ompF Expression Research Articles

Cloning of multidrug resistance gene pqrA from Proteus vulgaris

The multiple antibiotic resistance gene pqrA was cloned from the chromosomal DNA of a clinical isolate of Proteus vulgaris 881051 into Escherichia coli KY2563. The MICs of quinolones tetracycline, cephalosporin, and chloramphenicol for transformant strain DNS7020 were from 8 to 32 times higher than those for the parent strain, KY2563. The level of expression of outer membrane protein F (OmpF) by DNS7020 was lower than that of KY2563 but not as low as that of an OmpF-deficient control strain. The 1.4-kb fragment containing the pqrA gene had an open reading frame encoding a polypeptide of 122 amino acid residues with a molecular weight of about 14,000, which was consistent with the experimental value identified by the Maxicell method. The putative PqrA polypeptide showed significant amino acid sequence similarity to the E. coli proteins SoxS and MarA. These polypeptides are strongly conserved in predicted helix-turn-helix DNA binding domains. The MarA protein, which is responsible for multiple antibiotic resistance in E. coli, also decreases OmpF expression. Moreover, the SoxS protein, which is characterized as a superoxide response regulon of E. coli, has also been shown to increase resistance to many structurally unrelated antibiotics. The soxS gene increases superoxide dismutase levels in addition to decreasing OmpF expression. The expression level of superoxide dismutase with DNS7020 was about 1.5 times higher than that with KY2563. These findings suggest that the pqrA gene in P. vulgaris confers multidrug resistance in a way similar to that of the soxS and marA genes in E. coli.

The leucine-responsive regulatory protein of Escherichia coli negatively regulates transcription of ompC and micF and positively regulates translation of ompF.

The two major porins of Escherichia coli K-12 strains, OmpC and OmpF, are inversely regulated with respect to one another. The expression of OmpC and OmpF has been shown to be influenced by the leucine-responsive regulatory protein (Lrp): two-dimensional gel electrophoresis of proteins from strains with and strains without a functional Lrp protein revealed that OmpC expression is increased in an lrp strain, while OmpF expression is decreased. In agreement with these findings, we now present evidence that transcriptional (operon) fusions of lacZ+ to ompC and micF are negatively regulated by Lrp. Lrp binds specifically to the intergenic region between micF and ompC, as indicated by mobility shift assays and by DNase I footprinting. The expression of an ompF'-lacZ+ gene (translational) fusion is increased 3.7-fold in an lrp+ background compared with an lrp background, but expression of an ompF-lacZ+ operon fusion is not. Studies of in vivo expression of the outer membrane porins during growth on glucose minimal medium showed that the OmpF/OmpC ratio is higher in lrp+ strains than it is in isogenic lrp strains. The effect of Lrp was not seen in a strain containing a deletion of micF. Our studies suggest that the positive effect of Lrp on OmpF expression stems from a negative effect of Lrp on the expression of micF, an antisense RNA that inhibits ompF translation.

Effect of Hydrostatic Pressure on the Synthesis of Outer Membrane Proteins inEscherichia coli

We examined the effects of hydrostatic pressure on the synthesis of Escherichia coli outer membrane proteins, particularly for the osmoregulated ImpC and OmpF porins. It was found that the expression of ompC and ompF was markedly reduced during the growth at high pressure, most likely at the transcriptional level. However, the signal transduction processes through the regulatory proteins, EnvZ and OmpR, was not influenced by the environmental pressure. It was also found that the expression of a presumed novel outer membrane protein, named OmpX, was affected by both the medium osmolarity and pressure in a manner independent of the function of EnvZ and OmpR.

Mechanisms of resistance to fluoroquinolones: state-of-the-art 1992-1994.

