Regulation of Serratia marcescens ompF and ompC porin genes in response to osmotic stress, salicylate, temperature and pH

Abstract

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...

Nosocomial infection and its molecular mechanisms of antibiotic resistance

“…The oprD promoter breakdown appears as a result of deletions or insertions in the upstream region of oprD. It was reported that a deletion containing the putative promoter and initiation codon blocked transcription of oprD (69)(70)(71)(72). Based on previous research, IS1394 and an ISPa16-like insertion element have been proposed as an upstream region of the oprD in imipenem-resistant strains of P. aeruginosa showing down-regulated oprD expression (73,74).…”

Adaptive and Mutational Resistance: Role of Porins and Efflux Pumps in Drug Resistance

“…The authors of that study observed that this conferred increased tolerance to not only this quaternary ammonium compound but also antibiotics such as chloramphenicol, ciprofloxacin, nalidixic acid, ampicillin, and cefotaxime. In Serratia marcescens, the presence of the two major porins OmpF and OmpC varies according to osmotic stress, temperature, pH, and added salicylate (19). Salicylate is known to induce a multipleantibiotic-resistance phenotype in E. coli by promoting the expression of the marRAB operon (5).…”

Highly Solvent Tolerance in Serratia marcescens IBBPo15

“…For rep-PCR amplification, 1 μl of DNA extract was added to a final volume of 25 μl reaction mixture, containing: 5×GoTaq flexi buffer, MgCl 2 , dNTP mix, primers (REP 1R-Dt and REP 2-Dt, BOXA 1R, (GTG) 5 , 21 ), and GoTaq G2 hot start polymerase (Promega). The PCR program consisted in initial denaturation for 10 min at 94°C, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 40°C or 50°C for 1 min, extension at 65°C for 8 min, and a final extension at 65°C for 16 min.…”

“…Membrane transporters, such as porins or efflux pumps, are main filters regulating the internal accumulation of toxic compounds 4 . Porins OmpF (with molecular weight of 41 kDa) and OmpC (40 kDa) which exist in outer membrane of S. marcescens cells are two non-specific protein channels that serve to take in nutrients and toxic compounds, and export waste products 5,6 . Furthermore, the AcrAB-TolC efflux system is a tripartite complex and is a member of the resistance-nodulation-division (RND) family which is considered the major efflux system in members of Enterobacteriaceae family 7 responsible for tolerance to toxic compounds, including organic solvents 8 .…”