Expression Of OmpC Research Articles

Hierarchical and co-operative binding of OmpR to a fusion construct containing the ompC and ompF upstream regulatory sequences of Escherichia coli.

OmpR is a transcription factor that regulates the expression of the porin genes ompF and ompC in Escherichia coli. The phosphorylation state of OmpR, directed by the osmosensor EnvZ, determines its ability to bind to the upstream regulatory regions of these genes, a total of 14 phospho-OmpR binding sites. While it has been possible to study the stoichiometry and hierarchy of the OmpR-DNA interaction in the upstream regions of ompF and ompC, their disunited location on the bacterial chromosome has made it difficult to compare the individual binding affinities of respective sites. Using 1,10-phenanthroline-Cu+ footprinting on a fused construct containing both the ompF and ompC upstream regulatory sequences, and gel shift experiments on oligomers corresponding to individual sites, we have established a comparative hierarchy for OmpR binding, as F1, C1 > F2, F3 > C2 > C3. In addition, the binding patterns reveal an apparent co-operative relationship between OmpR molecules bound at several upstream motifs. Densitometric analyses of the footprinted regions provide support for these observations. Mutational analysis of this construct reveals that the alteration of a conserved cytidine in the F1 motif (-86) causes a loss of OmpR affinity and disrupts hierarchical OmpR-binding in the entire ompF region. The present results provide a unique view of the OmpR interaction with the two respective promoters, ompF and ompC, and an insight into the question of how the expression of ompF and ompC are reciprocally regulated by medium osmolarity.

Enhancement of Cell Death Due to Decrease in Mg2+Uptake by OmpC (Cation-Selective Porin) Deficiency in Ribosome Modulation Factor-Deficient Mutant

Ribosome modulation factor (RMF) is involved in stabilization of ribosomes during the transition from exponential growth to the stationary growth phase inEscherichia coli.A deficiency of RMF is known to reduce cell viability. Overaccumulation of spermidine also leads to a decrease in cell viability and to a decrease in the synthesis of RMF and of the cation-selective porin OmpC. Thus, a decrease in RMF levels may be involved in the decreased cell viability caused by excess spermidine. Because spermidine also influences the expression of OmpC, we examined whether OmpC deficiency enhances the cell death caused by RMF deficiency. TheompCmutant by itself did not affect protein synthesis or cell viability, but the doublermf ompCmutant produced a much larger decrease in protein synthesis and cell viability than did the singlermfmutant. There was also a decrease in the amount of ribosomes and in the Mg2+content in the doublermf ompCmutant, and cell viability could be partially restored by the addition of Mg2+to the growth medium. RMF deficiency was found to inhibit the synthesis of another cation-selective porin OmpF. Thus, the doublermf ompCmutant is deficient in both OmpC and OmpF, which probably accounts for the pronounced decrease in Mg2+uptake in this mutant. The results indicate that both RMF and Mg2+, acting through stabilization of ribosomes, are important for cell viability at the stationary growth phase.

Regulation of porin-mediated outer membrane permeability by nutrient limitation in Escherichia coli.

OmpF and OmpC porins were differentially regulated by nutrient limitation and growth rate in glucose- or nitrogen-limited chemostat cultures of Escherichia coli. Transcriptional and translational ompF fusions showed a sharp peak of expression under glucose limitation at D = 0.3 h-1, with lower amounts at lower and higher growth rates. The peak of OmpR-dependent transcriptional stimulation of ompF under glucose limitation in minimal salts media was about 20-fold above nutrient excess levels and 3-fold higher than that achieved with low osmolarity. Analysis of outer membrane protein levels and results of growth competition experiments with porin mutants were consistent with the enhanced role of OmpF under glucose limitation, but not N limitation. In contrast, OmpC was the major porin under N limitation but was increasingly expressed under glucose limitation at very low growth rates approaching starvation, when OmpF was downregulated. In summary, outer membrane permeability under N-limited, sugar-rich conditions is largely based on OmpC, whereas porin activity is a complex, highly sensitive function of OmpF, OmpC, and LamB glycoporin expression under different levels of glucose limitation. Indeed, the OmpF level was more responsive to nutrient limitation than to medium osmolarity and suggested a significant additional layer of control over the porin-regulatory network.

