Synthesis and turnover of leghaemoglobin in lupin root nodules.

1. The problem of whether leghaemoglobin is synthesized on plant of bacterial ribosomes in root nodules of yellow lupin has been examined. 2. Leghaemoglobin, soluble plant protein and soluble bacteroid protein were labelled with 14C administered by uptake of 14CO2. 3. Exposure of roots to 1 mM D-threo-chloramphenicol resulted in inhibition of soluble bacteroid protein synthesis, but leghaemoglobin synthesis and soluble plant protein synthesis were unaffected. This result is consistent with leghaemoglobin being synthesized on plant ribosomes. 4. After nitrogen-fixing plants had been supplied with a pulse of 14CO2, the decay of specific radioactivity of nodule protein fractions was observed. Leghaemoglobin had an apparent half-life of 18 days and is a stable protein in nitrogen-fixing yellow lupin nodules.

Amino acid composition and relationships of lupin and serradella leghaemoglobins.

The amino acid compositions of two lupin and two serradella leghaemoglobins were determined by conventional techniques. They are as follows: Lupin component I: Ala20, Arg1, (Asp + Asn)17, (Glu + Gln)17, Gly9, His4, Ile9, Leu15, Lys17, Met1, Phe8, Pro5, Ser12, Thr9, Trp3, Tyr2 and Val17. Lupin component II: Ala26, Arg1, (Asp + Asn)18, (Glu + Gln)20, Gly9, His5, Ile10, Leu16, Lys18, Met1, Phe8, Pro6, Ser12, Thr10, Trp3, Tyr2 and Val18. Serradella component I: Ala32, Arg1, (Asp + Asn)10, (Glu + Gln)18, Gly8, His3, Ile5, Leu5, Lys13, Phe8, Pro4, Ser14, Thr7, Try2, Tyr3 and Val12. Serradella component II: Ala37, Arg1, (Asp + Asn)11, (Glu + Gln)20, Gly9, His3, Ile6, Leu17, Lys15, Phe9, Pro5, Ser16, Thr8, Try2, Tyr3 and Val13. Notable features of these are (a) the absence of cyst(e)ine, which suggests that each is a single polypeptide chain (b) the presence of a methionine residue in the lupin leghaemoglobins, and (c) the high contents of alanine, aspartic acid plus asparagine, glutamic acid plus glutamine, leucine, lysine, serine and valine. The relationships of leghaemoglobins to each other and to other groups of haem-containing proteins have been investigated using a statistical procedure based on amino acid composition.

An alternative nitrogenase is not expressed in molybdenum-deficient legume root nodules.

Three legume root nodule bacteria systems (Medicago polymorpha L. -Rhizobium meliloti, Ornithopus sativus Brot. -Bradyrhizobium lupini and Trifolium subterraneum L.- Rhizobium leguminosarum by. trifolii) were grown in solution culture under conditions likely to lead to the production of alternative nitrogenases (molybdenum-deficient, or molybdenum-deficient but supplemented with vanadium). Addition of 1 µM molybdenum produced significant responses in both nodule and top weights while 2 µM vanadium did not. Ethane, which is produced as well as ethylene when acetylene is reduced by vanadium nitrogenase or nitrogenase-3 from Azotobacter, was not found in significant amounts during assays of acetylene reduction in either molybdenum-deficient or molybdenum-deficient, vanadium-supplemented treatments, suggesting that no non-molybdenum nitrogenase was produced by these root nodule bacteria.

Microscopic evidence on how iron deficiency limits nodule initiation in Lupinus angustifolius L.

Lupins (Lupinus angustifolius L. ev. Yandee), grown in solution culture, have been used to study the sites and process of infection by Bradyrhizobium sp. (Lupinus) and the impairment of nodulation by iron deficiency. Infection leading to nodulation occurred in an area of epidermal cells either lacking root hairs or with very young root hairs at the time of inoculation. Cells aged 13 h or over appeared not to be infected. Infection was initiated in the outer cortex. Rare, short infection threads were evident on day 4 after inoculation, 2 d after the initial division of cortical cells resulting from the bradyrhizobial inoculation. Bacteria had been released into the cytoplasm of cortical cells within 5 d after inoculation. Bacteroids multiplied in the cytoplasm, segregated passively and spread in the infection zones by repeated division of the invaded cells. Under iron deficiency, initial cell division occurred in the outer cortex of host roots, as in iron-sufficient plants after inoculation. Iron deficiency then limited further division of cortical cells. Only a few surviving infection sites developed nodules with normal structure but development was much slower than in iron-sufficient plants.

Impaired nodule function in Medicago polymorpha L. infected with alfalfa mosaic virus.

The effects of alfalfa mosaic virus (AMV) on growth, nodule formation and nodule function in the annual burr medic, Medicago polymorpha cv. Circle Valley, were investigated in glasshouse pot experiments. Systemically-infected plants from AMV-infected seed produced 21% less shoot dry weight and accumulated 24% less fixed nitrogen in shoots than healthy plants when harvested 53 d after germination. At day 75, infected plants showed similar shoot dry weight losses (22%), but the quantity of nitrogen fixed fell by only 15%. At day 53, soluble sugar, starch and bacteroid concentrations in nodules were unaffected by AMV infection, but nitrogenase specific activity was decreased by 25% and soluble amino acids by 20%. Although AMV infection resulted in no differences in the number of nodules formed in the first 11 d after germination or at any harvest thereafter, nodule mass was decreased by 23% for virus-infected plants at day 53. However this difference disappeared by day 75. Growth of AMV-infected plants was decreased probably because of impaired N2 fixation with nodule function affected rather than nodulation. Increased nodule mass relative to plant weight in virus-infected plants, seen at day 75, implied some degree of compensation for the limitation in N2 -fixing capacity. ELISAs for AMV antigen indicated that nodules were active sites of virus multiplication.

