Interrelationship of nitrogen fixation,hydrogen evolution and photoreduction in Rhodospirillum rubrum

Summary Rhodospirillum rubrum was grown: 1. photoheterotrophically on a medium containing dl-malate as the carbon source and ammonium chloride as the nitrogen source (medium No. 1); 2. phototrophically with N₂ and dl-malate (medium No. 2); 3. photoautotrophically with N₂, CO₂ and H₂ (medium No. 3).Resting cells derived from these cultures were tested for their ability to photoreduce CO₂, evolve H₂ and fix N₂. Only cells which were grown in medium No. 2 were able to perform all three gas exchanges. The activity pattern of gas exchanges altered in a characteristic way during the growth cycle of the bacterial culture. Cells newly transferred to medium No. 2 showed an enormous increase in the rate of H₂ evolution, which dropped sharply when all l-malate had been used up. The rate of photoreduction of CO₂ increased steadily and reached a maximum level after 120 h. The nitrogen fixing activity remained constant during the whole growth cycle.The yields of H₂ produced per mole of l-malate added were measured as a function of cell age. Only very young cultures gave appreciable yields, which dropped gradually with increasing age.The function of the carbon source is discussed as a regulating factor for photoreduction and hydrogen evolution.     

<Emphasis Type="SmallCaps">l</Emphasis>-Proline nutrition and catabolism in <Emphasis Type="Italic">Staphylococcus saprophyticus</Emphasis>

Staphylococcus saprophyticus strains ATCC 15305, ATCC 35552, and ATCC 49907 were found to require l-proline but not l-arginine for growth in a defined culture medium. All three strains could utilize l-ornithine as a proline source and contained l-ornithine aminotransferase and Δ¹-pyrroline-5-carboxylate reductase activities; strains ATCC 35552 and ATCC 49907 could use l-arginine as a proline source and had l-arginase activity. The proline requirement also could be met by l-prolinamide, l-proline methyl ester, and the dipeptides l-alanyl-l-proline and l-leucyl-l-proline. The bacteria exhibited l-proline degradative activity as measured by the formation of Δ¹-pyrroline-5-carboxylate. The specific activity of proline degradation was not affected by addition of l-proline or NaCl but was highest in strain ATCC 49907 after growth in Mueller–Hinton broth. A membrane fraction from this strain had l-proline dehydrogenase activity as detected both by reaction of Δ¹-pyrroline-5-carboxylate with 2-aminobenzaldehyde (0.79 nmol min⁻¹ mg⁻¹) and by the proline-dependent reduction of p-iodonitrotetrazolium (20.1 nmol min⁻¹ mg⁻¹). A soluble fraction from this strain had Δ¹-pyrroline-5-carboxylate dehydrogenase activity (88.8 nmol min⁻¹ mg⁻¹) as determined by the NAD⁺-dependent oxidation of dl-Δ¹-pyrroline-5-carboxylate. Addition of l-proline to several culture media did not increase the growth rate or final yield of bacteria but did stimulate growth during osmotic stress. When grown with l-ornithine as the proline source, S. saprophyticus was most susceptible to the proline analogues L-azetidine-2-carboylate, 3,4-dehydro-dl-proline, dl-thiazolidine-2-carboxylate, and l-thiazolidine-4-carboxylate. These results indicate that proline uptake and metabolism may be a potential target of antimicrobial therapy for this organism.     

Effect of amino acids on growth ofSphaerotilus discophorus

The effect of casein hydrolysate, of mixtures of amino acids and of individual amino acids on the growth of 4 strains ofSphaerotilus discophorus was determined. Growth was virtually completely inhibited by 1.0% Bacto Casamino Acids, 0.54% simulated casein hydrolysate and 0.2% of a uniform mixture of 18 amino acids. The latter were prepared withl amino acids except thatdl-serine,dl-valine anddl-threonine were present in the uniform amino acid mixture.Experiments designed to test the toxicity of the 18 individual amino acids at 0.018 – 0.36% concentration indicated that arginine, glutamic acid, leucine, lysine and proline were non-toxic. However, aspartic acid and methionine were moderately toxic; growth was greatly repressed at a concentration of 0.36%. The remaining 11 amino acids which included alanine, cystine, glycine, tyrosine, histidine, isoleucine, phenylalanine, serine, threonine, tryptophane and valine were the most toxic of the group. They prevented growth partially or completely, at a concentration of 0.18% or 0.36%.dl-Serine anddl-valine were especially toxic and prevented growth at a concentration of 0.018%. The toxicity of the individuall-amino acids can account for the toxicity of Casamino Acids and simulated casein hydrolysate. l-Methionine or cyanocobalamin (vitamin B₁₂) is required for the growth ofS. discophorus. Alsod- anddl-methionine can replace cyanocobalamin although they completely repress growth when used at the relatively high concentration of 200 µg per ml of medium.     

