Department of Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK1
Author for correspondence: Ian R. Booth. Tel: +44 1224 273152. Fax: +44 1224 273144. e-mail: gen118{at}abdn.ac.uk
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ABSTRACT |
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Keywords: enteric bacteria, amino acid synthesis, acetate, weak acids, metabolite toxicity
Abbreviations: pHi, intracellular pH; pHo, external pH
a Present address: Zoonotic and Animal Pathogens Research Group, Department of Veterinary Pathology, Teviot Place, Edinburgh EH8 9AG, UK.
b Present address: Department of Microbiology, National University of Ireland Galway, Galway, Ireland.
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INTRODUCTION |
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In order to understand how cells respond to weak acid treatment a number of recent studies have looked at the global effects of weak acids on gene transcription (Arnold et al., 2001 ; Pomposiello et al., 2001
) and protein expression (Blankenhorn et al., 1999
; Lambert et al., 1997
). These studies demonstrate the complexity of the cells response to weak acid treatment. For example, treatment of cells with 5 mM sodium salicylate caused growth inhibition, and the transcription of 134 genes was significantly modulated (Pomposiello et al., 2001
). In the case of acetate treatment the expression of 86 genes was found to change significantly (Arnold et al., 2001
), and many of the changes involved genes that also show altered expression during exposure to salicylate. Proteomic studies have also led to the identification of several proteins whose expression is altered during weak acid stress (Blankenhorn et al., 1999
; Lambert et al., 1997
). Once again the response is complex, involving the induction of general stress proteins (e.g. ClpB, DnaK, GroL, UspA), transcriptional regulators (e.g. Fur, H-NS) as well as proteins involved in metabolism (e.g. MalE, MalX, AceA, PtsH). Despite all the data obtained during these studies, however, the basis for inhibition of growth by weak acids remains uncertain.
In a previous study we looked at the behaviour of Escherichia coli cells treated with acetic acid (Roe et al., 1998 ). The pHi is reduced from 7·85 to 7·48 when cells are grown in the presence of 8 mM acetate in a defined medium with a pH (pHo) of 6·0. We also found that cells treated with inhibitory concentrations of acetate accumulate high levels of acetate anions in the cytoplasm and this accumulation is compensated for, at least in part, by a reduction in the size of the intracellular glutamate pool. Furthermore, recovery of pHi after the removal of acetate was dependent on an ability of the cells to synthesize glutamate. These data suggested that growth inhibition could result either from the observed reduction in pHi or from some consequence of the intracellular accumulation of the acetate anion. The question of how growth inhibition was achieved therefore remained unresolved.
Previous studies suggest that the methionine biosynthetic pathway in E. coli can be perturbed by environmental stresses leading to a reduced growth rate, which is caused by partial methionine auxotrophy. The growth of E. coli cells in minimal medium is inhibited at temperatures above 40 °C and this inhibition can be overcome by supplementation of the growth medium with methionine (Ron & Davis, 1971 ). This observation is explained by the heat sensitivity of MetA, the enzyme that catalyses the conversion of homoserine to O-succinylhomoserine (Fig. 1
). In addition, a mutation conferring a defective heat-shock response maps to glyA, which encodes serine hydroxymethyltransferase (Gage & Neidhardt, 1993
). This enzyme is required for the biosynthesis of glycine from serine and, because of its role in regenerating the pool of N5-methyltetrahydrofolate, is also required for the biosynthesis of methionine (Fig. 1
). It has also been observed that methionine can relieve the inhibitory effects of acetate, which accumulates as a metabolic end product during fermentations with E. coli growing on glucose (Han et al., 1993
). However, in this case no explanation for the relieving effect of methionine was offered. Since this observation had the potential to reveal more about how cells are inhibited by weak acids we investigated it further.
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METHODS |
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Bacterial strains.
E. coli strains Frag1 (F- thi rha lac gal) and Frag5 (Frag1, kdpABC5) (Epstein & Kim, 1971
) were from our frozen stocks. MJF556 (Frag1, metB zij-2009::Tn10) was created by transducing Frag1 to tetracycline resistance using a P1 lysate grown on strain DV62 (metB zij-2009::Tn10) (Vallari & Rock, 1987
). The co-transduction of the Tn10 marker and the metB allele was confirmed by demonstrating that MJF556 was auxotrophic for methionine. Strains RG62 (metK84), RG109 (metK85) and their isogenic parent strain (K12 wild-type) were donated by Ron Greene (Basic Science Laboratory, Department of Biochemistry, Duke Medical Centre, Durham, NC, USA). Strain DG232 (glyA127) was obtained from Fred Neidhardt (University of Michigan, Medical School, Ann Arbor, MI, USA). Strains MJF378 (MJF274 rpoS::Tn10) and MJF358 (MJF274 rpoS::Tn10-kan) were used as donors in the P1 transductions to generate rpoS derivatives of Frag1. The rpoS::Tn10 alleles were originally derived from E. coli strains RH90 (obtained from R. Hengge-Aronis) and ZK1000 (obtained from A. Martinez), respectively.
