N2-fixation in Erwinia herbicola

Four from 18 strains of Erwinia herbicola tested had nitrogenase activity and grew with N₂ as sole source of nitrogen under strict anaerobic conditions with a doubling time of 20–24 h. Nitrogenase activity started only 96–120 h after transfer to a special medium maintained under anaerobic conditions. A ten fold increase in protein per culture found after the maximum nitrogenase activity of 80–130 nmol C₂H₄. mg protein^-1·min^-1 was accompanied by a fall in pH of the medium (20 mM phosphate buffer and in 125 mM Tris-buffer) from pH 7.2 to 5.4 or less, but only to 6.8 in 100 mM phosphate buffer. In all cases we found a sharp curtailing of nitrogenase activity 48 h after the maximum. The bacteria utilized only 35–50% of the nitrogen fixed for growth. Erwinia herbicola strains differed from two strains of Enterobacter agglomerans in being unable to fix nitrogen on agar surfaces exposed to air. Specific nitrogenase activity in Erwinia herbicola is compared with data reported for other Enterobacteriaceae and is found to be higher than that reported for Klebsiella pneumoniae, Enterobacter cloacae or Citrobacter freundii.     

Nitrogen fixation by cellulolytic communities at aerobic-anaerobic interfaces in straw

Microbial communities involved in both cellulolysis and nitrogen fixation were isolated from decomposing straw. Cellulolytic activity appeared restricted to fungal isolates, predominantly species of Penicillium and Fusarium , growing in the presence of oxygen. Bacteria isolated under anaerobic conditions on nitrogen-free media were identified exclusively as Clostridium butyricum . Enterobacters isolated on media containing fixed nitrogen, particularly Enterobacter cloacae , might also be involved in nitrogen fixation when growing under conditions of restricted aeration. Anaerobic or facultative anaerobic nitrogen-fixing bacteria are thought to be sustained by the products of cellulolytic enzymes from aerobic fungi.     

Unicellular, aerobic nitrogen-fixing cyanobacteria of the genus Cyanothece.

Two marine, unicellular aerobic nitrogen-fixing cyanobacteria, Cyanothece strain BH63 and Cyanothece strain BH68, were isolated from the intertidal sands of the Texas Gulf coast in enrichment conditions designed to favor rapid growth. By cell morphology, ultrastructure, a GC content of 40%, and aerobic nitrogen fixation ability, these strains were assigned to the genus Cyanothece. These strains can use molecular nitrogen as the sole nitrogen source and are capable of photoheterotrophic growth in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and glycerol. The strains demonstrated a doubling time of 10 to 14 h in the presence of nitrate and 16 to 20 h under nitrogen-fixing conditions. Rapid growth of nitrogen-fixing cultures can be obtained in continuous light even when the cultures are continuously shaken or bubbled with air. Under 12-h alternating light and dark cycles, the aerobic nitrogenase activity was confined to the dark phase. The typical rates of aerobic nitrogenase activity in Cyanothece strains BH63 and BH68 were 1,140 and 1,097 nmol of C2H2 reduced per mg (dry weight) per h, respectively, and nitrogenase activity was stimulated twofold by light. Ultrastructural observations revealed that numerous inclusion granules formed between the photosynthetic membranes in cells grown under nitrogen-fixing conditions. These Cyanothece strains posses many characteristics that make them particularly attractive for a detailed analysis of the interaction of nitrogen fixation and photosynthesis in an aerobic diazotroph.     

Nitrogen Starvation and the Regulation of Glutamine Synthetase in Agmenellum quadruplicatum

The level of glutamine synthetase activity in Agmenellum quadruplicatum strain PR-6 was dependent on the nitrogen source used for growth and on the nutritional status of the cells. During exponential growth, glutamine synthetase activity was low in cells grown on ammonia, urea, or nitrate. During the transition from nitrogen replete to nitrogen starved growth, glutamine synthetase activity began to rise. With ammonia as a nitrogen source, glutamine synthetase activity as determined in whole cells increased from 1 nanomole per minute per milliliter during exponential growth to 22 nanomoles per minute per milliliter during severe nitrogen starvation. In cells grown on nitrate the increase was from 5 to 39 nanomoles per minute per milliliter, and in cells grown on urea the increase was from 4 to 31 nanomoles per minute per milliliter.     