Condensed phosphate deposition,sulfur amino acid use,and unidirectional transsulfuration in Synechococcus leopoliensis

Cells of the cyanobacterium, Synechococcus leopoliensis, have previously been shown to exhibit diminished growth, increased condensed phosphate accumulation, enlarged polyphosphate bodies, and severe chlorosis when cultured under conditions of sulfur deficiency. These characteristics were used to identify which of several sulfur amino acids and a tripeptide served as a sole sulfur source for this unicellular microorganism. Completely serving sulfur compounds were l-cystine, dl-lanthionine, l-djenkolic acid, and glutathione. Sulfur amino acids serving poorly or not at all were l-cystathionine, dl-homocystine, l-methionine, l-cysteic acid, and taurine. This pattern of use suggests that the unidirectional transsulfuration pathway demonstrated in enteric bacteria and green plants, i.e. l-cysteine to l-homocysteine, operates as well in cyanobacteria of the Synechococcus type.     

Surface-motility induction,attraction and hitchhiking between bacterial species promote dispersal on solid surfaces

The ability to move on solid surfaces provides ecological advantages for bacteria, yet many bacterial species lack this trait. We found that Xanthomonas spp. overcome this limitation by making use of proficient motile bacteria in their vicinity. Using X. perforans and Paenibacillus vortex as models, we show that X. perforans induces surface motility, attracts proficient motile bacteria and ‘rides'' them for dispersal. In addition, X. perforans was able to restore surface motility of strains that lost this mode of motility under multiple growth cycles in the lab. The described interaction occurred both on agar plates and tomato leaves and was observed between several xanthomonads and motile bacterial species. Thus, suggesting that this motility induction and hitchhiking strategy might be widespread and ecologically important. This study provides an example as to how bacteria can rely on the abilities of their neighboring species for their own benefit, signifying the importance of a communal organization for fitness.     

Initiation of Sperm Motility Induced by Cyclic AMP in Hamster and Boar

When the plasma membrane of hamster and boar spermatozoa was extraced by treatment with Triton X-100 and the demembranated spermatozoa were transferred to a reactivating medium containing only ATP, axonemes were initially immotile, and then gradually became motile. Under these experimental conditions, the cAMP content in the reactivating medium increased soon. This suggests that cAMP is synthesized from ATP by adenylate cyclase involved in incompletely removed or solubilized residual sperm membrane and that the autosynthesized cAMP causes the delay in motility initiation. This delayed initiation of motility did not occur when phosphodiesterase was added to the reactivating medium and the phosphodiesterase-dependent quiescent sperm became motile instantaneously at any time when excess cAMP was supplemented. Furthermore, demembranated sperm which were diluted in the reactivating medium containing ATP and cAMP, immediately became motile. cAMP levels in the cell increased during the initiation of sperm motility in both species. These results suggest that cAMP is the real factor indispensable for the initiation of sperm motility at ejaculation in mammals.     

Effect of organic cosolvent on kinetic resolution of tert-leucine by penicillin G acylase from Kluyvera citrophila

Penicillin G acylase (PGA) from Kluyvera citrophila immobilized on Amberzyml was used for enantioselective hydrolysis of N-phenylacetylated-dl-tert-leucine (N-Phac-dl-Tle) to produce l-tert-leucine (l-Tle). The effects of various organic cosolvents on hydrolysis of N-Phac-dl-Tle have been investigated in aqueous-cosolvent medium. It was founded that the rate of PGA-catalyzed reaction was significantly affected by the presence of 2% (v/v) organic cosolvent concentration. The initial rate fell with increasing logP of the cosolvent, but for logP values less than −0.24 the rate was faster than in purely aqueous medium. Additionally, the relative rate increases with the increase of dielectric constant (ε) of organic cosolvents. The yields of l-Tle in all aqueous-cosolvent systems were above 95% with the enantiomeric excess (ee) of >99%.     

