Catalytic-rate improvement of a thermostable malate dehydrogenase by a subtle alteration in cofactor binding.

The nucleotide-binding fold of many NAD(+)-dependent dehydrogenases contains a conserved acidic amino acid residue which hydrogen-bonds with the 2'- and 3'-hydroxy groups of the adenine-ribose of the cofactor. This residue is highly conserved as aspartate in malate dehydrogenases, except in the thermophilic enzyme from Thermus aquaticus B (TaqMDH), which has glutamic acid-41 in the equivalent position. The catalytic mechanism was dissected to investigate the functional significance of this difference in TaqMDH with respect to a mutant enzyme where glutamic acid-41 was replaced by aspartic acid. The mutant enzyme was found to retain a high degree of protein structural stability to both thermal and chemical denaturation. When compared with the wild-type enzyme the mutant had a higher Km and Kd for both reduced and oxidized cofactors (NADH and NAD+) and a 2-3-fold increase in steady-state kcat in both assay directions. The rate-determining step for the reduction of oxaloacetate by wild-type TaqMDH was shown to be the rate of NAD+ release, which was about 2.5-fold higher for the mutant enzyme. This correlates well with the 1.8-fold higher steady-state kcat of the mutant enzyme and represents an improvement in the steady-state kcat of a thermophilic enzyme at moderate temperature by a conservative amino acid substitution which increases the rate of product release.

Nitrogen Fixation Associated with Development and Localization of Mixed Populations of Cellulomonas sp. and Azospirillum brasilense Grown on Cellulose or Wheat Straw.

Mixed cultures of Cellulomonas sp. and Azospirillum brasilense were grown with straw or cellulose as the carbon source under conditions favoring the fixation of atmospheric nitrogen. Rapid increases in cell numbers, up to 10 cells per g of substrate, were evident after 4 and 5 days of incubation at 30 degrees C for cellulose and straw, respectively. Nitrogen fixation (detected by acetylene reduction measured on parallel cultures) commenced after 2 and 4 days of incubation for straw and cellulose, respectively, and continued for the duration of the experiment. Pure cultures of Cellulomonas sp. showed an increase in cell numbers, but CO(2) production was low, and acetylene reduction was not detected on either cellulose or straw. Pure cultures of A. brasilense on cellulose showed an initial increase in cell numbers (10 cells per g of substrate) over 4 days, followed by a decline presumably caused by the exhaustion of available carbon substrate. On straw, A. brasilense increased to 10 cells per g of substrate over 5 days and then declined slowly; this growth was accompanied by acetylene reduction. Scanning electron micrographs of straw incubated with a mixed culture under the above conditions for 8 days showed cells of both species in close proximity to each other. Evidence was furnished that the close spatial relationship of cells from the two species facilitated the mutually beneficial association between them and thus increased the efficiency with which the products of straw breakdown were used for nitrogen fixation.

Comparison of Two Cellulomonas Strains and Their Interaction with Azospirillum brasilense in Degradation of Wheat Straw and Associated Nitrogen Fixation.

A mutant strain of Cellulomonas sp. CS1-17 was compared with Cellulomonas gelida 2480 as the cellulolytic component of a mixed culture which was responsible for the breakdown of wheat straw to support asymbiotic nitrogen fixation by Azospirillum brasilense Sp7 (ATCC 29145). Cellulomonas sp. strain CSI-17 was more efficient than was C. gelida in cellulose breakdown at lower oxygen concentrations and, in mixed culture with A. brasilense, it supported higher nitrogenase activity (C(2)H(2) reduction) and nitrogen fixation with straw as the carbon source. Based on gravimetric determinations of straw breakdown and total N determinations, the efficiency of nitrogen fixation was 72 and 63 mg of N per g of straw utilized for the mixtures containing Cellulomonas sp. and C. gelida, respectively. Both Cellulomonas spp. and Azospirillum spp. exhibited a wide range of pH tolerance. When introduced into sterilized soil, the Cellulomonas sp.-Azospirillum brasilense association was more effective in nitrogen fixation at a pH of 7.0 than at the native soil pH (5.6). This was also true of the indigenous diazotrophic microflora of this soil. The potential implications of this work to the field situation are discussed.