This paper gives an update on the mechanisms of bacterial resistance to fluoroquinolones. The laboratory techniques currently used to determine the mechanism(s) of resistance are outlined, including the use of restriction fragment length polymorphism and single-stranded conformational polymorphism analysis of mutations in gyrA. Alterations in gyrA have continued to be the most reported cause of resistance, with high level resistance due to 2 or more mutations in this gene. Recently, mutations in gyrA of Mycobacterium tuberculosis and Campylobacter jejuni have been described. Complementation studies with plasmid encoded cloned gyrB from Escherichia coli suggest that high fluoroquinolone resistance (minimum inhibitory concentration = 32 mg/L) in Salmonella typhimurium can be due to mutation in both gyrA and gyrB. Decreased fluoroquinolone accumulation into E. coli has been shown to be due to mutations in a number of genes at different loci. Current interest has focused upon the marRAB and soxRS loci, with mutations in genes of either loci giving rise to decreased susceptibility to several unrelated drugs, including fluoroquinolones, tetracycline, chloramphenicol and some beta-lactams, and decreased expression of OmpF. The genetic characterisation of fluoroquinolone efflux from Staphylococcus aureus has shown that efflux occurs in both fluoroquinolone-susceptible and -resistant bacteria. The most likely cause of resistance is overexpression of NorA, giving rise to increased efflux. Recently, 2 efflux systems in Pseudomonas aeruginosa have been proposed, MexA-MexB-OprK and MexC-MexD-OprM, conferring decreased susceptibility to fluoroquinolones, tetracycline, chloramphenicol and some beta-lactams.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel device of bacterial signal transducers.

The osmoregulatory expression of ompC and ompF in Escherichia coli is mediated by a pair of bacterial signal transduction proteins, EnvZ (sensory kinase) and OmpR (response regulator). We isolated previously multicopy suppressors which can complement a defect in the phosphotransfer signal transduction caused by an envZ deletion mutation. Among such suppressors, arcB and barA are of particular interest because these gene products are unique in the sense that they contain both an autophosphorylated histidine site (or transmitter module) and a phospho-accepting aspartate site (or receiver module) in their primary amino acid sequences. Here we report that ArcB and BarA possess in the C-terminal region a phosphorylated histidine site which has never been noticed, in addition to the authentic one identified previously. This newly identified histidine in ArcB and BarA was demonstrated to play a crucial role in the observed multicopy suppression. Furthermore, it was demonstrated in vivo and in vitro for ArcB that the C-terminal domain containing the histidine can function as an alternative phosphodonor (or transmitter). This novel type of sensory kinase was therefore revealed to contain two independent phosphodonor sites, together with a phospho-accepting site. These findings suggest that this unique feature of ArcB and BarA, in terms of the signaling modules, make it possible for these sensory kinases to function as dual-signaling transducers.

MicF antisense RNA has a major role in osmoregulation of OmpF in Escherichia coli.

micF RNA, produced from a multicopy plasmid, was originally shown to be a major factor in negative osmoregulation of the OmpF outer membrane protein in Escherichia coli. However, subsequent experiments with a micF deletion strain suggested that chromosomal micF RNA was not a key component in this process. We report here that micF RNA is essential for the reduction in OmpF levels in cells grown in media of low-to-intermediate levels of osmolarity. Under these conditions, the amount of OmpF was reduced up to 60% in the parent strain while OmpF levels were not altered in the micF deletion mutant. In medium of higher osmolarity, OmpF synthesis was strongly inhibited in both strains. RNA measurements showed that micF RNA levels rose rapidly in cells grown in low-to-intermediate levels of osmolarity concomitant with the reduction in OmpF protein, while ompF mRNA decreased strongly only during high-osmolarity conditions. Taken together, these results strongly suggest that the negative osmoregulation of OmpF at low-to-intermediate osmolarity levels requires micF RNA and that this is masked at higher osmolarity by the known strong inhibition of OmpF transcription by OmpR. Results consistent with this model were also obtained by using procaine, a compound reported to inhibit ompF expression by a mechanism very similar to that involved in osmoregulation.

