Using interposon mutagenesis, we have generated strains of Pseudomonas aeruginosa which lack or overexpress the substrate-selective OprB porin of this species. A marked decrease or increase in the initial uptake of glucose by these strains verified the role of OprB in facilitating the diffusion of glucose across the outer membrane of P. aeruginosa. However, we also demonstrated that the loss or overexpression of OprB had a similar effect on the uptake of three other sugars able to support the growth of this bacterium (mannitol, glycerol, and fructose). This effect was restricted to carbohydrate transport; arginine uptake was identical in mutant and wild-type strains. These results indicated that OprB cannot be considered strictly a glucoseselective porin; rather, it acts as a central component of carbohydrate transport and is more accurately described as a carbohydrate-selective porin.Diffusion rates across the outer membrane of Pseudomonas aeruginosa are 100 to 500 times lower than those measured for Escherichia coli (37). The low outer membrane permeability of P. aeruginosa has been attributed to the unusual channel-forming properties of the OprF porin, the major, constitutively expressed protein in the outer membrane of this bacterium (9). OprF has some homology with the OmpA protein of enteric bacteria (33) and, like OmpA, shows only limited porin activity (28). In enteric bacteria, the low channel-forming activity of OmpA does not have a major influence on outer membrane permeability, as these bacteria also produce classical general diffusion porins (e.g., OmpF and OmpC). The more efficient permeation of solutes through OmpF and OmpC overshadows the diffusion properties of the OmpA protein. In contrast, the apparent lack of typical general diffusion porins in P. aeruginosa results in the diffusion properties of the outer membrane of this bacterium being determined largely by the relatively inefficient OprF porin.The low outer membrane permeability of P. aeruginosa is compensated for by the presence of several substrate-selective porins (14,21,29). Porins of this type are also present in enteric bacteria, but in these species, substrate-selective porins have generally been associated with the diffusion of relatively large substrates (e.g., LamB, maltodextrins [32]; ScrY, sucrose [25]; TolC, peptides [2]). In the absence of their respective porins, these substrates either do not diffuse or diffuse to only a limited extent, through nonspecific channels. In contrast, the substrate-selective porins of P. aeruginosa facilitate the diffusion of much smaller substrates. In E. coli, diffusion of small substrates, such as glucose or glycerol, is generally believed to occur via nonspecific channels. In P. aeruginosa, if facilitated diffusion across the outer membrane did not occur, the low outer membrane permeability would potentially limit the transport of almost all substrates, regardless of size.Four substrate-selective porins have been identified in P. aeruginosa. The OprD porin facilitates the diffusion of basic ...

The major outer membrane protein was extracted from Campylobacter coli by Triton X-100/EDTA fractionation of cell envelopes. This heat-modifiable protein was shown to have pore-forming activity in black lipid bilayers. The C. coli porin formed a relatively small cation-selective pore with a mean single-channel conductance of 0.53 + 0.16 nS in 1.0 M KCI. There was no evidence of oligomer formation, which suggested that each protein monomer formed a pore. Pore-forming activity of the C. coli porin and similarly prepared Campylobacterjejuni porin was also measured in liposome-sweiling assays. These results confirmed the cation selectivity of both pores. The C. coli porin formed a small pore, which hindered the penetration of solutes with a molecular weight of 262, and a larger pore, which hindered the penetration of solutes with a molecular weight of 340, in a protein-concentration-dependent manner. C. jejuni formed one size of pore that was slightly larger than the C. coli pore and just permitted the passage of solutes, with a molecular weight of 340.

Regulation ofSerratia marcescens is a Gram-negative enterobacterium that has become an important opportunistic pathogen, largely due to its high degree of natural antibiotic resistance. One factor contributing to this natural antibiotic resistance is reduced outer membrane permeability, which is controlled in part by OmpC and OmpF porin proteins. OmpF expression is regulated by micF, an RNA transcript encoded upstream of the ompC gene, which hybridizes with the ompF transcript to inhibit its translation. Regulation of S. marcescens porin gene expression, as well as that of micF, was investigated using b-galactosidase reporter gene fusions in response to 5, 8 and 10 % sucrose, 1, 5 and 8 mM salicylate, and different pH and temperature values. b-Galactosidase activity assays revealed that a lower growth temperature (28 6C), a more basic pH (pH 8), and an absence of sucrose and salicylate induce the transcription of the ompF gene, whereas the induction of ompC is stimulated at a higher growth temperature (42 6C), acidic pH (pH 6), and maximum concentrations of sucrose (10 %) and salicylate (8 mM). In addition, when multiple conditions were tested, temperature had the predominant effect, followed by pH. In this study, it was found that the MicF regulatory mechanism does not play a role in the osmoregulation of the ompF and ompC genes, whereas MicF does repress OmpF expression in the presence of salicylate and high growth temperature, and under low pH conditions. INTRODUCTIONSerratia marcescens is a Gram-negative enteric bacterium that has become an important opportunistic pathogen associated with a number of life-threatening diseases and nosocomial infections. In the last two decades, S. marcescens has gained much attention due to a high incidence of antibiotic resistance. b-Lactam resistance in this organism is due in part to b-lactamase enzymes (Sanders & Sanders, 1992); however, a reduction in the levels of outer membrane porins is also responsible for altering resistance levels by decreasing the outer membrane permeability (Gutmann et al., 1984).Our group has characterized two S. marcescens porins, OmpC and OmpF (Hutsul & Worobec, 1997), with molecular masses of 40 and 41 kDa, respectively. These porins are non-specific protein channels that serve to take in nutrients and antibiotics, such as b-lactams, and export waste products. OmpF is believed to have a slightly larger pore diameter, resulting in a faster rate of diffusion through OmpF than through OmpC. The structural genes for both OmpF and OmpC have been cloned and sequenced. S. marcescens OmpC and OmpF are 71 % and 68 % similar to Escherichia coli OmpC and OmpF, respectively, at the amino acid level (Hutsul & Worobec, 1997).The production of E. coli OmpF and OmpC is regulated by many environmental factors, such as osmotic pressure, temperature and pH. Production of the appropriate outer membrane porin is required for survival of the organism under widely differing conditions (Csonka, 1989). For example, in the human gut, where concentrations of both nutri...

