micF RNA in ompB mutants of Escherichia coli: different pathways regulate micF RNA levels in response to osmolarity and temperature change.

The repressor RNA, micF RNA, is regulated by temperature, osmolarity, and other stress conditions during growth of Escherichia coli. Northern (RNA) blot analyses showed that levels of micF RNA differ widely in various ompB mutant strains when cells are grown at 24 degrees C in LB broth. For example, relative to the parental strain MC4100, the ompR101 mutant strain (which contains no functional OmpR) had about a 10-fold reduction in micF RNA, whereas the envZ11 strain showed about a 5-fold increase. At 37 degrees C, however, micF RNA levels in the ompR101 and envZ11 strains and other ompB mutants differed by less than two-fold compared with the level in strain MC4100, thus indicating that a factor(s) independent of the ompB locus regulates micF RNA expression with temperature increase and that there is an additional control mechanism(s) which maintains the levels of micF RNA in these mutants close to that of the wild type during growth at high temperatures. In a plasmid strain containing the micF gene but without the upstream OmpR-binding site, steady-state levels of micF RNA increased with temperature increase but did not change with osmolarity increase. This showed that osmolal regulation but not temperature regulation of micF depends on these upstream sequences and suggested that while osmolal regulation of the micF gene depends on OmpR, thermal regulation does not.     

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.     

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.     

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.     

Re-examination of the role of the periplasmic domain of EnvZ in sensing of osmolarity signals in Escherichia coli

In Escherichia coli , EnvZ senses changes in the osmotic conditions of the growth environment and controls the phosphorylated state of the regulatory protein, OmpR. OmpR-phosphate regulates the expression of the porin genes, ompF and ompC . To investigate the role of the periplasmic domain of EnvZ in sensing of osmolarity signals, portions of this domain were deleted. Cells containing the EnvZ mutant proteins were able to regulate normally the production of OmpF and OmpC in response to changes in osmolarity. The periplasmic domain of EnvZ was also replaced with the non-homologous periplasmic domain of the histidine kinase PhoR of Bacillus subtilis . Osmoregulation of OmpF and OmpC production in cells containing the PhoR–EnvZ hybrid protein was indistinguishable from that in cells containing wild-type EnvZ. Identical results were obtained with an envZ – pta/ack strain, which could not synthesize acetyl phosphate. Thus, acetyl phosphate was not involved in the regulation of ompF and ompC observed in this study. These results indicate that the periplasmic domain of EnvZ is not essential for sensing of osmolarity signals.