Alleviation of aluminum toxicity to Rhizobium leguminosarum bv. viciae by the hydroxamate siderophore vicibactin.

Acid rain solubilises aluminum which can exert toxic effects on soil bacteria. The root nodule bacterium Rhizobium leguminosarum biovar viciae synthesises the hydroxamate siderophore vicibactin in response to iron limitation. We report the effect of vicibactin on the toxicity of aluminum(III) to R. leguminosarum and kinetic studies on the reaction of vicibactin with Al(III) and Fe(III). Aluminum (added as the nitrate) completely inhibited bacterial growth at 25 microM final concentration, whereas the preformed Al-vicibactin complex had no effect. When aluminum and vicibactin solutions were added separately to growing cultures, growth was partly inhibited at 25 microM final concentration of each, but fully inhibited at 50 microM final concentration of each. Growth was not inhibited at 50 microM Al and 100 microM vicibactin, probably reflecting the slow reaction between Al and vicibactin; this results in some aluminum remaining uncomplexed long enough to exert toxic effects on growth, partly at 25 microM Al and vicibactin and fully at 50 microM Al and vicibactin. At 100 microM vicibactin and 50 microM Al, Al was complexed more effectively and there was no toxic effect. It was anticipated that vicibactin might enhance the toxicity of Al by transporting it into the cell, but the Al-vicibactin complex was not toxic. Several explanations are possible: the Al-vicibactin complex is not taken up by the cell; the complex is taken up but Al is not released from vicibactin; Al is released in the cell but is precipitated immediately. However, vicibactin reduces the toxicity of Al by complexing it outside the cell.

Legume root nodule bacteria and acid pH.

In 1984 the Australian Wool Research Trust Fund called for expressions of interest in projects directed at using the developing techniques of molecular biology for application to agricultural problems. With our interests in legume root nodule bacteria and their physiology, we felt that the problems for legume nodulation and N2 fixation posed by soils which were already acid, or which were rapidly acidifying, required just such attention. Further, the finding body's request coincided with the highly successful introduction into Western Australian agriculture of acid-tolerant strains of the medic-nodulating bacteria Sinorhizobium meliloti originating from acid soils on Sardinia (see below). The existence of such strains made it obvious that acid tolerance was a genetically determined trait, and provided invaluable biologically diverse material with which to work. The biological bases for that trait of acid tolerance were totally obscure, and many remain so, but the following account provides some light in the darkness. The research that we have done since in pursuit of explanations for acid tolerance have been funded first by the Wool Research Trust Fund and the Rural Credits Development Fund, and later by the Australian Research Council, and we here record our appreciation for their support.

Problems of adverse pH and bacterial strategies to combat it.

This chapter aims to survey the problems faced by bacteria found in environments of adverse pH, to review strategies used to combat those problems and to ask how those strategies are implemented. At acid or alkaline pH, bacteria are challenged not just by excess of H+ or OH- but also by excess of metal ions (aluminium, heavy metals at acidic pH, Na+ at alkaline pH), as well as shortages. Bacteria attempt to maintain their intracellular pH by minimizing membrane permeability to H+ and other ions, buffering the cytoplasm, ameliorating the external pH through catabolism or selective substrate utilization, and developing ionic pumping systems. The amelioration of pH depends on the availability of substrate, and is unlikely in most naturally stressful environments. Ion pumping is expensive energetically, although the cost to growth is unknown. The response to adverse pH involves sensing systems and responsive regulatory systems. The adaptive acid tolerance response is now well known in and other bacteria, but is there a widespread adaptive alkali tolerance response? What and where are the sensors? Whether they sense intracellular pH, extracellular pH or delta pH is unclear, although an external sensory input seems essential. Is there one major sensory system responsive to pH or multiple systems with back-up mechanisms? What and where are the regulators? Is there one central regulator controlling all the responses or are there cascades of responses?

Acid tolerance in root nodule bacteria.

