ompF Promoter Research Articles

Salmonella T3SS effector SseK1 arginine-glycosylates the two-component response regulator OmpR to alter bile salt resistance

Type III secretion system (T3SS) effector proteins are primarily recognized for binding host proteins to subvert host immune response during infection. Besides their known host target proteins, several T3SS effectors also interact with endogenous bacterial proteins. Here we demonstrate that the Salmonella T3SS effector glycosyltransferase SseK1 glycosylates the bacterial two-component response regulator OmpR on two arginine residues, R15 and R122. Arg-glycosylation of OmpR results in reduced expression of ompF, a major outer membrane porin gene. Glycosylated OmpR has reduced affinity to the ompF promoter region, as compared to the unglycosylated form of OmpR. Additionally, the Salmonella ΔsseK1 mutant strain had higher bile salt resistance and increased capacity to form biofilms, as compared to WT Salmonella, thus linking OmpR glycosylation to several important aspects of bacterial physiology.

Structural basis for promoter DNA recognition by the response regulator OmpR

OmpR, a response regulator of the EnvZ/OmpR two-component system (TCS), controls the reciprocal regulation of two porin proteins, OmpF and OmpC, in bacteria. During signal transduction, OmpR (OmpR-FL) undergoes phosphorylation at its conserved Asp residue in the N-terminal receiver domain (OmpRn) and recognizes the promoter DNA from its C-terminal DNA-binding domain (OmpRc) to elicit an adaptive response. Apart from that, OmpR regulates many genes in Escherichia coli and is important for virulence in several pathogens. However, the molecular mechanism of the regulation and the structural basis of OmpR–DNA binding is still not fully clear. In this study, we presented the crystal structure of OmpRc in complex with the F1 region of the ompF promoter DNA from E. coli. Our structural analysis suggested that OmpRc binds to its cognate DNA as a homodimer, only in a head-to-tail orientation. Also, the OmpRc apo-form showed a unique domain-swapped crystal structure under different crystallization conditions. Biophysical experimental data, such as NMR, fluorescent polarization and thermal stability, showed that inactive OmpR-FL (unphosphorylated) could bind to promoter DNA with a weaker binding affinity as compared with active OmpR-FL (phosphorylated) or OmpRc, and also confirmed that phosphorylation may only enhance DNA binding. Furthermore, the dimerization interfaces in the OmpRc–DNA complex structure identified in this study provide an opportunity to understand the regulatory role of OmpR and explore the potential for this “druggable” target.

Construction of a bacterial surface display system based on outer membrane protein F

BackgroundBacterial surface display systems were developed to surface expose heterologous proteins or peptides for different applications, such as peptide libraries screening and live bacterial vaccine design. Various outer membrane proteins, such as outer membrane protein A (OmpA), OmpC and outer membrane pore protein E precursor (PhoE), have been used as carriers for surface display, fused to the proteins or peptides of interest in Gram-negative bacteria. Here, we investigated the utility of constitutively expressed OmpF for the display of foreign immune epitopes on the Escherichia coli cell surface and then compared it with plasmid-induced expression of OmpF and OmpC.ResultsEnhanced expression of OmpF was linked to a mutation in the OmpF promoter sequence. This mutation rendered OmpF an ideal carrier protein for the enriched display of a target of interest on the bacterial surface. To this end, we grafted two peptides, harboring important epitopes of the hepatitis B virus (HBV) S antigen and human papilloma virus (HPV) L2 protein, onto OmpF of E. coli by genome editing. The resultant fused OmpF proteins were constitutively expressed in the edited E. coli and purified by membrane component extraction. The epitope that displayed on the bacterial surface was verified by SDS-PAGE, western blotting, flow cytometry, and immunoelectron microscopy of the intact bacteria. We further compared this constitutive expression with plasmid-induced expression of OmpF and OmpC in bacterial cells using the same methods for verification. We found that plasmid-induced expression is much less efficient than constitutive expression of OmpF from the bacterial genome.ConclusionsEnhanced expression of OmpF in a plasmid-independent manner provides an amenable way to display epitopes on the bacterial surface and sheds light on ways to engineer bacteria for biotechnological applications.

