Effect of some herbicides on the production of lysine byAzotobacter chroococcum

Summary Production of lysine byAzotobacter chroococcum strain H23 was studied in chemically-defined media amended with different concentrations of alachlor, metolachlor, 2,4-D, 2,4,5-T and 2,3,6-TBA. The presence of 5, 10, and 50g/ml of alachlor or 2,3,6-TBA significantly decreased quantitative production of lysine. However, the presence 2,4-D or 2,4,5-T at concentrations of 10 and 50g/ml enhanced the production of lysine. Quantitative production of lysine was not affected as consequence of the addition of metolachlor to the culture medium, showing that the release lysine to the culture media byA. chroococcum was not affected by that herbicide.     

The effect of aspartate-derived amino acids (lysine,threonine, methionine) on the growth of excised embryos of wheat and barley

Excised wheat (Triticum aestivum L. var. Maris Freeman) and barley (Hordeum vulgare L. var. Maris Mink) embryos were grown on medium containing both nitrate and ammonium ions. Addition of lysine (1 mM) plus threonine (1 mM) caused a synergistic inhibition of growth measured by length of first leaf or dry weight. The inhibition was specifically relieved by methionine, homocysteine and homoserine. Threonine at 0.2–0.3 mM caused half-maximal inhibition of growth at all lysine concentrations whereas lysine increased the synergistic inhibition up to 3 mM. The inhibition is explained by a model in which lysine acts as a feedback inhibitor of aspartate kinase and threonine of homoserine dehydrogenase. This is compatible with published studies of the enzymes involved. The implications of these findings for using lysine plus threonine as a selection system for lysine-overproducing cereals are discussed.Abbreviations Lys Lysine - Thr Threonine - Met Methionine - Hser Homoserine - Hcys Homocysteine     

Production of amino acids by Azotobacter vinelandii and Azotobacter chroococcum with phenolic compounds as sole carbon source under diazotrophic and adiazotrophic conditions

Summary. Azotobacter vinelandii strain ATCC 12837 and Azotobacter chroococcum strain H23 (CECT4435) were tested to grow in N-free or NH₄Cl amended chemically defined media, with protocatechuic acid or sodium p-hydroxybenzoate as sole carbon (C) sources at a concentration of 2 mmol/L. Both substrates supported grow at similar rates than bacteria grown in control media amended with 2 mmol/L sodium succinate as C source. The two strains produced aspartic acid, serine, glutamic acid, glycine, hystidine, threonine, arginine, alanine, proline, cysteine, tyrosine, valine, methionine, lysine, isoleucine, leucine and phenylalanine after 72 h of growth in chemically defined media with 2 mmol/L of phenolic compounds or sodium succinate as sole C source amended or unamended with 0.1% (w/v) NH₄Cl. Qualitative and quantitative production of all amino acids was not affected by the use of different C and N substrates.     

Studies on lysine analogs,aspartate-derived amino acids,and attempted mutant selection on oat seedlings

The lysine analogs S-2-aminoethyl-L-cysteine (AEC) and DL--hydroxylysine (DHL) caused severe growth inhibition in dark-grown oat seedlings (Avena sativa L. and A. nuda L.) at similar concentrations while L-lysine methyl ester (LME) had little effect. Lysine, arginine, and ornithine reversed the inhibition caused by AEC and DHL, the order of effectiveness being lysine>arginine>ornithine. Of aspartate-pathway amino acids, tested individually and in combinations for inhibitory effects on seedling growth, lysine and combinations containing lysine were the most inhibitory, but the inhibition was much less than that produced by AEC. Only slight synergistic effects occurred when oat seedlings were grown in the presence of paired combinations of aspartatepathway endproduct amino acids.Ca. 54,000 seeds obtained from 3,463 plants grown from ethyl-methanesulfonate (EMS) treated seed were screened for resistance to AEC. Three resistant variants were identified but the resistance was not recovered among their self-pollinated progeny.Abbreviations AEC S-2-aminoethyl-L-cysteine - DHL DL--hydroxylysine - EMS ethyl methanesulfonate - LME L-lysine methyl ester Paper No. 10351, Scientific Journal Series, Minnesota Agricultural Experiment Station     

