Nitrate and nitrite reductase negative mutants of N2-fixingAzospirillum spp.

Chlorate resistant spontaneous mutants ofAzospirillum spp. (syn.Spirillum lipoferum) were selected in oxygen limited, deep agar tubes with chlorate. Among 20 mutants fromA. brasilense and 13 fromA. lipoferum all retained their functional nitrogenase and 11 from each species were nitrate reductase negative (nr^–). Most of the mutants were also nitrite reductase negative (nir^–), only 3 remaining nir⁺. Two mutants from nr⁺ nir⁺ parent strains lost only nir and became like the nr⁺ nir^– parent strain ofA. brasilense. No parent strain or nr⁺ mutant showed any nitrogenase activity with 10 mM NO ₃ ^– . In all nr^– mutants, nitrogenase was unaffected by 10 mM NO ₃ ^– . Nitrite inhibited nitrogenase activity of all parent strains and mutants including those which were nir^–. It seems therefore, that inhibition of nitrogenase by nitrate is dependent on nitrate reduction. Under aerobic conditions, where nitrogenase activity is inhibited by oxygen, nitrate could be used as sole nitrogen source for growth of the parent strains and one mutant (nr^– nir^–) and nitritite of the parent strains and 10 mutants (all types). This indicates the loss of both assimilatory and dissimilatory nitrate reduction but only dissimilatory nitrite reduction in the mutants selected with chlorate.     

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

Abstract 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.     

Isolation and characterization of nitrogenase-derepressed mutant strains of cyanobacterium Anabaena variabilis.

A positive selection method for isolation of nitrogenase-derepressed mutant strains of a filamentous cyanobacterium, Anabaena variabilis, is described. Mutant strains that are resistant to a glutamate analog, L-methionine-D,L-sulfoximine, were screened for their ability to produce and excrete NH4+ into medium. Mutant strains capable of producing nitrogenase in the presence of NH4+ were selected from a population of NH4+-excreting mutants. One of the mutant strains (SA-1) studied in detail was found to be a conditional glutamine auxotroph requiring glutamine for growth in media containing N2, NO3-, or low concentrations of NH4+ (less than 0.5 mM). This glutamine requirement is a consequence of a block in the assimilation of NH4+ produced by an enzyme system like nitrogenase. Glutamate and aspartate failed to substitute for glutamine because of a defect in the transport and utilization of these amino acids. Strain SA-1 assimilated NH4+ when the concentration in the medium reached about 0.5 mM, and under these conditions the growth rate was similar to that of the parent. Mutant strain SA-1 produced L-methionine-D,L-sulfoximine-resistant glutamine synthetase activity. Kinetic properties of the enzyme from the parent and mutant were similar. Mutant strain SA-1 can potentially serve as a source of fertilizer nitrogen to support growth of crop plants, since the NH4+ produced by nitrogenase, utilizing sunlight and water as sources of energy and reductant, respectively, is excreted into the environment.     

Symbiotic properties of antibiotic-resistant mutants of Rhizobium galegae

Mutagenesis provoked by exposure to increased concentration of antibiotics of five indigenous Rhizobium galegae strains resulted in the generation of several antibiotic-resistant mutants. The mutants differed from the wild type and one from another in respect to the nodulation capacity, the nitrogenase activity, the nodule ultrastructure, and the plant growth response. Galega plants inoculated with mutants resistant to streptomycin and rifampicin formed nodules with higher nitrogenase activity and accumulated more shoot dry biomass than plants inoculated with the parent strains. Resistance to kanamycin and nalidixic acid was associated with significant decrease of nitrogenase activity. A correlation between nitrogen-fixing efficiency and nodule infected cell ultrastructure was found. When the bacteroids occupied about 10 times higher area in infected cells of nodule than peribacteroid spaces and host cytosol had electron dense and homogenous structure, the nitrogenase activity was the highest. This revised version was published online in August 2006 with corrections to the Cover Date.     