E.coli PhoE porin has an opposite voltage-dependence to the homologous OmpF.

We used patch clamp analysis to compare the electrophysiological behavior of two related porins from Escherichia coli, the anion-specific PhoE and the cation-selective OmpF. Outer membrane fractions were obtained from strains expressing just one of these porin types, and the channels were reconstituted into liposomes without prior purification. We show that the orientation of the reconstituted channels is not random and is the same for both PhoE and OmpF. Like cation-selective porins, PhoE shows fast and slow gating to closed levels of various amplitudes, testifying that the channels visit multiple functional states and behave as cooperative entities. The voltage-dependence of PhoE closure is asymmetric, but strikingly, occurs at voltages of inverse polarity from those promoting closures of OmpC and OmpF. Both slow kinetics and inverse voltage-dependence are removed when 70 amino acids from the N-terminal of OmpF are introduced into the homologous region of PhoE. This novel observation regarding the voltage-dependence of the two channel types, along with published results on PhoE and OmpF mutants, allows us to propose a molecular mechanism for voltage sensing and sensor charge movements in bacterial porins. It also offers new cues on the possible physiological relevance in bacteria of this common form of channel modulation.

Differential expression of the OmpF and OmpC porin proteins in Escherichia coli K-12 depends upon the level of active OmpR.

We have generated a mutant form of the OmpR regulatory protein, OmpRD55E, that is active independent of the EnvZ kinase. Notably, the pattern of OmpF and OmpC expression can be altered simply by changing the level of this mutant protein in the cell. This result supports a key prediction of the current model of porin regulation, which states that the differential regulation of OmpF and OmpC is a direct consequence of the cellular level of the active form of OmpR.

Mutational analysis of the linker region of EnvZ, an osmosensor in Escherichia coli.

EnvZ, a transmembrane signal transducer, is composed of a periplasmic sensor domain, transmembrane domains, and a cytoplasmic signaling domain. Between the second transmembrane domain and the cytoplasmic signaling domain there is a linker domain consisting of approximately 50 residues. In this study, we investigated the functional role of the EnvZ linker domain with respect to signal transduction. Amino acid sequence alignment of linker regions among various bacterial signal transducer proteins does not show a high sequence identity but suggests a common helix 1-loop-helix 2 structure. Among several mutations introduced in the EnvZ linker region, it was found that hydrophobic-to-charged amino acid substitutions in helix 1 and helix 2 and deletions in helix 1, loop, and helix 2 (delta14, delta8, and delta7) resulted in constitutive OmpC expression. In the linker mutant EnvZ x delta7, both kinase and phosphatase activities were significantly reduced but the ratio of kinase to phosphatase activity increased, consistent with the constitutive OmpC expression. In contrast, the purified cytoplasmic fragment of EnvZ x delta7 possessed both kinase and phosphatase activities at levels similar to those of the cytoplasmic fragment of wild-type EnvZ. In addition, the linker mutations had no direct effect on EnvZ C-terminal dimerization. These results together with previous data suggest that the linker region is not directly involved in EnvZ enzymatic activities and that it may have a crucial role in propagating a conformational change to ensure correct positioning of two EnvZ molecules within a dimer during the transmembrane signaling.

Genetically probing the regions of ribose-binding protein involved in permease interaction.

