Specialized transducing bacteriophage lambda carrying the structural gene for a major outer membrane matrix protein of Escherichia coli K-12.

A specialized transducing phage lambda carrying the structural gene for the OmpF protein, an outer membrane matrix protein, was isolated. The phage carries the 20.5--21-min region of the Escherichia coli K-12 chromosome and carries asnS, ompF, and aspC genes.     

The function of micF RNA. micF RNA is a major factor in the thermal regulation of OmpF protein in Escherichia coli

The role of chromosomally derived micF RNA as a repressor of outer membrane protein OmpF of Escherichia coli was examined for various growth conditions. Levels of micF RNA as determined by Northern analyses are found to increase in response to cell growth at high temperature, in high osmolarity or in the presence of ethanol. After a switch to higher growth temperature, the levels of ompF mRNA and of newly synthesized OmpF decrease with time in E. coli strain, MC4100 but these decreases are not observed in isogenic micF deletion strain, SM3001. In addition, while levels of ompF mRNA are substantially reduced in both strains in response to high osmolarity or ethanol at 24 degrees C, the reduced levels in the parental strain are still 4-5-fold lower compared with the micF deletion strain. These findings indicate that chromosomally derived micF RNA plays a major role in the thermal regulation of OmpF and represses OmpF synthesis in response to several environmental signals by decreasing the levels of ompF mRNA. Analyses of the effect of a multicopy micF plasmid on the levels of OmpF and ompF mRNA after an increase in temperature indicated that multicopies of micF RNA markedly inhibited OmpF synthesis but did not accentuate ompF mRNA decrease. These data suggest that multicopy micF inhibits OmpF synthesis primarily through translational inactivation of ompF mRNA and that a limiting factor in addition to micF RNA is necessary to destabilize ompF mRNA.     

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.     

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 (EnvZ^X.n.) is composed of 342 amino acid residues, which is 108 residues shorter than EnvZ of E. coli (EnvZ^E.c.). Amino acid sequence comparison showed that the cytoplasmic domains of the EnvZ moleculsshared 57% sequence identity. In contrast, the large hydrophilic periplasmic domain of EnvZ^E.c. was absent in EnvZ^X.n., and was replaced by a shorter hydrophobic region. Although the periplasmic domains had diverged extensively, envZ^X.n. was able to complement a Δ 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 EnvZ^X.n. and OmpR of E. coli (OmpR^E.c.) accounted for the complementation of the Δ 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 changes in the osmolarity of the growth environment and properly regulate the levels of OmpR-phosphate in E. coli .     

Purification and characterization of the OmpR protein, a positive regulator involved in osmoregulatory expression of the ompF and ompC genes in Escherichia coli

The OmpR protein is a positive regulator involved in osmoregulatory expression of the ompF and ompC genes, which respectively code for major outer membrane proteins OmpF and OmpC of Escherichia coli. The OmpR protein has been purified to homogeneity from an overproducing strain harboring an ompR gene-carrying plasmid. Throughout the purification the OmpR protein behaved as a single entity. The molecular weight determined on sodium dodecyl sulfate-polyacrylamide gel, the total amino acid composition, and the NH2-terminal amino acid sequence of the purified protein were essentially the same as those deduced from the nucleotide sequence of the ompR gene. Molecular weight determination and cross-linking study on the native protein revealed that the purified protein exists as a monomer. The purified OmpR protein was specifically bound to the promoter regions of the ompC and ompF genes. Experiments with a series of upstream deletions of the ompC and ompF promoters revealed that the region upstream from the -35 region was indispensable for OmpR binding to both the ompC and the ompF promoters. Although it has been proposed that depending on the medium osmolarity the OmpR protein may exist in two alternative structures, which respectively regulate functioning of the ompC and the ompF promoters, the purified OmpR protein appeared to be homogeneous and interacted with both promoters to the same extent.     