Short-term ammonium inhibition of nitrate uptake by Azotobacter chroococcum

Addition of NH₄Cl at low concentrations to Azotobacter chroococcum cells caused an immediate cessation of nitrate uptake activity, which was restored when the added NH ₄ ⁺ was exhausted from the medium or by adding an NH ₄ ⁺ assimilation inhibitor, l-methionine-dl-sulfoximine (MSX) or l-methionine sulfone (MSF). In the presence of such inhibitors the newly-reduced nitrate was released into the medium as NH ₄ ⁺ . When the artificial electron donor system ascorbate/N-methylphenazinium methylsulfate (PMS), which is a respiratory substrate that was known to support nitrate uptake by A. chroococcum while inhibiting glutamine synthetase activity, was the energy source, externally added NH ₄ ⁺ had no effect on nitrate uptake. It is concluded that, in A. chroococcum cells, NH ₄ ⁺ must be assimilated to exert its short-term inhibitory effect on nitrate uptake. A similar proposal was previously made to explain the short-term ammonium inhibition of N₂ fixation in this bacterium.Abbreviations MOPS morpholinopropanesulfonic acid - MSX l-methionine-dl-sulfoximine - PMS N-methylphenazinium methylsulfate - MSF l-methionine sulfone     

Induction of motility and alteration of surface membrane polypeptides in lymphocytes by contact with autologous and allogeneic fibroblasts

Contact with cultured fibroblasts induced and maintained motile behavior in autologous and allogeneic human lymphocytes. After 3 h of contact with fibroblasts, 50 +/- 19% of the autologous lymphocytes were motile and after 24 h the corresponding figure was 49 +/- 18%. On a plastic surface the number of motile lymphocytes in the same individuals generally persisted below 15%. SDS-PAGE of iodine-labeled lymphocytes indicated that contact with fibroblasts but not with plastic for a 3-h period caused the appearance of a 300-kda band and the disappearance of several bands of lower molecular weight. During the course of T-lymphocyte activation by concanavalin A or allogeneic cells on a plastic surface, the number of motile forms did not reach a maximum (30 to 50% in separate individuals) until after 2 to 4 days in culture. Thus, in terms of both rate of development and number of motile forms, the fibroblast-dependent motility mechanism was more effective than conventional lymphocyte activation to blast transformation. Conditioned medium from fibroblasts did not induce motile behavior in the lymphocytes and did not provoke alteration of surface membrane polypeptides as revealed by iodination. Fibroblasts also triggered lymphocyte locomotion in serum-free medium, but their triggering effect was enhanced markedly by serum. The development and maintenance of lymphocyte motility required protein synthesis. These data suggest that during contact with fibroblasts lymphocytes acquire locomotor capacity by a mechanism different from activation to blast transformation.     

d-Alanine production from dl-alanine by Candida maltosa with asymmetric degrading activity

Summary To develop a practical process for d-alanine production from dl-alanine, we screened 107 yeasts for their asymmetric degrading activity against dl-alanine. Candida maltosa JCM1504 degraded the l-isomer ten times more rapidly than the d-isomer. The cells of this strain were used as a biocatalyst for eliminating the l-isomer. However, when the degradation reaction was conducted in the presence of a high concentration of dl-alanine, the pH of the reaction mixture was rapidly increased by the liberation of ammonia from l-alanine, and consequently the reaction stopped. This hindrance was overcome by controlling the pH value at 6.0 with H₂SO₄ during the reaction. Additionally, we found that the maximum rate of l-isomer degradation was obtained at 30° C and pH 6.0 under conditions of high aeration (1.0 vvm) and agitation (1200 rpm). Under the optimal conditions, the l-isomer of 200 g dl-alanine/l was completely degraded within 40 h and 90 g d-alanine/l remained in the reaction mixture. d-Alanine was easily isolated from the reaction mixture. The chemical and optical purity of the d-isomer product so obtained was 99.0% and 99.9% enantiomeric excess, respectively.Offprint requests to: I. Umemura     