The ribose-binding protein (RBP) of Escherichia coli, located in the periplasm, binds to ribose and mediates transport and chemotaxis. The regions on the tertiary structure of RBP that interact with the membrane permease, an ABC transporter, were genetically probed by screening a mutation using the chimeric receptor Trz. Trz is a hybrid protein between the periplasmic domain of chemoreceptor Trg and the cytoplasmic portion of osmosensor EnvZ, which provides a system for monitoring the chemotactic interaction of RBP on MacConkey agar plates when coupled with a reporter lacZ fused to an ompC gene. The expression of ompC can be increased by an interaction of ribose-bound RBP with Trz. A transport defect, either in the binding protein or in the membrane permease, causes a signalling-constitutive Lac+ phenotype of Trz even in the absence of ribose. This appears to be due to the presence of a small amount of ribose, which is normally taken up by the high-affinity transport system. By taking advantage of this, we have designed a system for genetic screening that permits a selection for mutations in the binding protein, causing specific defects in permease interaction but not in tactic interaction. Mutant RBPs that were isolated were unable to perform normal ribose uptake and to utilize ribose as a carbon source, while other functions such as taxis and sugar-binding properties were not substantially affected. The mutational changes were repeatedly found in several residues of RBP, concentrating on three surface regions and comprising two domains of the tertiary structure. We suggest that the two regions, including residues 52 and 166, are specifically involved in the permease interaction while the third region, including residues 72, 134, and others, recognizes both the permease and the chemosensory receptor.

H-NS regulates OmpF expression through micF antisense RNA in Escherichia coli.

H-NS is a major constituent of the Escherichia coli nucleoid. Expression of the major outer membrane proteins, OmpC and OmpF, is influenced by hns mutations such that OmpC expression increases whereas OmpF expression decreases irrespective of the osmolarity of the medium (K. A. Graeme-Cook, G. May, E. Bremer, and C. F. Higgins, Mol. Microbiol. 3:1287-1294, 1989). In this study we show that the effect of an hns::neo mutation on OmpF expression is largely diminished in a deletion mutant carrying the micF gene that encodes the ompF mRNA-specific antisense RNA. In addition, the micF transcript levels in the hns::neo mutation are high compared with transcript levels in wild-type cells. On the basis of these results, we provide evidence for a link between OmpC/OmpF expression and the regulatory function of H-NS. We suggest that H-NS most likely affects OmpC expression directly at the level of transcription, but OmpF expression is indirectly regulated by micF antisense RNA.

Isolation and characterization of the heat-responsive genes in Escherichia coli.

The ompC gene expression is induced by increasing temperature as well as osmotic pressure. In this study, a mutant (TD2) defective in this thermoresponse was isolated with transposon Tn10; the mutation was complemented by pMAN55 or pMAN56 containing micF and mapped at 48 min on Escherichia coli K-12. Furthermore, a new gene (hrsA) that suppressed the mutation was cloned. Its nucleotide sequence was analyzed and it was located close to the suc operon at 16.7 min corresponding to #18F11 (Kohara bank) on E. coli genome. In TD2 containing the hrsA on a multicopy plasmid, the ompC expression was induced and dependent on OmpR with increased temperature. The HrsA was found to have Enzyme IIA, IIB, and IIC domains that are homologous to Enzyme II, involved in the fructose-specific PTS (phosphotransferase system). The putative phosphorylation sites (His87 and Cys192) were also conserved in HrsA.

Characterization of the signal transduction via EvgS and EvgA in Escherichia coli.

evgS and evgA located at 51 min on the Escherichia coli chromosome were previously found as a new two-component system, which is highly homologous to bvgS and bvgA in Bordetella pertussis. In this study, the putative phosphorylation and phospho-transferred sites of EvgS and EvgA, respectively, were changed using modified primers during PCR amplification of their structural genes, and their functions were analyzed in vivo. The ompC expression was induced in a ΔenvZ E. coli in the presence of pSK-001, which contains evgS and evgA, but was not changed in the presence of pKM18 (evgS721Arg) or pKM12 (evgA52Ala). Moreover, phosphotransfer from phosphorylated EvgS to EvgA was observed in vitro using the membrane fraction containing EvgS and His-tagged EvgA. These results suggested that the signal transduction via EvgS and EvgA involves the previously reported phosphorelay in the two-component system via BvgS and BvgA.