Growth media.
The growth experiments in this study were all carried out in defined media based upon citrate/phosphate buffer at pHo 6·0 as used in previous studies (Roe et al., 1998 ). Measurements of growth were made by monitoring the OD650 of 1 ml samples. A single colony of E. coli was used as an inoculum for overnight growth under limiting glucose conditions (0·04%, w/v). Cells were subsequently supplemented with glucose (0·2 %, w/v) and allowed one cell-doubling step prior to dilution in fresh medium to an OD650 of 0·05. Methionine supplementation was performed using a 0·2 M (100x) methionine stock solution, prepared freshly and filter-sterilized on the day of the experiment. All specific growth rates are expressed as the mean of at least three independent growth experiments, ±SD.
pHi measurements.
The pHi was determined by using the distribution across the cell membrane of a radiolabelled weak acid following centrifugation (Kroll & Booth, 1981 ). In this method, bromododecane oil was used to separate the cell pellet from the supernatant, [7-14C]benzoic acid (4·5 µM; 0·1 mCi ml-1, 3·7 MBq ml-1) was used as the weak acid, and [3H]inulin (1·0 mM; 1·0 µCi ml-1, 1 kBq ml-1) was employed as an extracellular marker.
Pathway intermediate pool measurements.
Cells (OD650 0·6) were harvested for amino acid pool analysis by filtration of 1 ml through a Whatman membrane filter (cellulose nitrate; 0·45 µM pore size) under vacuum. Filters were washed immediately with 5 ml slightly hypertonic NaCl solution (medium +50 mM NaCl) added drop-wise to the filter. The filters carrying the cells were then placed in Eppendorf tubes containing 1 ml ice-cold 0·1% trifluoroacetic acid with norleucine (250 pmol ml-1) as an internal standard. The Eppendorf tubes were left on ice for 30 min to allow for extraction of the amino acids, the filters were removed, and the samples were then stored at -20 °C. The analysis of the amino acid pools was carried out as described previously (Amezaga et al., 1995 ; Roe et al., 1998
).
Overexpression of GlyA and MetE.
The glyA gene was PCR-amplified from E. coli strain Frag5 and cloned into vector pTrc99A (Amann et al., 1988 ) using standard techniques. Since there are two potential methionine start codons for GlyA both were used for cloning to produce plasmids pGlyA1 and pGlyA2. The 5' primers PG1 (gcccatggATGCGGATGTTAAAGCGTGAAATG) and PG2 (gcccatggATGTTAAAGCGTGAAATGACCATT) were designed to amplify products with alternative methionine start codons in conjunction with primer PGSTOP (ccctgcagGGCATGAACAACGAGCACATTGAC) at the 3' end of the glyA gene. E. coli Frag5 was used as a template for the PCR reaction, which followed standard conditions. Primers PG1 and PG2 incorporated NcoI sites and pGSTOP a PstI site that allowed the PCR products to be cloned into the region downstream of the inducible Trc promoter of pTrc99a. Cloned products were sequenced in both directions to ensure they were correct when compared with the published sequence of E. coli MG1655 (GenBank accession number NC000913).
The metE gene was overexpressed in Frag1 using plasmid pMetE, derived from pRSE562, which carries the metE and metR genes (Maxon et al., 1989 ). pMetE was constructed by removing the metR gene from pRSE562. This was achieved by digestion of pRSE562 with SalI and ligation of the gel-purified plasmid backbone using standard methods. The removal of metR was necessary since very high levels (40% of soluble protein) of MetE are produced from pRSE562 and this leads to growth inhibition (Gonzalez et al., 1992
). Overexpression of MetE was confirmed by SDS-PAGE analysis of crude extracts of Frag1 (pMetE).
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RESULTS |
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Methionine relief is independent of RpoS
The stress-inducible sigma factor, RpoS (s), is known to play an important role in acid tolerance in E. coli (Castanie-Cornet et al., 1999
) and it accumulates under conditions of weak-acid stress (Mulvey et al., 1990
; Schellhorn & Stones, 1992
). It was therefore possible that the relief of growth inhibition observed in the presence of methionine was dependent on this sigma factor. We examined this possibility by looking at the ability of methionine to relieve acetate-mediated growth inhibition in a strain lacking RpoS. Frag1 was transduced to either tetracycline resistance or kanamycin resistance with P1 lysates grown on strains MJF378 (MJF274 rpoS::Tn10) and MJF358 (MJF274 rpoS::Tn10-kan), generating Frag1 rpoS::Tn10 and rpoS::Tn10-kan, respectively. Western analysis confirmed that these strains did not express RpoS (data not shown). When these strains were grown at pH 6·0 in the presence of 8 mM acetate their growth was inhibited to the same extent as the wild-type parent. When methionine (2 mM) was included in the medium all three strains showed comparable relief (Table 2
). These data suggest that the relieving effect of methionine on weak-acid inhibition of growth is independent of RpoS.
