The effects of simultaneous expression of several efflux pumps on antibiotic resistance were investigated in Escherichia coli and Pseudomonas aeruginosa. Several combinations of efflux pumps have been studied: (i) simultaneous expression of a single-component efflux pump, which exports antibiotics into the periplasm, in combination with a multicomponent efflux pump that accomplishes efflux directly into the external medium; (ii) simultaneous expression of two single-component pumps; and (iii) simultaneous expression of two multicomponent pumps. It was found that when efflux pumps of different structural types were combined in the same cell (the first case), the observed antibiotic resistance was much higher than that conferred by each of the pumps expressed singly. Simultaneous expression of pairs of single-component or multicomponent efflux pumps (the second and third cases) did not produce strong increases in antibiotic resistance.Efflux of antibiotics out of cells is broadly recognized as a major component of bacterial resistance to many classes of antibiotics (26,28). This efflux occurs due to the activity of membrane transporter proteins, the so-called drug efflux pumps. Some efflux pumps selectively extrude specific antibiotics, while others, referred to as multidrug resistance (MDR) pumps, expel various structurally diverse antibiotics. While antibiotic-specific efflux pumps are usually encoded on transmissible plasmids and transposons, genes encoding many MDR pumps are normal constituents of bacterial chromosomes. Efflux pumps occur as either single-component or multicomponent systems. In gram-negative bacteria, single-component efflux pumps extrude their substrates into the periplasmic space (40). Examples of such single-component efflux pumps include the transposon-encoded tetracycline-and chloramphenicol-specific pumps, TetA and CmlA, respectively (2, 38), and the MDR pump MdfA, encoded in the chromosome of Escherichia coli (6). Multicomponent efflux pumps (which are found exclusively in gram-negative bacteria) traverse both inner and outer membranes. Examples include the MDR pumps AcrAB-TolC (19) and MexAB-OprM (34) from E. coli and Pseudomonas aeruginosa, respectively. Each pump contains a transporter located in the cytoplasmic membrane (as exemplified by AcrB or MexB), an outer membrane channel (TolC or OprM), and a periplasmic linker protein (AcrA or MexA), which is thought to bring into contact the other two components (42). This structural organization allows extrusion of substrates directly into the external medium, bypassing the periplasm and the outer membrane (27). The outer membrane of gram-negative bacteria serves as an efficient permeability barrier for both hydrophobic and hydrophilic antibiotics (29). Therefore, when antibiotics are extruded directly into the external medium, two independent mechanisms, efflux and low uptake through this permeability barrier, contribute to decreased intracellular accumulation of antibiotics (26).A single bacterial cell may contain multiple efflux pumps ...

To achieve an integrated understanding of the stem cell system of planarians at both the cellular and molecular levels, we developed a new method by combining ''fluorescent activated cell sorting (FACS) index sorting'' analysis and single-cell reverse transcription-polymerase chain reaction (RT-PCR) to detect the gene expression and cell cycle state of stem cells simultaneously. Single cells were collected using FACS, and cDNAs of each cell were used for semi-quantitative RT-PCR. The results were plotted on the FACS sorting profile using the ''index sorting'' function, which enabled us to analyze the gene expression in combination with cell biological data (such as cell cycle phase) for each cell. Here we investigated the adult stem cells of planarians using this method and obtained findings suggesting that the stem cells might undergo commitment during S to G2 ⁄ M phase. This method could be a powerful and straightforward tool for examining the stem cell biology of not only planarians but also other organisms, including vertebrates.

No abstract

Staphylococcus aureus is an important pathogen that adapts and survives in low-pH environments. One component of this adaptation involves the regulation of genes encoding bacterial transporters that could affect response to antibiotics under these conditions. We previously demonstrated that the transcriptional regulator MgrA in its phosphorylated form (MgrA-P) represses the expression of norB, encoding the NorB multidrug resistance efflux pump. In this study, we focused on changes in the expression of mgrA at the transcriptional and posttranslational levels, following a shift from pH 7.0 to pH 4.5. We then correlated those changes with modifications in transcript levels of norB and to resistance to moxifloxacin, a substrate of NorB. At pH 4.5, S. aureus MgrA increased 2-fold and MgrA-P decreased 4-fold, associated with an 8-fold increase in norB transcripts and a 6-fold reduction in bacterial killing by moxifloxacin, and the phenomenon was dependent on intact mgrA. Taken together, these new data showed that phosphoregulation of MgrA at low pH reverses its repression of norB expression, conferring resistance to moxifloxacin.