Growth of Pseudomonas aeruginosa mutants lacking glutamate synthase activity.

Mutant strains SU1, SU4, and US1 lacking glutamate synthase (GOGAT) activity were isolated from strains of P. aeruginosa for which histidine is a growth rate-limiting source of nitrogen. Strains SU1 and SU4 were unable to grow when a low concentration of ammonia and a variety of compounds, including histidine, were supplied as sole sources of nitrogen. A revertant of strain SU1, strain 39, produced no GOGAT but high levels of nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase and had restored ability to grow on a limited number of nitrogen sources. Strain US1 grew at the same rate in histidine medium as did its parent; it was derepressed for glutamine synthase synthesis, and histidase was less sensitive to repression by ammonia than in the parent strain. We conclude that GOGAT is not essential for growth on histidine but high levels of glutamine synthase are required nd high levels of nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase can sustain growth at low concentrations of ammonia in the absence of GOGAT.     

Mutational Engineering of the cyanobacterium Nostoc muscorum for resistance to growth-inhibitory action of LiCl and NaCl

The effect of NaCl on two vital processes of cyanobacterial metabolism, viz. N(2) fixation and oxygenic photosynthesis, was studied in the cyanobacterium Nostoc muscorum grown diazotrophically. An increase in NaCl concentration suppressed the formation of heterocyst and adversely affected the nitrogenase activity in the parent, whereas in Li(+)-R and Na(+)-R mutants NaCl stress did not cause any adverse effect. The rate of photosynthetic O(2)-evolution was also adversely affected by the NaCl stress, but the magnitude was less than that of nitrogenase activity. L-Proline, the well-known osmoprotectant, provided protection to the cyanobacterium against NaCl stress. The parent strain utilized L-proline as a nitrogen source and suppressed heterocyst formation and nitrogenase activity, while mutants showed normal heterocyst frequency and nitrogenase activity. Therefore, it may be that the proline metabolism is altered as a result of mutation. The intracellular levels of proline in the parent were enhanced about threefold in the medium containing 1 mol x m(-3) proline, while in mutants there was no significant increase in the intracellular level of proline. In the medium containing both NaCl and proline, the intracellular level of proline was enhanced in the parent as well as in both mutant strains. This suggests that the parent strain possessed both normal proline uptake and salt-induced proline uptake systems, whereas the mutant strains were defective in normal proline uptake and had only salt-induced proline uptake. The over-accumulation of proline in the presence of NaCl stress is due either to the loss of proline oxidase activity or to the accumulation of exogenous proline.     

Mutant Strain of Bradyrhizobium japonicum with Increased Symbiotic N(2) Fixation Rates and Altered Mo Metabolism Properties

Mutant strains of Bradyrhizobium japonicum that required higher levels of molybdate than the wild-type strain for growth on NO(3)-containing medium were obtained after transposon Tn5 mutagenesis of the wild-type strain. The mutant strains expressed more than fivefold-greater nitrate reductase activities in the range of 0.1 to 1.0 mM added molybdate compared with activities expressed upon incubation in non-Mo-supplemented medium, whereas the nitrate reductase activity of the wild-type strain (JH) was not markedly influenced by Mo supplementation. In free-living culture, mutant strains JH310 and JH359 expressed substantial nitrogenase activity, even in medium treated to remove molybdate, and nitrogenase activity was influenced little by Mo supplementation, whereas the wild-type strain required 100 nM added Mo for highest nitrogenase activity. Double-reciprocal plots of Mo uptake rates versus Mo concentration showed that both bacteroids and free-living cells of mutant strain JH359 had about the same affinity for Mo as did the parent strain. Bacteroids of both the mutants and the wild type also exhibited similar Mo accumulation rates over a 9-min period under very-low-Mo (4 nM) conditions. Nitrogenase activities for strain JH359 and for the wild-type strain in free-living culture were both strongly inhibited by tungsten; thus, the nitrogenase activities of both strains are probably the result of a "conventional" Mo-containing nitrogenase. Soybeans inoculated with strain JH359 and grown under either Mo-supplemented or Mo-deficient conditions had greater specific acetylene reduction rates and significantly greater plant fresh weight than those inoculated with the wild-type strain. Under Mo-deficient conditions, the acetylene reduction rates and plant fresh weights were up to 35 and 58% greater, respectively, for mutant-nodulated plants compared with wild-type-strain-nodulated plants.     