Radiometric assay for carboxypeptidase H (EC 3.4.17.10) and other carboxypeptidase B-like enzymes

Carboxypeptidase H, EC 3.4.17.10, also known as enkephalin convertase, carboxypeptidase E, and crino carboxypeptidase B, is an important enzyme involved in the biosynthesis of bioactive peptides. To assay the enzyme, tissues are homogenized in at least 20 vol (ml/g) of 0.025 M Tris-HCl buffer, pH 8, with 5 mg/ml of bovine serum albumin. After centrifugation, the supernatant is brought to pH 5.6 and centrifuged again. Following a 20-min preincubation in 2 mM CoCl2, the supernatant is incubated with 0.1 mM (final concentration) of the radioactive substrate [3H]benzoyl-Phe-Ala-Arg. The 100-microliters assay is stopped by the addition of 680 microliters of acetonitrile/0.25 M HCl (0.7/1). The 1.5-ml tube is transferred into a scintillation vial and is flushed with 4 ml of Econofluor, a water-immiscible scintillation fluid. The product, [3H]benzoyl-Phe-Ala, recovered in the organic phase, is counted directly with no interference from the substrate remaining in the aqueous phase. The blank is below 1%. Expressed in nanomoles per minute per milligram of tissue, the activity of the soluble enzyme in rat is 0.34 for striatum, 21.0 for pancreatic islet, 16.6 for anterior pituitary, 46.0 for intermediate pituitary, and 10.9 for neural pituitary. In every case 25 microM guanidinoethylmercaptosuccinic acid, an active site-directed inhibitor of carboxypeptidase H, completely inhibits the activity.     

Relationship of Ribulose-1,5-bisphosphate Carboxylase-Oxygenase Specific Activity to Subunit Composition

Ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBPCase, EC 4.1.1.39) was isolated from Nicotiana sylvestris and from two cultivars and three nuclear substitution lines of Nicotiana tabacum. Isoelectric focusing patterns, supported by amino acid analyses and tryptic peptide mapping, were used to divide these enzymes into two categories: (a) RuBPCase with variable large subunits and identical small subunits; and (b) RuBPCase with identical large but different small subunits. Specific activities for both the carboxylation and oxygenation reactions were determined for all six RuBPCase enzymes under standard conditions of activation and assay. High, intermediate, and low levels of carboxylase (880, 530, and 340 nanomoles HCO₃⁻ per milligram per minute) and oxygenase (66, 45, and 35 nanomoles O₂ per milligram per minute) activity were noted. The carboxylase to oxygenase ratios ranged from 9 to 14.     

Enumeration and identification of nitrogen-fixing bacteria from forage grass roots

Root-soil cores were collected from forage grasses growing in a subtropical region of Texas and tested for acetylene reduction activity. The population density of nitrogen-fixing bacteria was measured, using various media and incubation conditions. Bacteria were confirmed as nitrogen fixing, using the acetylene reduction assay, and were classified according to standard biochemical and cultural methods. The majority of the nitrogen-fixing bacteria isolated from roots were Enterobacter cloacae or Klebsiella pneumoniae. Root-associated, nitrogen-fixing bacteria were isolated from 21 of 24 root-soil cores. The population densities of nitrogen-fixing bacteria ranged from approximately 10 to 3 x 10 per g of root. Population density on roots was significantly correlated with the rate of acetylene reduction but the relationship was not linear.     

