MotA and MotB are integral membrane proteins of Escherichia coli that form the stator of the proton-fueled flagellar rotary motor. The motor contains several MotA/MotB complexes, which function independently to conduct protons across the cytoplasmic membrane and couple proton flow to rotation. MotB contains a conserved aspartic acid residue, Asp32, that is critical for rotation. We have proposed that the protons energizing the motor interact with Asp32 of MotB to induce conformational changes in the stator that drive movement of the rotor. To test for conformational changes, we examined the protease susceptibility of MotA in membrane-bound complexes with either wild-type MotB or MotB mutated at residue 32. Small, uncharged replacements of Asp32 in MotB (D32N, D32A, D32G, D32S, or D32C) caused a significant change in the conformation of MotA, as evidenced by a change in the pattern of proteolytic fragments. The conformational change does not require any flagellar proteins besides MotA and MotB, as it was still seen in a strain that expresses no other flagellar genes. It affects a cytoplasmic domain of MotA that contains residues known to interact with the rotor, consistent with a role in the generation of torque. Influences of key residues of MotA on conformation were also examined. Pro173 of MotA, known to be important for rotation, is a significant determinant of conformation: Dominant Pro173 mutations, but not recessive ones, altered the proteolysis pattern of MotA and also prevented the conformational change induced by Asp32 replacements. Arg90 and Glu98, residues of MotA that engage in electrostatic interactions with the rotor, appear not to be strong determinants of conformation of the MotA/MotB complex in membranes. We note sequence similarity between MotA and ExbB, a cytoplasmic-membrane protein that energizes outer-membrane transport in Gram-negative bacteria. ExbB and associated proteins might also employ a mechanism involving proton-driven conformational change.

Small, hydrophilic compounds such as β-lactams diffuse across the outer membrane of Gram-negative bacteria through porin channels, which were originally thought to be nonspecific channels devoid of any specificity. However, since the discovery of an ampicillin-binding site within the OmpF channel in 2002, much attention has been focused on the potential specificity of the channel, where the binding site was assumed either to facilitate or to retard the penetration of β-lactams. Since the earlier studies on porin permeability were done without the knowledge of the contribution of multidrug efflux pumps in the overall flux process across the cell envelope, in this study we have carefully studied both the porin permeability and active efflux of ampicillin and benzylpenicillin. We found that the influx occurs apparently by a spontaneous passive diffusion without any indication of specific binding within the concentration range relevant to the antibiotic action of these drugs, and that the higher permeability for ampicillin is totally as expected from the gross property of this drug as a zwitterionic compound. The active efflux by AcrAB was more effective for benzylpenicillin due to the stronger affinity and high degree of positive cooperativity. Our data now give a complete quantitative picture of the influx, efflux, and periplasmic degradation (catalyzed by AmpC β-lactamase) of these two compounds, and correlate closely with the susceptibility of Escherichia coli strains used here, thus validating not only our model but also the parameters obtained in this study.AcrB | OmpC

The stator of the bacterial flagellar motor is formed from the membrane proteins MotA and MotB, which associate in complexes with stoichiometry MotA(4)MotB(2) (Kojima, S., and Blair, D. F., preceding paper in this issue). The MotA/MotB complexes conduct ions across the membrane, and couple ion flow to flagellar rotation by a mechanism that appears to involve conformational changes within the complex. MotA has four membrane-crossing segments, termed A1-A4, and MotB has one, termed B. We are studying the organization of the 18 membrane segments in the MotA(4)MotB(2) complex by using targeted disulfide cross-linking. A previous cross-linking study showed that the two B segments in the complex (one from each MotB subunit) are arranged as a symmetrical dimer of alpha-helices. Here, we extend the cross-linking study to segments A3 and A4. Single Cys residues were introduced by mutation in several consecutive positions in segments A3 and A4, and double mutants were made by pairwise combination of subsets of the Cys replacements in segments A3, A4, and B. Disulfide cross-linking of the single- and double-Cys proteins was studied in whole cells, in membranes, and in detergent solution. Several combinations of Cys residues in segments A3 and B gave a high yield of disulfide-linked MotA/MotB heterodimer upon oxidation with iodine. Positions of efficient cross-linking identify a helix face on segment A3 that is in proximity to segment(s) B. Some combinations of Cys residues in segments A4 and B also gave a significant yield of disulfide-linked heterodimer, indicating that segment A4 is also near segment(s) B. Certain combinations of Cys residues in segments A3 and A4 cross-linked to form MotA tetramers in high yield upon oxidation. The high-yield positions identify faces on A3 and A4 that are at an interface between MotA subunits. Taken together with mutational studies and patterns of amino acid conservation, the cross-linking results delineate the overall arrangement of 10 membrane segments in the MotA/MotB complex, and identify helix faces likely to line the proton channels.