OprB, a glucose-inducible porin of P. aeruginosa, was characterized by black lipid bilayer analysis and circular dichroism spectroscopy. Black lipid bilayer analysis of OprB revealed a single-channel conductance of 25 pS, the presence of a glucose binding site with a Ks for glucose of 380 +/- 40 mM, and the formation of channels with a strong selection for anions. Analysis of P. aeruginosa OprB circular dichroism spectra revealed a high beta sheet content (40%) which is within the range of that determined for other porins. Values obtained from black lipid bilayer analysis were compared to those previously obtained for OprB of P. putida [Saravolac et al. (1991). J. Bacteriol. 173, 4970-4976] and indicated extensive similarities in the single-channel conductance and glucose-binding properties of these two porins. Immunological and amino terminal sequence analysis revealed a high degree of homology. Of the first 14 amino terminal residues, 12 were identical. A major difference between the two porins was found in their ion selectivity. Whereas P. aeruginosa OprB is anion selective, P. putida OprB and other carbohydrate selective porins are known to be cation selective.

Serratia marcescens is an important nosocomial agent known for causing various infections in immunocompromised individuals. Resistance of this organism to a broad spectrum of antibiotics makes the treatment of infections very difficult. This study was undertaken to identify multidrug resistance efflux pumps in S. marcescens. Three mutant strains of S. marcescens were isolated in vitro by the serial passaging of a wild-type strain in culture medium supplemented with ciprofloxacin, norfloxacin, or ofloxacin. Fluoroquinolone accumulation assays were performed to detect the presence of a proton gradient-dependent efflux mechanism. Two of the mutant strains were found to be effluxing norfloxacin, ciprofloxacin, and ofloxacin, while the third was found to efflux only ofloxacin. A genomic library of S. marcescens wild-type strain UOC-67 was constructed and screened for RND pump-encoding genes by using DNA probes for two putative RND pump-encoding genes. Two different loci were identified: sdeAB, encoding an MFP and an RND pump, and sdeCDE, encoding an MFP and two different RND pumps. Northern blot analysis revealed overexpression of sdeB in two mutant strains effluxing fluoroquinolones. Analysis of the sdeAB and sdeCDE loci in Escherichia coli strain AG102MB, deficient in the RND pump (AcrB), revealed that gene products of sdeAB are responsible for the efflux of a diverse range of substrates that includes ciprofloxacin, norfloxacin, ofloxacin, chloramphenicol, sodium dodecyl sulfate, ethidium bromide, and n-hexane, while those of sdeCDE did not result in any change in susceptibilities to any of these agents.Serratia marcescens is an important nosocomial agent that is frequently reported as the causative agent of a variety of infections, including respiratory tract infections, urinary tract infections, septicemia, meningitis, endocarditis, and wound infections. This organism is commonly isolated from the urine of patients with indwelling catheters and is considered significant in patients with serious underlying disease, particularly neonates, patients requiring treatment in intensive care units, neutropenic patients, or those with disseminated malignancies (8). The high intrinsic resistance of this organism to a variety of antibiotics makes the treatment of infections very difficult. S. marcescens has been found to be resistant to ␤-lactams, aminoglycosides, and quinolones. The resistance of this organism to quinolones was reported shortly after the initial clinical use of these antibiotics (5).An important mechanism of resistance to quinolones is the decreased accumulation of the antibiotics inside the cell, as mediated by efflux pumps. Efflux-mediated resistance to fluoroquinolones is widespread among various bacterial pathogens, including Escherichia coli (11), Pseudomonas aeruginosa (14), and Salmonella enterica serovar Typhimurium (6). In this mechanism of resistance, bacteria pump out antibiotic molecules against a concentration gradient in an energy-dependent manner. In gram-negative bacteria, efflux pumps belon...