Biosorption of metal ions byAzotobacter vinelandii

Azotobacter vinelandii was better than eitherDerxia gummosa orRhizobium trifolii for sorption of UO ₂ ²⁺ . Its maximum binding capacity was 0.25 mmol UO ₂ ²⁺ /g dry biomass with an affinity constant of 333 l/mmol at pH 4.1 according to the Langmuir model. In a semisynthetic medium,A. vinelandii showed the highest sorption capacity in the early stationary phase. The binding of UO ₂ ²⁺ , Cu²⁺, Ca²⁺ and Zn²⁺ was affected by the pH of the solution. With HCl as eluent, virtually all the sorbed UO ₂ ²⁺ was released. The presence of Cu²⁺, Cd²⁺, Ca²⁺, and Zn²⁺ inhibited the UO ₂ ²⁺ biosorption whereas Mg²⁺ and K⁺ had no effect.     

Effect of O2 limitation on growth and respiration of the wild type and an ascorbate-tetramethyl-p-phenylenediamine-oxidase-negative mutant strain ofAzotobacter vinelandii

Azotobacter vinelandii strain AVOP (wild type) and an ascorbate-N,N,N,N-tetramethylene-p-phenylenediamine oxidase-negative mutant (AV11) were each grown in O₂-limited chemostat cultures. The results showed that the mutant strain grew and used O₂ less efficiently than the wild-type strain. Respiration rates of membrane particles with NADH or malate as the substrate were similar for each strain. Succinate oxidase activity was about fourfold lower in membrane particles prepared from mutant than from wild-type strain. Cyanide at a concentration that completely inhibited ascorbate-TMPD oxidase activity resulted in a 50% inhibition of NADH oxidase activity in membrane particles of AVOP. These data suggest that the cytochromeo,a ₁, oxidase branch of the respiratory chain may be important in the physiology ofA. vinelandii under O₂-limiting growth conditions.     

Seed inoculation withAzospirillum brasilense andAzotobacter chroococcum and the root biomass of rice (Oryza sativa L.)

Summary The root biomass of rice seedlings increased due to inoculation withAzospirillum brasilense andAzotobacter chroococcum singly or in combination. In general, the increase in the biomass of roots was better in unsterilized soil than in sterilized one with or without inorganic nitrogen in the form of urea.     

Ammonia excretion byAzotobacter chroococcum in liquid culture and soil in the presence of manganese and clay minerals

Summary Ammonia excretion was studied in the presence of Mn⁺⁺ in liquid culture and soil with amendments of the minerals using two ammonia excreting strains ofAzotobacter chroococcum (AC2 and AC50) and a non-excreting strain (AC45) under sterilized as well as unsterilized conditions. Ammonia excretion increased in the presence of 1×10⁻⁷M Mn⁺⁺ in liquid culture as well as in soil. But there was a decrease in ammonia excretion with increasing clay content.     

Formation of protoplasts in Azotobacter vinelandii

Protoplasts of Azotobacter vinelandii were formed by incubating whole cells in lysozyme and EDTA in Tris-HCl buffer (0.05 M, pH 8.0) supplemented with sucrose (15% w/v). This appeared to be related to the special chelating ability of EDTA and Tris-HCl since substitution of the former by nitrilotriacetic acid or by trisodium citrate and the latter by veronal-acetate buffer or tris-maleate buffer over a pH range of 5.2 to 8.6 yielded only spheroplasts. Of nine strains of Azotobacter studied, only A. vinelandii strain 12837 and strain 0 formed protoplasts.     

