The role of soil microbes in plant sulphur nutrition

Chemical and spectroscopic studies have shown that in agricultural soils most of the soil sulphur (>95%) is present as sulphate esters or as carbon-bonded sulphur (sulphonates or amino acid sulphur), rather than inorganic sulphate. Plant sulphur nutrition depends primarily on the uptake of inorganic sulphate. However, recent research has demonstrated that the sulphate ester and sulphonate-pools of soil sulphur are also plant-bioavailable, probably due to interconversion of carbon-bonded sulphur and sulphate ester-sulphur to inorganic sulphate by soil microbes. In addition to this mineralization of bound forms of sulphur, soil microbes are also responsible for the rapid immobilization of sulphate, first to sulphate esters and subsequently to carbon-bound sulphur. The rate of sulphur cycling depends on the microbial community present, and on its metabolic activity, though it is not yet known if specific microbial species or genera control this process. The genes involved in the mobilization of sulphonate- and sulphate ester-sulphur by one common rhizosphere bacterium, Pseudomonas putida, have been investigated. Mutants of this species that are unable to transform sulphate esters show reduced survival in the soil, indicating that sulphate esters are important for bacterial S-nutrition in this environment. P. putida S-313 mutants that cannot metabolize sulphonate-sulphur do not promote the growth of tomato plants as the wild-type strain does, suggesting that the ability to mobilize bound sulphur for plant nutrition is an important role of this species.     

Importance of organosulfur utilization for survival of Pseudomonas putida in soil and rhizosphere

The sulfur present in both agricultural and uncultivated soils is largely in the form of sulfonates and sulfate esters and not as free, bioavailable inorganic sulfate. Desulfurization of the former compounds in vitro has previously been studied in Pseudomonas putida, a common rhizosphere inhabitant. Survival of P. putida strains was now investigated in three sulfur-deficient Danish soils which were found to contain 60 to 70% of their sulfur in sulfonate or sulfate ester form, as determined by X-ray near-edge spectroscopy. The soil fitness of P. putida S-313 was compared with that of isogenic strains with mutations in the sftR and asfA genes (required for in vitro desulfurization of sulfate esters and arylsulfonates, respectively) and in the ssu locus (required in vitro for the desulfurization of both sulfonates and sulfate esters). asfA or sftR mutants showed significantly reduced survival compared to the parent strain in bulk soil that had been enriched with carbon and nitrogen to mimic rhizosphere conditions, but this reduced survival was not observed in the absence of these additives. In a tomato rhizosphere grown in compost, survival of sftR and ssu mutants was reduced relative to the parent strain. The results demonstrate that the ability to desulfurize sulfonates and sulfate esters is critical for survival of bacteria in the rhizosphere but less so in bulk soils outside the influence of plant roots, where carbon is the limiting nutrient for growth.     

Substrate specificity and other properties of the inducible S3 secondary alkylsulphohydrolase purified from the detergent-degrading bacterium Pseudomonas C12B.

The inducible S3 secondary alkylsulphohydrolase of the soil bacterium Pseudomonas C12B was purified to homogeneity (683-fold from cell-free extracts by a combination of column chromatography on DEAE-cellulose. Sephadex G-100 and Blue Sepharose CL-6B. The enzyme has a molecular weight in the region of 40000--46000, and is active over a broad range of pH from 5 to 9, with maximum activity at pH 8.2. The preferred substrates of the enzyme are the symmetrical secondary alkylsulphate esters such as heptan-4-yl sulphate and nonan-5-yl sulphate and the asymmetric secondary octyl and nonyl sulphate esters with the sulphate group attached to C-3 or C-4. However, for each asymmetric ester, the L-isomer is much more readily hydrolysed than the D-isomer. This specificity is interpreted in terms of a three-point attachment of the substrate to the enzyme's active site. The alkyl chains on either side of the esterified carbon atom are bound in two separate sites, one of which can only accommodate alkyl chains of limited size. The third site binds the sulphate group. Enzymic hydrolysis of this group is accompanied by complete inversion of configuration at the asymmetric carbon atom. The implied cleavage of the C--O bond of the C--O--S ester linkage was confirmed by 18O-incorporation studies.     

