Plant and Soil 171: 63-69, 1995. (~) 1995 Kluwer Academic Publishers. Printed in the Netherlands.

A mechanism of aluminium toxicity to soil bacteria and possible ecological implications

Martin Wood Department of Soil Science, University of Reading, London Road, Reading, RGI 5AQ, UK

Key words: aluminium, mutations, Pseudomonasfluorescens, Rhizobium, soil pH, toxicity mechanism

Abstract

Pseudomonasfluorescens and Rhizobium showed similar responses to A1 in defined medium. Evidence that DNA is a site of action of A1 in Rhizobium, and for mutagenic effects of AI in laboratory media, is reviewed. Preliminary data are presented which indicate that A1 tolerance is not a plasmid-borne trait in these bacteria. The possible role of A1 in the ecology of bacteria in soil is discussed in relation to the neutralisation of A1 brought about by changes in soil pH by bacterial metabolism.

Introduction

Soil is an environment rich in its diversity of bacterial species; it has been estimated that half of the known bacterial genera contain species which can be consid- ered as soil bacteria (Clark, 1967). Soil also contains aluminium in a variety of forms such as aluminosili- cate minerals, and exchangeable or soluble A1 (Sposito, 1989). For soil microorganisms AI is therefore a ubiq- uitous component of the environment, and although poorly understood, the relationship between the two may extend back to the origin of life itself (Cairns- Smith, 1985). The physical interaction between con- temporary soil microorganisms and clay minerals has received much attention (Stotzky, 1986).

However, as soils become more acid (a con- sequence of both natural weathering processes and anthropogenic factors) A1 becomes more soluble and potentially toxic to plants (e.g. Foy and Brown, 1963) and microorganisms (e.g. Keyser and Munns, 1979). Although there is much information on the possible mechanisms of A1 toxicity to plants (Taylor, 1988) AI toxicity to microorganisms has received little attention (Flis et al., 1993).

We have studied the effects of A1 on Rhizobium, and to a lesser extent on Pseudomonasfluorescens, and have attempted to elucidate a mechanism of toxicity, and to consider the ecological implications of these findings.

The aims of this paper are to present

• data on AI toxicity to Pseudomonasfluorescens;

• evidence for DNA as a site of action for Al in Rhizobium;

• evidence for mutagenic effects of A1 on Rhizobium;

• preliminary indications of the genetic basis of A1 tolerance in Rhizobium and Pseudomonas fluo- rescens;

• possible ecological implications arising from the effects of A1 on soil bacteria.

Materials and methods

A I toxicity to Pseudomonas fluorescens

Pseudomonasfluorescens strains 1G and 1 E, obtained from the Department of Microbiology, University of Reading were grown on King's B medium (King et al., 1954) and checked for fluorescence under ultra-violet light. A defined glutamate-mineral salts liquid medi- um was used for AI studies with the following com- position (#M) : CaCI2. 6H20, 1000; MgSO4.7H20, 500; KC1, 50; FeEDTA, 25; KHzPO4, 10; H3BO3, 10; MnSO4.4H20, 1; ZnSO4.7H20, 0.5; CuSO4.5H20, 0.1; Na2MoO4, 0.025; COC12.6H20, 0.005. Sodium glutamate (1.8 g L -I) was added before autoclav- ing and the pH value of the medium adjusted with hydrochloric acid (SG 1.18). Where required, A1 was added after autoclaving as a filter-sterilised solution of A1K(SO4)2.12H20. The medium was dispensed in 25 mL aliquots into 50 mL Erlenmeyer flasks. Solid

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defined medium was prepared as above with the addi- tion of agar (1% w/v).

Strains IG and 1E were grown to >107 cfu mL -1 l in liquid medium at pH 6.7, and after dilution in

strength Ringer's solution, used to inoculate 25 mL aliquots of liquid medium at pH 6.7, 5.5, 5.0 or 4.5. Duplicate flasks were incubated at 25 °C. The pH was measured each day for eight days. Viable counts were made each day on nutrient agar using the technique of Miles and Misra (1938).

The experiment was repeated using 0, 10, 20 and 50 #M A1 at pH 5.5 for both strains and at pH 5.0 for strain 1E.