钙对蓝藻固氮受氯化钠胁迫的缓解效应及其与生理条件的关系

氯化钠胁迫导致蓝藻固氮活性的下降,可因加人适当浓度的氯化钠而有一定程度的缓解．在光合作用受抑（暗处理或添加光合抑制剂）、厌氧（Ａｒ或Ｎ２中）和有分子氧的情况下,此种缓解作用减弱．光合作用、需氧代谢（通气）和羟化反应（同时供给氢和氧）正常进行以及碳架（添加外源蔗糖或提高ＣＯ２浓度）供应良好时,钙对氯化钠胁迫的缓解效应增大．改善合成固氮酶的物质基础供应（同时供应ＣＯ２和Ｎ２）对此也有一定的正效应．     

Denitrification under various aeration conditions in Comamonas sp., strain SGLY2

Abstract A number of bacteria were isolated from different anoxic reactors. Those having denitrifying potential were tested for their ability to denitrify under aerobic conditions. The activity of their denitrifying enzymes varied from partial inactivation by oxygen (strains NO2B9 and TCET1) to oxygen-independent activity in a strain named SGLY2 which was tentatively identified as Comamonas sp. The effect of different aeration conditions on growth and on denitrification of SGLY2 was studied more extensively. This strain was able to consume oxygen and nitrate simultaneously with the production of nitrogen and without build-up of nitrite. The dissimilatory nitrate-reductase of nitrate-adapted cells was found to be more active in the presence of oxygen than in micro-aerobic or strictly anaerobic conditions.     

Localization of enzymes of ureide biosynthesis in peroxisomes and microsomes of nodules

The intracellular location of enzymes involved in the synthesis of the ureides, allantoin and allantoic acid, was investigated in nodules of Glycine max L. Merr. Cellular organelles were separated on isopycnic sucrose density gradients. Xanthine dehydrogenase activity (270 nanomoles per min per gram fresh weight) was totally soluble, whereas approximately 15% of the total uricase and catalase activities (1 and 2000 micromoles per minute per gram fresh weight, respectively) was in the fraction containing intact peroxisomes. Allantoinase activity (680 nanomoles per minute per gram fresh weight) was associated with the microsomal fraction, which apparently originates from the endoplasmic reticulum.     

Isolation and characterization of enteric bacteria from the hindgut of Formosan termite

The Formosan subterranean termite, Coptotermes formosanus Shiraki, is an aggressive, invasive termite species that has caused billions of dollars of damage across the United States for the past 50 years. Termites depend on intestinal microorganisms for cellulose digestion. Symbiotic microorganisms in the termite gut play key physiological functions such as cellulose and hemicellulose digestion, acetogenesis, hydrogenesis, methanogenesis, sulfate reduction, and nitrogen fixation. Additionally, intestinal microbes create suitable conditions for symbiotic protozoans through the production of nutrients and the maintenance of the pH and the anaerobic conditions in the gut. Although extensive research has been done on the symbiotic relationship of these termites and the microbes found in its gut, there is little information available on the role of facultative anaerobes in the gut. We isolated four enteric bacteria from the hindgut of Formosan subterranean termite, C. formosanus. All isolates were facultative anaerobes and G-. The isolates were identified as Serratia marcescens, Enterobacter aerogens, Enterobacter cloacae, and Citrobacter farmeri by using BIOLOG assay and fatty acid methyl ester analysis (FAME). Each isolate was characterized using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and biochemical study. This is the first report on the presence of facultative microbes in termite gut. Results of this first study on facultative microbes in the termite gut indicate that the role of facultative organisms in the Formosan termite gut may be to scavenge oxygen and create anaerobic conditions for the anaerobic microorganisms, which are essential for digestion of cellulose consumed by the termite.     

Respiration supported nitrogenase activity of isolated Rhizobium meliloti bacteroids