Effect of salicylate, bismuth, osmolytes, and tetracycline resistance on expression of fimbriae by Escherichia coli

Adherence of Escherichia coli is facilitated by fimbriae and several outer membrane proteins (OMPs). Hypertonic conditions, salicylate, and Mar mutations are known to reduce OmpF expression. We speculated that OMPs involved in export or assembly of fimbrial subunits might be similarly affected. To explore this hypothesis, E. coli expressing P, type 1, S, colonization factor antigen I (CFA/I), or CFA/II fimbriae was grown in the presence of salicylate, bismuth salts, NaCl, and nonfermented sugars. Tetracycline-resistant clones were derived from several P-fimbriated strains. The bacteria were tested for the ability to agglutinate erythrocytes, yeast cells, and alpha-D-Gal(-4)-beta-D-Gal-bonded latex (Gal-Gal) beads and were examined for fimbriae by electron microscopy. Hyperosmolar conditions decreased fimbrial expression for all strains. Expression of P fimbriae by pyelonephritic strains, all of which were OmpF+, was reversibly repressed by salicylate and bismuth salts. CFA strains were similarly affected. Tetracycline-resistant P-fimbriated strains were OmpF deficient, were unable to agglutinate erythrocytes and Gal-Gal beads, and lacked fimbriae as observed by electron microscopy. Strains with plasmid-encoded P-fimbrial genes did not demonstrate OmpF on polyacrylamide gel electrophoresis profiles and were not affected by salicylate. The type 1-fimbriated phenotype was not affected by salicylate or bismuth unless the strains also expressed P fimbriae. S-fimbriated strains were not affected. The mechanism by which salicylates, bismuth salts, and tetracycline resistance inhibit or modulate the expression of P fimbriae may be mediated through OmpF and other OMPs.

The OmpR protein of Escherichia coli binds to sites in the ompF promoter region in a hierarchical manner determined by its degree of phosphorylation.

In Escherichia coli the ompF gene encodes a major outer membrane porin protein that is differentially regulated by the OmpR protein. OmpR acts as a positive as well as a negative regulator of ompF expression by binding to DNA sequences in the ompF promoter region. The DNA binding activity of OmpR is itself regulated by phosphorylation through the kinase protein EnvZ. Phosphorylation is believed to change the function of OmpR from an activator to a repressor molecule. By using purified OmpR and various regions of the ompF promoter we show that phosphorylation causes binding of OmpR to a DNA region between the -40 to -100 region of the ompF promoter previously shown to be important for ompF expression. As the amount of OmpR-phosphate increases, a binding site located at a further upstream -360 to -380 region was occupied. This latter site has been reported to be important for ompF repression. Further experiments indicate that the -70 to -100 region is a high affinity site, while the -45 to -60 and -360 to -380 regions are low affinity sites. We also provide evidence that OmpR binding at the -360 to -380 region requires previous binding at downstream sequences, which is indicative of long range interactions between OmpR molecules. We interpret our results in terms of a model for ompF regulation involving hierarchical binding by phosphorylated OmpR and potential DNA looping.

A distant upstream site involved in the negative regulation of the Escherichia coli ompF gene

The two-component regulatory system, OmpR-EnvZ, of Escherichia coli K-12 regulates the expression of the major outer membrane porin protein, OmpF. OmpR is a DNA-binding protein which acts as both an activator and a repressor to control ompF transcription. In this article, we describe a new OmpR-binding site that is located between 384 to 351 bp upstream from the ompF start point of transcription. Inactivation of this site by insertion of a 22-bp fragment prevents the repression of ompF expression conferred by the dominant negative mutation, envZ473. On the basis of the location of this binding site, the presence of bent DNA in the ompF regulatory region (T. Mizuno, Gene 54:57-64, 1987), and the fact that mutations altering integration host factor result in constitutive ompF expression (P. Tsui, V. Helu, and M. Freundlich, J. Bacteriol. 170:4950-4953, 1988), we propose that the negative regulation of ompF involves a DNA loop structure.

