Flagella act as semirigid helical propellers that are powered by reversible rotary motors. Two membrane proteins, MotA and MotB, function as a complex that acts as the stator and generates the torque that drives rotation. The genome sequence of Pseudomonas aeruginosa PAO1 contains dual sets of motA and motB genes, PA1460-PA1461 (motAB) and PA4954-PA4953 (motCD), as well as another gene, motY (PA3526), which is known to be required for motor function in some bacteria. Here, we show that these five genes contribute to motility. Loss of function of either motAB-like locus was dispensable for translocation in aqueous environments. However, swimming could be entirely eliminated by introduction of combinations of mutations in the two motAB-encoding regions. Mutation of both genes encoding the MotA homologs or MotB homologs was sufficient to abolish motility. Mutants carrying double mutations in nonequivalent genes (i.e., motA motD or motB motC) retained motility, indicating that noncognate components can function together. motY appears to be required for motAB function. The combination of motY and motCD mutations rendered the cells nonmotile. Loss of function of motAB, motY, or motAB motY produced similar phenotypes; although the swimming speed was only reduced to ϳ85% of the wild-type speed, translocation in semisolid motility agar and swarming on the surface of solidified agar were severely impeded. Thus, the flagellar motor of P. aeruginosa represents a more complex configuration than the configuration that has been studied in other bacteria, and it enables efficient movement under different circumstances.

Thirteen derivatives of the Streptococcus pneumoniae TIGR4 strain in which putative drug efflux pumps were genetically inactivated were constructed and characterized. The results indicate that two linked genes encoding the ABC-type transporters SP2073 and SP2075 function together to confer intrinsic resistance to a series of structurally unrelated compounds, including certain fluoroquinolones.Multidrug resistance efflux pumps are important components of innate and acquired bacterial resistance to antimicrobial agents (5). Herein we report the first systematic inactivation of genes encoding all nonessential candidate drug efflux systems in a gram-positive virulent pathogen, Streptococcus pneumoniae TIGR4 (12), and the characterization of the resulting mutants in terms of altered antimicrobial susceptibility.Fourteen putative drug efflux transporters, identified by a combination of internal bioinformatics and by reference to the analysis of Paulsen and associates (http://www.membranetransport .org/index.html), were targeted for gene disruption (Table 1) using linear DNA fragments containing antibiotic resistance cassette-disrupted versions of the individual gene to be inactivated. Following the induction of genetic competence with competence-stimulatory peptide 2 (CSP-2), strains in which the targeted gene had been inactivated by allelic replacement were recovered by plating for the antibiotic resistance encoded by the marker used for gene disruption (as described previously in reference 8) (Table 1). Of the gene disruptions attempted, 13 yielded the anticipated insertion mutations, as verified by diagnostic PCR analysis (data not shown). In repeated attempts, mutants disrupted in open reading frame (ORF) SP1435 were not recovered, possibly indicating that either SP1435 is essential for growth or lethality resulted from the polar effect(s) on the expression of adjacent loci. Additional experiments would be necessary to resolve this issue.Thirteen strains carrying disruptions in putative drug efflux pumps and a strain (CB318) lacking both SP2073 and SP2075 showed normal colony size and morphology relative to those of the parent strain TIGR4 on TSA II blood agar. These strains also exhibited similar growth kinetics in Todd-Hewitt broth supplemented with 5% (wt/vol) yeast extract and 0.1 mg of bovine catalase per ml. These 14 strains and the parent strain, TIGR4, were subsequently characterized in terms of altered susceptibility to 31 antimicrobial substances (7). Eleven strains, including a strain carrying a disruption in the SP0972 ORF encoding the previously described PmrA efflux pump (3), did not show appreciable changes in susceptibility to the compounds tested (data not shown). The lack of effect of inactivation of SP0972 (pmrA) in this study indicates that PmrA is not intrinsically active in S. pneumoniae TIGR4, which is consistent with literature reports that indicate that PmrA is derepressed at the transcriptional level in clinical strains with PmrA-based efflux resistance (9, 10). The lack of effect followi...

