Angelika Jellen-Ritter

Fluoroquinolone-resistant mutants, selected from a wild-type Escherichia coli K-12 strain and its Mar mutant by exposure to increasing levels of ofloxacin on solid medium, were analyzed by Northern (RNA) blot analysis, sequencing, and radiolabelled ciprofloxacin accumulation studies. Mutations in the target gene gyrA (DNA gyrase), the regulatory gene marR, and additional, as yet unidentified genes (genes that probably affect efflux mediated by the multidrug efflux pump AcrAB) all contributed to fluoroquinolone resistance. Inactivation of the acrAB locus made all strains, including those with target gene mutations, hypersusceptible to fluoroquinolones and certain other unrelated drugs. These studies indicate that, in the absence of the AcrAB pump, gyrase mutations fail to produce clinically relevant levels of fluoroquinolone resistance.

The development of fluoroquinolone resistance in Escherichia coli may be associated with mutations in regulatory gene loci such as marRAB that lead to increased multidrug efflux, presumably through activation of expression of the AcrAB multidrug efflux pump. We found that multidrug-resistant (MDR) phenotypes with enhanced efflux can also be selected by fluoroquinolones from marRAB- or acrAB-inactivated E. coli K-12 strains having a single mutation in the quinolone-resistance-determining region of gyrA. Mutant 3-AG100MKX, obtained from a mar knockout strain after two selection steps, showed enhanced expression of acrB in a reverse transcriptase PCR associated with insertion of IS186 into the AcrAB repressor gene acrR. In vitro selection experiments with acrAB knockout strains yielded MDR mutants after a single step. Enhanced efflux in these mutants was due to increased expression of acrEF and associated with insertion of IS2 into the upstream region of acrEF, presumably creating a hybrid promoter. These observations confirm the importance of efflux-associated nontarget gene mutations and indicate that transposition of genetic elements may have a role in the development of fluoroquinolone resistance in E. coli.

Mutations in loci other than genes for the target topoisomerases of fluoroquinolones, gyrA and parC, may play a role in the development of fluoroquinolone resistance in Escherichia coli. A series of mutants with increasing resistance to ofloxacin was obtained from an E. coli K-12 strain and five clinical isolates. First-step mutants acquired a gyrA mutation. Second-step mutants reproducibly acquired a phenotype of multiple antibiotic resistance (Mar) and organic solvent tolerance and showed enhanced fluoroquinolone efflux. None of the second-step mutants showed additional topoisomerase mutations. All second-step mutants showed constitutive expression of marA and/or overexpressed soxS. In some third-step mutants, fluoroquinolone efflux was further enhanced compared to that for second-step mutants, even when the mutant had acquired additional topoisomerase mutations. Attempts to circumvent the second-step Mar mutation by induction of the mar locus with sodium salicylate and thus to select for pure topoisomerase mutants at the second step were not successful. At least in vitro, non-target gene mutations accumulate in second- and third-step mutants upon exposure to a fluoroquinolone and typically include, but do not appear to be limited to, mutations in the mar or sox regulons with consequent increased drug efflux.

To elucidate the role of small noncoding RNAs (sRNAs) in archaea we applied RNomics to identify sRNAs in the halophilic archaeon Haloferax volcanii. Using a size-selected cDNA library, 39 different previously uncharacterized sRNAs were identified ranging in size from 130 to 460 nucleotides. Twenty-one of these sRNAs are located in intergenic regions and 18 in antisense orientation. One of the intergenic sRNAs codes for a peptide. Only a minor fraction of sRNA genes were preceded by promoter elements (15 of 39), indicating that the majority might be generated by processing from larger precursors. Northern blot analyses of the intergenic sRNAs revealed differential expression for several sRNAs. Deletion mutants of two sRNAs were constructed, demonstrating that this approach is suitable to elucidate their biological function. Both mutant strains showed a defined phenotype: sRNA(30) gene deletion mutant was less resistant to higher temperatures and sRNA(63) gene deletion mutant resulted in a severe growth defect at low salt concentrations. Proteome analyses revealed clear differences between wildtype and deletion strains. These results represent the first reported examples of experimentally characterizing the function of sRNAs, excepting snoRNAs, in archaea. Taken together, we showed that haloarchaea encode sRNAs, some of which are differentially expressed and which have the potential to fulfil important biological functions in vivo.