Iron-deficiency specifically limits nodule development in peanut inoculated with Bradyrhizobium sp.

Severely iron-deficient peanuts (Arachis hypogaaea L.) grown on calcareous soils in central Thailand failed to nodulate until given foliar iron applications. Glasshouse experiments were conducted on two cultivars (Tainan 9 and Robut 33-1) to identify which stage of the nodule symbiosis was most sensitive to iron-deficiency. Iron-deficiency did not limit growth of soil or rhizosphere populations of peanut liradyrhizobium. Similar numbers of root nodule initials formed in the roots of both control and iron-sprayed plants, showing that iron-deficiency did not directly affect root infection and nodule initiation. Plants sprayed with iron produced greater numbers of excisable nodules and carried a greater nodule mass than untreated plants. Five days after iron application, nodules on sprayed plants of CV. Tainan 9 contained 200-fold higher bacteroid numbers per unit weight and 14-fold higher concentrations of leghaemoglobain. The onset of nitrogenase activity was also delayed by iron deficiency in both cultivars. Tainan 9 appeared more sensitive to iron-deficiency than Robut 33-1 in terms of nodule mass produced, but both cultivars showed the same effect of iron-deficiency on nitrogenase activity per plant. It is concluded that the failure of the infecting rhizobia to obtain adequate amounts of iron from the plant results in arrested nodule development and a failure of nitrogen fixation.

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.

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.

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).

Inhibition of protein synthesis by D-threo-chloramphenicol in the laboratory and nodule forms of Rhizobium lupini.

Protein synthesis by both laboratory-grown bacteria and isolated nodule bacteroids of Rhizobium lupini (strain WU8) is inhibited by D-threo-chloramphenicol, the bacteroid form being the more sensitive to the antibiotic. A comparison between the two forms of the uptake of [14C]chloramphenicol showed that the bacteria always attained a lower intracellular chloramphenicol concentration. It is proposed that the sensitivity difference is due to a difference in membrane permeability between the two forms.

Ammonia assimilation by rhizobium cultures and bacteroids.

The enzymes involved in the assimilation of ammonia by free-living cultures of Rhizobium spp. are glutamine synthetase (EC. 6.o.I.2), glutamate synthase (L-glutamine:2-oxoglutarate amino transferase) and glutamate dehydrogenase (ED I.4.I.4). Under conditions of ammonia or nitrate limitation in a chemostat the assimilation of ammonia by cultures of R. leguminosarum, R. trifolii and R. japonicum proceeded via glutamine synthetase and glutamate synthase. Under glucose limitation and with an excess of inorganic nitrogen, ammonia was assimilated via glutamate dehydrogenase, neither glutamine synthetase nor glutamate synthase activities being detected in extracts. The coenzyme specificity of glutamate synthase varied according to species, being linked to NADP for the fast-growing R. leguminosarum, R. melitoti, R. phaseoli and R. trifolii but to NAD for the slow-growing R. japonicum and R. lupini. Glutamine synthetase, glutamate synthase and glutamate dehydrogenase activities were assayed in sonicated bacteroid preparations and in the nodule supernatants of Glycine max, Vicia faba, Pisum sativum, Lupinus luteus, Medicago sativa, Phaseolus coccineus and P. vulgaris nodules. All bacteroid preparations, except those from M. sativa and P. coccineus, contained glutamate synthase but substantial activities were found only in Glycine max and Lupinus luteus. The glutamine synthetase activities of bacteroids were low, although high activities were found in all the nodule supernatants. Glutamate dehydrogenase activity was present in all bacteroid samples examined. There was no evidence for the operation of the glutamine synthetase/glutamate synthase system in ammonia assimilation in root nodules, suggesting that ammonia produced by nitrogen fixation in the bacteroid is assimilated by enzymes of the plant system.

Hydrazine is a product of dinitrogen reduction by the vanadium-nitrogenase from Azotobacter chroococcum.

During the enzymic reduction of N2 to NH3 by Mo-nitrogenase, free hydrazine (N2H4) is not detectable, but an enzyme-bound intermediate can be made to yield N2H4 by quenching the enzyme during turnover [Thorneley, Eady & Lowe (1978) Nature (London) 272, 557-558]. In contrast, we show here that the V-nitrogenase of Azotobacter chroococcum produces a small but significant amount of free N2H4 (up to 0.5% of the electron flux resulting in N2 reduction) as a product of the reduction of N2. The amount of N2H4 formed increased 15-fold on increasing the assay temperature from 20 degrees C to 40 degrees C. Activity cross-reactions between nitrogenase components of Mo- and V-nitrogenases showed that the formation of free N2H4 was associated with the VFe protein. These data provide the first direct evidence for an enzyme intermediate at the four-electron-reduced level during the reduction of N2 by V-nitrogenase.