Osmotic adaptation of Escherichia coli with a negligible proton motive force in the presence of carbonyl cyanide m-chlorophenylhydrazone

Ohyama, Takao; Mugikura, Shigeru; Nishikawa, M; Igarashi, Kazuei; Kobayashi, Hiroshi

doi:10.1128/jb.174.9.2922-2928.1992

Cited by 18 publications

(22 citation statements)

References 35 publications

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“…The growth rate of the culture exposed to 1.0 mM DNP increased somewhat but never reached the pre-DNP growth rate. A similar growth response has been noted when E. coli is exposed to inhibitory concentrations of CCCP (18). PQQ glucose dehydrogenase (12).…”

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confidence: 51%

Adaptation of Escherichia coli to the uncoupler of oxidative phosphorylation 2,4-dinitrophenol

Gage

Neidhardt

1993

J Bacteriol

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Escherichia coli was found to adapt to the uncoupler of oxidative phosphorylation 2,4-dinitrophenol. The rates of synthesis of 53 proteins were increased following exposure to 2,4-dinitrophenol. Adaptation was accelerated when the cofactor pyrroloquinoline quinone was provided in the growth medium.In Escherichia coli, energy associated with the oxidation of an energy source is conserved as an electrochemical potential difference of hydrogen ions across the cytoplasmic membrane and is termed the proton motive force (Ap). The electrical potential (At) together with the difference in proton concentration between the periplasm and cytoplasm (zApH) make up the proton motive force, which is typically maintained at a steady-state value of -150 to -200 mV (14). Ap is harnessed to drive nutrient transport, ATP synthesis, and other endergonic reactions at the cell membrane.Uncouplers of oxidative phosphorylation, such as 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), are able to transport protons across biological membranes, which usually have extremely low proton conductance (16,17). By transporting protons down the electrochemical potential gradient, these agents collapse Ap. The collapse of Ap has detrimental effects on the bacterial cell: (i) the rate of proton-coupled ATP synthesis declines; (ii) the pH of the cytoplasm, typically maintained near 7.8 (3), approaches that of the external medium (8); and (iii) nutrient transport is inhibited, as is the translocation of proteins destined for insertion into the cytoplasmic membrane (1, 7, 10).Here we show that wild-type E. coli is able to adapt to the uncoupler of oxidative phosphorylation DNP and that the rate of adaptation is accelerated by the cofactor pyrroloquinoline quinone (PQQ). In addition, we show that E. coli induces approximately 50 proteins, including the heat shock proteins, upon exposure to DNP. E. coli adapts to DNP. E. coli W3110 growing in glucose-rich morpholinepropanesulfonic acid (MOPS) medium (GRM) without methionine (9) at 28°C was exposed to 0.5, 0.75, and 1.0 mM DNP. Following challenge with these concentrations of DNP, there were immediate cutbacks in the growth rate of 45, 75, and 90%, respectively (Fig. 1). Following the cutback in growth rate, cultures exposed to 0.5 and 0.75 mM DNP increased their growth rates and nearly reached the rate at which they grew before exposure to DNP. The growth rate of the culture exposed to 1.0 mM DNP increased somewhat but never reached the pre-DNP growth rate. A similar growth response has been noted when E. coli is exposed to inhibitory concentrations of CCCP (18 (Fig. 2). PQQ did not increase the growth rate of a culture growing in medium without DNP, indicating that PQQ did not act as a general growth enhancer. It was shown that glucose dehydrogenase, and not some other enzyme which utilizes PQQ as a cofactor, was responsible for the accelerated adaptation to DNP by conducting the same experiment using isogenic strain DG300 (gcd::cat), which contains a chloramphenicol resistan...

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confidence: 51%

Adaptation of Escherichia coli to the uncoupler of oxidative phosphorylation 2,4-dinitrophenol

Gage

Neidhardt

1993

J Bacteriol

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“…Ohyama et al (10) reported that E. coli can adapt itself to a hyperosmotic medium with a negligible proton motive force in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) and that glutamate was accumulated similarly in the presence and in the absence of this drug. We measured again the accumulation of glutamate of cells stressed osmotically by 0.6 M sucrose in the presence and in the absence of CCCP, and the results sometimes showed that the hyperosmosis-stressed cells accumulated a smaller amount of glutamate in the presence of CCCP than in its absence.…”

Section: Resultsmentioning

confidence: 99%

“…Amino acids and ␥-aminobutyrate were extracted from cells by heat treatment with 5% trichloroacetic acid, and their contents in the extract were analyzed with an amino acid analyzer (Hitachi 835) as described previously (10). The standard deviation of this measurement was less than 10%.…”

