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In Escherichia coli membranes, three major phospholipids are formed: phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL). We report here the survival of mutants lacking either PE or both PG and CL at an acid pHo and during long-term survival experiments. Stationary phase cultures of E. coli lacking PE are much more sensitive to acid shock (pHo 3) than the wild-type strain. Moreover, in the strain lacking PE, long-term survival in stationary phase is impaired and after 5 days no viable cells are recovered. The survival of an exponential phase culture to acid shock is known to be increased if the culture is exposed to moderately acid conditions (pHo 5) prior to a shift to pHo 3. If either PE or both PG and CL are missing, the exposure to pHo 5 does not increase the survival at pHo 3.

The expression of the maltose system in Escherichia coli is regulated at both transcriptional and translational levels by the pH of the growth medium (pHo). With glycerol as the carbon source, transcription of malT , encoding the transcriptional activator of the maltose regulon, is weaker in acidic medium than in alkaline medium. malT transcription became high, regardless of the pHo, when glycerol-3-phosphate or succinate was used as the carbon source. Conversely, malT expression was low, regardless of the pHo, when maltose was used as the carbon source. The increase in malT transcription, associated with the pHo, requires the presence of glycerol in the growth medium and the expression of the glycerol kinase (GlpK). Changes in the level of glpK transcription had a great effect on malT transcription. Indeed, a glpFKX promoter-down mutation has been isolated, and in the presence of this mutation, malT expression was increased. When glpK was expressed from a high-copy-number plasmid, the glpK -dependent reduced expression of the maltose system became effective regardless of the pHo. Analysis of this repression showed that a malTp1 malTp10 promoter, which is independent of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex, was no longer repressed by glpFKX amplification. Thus, GlpK-dependent repression of the maltose system requires the cAMP-CRP complex. We propose that the pHo may affect a complex interplay between GlpK, the phosphotransferase-mediated uptake of glucose, and the adenylate cyclase.

The porin composition of the Escherichia coli cell envelope was analyzed during growth at different external pHs (pHo) as a function of the acetyl phosphate (AcP) level (Δ ackA pta or ackA mutant, pyruvate or glucose as the carbon source) in the presence or absence of EnvZ. Our results indicate that the AcP level is influenced by the pHo, leading to modulation of the amount of OmpR-P and subsequent pHo-dependent expression of ompF and ompC . We also propose the existence of a specific signal, independent of EnvZ and AcP, leading to OmpR phosphorylation in response to pyruvate.

Summary: The Escherichia coli maltose regulon consists of five operons under the control of the MalT transcriptional activator. lac operon fusions were constructed in vitro with the MalT-dependent promoter and with the malT promoter itself. -Galactosidase activity displayed by these fusions during growth at different external pH (pHo) revealed that growth at a pHo higher than 6 stimulates the transcription of malT- and MalT-controlled genes in the absence or presence of maltose. Using a malTp1 malTp10 promoter that is cAMP-CRP (cAMP receptor protein)-independent, it was demonstrated that CRP is essential for malT pHo regulation and that the pHo-dependent activity of malKp is a direct consequence of malT regulation. The pHo regulation displayed by a deleted but still functional malT promoter fused to lacZ demonstrates that this minimal promoter contains all the regulatory regions for establishing pHo regulation. In the absence of Mlc, a repressor of malT expression, the pHo regulation of malT was still effective. It is proposed that binding of cAMP-CRP at malTp may be affected by malTp topology induced by pHo or that a pHo-dependent effector may act in concert with the cAMP-CRP complex.

We have identified a conditional mutation which confers a pleiotropic phenotype to Escherichia coli cells: no growth at temperature higher than 36 degrees C, an altered control of the synthesis of several phosphate-regulated polypeptides (including alkaline phosphatase, sn-glycerol-3-phosphate binding protein, phosphate binding protein and outer membrane porin protein PhoE) after growth at 36 degrees C and a wild-type phenotype at 30 degrees C. This mutation was located at minute 89.5 on the E. coli chromosome in a gene we have called cpr for conditional phosphate-regulated.

Summary Using a set of gene fusions generated with TnphoA, we previously identified the phmA locus, whose expression is modulated as a function of external pH (pHo). The phmA::phoA fusion was cloned and sequenced and the phmA locus was identified with the nmpC gene. This gene lies within the defective lambdoid prophage qsr′ and NmpC is an outer membrane protein which functions as a porin. We demonstrated that nmpC is sensitive to catabotite repression and dependent on the CRP—cAMP complex. However, cAMP is not a signal for the pHo-dependent expression of nmpC. By generating step deletions in the sequence 5′ to the nmpC coding region, we identified a DNA region in position —345 to —127 which is involved in nmpC repression, mainly during growth at acid pHo. Four regions with strong homologies and a very well-conserved organization of the functional sequence were found in the nmpC and ompF promoters. We propose that the negative regulation of nmpC during growth at low pHo might involve DNA looping of the nmpC promoter.

Analysis of porin gene expression as a function of extracellular pH (pHo) showed that ompF and lamB expression passed through a maximum, whereas ompC expression passed through a minimum at pH 8. This pH-dependent control operated at both transcriptional and post-transcriptional levels of ompF, but only at the transcriptional level on ompC. Similar studies carried out with tolA, tol B and excC mutant strains showed that the TolA, TolB and ExcC proteins are involved in the pH regulation of porin genes.

Electrophoretic 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.

A 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.