Plasmid-curing of strains 1G and 1E was carried out using either heat (Zurkowski and Lorkiewicz, 1978) or mitomycin C (Chakrabarty, 1972). Four mL aliquots of defined medium containing 108 cfu mL-1 of either strain were incubated in test tubes without mitomycin C at25 °C, 31 °C or 37 °C and with 5, 10or 15/lg mL -1 mitomycin C at 25 °C. After 4 days samples from each treatment were diluted to give approximately 100 cfu when plated on solid defined medium and incubated for 3 days at 25 °C. Four replicate plates were used for each curing treatment. Ninety six colonies from the 25 °C treatment without mitomycin C (control) and 192 colonies from all other treatments were then trans- ferred to individual wells in microtitre plates (Sterilin) containing 200 pL of defined medium at pH 6.7. After incubation for 4 days at 25 °C, approximately 103 cells were transferred from each well, using a multi-point inoculator, to fresh defined medium at pH 5.5 contain- ing either no AI or 20 pM AI in microtitre plates. The appearance of visible turbidity within 5 days at 25 °C was the criterion for tolerance.

Modification of soil pH by Rhizobium

The model described by Darrah et al. (1987) which was originally used to study pH changes associated with nitrification was used here to predict the localised changes in soil pH due to metabolism of soluble organ- ic compounds by Rhizobium loti in soil solution.

The following data were used for the model to pre- dict the changes in pH values with time and distance, under two different cell distributions: (i) the cells were assumed to be uniformly and singly distributed (occu- pying 99% of the soil volume); (ii) the cells were assumed to be distributed in clusters (occupying 1% of the soil volume) within soil. Number of clusters cm -3 soil: 50,000 Stoichiometry of acid/alkali production: -0.97 × 10 -3

8

. ' " 6

,,-1

2

/ ~ IF . . . . . . • . . . . . . • . . . . . . • . . . . . . • . . . . . . - I I . . . . . . . • •

1 2 3 4 5 6 7 8 T i m e (days )

Fig. 1. Multiplication (solid lines) and changes in acidity (broken lines) for P. fluorescens strain IG at initial pH values of 5.5 (O) and 4.5 ( i ) in defined glutamate-mineral salts medium.

Volume fraction of soil occupied by biomass: 1%, 99% Initial soil pH:5.0 pH buffer capacity (mol dm -3 soil): 0.35 × 10 -1 Initial biomass index (mol dm -3 soil): 0.0014 Maximum specific growth rate at optimum pH (h-l): 0.27 Volumetric moisture content (dm 3 dm-3): 0.3 Impedance factor: 0.3 Incubation time (h): 350

The data were based on the pH changes brought about by Rhizobium loti strain NZP 2037 grown in soil solution which had been extracted from Rowland series soil (a sandy loam) by the method of Edmunds and Bath (1976), fiiter-sterilised, adjusted to pH 5, inoculated with NZP2037 at an initial cell density of 1000 cfu mL - t , and incubated at 25 °C to a final density of 108 cfu mL -1 in the extracted soil solution.

Results and discussion

Al toxicity to Pseudomonas

Both strains of Pseudomonas multiplied to >107 cfu mL -1 at pH 6.7 and 5.5, but died at pH 4.5 (Fig. 1). At pH 5.0 strain 1E multiplied, but strain 1G died. Growth rate was reduced as the pH value decreased. The pH of the defined medium remained constant until the cell density reached approximately 108 cfu mL -1 (Fig. 1), thereafter the buffering capacity was exceeded and the pH increased. Therefore, the data indicated the ability of the organism to multiply at that pH value and

8

7

~6 45

3

2

1 I I I I I t t I 2 3 4 5 6 7

T i m e ( d a y s )

Fig. 2. Multiplication of P fluorescens strain IG at pH 5.5 with 0 (Q)), 10 (&), 20 (0) or 50 #M, AI (11) in defined glutamate-mineral salts medium.

not merely to produce more favourable conditions of acidity before multiplication occurred. P. fluorescens is generally considered to be sensitive to pH values less than 5 (Stanier et al., 1966), however, these results indicate inter-strain variation in response to pH 5 and other strains (not reported here) are tolerant of pH 4.5 in glutamate-mineral salts medium.

