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.     

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 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.     

Control of the synthesis of alkaline phosphatase and the phosphate-binding protein in Escherichia coli.

Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunological techniques, we have compared the synthesis of the phoA protein (alkaline phosphatase) and the phoS protein (phosphate-binding protein) in response to the level of phosphate in the medium in different genetic backgrounds containing the known alkaline phosphatase control mutations. Both proteins are produced in excess phosphate media in a phoR1a- strain, whereas neither protein is produced in a phoB- strain even under derepression conditions. In four different phoR1c- strains, however, the phoA product cannot be detected in extracts of cells obtained from any growth condition, whereas the phoS product is produced in both excess and limiting phosphate media. It is not yet known if phoR1c- mutants are a special class of mutations within the phoB gene or whether they occur in a separate cistron involved in alkaline phosphatase regulation. From these results we conclude that the expression of the phoA gene is not always co-regulated with expression of the phoS gene product. We have determined that the phoS protein is a component of periplasmic protein band P4 described by Morris et al. (1974). The phoS product lacks sulfur-containing amino acids and is extractable by treatment with polymyxin sulfate. The other component of band P4 contains methionine and/or cysteine and is not extracted by polymyxin sulfate treatment. Like the phoS and phoA proteins, its synthesis is sensitive to the concentration of phosphate in the growth medium. In addition, the existence of a new class of periplasmic proteins synthesized at maximum rate in high phosphate media is demonstrated.     

Effect of changes in the osmolarity of the growth medium on Vibrio cholerae cells.

The rate and extent of lysis of Vibrio cholerae cells under nongrowing conditions were dependent on the osmolarity of the growth medium. Gross alterations in cellular morphology were observed when V. cholerae cells were grown in media of high and low osmolarity. The rate of lysis of V. cholerae cells under nongrowing conditions increased after treatment with chloramphenicol. Chloramphenicol-treated V. cholerae 569B cells showed formation of sphaeroplast-like bodies in medium of high osmolarity, but not in low osmolarity. Changes in the osmolarity of the growth medium also regulated the expression of the outer membrane proteins. This regulation was abolished if V. cholerae cells were grown in Pi-depleted medium. Analysis of the lytic behavior and composition of outer membrane proteins of an osmotically fragile mutant strain revealed a similar dependence on the osmolarity of the growth medium.     

Effects of pho regulatory mutations and phoA gene amplification on alkaline phosphatase synthesis and release by lky mutants of Escherichia coli K12

lky mutants of Escherichia coli K12 spontaneously released alkaline phosphatase (APase) into the extracellular medium to give up to 300 units ml-1. APase is a phosphate repressible periplasmic enzyme encoded by the gene phoA. With a view to establishing a method of easy purification, we have analysed APase synthesis and release patterns of isogenic lky strains containing either a constitutive pho regulatory mutation, or a hybrid plasmid carrying the structural gene phoA+ and pho regulatory genes, or a transducing phi 80 phoA+ phage. In the presence of the phoS2333 mutation, F- lky strains lysogenized with phi 80 phoBin phoA+ phage and grown in high phosphate medium were able to release eight times more APase activity (2300 units ml-1) than haploid strain 2336 (phoS+ lky) grown in low phosphate medium. Neither protein synthesis, the cell export machinery nor leakage mechanisms were limiting for APase release. Sufficient APase was released into the medium to facilitate its purification.     

The physiological state of suspension cultured cells affects the expression of the β-glucuronidase gene following transformation of tall fescue (Festuca arundinacea) protoplasts

Both the stage of the growth cycle and the age of the cell culture used to isolate protoplasts had a pronounced effect on both transient and stable expression of the GUS gene. A level of GUS gene transient expression of 9000 pmol 4MU/μg protein/h and a frequency of GUS gene stable expression of 5.72% were obtained with protoplasts isolated from suspension cultures 10–20 weeks after initiation and 3–4 days after subculturing when an optimized transformation protocol and a rice actin 1 promoter-uidA gene construct were used. The effect of the cell growth cycle on GUS gene transient expression was closely correlated with the growth rate and the rate of protein synthesis in cell cultures whereas prolonged subculturing of the cells resulted in a gradual decline in both transient and stable expression. The length of time cells were digested in cell wall digestion enzyme and the osmolarity of the transformation medium were found to critically affect both the level of transient and stable GUS gene expression. The composition and osmolarity of the protoplast culture medium was less critical for transient GUS gene expression although the osmolarity of the medium was shown to have a significant effect on stable expression of the GUS gene.     

Turgor-controlled K+ fluxes and their pathways in Escherichia coli

Escherichia coli like most gram-negative bacteria with walls maintains a cytoplasmic osmolarity exceeding that of the medium; the resulting hydrostatic pressure (turgor pressure) pushes the cytoplasmic membrane against the peptidoglycan and creates a tension in the two envelopes. Potassium is the only cation which takes part in the regulation of cellular osmolarity. The adaptation of intracellular K+ concentration to external osmolarity involves K+ turgor-controlled fluxes. When the medium osmolarity is raised an osmodependent influx of K+ can be observed; this is carried out by the K+ transport system TrkA which can also taken up rubidium. A specific and unidirectional pathway allows K+ ions to flow out of the cell when the medium osmolarity is decreased; this pathway reveals two characteristics: it has no affinity for rubidium and it can be blocked by the blockers of eukaryotic K+ channels. Osmodependent fluxes are turned on immediately after the medium osmolarity is disturbed; in contrast, they are turned off gradually as the rate of K+ fluxes approach zero. The rate of K+ influx seems to depend on the level of internal osmolarity and not on the extent of the increase in medium osmolarity. The rate of the efflux is directly proportional to the decrease in medium osmolarity and is independent on the level of internal osmolarity.     