Bacteroids having a high level of respiration-supported nitrogenase activity were isolated from nitrogen-fixing alfalfa root nodules. Gentle maceration under anaerobic conditions in the presence of sodium succinate and a fatty acid scavenging agent were employed in this method. A large proportion of isolated bacteroids retained a triple membrane structure as shown by transmission electron microscopy. Dicarboxylic acids of the TCA cycle (malate, fumarate, succinate), but not glutamate or aspartate, supported sufficient respiratory activity to supply the nitrogenase system with ATP and reducing equivalents and to protect the nitrogenase system from inactivation by 4% oxygen over a period of 20-30 min. Sugars did not support nitrogenase activity in intact bacteroids. The properties of the isolated bacteroids were ascribed to minimal damage to the cytoplasmic membrane and peribacteroidal membrane during isolation. With succinate as substrate and oxygen as terminal electron acceptor, initial nitrogenase activity was determined at 4% oxygen in the gas phase of the assay system employed. At this oxygen concentration, the sustained rate of acetylene reduction by respiring bacteroids was linear up to 30 min. Bacteroid activity declined rapidly with time of exposure to oxygen above 4% in the gas phase. The optimum temperature range for this activity was 10-20 degrees C. Nitrogenase activity was measurable at incubation temperatures below 10 degrees C under 4% oxygen. Functionally intact bacteroids had little nitrogenase activity under anaerobic conditions in the presence of an external source of ATP and reductant. Treatment of the bacteroids with chlorpromazine eliminated respiration-supported activity and rendered the bacteroid cell membrane permeable to external ATP. Bacteroids treated with chlorpromazine had high acetylene reducing activity with external ATP and dithionite in the absence of oxygen.     

Biochemical and Antibiotic Susceptibility Studies of H2S-Negative Citrobacter

Ninety-four strains of H(2)S-negative Citrobacter were biochemically characterized and their antibiograms were determined. The antibiograms demonstrated not only a difference from Enterobacter cloacae but also a difference within the Citrobacter group between the indole-negative and indole-positive strains. These differences were statistically significant and emphasize the importance of the indole reaction as an aid to speciation of the H(2)S-negative Citrobacter.     

Gas exchange in the filamentous cyanobacterium Nostoc punctiforme strain ATCC 29133 and Its hydrogenase-deficient mutant strain NHM5

Nostoc punctiforme ATCC 29133 is a nitrogen-fixing, heterocystous cyanobacterium of symbiotic origin. During nitrogen fixation, it produces molecular hydrogen (H(2)), which is recaptured by an uptake hydrogenase. Gas exchange in cultures of N. punctiforme ATCC 29133 and its hydrogenase-free mutant strain NHM5 was studied. Exchange of O(2), CO(2), N(2), and H(2) was followed simultaneously with a mass spectrometer in cultures grown under nitrogen-fixing conditions. Isotopic tracing was used to separate evolution and uptake of CO(2) and O(2). The amount of H(2) produced per molecule of N(2) fixed was found to vary with light conditions, high light giving a greater increase in H(2) production than N(2) fixation. The ratio under low light and high light was approximately 1.4 and 6.1 molecules of H(2) produced per molecule of N(2) fixed, respectively. Incubation under high light for a longer time, until the culture was depleted of CO(2), caused a decrease in the nitrogen fixation rate. At the same time, hydrogen production in the hydrogenase-deficient strain was increased from an initial rate of approximately 6 micro mol (mg of chlorophyll a)(-1) h(-1) to 9 micro mol (mg of chlorophyll a)(-1) h(-1) after about 50 min. A light-stimulated hydrogen-deuterium exchange activity stemming from the nitrogenase was observed in the two strains. The present findings are important for understanding this nitrogenase-based system, aiming at photobiological hydrogen production, as we have identified the conditions under which the energy flow through the nitrogenase can be directed towards hydrogen production rather than nitrogen fixation.     

A novel strategy of oxygen restriction by some diazotrophic enteric bacteria

Protection of nitrogenase against oxygen inactivation in diazotrophs involves numerous strategies. Glutathione is known to play an important role in scavenging oxyradicals in many living systems. The involvement of glutathione (reduced) (GSH), glutathione peroxidase (GPX) and glutathione reductase (GR) in the protection of nitrogenase in free living diazotrophs is reported here for the first time. Reduced glutathione content and the activity of glutathione peroxidase and glutathione reductase increased with increase in oxygen concentration under nitrogen fixing conditions but decreased under anaerobic and nitrogenase repressed conditions. This correlation is used to postulate a protecting role for GSH-GPX-GR system against oxygen inactivation of nitrogenase.     

