The mtr (multiple transferable resistance) system of Neisseria gonorrhoeae determines levels of gonococcal resistance to hydrophobic agents (HAS), including detergent-like fatty acids and bile salts that bathe certain mucosal surfaces. The genetic organization of the mtr system was determined and found t o consist of the mtrR gene, which encodes a transcriptional regulator (MtrR), and three tandemly linked genes termed mtrCD€. The mtrCD€ genes were organized in the same apparent transcriptional unit, upstream and divergent from the mtrR gene. The mtKD€-encoded proteins of N. gonorrhoeae were analogous to a family of bacterial eff luxhransport proteins, notably the MexABOprK proteins of Pseudomonas aenrginosa and the AcrAE and EnvCD proteins of Escherichia coli, that mediate resistance to drugs, dyes, and detergents. Inactivation of the mtrC gene resulted in loss of the MtrC lipoprotein and rendered gonococci hypersusceptible to structurally diverse HAS; this revealed the importance of the mtr system in determining HAR in gonococci. Further support for a role of the mtrCD€ gene complex in determining levels of HAR in gonococci was evident when transformants bearing mutations in the mtrR gene were analysed. In this respect, missense and null mutations in the mtrR gene were found to result in increased levels of MtrC and HAR. However, high levels of MtrC and HAR, similar to those observed for clinical isolates, were associated with a single bp deletion in a 13 bp inverted repeat sequence that intervened the divergent mtrR and mtrC genes. We propose that the 13 bp inverted-repeat sequence represents a transcriptional control element that regulates expression of the mtrRCDE gene complex, thereby modulating levels of gonococcal susceptibility to HAS.

The paradigm for differential antigen expression in Borrelia burgdorferi, the agent of Lyme disease, is the reciprocal expression of its outer surface (lipo)proteins (Osp) A and C; as B. burgdorferi transitions from its arthropod vector into mammalian tissue, ospC is upregulated, and ospA is downregulated. In the current study, using B. burgdorferi cultivated under varying conditions in BSK‐H medium, we found that a decrease in pH, in conjunction with increases in temperature (e.g. 34°C or 37°C) and cell density, acted interdependently for the reciprocal expression of ospC and ospA. The lower pH (6.8), which induced the reciprocal expression of ospC and ospA in BSK‐H medium, correlated with a drop in pH from 7.4 to 6.8 of tick midgut contents during tick feeding. In addition to ospC and ospA, other genes were found to be regulated in reciprocal fashion. Such genes were either ospC‐like (e.g. ospF, mlp‐8 and rpoS) (group I) or ospA‐like (lp6.6 and p22) (group II); changes in expression occurred at the mRNA level. That the expression of rpoS, encoding a putative stress‐related alternative sigma factor (σs), was ospC‐like suggested that the expression of some of the group I genes may be controlled through σs. The combined results prompt a model that allows for predicting the regulation of other B. burgdorferi genes that may be involved in spirochaete transmission, virulence or mammalian host immune responses.

The capacity of Neisseria gonorrhoeae to resist structurally diverse hydrophobic agents (HAs) because of the mtr (multiple transferrable resistance) efflux system was found to be regulated at the level of transcription by two distinct mechanisms. This was surmised because a deletion that removed >90% of the coding sequence of the mtrR (multiple transferrable resistance regulator) gene or a single-base-pair deletion within a 13-bp inverted repeat sequence located in its promoter resulted in altered expression of the mtrC gene; mtrC encodes a 44-kDa membrane lipoprotein essential for the efflux of HAs. However, the single-base-pair deletion had the more significant impact on gene expression since it resulted in the loss of expression of mtrR and a threefold increase in the expression of mtrC. Hence, the mtr efflux system in gonococci is subject to both MtrR-dependent and MtrR-independent regulation, and the levels of mtrC mRNA correlate well with HA resistance levels in gonococci.The genetic organization of the mtr system in gonococci (17) was recently determined (4, 12) and was found to be remarkably similar to that of the mexAB-oprK efflux system of Pseudomonas aeruginosa (13) as well as those of the acrAE (7) and envCD (5) efflux pumps possessed by Escherichia coli; the E. coli efflux operons have been renamed acrAB and acrEF, respectively (8). Recent studies confirmed (6) the energy-dependent efflux action capacity of the mtr system. As was emphasized in a recent review by Nikaido (11), efflux systems have importance for bacterial resistance to structurally diverse antimicrobial agents, including antibiotics, dyes, and detergents.