Nutritional improvement of the aspartate family of amino acids in edible crop plants

Summary Plants are the primary source of protein for man and livestock, however, not all plants produce proteins which contain a balance of amino acids for the diet to ensure proper growth of livestock and humans. Alteration of the amino acid composition of plants may be accomplished using techniques of molecular biology and genetic engineering. Genes encoding key enzymes regulating the synthesis of lysine and threonine have been cloned from plants andE. coli and are available for modification and transformation into plants. Genes encoding seed storage proteins have been cloned and modified to encode more lysine residues for developing transgenic plants with higher seed lysine. Genes encoding seed storage proteins naturally higher in methionine have been cloned and expressed in transgenic plants, increasing methionine levels of the seed. These and other approaches hold great promise in their application to increasing the content of essential amino acids in plants.Abbreviations: AK = aspartokinase; HSDH = homoserine dehydrogenase; DS = dihydrodipicolinic acid synthase; AEC = S-(2-aminoethyl)-L-cysteineMention of trademark, proprietary product or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may be suitable.     

Characterization of the pyoverdines ofAzotobacter vinelandii ATCC 12 837 with regard to heterogeneity

Summary Azotobacter vinelandii strain ATCC 12 837 produces peptide siderophores of the general class known as pyoverdines. In the past, it was assumed that a single well-defined pyoverdine was produced by each parent microorganism. However, there are a number of reports of incompletely characterized pyoverdines that demonstrate heterogeneity in pyoverdine preparations obtained from a single organism, but the nature of this phenomena has not been explained. This study shows thatA. vinelandii does indeed produce more than one pyoverdine and that these compounds differ in their peptide components. The metabolism of these siderophores suggests that only one of them is a true siderophore while the others are metabolic byproducts. It was demonstrated that this phenomenon is likely due to intrinsic limitations of the synthetase complex involved in the biosynthesis of these compounds. Characterization of two of the major pyoverdines produced demonstrated that they are novel compounds, although they belonged to theAzotobacter-type family of pyoverdines.     

Influence of short chain fatty acids and lysine on Salmonella typhimurium cadA expression

In Salmonella typhimurium, cadA has a role in virulence expression and is an inducible gene that responds to external lysine concentration. In this study, a strain of S. typhimurium carrying a cadA: lacZ fusion was used to determine if the induction of cadA occurred under different lysine concentrations and mildly acid conditions in the presence of short chain fatty acids. Aliquots of an 18-h culture of S. typhimurium were placed on fresh media containing different lysine concentrations at pH 5.8 adjusted by addition of HCl or by 1 M short chain fatty acids (SCFA, acetic, propionic and butyric acid) stock solution. After an induction period of 2 h, -galactosidase activities were assayed. Expression of cadA in rich medium was significantly higher than that of minimal medium at neutral pH and different lysine concentrations. In contrast, at pH 5.8, there was a significant increase in cadA expression, particularly when pH was adjusted using HCl at all lysine levels. Addition of a mixture of organic acids yielded an overall lower cadA expression at all lysine levels studied when compared to HCl. However, each SCFA challenge (individual or as a mixture) caused a high level of expression, both at neutral and acidic pH. Based on these results it is apparent that in the presence of external lysine, SCFA and nutrient availability can influence cadA expression in S. typhimurium.     

Comparative study of the response ofAzotobacter vinelandii andAcetobacter diazotrophicus to changes in pH

Summary Curves were established for the pH response of respiration on eleven substrates byAzotobacter vinelandii andAcetobacter diazotrophicus. With every substrate the optimal pH forA. diazotrophicus was lower than forA. vinelandii. The optimal hydrogen ion concentration forA. diazotrophicus was 5 fold to 365 fold greater than forA. vinelandii depending upon the substrate. In general,A. diazotrophicus supports respiration over a wider pH range than doesA. vinelandii.Dedicated to the memory of Professor John G. Torrey     

Production of amino acids by free-living heterotrophic nitrogen-fixing bacteria

Summary Interest of microbial production of amino acids has been increased greatly since development of biotechnological methods. These methods represent a perspective way applied in a future large-scale manufacture of inexpensive amino acids. In this context, the isolation of producing organisms that may be exploited in the desing of alternative methods for the production of amino acids could be of primary importance.In this review we will describe the liberation of amino acids (methionine, lysine, arginine, tryptophane and glutamic acid) byAzotobacter andAzospirillum during growth in culture media with different carbon sources under diazotrophic and adiazotrophic conditions. These organisms may be useful in developing new methods for the industrial production of amino acids.     