Nitrogen Fixation Associated with Rinsed Roots and Rhizomes of the Eelgrass Zostera marina

Nitrogen fixation was associated with the rinsed roots and rhizomes of the seagrass, Zostera marina L. Nitrogenase activity (acetylene reduction) was greater on rhizomes compared to roots, and on older roots and rhizomes relative to younger tissue. Compared to aerobic assays, anaerobic or microaerobic conditions enhanced the rate of acetylene reduction by rhizomes with attached roots, with the highest activity (100 nanomoles per gram dry weight per hour) occurring at pO₂ = 0.01 atmosphere. Addition of glucose, sucrose, or succinate also increased the rate of acetylene reduction under anaerobic conditions, with glucose providing the most stimulation. In one experiment, comparison of acetylene reduction assays with ¹⁵N₂ incorporation yielded a ratio of about 2.6:1. Seagrass communities are thought to be limited by the availability of nitrogen and, therefore, nitrogenase activity directly associated with their roots and rhizomes suggests the possibility of a N₂-fixing flora which may subsidize their nutritional demand for nitrogen.     

Persistense of mono- and associated cultures of conditionally pathogenic enterobacteria

The comparative study of adhesive, hemolytic, DNA-ase, lecithinase, antilysozymic, anticomplementary activities of mono- and associated cultures of 57 Enterobacter spp., 61 Citrobacter spp. and 55 Serratia spp. strains, isolated from patients with pyoinflammatory, intestinal and urological diseases is carried out. Different variations of cocultivated bacteria including Enterobacter and Citrobacter, Enterobacter and Serratia, Citrobacter and Serratia are used. It was shown, that cocultivated Enterobacter, Citrobacter and Serratia bacteria increased the persistent properties of mixt cultures.     

Nitrogen fixation by Bacillus strains isolated from the rhizosphere of Ammophila arenaria

Summary Several Bacillus strains, from the rhizosphere of Ammophila arenaria, appeared on ‘nitrogen-free’ agar plates. They were able to grow in nitrogen-poor medium to which 0.1% yeast extract was added. Three of these bacilli were tested for their ability to fix nitrogen using the acetylene reduction assay. The C₂H₂-reducing activity was determined at 8-hour intervals during their growth cycle. C₂H₂ reduction (and accordingly N₂ fixation) was greater under anaerobic than aerobic conditions. Additions of 0.1% CaCO₃ significantly increased the C₂H₂-reducing activity under both conditions. Characterisation suggests that these strains are new nitrogen-fixing Bacillus species. re]19740121     

Heterotrophic bacteria present in hindguts of wood-eating termites [Reticulitermes flavipes (Kollar)]

Strict anaerobic culture techniques were used to quantitate heterotrophic bacteria present in hindguts of Reticulitermes flavipes. The grand mean number of viable cells per hindgut was 0.4 X 10(5) (first-instar larvae), 1.3 X 10(5) (third-instar larvae), 3.5 X 10(5) (workers), and 1.5 X 10(5) (soldiers). Of a total of 344 isolates, 66.3% were streptococci that were always obtained regardless of the origin of termites, their developmental stage or caste, or their length of captivity. Most of the remaining isolates were strains of Bacteroides and Enterobacteriaceae. A small percentage were strains of Lactobacillus, Fusobacterium, and unidentified anaerobic gram-positive rods. Recovery of bacteria from worker hindguts was 13.0% of the direct microscopic count. Isolations performed aerobically failed to reveal strict aerobes. Attempts to isolate cellulolytic bacteria were uniformly unsuccessful. Of 145 streptococcal strains isolated from freshly collected termites, almost all were Streptococcus lactis and S. cremoris. Enterobacteriaceae isolates from the same termite specimens were indole-positive Citrobacter, citrate-negative Citrobacter, and Enterobacter cloacae. The possibility of in situ interspecies lactate transfer, between lactate producers (e.g., streptococci) and lactate fermenters (Bacteroides), is discussed.