Regulatory mutations conferring constitutive synthesis of major outer membrane proteins (OmpC and OmpF) in Escherichia coli.

An ompB strain of Escherichia coli K-12 lacking major outer membrane proteins OmpC and OmpF was used to isolate a pair of mutants that have restored the ability to synthesize either OmpC or OmpF protein. These mutants were found to produce the respective proteins constitutively under the several conditions where the synthesis in the wild-type strain was markedly repressed; namely, in the absence of the ompB gene function, under restrictive medium conditions, or upon lysogenization with phage PA-2. The mutations ompCp1 and ompFp9 responsible for such synthesis were shown to be located in the close vicinity of the corresponding structural genes, ompC and ompF. Moreover, the mutations affect the expression of these genes in a cis-dominant fashion. Taken together with other evidence, it was suggested that ompCp1 and ompFp9 represent regulatory site mutations occurring at the promoter regions of ompC and ompF respectively. Relevance of these findings to the genetic control of outer membrane protein synthesis is discussed.     

Complementation analysis of the wild-type and mutant ompR genes exhibiting different phenotypes of osmoregulation of the ompF and ompC genes of Escherichia coli

Expression of the ompF and ompC genes, which encode the major outer membrane proteins, OmpF and OmpC, respectively, is affected in a reciprocal manner by the osmolarity of the growth medium. This osmoregulation is mediated by the OmpR protein, a positive regulator of both genes, which is encoded by the ompR gene. Structural and functional properties of this regulatory protein were studied through complementation analysis of the wild-type and five mutant ompR genes that exhibited differences in osmoregulation of the expression of the OmpF and OmpC proteins. Complementation was carried out with combinations of a host strain and a plasmid, each of which carried either the wild-type or a mutant ompR gene. In some combinations, negative complementation was observed. For example, ompR1, a deletion mutation with an OmpF- OmpC- phenotype, was dominant to OmpF+ or OmpC+ phenotypes conferred by other ompR genes. Positive complementation of two mutant ompR genes was also observed in other combinations, when the two mutations were distantly located from each other on the OmpR protein. These results, together with other observations, support the view that the OmpR protein has a two-domain structure, each domain exhibiting a different role in the expression of the OmpF and OmpC proteins, and that this protein takes a multimeric structure as a functional unit.     

The regulation of porin expression in Escherichia coli: effect of turgor stress

The OmpF and OmpC porins are major outer membrane proteins of Escherichia coli. Their expression is affected by medium osmolarity such that OmpF is normally produced at low osmolarity and OmpC at high osmolarity. Potassium ion accumulation is a major means by which cells maintain their internal osmolarity in high osmolarity medium in the absence of organic osmolytes such as glycine-betaine. Starvation for potassium causes cells to become turgor stressed. The effect of turgor stress and potassium ion concentration on OmpF and OmpC expression was examined. It was found that ompF gene expression was switched off by turgor stress but there was no concomitant increase in OmpC. Instead, ompC expression responded to the accumulation of potassium ions by the cell in high osmolarity medium.     

Mutation causing reverse osmoregulation of synthesis of OmpF, a major outer membrane protein of Escherichia coli.

Supplementation of growth media with high concentrations of substances like sucrose results in the induction of OmpC synthesis and the suppression of OmpF synthesis. We isolated a novel mutant in which OmpF synthesis is in the opposite direction from normal osmoregulation. By transductional mapping, the mutation was localized at 75 min between malA and aroB on the Escherichia coli chromosome map where the ompR-envZ region is. The mutation was suppressed by a plasmid carrying the ompR gene but not by a plasmid carrying the envZ gene alone. The mutation also resulted in the almost complete suppression of OmpC synthesis. However, the remaining OmpC synthesis was osmoregulated normally. Based on these observations, the mechanism of osmoregulation of OmpF-OmpC synthesis is discussed.     

OmpC and OmpF are required for growth under hyperosmotic stress above pH 8 in Escherichia coli

AIMS: To investigate the requirement of outer membrane porins for osmotic adaptation at alkaline pH in Escherichia coli. METHODS AND RESULTS: Escherichia coli mutants deficient in ompC, ompF and both genes were constructed and the growth of these mutants was observed at alkaline pH. The growth rate of the mutant deficient in both ompC and ompF was slower than that of the wild type and mutants deficient in one of these genes under hyperosmotic stress at pHs above 8.0. The decreased rate was recovered when a cloned ompC was introduced to the mutant, but the growth recovery with a cloned ompF was partial. Such growth diminution was not observed at pHs below 8.0. CONCLUSION: OmpC and OmpF were shown to participate in hyperosmotic adaptation at alkaline pH in E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report to demonstrate that OmpC and OmpF are required for hyperosmotic adaptation at pHs above 8.0, but not below 8.0.     