Cellulose Decomposition and Associated Nitrogen Fixation by Mixed Cultures of Cellulomonas gelida and Azospirillum Species or Bacillus macerans.

Mixed cultures of Cellulomonas gelida plus Azospirillum lipoferum or Azospirillum brasilense and C. gelida plus Bacillus macerans were shown to degrade cellulose and straw and to utilize the energy-yielding products to fix atmospheric nitrogen. This cooperative process was followed over 30 days in sand-based cultures in which the breakdown of 20% of the cellulose and 28 to 30% of the straw resulted in the fixation of 12 to 14.6 mg of N per g of cellulose and 17 to 19 mg of N per g of g straw consumed. Cellulomonas species have certain advantages over aerobic cellulose-degrading fungi in being able to degrade cellulose at oxygen concentrations as low as 1% O(2) (vol/vol) which would allow a close association between cellulose-degrading and microaerobic diazotrophic microorganisms. Cultures inoculated with initially different proportions of A. brasilense and C. gelida all reached a stable ratio of approximately 1 Azospirillum/3 Cellulomonas cells.

Straw and Xylan Utilization by Pure Cultures of Nitrogen-Fixing Azospirillum spp.

Azospirillum spp. were shown to utilize both straw and xylan, a major component of straw, for growth with an adequate combined N supply and also under N-limiting conditions. For most strains examined, a semisolid agar medium was satisfactory, but several strains appeared to be capable of slow metabolism of the agar. Subsequently, experiments were done with acid-washed sand supplemented with various carbon sources. In these experiments, authenticated laboratory strains, and all 16 recent field isolates from straw-amended soils, of both A. brasilense and A. lipoferum possessed the ability to utilize straw and xylan as energy sources for nitrogen fixation. Neither carboxymethyl cellulose nor cellulose was utilized. The strains and isolates differed in their abilities to utilize xylan and straw and in the efficiency of nitrogenase activity (CO(2)/C(2)H(2) ratio). Reasonable levels of activity could be maintained for at least 14 days in the sand cultures. Nitrogenase activity (acetylene reduction) was confirmed by N(2) incorporation. The level of nitrogenase activity observed was dependent on the time of the addition of acetylene to the culture vessels.

Isolation and characterization of an Escherichia coli K-12 mutant defective in tyrosine- and phenylalanine-specific transport systems.

A mutant strain of Escherichia coli K-12 that is defective in both the tyrosine-specific and phenylalanine-specific transport systems was isolated. The defects in these systems were shown to be due to mutations in two distinct loci, tyrP and pheP, respectively.

Effects of certain cations on the formation and infectivity of Phytophthora zoospores. 2. Effects of copper, boron, cobalt, manganese, molybdenum, and zinc ions.

The effects of copper, boron, cobalt, manganese, molybdenum, and zinc on the production of zoosporangia by P. cinnamomi and P. drechsleri in the presence of favourable concentrations of calcium, magnesium, potassium, and iron were investigated. Copper ions were the most effective in reducing the numbers of zoosporangia formed by both fungal species. Molybdenum was also slightly inhibitory. Total inhibition of mycelial growth occurred between 1 and 5 X 10(-5) M Cu2+ whereas total inhibition of sporangial formation occurred between 1 and 5 X 10(-7) M Cu2+. At copper concentrations between 10(-5) M and 5 X 10(-7) M, many P. drechsleri zoosporangia were abnormal in appearance and nonviable. Infection of eucalypt cotyledons by P. drechsleri zoospores was inhibited by 10(-6) M Cu2+ but this inhibition was reversed by EDTA (10(-4) M). There was no evidence for interaction between Cu2+ and Ca2+, Mg2+, K+, or Fe2+ present in the solutions used in the axenic production of zoospores. Preliminary pot trials indicated that CuSO4 had a protective action for safflower seedlings to infection by P. drechsleri when CuSO4 was applied as a dilute solution over the infection period.

