Exhibited Nitrogenase Activity Research Articles

Role of bacterial polysaccharides in the derepression of ex-planta nitrogenase activity with rhizobia

Since bacterial polysaccharides may limit the availability of oxygen to the cells, we have investigated the role of rhizobial extracellular polysaccharides (EPS) and the non-rhizobial polyscharide, xanthan, in the depression of ex-planta nitrogenase activity with rhizobia in liquid medium. Two rhizobial strains known to exhibit ex-planta nitrogenase activity on solid media were used; the slow-growing Bradyrhizobium japonicum USDA 110 and the arctic Rhizobium strain N31, both being prolific EPS producers. In low nitrogen mannitol (LNM) liquid medium strain N31 exhibited nitrogenase activity only after 15 days, when sufficient EPS had accumulated in the medium, and activity was correlated with EPS production. When rhizobial EPS from an old culture was added to the LNM medium, nitrogenase activity was detected after 48 h incubation, indicating that EPS of the medium decreased oxygen diffusion to cells to a level that depressed nitrogenase activity. In modified LNM medium with xanthan nitrogenase activity was readily depressed. In both strains activity increased with increased xanthan concentration, but decreased sharply at higher concentrations. Strain N31 exhibited a narrower range of polysaccharide concentration for nitrogenase activity than the slow strain USDA 110. Thus, the condition for derepression of nitrogenase might be a careful balancing of the oxygen concentration surrounding the cells, and this condition is met when a balancing of polsaccharide, either synthesized by the rhizobia or added to the medium, can permit oxygen diffusion to within the narrow range required for the depression and expression of nitrogenase.

Role of bacterial polysaccharides in the derepression of ex-planta nitrogenase activity with rhizobia

Since bacterial polysaccharides may limit the availability of oxygen to the cells, we have investigated the role of rhizobial extracellular polysaccharides (EPS) and the non-rhizobial polyscharide, xanthan, in the depression of ex-planta nitrogenase activity with rhizobia in liquid medium. Two rhizobial strains known to exhibit ex-planta nitrogenase activity on solid media were used; the slow-growing Bradyrhizobium japonicum USDA 110 and the arctic Rhizobium strain N31, both being prolific EPS producers. In low nitrogen mannitol (LNM) liquid medium strain N31 exhibited nitrogenase activity only after 15 days, when sufficient EPS had accumulated in the medium, and activity was correlated with EPS production. When rhizobial EPS from an old culture was added to the LNM medium, nitrogenase activity was detected after 48 h incubation, indicating that EPS of the medium decreased oxygen diffusion to cells to a level that depressed nitrogenase activity. In modified LNM medium with xanthan nitrogenase activity was readily depressed. In both strains activity increased with increased xanthan concentration, but decreased sharply at higher concentrations. Strain N31 exhibited a narrower range of polysaccharide concentration for nitrogenase activity than the slow strain USDA 110. Thus, the condition for derepression of nitrogenase might be a careful balancing of the oxygen concentration surrounding the cells, and this condition is met when a balancing of polsaccharide, either synthesized by the rhizobia or added to the medium, can permit oxygen diffusion to within the narrow range required for the depression and expression of nitrogenase.

Physiological differentiation of flooded N 2-fixing grass-diazotroph associations from non-flooded, non-N 2-fixing ones

N fixation by grass-associated diazotrophs is more active under flooded than under nonflooded conditions. Roots cut from Spartina alterniflora Loisel, rice or oats that were flooded exhibited nitrogenase activity (NA, assessed by acetylene reduction) that was at least ten times as high as that of roots from plants growing in non-flooded soils. O 2 inhibition of NA or lack of appropriate NA-supporting nutrients did not account for the low activity associated with the roots of the non-flooded plants. The roots of the plants from the non-flooded soils contained a factor that inhibited NA. This factor was not detected in roots of plants from flooded soils. The factor was soluble, heat stable (100°C) and passed through 1000 m.w. cut-off dialysis membrane. Analogous NA-inhibiting factors may serve to differentiate other N 2-fixing systems from non-fixing ones.