Effect of outer membrane permeability on chemotaxis in Escherichia coli.

The relationship between outer membrane permeability and chemotaxis in Escherichia coli was studied on mutants in the major porin genes ompF and ompC. Both porins allowed passage of amino acids across the outer membrane sufficiently to be sensed by the methyl-accepting chemotaxis proteins, although OmpF was more effective than OmpC. A mutant deleted for both ompF and ompC, AW740, was almost completely nonchemotactic to amino acids in spatial assays. AW740 required greater stimulation with L-aspartate than did the wild type to achieve full methylation of methyl-accepting chemotaxis protein II. Induction of LamB protein allowed taxis to maltose but not to L-aspartate, which indicates that the maltoporin cannot rapidly pass aspartate. Salt taxis was less severely inhibited by the loss of porins than was amino acid taxis, which implies an additional mechanism of outer membrane permeability. These results show that chemotaxis can be used as a sensitive in vivo assay for outer membrane permeability to a range of compounds and imply that E. coli can regulate chemotactic sensitivity by altering the porin composition of the outer membrane.     

Isolation and characterization of mutations altering expression of the major outer membrane porin proteins using the local anaesthetic procaine

Mutations at several different chromosomal locations affect expression of the major outer membrane porin proteins (OmpF and OmpC) of Escherichia coli K12. Those that map at 21 and 47 minutes define the structural genes for OmpF and OmpC, respectively. A third locus, ompB, is defined by mutations that map at 74 minutes. The ompB locus contains two genes whose products regulate the relative amounts of ompF and ompC expression. One of these genes, ompR, encodes a positive regulatory protein that interacts at the ompF and ompC promoters. Mutations in ompR exhibit an OmpF- OmpC- or an OmpF+ OmpC- phenotype. The product of the second gene, envZ, affects regulation of the porin proteins in an unknown manner. Previously isolated mutations in envZ exhibit an OmpF- OmpC+ phenotype and also have pleiotropic effects on other exported proteins. In the presence of local anaesthetics such as procaine, wild-type strains exhibit properties similar to these envZ mutants, i.e. OmpF- OmpC+. Using ompF-lac fusion strains, we have exploited this procaine effect to isolate two new classes of envZ mutations. One of these classes exhibits an OmpF+ OmpC- phenotype. The other allows expression of both OmpF and OmpC but alters the relative amounts found under various growth conditions. Like previously isolated envZ mutations, these also affect regulation of other exported proteins, such as lambda receptor. These results permit a more detailed analysis of the omp regulon and they may shed light on one of the mechanisms by which local anaesthetics exert their effect.     

Acquisition of Escherichia coli outer membrane proteins by Bdellovibrio sp. strain 109D

The ability of Bdellovibrio sp. to acquire the OmpF major outer membrane protein from its Escherichia coli prey was examined to determine if there were other outer membrane proteins which could or could not be acquired. Growth of bdellovibrios on mutant prey which were defective in the expression of outer membrane proteins revealed that Bdellovibrio sp. could acquire the OmpC protein in the absence of the OmpF protein. However, the OmpA, LamB, and protein 2 proteins could not be found in the Bdellovibrio Triton-insoluble outer membrane. The disappearance of the OmpF and OmpC proteins from the bdelloplast surface was measured, and it was determined that Bdellovibrio sp. exhibited a kinetic and temporal preference for the OmpF protein. Bdellovibrios could be grown on porin-deficient prey, and the progeny bdellovibrios possessed outer membranes with a protein mass deficiency.     

OmpF assembly mutants of Escherichia coli K-12: isolation, characterization, and suppressor analysis.