Identification of the genes in multicopy plasmids affecting ompC and ompF expression in Escherichia coli

Osmoregulation of the porin genes, ompF and ompC of Escherichia coli, occurs at the level of transcription through the action of EnvZ and OmpR proteins as well as at the level of translation through micF antisense RNA. In this study, we used a genetic screening approach to identify new genes which interfere with the expression of ompC or ompF. Using an E. coli genomic library in pUC19, we identified three clones whose products altered expression of ompC and ompF in response to medium osmolarity. One clone carrying the secB gene was found to block ompC and inhibit ompF expression. One clone carrying gcvA, a transcriptional regulator for the gvcA operon, was found to block ompF expression at high osmolarity and elevate ompC expression at low osmolarity. One clone carrying rbsR, a repressor for the rbs operon, was found to block ompF expression at both low and high osmolarities and elevate ompC expression at low osmolarity. These results suggest that ompF and ompC expression is associated with other physiological regulating systems in addition to osmoregulation.

Heterologous expression of Escherichia coli porin genes in Salmonella typhi Ty2: regulation by medium osmolarity, temperature and oxygen availability

Electrophoretic analysis of outer membrane proteins showed that Salmonella typhi OmpC expression is not reciprocally regulated relative to OmpF as described for Escherichia coli and S. typhimurium. When bacteria were grown in minimal media, both OmpC and OmpF were repressed as the osmolarity increased. However, in Luria broth, expression of OmpC was slightly induced by osmolarity up to 0.3 osmM. Plasmids bearing E. coli ompC-lacZ or ompF-lacZ gene fusions were studied for their expression in S. typhi and E. coli. Under anaerobic growth conditions, expression of ompC-lacZ in S. typhi was maximal at 0.16 osmM, while in E. coli expression was maximal at 0.7 osmM. ompF-lacZ expression was similarly repressed by medium osmolarity and anaerobiosis in both species. In contrast, a drastic difference in the regulation of OmpF by temperature was observed; at 37 °C ompF-lacZ expression was repressed in E. coli. while in S. typhi it was induced.

Identification of the genes in multicopy plasmids affecting ompC and ompF expression in Escherichia coli.

Osmoregulation of the porin genes, ompF and ompC of Escherichia coli, occurs at the level of transcription through the action of EnvZ and OmpR proteins as well as at the level of translation through micF antisense RNA. In this study, we used a genetic screening approach to identify new genes which interfere with the expression of ompC or ompF. Using an E. coli genomic library in pUC19, we identified three clones whose products altered expression of ompC and ompF in response to medium osmolarity. One clone carrying the secB gene was found to block ompC and inhibit ompF expression. One clone carrying gcvA, a transcriptional regulator for the gvcA operon, was found to block ompF expression at high osmolarity and elevate ompC expression at low osmolarity. One clone carrying rbsR, a repressor for the rbs operon, was found to block ompF expression at both low and high osmolarities and elevate ompC expression at low osmolarity. These results suggest that ompF and ompC expression is associated with other physiological regulating systems in addition to osmoregulation.

Heterologous expression of Escherichia coli porin genes in Salmonella typhi Ty2: regulation by medium osmolarity, temperature and oxygen availability.

Electrophoretic analysis of outer membrane proteins showed that Salmonella typhi OmpC expression is not reciprocally regulated relative to OmpF as described for Escherichia coli and S. typhimurium. When bacteria were grown in minimal media, both OmpC and OmpF were repressed as the osmolarity increased. However, in Luria broth, expression of OmpC was slightly induced by osmolarity up to 0.3 osmM. Plasmids bearing E. coli ompC-lacZ or ompF-lacZ gene fusions were studied for their expression in S. typhi and E. coli. Under anaerobic growth conditions, expression of ompC-lacZ in S. typhi was maximal at 0.16 osmM, while in E. coli expression was maximal at 0.7 osmM. ompF-lacZ expression was similarly repressed by medium osmolarity and anaerobiosis in both species. In contrast, a drastic difference in the regulation of OmpF by temperature was observed; at 37 degrees C ompF-lacZ expression was repressed in E. coli, while in S. typhi it was induced.