The evaluation of plant-available sulphur in soils. II

Summary The availability to oats of adsorbed sulphate in soils and of sulphate impurity in calcium carbonate was studied in pot-culture experiments.When calcium carbonate was added to soils with pH values ranging from 5.7 to 7.4 the uptake of sulphur by oats was increased, due probably to enhanced mineralization of soil organic sulphur. When the calcium carbonate contained sulphate impurity the uptake of sulphur was further increased by an amount comparable with the release of sulphate which could be expected from a reaction of the calcium carbonate with the exchangeable hydrogen of the soil. Sulphate in excess of this amount appeared to be largely unavailable. Uptake of sulphur by oats from calcareous sands containing large amounts of insoluble sulphate associated with calcium carbonate also suggested that soil sulphur in this form had very low availability to plants.Substantial increases in the amounts of sulphur extracted by reagents commonly used for the determination of adsorbed sulphate in soils occurred when soils were airdried at about 20°C. Decreases in adsorbed sulphate in soils following the growth of oats in pot culture confirmed that adsorbed sulphate is readily available to plants.     

Sulphur mineralization and adsorption in soils

Summary Studies were conducted to determine the comparative sulphur mineralizing capacity of selected Malaysian and Iowan soils and to determine the amounts of available and adsorbed sulphate in a number of Malaysian soils. Results of the mineralization study indicated that more sulphur mineralised from Malaysian soils although their average contents of total sulphur were lower compared to Iowan Soils. For both sets of soils, significant correlations between contents of organic carbon and total sulphur existed indicating that most of the sulphur was in organic combination. Phosphate solution consistently extracted higher quantities of sulphate in comparison to chloride solution in the Malaysian surface soils implying that a portion of the sulphate existed in adsorbed form. Adsorption of sulphate in soils was found to be dependent on concentration of sulphate added and followed the Langmuir adsorption isotherm.     

The evaluation of plant-available sulphur in soils

Summary Examination of a range of naturally occurring calcium carbonates and calcareous soils has shown that insoluble sulphate associated with calcium carbonate may comprise an important fraction of soil sulphur. One soil contained as much as 93 per cent of its sulphur in this form. It seems likely that this sulphate occurs as a co-precipitated or co-crystallized impurity in the calcium carbonate.Most surface soils had only low capacity to adsorb sulphate and contained only small amounts of sulphur in this form. Two acid surface soils and many acid subsoils, however, adsorbed sulphate quite strongly and in some acid subsoil clays adsorbed sulphate made up an important fractions of the total sulphur.Sulphate adsorption was found to be negligible above pH 6.5 and adsorbed sulphate may be determined by aqueous extraction after increasing the pH above this value by addition of solid calcium carbonate. Adsorption of sulphate during acid extraction of soils can lead to low values in the determination of acid-soluble sulphates. Sulphate so adsorbed can be determined by a second extraction with water after the addition of solid calcium carbonate to increase the pH to a value greater than 6.5.     

Sulphur oxidation by soil fungi including some species of mycorrhizae and wood-rotting basidiomycetes

Abstract The mycorrhizal fungi Amanita muscaria, Paxillus involutus, Hymenoscyphus ericae, Pisolithus tinctorius, Rhizopogon roseolus , and Suillus bovinus oxidized elemental sulphur to thiosulphate and sulphate in vitro. In some, but not all cases, tetrathionate was also formed. Limited oxidation of elemental sulphur by R. roseolus also occurred when growing in association with Pinus contorta in unsterilized peat. Although yeasts capable of oxidizing sulphur could not be isolated from a wide range of soils, a yeast-like fungus ( Monilia sp.) isolated from deciduous woodland soil oxidized elemental sulphur to sulphate, forming thiosulphate, but not tetrathionate. This fungus also oxidized tetrathionate to sulphate but showed only limited ability to oxidize thiosulphate to tetrathionate. Both Aspergillus niger and Trichoderma harzianum oxidized elemental sulphur in mixed culture with Mucor flavus . Larger amounts of sulphate were initially formed in mixed, compared to single culture; but by week 5 of the incubation period sulphate formation was greatest in single culture. The wood-rotting fungi, Hypholoma fasciculare and Phanerochaete velutina showed a limited ability to oxidize elemental sulphur in vitro but were incapable of oxidizing the element when growing as mycelial cords in non-sterilized soils. The relevance of these results to the possibility that fungi play a role in sulphur oxidation in soils is commented upon.     