THE ADAPTATION OF FUNGI TO FUNGICIDES: ADAPTATION TO COPPER AND MERCURY SALTS

The susceptibility of Botrytis cinerea to copper sulphate in liquid media increased when the volume, and therefore the depth, of the medium in culture bottles exceeded certain values; when the volume was 40 ml. the maximum concentration allowing growth was 300 p.p.m.
By growing mycelium in media containing progressively higher concentrations of copper sulphate a strain was produced which grew at a concentration of 750 p.p.m.
In high concentrations of copper sulphate growth always started at the edge of the liquid, and inocula grew only if they were placed in this position.
In germination tests spores from the resistant strain were more resistant to copper sulphate than were spores of the parent strain.
The resistance of mycelium, and to a lesser extent of spores, was retained after growth of the resistant strain for six months in fungicide-free media.
Spore and mycelial inocula grew in much higher concentrations of copper sulphate when nutrient media were solidified with agar.
The strain resistant in liquid media was no more resistant than the parent strain on agar media.
The resistance of the fungus was not increased after growth for long periods on agar containing high concentrations of copper sulphate. The resistance of the strain resistant in liquid media was not lost after growth on agar media for 3 months.
Attempts to produce strains more resistant than the parent to mercuric chloride were unsuccessful.
The results obtained with phenyl-mercuric acetate were essentially similar to those obtained with copper sulphate, but relatively much more resistant strains were produced.     

Mechanism of nitrogenase switch-off by oxygen.

Oxygen caused a reversible inhibition (switch-off) of nitrogenase activity in whole cells of four strains of diazotrophs, the facultative anaerobe Klebsiella pneumoniae and three strains of photosynthetic bacteria (Rhodopseudomonas sphaeroides f. sp. denitrificans and Rhodopseudomonas capsulata strains AD2 and BK5). In K. pneumoniae 50% inhibition of acetylene reduction was attained at an O2 concentration of 0.37 microM. Cyanide (90 microM), which did not affect acetylene reduction but inhibited whole-cell respiration by 60 to 70%, shifted the O2 concentration that caused 50% inhibition of nitrogenase activity to 2.9 microM. A mutant strain of K. pneumoniae, strain AH11, has a respiration rate that is 65 to 75% higher than that of the wild type, but its nitrogenase activity is similar to wild-type activity. Acetylene reduction by whole cells of this mutant was inhibited 50% by 0.20 microM O2. Inhibition by CN- of 40 to 50% of the O2 uptake in the mutant shifted the O2 concentration that caused 50% inhibition of nitrogenase to 1.58 microM. Thus, when the respiration rates were lower, higher oxygen concentrations were required to inhibit nitrogenase. Reversible inhibition of nitrogenase activity in vivo was caused under anaerobic conditions by other electron acceptors. Addition of 2 mM sulfite to cell suspensions of R. capsulata B10 and R. sphaeroides inhibited nitrogenase activity. Nitrite also inhibited acetylene reduction in whole cells of the photodenitrifier R. sphaeroides but not in R. capsulata B10, which is not capable of enzymatic reduction of NO2-. Lower concentrations of NO2- were required to inhibit the activity in NO3- -grown cells, which have higher activities of nitrite reductase.(ABSTRACT TRUNCATED AT 250 WORDS)     

An azide-resistant mutant of the blue-green algaNostoc muscorum producing heterocyst and nitrogenase in the presence of fixed inorganic nitrogen source