Drug efflux systems contribute to the intrinsic resistance of Pseudomonas aeruginosa to many antibiotics and biocides and hamper research focused on the discovery and development of new antimicrobial agents targeted against this important opportunistic pathogen. Using a P. aeruginosa PAO1 derivative bearing deletions of opmH, encoding an outer membrane channel for efflux substrates, and four efflux pumps belonging to the resistance nodulation/cell division class including mexAB-oprM, we identified a small-molecule indole-class compound (CBR-4830) that is inhibitory to growth of this efflux-compromised strain. Genetic studies established MexAB-OprM as the principal pump for CBR-4830 and revealed MreB, a prokaryotic actin homolog, as the proximal cellular target of CBR-4830. Additional studies establish MreB as an essential protein in P. aeruginosa, and efflux-compromised strains treated with CBR-4830 transition to coccoid shape, consistent with MreB inhibition or depletion. Resistance genetics further suggest that CBR-4830 interacts with the putative ATP-binding pocket in MreB and demonstrate significant cross-resistance with A22, a structurally unrelated compound that has been shown to promote rapid dispersion of MreB filaments in vivo. Interestingly, however, ATP-dependent polymerization of purified recombinant P. aeruginosa MreB is blocked in vitro in a dosedependent manner by CBR-4830 but not by A22. Neither compound exhibits significant inhibitory activity against mutant forms of MreB protein that bear mutations identified in CBR-4830-resistant strains. Finally, employing the strains and reagents prepared and characterized during the course of these studies, we have begun to investigate the ability of analogues of CBR-4830 to inhibit the growth of both efflux-proficient and efflux-compromised P. aeruginosa through specific inhibition of MreB function.The activity of efflux-based drug extrusion mechanisms impact the overall effectiveness of current antimicrobial therapies and impair efforts to discover new chemotherapeutic treatments for many serious bacterial diseases (4,42,43,65). Few bacterial pathogens exemplify the importance of the interplay between xenobiotic efflux extrusion systems and the exclusion of antimicrobial agents at the level of the poorly permeable gram-negative outer membrane quite like Pseudomonas aeruginosa (46, 58). Indeed, this organism has the capacity to encode more than 50 potential multidrug transporters including 12 resistance nodulation/cell division (RND)-class transporters, which to date, represent the only clinically significant drug transporters that have been experimentally validated for P. aeruginosa (20,58).The MexAB-OprM tripartite pump confers basal resistance to P. aeruginosa to a number of chemically distinct antibiotics (58) and through gain-of-function mutations, including those in regulatory proteins (e.g., mexR or mexZ), may overproduce MexAB-OprM or other RND-class pumps which are significant contributors to multidrug resistance in clinical P. areuginosa isolates...

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Rifamycins have proven efficacy in the treatment of persistent bacterial infections. However, the frequency with which bacteria develop resistance to rifamycin agents restricts their clinical use to antibiotic combination regimens. In a program directed toward the synthesis of rifamycins with a lower propensity to elicit resistance development, a series of compounds were prepared that covalently combine rifamycin and quinolone pharmacophores to form stable hybrid antibacterial agents. We describe modeof-action studies with Staphylococcus aureus of CBR-2092, a novel hybrid that combines the rifamycin SV and 4H-4-oxo-quinolizine pharmacophores. In biochemical studies, CBR-2092 exhibited rifampin-like potency as an inhibitor of RNA polymerase, was an equipotent (balanced) inhibitor of DNA gyrase and DNA topoisomerase IV, and retained activity against a prevalent quinolone-resistant variant. Macromolecular biosynthesis studies confirmed that CBR-2092 has rifampin-like effects on RNA synthesis in rifampin-susceptible strains and quinolone-like effects on DNA synthesis in rifampin-resistant strains. Studies of mutant strains that exhibited reduced susceptibility to CBR-2092 further substantiated RNA polymerase as the primary cellular target of CBR-2092, with DNA gyrase and DNA topoisomerase IV being secondary and tertiary targets, respectively, in strains exhibiting preexisting rifampin resistance. In contrast to quinolone comparator agents, no strains with altered susceptibility to CBR-2092 were found to exhibit changes consistent with altered efflux properties. The combined data indicate that CBR-2092 may have potential utility in monotherapy for the treatment of persistent S. aureus infections.