Raymond Portalier

Regulation of major outer membrane porin proteins of Escherichia coli K 12 by pH.

SummaryElectrophoretic analysis of total membrane proteins of Escherichia coli cells grown at neutral or acid pH showed that ompF, ompC and lamB porin gene expression was regulated by changes in extracellular pH. Growth at acid pH was correlated with a decrease in outer membrane proteins OmpF and LamB and an increase in protein OmpC. pH-induced effects were confirmed and quantitatively estimated by using strains carrying ompF-lacZ, ompC-lacZ and lamB-lacZ fusions. Our studies showed that the pH-dependent regulation acted at a transcriptional level on ompF and ompC gene expression and also at a post-transcriptional level on ompF gene expression. Similar studies carried out with envZ strains showed that the pH-controlled transducing signal was mediated via the EnvZ protein, although other pH-dependent pathways were also involved.

tolA, tolB and excC, three cistrons involved in the control of pleiotropic release of periplasmic proteins by Escherichia coli K12

SummaryMutants of Escherichia coli K12 carrying exc mutations inducing the release of the plasmid pBR322-encoded β-lactamase (EC 3.5.2.6) into the extracellular medium were analysed and compared with previously described excretory mutants carrying lky mutations associated with the release of alkaline phosphatase and to tolA and tolB mutants, originally described as tolerant towards various colicins. The exc, lky and tol mutations mapped near the gal operon at min 16.5 of the E. coli linkage map. A genetic analysis presented in this paper showed that some exc and lky mutations belonged to the tolA and tolB complementation groups. Furthermore, we identified a third cistron, excC, involved in the excretion of periplasmic enzymes but distinct from the two others.

Production of extracellular alkaline phosphatase by Escherichia coli K-12 periplasmic-leaky mutants carrying phoA+ Plasmids

SummaryCol E1 hybrid plasmids carrying the phoA+ structural gene of alkaline phosphatase, a periplasmic enzyme of Escherichia coli K-12, were identified from the Clarke and Carbon genomic bank. Wild-type (lky+) phoA+ plasmid-bearing strains synthesized 14 times more intracellular enzyme than the haploid lky+ strain. Phosphate-induced repression was maintained in transformed strains. PhoA+ plasmids carrying the phoB and phoR regulatory genes were introduced into a periplasmic-leaky (lky) recipient strain able to release alkaline phosphatase into the extracellular medium. Transformed lky mutants excreted up to 90% of total enzyme activity which corresponded to 3.5 times the amount of intracellular alkaline phosphatase made by the haploid lky+ strain. The protein composition analysis of periplasmic and extracellular fractions showed that: (i) wild-type phoA+ hybrid plasmidbearing clones did not excrete alkaline phosphatase but had a modified periplasmic content; (ii) alkaline phosphatase was the major excreted protein by transformed lky mutants. The use of periplasmic-leaky phoA+ hybrid plasmid-bearing mutants for an easier production and purification of alkaline phosphatase is discussed.

Overproduction and excretion of beta lactamase and alkaline phosphatase by escherichia coli olp mutants

We have isolated spontaneousolp mutants ofEscherichia coli K-12 overproducing the periplasmic enzymes β-lactamase (Bla) and alkaline phosphatase (PhoA). Enzyme overproduction was maintained inolp strains transformed with plasmids carryingbla + andphoA + structural genes, and synthesizing high levels of Bla and PhoA. Transformedolp strains excreted up to 40% of these enzymes into the growth medium. The introduction of atolA excretory mutation intoolp strains led to an increase of enzyme overproduction and a release of 85% of Bla and PhoA enzyme activities into the culture medium.

Regulation of the lkyB gene expression in Escherichia coli K-12 strains carrying an lkyB-lacZ gene fusion

SummaryPhage MudII301 was used to isolate new periplasmic-leaky mutants of Escherichia coli K12 carrying an lkyB-lacZ gene fusion. The properties of strain JC2299 carrying the lkyB-2299 insertion mutation were identical to those of strain JC207 carrying the previously described lkyB-207 mutation. The LkyB-beta-galactosidase hybrid protein was partially extracellular and membrane bound. It was shown that both a nonsense (envZ-22) and a polar (ompR::Tn10) mutation in the ompB operon led to an increase of beta-galactosidase activity in the lkyB-lacZ fusion strain. On the other hand, mutations in the phoB, phoR, phoS, phoT, malT or envY genes had no effect on lkyB gene expression.

Cloning of the lkyB (tolB) gene of Escherichia coli K12 and characterization of its product

SummaryThe lkyB gene of Escherichia coli K12 has been cloned from the Clarke and Carbon colony bank by selecting a ColE1 plasmid conferring cholic acid resistance to lkyB mutants. The lkyB gene was localized on hybrid plasmid pJC778 by analysis of mutated plasmids generated by Tn5 insertions. Restriction analysis and complementation studies indicated that plasmid pJC778 carried genes nadA, lkyB and sucA which mapped at min 16.5; the lkyB+ allele was dominant over the lkyB207 mutant allele. Analysis of cell envelope proteins from strains carrying plasmids pJC778 (lkyB+), pJC2578 or pJC2579 (lkyB::Tn5), as well as plasmid-coded proteins in a maxicell system, made it likely that the lkyB gene product was a membrane protein of molecular weight 42,000.

Cell localization of EcoRI endonuclease in Escherichia coli K-12

SummaryCell compartmentation of EcoRI endonuclease was analyzed using either parental or tolA excretory strains of Escherichia coli. Cells were subjected to various fractionation procedures such as osmotic shock or spheroplast formation. Our results showed that EcoRI activity was almost entirely recovered into cytoplasmic fractions and consequently was not released into the extracellular medium by a tolA mutant. These results did not support previous reports suggesting a periplasmic location for the EcoRI enzyme and did not allow to develop a simple method for EcoRI purification from culture supernatants of excretory mutants.

Isolation and Characterization of a New Type of Escherichia coli K12 phoB Mutants

SummaryA new type of Escherichia coli K12 phoB mutant was isolated as 5-fluorouracil-plus-adenosine-resistant derivatives of a upp phoS, T strain. Such phoB mutants (called type III) differ from previously described phoB mutants (types I and II) in the synthesis pattern of phosphate-regulated periplasmic proteins P4a and 30.5 K, sensitivity to toxic compounds, and several other phenotypic characters. The analysis of isogenic strains carrying phoB mutations (type I, II or III) showed that; the phoB gene product exerted (i) a positive control over the synthesis of periplasmic proteins 30.5 K, 11.5 K, and 9 K, inner membrane proteins 32 K and 17.5 K, and outer membrane protein Tsx, (ii) and a direct or indirect negative control over the synthesis of a 20 K phosphate-inducible periplasmic protein.

Overproduction and excretion ofβ-lactamase and alkaline phosphatase byEscherichia coli olp mutants

SummaryWe have isolated spontaneousolp mutants ofEscherichia coli K-12 overproducing the periplasmic enzymes β-lactamase (Bla) and alkaline phosphatase (PhoA). Enzyme overproduction was maintained inolp strains transformed with plasmids carryingbla+ andphoA+ structural genes, and synthesizing high levels of Bla and PhoA. Transformedolp strains excreted up to 40% of these enzymes into the growth medium. The introduction of atolA excretory mutation intoolp strains led to an increase of enzyme overproduction and a release of 85% of Bla and PhoA enzyme activities into the culture medium.