but the sensitivity for Vero cells was variable . The proportion of isolates demonstrating a high invasiveness potential (>1,000 CFU ml-1) or Vero cell cytotoxicity was significantly higher for human than for poultry isolates . Invasiveness was associated with Campylobacter jejuni isolates of biotypes 1 and 2, whereas CHO and INT-407 cell cytotoxicity was associated with C . jejuni isolates of biotypes 3 and 4 . Cytotoxic isolates were also clustered according to pulsed-field gel electrophoresis profiles .
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