Short communication
Substrate selection as a possible strategy for amelioration of acid
pH by
Rhizobium leguminosarum
biovar
viciae
Stella M. Castro
a, Andrew R. Glenn
b, Michael J. Dilworth
c,*
a
Facultdad de Ciencias Exactas, FõÂsico-QuõÂmicas y Naturales, Universidad Nacional de RõÂo Cuarto, CoÂrdoba, Argentina
b
Oce of the Pro Vice-Chancellor (Research), University of Tasmania, Hobart, Tasmania 7001, Australia
c
Centre for Rhizobium Studies, School of Biological Sciences and Biotechnology, Murdoch University, Murdoch, WA 6150, Australia
Accepted 12 January 2000
Abstract
A possible mechanism for bacteria to ameliorate an unfavorably acidic external pH would be to select for catabolism of those substrates in a mixture that would alkalinize the medium. This hypothesis was tested with cultures ofRhizobium leguminosarum
biovarviciaeWSM710 grown at pH 7.0 or 5.5 on binary mixtures of glucoseplusfumarate or glucoseplushistidine. The results showed no signi®cant increase in the absolute or relative utilization of the alkalinizing substrates (fumarate or histidine) at pH 5.5. 72000 Elsevier Science Ltd. All rights reserved.
Keywords:Acid pH amelioration; Root nodule bacteria; Substrate selection
Bacteria alter the pH of their environment (pHe)
during substrate metabolism. Sugar or sugar alcohol utilization often results in acidi®cation with organic acids produced as partial oxidation products, while or-ganic or amino acid use leads to alkalinization, either because of organic acid/OH-exchange or from ammo-nia release.
Under acidic conditions, amino acids such as lysine or arginine can be decarboxylated as a controlled re-sponse to pHe (Gale and Epps, 1942), involving the
conversion of lysine to cadaverine in Escherichia coli (the cad system; Meng and Bennett, 1992; Watson et al., 1992) or of arginine to agmatine and putrescine (the adi system; Stim and Bennett, 1993), and increas-ing pHe. In Clostridium acetobutylicum, a decrease in
pHe caused by the fermentation of sugar to acetic and
butyric acids results in a metabolic shift to reduction of butyric acid to butanol (Huang et al., 1986), while
low pHe causesLactobacillus plantarumto switch from
acidifying lactic acid production from sugars to neutral acetoin formation (Tsau et al., 1992).
While inducible systems are clearly harnessed to modify pHe, can organisms when given a mixture of
substrates, whose individual catabolism would inevita-bly lead to acid or alkali production, elect at acidic pH to selectively use those substrates that would result in alkalinization?
R. leguminosarum biovar viciae co-oxidises both components of a binary mixture of substrates at pH 7.0 (Dilworth et al., 1983) rather than showing the strict catabolite repression and diauxic growth of enterobacteria, and R. meliloti (Ucker and Signer, 1978) andR. leguminosarumbiovartrifolii(de Hollaen-der and Stouthamer, 1979) behave in a similar manner. A pHe change can be prevented by using a suitable
mixture of an acidifying substrate (glucose) and a sub-strate producing alkali (fumarate) (Glenn and Dil-worth, 1994). If low pH were to modify selection of substrates by cells to include a greater proportion of alkalinizing substrates, pHecould be increased. Such a
mechanism might operate in acid soils to create less Soil Biology & Biochemistry 32 (2000) 1039±1041
0038-0717/00/$ - see front matter72000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 0 0 8 - 0
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* Corresponding author. Tel.: 2112; fax: +61-8-9360-6486.
acidic microhabitats and allow organisms to exist (Richardson and Simpson, 1988) in conditions where bulk soil pH would predict that they would not. Whether soil carbon reserves are sucient for metab-olism-induced pH amelioration is, however, question-able. This work sought to determine if acidic pH resulted in greater catabolism of an alkali-producing substrate by R. leguminosarum biovar viciae from a mixture of alkali and acid producing substrates.