Effect of amino acids on production of xylanase and pectinase from Streptomyces sp. QG-11-3

Xylanase and pectinase production by Streptomyces sp. QG-11-3 was stimulated by DL-norleucine, L-leucine, DL-isoleucine, L-lysine monohydrochloride and DL--phenylalanine by up to 3.72- and 2.78-fold, respectively, whereas the combination of DL-norleucine, L-leucine and DL-isoleucine synergistically stimulated the xylanase and pectinase production by up to 6.72- and 5.62-fold, respectively. Glycine, DL-norvaline, DL-methionine, and DL-aspartic acid showed no significant stimulatory effect on enzyme production.     

Influence of methionine sulfoximine on the photosynthesis of isolated chloroplasts

Methionine sulfoximine provided at a concentration which inhibits photosynthesis in intact leaves (10 mM) had no significant influence on the rate of photosynthesis of isolated pea leaf chloroplasts. In contrast, ammonium, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and D,L-glyceraldehyde all strongly inhibited the photosynthesis of isolated chloroplasts. We conclude that low concentrations of methionine sulfoximine (up to 10 mM) have no direct effect on the photosynthetic process.     

Effect of Dissolved Oxygen on Continuous Production of Methionine

Methionine production by a mutant strain of Corynebacterium lilium was studied at different dilution rates and different dissolved oxygen concentrations, based on a statistical central composite design. The effect of dissolved oxygen levels between 2 % and 60 % and dilution rate levels between 0.04 to 0.29 on continuous production of methionine was studied. The process is described by the Leudeking‐Piret model. The experimentally obtained maxima for methionine and biomass productivities, observed at the same dilution rate of 0.17 h^–1, also corresponds to the theoretical prediction based on this model. This experimental observation is further supported by the surface response prediction of a dilution rate of 0.16 h^–1 for maximum methionine productivity and 0.15 h^–1 for cell mass productivity. Also, beyond the critical value of 30 % dissolved oxygen, the apparent benefits of oxygen transfer become less significant.     

Influence of nitrogen source on growth and nitrogenase activity inAzotobacter vinelandii

Growth and nitrogenase activity were studied in cultures ofAzotobacter vinelandii growing with dinitrogen, ammonium sulfate, aspartic acid or yeast extract. Nitrogenase activity was measured by means of the C₂H₂ reduction test.In the presence of ammonium sulfate nitrogenase is completely repressed. After exhaustion of ammonia its activity is restored following a diauxic lag period of 30 min. With aspartic acid nitrogenase activity is partially repressed, and growth yield is higher than in the culture growing with N₂ only. This is due to simultaneous use of dinitrogen and aspartate. Fluctuations of nitrogenase activity occurring during exponential growth and the mechanism of their regulation are discussed.Abbreviations NA nitrogenase activity - BNF Burk's nitrogen free medium     

Isolation and characterization of nif::kanamycin and nitrogen fixation proficient Azotobacter vinelandii strain, and its implication on the status of multiple chromosomes in Azotobacter

Several lines of experimental analyses on the ploidy status of Azotobacter vinelandii genome lead to the conclusion that it contains more than 40 copies of its chromosome and therefore it is a polyploid organism. The genetic evidence argues against the existence of polyploidy in these cells since the segregation pattern of genetic markers under lack of selection pressure mimic that of haploids. However, when A. vinelandii was made Nif⁻ by inserting a kanamycin resistance marker gene in the nifDK sequence and the cells were selected for kanamycin resistance and Nif⁺ phenotype, we were able to score colonies that are both kanamycin resistant and Nif⁺. Therefore, when the cells were subjected to forced double selection of the same locus, they behaved as if they carried at least two chromosomes, one carrying the kanamycin resistance marker in the nifDK genes and the other carrying the intact nifDK genes. These analyses suggested that at least a diploidy status can be induced in these cells under selection pressure. This revised version was published online in July 2006 with corrections to the Cover Date.