Bacterial assimilation of d- and l-2-chloropropionates and occurrence of a new dehalogenase

The occurrence of a new bacterial dehalogenase acting on both the optical isomers of 2-halogenated alkanoic acids was demonstrated. When the haloalkanoic acid-utilizing bacteria were screened in a medium containing dl-2-chloropropionate as a sole carbon source, two types of bacteria were isolated: (1) a few strains utilizing both d- and l-isomers of 2-chloropropionate and (2) strains utilizing only the l-isomer. A dehalogenating enzyme was obtained from the cells of Pseudomonas sp. which is able to utilize both isomers. The crude enzyme catalyzed the dehalogenation of d- and l-2-chloropropionates to yield l- and d-isomers of lactate, respectively. The enzyme showed the same pH optimum and heat inactivation rate for the d- and l-isomers. Apparent K _m values for d- and l-2-chloropropionates were 4.5 and 1.0 mM, respectively. The enzyme acted specifically on 2-haloalkanoic acids. Activity staining of disc-gels electrophoresed witg the crude enzyme preparation showed that the dehalogenation of d- and l-2-chloropropionates, monochloroacetate, dichloroacetate, 2,2-dichloropropionate, and dl-2-chlorobutyrate is due to a single protein.Abbreviations MCA monochloroacetic acid - DCA dichloroacetic acid - TCA trichloroacetic acid - 2 MCPA 2-monochloropropionic acid - 22 DCPA 2,2-dichloropropionic acid - 3 MCPA 3-monochloropropionic acid - 2 MCBA 2-monochlorobutyric acid - 3 MCBA 3-monochlorobutyric acid - 4 MCBA 4-monochlorobutyric acid     

Membrane-bound <Emphasis Type="SmallCaps">l</Emphasis>- and <Emphasis Type="SmallCaps">d</Emphasis>-lactate dehydrogenase activities of a newly isolated <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> strain

Pseudomonas stutzeri SDM was newly isolated from soil, and two stereospecific NAD-independent lactate dehydrogenase (iLDH) activities were detected in membrane of the cells cultured in a medium containing dl-lactate as the sole carbon source. Neither enzyme activities was constitutive, but both of them might be induced by either enantiomer of lactate. P. stutzeri SDM preferred to utilize lactate to growth, when both l-lactate and glucose were available, and the consumption of glucose was observed only after lactate had been exhausted. The Michaelis–Menten constant for l-lactate was higher than that for d-lactate. The l-iLDH activity was more stable at 55°C, while the d-iLDH activity was lost. Both enzymes exhibited different solubilization with different detergents and different oxidation rates with different electron acceptors. Combining activity staining and previous proteomic analysis, the results suggest that there are two separate enzymes in P. stutzeri SDM, which play an important role in converting lactate to pyruvate. Ma and Gao contributed equally to this work.     

In vivo nuclear-magnetic-resonance analysis of polyamine and alkaloid metabolism in transformed root cultures of Datura stramonium L.: evidence for the involvement of putrescine in phytohormone-induced de-differentiation

The utilisation and accumulation of ¹⁵N-labeled metabolites by a ¹⁵N-labeled transformed root culture of Daturastramonium L. was investigated by in vivo ¹⁵N-nuclear-magnetic-resonance (NMR) spectroscopy. After resuspension in spent growth medium, the pools of [¹⁵N]glutamate and [¹⁵N]glutamine were rapidly depleted and there was an increase in the ¹⁵N-NMR signals from conjugated putrescines and hyoscyamine. The signal from the conjugated putrescines passed through a maximum 2 d after the roots were resuspended, and it was concluded that putrescine could be stored as putrescine conjugates prior to its utilisation in other pathways. The transient accumulation of ¹⁵N-label in the hydroxy-cinnamoylputrescines was reduced when the de-differentiation of the root cultures into a suspension culture was initiated by exposure to a medium containing α-napthaleneacetic acid and kinetin. This led to the hypothesis that phytohormone-induced de-differentiation of the root cultures required the presence of free polyamines, and this was tested using two potent inhibitors of putrescine biosynthesis, dl-α-difluoromethylarginine and dl-α-difluoromethylornithine. In-vivo ¹⁵N-NMR spectra of roots grown in ¹⁵N-enriched medium supplemented with these inhibitors showed that the ¹⁵N-labelling of the conjugated polyamines and hyoscyamine was markedly reduced. dl-α-difluoromethylarginine also prevented the phytohormone induced de-differentiation of the root cultures, and this effect could be reversed by the supply of exogenous putrescine. Thus the supply of putrescine appears to play a crucial role in mediating the phytohormone induced de-differentiation of the root culture. Received: 13 September 1997 / Accepted: 12 November 1997     