Studies on N2-fixing bacteria associated with the salt-tolerant grass,Leptochloa fusca (L.) Kunth

N2-fixing bacteria were isolated from the rhizosphere of naturally grown salt tolerant grass (Leptochloa fusca). A broad spectrum of diazotrophs was found to be associated with the roots ofL. fusca. the systematic position of the three isolates, NIAB-1, C-2 and Iso-2 was determined by morphological, biochemical and mol % (G+C) DNA contents. Two isolates were identified asKlebsiella pneumoniae (NIAB-1) andBeijerinckia sp. (Iso-2).15N enrichment studies confirmed the nitrogen fixing ability of the isolates. The effects of different levels of combined nitrogen (NO 3 − & NH 4 + ), pH (5.5–9.0) and salt (NaCl) on nitrogenase activity of the isolates were determined at various time intervals. All isolates exhibited nitrogenase activity even in the presence of 5 mmol/l NO 3 − or NH 4 + in a semi-solid medium after 24 h of growth. Maximum nitrogenase activity was observed at alkaline pH and all isolates were able to tolerate up to 3% NaCl in the medium.

Isolation and Characterization of Partially Derepressed Mutants of Cyanobacterium Aulosira spec.

Summary The chlorate and L-methionine DL-Sulfoximine resistant mutants were isolated in rice field cyanobacterium Aulosira spec. after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. MSX-resistant mutant was partially blocked in the glutamine synthetase (GS) and exhibited 44.1 % GS transferase activity compared to the parent grown on N2. Unlike its parent, the mutant showed nitrogenase activity of 2.19 nmol/µ g chlorophyll a/h when grown in ammonia. The mutant resistant to both chlorate and JISX (ClrR MSXR) was defective in both nitrate reductase and GS. It possessed only 39.4 % GS transferase activity in NZ grown culture and 6.2 % of nitrate reductase activity in nitrate grown culture as compared to the parent. Unlike its parent this mutant exhibited nitrogenase activity of 2.22 and 4.34 nmol/µg chl - a/h when grown in nitrate and ammonia respectively. The maximum of the GS transferase and nitrogenase activities were observed on N2 grown cultures of parent, MSXR and ClrR MSXR mutants whereas minimum level of GS transferase was noticed in glutamine grown culture.

Host–endophyte interactions in effective and ineffective nodules induced by the endophyte of Myrica gale

Suspensions of crushed root nodules of Myrica gale containing the actinomycete Frankia induced nodule formation on roots of seedlings of M. gale and Comptonia peregrina grown in nutrient water culture. Nodules formed on M. gale were normal in structure and exhibited nitrogenase activity (measured as acetylene reduction) and provided the necessary nitrogen for seedling development. These effective nodules showed typical external and internal structure with the endophyte developing both vesicles and sporangia within cortical cells of the host tissue. Small nodules formed on C. peregrina representing the primary nodule stage. They lacked nitrogenase activity and were termed ineffective. Vesicles failed to develop within these ineffective nodules. However, sporangia were formed in infected cells and within intercellular spaces of the nodule cortical tissue. In addition, prominent amyloplasts occurred in infected cells of the ineffective nodules, a feature lacking in effective nodules. Exogenously supplied combined nitrogen increased seedling growth but did not improve nodule development or endophyte morphogenesis in the ineffective nodules.

Associative symbiosis of Azospirillum lipoferum with dicotyledonous succulent plants of the Indian desert

The roots of several dicotyledonous xerophytic plants exhibited nitrogenase activity. Azospirillum lipoferum was isolated from the roots of these plants including several species of Opuntia having crassulacean acid metabolism. These isolates showed high rates of acetylene reduction; maximum nitrogenase activity was observed with the isolate from Opuntia vulgaris. Organic acids rather than sugars were preferred as carbon source for two selected isolates studied. However, no activity was detected with formic, oxalic, or lactic acid. Inorganic nitrogen sources caused significant reduction in nitrogenase activity, while organic sources such as amino acids and proteins either stimulated or did not significantly inhibit the activity. However, urea completely inhibited formation of nitrogenase activity. Growth measured as optical density was enhanced with all the nitrogen sources except potassium nitrite.