Some factors affecting the mineralization of organic sulphur in soils

Summary Factors affecting the release of sulphate from a number of eastern Australian soils were studied.All of the soils released sulphate when dried. The amounts released were influenced by the manner in which the soil was dried. Air-drying in the laboratory at 20°C released least sulphate.Sulphate was mineralized in all soils by incubation at 30°C but the amounts mineralized could not be related to soil type or any single soil property. The ratio of nitrogen mineralized: sulphur mineralized varied widely between soils and was generally appreciably greater than the ratio of total nitrogen: organic sulphur in the soils.A rapid flush of mineralization of both sulphur and nitrogen took place when some of the soils were rewetted and incubated after they had been dried in the laboratory and stored for 4 to 5 months. Following this, the rate of mineralization was similar to that in the original undried soil. During this flush, the enhancement of sulphur mineralization was relatively greater than that of nitrogen so that the ratio of nitrogen mineralized: sulphur mineralized was considerably smaller than that during later phases of the incubation or that of the original moist soil. Soils collected after they had remained dry in the field for a similar period of time did not show this type of mineralization although they had initially done so when collected moist and air-dried in the laboratory.The effects of temperature, soil moisture, toluene and formaldehyde, and the addition of calcium carbonate to soils on the mineralization of sulphur were similar to their effects on the mineralization of nitrogen.     

Effects of organic matter on sulphur oxidation in soil and influence of sulphur oxidation on soil nitrification

Summary The effects of wheat straw and pressed sugar beet pulp on sulphur oxidation were determined in a loam soil amended with 1% (w/w) elemental sulphur. Wheat straw stimulated the oxidation of elemental sulphur over the first 2 to 3 weeks of the incubation period, resulting in an increase in LiCl-extractable sulphate. After 4 to 7 weeks incubation however, the only significant increase in soil sulphate followed the 1% straw addition, while at week 7 sulphate concentrations in the 0.25% and 5.0% straw amended soils were lower than the control. Pressed sugar beet pulp (1% w/w) initially stimulated the oxidation of elemental sulphur in the soil, but by weeks 3 to 7 of the incubation period rates of oxidation in pulp-amended soils were lower than the control. Towards the end of the incubation period however, sulphate concentrations in the amended soils exceeded the control values, significantly so by week 11. The concentration of thiosulphate and tetrathionate also increased in soils receiving sugar beet pulp. Nitrification was inhibited in soils in which sulphur oxidation was actively occurring. Although possible alternatives are mentioned, such inhibition appears to result from a decrease in soil pH brought about by the oxidation of elemental sulphur to sulphuric acid.     

Sulphur‐oxidizing and sulphate‐reducing communities in Brazilian mangrove sediments

Mangrove soils are anaerobic environments rich in sulphate and organic matter. Although the sulphur cycle is one of the major actors in this ecosystem, little is known regarding the sulphur bacteria communities in mangrove soils. We investigated the abundance, composition and diversity of sulphur‐oxidizing (SOB) and sulphate‐reducing (SRB) bacteria in sediments from three Brazilian mangrove communities: two contaminated, one with oil (OilMgv) and one with urban waste and sludge (AntMgv), and one pristine (PrsMgv). The community structures were assessed using quantitative real‐time polymerase chain reaction (qPCR), polymerase chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE) and clone libraries, using genes for the enzymes adenosine‐5′‐phosphosulphate reductase (aprA) and sulphite reductase (Dsr) (dsrB). The abundance for qPCR showed the ratio dsrB/aprA to be variable among mangroves and higher according to the gradient observed for oil contamination in the OilMgv. The PCR‐DGGE patterns analysed by Nonmetric Multidimensional Scaling revealed differences among the structures of the three mangrove communities. The clone libraries showed that Betaproteobacteria, Gammaproteobacteria and Deltaproteobacteria were the most abundant groups associated with sulphur cycling in mangrove sediments. We conclude that the microbial SOB and SRB communities in mangrove soils are different in each mangrove forest and that such microbial communities could possibly be used as a proxy for contamination in mangrove forests.     

