External-pH-dependent expression of the maltose regulon and ompF gene in Escherichia coli is affected by the level of glycerol kinase, encoded by glpK

Abstract

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.

FIG. 1

Schematic representation of the glpFKX operon cloned in pACYC177 and sequence of the parental and mutant glp promoter regions. (A) Open reading frames within the glpFKX operon, with the direction of transcription, are indicated by open arrows. Restriction sites: EI, EcoRI; BI, BamHI; R, RsrII; EV, EcoRV; BII, BstEII; A, AatII. The glp transcriptional start site is indicated with an arrow upstream of glpF (32). The primers used in PCR amplification are indicated with triangles. (B) Grey boxes indicate the binding site of the GlpR repressor. Striped boxes indicate the cAMP-CRP binding site. The transcriptional start point is at position +1 (32). Putative −10 and −35 sequences are underlined.

FIG. 2

pHo regulation of malT and malK during growth with different carbon sources. β-Galactosidase activity was assayed during growth at 30°C in MM adjusted to different pH values and supplemented with ampicillin and the indicated carbon source. (A) (malT′-lacZ⁺)1 operon fusion on pJEL250 (GPH8881); (B) (malK′-lacZ⁺)1 operon fusion on pJEL250 (GPH1768). (Inset) Induced malK expression with maltose as the carbon source. Error bars show standard deviations. The absence of error bars indicates that the deviation fell below the resolution limit of the graphing program.

FIG. 3

Influence of the iea18 mutation on malK, malP, and malT pHo regulation. β-Galactosidase activity was assayed during growth at 30°C in MM adjusted to different pHs and supplemented with ampicillin and glycerol (A and C) or glycerol and maltose (B). ⧫, parental strain; □, iea18 strain. (A) (malK′-lacZ⁺)1 operon fusion on pJEL250 (GPH1768 and GPH8840); (B) (malP′-lacZ⁺)1 operon fusion on pJEL250 (GPH9463 and GPH9473); (C) (malT′-lacZ⁺)1 operon fusion on pJEL250 (GPH8881 and GPH9401). Error bars are as in Fig. 2.

FIG. 4

ompF and ompC expression at pHo 5 and 7 in parental and iea18 strains. Strains were grown at 30°C in MM adjusted to pH 5 or 7 and supplemented with glycerol as the carbon source. (A) GPH9604 parental strain and GPH9605 iea18 strain; (B) GPH9910 parental strain and GPH9909 iea18 strain. Error bars are as in Fig. 2.

FIG. 5

Parental and mutant glp promoter activities according to the pHo. β-Galactosidase activity was assayed during growth at 30°C in MM adjusted to different pHs and supplemented with glycerol and ampicillin. (A) (glpF′-lacZ⁺)1 operon fusion on pJEL250 (pGPH11247 in MC4100); (B) (glpFp18′-lacZ⁺)1 operon fusion on pJEL250 (pGPH11248 in MC4100). Error bars are as in Fig. 2.