The α subunit of RNA polymerase specifically inhibits expression of the porin genes ompF and ompC in vivo and in vitro in Escherichia coli

The α subunit of RNA polymerase specifically inhibits expression of the porin genes ompF and ompC in vivo and in vitro in Escherichia coli

Identification of a phosphorylation site and functional analysis of conserved aspartic acid residues of OmpR, a transcriptional activator for ompF and ompC in Escherichia coli

In Escherichia coli the OmpR and EnvZ proteins regulate the expression of the outer membrane porin proteins OmpC and OmpF. EnvZ and OmpR belong to a family of sensor/effector protein pairs that control adaptation to a variety of environmental conditions. EnvZ acts as the sensor protein that phosphorylates OmpR, which in turn regulates porin gene expression. The level of phosphorylated OmpR appears to be a determining factor for ompC and ompF regulation. Phosphorylation of OmpR is considered to occur at one or more aspartic acid residues (Asp-11, Asp-12 and/or Asp-55) that are highly conserved among the effector proteins. In this report we biochemically characterized the aspartic acid residue(s) in OmpR that were phosphorylated by EnvZ. Reduction of aspartyl phosphate residues in the amino-terminal domain of OmpR with [3H]-NaBH4 indicated that Asp-55 was a primary site of modification. We further studied the role of the highly conserved aspartate residues by creating OmpR mutants having aspartate to alanine substitutions at positions 11 (D11A), 12 (D12A) and 55 (D55A). Studies of ompF and ompC expression as well as in vivo and in vitro phosphorylation experiments also demonstrated that while Asp-55 is the primary phosphate acceptor site in OmpR, Asp-11 may also serve as a phosphorylation site, particularly in the absence of Asp-55.

Mutations in the alpha subunit of RNA polymerase that affect the regulation of porin gene transcription in Escherichia coli K-12

The two-component regulatory system consisting of OmpR and EnvZ controls the differential expression of major outer membrane porin proteins OmpF and OmpC of Escherichia coli K-12. We have isolated and characterized two mutations in rpoA, the gene encoding the alpha subunit of RNA polymerase, that decrease the expression of OmpF. These mutations have a number of properties that distinguish them from previously isolated rpoA mutations that affect porin expression. The rpoA203 mutation decreases the expression of porin genes ompF and ompC and also decreases the expression of the malE and phoA genes. In contrast, rpoA207 decreases the expression of ompF but does not affect ompC, malE, or phoA transcription. Our results suggest that mutations at various positions in the alpha subunit may affect the OmpR-dependent transcription of ompF and ompC differently and may be useful for analyzing the mechanism underlying their differential expression in response to medium osmolarity.

Overexpression of the MarA positive regulator is sufficient to confer multiple antibiotic resistance in Escherichia coli.

A genetic approach was undertaken to identify normal bacterial genes whose products function to limit the effective concentration of antibiotics. In this approach, a multicopy plasmid library containing cloned Escherichia coli chromosomal sequences was screened for transformants that showed increased resistance to a number of unrelated antibiotics. Three such plasmids were identified, and all contained sequences originating from the mar locus. DNA sequence analysis of the minimal complementation unit revealed that the resistance phenotype was associated with the presence of the marA gene on the plasmids. The putative marA gene product is predicted to contain a helix-turn-helix DNA binding domain that is very similar to analogous domains found in three other E. coli proteins. One such similarity was to the SoxS gene product, the elevated expression of which has previously been associated with the multiple antibiotic resistance (Mar) phenotype. Constitutive expression of marA conferred antibiotic resistance even in cells carrying a deletion of the chromosomal mar locus. We have also found that transformants bearing marA plasmids show a significant reduction in ompF translation but not transcription, similar to previously described mar mutants. However, this reduction in ompF expression plays only a minor role in the resistance mechanism, suggesting that functions encoded by genes unlinked to mar must be affected by marA. These results suggest that activation of marA is the ultimate event that occurs at the mar locus during the process that results in multiple antibiotic resistance.

Naturally-occurring, osmo-remedial variants of Escherichia coli.