A Novel Regulatory Cascade Involving BluR, YcgZ, and Lon Controls the Expression of Escherichia coli OmpF Porin

In Escherichia coli, OmpF is an important outer membrane protein, which serves as a passive diffusion pore for small compounds including nutrients, antibiotics, and toxic compounds. OmpF expression responds to environmental changes such as temperature, osmolarity, nutrients availability, and toxic compounds via complex regulatory pathways involving transcriptional and post-transcriptional regulation. Our study identified a new regulatory cascade that controls the expression of OmpF porin. This pathway involves BluR, a transcriptional regulator repressing the expression of the ycgZ-ymgABC operon. We showed that BluR was responsible for the temperature-dependent regulation of the ycgZ-ymgABC operon. Furthermore, our results showed that independent expression of YcgZ led to a decreased activity of the ompF promoter, while YmgA, YmgB, and YmgC expression had no effect. We also determined that YcgZ accumulates in the absence of the Lon protease. Thus, mutation in bluR leads to de-repression of ycgZ-ymgABC transcription. With a second mutation in lon, YcgZ protein accumulates to reach levels that do not allow increased expression of OmpF under growth conditions that usually would, i.e., low temperature. With BluR responding to blue-light and temperature, this study sheds a new light on novel signals able to regulate OmpF porin.

The Salmonella enterica serovar Typhi ltrR‐ompR‐ompC‐ompF genes are involved in resistance to the bile salt sodium deoxycholate and in bacterial transformation

A characterization of the LtrR regulator, an S. Typhi protein belonging to the LysR family is presented. Proteomics, outer membrane protein profiles and transcriptional analyses demonstrated that LtrR is required for the synthesis of OmpR, OmpC and OmpF. DNA-protein interaction analysis showed that LtrR binds to the regulatory region of ompR and then OmpR interacts with the ompC and ompF promoters inducing porin synthesis. LtrR-dependent and independent ompR promoters were identified, and both promoters are involved in the synthesis of OmpR for OmpC and OmpF production. To define the functional role of the ltrR-ompR-ompC-ompF genetic network, mutants in each gene were obtained. We found that ltrR, ompR, ompC and ompF were involved in the control of bacterial transformation, while the two regulators and ompC are necessary for the optimal growth of S. Typhi in the presence of one of the major bile salts found in the gut, sodium deoxycholate. The data presented establish the pivotal role of LtrR in the regulatory network of porin synthesis and reveal new genetic strategies of survival and cellular adaptation to the environment used by Salmonella.

A phosphorylation site mutant of OmpR reveals different binding conformations at ompF and ompC

In Escherichia coli, the two-component regulatory system that controls the expression of outer membrane porins in response to environmental osmolarity consists of the sensor kinase EnvZ and the response regulator OmpR. Phosphorylated OmpR activates expression of the OmpF porin at low osmolarity, and at high osmolarity represses ompF transcription and activates expression of OmpC. We have characterized a substitution in the amino-terminal phosphorylation domain of OmpR, T83I, its phenotype is OmpF− OmpC−. The mutant protein is not phosphorylated by small molecule phosphodonors such as acetyl phosphate and phosphoramidate, but it is phosphorylated by the cognate kinase EnvZ. Interestingly, the active site T83I substitution alters the DNA binding properties of the carboxyl-terminal effector domain. DNase I protection assays indicate that DNA binding by the mutant protein is similar to wild-type OmpR at the ompF promoter, but at ompC, the pattern of protection is different from OmpR. Our results indicate that all three of the OmpR binding sites at the ompC promoter must be filled in order to activate gene expression. Furthermore, it appears that OmpR-phosphate must adopt different conformations when bound at ompF and ompC. A model is presented to account for the reciprocal regulation of OmpF and OmpC porin expression.

A role for the Escherichia coli H-NS-like protein StpA in OmpF porin expression through modulation of micF RNA stability.