Induction of primary alkylsulphatases and metabolism of sodium hexan-1-yl sulphate by Pseudomonas C12B

Sodium hexan-1-yl sulphate and certain related alkyl sulphate esters have been shown to serve as inducers of the formation of primary alkylsulphatases (designated as P1 and P2) in Pseudomonas C12B. When the organism is grown on sodium hexan-1-yl [(35)S]sulphate as the sole source of sulphur or as the sole source of carbon and sulphur only the P2 alkylsulphatase is formed and inorganic (35)SO(4) (2-) is liberated into the media. Cell extracts contain this anion as the major (35)S-labelled metabolite although two unidentified labelled metabolites as well as choline O-[(35)S]sulphate occur in trace quantities in some extracts. Dialysed cell extracts are capable of liberating inorganic (35)SO(4) (2-) from sodium hexan-1-yl [(35)S]sulphate without the need to include cofactors known to be required for the bacterial degradation of n-alkanes. The collective results suggest that sodium hexan-1-yl sulphate can act as an inducer of P1 alkylsulphatase formation without the need for prior metabolic modification of the carbon moiety of the ester.     

Purification and some properties of the D-lactate-2-sulphatase of Pseudomonas syringae GG.

A soil bacterium grown on propan-2-yl sulphate as sole source of carbon and sulphur yielded extracts containing an enzyme capable of liberating sulphate from racemic lactate-2-sulphate. The enzyme was purified to homogeneity by a combination of streptomycin sulphate precipitation of nucleic acids, batch treatment with DEAE-cellulose, and chromatography on columns of DEAE-cellulose, Sephacryl S-300 and butyl-agarose. The protein was monomeric with an Mr of 55 000-60 000. The enzyme activity was specific for D-lactate-2-sulphate (Km 6.6 nM; maximal specific activity 14.3 mumol/min per mg of protein) and showed no activity towards the L-isomer. The products of the enzyme's action were inorganic sulphate and D-lactate which were released in equimolar amounts and stoicheiometrically with the amount of ester hydrolysed. No L-lactate was formed. Retention of configuration implied cleavage of the O-S bond of the C-O-S ester link and this was confirmed by 18O-incorporation experiments in which 18O from 18O-enriched water in the incubation medium was incorporated exclusively and quantitatively into inorganic sulphate. Only two other esters (serine-O-sulphate and p-nitrophenyl sulphate) of a total of 29 compounds tested were substrates for the enzyme. D-Lactate, L-lactate-2-sulphate and the substrate analogues glycollate-2-sulphate and butyrate-2-sulphate were significantly inhibitory.     

The effect of sodium chloride and sulphate on sulphur oxidation in soil

Summary The effect of sodium chloride on sulphur oxidation in Terra Rossa and Rendzina soils was studied by incubation and perfusion techniques. Sulphur oxidation was observed at concentrations up to 8 per cent NaCl, but was completely arrested at 10 per cent sodium chloride. Sodium chloride caused a delay in the onset of sulphur oxidation, its rate being only slightly affected. A relationship between sulphate appearance and decrease in pH was observed only in sulphur-amended Terra Rossa soil. Under optimal conditions, 53 and 54 per cent of added sulphur (5000 ppm) was recovered as SO₄-S from the Terra Rossa and Rendzina soils, respectively. This maximal level of sulphate production was only slightly affected by the addition of sulphate up to 3000 ppm S.It was concluded that inhibition in further sulphur oxidation was not caused by sulphate accumulation.     