Both strains multiplied with 10 and 20/~M A1 at pH 5.5 and the more acid-tolerant strain 1E also mul- tiplied with these levels of A1 at pH 5.0. Both strains died with 50 #M A1 at pH 5.5 and 5.0. A1 caused an increase in lag time and reduced growth rate (Fig. 2). Rhizobium leguminosarum biovar trifolii is also sensi- tive to pH 4.5, and to 5.0 ~uM A1 at higher pH values (Wood and Cooper, 1985). However, Rhizobium loti is tolerant of these levels of acidity and AI (Wood et al., 1988). Pseudomonas therefore appears to be as sus- ceptible/tolerant to A1 as Rhizobium leguminosarum biovar trifolii.

Mechanism of A1 toxicity to Rhizobium

Flis et al. (1993) have provided an overview of the possible mechanisms involved in A1 toxicity to root- nodule bacteria. We were particularly interested in the interaction between A1 and DNA, and the conse- quences of this for Rhizobium. Early work reviewed by Wood (1986) attempted to reconcile limited knowledge of the chemistry of AI in complex solutions such as growth media with effects on cell viability. An appar- ent anomaly in this work was the observation that AI in defined medium at pH 5.5 was inhibitory despite

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the appearance of a visible precipitate of aluminium hydroxide (Wood and Cooper, 1984). This was taken as an indication that either AI was indirectly affect- ing the rhizobia by reducing the supply of an essential nutrient such as phosphate, or that polymeric forms of A1, presumed to be present at pH 5.5, were directly tox- ic to the bacteria. However, there was no convincing evidence to support these hypotheses (Wood, 1986).

Munns and Keyser ( 1981) showed that cells of Rhi- zobium were most sensitive to AI at the moment of cell division, and Wood and Cooper (1988) reported that cells undergoing rapid cell division (during the expo- nential phase of growth) were more susceptible to A1 than cells in stationary phase. There was also evidence that A1 could bind to mammalian DNA in vitro (Kar- lick et al., 1980) and to plant DNA in vitro (Morimura and Matsumoto, 1978). Could DNA also be the target of A1 in prokaryotic cells?

Preliminary evidence came from a microscopy study of Rhizobium after exposure to AI; cells were elongated as if replication, but not cell growth, had been halted. Furthermore these symptoms resembled those caused by mitomycin C (Johnson, 1988), an antibiotic which acts by cross-linking the DNA duplex (Franklin and Snow, 1981).

More detailed in vivo studies on A1 toxicity involv- ing the relatively tolerant Rhizobium Ioti and the rela- tively sensitive Rhizobium leguminosarum biovar tri- folii were reported by Johnson and Wood (1990). Alu- minium analysis of nucleic acid extracted from cells exposed to A1 in defined medium showed that A1 could (a) penetrate the cell envelope, (b) associate with nucle- ic acid, of both sensitive and tolerant strains. Gel filtra- tion of the nucleic acid and treatment with DNase and RNase prior to filtration showed that the A1 bound to DNA of sensitive and tolerant strains. Once inside the cell the AI would form predominantly polymeric com- plexes in response to the near-neutral pH (O'Hara et al., 1989). These positively-charged polymers could cross- link the phosphate groups of the two DNA strands as suggested for plant DNA by Morimura and Matsumoto (1978).

Despite causing a reduction in viability, AI caused an increase in DNA synthesis in the AI sensitive strain, as indicated by [3H] thymidine incorporation (Johnson and Wood, 1990). DNA synthesis was unaffected in the A1 tolerant strain. The increased DNA synthesis in the sensitive strain may have been associated with an exci- sion repair response by the cells to the damage caused by the A1, similar to that reported for Escherichia coli (Walker, 1985). The drastic repair response in the sen-

66

sitive strain may in itself have contributed to the loss of viability of the cells.

The mechanisms involved in the differential tol- erance to AI by these two species of Rhizobium are not known; it may be that the tolerant strain is able to restrict the amount of AI binding to its DNA, or it is able to remove the AI bound to its DNA more effectively than the sensitive strain. However, if the external concentration of A1 is increased then the tol- erant strain becomes susceptible; data for mutagenic effects presented below indicate that an error-prone repair mechanism is then induced.