Section: Methodsmentioning

confidence: 99%

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Accumulation of glutamate by osmotically stressed Escherichia coli is dependent on pH

et al. 1995

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In the present study, we measured the accumulation of glutamate after hyperosmotic shock in Escherichia coli growing in synthetic medium. The accumulation was high in the medium containing sucrose at a pH above 8 and decreased with decreases in the medium pH. The same results were obtained when the hyperosmotic shock was carried out with sodium chloride. The internal level of potassium ions in cells growing at a high pH was higher than that in cells growing in a neutral medium. A mutant deficient in transport systems for potassium ions accumulated glutamate upon hyperosmotic stress at a high pH without a significant increase in the internal level of potassium ions. When the medium osmolarity was moderate at a pH below 8, E. coli accumulated ␥-aminobutyrate and the accumulation of glutamate was low. These data suggest that E. coli uses different osmolytes for hyperosmotic adaptation at different environmental pHs.Since bacterial habitats encompass a wide range of environmental conditions, bacteria are useful species for investigating the environmental adaptation of living things. Osmolarity of growth medium is one of the important conditions for bacterial growth, and there have been numerous reports concerning bacterial adaptation to changes in external osmolarity (1).Many materials have been identified as being accumulated after hyperosmotic shock in order to increase the cytoplasmic osmolarity or to protect cellular metabolism under high osmotic pressure. For example, potassium ion, proline, betaine, trehalose, glutamate, glutamine, glutamyl peptide, and taurine, etc., have been reported to function as compatible solutes or osmoprotectants (1, 5-8). Some of them are probably not absolutely essential for osmotic adaptation, but they may increase the growth rate of hyperosmosis-stressed cells. Thus, in order to clarify the mechanism of bacterial osmotic adaptation, examination of the minimum requirement for the adaptation would be required.Potassium glutamate is known to be a major osmolyte of Escherichia coli growing in hyperosmotic minimal medium (1, 7). In the present study, we investigated the accumulation of glutamate at various pH values of the minimal medium and found that the internal level of glutamate was increased at a pH above 8. ␥-Aminobutyrate was accumulated when the glutamate accumulation was low upon moderate hyperosmotic shock at a pH below 8. Thus, E. coli seems to use different osmolytes at different pH levels. MATERIALS AND METHODSBacterial strains and growth media. E. coli W3110 and TK2642 [F Ϫ thi lac rha trkA405 trkD1 del(kdpABC)54 nagA malT] were generously supplied by Y. Anraku (Tokyo University) and W. Epstein (Chicago University), respectively. E. coli was grown at 37ЊC in minimal medium containing 5 mM K 2 PO 4 , 20 mM (NH 4 ) 2 SO 4 , 1 mM MgCl 2 , 0.1 mM CaCl 2 , 1% glucose, and thiamine (2 g/ml). Tricine [tris(hydroxymethyl)methylglycine] (60 mM) or HEPES (N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic acid) was used as a buffer, and the medium pH was adjusted by the addition of KO...

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“…GDH activity in vitro is also stimulated by K ϩ (38), a finding which has been interpreted as the possible basis for increased glutamate levels in high-osmolarity-grown cultures. However, Ohyama et al (41) have shown that glutamate accumulation at elevated osmolarity is unaffected even in cells that fail to accumulate K ϩ . In this report, we describe the identification of a gltBD mutant derivative of E. coli (in which glutamate synthesis can proceed only via GDH) that is osmosensitive even at high [NH 4 ϩ ].…”

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confidence: 99%

Roles of SpoT and FNR in NH4+ assimilation and osmoregulation in GOGAT (glutamate synthase)-deficient mutants of Escherichia coli

Saroja

Gowrishankar

1996

J Bacteriol

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An osmosensitive mutant of Escherichia coli was isolated and shown to harbor two mutations that were together necessary for osmosensitivity. One (ossB) was an insertion mutation in the gltBD operon, which encodes the enzyme glutamate synthase (GOGAT), involved in ammonia assimilation and L-glutamate biosynthesis. The other (ossA) was in the fnr gene, encoding the regulator protein FNR for anaerobic gene expression. Several missense or deletion mutations in fnr and gltBD behaved like ossA and ossB, respectively, in conferring osmosensitivity. A mutation affecting the DNA-binding domain of FNR was recessive to fnr ؉ with respect to the osmotolerance phenotype but was dominant-negative for its effect on expression of genes in anaerobic respiration. Our results may most simply be interpreted as suggesting the requirement for monomeric FNR during aerobic growth of E. coli in high-osmolarity media, presumably for L-glutamate accumulation via the GOGAT-independent pathway (catalyzed by glutamate dehydrogenase [GDH]), but the mechanism of FNR action is not known. We also found that the spoT gene (encoding guanosine 3,5-bispyrophosphate

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Osmotic adaptation of Escherichia coli with a negligible proton motive force in the presence of carbonyl cyanide m-chlorophenylhydrazone

Cited by 18 publications

References 35 publications

Adaptation of Escherichia coli to the uncoupler of oxidative phosphorylation 2,4-dinitrophenol

Adaptation of Escherichia coli to the uncoupler of oxidative phosphorylation 2,4-dinitrophenol

Accumulation of glutamate by osmotically stressed Escherichia coli is dependent on pH

Roles of SpoT and FNR in NH4+ assimilation and osmoregulation in GOGAT (glutamate synthase)-deficient mutants of Escherichia coli

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