R. leguminosarum biovar viciae WSM710, a strain isolated from Pisum sativum in Japan, was obtained from Agriculture WA, Perth, Western Australia. Cells were grown with rotary mixing at 288C in the liquid minimal medium of Brown and Dilworth (1975) with 10 mM NH4Cl as the nitrogen source and 5 mM
glu-cose as carbon source to an A600 nm of approximately
1.They were then centrifuged and resuspended in buf-fered minimal salts at pH 7.0. An inoculum (1 ml) of this suspension was added to 50 ml buered minimal salts containing 10 mM NH4Cl at dierent pH values
and the following carbon sources: (a) 5 mM glucose; (b) 5 mM fumarate; (c) 5 mM L-histidine; (d) 5 mM glucose plus 5 mM fumarate; (e) 5 mM glucoseplus 5 mML-histidine. The buers used were 20 mM HEPES ((N-[2-hydroxyethyl]piperazine-N'-[2-ethane sulfonic acid]) at pH 7.0 or 20 mM MES (2-[N -morpholino]-ethane sulfonic acid]) at pH 5.5. These two pH values were selected to give a signi®cant pH dierence with-out moving into the ``transition'' zone for WSM710 (Glenn and Dilworth, 1994), where growth rate falls rapidly over a narrow pH interval. Growth of WSM710 on glucose as a sole carbon source acidi®es unbuered media, whereas growth on fumarate or his-tidine raises the pHe.
Growing cultures were sampled at 1-h intervals for 8 h of logarithmic growth, their absorbance measured at 600 nm (with dilution if necessary) and a culture supernatant retained for substrate assay. Adequacy of buering was con®rmed by lack of pH change during the 8 h growth period.
Glucose concentrations were measured by the glu-cose oxidase method with 4-aminophenazone (Trinder, 1969), fumarate concentration from its absorbance at 253 nm in 40 mM HEPES buer (pH 7.2) and histi-dine concentrations by the method of Ray (1967). To compensate for bacterial growth, the areas under the growth curves were calculated, and substrate concen-trations plotted against these values of A600dt,
result-ing in linear plots from which consumption rates could be calculated (de Hollaender and Stouthamer, 1979). These were converted to mmol hÿ1
(mg dry weight)ÿ1
using an experimentally determined value of 0.329 mg dry weight mlÿ1
for a culture with A6001:Each con-sumption rate, therefore, represents a statistically sig-ni®cant regression line (p < 0.001 in all cases). The data were then analysed statistically in two dierent
ways: the ®rst using an analysis of variance of two replicate consumption rates for each treatment and cal-culation of the probability that any two means were signi®cantly dierent; and in the second, the 16 data points for the two replicates of each treatment were combined into one linear regression line and the result-ing slopes compared in pairs. The two approaches yielded the same pattern of signi®cant dierences.
R. leguminosarum biovar viciae WSM710 grows rapidly on either glucose or fumarate as sole carbon source, but more slowly with histidine (Table 1). Both pH values are above the ``transition'' zone (Glenn and Dilworth, 1994) for this organism, and the adverse eect of lowered pH is, with the possible exception of histidine as a substrate, generally small.
The consumption rates for glucose or fumarate at pH 7.0 are similar (Table 2). For these single sub-strates at pH 5.5, there is a ca. 33% increase in the rate of glucose catabolism, but no statistically signi®-cant change in the rate of fumarate consumption.
When both the substrates are present at pH 7.0, both are catabolized simultaneously, with the decrease in the rate of glucose oxidation being greater than the decrease in the rate of fumarate oxidation. Shifting the pH to 5.5, however, results in a 44% decrease in the rate of fumarate consumption, but no change in the rate of glucose consumption relative to the respective values at pH 7.0. Contrary to what might have been expected if the cells were trying to moderate the acidic pH towards a more neutral one, the balance of sub-strate consumption is seen to shift towards a greater relative consumption of the acidifying substrate (glu-cose). The molar ratio between glucose and fumarate consumption rates changes from 0.74 at pH 7.0 to 1.33 at pH 5.5.
Growth was signi®cantly slower when histidine was the sole carbon source (Table 1) at pH 7.0; at pH 5.5 the rate of histidine utilization fell about 25% (but not signi®cantly) (Table 2). The combination of glucose with histidine resulted in a much decreased rate of his-tidine consumption at pH 7.0, although co-utilization
Table 1
Mean generation times for cells of R. leguminosarum WSM710 grown on various substrates or substrate mixtures
Growth substrate Mean generation time (h)a
pH 5.5 pH 7.0
Values represent the means2SEM of 2±4 determinations.
b
Signi®cantly dierent (p< 0.05).