Evidence for mutagenic effects of Al

The studies on the mechanism of A1 toxicity dis- cussed above produced circumstantial evidence that A1 is mutagenic:

(i) The effects of A1 on cell viability and morphology were similar to those of mitomycin C (Johnson, 1988) a known mutagen (Moseley and Copland, 1978).

(ii) Associated with a decrease in viability of Rhizobi- um leguminosarum biovar trifolii strain RDG 2002, AI caused an increase in DNA synthesis (John- son and Wood, 1990). In Escherichia coli such a response is associated with a mutagenic SOS-type repair response (Walker, 1985).

Octive et al. (1991) provided further evidence for mutagenic effects of AI; exposure of Rhizobium legu- minosarum biovar trifolii and Rhizobium loti to a con- centration of A1 which inhibited multiplication of both strains caused an increase in rifampicin resistance in both strains. There were several similarities between the response of the strains to A1 and to mitomycin C; greater tolerance to AI in Rhizobium loti was associat- ed with greater tolerance to mitomycin C, and a lower incidence of mutation in response to A1 was associated with a lower mutation rate in response to mitomycin C. This suggested that the more tolerant Rhizobium loti had a DNA repair system which was less prone to error than that in the more sensitive Rhizobium legumi- nosarum biovar trifolii. The mechanism of DNA repair in Rhizobium is not known, but the recA gene which is involved in both DNA repair and mutagenesis has been identified in Rhizobium meliloti (Better and Helinski, 1983).

Table 1. The response of Pseudomonas fluorescens strain 1G to 20 /zM AI at pH 5.5 in liquid medium following treatment with either heat or mitomycin C

Temp Mitomycin C No. colonies No. colonies tolerant (o C) (Izg mL- l ) tested of 20 p.M A1 at pH 5.5

25 0 96 96 25 5 192 192 25 10 192 192 25 15 192 192 31 0 192 192 37 0 192 192

Genetic basis of Al tolerance in Rhizobium and Pseudomonas

There is no information on the genetic basis of AI tolerance in Rhizobium or Pseudomonas, although tol- erance of heavy metals is known to be a plasmid-borne trait in pseudomonas (Chakrabarty, 1976). Two lines of preliminary evidence are presented here for the lack of involvement ofplasmids in A1 tolerance in these soil bacteria.

Firstly, Wood et al. (1988) reported that Rhizobium loti strain NZP2042 showed a response to pH and A1 similar to that of NZP2037 referred to above. Howev- er, Pankhurst et al. (1986) showed that strain NZP2042 does not have plasmids. Tolerance of acidity and A1 in this strain (and there are others similar) cannot there- fore involve genes carried on plasmids.

Secondly, two techniques were used in an attempt to remove plasmids from Pseudomonas. All of the colonies which had been treated with either heat or mitomycin C responded to 20/~M A1 at pH 5.5 in the same way as colonies which had not received these treatments (Table 1). Mitomycin C removes plasmids from P.fluorescens with an efficiency of 99% (Bopp et al., 1983). The curing efficiency of heat at 35 °C varies from 0-75% for Rhizobium leguminosarum biovar tri- folii (Zurkowski and Lorkiewicz, 1978). These high efficiencies for plasmid curing together with the large number of colonies screened (192 for each treatment) suggest that the genes for A1 tolerance in P. fluorescens are not carried on plasmids.

Ecological aspects of Al toxicity

The finding that A1 can act as a mutagen has potential- ly important implications for soil microorganisms. In addition to changes in rifampicin resistance (Octive et al., 1991), not in itself likely to be of major ecolog- ical importance, previous exposure of Rhizobium and Bradyrhizobium to A1 in laboratory media has been shown to cause permanent changes in motility, nodu- lating ability and effectiveness (Octive et al., 1994). It is not known if such changes occur in soils in which bacteria are exposed to soluble AI.

From the evidence presented it does seem likely that A1 is a major factor in the ecology of root-nodule bacteria and other microorganisms in acid soils. There might be a link between the amounts of soluble or exchangeable A1 in soils and either the size or func- tion of a microbial population. Wood et al. (1985) reported that numbers of clover rhizobia in a range of Northern Ireland pasture soils were not correlated with the exchangeable A1, but were correlated with the exchangeable A1 expressed as a proportion of the effective cation exchange capacity. A1 saturation is believed to provide a more accurate estimate of the potential toxicity of A1 in soils than is exchangeable A1 alone (Kamprath, 1970).