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Three intermediates, homoserine, cystathionine and homocysteine (Fig. 1), were tested for their potential to relieve acetate-mediated growth inhibition. Homocysteine and homoserine were themselves found to inhibit the growth of Frag1, even in the absence of weak acid. The effect was most marked for homocysteine; growth of Frag1 in the absence of acetate was inhibited by approximately 70% when 2 mM homocysteine was included in the growth medium (Table 4
). Addition of acetate further increased the inhibition of growth (Table 4
). These inhibitory effects made it impossible to determine whether homoserine or homocysteine could bypass a potential acetate-mediated blockage in the methionine biosynthetic pathway. However, cystathionine, which feeds into the pathway after the MetB enzyme (cystathionine-
-synthase), was not inhibitory to the growth of Frag1 and we were unable to demonstrate relief from acetate inhibition when it was added to the growth medium at a concentration of 2 mM (Table 4
). This result was not due to a failure of cystathionine to support the biosynthesis of methionine in Frag1, since a Frag1-derived metB mutant (MJF556; Frag1, metB zij-2009::Tn10) could grow in methionine-deficient medium supplemented with cystathionine. Furthermore, the relief of acetate-mediated growth inhibition was only seen in MJF556 (metB) when methionine, but not cystathionine, was present in the growth medium (data not shown). These data suggest that inhibition by acetate might arise by blockage of a step after MetB in the methionine biosynthetic pathway.
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DISCUSSION |
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The data we have presented here suggest that in the presence of organic acids, at mildly acidic pH, the methionine biosynthetic pathway becomes inhibited at one of the steps below homocysteine. This has two effects on the cell: depletion of the intracellular methionine pool and accumulation of homocysteine, the immediate precursor of methionine in the biosynthetic pathway. Since methionine is essential for protein biosynthesis as well as being required for the biosynthesis of S-adenosylmethionine, which is itself required for several important cellular functions (Sekowska et al., 2000 ), it is likely that this will perturb the growth rate of cells.
How does the inclusion of methionine in the growth medium protect cells against inhibition by the weak acid acetic acid? First, methionine supplementation will overcome any growth inhibition arising from depletion of the intracellular methionine pool. Secondly, the presence of methionine in the medium is known to repress the methionine biosynthetic genes (Greene, 1996 ) and feedback-inhibit the activity of homoserine succinyltransferase (MetA), the first enzyme in the methionine biosynthetic pathway (Fig. 1
; Lee et al., 1966
), which will prevent synthesis of homocysteine. The mechanism by which homocysteine exerts its toxic effect is unclear. However, Hahn & Brown (1967)
showed that in Sarcina lutea homocysteine acts as a competitive inhibitor of methionyl-tRNA synthetase. When the intracellular ratio of homocysteine to methionine increases, which it is predicted to do when E. coli is treated with organic acids, such as acetate (Table 5
), the levels of tRNAMet will decline and growth inhibition will result. Relief in the presence of methionine could then occur by decreasing the homocysteine/methionine ratio, leading to restoration of the tRNAMet pools.
Previously we have considered the effects of organic acids primarily in terms of their lowering of cytoplasmic pH. At pH values close to the pK of the organic acid inhibition is multifactorial as was indicated above. It is clear that effects on cytoplasmic pH, perturbation of membrane lipids and effects of anion accumulation are likely to be maximal at these low pH values. However, at mildly acidic pH (pH 6) it is clear that the reduction in cytoplasmic pH by acetate is insufficient to cause profound growth inhibition, since in the presence of methionine the growth rate is restored to around 80% of the untreated control. Whether the enzymes of methionine synthesis are themselves sensitive to the lowered pH, organic anions or a combination of both is not clear. Incubation with acetate at pH 6 causes the accumulation of approximately 240 mM acetate anions in the cytoplasm (Roe et al., 1998 ). This accumulation is partially compensated for by the depletion of the glutamate pools. However 75% of the acetate anion pool must be compensated for by lowering the intracellular concentration of other anions, whose identity remains unknown (Roe et al., 1998
). It is conceivable that it is the depletion of these anions, which may represent important metabolic intermediates, that results in inhibition of methionine biosynthesis.
It is clear that methionine biosynthesis is unlikely to be the only pathway affected by incubation of cells with organic acids, since restoration of growth by methionine is not complete. Thus, we need to keep in mind that organic acids may exert their effects via changes in the activity of particularly sensitive pathways, such as that for methionine biosynthesis, and the more general inhibition arising from the change in cytoplasmic pH and anion composition. Clearly, in E. coli K-12 the inhibition arising from inclusion of acetate in the growth medium at mildly acidic external pH is more complex than simply altered cytoplasmic pH.
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ACKNOWLEDGEMENTS |
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Received 22 November 2001;
revised 11 March 2002;
accepted 22 March 2002.