Phosphorylation-dependent conformational changes in OmpR, an osmoregulatory DNA-binding protein of Escherichia coli.

Osmoregulated porin gene expression in Escherichia coli is controlled by the two-component regulatory system EnvZ and OmpR. EnvZ, the osmosensor, is an inner membrane protein and a histidine kinase. EnvZ phosphorylates OmpR, a cytoplasmic DNA-binding protein, on an aspartyl residue. Phospho-OmpR binds to the promoters of the porin genes to regulate the expression of ompF and ompC. We describe the use of limited proteolysis by trypsin and ion spray mass spectrometry to characterize phospho-OmpR and the conformational changes that occur upon phosphorylation. Our results are consistent with a two-domain structure for OmpR, an N-terminal phosphorylation domain joined to a C-terminal DNA-binding domain by a flexible linker region. In the presence of acetyl phosphate, OmpR is phosphorylated at only one site. Phosphorylation induces a conformational change that is transmitted to the C-terminal domain via the central linker. Previous genetic analysis identified a region in the C-terminal domain that is required for transcriptional activation. Our results indicate that this region is within a surface-exposed loop. We propose that this loop contacts the alpha subunit of RNA polymerase to activate transcription. Mass spectrometry also reveals an unusual dephosphorylated form of OmpR, the potential significance of which is discussed.

Molecular analysis of the two-component genes, ompR and envZ, in the symbiotic bacterium Xenorhabdus nematophilus.

In Escherichia coli the histidine kinase sensor protein, EnvZ, undergoes autophosphorylation and subsequently phosphorylates the regulatory protein, OmpR. Modulation of the levels of OmpR-phosphate controls the differential expression of ompF and ompC. While the phosphotransfer reaction between EnvZ and OmpR has been extensively studied, the domains involved in the sensing function of EnvZ are not well understood. We have used a comparative approach to study the sensing function of EnvZ. During our search of numerous bacteria we found that the symbiotic/pathogenic bacterium Xenorhabdus nematophilus contained the operon encoding both ompR and envZ. Nucleotide sequence analysis revealed that EnvZ of X. nematophilus (EnvZX.n.) is composed of 342 amino acid residues, which is 108 residues shorter than EnvZ of E. coli (EnvZE.c.). Amino acid sequence comparison showed that the cytoplasmic domains of the EnvZ molecules shared 57% sequence identity. In contrast, the large hydrophilic periplasmic domain of EnvZE.c. was absent in EnvZX.n., and was replaced by a shorter hydrophobic region. Although the periplasmic domains had diverged extensively, envZX.n. was able to complement a delta envZ strain of E. coli. OmpF and OmpC were differentially produced in response to changes in medium osmolarity in this strain. Further genetic analysis established that heterologous phosphorylation between EnvZX.n. and OmpR of E. coli (OmpRE.c.) accounted for the complementation of the delta envZ strain. In addition we show that the OmpR molecules of X. nematophilus and E. coli share 78% amino acid sequence identity. These results indicate that the EnvZ protein of X. nematophilus was able to sense these changes in the osmolarity of the growth environment and properly regulate the levels of OmpR-phosphate in E. coli.

Isolation and characterization of the IS3-like element from Thermus aquaticus.

We have cloned and characterized a genetic element (1187 bp) that is responsible for the induction of thermotolerance as well as ompC expression in E. coli. This element (ISLtaq1) was isolated from Thermus aquaticus. DNA and protein data bases were searched with this element (ISLtaq1), which suggested it to be very similar to IS150 belonging to the IS3 family. ORF1, found on ISLtaq1, which encodes 100 amino acids (aa), had a DNA-binding motif: a helix-turn-helix and a leucine zipper. In fact, when the ORF1 protein was overproduced in E. coli, thermotolerance as well as ompC expression was induced.

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.

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.