Secretion expression of recombinant glucagon inEscherichia coli

A novel approach for the preparation of recombinant human glucagon was described. An expression vector pAGluT, containing phoA promoter, phoA signal peptide and glucagon gene, was constructed by means of genetic engineering.Escherichia coli strain YK537 was transformed with pAGluT. High-level secretory expression of recombinant human glucagon was achieved. The expression yield of recombinant human glucagon was found to be 80 mg/L, approximately 30% of the total proteins in supernatant. The biological activities and the physicochemical properties of the purified recombinant human glucagon were found to be the same as that of native glucagon. In addition, our results suggested that phoA expression system may be suitable for the expression of other small peptides.     

Secretion expression of recombinant glucagon inEscherichia coli

A novel approach for the preparation of recombinant human glucagon was described. An expression vector pAGluT, containing phoA promoter, phoA signal peptide and glucagon gene, was constructed by means of genetic engineering.Escherichia coli strain YK537 was transformed with pAGluT. High-level secretory expression of recombinant human glucagon was achieved. The expression yield of recombinant human glucagon was found to be 80 mg/L, approximately 30% of the total proteins in supernatant. The biological activities and the physicochemical properties of the purified recombinant human glucagon were found to be the same as that of native glucagon. In addition, our results suggested that phoA expression system may be suitable for the expression of other small peptides.     

betaine modulates intracellular thiol and potassium levels in Escherichia coli in medium with high osmolarity and alkaline pH

Glycine betaine stimulates the growth rate of various bacteria in high osmolarity medium. In our studies, glycine betaine stimulated the growth rate of Escherichia coli K 12 in minimal medium with normal osmolarity at alkaline pH (pH 8.2). Betaine also caused a reduction in the intracellular pools of K⁺ and low molecular weight thiols in E. coli growing both in medium with high osmolarity and at alkaline pH. These effects of betaine were absent at pH 7.0. In cells growing in high osmolarity medium, 10 mM sodium acetate or 10 M N-ethylmaleimide reduced expression of the osmosensitive gene proU to the same extent as treatment with betaine; however, under these conditions, sodium acetate and N-ethylmaleimide did not stimulate the growth of E. coli. It is proposed that low molecular weight thiols and intracellular pH may participate in the response of E. coli to betaine.     

Osmoregulation of the maltose regulon in Escherichia coli.

The maltose regulon consists of four operons that direct the synthesis of proteins required for the transport and metabolism of maltose and maltodextrins. Expression of the mal genes is induced by maltose and maltodextrins and is dependent on a specific positive regulator, the MalT protein, as well as on the cyclic AMP-catabolite gene activator protein complex. In the absence of an exogenous inducer, expression of the mal regulon was greatly reduced when the osmolarity of the growth medium was high; maltose-induced expression was not affected, and malTc-dependent expression was only weakly affected. Mutants lacking MalK, a cytoplasmic membrane protein required for maltose transport, expressed the remaining mal genes at a high level, presumably because an internal inducer of the mal system accumulated; this expression was also strongly repressed at high osmolarity. The repression of mal regulon expression at high osmolarity was not caused by reduced expression of the malT, envZ, or crp gene or by changes in cellular cyclic AMP levels. In strains carrying mutations in genes encoding amylomaltase (malQ), maltodextrin phosphorylase (malP), amylase (malS), or glycogen (glg), malK mutations still led to elevated expression at low osmolarity. The repression at high osmolarity no longer occurred in malQ mutants, however, provided that glycogen was present.     

A model for the regulation of expression of the potassium-transport operon,kdp, inEscherichia coli

The intracellular concentration of K+in Escherichia coli is known to be determined by osmolarity of the growth medium, and it is believed that the expression of the potassium-transport operon, kdp, is controlled by the turgor pressure differential between the cytoplasm and the extracellular environment. Several lines of evidence, however, argue against a strict turgor-regulation model for kdp expression. Instead, it is proposed here that kdp is controlled by one fraction of intracellular [K+], and that the size of this fraction is independent of the osmolarity of the culture medium.     

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

Abstract 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.     

Influence of Osmolarity and the Presence of an Osmoprotectant on Lactococcus lactis Growth and Bacteriocin Production

Growth inhibition of Lactococcus lactis provoked by increasing osmolarity is reversed when glycine betaine (GB) or its analogs are added to a defined medium. Lacticin 481 production increased sharply with growth medium osmolarity in the absence of osmoprotectant but remained unaffected when GB was supplied in media of increasing osmolarity.     

Osmotic effects on the plasma membrane of thymocytes

Rat thymocytes were treated with media of different hypotonic osmolarity and the resulting damage of the plasma membrane was monitored by measuring the permeation of biochemical markers (K⁺, fluorescein, lactate dehydrogenase) and by observation of morphological changes in the plasma membrane using the freeze-etch technique. Release is dependent on the molecular weight of the markers; those with higher molecular weights permeate predominantly at a lower osmolarity of the medium and those with lower molecular weights permeate at a higher osmolarity. The marker release is temperature dependent: at 4°C it takes place at a lower osmolarity of the medium than at 37°C.With decreasing osmolarity of the medium at 4°C a decrease in the number of the intramembranous particles and increasing aggregation of the intramembranous particles occurs.