Effects of certain cations on the formation and infectivity of Phytophthora zoospores. 1. Effects of calcium, magnesium, potassium, and iron ions.

The effects of four cations, Ca2+, Mg2+, K+, and Fe3+, upon the production of zoosporangia by isolates of four species of Phytophthora in axenic culture have been investigated. A response surface design was used to examine main effects and possible interactions. Responses to Ca2+ and Fe3+ were strongly quadratic with the higher concentrations inhibiting sporangial production in P. cinnamomi and P. drechsleri isolates. Responses to Mg2+ and K+ were weaker and, in the case of magnesium, were linear rather than quadratic. There was no significant interaction between cations except in the case of P. cambivora where the interaction between Mg2+ and K+ was significant at the 5% level. For each cation, the optimum concentration for zoosporangial production was calculated. The processes of zoospore release and infection of plant material by P. drechsleri showed a strong linear and weak quadratic response to Ca2+, higher concentrations of Ca2+ favouring these processes. A strong quadratic response was obtained to Mg2+, K+, and Fe3+. Interaction between Ca2+ and Mg2+ was evident for all three host materials used; Mg2+ and K+ showed an interaction when Pinus radiata was used as the host material. The implications and limitations of these results are discussed.

Specificity of cytoplasmic and cell-wall antigens from four species of Phytophthora.

Cytoplasmic and cell-wall antigens and antisera were prepared from four Phytophthora species, and cell-wall antigens were prepared from two Pythium species. Immunodiffusion of the Pythium and Phytophthora cell-wall antigens showed that the two Pythium species did not cross-react with the Phytophthora cell-wall antisera. Immunodiffusion analysis of both cell-wall and cytoplasmic antigens of Phytophthora revealed some degree of specificity between species but not between A1 and A2 mating types in Phytophthora cinnamomi. Species specificity was improved by using indirect fluorescent antibody techniques and by the use of cross-absorbed sera. Agglutination and quantitative precipitation techniques did not significantly improve specificity. It was possible to distinguish serologically between Phytophthora cinnamomi and Phytophthora cambivora and the Phytophthora cryptogea-Phytophthora drechsleri group. The absence of consistent serological variation between P. cryptogea and P dreschsleri is consistent with the suggestion (Bumbieris, 1974) that P. cryptogea and P. drechsleri should be considered as one species.

Zoospore chemotaxis in Australian isolates of Phytophthora species.

Zoospores of Australian isolates of Phytophthora drechsleri, P. cryptogea, P. cinnamomi, P. nicotianae var. parasitica, and P. citricola were examined for their chemotactic responses to asparagine, glutamine, aspartate, glutamate, and structurally related compounds. Structural requirements for attraction include the alpha-amino-acid group with a short carbon chain terminating in an amide group. The one American isolate tested gave a different result and possible reasons for this are discussed. The pH of the environment was important, a neutral-charged molecule was more attractive than a negatively charged molecule, hence glutamine and aspartate were more attractive at pH 3.0 than pH 5.0. Zoospores tended to move away from regions with a high hydrogen ion concentration. Compounds other than amino acids were slightly attractive including several sugars and ethanol. Synergistic interactions between amino acids, ethanol, and sucrose were observed and may account for the high levels of attraction of zoospores to root exudates and extracts.

Overproduction of lysine by mutant strains of Escherichia coli with defective lysine transport systems.

Mutants selected on the basis of their resistance to S-(beta-aminoethyl) cysteine and overproduction of lysine were found to be defective in the lysine transport system. The overproduction of lysine was not due to mutation affecting either of the two regulatory enzymes aspartokinase and dihydrodipicolinic acid synthetase. Uptake of labeled lysine by the lysine-specific transport system was reduced to a negligible level, while uptake by the lysine, ornithine, arginine system was also affected. A hypothesis regarding the nature of these mutations and their effects on the regulation of lysine biosynthesis is discussed.