Biological nitrogen fixation, especially via the legume Rhizobium symbiosis, is important for world agriculture. The productivity of legume crops and pastures is significantly affected by soil acidity; in some cases it is the prokaryotic partner that is pH sensitive. Growth of Rhizobium is adversely affected by low pH, especially in the 'acid stress zone'. Rhizobia exhibit an adaptive acid tolerance response (ATR) that is influenced by calcium concentration. Using Tn5-mutagenesis, gusA fusions and 'proteome' analysis, we have identified a range of genes that are essential for growth at low pH (such as actA, actP, exoR, actR and actS). At least three regulatory systems exist. The two-component sensor-regulator system, actSR, is essential for induction of the adaptive ATR. Two other regulatory circuits exist that are independent of ActR. One system involves the low pH-induced regulator gene, phrR, which may control other low pH-regulated genes. The other circuit, involving a regulator that is yet unidentified, controls the expression of a pH-regulated structural gene (lpiA). We have used pH-responsive gusA fusions to identify acid-inducible genes (such as lpiA), and then attempted to identify the regulators of these genes. The emerging picture is of a relatively complex set of systems that respond to external pH.

A helicase gene (helO) in Rhizobium meliloti WSM419.

A 2.8 kb BamHI DNA fragment adjacent to a BamHI fragment containing actR-actS (a sensor/regulator pair required for low pH tolerance in Rhizobium meliloti WSM419) was cloned and sequenced. A computer predicted protein of 821 amino acids, designated HelO, showed extensive similarity with 'DEAH' motif helicases. Expression of helO was higher at pH 7.0 than pH 5.8 and it did not require the product of the actR gene. Inactivation of helO by insertion of a omega interposon at codon 40 did not affect nodulation, growth or tolerance to low pH, high temperature, osmolarity or elevated levels of copper or zinc.

Acid tolerance in Rhizobium meliloti strain WSM419 involves a two-component sensor-regulator system.

An acid-sensitive mutant, TG5-46, derived from Rhizobium meliloti WSM419 by Tn5 mutagenesis, fails to grow below pH 6.0 whereas the parent strain grows at pH 5.7. The DNA sequence of a 2.2 kb rhizobial DNA region flanking Tn5 in TG5-46 contains two open reading frames, ORF1 (designated actS) and ORF2 (designated actR), having high similarity to the sensor-regulator pairs of the two-component systems involved in signal transduction in prokaryotes. Insertion of an omega interposon into actS in R. meliloti WSM419 resulted in an acid-sensitive phenotype. A DNA fragment from the wild-type complemented the acid-sensitive phenotype of RT295 (ActS-) and TG5-46 (ActR-), while fragments containing only actR or actS complemented TG5-46 and RT295, respectively. The presence of multiple copies of actR complemented not only TG5-46 but also RT295. Cloning DNA upstream from actR and actS into a broad-host-range lacZ expression vector and measuring beta-galactosidase activities showed that both genes are constitutively expressed regardless of the external pH. Genomic DNA from all strains of R. meliloti, but no other bacteria tested, hybridized with an actRS probe at high stringency. These data implicate a two-component sensor-regulator protein pair in acid tolerance in R. meliloti and suggest their involvement in pH sensing and/or response by these bacteria.

Cloning and sequencing show that 4-hydroxybenzoate hydroxylase (PobA) is required for uptake of 4-hydroxybenzoate in Rhizobium leguminosarum.

Mutants of Rhizobium leguminosarum bv. viciae MNF300 and R. leguminosarum bv. trifolii WU95 unable to accumulate 4-hydroxybenzoate lack 4-hydroxybenzoate hydroxylase. The capacity of these mutants to take up and grow on 4-hydroxybenzoate was restored by a 2.0 kb EcoRI-PstI DNA fragment. This contained only one ORF which had over 60% DNA sequence similarity with the structural gene for 4-hydroxybenzoate hydroxylase (pobA) from Pseudomonas spp. and Acinetobacter. Reported effects of metabolic inhibitors and substrate analogues on the apparent uptake of 4-hydroxybenzoate have now been shown to be due to their direct effect on 4-hydroxybenzoate hydroxylase. We propose that uptake of 4-hydroxybenzoate is via a metabolic 'drag' mechanism dependent on the activity of the pobA gene product.

The adaptive acid tolerance response in root nodule bacteria and Escherichia coli.