The N₂, NO ₃ ^– , NO ₂ ^– , NH ₄ ⁺ and glutamine growing cultures of parentNostoc muscorum are found more or less equally sensitive to azide inhibition of growth. A mutant strain resistant to sodium azide was isolated from the parent strain in NO ₃ ^– medium and the two strains were compared with regard to their heterocyst formation and nitrogenase activity in NO ₃ ^– , NO ₂ ^– , NH ₄ ⁺ and glutamine media. While the parent strain stops production of both heterocyst and nitrogenase in all the fixed nitrogen media, the azide resistant strain forms both in the fixed inorganic nitrogen media but only heterocyst and no nitrogenase in the glutamine medium. Clearly a single genetic determinant of regulatory nature appears to mediate azide-resistance as well as relief of heterocyst and nitrogenase formation from inhibition by the fixed inorganic nitrogen source. The results of glutamine effect on the heterocyst and nitrogenase formation of the two strains indicate the operation of two levels of glutamine-sensitive regulation, one which operates through the common genetic determinant of heterocyst and nitrogenase regulation and the other exclusive to nitrogenase regulation. The in vivo functional nitrogenase does not appear to be the reason for azide-resistance and neither ammonia nor glutamine or its close metabolic product seems to function in the control of heterocyst spacing.     

Evidence for ammonia as an inhibitor of heterocyst and nitrogenase formation in the cyanobacterium Anabaena cycadeae

Growth and regulation of heterocyst and nitrogenase by fixed nitrogen sources were studied comparatively in parent and glutamine auxotrophic mutant of Anabaena cycadeae. The parent strain grew well on N2, NH+4 or glutamine while the mutant strain grew on glutamine but not on N2 or NH+4. The total lack of active glutamine synthetase in the mutant strain thus appears to be the reason for its observed lack of growth in N2 or NH+4, which explains why it is a glutamine auxotroph and at the same time shows glutamine synthetase to be the sole primary ammonia assimilating enzyme. NH+4 repression of heterocyst and nitrogenase in the mutant and the parental strains and their derepression by L-methionine-DL-sulfoximine suggest that NH+4 per se and not glutamine synthetase mediated pathway of ammonia assimilation is the initial repressor signal of heterocyst and nitrogenase in A. cycadeae.     

The roles of the nifW, nifZ and nifM genes of Klebsiella pneumoniae in nitrogenase biosynthesis

Active Fe protein of nitrogenase was synthesised in a non-nitrogen fixing organism when Escherichia coli was transformed with a plasmid encoding only two nif-specific genes, nifH and nifM of Klebsiella pneumoniae. Hence proteins NifH and NifM are sufficient to produce active Fe protein in E. coli. K. pneumoniae strains carrying chromosomal nifW- and nifZ- mutations were constructed and shown to be significant C2H2-reducing activity and to grow on N-free plates. Nevertheless, derepressing cultures of the mutant strains had reduced levels of MoFe protein activity, and consequently significantly lower levels of nitrogenase activity, than the nif+ parent strain. NifW and NifZ therefore appear to be involved in the formation or accumulation of active MoFe protein, but are not essential for nitrogen fixation in K. pneumoniae under the conditions tested.     

Toxic effects of chlorinated and brominated alkanoic acids on Pseudomonas putida PP3: selection at high frequencies of mutations in genes encoding dehalogenases

Mutant strains of Pseudomonas putida PP3 capable of utilizing monochloroacetate (MCA) and dichloroacetate (DCA) as the sole sources of carbon and energy were isolated from chemostat cultures. The mutants differed from the parent strain in that they could grow on products of MCA and DCA dehalogenation (catalyzed by inducible dehalogenases I and II) and were resistant to growth inhibition by the two substrates. The growth inhibition of strain PP3 by MCA, DCA, and other halogenated alkanoic acids was studied. Sensitivity to dehalogenase substrates was related to the expression of the dehalogenase genes. For example, mutants producing elevated levels of one or both of the dehalogenases were sensitive to 2-monochloropropionate and 2-monochlorobutanoate at concentrations which did not affect the growth of strain PP3. P. putida PP1, the parent of strain PP3, was resistant to the inhibitory effects of MCA and DCA. Spontaneous mutants of strain PP3, also resistant to MCA and DCA, were selected at high frequency, and four different classes of these strains were distinguished on the basis of dehalogenase phenotype. All dehalogenase-producing mutants were inducible; no constitutive mutant has yet been isolated. Most of the resistant mutants examined did not produce one or both of the dehalogenase, and over half of those tested failed to revert back to the parental (strain PP3) phenotype, indicating that the observed mutations involved high-frequency deletion of DNA base sequences affecting expression of genes encoding dehalogenases and associated permease(s).     