Two clones of Escherichia coli O27:K1:H31 and O2:H7, isolated from patients with urinary tract infection or bacteraemia, failed to grow in a synthetic minimal medium (MM) of low osmolality. They were considered to be osmo-remedial because they grew well when sufficient amounts of NaCl, mannitol or sucrose were added to raise the osmolality of the medium to > 300 mOsm/kg. The defect could also be corrected by nicotinamide or its precursors quinolinic and aspartic acids. Each clone had a unique DNA restriction enzyme profile, fimbriae and antibiotic susceptibility patterns. The osmo-remedial variants were unstable and underwent phenotypic modulation to form mixtures with osmo-tolerant forms when grown in MM. They tended to form satellites of small colonies around large colonies of osmo-tolerant cells on MM agar plates. The penicillin method of Davis was used to separate the two forms. Nicotinamide induced the expression of ompF when the osmo-remedial strains were grown under conditions of low osmolality. It is possible that the variants are defective in the synthesis of membrane-derived oligosaccharides or outer-membrane proteins, but this has yet to be determined.

Mutations in a central highly conserved non-DNA-binding region of OmpR, an Escherichia coli transcriptional activator, influence its DNA-binding ability.

OmpR is a transcriptional activator for the expression of outer membrane porin genes ompF and ompC in Escherichia coli. Its C-terminal half has been identified as the DNA-binding domain (K. Tsung, R. Brissette, and M. Inouye, J. Biol. Chem. 264:10104-10109, 1989). Recent studies have indicated that the N-terminal non-DNA-binding domain of OmpR is involved in modulating OmpR function through interaction with the EnvZ protein, a kinase and phosphatase for OmpR. We isolated and characterized two mutations, G94D and E111K, in the N-terminal domain of OmpR and one mutation, R182C, in the DNA-binding domain of OmpR. All three mutations abolished the ability of OmpR to bind to the ompF and ompC promoters in vivo, thus giving an OmpF- OmpC- phenotype. The decreased DNA-binding ability of the mutant OmpRs was not due to diminished phosphorylation of their N termini, since all the mutant OmpRs were found to be normally phosphorylated by EnvZ in vitro. The mutant OmpRs produced from multicopy plasmids were also found to inhibit completely the production of OmpF and OmpC in wild-type cells, and the complete inhibition depended on the function of EnvZ which was produced in cis or in trans from plasmids. The relationship of the possible alterations in OmpR by the mutations with the observed diminished binding ability is discussed.

Molecular analysis of the signaling pathway between EnvZ and OmpR in Escherichia coli.

OmpR is a DNA-binding protein that regulates transcription of ompF and ompC. The activity of OmpR is controlled by the inner membrane osmosensor, EnvZ. In order to study the signaling process between EnvZ and OmpR, we analyzed two different envZ strains: the envZ473 strain, in which OmpC is constitutively produced and OmpF is fully repressed, and the envZ3 strain, in which the production of OmpC is greatly reduced and OmpF is not fully repressed by high-osmolarity growth conditions. Using direct sequencing of DNA derived from the polymerase chain reaction amplification method, we identified the mutation in the envZ473 strain as a Val-241-to-Gly substitution and the mutation in the envZ3 as an Ala-219-to-Val substitution. The relative DNA-binding affinity of OmpR derived from the envZ473 strain was dramatically increased for the upstream sequence of both ompF and ompC. In contrast, OmpR derived from the envZ3 strain was not converted to the high-affinity form. The intracellular levels of OmpR-phosphate, as analyzed by the in vivo phosphorylation approach, significantly increased in the envZ473 strain, while in the envZ3 strain the levels were considerably reduced, relative to those found in the parent strain. The intracellular level of OmpR protein in the envZ473 strain was also found to be markedly elevated relative to that of the parent strain. These results are discussed in relation to the role of phosphorylation and relative DNA-binding affinity of OmpR in the expression of ompF and ompC.

A novel sensor-regulator protein that belongs to the homologous family of signal-transduction proteins involved in adaptive responses in Escherichia coli.