Root nodule bacteria and Escherichia coli show an adaptive acid tolerance response when grown under mildly acidic conditions. This is defined in terms of the rate of cell death upon exposure to acid shock at pH 3.0 and expressed in terms of a decimal reduction time, D. The D values varied with the strain and the pH of the culture medium. Early exponential phase cells of three strains of Rhizobium leguminosarum (WU95, 3001 and WSM710) had D values of 1, 6 and 5 min respectively when grown at pH 7.0; and D values of 5, 20 and 12 min respectively when grown at pH 5.0. Exponential phase cells of Rhizobium tropici UMR1899, Bradyrhizobium japonicum USDA110 and peanut Bradyhizobium sp. NC92 were more tolerant with D values of 31, 35 and 42 min when grown at pH 7.0; and 56, 86 and 68 min when grown at pH 5.0. Cells of E. coli UB1301 in early exponential phase at pH 7.0 had a D value of 16 min, whereas at pH 5.0 it was 76 min. Stationary phase cells of R. leguminosarum and E. coli were more tolerant (D values usually 2 to 5-fold higher) than those in exponential phase. Cells of R. leguminosarum bv. trifolii 3001 or E. coli UB1301 transferred from cultures at pH 7.0 to medium at pH 5.0 grew immediately and induced the acid tolerance response within one generation. This was prevented by the addition of chloramphenicol. Acid-adapted cells of Rhizobium leguminosarum bv. trifolii WU95 and 3001; or E. coli UB1301, M3503 and M3504 were as sensitive to UV light as those grown at neutral pH.

Siderophore and organic acid production in root nodule bacteria.

Nineteen strains of root nodule bacteria were grown under various iron regimes (0.1, 1.0 and 20 microM added iron) and tested for catechol and hydroxamate siderophore production and the excretion of malate and citrate. The growth response of the strains to iron differed markedly. For 12 strains (Bradyrhizobium strains NC92B and 32H1, B. japonicum USDA110 and CB1809, B. lupini WU8, cowpea Rhizobium NGR234, Rhizobium meliloti strains U45 and CC169, Rhizobium leguminosarum bv viciae WU235 and Rhizobium leguminosarum bv trifolii strains TA1, T1 and WU95) the mean generation time showed no variation with the 200-fold increase in iron concentration. In contrast, in Bradyrhizobium strains NC921, CB756 and TAL1000, B. japonicum strain 61A76 and R. leguminosarum bv viciae MNF300 there was a 2-5 fold decrease in growth rate at low iron. R. meliloti strains WSM419 and WSM540 showed decreased growth at high iron. All strains of root nodule bacteria tested gave a positive CAS (chrome azurol S) assay for siderophore production. No catechol-type siderophores were found in any strain, and only R. leguminosarum bv trifolii T1 and bv viciae WU235 produced hydroxamate under low iron (0.1 and 1.0 microM added iron). Malate was excreted by all strains grown under all iron regimes. Citrate was excreted by B. japonicum USDA110 and B. lupini WU8 in all iron concentrations, while Bradyrhizobium TAL1000, R. leguminosarum bv viciae MNF300 and B. japonicum 61A76 only produced citrate under low iron (0.1 and/or 1.0 microM added iron) during the stationary phase of growth.

Cloning, characterization, and complementation of lesions causing acid sensitivity in Tn5-induced mutants of Rhizobium meliloti WSM419.

Four Tn5-induced mutants of Rhizobium meliloti WSM419 were unable to grow or maintain intracellular pH at an external pH of 5.6. Restriction analysis of DNA fragments carrying Tn5 and flanking sequences cloned from these mutants indicated that all four cloned mutations are unique and that the two strains (TG1-6 and TG1-11) carry Tn5 insertions which are within 4.4 kilobases of each other on a single EcoRI fragment. Southern analysis of total mutant DNA indicated a single copy of Tn5 in each mutant. A limited cosmid gene bank of wild-type WSM419 DNA was probed for homology to mutant DNA cloned from the acid-sensitive mutants. Dot hybridization experiments identified one cosmid element within this bank carrying wild-type DNA sequences corresponding to DNA implicated in acid tolerance. This cosmid was able to complement defects in growth and intracellular pH maintenance in TG1-11 but not TG1-6.

Micro-organisms as fertilizers and pest control agents in agricultural crops.

Micro-organisms can potentially supplement or replace the use of chemical fertilizers and pesticides in many agricultural cropping systems. Their increased use may reduce the need for nitrogen and phosphorus fertilizers, particularly in legume and cereal crops, and provide a more ecologically sound method of controlling pests and phytopathogens. Recombinant DNA technology offers the potential to extend and improve these applications. However, genetically modified micro-organisms may occasionally themselves pose environmental risks. Model ecosystems such as intact soil-core microcosms may be useful for both testing efficacy and evaluating environmental risk prior to proceeding with field trials.