When a wild-type strain of Escherichia coli and its stpA, hns and stpA hns mutant derivatives were compared by two-dimensional protein gel electrophoresis, the levels of expression of several proteins were found to vary. One of these was identified as the outer membrane porin protein, OmpF. In the stpA hns double mutant, the level of OmpF was downregulated dramatically, whereas in hns or stpA single mutants, it was affected only slightly. Transcription from the ompF promoter was reduced by 64% in the double mutant; however, the level of ompF mRNA was reduced by 96%. This post-transcriptional expression was found to result from a strong reduction in the half-life of ompF message in the double mutant. The micF antisense RNA was shown to be involved in OmpF regulation by StpA using a strain deleted for micF. Moreover, micF antisense RNA accumulated considerably in an stpA hns background. Transcriptional data from a micF-lacZ fusion and measurements of micF RNA half-life confirmed that this was caused by transcriptional derepression of micF as a result of the hns lesion and increased micF RNA stability due to the absence of StpA (a known RNA chaperone). These data suggest a novel facet to the regulation of OmpF expression, namely destabilization of micF RNA by StpA.

Nucleoside-diphosphate Kinase-mediated Signal Transduction via Histidyl-Aspartyl Phosphorelay Systems in Escherichia coli

Nucleoside-diphosphate kinase (NDP kinase), a key enzyme in nucleotide metabolism, is also known to be involved in growth and developmental control and tumor metastasis suppression. Interestingly, we find that coexpression of NDP kinase with Taz1, a Tar/EnvZ chimera, in the absence of its native signal, can activate a porin gene ompC-lacZ expression in Escherichia coli. Further studies show that NDP kinase can act as a protein kinase to phosphorylate histidine protein kinases such as EnvZ and CheA which are members of the His-Asp phosphorelay signal transduction systems in E. coli. Instead of ATP, the exclusive phosphodonor for histidine kinases, GTP can be utilized in vitro in the presence of NDP kinase to phosphorylate EnvZ and CheA, which then transfer the phosphoryl group to OmpR and CheY, the respective response regulators. The direct involvement of GTP for the phosphorylation of EnvZ through NDP kinase was further demonstrated by the use of a mutant EnvZ, which lost ability to be autophosphorylated with ATP. Phospho-OmpR thus formed can bind specifically to an ompF promoter sequence. These results suggest that NDP kinase may play a physiological role in signal transduction.

Periplasmic expression of part of the major rotavirus capsid protein VP7 containing all the three antigenic regions in Escherichia coli

Part of the porcine rotavirus outer capsid protein VP7 containing all the three antigenic regions was expressed as a chimeric protein with bacterial alkaline phosphatase (AP) in E. coli. The construct contains an ompF promoter, the DNA encoding the signal sequence and the first 12 amino acids of mature OmpF, part of vp7 and the DNA encoding mature AP. The chimeric protein is stable, retains the biological property of AP and ability to react with polyclonal antiserum against the virus, and can be exported through the bacterial inner membrane into the periplasm.

Gene activation by the Escherichia coli positive regulator OmpR: a mutational study of the DNA-binding domain of OmpR.

The Escherichia coli DNA-binding protein, OmpR, is one of the best characterized of the bacterial positive regulators that enhance the transcriptional ability of RNA polymerase. OmpR, consisting of 239 amino acids, binds to specific sequences located upstream of the cognate ompC and ompF promoters. The C-terminal half of OmpR, consisting of about 120 amino acids, exhibits an inherent DNA-binding ability. To address the issue of DNA binding by OmpR, we selected a set of OmpR mutants, each of which has a single amino acid substitution in the C-terminal half of OmpR. In particular, we characterized a number of OmpR mutants which are defective in DNA binding and thereby result in an OmpF- OmpC phenotype. Among them, a putative positive control OmpR mutant was also obtained, which appears to be defective in phosphorylation-dependent transcriptional activation, but not in DNA binding. These results are discussed with general emphasis on DNA recognition by the E. coli family of OmpR-like regulatory proteins.

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.

Activation of the osmoregulated ompF and ompC genes by the OmpR protein in Escherichia coli: a study involving chimeric promoters.