Use of protoplast fusion to introduce methionine overproduction into Saccharomyces cerevisiae

Summary dl-Ethionine-resistant mutants of Saccharomyces uvarum ATCC 26602 were found to overproduce exogenous l-methionine. dl-Ethionine-resistant mutant ER 108, carrying a mutation to chloramphenicol resistance was converted to petite form, and protoplasts obtained from it were fused with protoplasts from antibiotic-sensitive S. cerevisiae X2928 carrying six auxotrophies. The resulting fusants maintained four auxotrophies and were capable of overproducing l-methionine. These fusants were stable after ten passages on complete medium.     

Differential effects of amino acid analogs on growth and heterocyst differentiation in two nitrogen-fixing blue-green algae

Effects of a few amino acid analogs on growth and heterocyst differentiation have been studied in two nitrogen-fixing species ofAnabaena. All the analogs except α-methyl-dl-aspartic acid inhibited growth. Exposure ofAnabaena doliolum, todl-5-fluorotryptophan anddl-p-fluorophenylalanine caused pronounced fragmentation of filaments into single cells. At low concentrations (0.01 mM), α-methyl-dl-aspartic acid stimulated growth of the strain ofA. doliolum as well as the strain of the second (unidentified)Anabaena species. Ethionine,dl-p-fluorophenylalanine,dl-5-fluorotryptophan, and canavanine blocked heterocyst differentiation, whereas α-methyl-dl-aspartic acid, α-methyl-dl-methionine,N-o-nitrophenylsulfenyl-l-tryptophan, norleucine, andS-2-aminoethyl-l-cysteine did not show any significant effect. Treatment with 7-azatryptophan,dl-β-hydroxynorvaline,l-methionine-dl-sulfoximine,l-methionine sulfone, and β-2-thienyl-dl-alanine led to a twofold increase in heterocyst frequency. Possible modes of action of the analogs in growth inhibition and changes in heterocyst frequency are discussed.     

Glutamine synthetase activity,ammonia assimilation and control of nitrate reduction in the unicellular red algaCyanidium caldarium

Addition ofl-methionine-dl-sulphoximine to cells ofCyanidium caldarium brings about a loss of glutamine synthetase activity. Concomitantly ammonia assimilation is prevented.Under physiological conditions nitrate reductase [NAD(P)H: nitrate oxidoreductase EC 1.6.6.2] is reversibly converted into an inactive enzyme upon addition of ammonia. In the presence of methionine sulphoximine, when glutamine synthetase activity is lost, nitrate reductase is no longer inactivated by ammonia. It is suggested that ammonia itself is not the actual effector of nitrate reductase inactivation.Concomitantly with the failure of nitrate reductase to undergo ammonia-inactivation, in the presence of methionine sulphoximine nitrate reduction is an uncontrolled process, thus, in media with nitrate ammonia continues to be produced and excreted into the external medium at a constant rate.Abbreviations NR Nitrate reductase - GS Glutamine synthetase - GOGAT Glutamate syntase - MSX l-methionine-dl-sulphoximine     

Cloning, expression, characterization and application of atcA, atcB and atcC from Pseudomonas sp. for the production of L-cysteine