More detailed studies showed no relationship between soil pH (a good general indicator of the poten- tial for A1 toxicity) and tolerance of either 5 pM AI at pH 5.5 (Wood and Cooper, 1985) or 10 pMAI at pH 4.5 (Wood and Shepherd, 1987) for clover rhizobia. Sim- ilar findings were reported for bean rhizobia isolated from Kenyan soils (Karanja and Wood, 1988).

Bulk soil properties such as pH may not be wholly relevant in defining the environment around a bacteri- um or bacterial colony in soil. Such localised changes in soil pH have been well established for the soil around plant roots (Nye, 1981), and have been invoked to explain discrepancies observed between acid tolerance as determined in pure culture studies and population distribution in acid soils for nitrifiers (Hankinson and Schmidt, 1984) and for streptomyces (Williams and Mayfield, 1971).

Norris (1965) attributed a major ecological role to the production of acid or alkali by root-nodule bacte- ria grown in laboratory media. In order to obtain an estimate of the ability of rhizobia to increase the pH of the soil as a result of metabolism of soluble organ- ic compounds present in soil, data for Rhizobium loti strain NZP2037 grown in a soil solution extracted from Rowland series soil were used with the model of Dar-

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pit

5.9

5.8

5.7

5.6

5.5

5.4

5.3

5.2

5.1

5

Clusler = 1% /

(a) /

. . . . Cluster = 99

50 100 150 200 250 300 350

Time (h)

5.8 Cluster= 1%

| 5.7

5.6

5.5

pH 5.4

5.3

5.2

5.1

5 , ~ , i , 1 , i , i ~ ,

100 200 300 400 500 600 700 800

Distance from the cluster ~tm)

Fig. 3. Predicted changes in pH values of Rowland series soil brought about by Rhizobium Ioti strain NZP'2037, based upon data obtained from growth in soil solution, using the model of Darrah et al. (1987) (a) changes in pH with time assuming the cells are either distributed as a cluster occupying 1% of the soil volume, or uniformly distributed in the soil occupying 99% of the soil volume (b) for the ceils assumed be clustered changes in pH with distance from the cluster.

rah et al. (1987). The results (Fig. 3a) for cells which were assumed to be evenly distributed in soil showed that the bacteria could increase the pH value of the soil only from a value of 5.0 to 5.06 after 350 h. Such a small increase in pH is unlikely to have any significant effect of A1 toxicity. However, if it is assumed that the cells are clustered and occupy only 1% of the soil volume then the pH value of the soil occupied by that cluster could be increased from 5.0 to 5.8 during 350 h (Fig. 3a). Such an increase in soil pH would cause a large reduction in the toxicity of AI in an otherwise acid soil.

The model was also used to predict the influence that the rhizobia would have on pH with distance from the cluster. In Rowland series soil it is predicted

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that the influence of the clustered cells only extends between 200 and 300 #m from the cluster (Fig. 3b). Such changes in soil pH would not be detected using conventional analytical techniques as illustrated by the calculation that the average pH of a soil sample with 99% of its volume at pH 5.0 and 1% at pH 5.8 would be 5.02 (Stanway, 1989).

Conclusions

There'is considerable laboratory-based evidence of a major role for A1 in the ecology of microorganisms under acid conditions both in terms of viability and mutagenic effects. However, the extent to which these processes occur in acid soils will be determined by factors such as the chemistry and mineralogy of the soil, the physiology of organisms in the soil and the extent to which those organisms are able to modify their local pH and neutralise the potential threat from AI.

Acknowledgements

I am grateful to Jim Cooper, Andrew Johnson, Andrew Stanway, Jerome Octive and Bhavna Solanki for stim- ulating ideas and carrying out many of the experiments which have contributed to this paper. Special thanks to Peter Darrah for carrying out the simulation modelling of the soil pH changes.

I would like to dedicate this paper to the memory of John Holding who encouraged me to pursue these studies.

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