Mutant Strain of Bradyrhizobium japonicum with Increased Symbiotic N2 Fixation Rates and Altered Mo Metabolism Properties

Mutant strains of Bradyrhizobium japonicum that required higher levels of molybdate than the wild-type strain for growth on NO₃-containing medium were obtained after transposon Tn5 mutagenesis of the wild-type strain. The mutant strains expressed more than fivefold-greater nitrate reductase activities in the range of 0.1 to 1.0 mM added molybdate compared with activities expressed upon incubation in non-Mo-supplemented medium, whereas the nitrate reductase activity of the wild-type strain (JH) was not markedly influenced by Mo supplementation. In free-living culture, mutant strains JH310 and JH359 expressed substantial nitrogenase activity, even in medium treated to remove molybdate, and nitrogenase activity was influenced little by Mo supplementation, whereas the wild-type strain required 100 nM added Mo for highest nitrogenase activity. Double-reciprocal plots of Mo uptake rates versus Mo concentration showed that both bacteroids and free-living cells of mutant strain JH359 had about the same affinity for Mo as did the parent strain. Bacteroids of both the mutants and the wild type also exhibited similar Mo accumulation rates over a 9-min period under very-low-Mo (4 nM) conditions. Nitrogenase activities for strain JH359 and for the wild-type strain in free-living culture were both strongly inhibited by tungsten; thus, the nitrogenase activities of both strains are probably the result of a “conventional” Mo-containing nitrogenase. Soybeans inoculated with strain JH359 and grown under either Mo-supplemented or Mo-deficient conditions had greater specific acetylene reduction rates and significantly greater plant fresh weight than those inoculated with the wild-type strain. Under Mo-deficient conditions, the acetylene reduction rates and plant fresh weights were up to 35 and 58% greater, respectively, for mutant-nodulated plants compared with wild-type-strain-nodulated plants.     

Enhanced thermotolerance and ethanol tolerance in Saccharomyces cerevisiae mutated by high-energy pulse electron beam and protoplast fusion

To increase thermotolerance and ethanol tolerance in Saccharomyces cerevisiae strain YZ1, the strategies of high-energy pulse electron beam (HEPE) and three rounds of protoplast fusion were explored. The YF31 strain had the characteristics of resistant to high-temperature, high-ethanol tolerance, rapid growth and high yield. The YF31 could grow on plate cultures up to 47?°C, containing 237.5?g?L^?1 of ethanol. In particular, the mutant strain YF31 generated 94.2?±?4.8?g?L^?1 ethanol from 200?g glucose L^?1 at 42?°C, which was 2.48 times the production of the wild strain YZ1. Results demonstrated that the variant phenotypes from the strains screening by HEPE irradiation could be used as parent stock for yeast regeneration and the protoplast fusion technology is sufficiently powerful in combining suitable characteristics in a single strain for ethanol fermentation.     

Effect of cadmium, chromium and copper on symbiotic and free-living Rhizobium leguminosarum biovar trifolii

Abstract Plasmid-minus derivatives of Rhizobium leguminosarum biovar trifolii have been isolated. Cured strains lacked symbiotic properties, however they showed increased heavy metal resistance. In the presence of 70 ppm chromium the parent strain, unlike cured derivatives, is unable to grow explanta but can nevertheless nodulate clover.
We propose that rhizobia can circumvent exposure to the heavy metal by entering the plant roots.
Acetylene reduction tests showed that nodulated plants, grown in the presence of 10 ppm of chromium, had an increased nitrogenase activity compared to the control plants.     