Klebsiella pneumoniae, one of the most important nosocomial pathogens, is becoming a major problem in health care because of its resistance to multiple antibiotics, including cephalosporins of the latest generation and, more recently, even carbapenems. This is largely due to the spread of plasmid-encoded extended-spectrum ␤-lactamases. However, antimicrobial agents must first penetrate the outer membrane barrier in order to reach their targets, and hydrophilic and charged ␤-lactams presumably diffuse through the porin channels. Unfortunately, the properties of K. pneumoniae porin channels are largely unknown. In this study, we made clean deletions of K. pneumoniae porin genes ompK35 and ompK36 and examined the antibiotic susceptibilities and diffusion rates of ␤-lactams. The results showed that OmpK35 and OmpK36 produced larger more permeable channels than their Escherichia coli homologs OmpF and OmpC; OmpK35 especially produced a diffusion channel of remarkably high permeability toward lipophilic (benzylpenicillin) and large (cefepime) compounds. These results were also confirmed by expressing various porins in an E. coli strain lacking major porins and the major multidrug efflux pump AcrAB. Our data explain why the development of drug resistance in K. pneumoniae is so often accompanied by the mutational loss of its porins, especially OmpK35, in addition to the various plasmid-carried genes of antibiotic resistance, because even hydrolysis by ␤-lactamases becomes inefficient in producing high levels of resistance if the bacterium continues to allow a rapid influx of ␤-lactams through its wide porin channels. IMPORTANCEIn Gram-negative bacteria, drugs must first enter the outer membrane, usually through porin channels. Thus, the quantitative examination of influx rates is essential for the assessment of resistance mechanisms, yet no such studies exist for a very important nosocomial pathogen, Klebsiella pneumoniae. We found that the larger channel porin of this organism, OmpK35, produces a significantly larger channel than its Escherichia coli homolog, OmpF. This makes unmodified K. pneumoniae strains more susceptible to relatively large antibiotics, such as the third-and fourth-generation cephalosporins. Also, even the acquisition of powerful ␤-lactamases is not likely to make them fully resistant in the presence of such an effective influx process, explaining why so many clinical isolates of this organism lack porins. K lebsiella pneumoniae is currently one of the most important Gram-negative nosocomial pathogens, which are often carbapenem resistant (1), and its wide dissemination is helped by its capacity to survive in the hospital environment (2), presumably aided by the production of thick capsules. A remarkable feature of multidrug-resistant isolates of this species is the absence of porin(s) (for an early report, see reference 3, and for reviews, see references 4 and 5). K. pneumoniae produces two classical trimeric porins, OmpK35 and OmpK36, which are homologs of OmpF and OmpC of Escherichia col...

Background: OmpC porin is less permeable than OmpF, but there is little difference in channel size. Results: There are more charged residues in the OmpC channel, and mutating them or increasing ionic strength made it more permeable and OmpF-like. Conclusion: Charged residues produce the low permeability of OmpC. Significance: OmpC, produced in a high ionic strength environment, has properties optimized for this environment.