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 mtrR gene of Neisseria gonorrhoeae controls the level of susceptibility to hydrophobic antibiotics and detergents. The mtrR gene was cloned and shown to encode a putative transcriptional repressor. The mtr region was homologous to the envCD and acrAB regions of Escherichia coli, which are also involved in susceptibility to hydrophobic compounds. A homologous repressor protein was encoded by a previously unrecognized open reading frame within both the envCD and acrAB regions. Deletion of mtrR resulted in increased resistance to antibiotics and detergents: the mtrR mutations in two penicillin-resistant clinical isolates resulted in a change of His-105 to Tyr. We propose that the mtrR repressor allows gonococci to regulate the permeability of its cell envelope in response to environmental signals, so that they can grow in the presence of toxic faecal lipids in the rectum as well as in the genital tract.

Disruption of gene HI0894 or HI0895 in Haemophilus influenzaeRd, homologs of Escherichia coli acrAB multidrug efflux genes, caused hypersusceptibility to erythromycin, rifampin, novobiocin, and dyes such as ethidium bromide and crystal violet and increased accumulation of radioactive erythromycin, showing that these genes are expressed and contribute to the baseline level resistance of this organism through active drug efflux. The gene disruption did not produce detectable changes in susceptibility to several other antibiotics, possibly because rapid influx of small antibiotic molecules through the large H. influenzae porin channels counterbalances their efflux.Haemophilus influenzae is a well-known pathogen that colonizes the upper respiratory tract and invades the respiratory mucous membranes. H. influenzae strains frequently exhibit multiple antibiotic resistance, usually attributed to the presence of large R plasmids containing several resistance genes, each directed toward a specific antibiotic (23). However, a more general mechanism for baseline and increased levels of resistance, such as that conferred by the synergistic interaction of the outer membrane barrier and the multidrug efflux pumps (19), should also be considered.H. influenzae has only one porin, Omp2, which produces large, highly permeable channels. The large size of the channel is evident from the observations that it allowed the penetration of a 1,845-Da oligosaccharide (25) (in contrast to Escherichia coli porins, which were impermeable to 666-Da stachyose [17]) and that the intact cells showed outer membrane permeability for ␤-lactams that was much higher than that observed in E. coli (3,22). Planar lipid membrane experiments gave a singlechannel conductance of 1.1 nS in 1 M KCl (24), and the apparently low level of this conductance, in comparison with the 2.0-nS conductance obtained in the same study with E. coli OmpF porin, was a puzzle. However, it has since become clear that the 1.2-to 2.0-nS "single-channel" conductance of E. coli porin is caused by the insertion of three channels of porin trimer (18). (Furthermore, a more recent study showed that the single-channel conductance steps of H. influenzae porin had a population centered at 2.3 nS [2], although this could have resulted from the insertion of oligomers.) Thus, all assays, including the planar lipid bilayer assay, show that the H. influenzae Omp2 porin produces a much wider channel than those of E. coli porins.

Resistance of Neisseria gonorrhoeae to antimicrobial hydrophobic agents (HAS) has been ascribed to the mtr (multiple transferable resistance) operon. This operon is composed of the mtrR gene, which encodes a transcriptional repressor (MtrR), and a three-gene complex (mtrcDE), which encodes cell envelope proteins (MtrC-MtrD-MtrE) that form an energy-dependent efflux pump. HA-hypersusceptible strains are often isolated from patients, but the genetic basis for such hypersusceptibility was heretofore unknown. The genetic basis of HA hypersusceptibility in laboratory-derived strains BR54 and BR87 was studied to learn if this trait could be linked to mutations in the mtr operon. Mutations in the mtrR gene of these strains that could be phenotypically suppressed by mutations in their mtrC or mtrD genes were identified. Thus, small deletions (4-10 bp) in the mtrC or mtrD genes of strains BR87 and BR54 that would result in the production of truncated efflux pump proteins that serve as a membrane fusion protein (MtrC) or transporter of HAS (MtrD) were found to be responsible for their HA-hypersusceptible property.

Groundwater from a xylene‐contaminated acquifer was enriched in the laboratory in the presence of toluene, xylenes, ethylbenzene, and benzene. A pure culture that degrades toluene and m‐xylene under nitrate‐reducing conditions was isolated. Fatty acid analysis, 16S rRNA sequencing, and morphological traits indicate that the isolate was a strain of Azoarcus tolulyticus. The kinetics of toluene degradation under nitrate‐reducing conditions by this isolate was determined. Nitrate reduction does not proceed beyond nitrite. Nitrate and toluene are substrate limiting at low concentrations, whereas toluene, nitrate, and nitrite are inhibitory at high concentrations. Several inhibition models were compared to experimental data to represent inhibition by these substrates. A kinetic model for toluene and nitrate degradation as well as for cell growth and nitrite production was developed and compared to experimental data. The results of this work may find important application in the remediation of groundwater aquifers contaminated with aromatic hydrocarbons. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 82–90, 1997.

tions. Several inhibition models were compared to experimental data to represent inhibition by these substrates. 1994). However, due to the low solubility of oxygen inA kinetic model for toluene and nitrate degradation as water it is not always possible or economically viable well as for cell growth and nitrite production was develto aerate a contaminated groundwater aquifer.