Mutants of Rhizobium capable of fixing N2 in the free-living condition

Six mutant strains of Rhizobium were isolated after UV treatment which could exhibit nitrogenase activity in Burk's N-free medium without any supplement. The activity ranged between 99.5 and 113 nmol/mg cell dry weight and hour. Two of the parent strains belonged to soybean, and one each to mungbean and Sesbania sp. Both the parent and mutant strains exhibited nitrogenase activity in CS 7 medium. One of the mutants retained its capacity to produce nodules on soybean roots.

Utility of some nitrogen-fixing microorganisms in the phyllosphere of crop plants

The utility of spraying some known N2-fixing microorganisms on rice leaves grown both in N-less sand culture and under field conditions was examined. The effect was compared with that of spraying a phyllosphere N2-fixing isolate of Klebsiella, KUPBR2, and application of nitrogenous fertilizers. All the growth parameters studied including dry weight and N-content were enhanced. Under field conditions number of tillers was increased by 26% withKlebsiella pneumoniae M5al and by 65% with Aphanothece. The dry weight of the plants was enhanced by 61–119%. The yield per 10 m2 was almost doubled with Aphanothece, Beijerinckia 8007,Mycobacterium flavum, K. pneumoniae M5al and KUPBR2. The increases observed withStreptomyces sp. G12 though less spectacular was significant at 1% level with respect to several growth parameters.K. pneumoniae M5al,M. flavum andStreptomyces sp. G12 exhibited nitrogenase activity both in laboratory culture and in association with rice plants.

Occurrence of aerobic nitrogen fixing bacteria in wetland and dryland plants

Abstract We recently suggested that N2-fixing bacteria other than Azospirillum are present in greater abundance in the roots of wetland rice (2). Using a low concentration of tryptic soy agar, we isolated aerobic heterotrophic bacteria that required low levels of combined nitrogen for nitrogenase activity from wetland rice roots (1). In a liquid medium under batch culture conditions, the bacteria were unable to grow without combined nitrogen under either aerobic or anaerobic conditions. Neither could they grow nor exhibit nitrogenase activity in a semi-solid N-free medium unless the medium was supplemented with a small amount of yeast extract, casamino acids or any amino acid. Among the nitrogenase positive aerobic heterotrophic isolates from the inner tissue of rice roots that accounted for 80% of the isolated heterotrophs, about 90% were found to be Pseudomonas-like, which require a low level of combined nitrogen. We further report here the occurrence of aerobic nitrogen fixing bacteria in association w...

The association of N2-fixing bacteria with sea urchins

Dinitrogen fixation associated with bacteria in the gastrointestinal tract of sea urchins appears to be a widespread phenomenon: sea urchins from the tropics (Diadema antillarum, Echinometra lacunter, Tripneustes ventricosus), the temperature zone (Strongylocentrotus droebachiensis) and the arctic (S. droebachiensis) exhibited nitrogenase activity (C2H2 reduction). Pronounced seasonal variation was found in nitrogenase activity of temperate sea urchins feeding on kelp (Laminaria spp.) and eelgrass (Zostera marina). The mean monthly nitrogenase activity was inversely correlated with the nitrogen content of the sea urchin's food, which varied up to fivefold over the course of a year. The highest rate of nitrogenase activity recorded for a temperate sea urchin during the 14 month sampling period was 11.6μg N fixed g wet wt-1 d-1, with a yearly mean activity of 1.36 μg N fixed g wet wt-1 d-1. Studies with 15N confirmed the C2H2 reduction results and showed incorporation of microbially-fixed nitrogen into S. droebachiensis demonstrating that N2 fixation can be a source of N for the sea urchin. Laboratory experiments indicated that part of the sea urchin's (S. droebachiensis) normal gastrointestinal microflora is responsible for the observed nitrogenase activity.