Gonococcal resistance to antimicrobial hydrophobic agents (HAs) is due to energy-dependent removal ofHAs from the bacterial cell by the MtrCDE membrane-associated efflux pump. The mtrR (multiple transferrable resistance Regulator) gene encodes a putative transcriptional repressor protein (MtrR) believed to be responsible for regulation of mtrCDE gene expression. Gel mobility shift and DNase I footprint assays that used a maltose-binding protein (MBP)-MtrR fusion protein demonstrated that the MtrR repressor is capable of specifically binding the DNA sequence between the mtrR and mtrC genes. This binding site was localized to a 26-nucleotide stretch that includes the promoter utilized for mtrCDE transcription and, on the complementary strand, a 22-nucleotide stretch that contains the ؊35 region of the mtrR promoter. A single transition mutation (A3G) within the MtrR-binding site decreased the affinity of the target DNA for MtrR and enhanced gonococcal resistance to HAs when introduced into HA-susceptible strain FA19 by transformation. Since this mutation enhanced expression of the mtrCDE gene complex but decreased expression of the mtrR gene, the data are consistent with the notion that MtrR acts as a transcriptional repressor of the mtrCDE efflux pump protein genes.Resistance of Neisseria gonorrhoeae to structurally diverse hydrophobic agents (HAs) has been the subject of several recent investigations (5,6,10,20,23). Although earlier studies indicated that the mtr (multiple transferrable resistance) locus modifies the permeability barrier of the gonococcal cell envelope (3,4,13,16,22,24), more recent studies revealed that mtr encodes an energy-dependent efflux system (5,6,10,20,23). The membrane proteins (MtrC, MtrD, and MtrE) forming the efflux pump share considerable amino acid sequence similarity with other efflux proteins in Escherichia coli (12,17) and Pseudomonas aeruginosa (21). Active removal of toxic compounds by bacteria is an important mechanism of multiple antibiotic resistance (8, 17) and may confer a selective advantage on organisms, such as gonococci, when they colonize mucosal sites bathed in fluids containing antibacterial fatty acids and bile salts or encounter other environmental stresses (8,11,12,(16)(17)(18)23).Production of the MtrCDE efflux proteins is controlled at the level of transcription by both cis-and trans-acting factors involving the mtrR gene (5,6,20,23). The mtrR gene is located 250 bp upstream of the mtrCDE gene complex and transcribed divergently (5,6,20). It encodes a 210-amino-acid protein with a molecular mass of approximately 23 kDa that contains a putative helix-turn-helix motif near its N terminus (5,20,23). MtrR has considerable amino acid sequence similarities to several transcriptional repressors, most notably, the tetracycline repressor of pSC101 (1,7,20). Recent investigations have revealed that missense or deletion mutations within the mtrR coding region result in enhanced mtrCDE transcription and gonococcal resistance to HAs (6, 23). Thus, the available genetic dat...

Francisella tularensis is a gram-negative coccobacillus that is capable of causing severe, fatal disease in a number of mammalian species, including humans. Little is known about the proteins that are surface exposed on the outer membrane (OM) of F. tularensis, yet identification of such proteins is potentially fundamental to understanding the initial infection process, intracellular survival, virulence, immune evasion and, ultimately, vaccine development. To facilitate the identification of putative F. tularensis outer membrane proteins (OMPs), the genomes of both the type A strain (Schu S4) and type B strain (LVS) were subjected to six bioinformatic analyses for OMP signatures. Compilation of the bioinformatic predictions highlighted 16 putative OMPs, which were cloned and expressed for the generation of polyclonal antisera. Total membranes were extracted from both Schu S4 and LVS by spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation, which separated OMs from cytoplasmic (inner) membrane and other cellular compartments. Validation of OM separation and enrichment was confirmed by probing sucrose gradient fractions with antibodies to putative OMPs and inner membrane proteins. F. tularensis OMs typically migrated in sucrose gradients between densities of 1.17 and 1.20 g/ml, which differed from densities typically observed for other gram-negative bacteria (1.21 to 1.24 g/ml). Finally, the identities of immunogenic proteins were determined by separation on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analysis. This is the first report of a direct method for F. tularensis OM isolation that, in combination with computational predictions, offers a more comprehensive approach for the characterization of F. tularensis OMPs.