The expression of each of the Escherichia coli ompF and ompC genes is activated by the common positive regulator, OmpR, in response to the medium osmolarity. The promoters for these genes consist of canonical -35 and -10 regions, and upstream OmpR-binding sites. In this study, we constructed a functional ompF-ompC chimeric promoter consisting of the OmpR-binding site of ompF, and the -35 and -10 regions of ompC, which was fused to the lacZ gene on a low-copy-number plasmid. It is known that the ompF and ompC genes are expressed in a different manner in cells with mutations in the ompR gene. In this respect, it was found that the ompF-ompC chimeric promoter behaves just like ompF promoter with respect to an ompR mutation (ompR472). It was revealed that, in this chimeric promoter, the OmpR-binding site must be located stereospecifically with respect to the -35 and -10 regions for OmpR-dependent transcription of the ompF-ompC chimeric promoter to occur. These results are discussed in relation to the structures and functions of the ompF and ompC promoters, the occurrence of a DNA curvature in the promoter regions being implied, which may be an important parameter for the transcription activation by the OmpR protein.

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.

Cis-acting ompF mutations that result in OmpR-dependent constitutive expression.

OmpR and EnvZ differentially control the transcription of the major outer membrane porin genes, ompF and ompC, in Escherichia coli in response to the osmolarity of the medium. We have previously provided evidence that OmpR works both positively and negatively at the ompF promoter to give the characteristic switch from OmpF to OmpC production with increasing osmolarity. Here, we describe the isolation of cis-acting ompF mutations that affect negative regulation by OmpR by affecting the three-dimensional structure of the promoter region as measured by agarose gel mobility. These results further clarify the mechanism by which OmpR negatively regulates ompF expression, suggesting a model in which OmpR forms a repressive loop in the ompF promoter region.

Enhancement of RNA polymerase binding to promoters by a transcriptional activator, OmpR, in Escherichia coli: its positive and negative effects on transcription.

The OmpR binding sequence (OBS) in the upstream region of the ompF promoter of Escherichia coli was fused to 27 synthetic promoters. Transcription from a number of weak promoters, regardless of their sequences, was dramatically activated in the presence of OmpR, a transcriptional activator. In vivo DNA footprinting revealed that OmpR enhanced the binding of RNA polymerase to the promoters. This enhancement was essential for transcription of weak promoters, while OmpR binding to the OBS fused to a strong promoter was inhibitory for transcription. These results indicate that OmpR stabilizes the formation of an RNA polymerase-promoter complex, possibly a closed promoter complex, and that a transcription activator can serve not only as a positive but also as a negative regulator for gene expression.

Phosphorylation and dephosphorylation of a bacterial transcriptional activator by a transmembrane receptor.

Signal transduction in the bacterial Omp, Che, and Ntr systems involves the phosphorylation and dephosphorylation of response regulators (OmpR, CheY and CheB, NRI) that share a homologous domain. We show that in the Omp system, the transmembrane sensor EnvZ, catalyzes both the phosphorylation of OmpR and the dephosphorylation of OmpR-P. The phosphorylation reaction proceeds by a mechanism shared with the Ntr and Che kinases, NRII, and CheA. EnvZ can phosphorylate NRI and can stimulate transcription from the glnAp2 promoter, and similarly, CheA can phosphorylate OmpR and can stimulate transcription from the ompF promoter. OmpR-P formed by either CheA or EnvZ is much more stable than CheY-P and NRI-P, but is rapidly hydrolyzed to OmpR and Pi by EnvZ in the presence of ATP, ADP, or nonhydrolyzable analogs of ATP. Because EnvZ is normally a transmembrane receptor with a periplasmic sensory domain, our results suggest that the role of EnvZ may be to control the intracellular concentration of OmpR-P in response to environmental signals.