This paper describes a novel genetic method used to isolate mutations that alter proper assembly of OmpF in the outer membrane. The thermolabile nature of assembly intermediates allowed selection of temperature-sensitive mutations within the ompF gene. A variant allele of ompF (ompF-Dex) was used because it provided a convenient selectable phenotype (Dex+). Assembly mutants were isolated in two steps. First, amber mutations were obtained that mapped in ompF-Dex. This resulted in a Dex- phenotype. Starting with these Dex- strains, Dex+ revertants were isolated. Mutants that displayed a temperature-sensitive Dex+ phenotype were further characterized. Three such mutants possessed a single substitution within ompF that reverted the nonsense codon to a sense codon which replaced W214 with either an E or Q and Y231 with a Q residue in the mature OmpF protein. All three mutant OmpF proteins showed an assembly defect. This defect led to a substantial reduction in the amount of stable OmpF trimers with the concomitant increase of a high-molecular-weight form of OmpF which migrated at the top of the gel. Suppressor mutations were sought that corrected the assembly defect of OmpF. These extragenic suppressor mutations were mapped at 45 min on the Escherichia coli chromosome. The suppressor mutations displayed no allele specificity and were recessive to the wild-type allele. In the presence of a suppressor, mutant stable trimers appeared in an almost normal manner. The appearance of stable trimers concurred with a substantial loss of the high-molecular-weight OmpF species. At this stage, it is not clear whether the high-molecular-weight species of OmpF is a normal assembly intermediate or a dead-end assembly product. The results presented in this study raise the intriguing possibility of a chaperone-like activity for the wild-type suppressor gene product.     

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.     

Selection for mutants altered in the expression or export of outer membrane porin OmpF.

Strains in which the lacZ gene (which specifies beta-galactosidase) is fused to a gene encoding an envelope protein often exhibit a phenotype termed overproduction lethality. In such strains, high-level synthesis of the cognate hybrid protein interferes with the process of protein export, and this leads ultimately to cell death. A variation of this phenomenon has been discovered with lacZ fusions to the gene specifying the major outer membrane porin protein OmpF. In this case, we find that lambda transducing phage carrying an ompF-lacZ fusion will not grow on a host strain that constitutively overexpresses ompF. We have exploited this observation to develop a selection for ompF mutants. Using this protocol, we have isolated mutants altered in ompF expression and have identified mutations that block OmpF export. Our results suggest that it should be possible to adapt this selection for use with other genes specifying exported proteins.     

Phosphorylation of a bacterial activator protein, OmpR, by a protein kinase, EnvZ, results in stimulation of its DNA-binding ability

The Escherichia coli OmpR protein is an activator protein specific for the ompF and ompC genes, which respectively encode the outer membrane proteins, OmpF and OmpC. The EnvZ protein is a protein kinase specific for the OmpR protein. In this study, we compared the in vitro DNA-binding ability of the phosphorylated form of the OmpR protein with that of the non-phosphorylated form by means of non-denaturing gel retardation analysis and DNase I footprinting analysis. The results indicate that the phosphorylation of the OmpR protein results in stimulation of its in vitro DNA-binding ability as to both the ompF and ompC promoter DNAs.     

Osmoregulatory expression of porin genes in Escherichia coli: a comparative study on strains B and K-12

Escherichia coli K-12 produces both the OmpF and OmpC porins, the relative amounts of which in the outer membrane are affected in a reciprocal manner by the osmolarity of the growth medium. In contrast, E. coli B produces only the OmpF porin, regardless of the medium osmolarity. In this study, it was revealed that there is an extensive deletion within the ompC locus of the E. coli B chromosome. Cloning and nucleotide sequencing of the regulatory gene, ompR , of E. coli B revealed that there are two amino acid alterations (Lys-6 to Asn and Ala-130 to Thr) in the amino acid sequence of the OmpR protein, as compared with that of E. coli K-12. It is suggested that these particular amino acid alterations are responsible for the constitutive expression of the ompF gene observed in E. coli B.     

Promoter exchange between ompF and ompC, genes for osmoregulated major outer membrane proteins of Escherichia coli K-12.

Expression of the ompF and ompC genes coding for major outer membrane proteins OmpF and OmpC is regulated in opposite directions by medium osmolarity. Chimera genes were constructed by a reciprocal exchange of the promoter-signal sequence region between the two genes. The chimera gene construction was designed so that the proteins synthesized by these genes were essentially the same as the OmpC and OmpF proteins. Studies with the chimera genes demonstrated that the osmoregulation of the OmpF-OmpC synthesis was promoter dependent. They also showed that cells grew normally even when the osmoregulation took place in opposite directions. The effects of the ompR2 and envZ mutations, which suppress ompC and ompF expression, respectively, also became reversed. The reduced expression was still subject to the promoter-controlled osmoregulation. Based on these observations, the mechanism of regulation of the ompF-ompC gene expression and its physiological importance are discussed.