Availability of different forms of sulphur fertilisers to wheat and oilseed rape

A pot experiment was conducted to compare the availability and efficiency of three sulphur (S) fertilisers to wheat in the first year and oilseed rape in the second year, using six agricultural soils. Four treatments were applied in the initial year: control (no S), two forms of elemental S (either micronised S° particles or a bentonite + S° mixture) and a sulphate fertiliser (ammonium sulphate). In the first year, the micronised S° was as effective as the sulphate fertiliser, both producing similar increases of wheat grain yield (on average 36%) and S uptake (on average 164%) over the control. In contrast, responses to the bentonite + S° form were minimal, indicating a limited S supply. In the second year the control treatment failed to produce seeds in most soils, whereas the micronised S° and sulphate treatments increased seed yields of oilseed rape to an average of 13.4 and 12.9 g pot^-1, respectively. The performance of the bentonite + S° varied between soils: two soils produced yields similar to those of the other S fertilisers, while the remaining soils had low yields. To test whether the poor performance of the bentonite clay + S° fertiliser was due to the lack of exposure of the prills to physical weathering in the glasshouse, the effect of freeze-thaw action on the fertilisers performance was assessed in a separate pot experiment. The responses in wheat yield and S uptake showed that freeze-thaw did not enhance the physical disruption of the prills or fertiliser effectiveness. These results suggest that the release of available S from the bentonite + S° mixture was too slow to meet the requirement of wheat and oilseed rape. This revised version was published online in June 2006 with corrections to the Cover Date.     

A sulphur deficiency-induced gene, sdi1, involved in the utilization of stored sulphate pools under sulphur-limiting conditions has potential as a diagnostic indicator of sulphur nutritional status

A sulphate deficiency-induced gene, sdi1 , has been identified by cDNA-amplified fragment length polymorphism (AFLP) analysis utilizing field-grown, nutrient-deficient wheat ( Triticum aestivum var. Hereward). The expression of sdi1 was specifically induced in leaf and root tissues in response to sulphate deficiency, but was not induced by nitrogen, phosphorus, potassium or magnesium deficiency. Expression was also shown to increase in plant tissues as the external sulphate concentration in hydroponically grown plants was reduced from 1.0 to 0.0 m m . On this basis, sdi1 gene expression has potential as a sensitive indicator of sulphur nutritional status in wheat. Genome-walking techniques were used to clone the 2.7-kb region upstream of sdi1 from genomic DNA, revealing several cis -element motifs previously identified as being associated with sulphur responses in plants. The Arabidopsis thaliana gene most highly homologous to sdi1 is At5g48850, which was also demonstrated to be induced by sulphur deficiency, an observation confirmed by the analysis of microarray data available in the public domain. The expression of Atsdi1 was induced more rapidly than previously characterized sulphur-responsive genes in the period immediately following the transfer of plants to sulphur-deficient medium. Atsdi1 T-DNA 'knockout' mutants were shown to maintain higher tissue sulphate concentrations than wild-type plants under sulphur-limiting conditions, indicating a role in the utilization of stored sulphate under sulphur-deficient conditions. The structural features of the sdi1 gene and its application in the genetic determination of the sulphur nutritional status of wheat crops are discussed.     

The use of 17O-NMR in the study of bond cleavage during the hydrolysis of sulphate esters

The use of 17O-NMR to investigate bond cleavage during the hydrolysis of sulphate esters in water enriched in 17O is described. Despite the inherent disadvantages of 17O for NMR studies, this work shows that, in favourable cases, 17O-NMR of 17O-enriched species is a powerful and sensitive tool for mechanistic studies. It is particularly useful when O-S cleavage occurs, resulting in the formation of S17O16O3(2-) (5% 17O), which can easily be detected at the biologically relevant mumole level. The method complements those using H2(18)O and has the advantage that in principle 17O can be detected in either of the hydrolysis products with little or no purification. It has been shown that sulphatase A (aryl-sulphate sulphohydrolase, EC 3.1.6.1) cleaves the O-S bond while functioning as a cerebroside sulphatase, as it does when functioning as an aryl- or glycosulphatase.     