Characterization of OmpR binding sequences in the upstream region of the ompF promoter essential for transcriptional activation

In Escherichia coli the expression of the outer membrane porin gene ompF requires the transcriptional activator protein OmpR. Previous DNase I footprinting experiments with purified OmpR localized the OmpR binding site from positions -105 to -60 (relative to the transcriptional start site) in the ompF promoter, and three tandem 10-base pair sequences elements (Fa, Fb, and Fc) within this region were proposed to be important for OmpR recognition. In order to elucidate the roles of the F boxes for transcriptional activation of ompF, various F box deletions and point mutations were constructed and analyzed for their effects on ompF-lacZ expression and OmpR binding. Removal of 102 nucleotides, which included a portion of the OmpR binding region (the Fa box), evidenced the largest decrease in transcriptional activation and significantly reduced OmpR binding. Additional deletion of four more base pairs in this target site (representing half of the Fb box) further reduced ompF expression. OmpR interactions with DNA sequences representing the OmpR binding region were analyzed by DNA mobility shift experiments. A 43-base pair ompF oligonucleotide containing the Fa, Fb, and Fc regions was sufficient for OmpR-dependent DNA binding using either purified OmpR or cell supernatants. The central C residue in each F box was changed to a T and unique patterns of protein-DNA complexes were observed that were different from that of the wild type binding site. The most dramatic effect on OmpR binding was observed when the C to T transversion occurred in the Fb box, and this mutation also reduced the level of ompF-lacZ expression. Our results indicate that the F boxes play important roles in the activation of ompF expression, and we suggest that OmpR may interact cooperatively with these boxes.

Location of phosphorylation site and DNA-binding site of a positive regulator, OmpR, involved in activation of the osmoregulatory genes of Escherichia coli

The OmpR protein of Escherichia coli is a positive regulator involved in activation of the ompF and ompC genes which encode the major outer membrane proteins OmpF and OmpC, respectively. By employing recombinant DNA techniques, we isolated the N- and C-terminal halves of the OmpR molecule. From the results of biochemical analyses of these fragments, it was concluded that the N-terminal portion contains a site involved in phosphorylation by an OmpR-specific protein kinase EnvZ, whereas the C-terminal part possesses a DNA-binding site for the ompC and ompF promoters.

A bacterial environmental sensor that functions as a protein kinase and stimulates transcriptional activation.

Transcription of the genes that encode the major outer membrane porin proteins OmpF and OmpC of Escherichia coli is regulated in response to changes in medium osmolarity by EnvZ and OmpR. EnvZ functions to sense environmental conditions and to relay this information to the DNA-binding protein OmpR. We have used a truncated EnvZ protein (EnvZ115), which is defective in sensory function but able to communicate with OmpR, to study the biochemical interactions between these two proteins and their effects on transcription from the ompF promoter. We show that purified EnvZ115 can phosphorylate OmpR in the presence of ATP. In addition, EnvZ115 stimulates the ability of OmpR to activate ompF transcription in vitro. Using antibodies specific for EnvZ, we have purified the wild-type protein and have shown that it is also an OmpR kinase. These results provide a prokaryotic example of a transmembrane sensory protein that functions as a protein kinase and suggest a mechanism by which EnvZ communicates with OmpR in signal transduction.

High osmolarity is a signal for enhanced algD transcription in mucoid and nonmucoid Pseudomonas aeruginosa strains

Chronic lung infection with mucoid, alginate-producing strains of Pseudomonas aeruginosa is a major cause of mortality in cystic fibrosis (CF) patients. Transcriptional activation of the P. aeruginosa algD gene, which encodes GDPmannose dehydrogenase, is essential for alginate synthesis. Activation of algD is dependent on the product of the algR gene. Sequence homology between the P. aeruginosa algR gene and the Escherichia coli ompR gene, which regulates the cellular response to changes in osmolarity of the growth medium, together with the abnormally high levels of Na+ and Cl- in respiratory tract fluid in CF patients suggested that high osmolarity in the lung of the CF patient might be a signal contributing to the induction of alginate synthesis (mucoidy) in infecting P. aeruginosa. In both mucoid and nonmucoid P. aeruginosa strains (containing a functional algR gene), transcriptional activation of algD increased as the osmolarity of the culture medium increased. The increased activation of algD at high osmolarity was not in itself sufficient to induce alginate synthesis in nonmucoid strains, however, suggesting that other environmental factors are involved in full activation of the alginate genes. The targets of AlgR and OmpR, the algD promoter and the ompC and ompF promoters, respectively, were found to have appreciable sequence homology in the -60 to -110 regions. In E. coli, OmpR was capable of activating the algD promoter nearly as well as AlgR, but in both cases, activation occurred only under conditions of high osmolarity.