Ammonia movements in rhizobia.

When free-living rhizobia are grown under N-excess conditions they appear to take up ammonia by a diffusive mechanism. Under low or limiting N, they derepress an ammonium permease which serves to scavenge NH4+. Current data suggest that N2-fixing bacteroids lose ammonia by a diffusive movement sustained by the continual removal of ammonia via the plant ammonia assimilatory system(s).

Selenite uptake and incorporation by Selenomonas ruminantium.

Selenite uptake and incorporation in Selenomonas ruminantium was constitutive with an inducible component. It was distinct from sulphate or selenate transport, since sulphate and selenate did not inhibit uptake, nor could sulphate or selenate uptake be demonstrated. Selenite uptake had an apparent Km of 1.28 mM and a Vmax of 148 ng Se min-1 mg-1 protein. Uptake was sensitive to inhibition by 2,4-dinitrophenol (DNP), carbonyl cyanide m-chlorophenyl hydrazone (CCCP), azide, iodoacetic acid (IAA) and N-ethylmaleimide (NEM), but not chlorpromazine (CPZ), N,N'-dicyclohexyl-carbodiimide (DCCD), quinine, arsenate, or fluoride. Treatment of cells accumulating 75[Se]-Selenite with 2,4,DNP inhibited uptake, but did not cause efflux. Transport of selenite was inhibited by sulphite and nitrite, but not by nitrate, phosphate, sulphate of selenate. 75[Se]-Selenite was incorporated into selenocystine, selenoethionine, selenohomocysteine, and selenomethionine and was also reduced to red elemental selenium.

A light and electron microscope study of the interaction of soil bacteria with Phytophthora cinnamomi Rands.

Light- and electron-microscopic examination showed that bacteria became associated with the hyphae and asexual reproductive structures of P. cinnamomi in soil. In suppressive soils this association appears to be correlated with hyphal lysis, inhibition of zoospore production, and sporangial breakdown. One notable feature of the microbial association between P. cinnamomi and soil bacteria is the formation of extensive slime material. Many of the bacteria isolated from the fungal hyphosphere display antagonism to the growth of P. cinnamomi in vitro. The bacteria are morphologically varied and include Pseudomonas, Bacillus, and Streptomyces spp. These observations suggest that the appropriate manipulation of the antagonistic bacteria may provide a means of biological control of P. cinnamomi.

Cytochemical studies on alkaline phosphatase production during sporulation in Bacillus subtilis.

During sporulation of Bacillus subtilis 168 there is an increase in activity of alkaline phosphatase in the presence of Pi. This enzyme was shown by cytochemical techniques to be associated with the cytoplasmic membrane of the mother cell and also with the membranes of the developing prespore. There is a strong correlation between an increasing number of electron-dense deposits due to phosphatase activity and the formation of the spore septum, i.e. stage II of sporulation. Cytochemical and biochemical evidence shows that cells well advanced in spore formation can be derepressed to produce the very much higher amounts of alkaline phosphatase characteristic of phosphate-starved vegetative cells.

Effect of haemin on endogenous protein synthesis in oocytes of the Queensland cane toad Bufo marinus.

Although general protein synthesis as measured by L-[3H]leucine incorporation is unaffected by injection of haemin into oocytes of B. marinus, the synthesis of at least two proteins is repressed. It appears that haemin is capable of exerting inhibitory effects on eukaryote protein synthesis as well as stimulating it as previously reported by other workers.

Accumulation of messenger ribonucleic acid specific for extracellular protease in Bacillus subtilis 168.

Production of extracellular protease by Bacillus subtilis 168 in a medium containing low concentrations of amino acids is essentially linear, whereas in a medium containing high levels of amino acids the time course of production is biphasic. Cells harvested from the growth medium are capable of secreting enzyme for 30 min in the presence of rifampin, but the appearance of the enzyme is sensitive to chloramphenicol and pactamycin. The protease messenger ribonucleic acid (mRNA), nevertheless, appears to have a short half-life typical of bacterial messengers, and this indicates that these cells contain a relatively large pool of protease-specific mRNA. This pattern of results is identical to that observed previously with B. amyloliquefaciens. Because it has now been found in two distinct organisms, it is concluded that the accumulation of mRNA for extracellular protease, supported by rapid transcription, is a biologically meaningful phenomenon related to extracellular enzyme synthesis rather than aberrant behavior due to a transcriptional control mutation.