脱落酸产生菌的遗传育种

从南京蔬菜研究所植物致病株中分离到3株脱落酸（ABA）产量较高菌株,经初步鉴定均为灰葡萄孢（BolrytiscinereaPers．）。分别命名为A23,A101,A160通过紫外诱变及菌株生长状况比较,选出一株菌A101作为出发菌,经紫外,硫酸二乙酯多次诱变,在ANA合成代谢关键酶抑制剂平板上定向选育出一株ANA高产菌UUD－1,该菌固体发酵可产生ABA374μg／ml,是出发菌株产量的14倍,且其产量连续培养5代稳定。     

Bradyrhizobium japonicum mutants defective in nitrogen fixation and molybdenum metabolism.

Bradyrhizobium japonicum JH mutants deficient in molybdenum metabolism into the enzymes nitrogenase and nitrate reductase were isolated by using the vector pSUP1011, which carries transposon Tn5 (streptomycin and kanamycin resistance). Mutants in Mo metabolism were obtained at a frequency of 3.6 X 10(-3) (per Kan Strr colony). The mutants were detected by their poor ability to grow in nitrate-containing medium without added Mo. One of the mutant types required 10(5) times more molybdate than the wild type to obtain maximal nitrogen fixation activity. Double-reciprocal plots of Mo uptake versus concentration indicated that the wild-type strain had a high- and a lower-affinity component for Mo binding. Mutant strains JH-90 and JH-119 lacked the high-affinity Mo uptake component and were also clearly deficient in Mo accumulation into a nonexchangeable form. Nitrogenase activity as well as Mo uptake ability could be restored in strains JH-90 and JH-119 by the addition of the sterile supernatant fraction of the wild type. Therefore, mutant strains JH-90 and JH-119 appeared to be deficient in an extracellular Mo-binding factor produced by the wild type. Mutant strains JH-14 and JH-143 had Mo uptake kinetics like those of the wild type (both high- and low-affinity binding for Mo) and appeared to be deficient in intracellular Mo metabolism processes. The addition of the wild-type supernatant did not restore Mo uptake or nitrogenase activity in these strains.     

Competitiveness of a nif− Bradyrhizobium japonicum mutant against the wild-type strain

Abstract By mixed inoculation experiments, the competitive ability of a nifD ::Tn 5 mutant of Bradyrhizobium japonicum was compared to its effective, isogenic parent strain. When the strains were inoculated in a 1:1 ratio at high concentration, the mutant was found to colonize almost as many nodules as the wild type. Thus, lack of expression of a functional nitrogenase system does not severely reduce competitiveness. In such experiments the majority of the nodules (> 60%) were infected by both wild-type and mutant strains. From statistical analysis it was concluded that a mean number of 2–4 bacteria have successfully elicited one nodule under the described conditions. Visual and microscopic observations of sections from mixed infected nodules revealed separated sectors containing effective or ineffective bacteroids.     

Modeling growth and biochemical activities of Azospirillum spp.

An unstructured mathematical model was developed and used in the evaluation of biochemical activities of four Azospirillum spp. strains grown in batch cultures in a high C/N-ratio medium. The strains were evaluated for their ability to grow on fructose and produce exo-polysaccharide, and to sustain nitrogenase activity by using fructose or polysaccharides. Quantitative expression of the regulation of polysaccharide synthesis and nitrogenase (acetylene reduction) activity from the mineral nitrogen and sugar concentration in the culture medium was achieved. It was found that, during growth, Azospirillum spp. produced significant quantities of exocellular and capsular polysaccharide, whereas after depletion of the carbon source from the culture medium polysaccharides were consumed, especially in A. lipoferum strains. Significant nitrogenase activity was detected during polysaccharide degradation. Oxygen uptake was high during assimilation of fructose and low during polysaccharide degradation.