Expression of hydrogenase activity in free-living Rhizobium japonicum

A medium is described on which selected Rhizobium japonicum strains express hydrogenase (H(2) uptake) activity under free-living conditions. Low concentrations of carbon substrates, decreased oxygen tension, and the quantity of combined nitrogen in the medium were major factors influencing hydrogenase expression. Hydrogenase activity was dependent upon a preincubation period in the presence of H(2) under conditions such that the cells did not exhibit nitrogenase activity. H(2) uptake rates were easily measured amperometrically in aerobically or anaerobically prepared suspensions from free-living cultures. Six R. japonicum strains that formed nodules with the ability to utilize H(2) oxidized this gas when grown in free-living cultures. In comparison six randomly chosen strains forming nodules that lost H(2) in air either showed no or low capacity to take up H(2) under free-living conditions. The reduction of triphenyltetrazolium chloride in an agar medium was used to detect strains capable of oxidizing H(2). This method has enabled us to isolate a spontaneous R. japonicum mutant strain that has lost the ability to utilize H(2). This mutant strain forms nodules that evolve H(2) but other symbiotic characteristics appear normal. This strain will be useful in evaluating the importance of the hydrogenase system in the nitrogen-fixing process of legumes.

N2‐Fixation by roots and rhizosphere of sand dune plants

AbstractNitrogenase activity (by acetylene reduction assay) has been found on the roots and in the rhizosphere‐soil of various plant species collected from dune soils (Table 1.) located on the west coast of Denmark. Out of twelve plant species, five plant species (Deschampsia flexuosa, Ammophila arenaria, Calluna vulgaris, Empetrum nigrum and Corynephorus canescens) revealed a positive nitrogenase activity. Among these five, three were temperate grasses. Except Ammophila arenaria, all four plant species were collected from old sand dune and showed nitrogenase activity on roots as well as in their rhizosphere soil, Ammophila arenaria was collected from fresh sand dune and exhibited nitrogenase activity only on roots (Table 2).

Nitrogen Fixation (Acetylene Reduction) Associated with Sea Urchins (Strongylocentrotus droebachiensis) Feeding on Seaweeds and Eelgrass

Sea urchins feeding in different macrophyte zones in St. Margaret's Bay, Nova Scotia, in November and December 1975 were examined for nitrogenase activity using the acetylene reduction technique. For sea urchins feeding on Laminaria digitata and L. longicruris, the average rate of acetylene reduction was equivalent to N2 fixation of 55 μg N2 per sea urchin per day assuming a 3:1 molar ratio of C2H2 reduction to N2 fixation. Lower nitrogenase activities were observed for sea urchins feeding on Agarum cribrosum and Zostera marina, and no nitrogenase activity was observed for sea urchins taken from bare rock substrate on which kelp had been absent for 4 yr. Samples of separated digestive tracts but not the degutted bodies of sea urchins exhibited nitrogenase activity, suggesting that the digestive tract is the site of nitrogen fixation in whole sea urchins.

Isolation and Characterization of Aquaspirillum fasciculus sp. nov., a Rod-shaped, Nitrogen-Fixing Bacterium Having Unusual Flagella