An isolate of a Pseudomonas sp. uses the l-NCC (N-carbamoyl-l-cysteine) pathway to convert dl-2-amino-Δ²-thiazoline-4-carboxylic acid (dl-ATC) to l-cysteine. Genes encoding ATC racemase (AtcA), l-ATC hydrolase (AtcB) and l-NCC amidohydrolase (AtcC), involved in this pathway, were cloned from the Pseudomonas sp. and expressed in Escherichia coli BL21 via pET-28a(+). The resulting enzymes were purified, their functions identified, and their biochemical properties are described. In vitro catalysis experiments, using these enzymes, revealed that the bioconversion rate of l-cysteine from dl-ATC in the presence of AtcA was more efficient than in the absence of AtcA. This is the first report describing simultaneous cloning and expression of atcA, atcB and atcC and characterization of their enzymes for l-cysteine production from dl-ATC via the l-NCC pathway, enabling the complete l-NCC pathway to be elucidated.     

WIPF2 promotes Shigella flexneri actin‐based motility and cell‐to‐cell spread

Shigella flexneri is an intracellular pathogen that disseminates in colonic epithelial cells through actin‐based motility and formation of membrane protrusions at cell–cell contacts, that project into adjacent cells and resolve into vacuoles, from which the pathogen escapes, thereby achieving cell‐to‐cell spread. Actin nucleation at the bacterial pole relies on the recruitment of the nucleation‐promoting factor N‐WASP, which activates the actin nucleator ARP2/3. In cells, the vast majority of N‐WASP exists as a complex with WIP. The involvement of WIP in N‐WASP‐dependent actin‐based motility of various pathogens, including vaccinia virus and S. flexneri, has been highly controversial. Here, we show that WIPF2 was the only WIP family member expressed in the human colonic epithelial cell line HT‐29, and its depletion impaired S. flexneri dissemination. WIPF2 depletion increased the number of cytosolic bacteria lacking actin tails (non‐motile) and decreased the velocity of motile bacteria. This correlated with a decrease in the recruitment of N‐WASP to the bacterial pole, and among N‐WASP‐positive bacteria, a decrease in actin tail‐positive bacteria, suggesting that WIPF2 is required for N‐WASP recruitment and activation at the bacterial pole. In addition, when motile bacteria formed protrusions, WIPF2 depletion decreased the number of membrane protrusions that successfully resolved into vacuoles.     

Racemic Resolution of some <Emphasis Type="SmallCaps">dl</Emphasis>-Amino Acids using <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis><Emphasis Type="SmallCaps">l</Emphasis>-Amino Acid Oxidase

The ability of Aspergillus fumigatus l-amino acid oxidase (l-aao) to cause the resolution of racemic mixtures of dl-amino acids was investigated with dl-alanine, dl-phenylalanine, dl-tyrosine, and dl-aspartic acid. A chiral column, Crownpak CR+ was used for the analysis of the amino acids. The enzyme was able to cause the resolution of the three dl-amino acids resulting in the production of optically pure d-alanine (100% resolution), d-phenylalanine (80.2%), and d-tyrosine (84.1%), respectively. The optically pure d-amino acids have many uses and thus can be exploited industrially. This is the first report of the use of A. fumigatus l-amino acid oxidase for racemic resolution of dl-amino acids.     

Long Lag Times and High Velocities in the Motility of Natural Assemblages of Marine Bacteria

The motility characteristics of natural assemblages of coastal marine bacteria were examined. Initially, less than 10% of the bacteria were motile. A single addition of tryptic soy broth caused an increase in the motile fraction of cells but only after 7 to 12 h. Motility peaked at 15 to 30 h, when more than 80% of cells were motile. These results support the proposal that energy limits motility in the marine environment. Cell speeds changed more than an order of magnitude on timescales of milliseconds and hours. The maximum community speed was 144 (mu)m s(sup-1), and the maximum individual burst velocity was 407 (mu)m s(sup-1). In uniform medium, speed was an inverse function of tryptic soy broth concentration, declining linearly over 0.001 to 1.0%. In media where concentration gradients existed, the mean speed was a function of position in a spatial gradient, changing from 69 to 144 (mu)m s(sup-1) over as little as 15 to 30 (mu)m. The results suggest that marine bacteria are capable of previously undescribed quick shifts in speed that may permit the bacteria to rapidly detect and keep up with positional changes in small nutrient sources. These high speeds and quick shifts may reflect the requirements for useful motility in a turbulent ocean.