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Ciprofloxacin Dimers Target Gyrase in Streptococcus pneumoniae. Katherine A. Gould, 2004.We have examined the antipneumococcal activities of novel quinolone dimers in which ciprofloxacin was tethered to itself or to pipemidic acid by linkage of C-7 piperazinyl rings . Symmetric 2,6-lutidinyl- and trans-butenyl-linked ciprofloxacin dimers (dimers 1 and 2, respectively) and a pipemidic acid-ciprofloxacin dimer (dimer 3) had activities against Streptococcus pneumoniae strain 7785 that were comparable to that of ciprofloxacin, i.e., MICs of 2, 1, and 4 to 8 µg/ml versus an MIC of 1 to 2 µg/ml, respectively . Surprisingly, unlike ciprofloxacin (which targets topoisomerase IV), several lines of evidence revealed that the dimers act through gyrase in S . pneumoniae . First, ciprofloxacin-resistant parC mutants of strain 7785 remained susceptible to dimers 1 to 3, whereas a gyrA mutation conferred a four- to eightfold increase in the dimer MIC but had little effect on ciprofloxacin activity . Second, dimer 1 selected first-step gyrA (S81Y or S81F) mutants (MICs, 8 to 16 µg/ml) that carried wild-type topoisomerase IV parE-parC genes . Third, dimers 1 and 2 promoted comparable DNA cleavage by S . pneumoniae gyrase and topoisomerase IV, whereas ciprofloxacin-mediated cleavage was 10-fold more efficient with topoisomerase IV than with gyrase . Fourth, the GyrA S81F and ParC S79F enzymes were resistant to dimers, confirming that the resistance phenotype is largely silent in parC mutants . Although a dimer molecule could bind very tightly by bridging quinolone binding sites in the enzyme-DNA complex, the greater potency of ciprofloxacin against gyrase and topoisomerase IV suggests that dimers 1 to 3 bind in a monomeric fashion . The bulky C-7 side chain may explain dimer targeting of gyrase and activity against efflux mutants . Tethered quinolones have potential as mechanistic tools and as novel antimicrobial agents . Production of Minicellulosomes from Clostridium cellulovorans in Bacillus subtilis WB800. Hee-Yeon Cho, 2004. Rubredoxins Involved in Alkane Oxidation. Jan B. van Beilen, 2002.Rubredoxins (Rds) are essential electron transfer components of bacterial membrane-bound alkane hydroxylase systems . Several Rd genes associated with alkane hydroxylase or Rd reductase genes were cloned from gram-positive and gram-negative organisms able to grow on n-alkanes (Alk-Rds) . Complementation tests in an Escherichia coli recombinant containing all Pseudomonas putida GPo1 genes necessary for growth on alkanes except Rd 2 (AlkG) and sequence comparisons showed that the Alk-Rds can be divided in AlkG1- and AlkG2-type Rds . All alkane-degrading strains contain AlkG2-type Rds, which are able to replace the GPo1 Rd 2 in n-octane hydroxylation . Most strains also contain AlkG1-type Rds, which do not complement the deletion mutant but are highly conserved among gram-positive and gram-negative bacteria . Common to most Rds are the two iron-binding CXXCG motifs . All Alk-Rds possess four negatively charged residues that are not conserved in other Rds . The AlkG1-type Rds can be distinguished from the AlkG2-type Rds by the insertion of an arginine downstream of the second CXXCG motif . In addition, the glycines in the two CXXCG motifs are usually replaced by other amino acids . Mutagenesis of residues conserved in either the AlkG1- or the AlkG2-type Rds, but not between both types, shows that AlkG1 is unable to transfer electrons to the alkane hydroxylase mainly due to the insertion of the arginine, whereas the exchange of the glycines in the two CXXCG motifs only has a limited effect . Fnr Is Involved in Oxygen Control of Herbaspirillum seropedicae N-Truncated NifA Protein Activity in Escherichia coli. Rose A. Monteiro, 2003.Herbaspirillum seropedicae is an endophytic diazotroph belonging to the ß-subclass of the class Proteobacteria, which colonizes many members of the Gramineae . The activity of the NifA protein, a transcriptional activator of nif genes in H . seropedicae, is controlled by ammonium ions through its N-terminal domain and by oxygen through mechanisms that are not well understood . Here we report that the NifA protein of H . seropedicae is inactive and more susceptible to degradation in an fnr Escherichia coli background . Both effects correlate with oxygen exposure and iron deprivation . Our results suggest that the oxygen sensitivity and iron requirement for H . seropedicae NifA activity involve the Fnr protein .
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