Expression of the Escherichia coli outer membrane porins, OmpC and OmpF, is regulated in response to changes in the medium osmolarity through the functions of the regulatory factors, EnvZ and OmpR. A 3.0 kilobase pair DNA fragment cloned from E. coli is able phenotypically to suppress the defect in ompC and ompF expression caused by an envZ deletion mutation, provided that a certain gene located in this fragment is expressed on a high copy-number plasmid. Nucleotide sequencing revealed that the putative gene encodes a protein of 102,452 Da. The deduced amino acid sequence of the protein shows a high degree of homology to those of both EnvZ and OmpR, i.e. it contains both a 'sensory kinase domain' and a 'response regulator domain' in its primary amino acid sequence. The protein identified in this study is probably a novel member of the homologous family of proteins involved in bacterial adaptive responses. Hence, the gene encoding this novel sensor-regulator protein was designated as barA (bacterial adaptive responses) and mapped at 60 min on the E. coli genetic map. The BarA protein in isolated membranes was demonstrated in vitro to undergo phosphorylation in the presence of ATP.

Membrane-derived oligosaccharides affect porin osmoregulation only in media of low ionic strength.

Gram-negative bacteria grown under conditions of low osmolarity accumulate significant amounts of periplasmic glucans, membrane-derived oligosaccharides (MDO) in Escherichia coli and cyclic glucans in members of the family Rhizobiaceae. It was reported previously (W. Fiedlder and H. Rotering, J. Biol. Chem. 263:14684-14689, 1988) that mdoA mutants unable to synthesize MDO show a number of altered phenotypes, among them a decreased expression of OmpF and an increased expression of OmpC, when grown in a Bacto Peptone medium of low osmolarity and low ionic strength. Although we confirm the findings of Fiedler and Rotering, we find that the regulation of OmpF and OmpC expression in mdoA mutants is normal in cells grown on other low-osmolarity media, eliminating the possibility that MDO itself might control porin expression. Our data suggest that a certain minimal ionic strength in the periplasm is needed for normal porin regulation. In media containing very low levels of salt, this may be contributed by anionic MDO.

In vitro interactions of integration host factor with the ompF promoter-regulatory region of Escherichia coli

Previous work has shown that integration host factor (IHF) mutants have increased expression and altered osmoregulation of OmpF, a major Escherichia coli outer membrane protein. By in vitro analysis the possibility was investigated that IHF interacts directly with the ompF promoter region. Gel retardation assays and DNase I protection experiments showed that IHF binds to two sites in the ompF promoter region centered at positions -180 and -60 relative to the start of transcription. Gel electrophoresis studies with circularly permuted ompF promoter fragments indicated that IHF binding strongly increased a small intrinsic bend in the ompF promoter region. The addition of IHF to a purified in vitro transcription system strongly and specifically inhibited ompF transcription. This inhibition was reversed by increasing the concentration of OmpR, a positive activator required for ompF expression, suggesting that IHF may inhibit ompF transcription by altering how OmpR interacts with the ompF promoter.

Procaine, a local anesthetic, signals through the EnvZ receptor to change the DNA binding affinity of the transcriptional activator protein OmpR

Local anesthetics are known to reduce the level of OmpF and increase the synthesis of OmpC in the outer membrane of Escherichia coli K-12. It has been shown that the anesthetics procaine and phenethyl alcohol (PEA) act at the transcriptional level for ompF and ompC and that in the case of procaine, its action is dependent on EnvZ, the membrane-bound signal transducer required for ompF and ompC expression. In an effort to further understand how anesthetics regulate ompF and ompC expression, we have analyzed the DNA binding properties of OmpR (the transcriptional activator protein for ompF and ompC genes) from cells treated with procaine or PEA. Treatment of a wild-type cell with either anesthetic converted OmpR from a low-affinity DNA binding form to a high-affinity DNA binding form. The change in DNA binding affinity was correlated with alterations in outer membrane porin profiles and could occur in the absence of protein synthesis. A strain lacking EnvZ was unable to respond to procaine to produce either the shift in the OmpR DNA binding property or cause any change in the outer membrane porin profile. PEA treatment was also dependent on EnvZ for the alteration in the OmpR DNA binding property, but it could induce ompC expression in the absence of EnvZ. Further studies suggest that the amino-terminal region of EnvZ is responsible for the procaine signalling. Our results indicate that procaine and PEA regulate ompF and ompC expression by modifying the DNA binding properties of OmpR through EnvZ signal transduction.