The control of sulphate reduction in Escherichia coli by O-acetyl-l-serine

1. Extracts of Escherichia coli A.T.C.C. 9723 and K(12)703 contain serine transacetylase and O-acetylserine sulphhydrase. Synthesis of the latter enzyme is repressed by growth on l-cyst(e)ine and other sulphur compounds. 2. O-Acetyl-l-serine added to cells growing on glutathione or sulphate as source of sulphur induces the enzymes that catalyse (a) the activation of sulphate to adenosine 3'-phosphate 5'-sulphatophosphate (EC 2.7.7.4 and 2.7.1.25), (b) the reduction of adenosine 3'-phosphate 5'-sulphatophosphate to sulphite and (c) the reduction of sulphite to sulphide (EC 1.8.1.2). Hydrogen sulphide is liberated from cultures growing on sulphate as source of sulphur and in the presence of O-acetylserine. 3. The cysE mutants of E. coli K(12) lack serine transacetylase. Addition of O-acetylserine permits growth on sulphate as source of sulphur; at the same time the enzymes of sulphate reduction, previously absent, are synthesized. Such mutants have no detectable intracellular cyst(e)ine when starved of sulphur. 4. These results suggest that O-acetylserine is necessary for synthesizing the enzymes of sulphate reduction in E. coli. Its action does not appear to be by interference with the repressive control exerted over these enzymes by cyst(e)ine.     

The glycosulphatase of Trichoderma viride

The growth of the mould Trichoderma viride on a defined medium containing either potassium d-glucose 6-O-sulphate or potassium d-galactose 6-O-sulphate as sole sources of both carbon and sulphur is marked by the production of an enzyme system capable of liberating inorganic SO(4) (2-) ions from either of the sulphate esters. The enzyme is not produced when the organism is grown with glucose (or galactose) and potassium sulphate or with glucose and methionine as sole sources of carbon and sulphur. Experimental conditions are described whereby inorganic SO(4) (2-) ions liberated from potassium glucose 6-O-sulphate by the growing mould appear in the culture medium after a constant lag period of 21-24hr. The enzyme has been shown to be a simple glycosulphatase that is active towards the 6-O-sulphate esters of d-glucose and d-galactose but not towards potassium glucose 3-O-sulphate. The properties of the crude glycosulphatase show the enzyme to be appreciably different from analogous molluscan enzymes that can degrade monosaccharide sulphate esters.     

Sulphur and nitrogen changes in forest soils of East Africa

Summary Sulphur measurements made on soils in East Africa revealed the unexpected occurrence of sulphates in forest subsoils. Soil profiles from plantation and natural forests were analysed and a general pattern of sulphur distribution was found.It has been found that in addition to the sulphur cycle dependent upon microbiological oxidation of organic sulphur there exists a cycle whereby sulphate is circulated as such between trees and soil.The biological oxidation of organic sulphur in the soil is an extremely slow process compared with that of carbon and nitrogen. Treatment with calcium carbonate, nitrates and non-sulphur-containing amino acids had no effect upon the subsequent sulphate production. Incubation with either S-containing amino acids or with calcium sulphate however, resulted in an increased rate of soil-sulphur oxidation and this effect is being further investigated.An aqueous extract of wattle leaves proved to inhibit nitrification in the soil for short periods and evidence is presented which suggests that methionine may be involved in this effect.     

Catalytic and regulatory properties of sulphur metabolizing enzymes in cyanobacterium Synechococcus elongatus PCC 7942

Synechococcus elongatus PCC 7942 was able to grow with several S sources. The sulphur metabolizing enzymes viz. ATP sulphurylase, cysteine synthase, thiosulphate reductase and L- and D-cysteine desulphydrases were regulated by sulphur sources, particularly by sulphur amino acids and organic sulphate esters. Sulphur starvation reduced ATP sulphurylase and cysteine synthase whereas reduced glutathione appreciated Cys degradation activity. With partially purified enzymes apparent Km values for sulphate, ATP, D- and L-Cys, thiosulphate, sulphide and O-acetyl serine were in a range of 12-50 microM. p-Nitrophenyl sulphate inhibited ATP sulphurylase competitively. Met was a feedback inhibitor of several key enzymes.