Expression of outer membrane proteins in Escherichia coli growing at acid pH

It is generally accepted for Escherichia coli that (i) the level of OmpC increases with increased osmolarity when cells are growing in neutral and alkaline media, whereas the level of OmpF decreases at high osmolarity, and that (ii) the two-component system composed of OmpR (regulator) and EnvZ (sensor) regulates porin expression. In this study, we found that OmpC was expressed at low osmolarity in medium of pH below 6 and that the expression was repressed when medium osmolarity was increased. In contrast, the expression of ompF at acidic pH was essentially the same as that at alkaline pH. Neither OmpC nor OmpF was detectable in an ompR mutant at both acid and alkaline pH values. However, OmpC and OmpF were well expressed at acid pH in a mutant envZ strain, and their expression was regulated by medium osmolarity. Thus, it appears that E. coli has a different mechanism for porin expression at acid pH. A mutant deficient in ompR grew slower than its parent strain in low-osmolarity medium at acid pH (below 5.5). The same growth diminution was observed when ompC and ompF were deleted, suggesting that both OmpF and OmpC are required for optimal growth under hypoosmosis at acid pH.     

Expression of micF involved in porin synthesis in Escherichia coli: two distinct cis-acting elements respectively regulate micF expression positively and negatively

micF RNA, whose sequence is highly complementary to a 5'-portion of ompF mRNA, has been implicated in the osmoregulation and thermoregulation of the ompF porin gene in Escherichia coli. To define and characterize cis-acting regulatory regions upstream of the micF promoter, a series of deletions of the micF promoter fused to the lacZ gene were constructed. Two distinct regions, which function differently, were identified as cis-acting regulatory elements, namely, one responsible for OmpR-dependent activation and the other for OmpR-independent repression of micF expression. The former contains the OmpR-binding site, which simultaneously regulates both the genes, micF and ompC, in response to the medium osmolarity. The latter may be involved in an unknown regulatory process of micF expression.     

Asparaginyl-tRNA synthetase from Escherichia coli has significant sequence homologies with yeast aspartyl-tRNA synthetase

The Escherichia coli asnS gene codes for asparaginyl-tRNA synthetase (NRSEC). We have sequenced the asnS region, including 382 bp of the 5'-untranslated region, 1398 bp of the coding region and 280 bp of the 3'-untranslated region. The DNA-derived NRSEC amino acid (aa) sequence was confirmed by direct aa sequencing of the N-terminal parts of the native protein and of a 28-kDa internal fragment generated by trypsin digestion. The asnS gene product has been purified to homogeneity using three chromatographic steps. Sequence comparison of the deduced NRSEC sequence with all aminoacyl-tRNA synthetase sequences showed significant homologies with the yeast aspartyl-tRNA synthetase and weaker relationships with other aminoacyl-tRNA synthetases for aa with an XAX codon.     

Characterization and environmental regulation of outer membrane proteins in Xenorhabdus nematophilus.

We have examined the production of the outer membrane proteins of the primary and secondary forms of Xenorhabdus nematophilus during exponential- and stationary-phase growth at different temperatures. The most highly expressed outer membrane protein of X. nematophilus was OpnP. The amino acid composition of OpnP was very similar to those of the porin proteins OmpF and OmpC of Escherichia coli. N-terminal amino acid sequence analysis revealed that residues 1 to 27 of the mature OpnP shared 70 and 60% sequence identities with OmpC and OmpF, respectively. These results suggest that OpnP is a major porin protein in X. nematophilus. Three additional proteins, OpnA, OpnB, and OpnS, were induced during stationary-phase growth. OpnB was present at a high level in stationary-phase cells grown at 19 to 30 degrees C and was repressed in cells grown at 34 degrees C. OpnA was optimally produced at 30 degrees C and was not present in cells grown at lower and higher temperatures. The production of OpnS was not dependent on growth temperature. In contrast, another outer membrane protein, OpnT, was strongly induced as the growth temperature was elevated from 19 to 34 degrees C. In addition, we show that the stationary-phase proteins OpnA and OpnB were not produced in secondary-form cells.     

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.