S.M. Castro et al. / Soil Biology & Biochemistry 32 (2000) 1039±1041
still occurred, as previously reported (Dilworth et al., 1983). In the binary glucose±histidine mixture at pH 5.5, there was a signi®cant increase in the rate of glu-cose consumption compared to that at pH 7.0 (+45%), but a lesser and non-signi®cant change in the rate of histidine utilization (Table 2). The ratio between the rates of glucose and histidine consumption changed from 3.7 at pH 7.0 to 4.1 at pH 5.5. As with the glucose±fumarate substrate mixture, therefore, there was a relative increase in the consumption of the acidifying substrate.
We conclude that R. leguminosarum biovar viciae WSM710 does not select among the substrates tested for compounds whose catabolism would lead to modi-®cation of pHe to a more favorable value. It remains
to be determined if this ®nding in WSM710 may hold true for a wider variety of substrate pairs, or indeed for other bacteria able to co-utilize acid- and alkali-producing substrates.
References
Brown, C.M., Dilworth, M.J., 1975. Ammonia assimilation by Rhizobium cultures and bacteroids. Journal of General Microbiology 86, 39±48.
de Hollaender, J.A., Stouthamer, A.H., 1979. Multicarbon-substrate growth of Rhizobium trifolii. FEMS Microbiology Letters 6, 57± 59.
Dilworth, M.J., McKay, I.A., Franklin, M., Glenn, A.R., 1983. Catabolite eects on enzyme induction and substrate utilization in Rhizobium leguminosarum. Journal of General Microbiology 129, 359±366.
Gale, E.F., Epps, H.M.R., 1942. The eect of the pH of the medium
on the enzymic activities of bacteria (Escherichia coli and Micrococcus lysodeikticus) and the logical signi®cance of the changes produced. Biochemical Journal 36, 600±619.
Glenn, A.R., Dilworth, M.J., 1994. The life of root nodule bacteria in the acidic underground. FEMS Microbiology Letters 123, 606± 610.
Huang, L., Forsberg, C.W., Gibbins, L.N., 1986. In¯uence of exter-nal pH and fermentation products on Clostridium acetobutylicum intracellular pH and cellular distribution of fermentation pro-ducts. Applied and Environmental Microbiology 51, 1230±1234. Meng, S., Bennett, G.N., 1992. Regulation of the Escherichia coli
cad operon: a system for neutralization of low extracellular pH. Journal of Bacteriology 174, 2659±2669.
Ray, W.J., 1967. Photochemical oxidation. Methods in Enzymology 11, 490±497.
Richardson, A.R., Simpson, R.J., 1988. Enumeration and distri-bution of Rhizobium trifolii under a subterranean clover-based pasture growing in an acid soil. Soil Biology and Biochemistry 20, 431±438.
Stim, K.P., Bennett, G.N., 1993. Nucleotide sequence of the adiA gene, which encodes the biodegradative acid-induced arginine decarboxylase of Escherichia coli. Journal of Bacteriology 175, 1221±1234.
Trinder, P., 1969. Determination of glucose using glucose oxidase with an alternative oxygen acceptor. Annals of Clinical Biochemistry 6, 24±27.
Tsau, J.-L., Guanti, A.A., Montville, T.T., 1992. Conversion of pyruvate to acetoin helps to maintain pH homeostasis in Lactobacillus plantarum. Applied and Environmental Microbiology 58, 891±894.
Ucker, D.S., Signer, E.R., 1978. Catabolite-repression-like phenom-enon in Rhizobium meliloti. Journal of Bacteriology 136, 1197± 1200.
Watson, N., Dunyak, D.S., Slonczewski, J.L., Olsen, E.R., 1992. Identi®cation of elements involved in transcriptional regulation of the Escherichia coli cad operon by external pH. Journal of Bacteriology 174, 530±540.
Table 2
Consumption rates (mmol hÿ1 mg dry weightÿ1) for single substrates and binary mixtures during growth of R. leguminosarumbiovar viciae
WSM710a
Substrate
Glucose + fumarate Glucose +L-histidine
pH of medium Fumarate Fumarate Glucose Glucose Glucose L-Histidine L-Histidine
pH 7.0 6.89 ns 5.90 4.35 6.75 5.35 1.46 5.59
ns ns ns ns
pH 5.5 6.25 3.33 4.40 9.40 7.77 1.90 3.50
aEach value in the table is derived from the linear regression for combined data (0±7 h) from two replicate measurements of the consumption
rate. Symbols between the values indicate the statistical signi®cance of the dierence between them: ns, not signi®cant;,p< 0.05;,p< 0.01; ,p< 0.001.