In 1971, Strength and Krieg reported the isolation of a gram-negative freshwater rod which exhibited bipolar flagellar fascicles clearly visible by dark-field microscopy. The flagellar fascicles exhibited helical wave propagation, basal bending, and an ability to coil up like springs. Despite the flagellar activity, the cells were apparently unable to swim freely. Such organisms appeared to be similar morphologically to an organism previously described by Houwink in 1953 and Jarosch in 1969. The present report describes a reliable isolation method for such organisms based on the use of L-proline and semisolid agar. Upon isolation, the organisms grew in flocs, from which a highly viscous matrix could be separated by high-speed centrifugation. After many transfers, the growth gradually became homogeneous and turbid, and the viscous substance could no longer be demonstrated. Under certain conditions of growth, steady straight-line motility could be observed and photographed within viscous flocs. Straight-line, free-swimming motility occurred in viscous suspensions of cells prepared by homogenization of flocs. In 8- to 12-h-old cultures in the nonviscous homogeneous condition, some cells could swim slowly in irregular, circular paths; other could move about on surfaces. When the viscosity of the medium was increased, nearly every cell could swim freely and steadily in straight paths. A viscosity of 200 centipoise was optimal for strain XI, whereas 10 centipoise was optimal for strains X and XII. These results suggest that the organisms may be highly adapted to life within viscous flocs. The organism exhibited nitrogenase activity when tested by methods developed by Döbereiner and her colleagues for “Spirillum” lipoferum; Aquaspirillum peregrinum also was found to possess nitrogenase activity. Investigation of the physiology and deoxyribonucleic acid base composition of strains X, XI, and XII has indicated that even though the organisms are straight rods, they are probably members of the genus Aquaspirillum. Important taxonomic considerations include: Coccoid body or “microcyst” formation, possession of a “polar membrane” similar to that occurring in certain spirilla, bipolar tufts of flagella, a strictly respiratory metabolism, inability to attack carbohydrates, positive catalase and oxidase reactions, and a deoxyribonucleic acid base composition of 62 to 65 mol% guanine plus cytosine. The organisms were assigned to a new species, Aquaspirillum fasciculus, and the type strain was deposited with the American Type Culture Collection under the number 27740.

Nitrogenase activity and photosynthesis in Plectonema boryanum.

Nitrogen-starved Plectonema boryanum 594 cultures flushed with N(2)/CO(2) or A/CO(2) (99.7%/0.3%, vol/vol) exhibited nitrogenase activity when assayed either by acetylene reduction or hydrogen evolution. Oxygen evolution activities and phycocyanin pigments decreased sharply before and during the development of nitrogenase activity, but recovered in the N(2)/CO(2) cultures after a period of active nitrogen fixation. Under high illumination, the onset of nitrogenase activity was delayed; however, the presence of 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU) eliminated this lag. Oxygen was a strong and irreversible inhibitor of nitrogenase activity at low (>0.5%) concentrations. In the dark, low oxygen tensions (0.5%) stimulated nitrogenase activity (up to 60% of that in the light), suggesting a limited but significant respiratory protection of nitrogenase at low oxygen tensions. DCMU was not a strong inhibitor of nitrogenase activity. A decrease in nitrogenase activity after a period of active nitrogen fixation was observed in the N(2)/CO(2-), but not in the A/CO(2-), flushed cultures. We suggest that this decrease in nitrogenase activity is due to exhaustion of stored substrate reserves as well as inhibition by the renewed oxygen evolution of the cultures. Repeated peaks of alternating nitrogenase activity and oxygen evolution were observed in some experiments. Our results indicate a temporal separation of these basically incompatible reactions in P. boryanum.

Nitrogen fixation by Klebsiella grown in the presence of oxygen.

Several facultative asymbiotic N2-fixing bacteria exhibited nitrogenase activity when grown in the presence of air and a limiting amount of fixed nitrogen. One isolate (S-4) identified as Klebsiella was examined in more detail for oxygen effects. Nitrogenase was not detectable in highly aerated submerged cultures but was detectable in shaken and continuous cultures at dissolved oxygen tensions of 3 and 10 mm of Hg respectively. No nitrogenase activity was found in cells from continuous cultures maintained at a dissolved oxygen tension of 15 mm of Hg or above. The nitrogenase activity in whole cells was not oxygen dependent but was oxygen tolerant.