Corynebacterium Autotrophicum Research Articles

Phylogenetic perspectives of nitrogen-fixing actinobacteria

It was assumed for a long time that the ability to catalyze atmospheric nitrogen (diazotrophy) has a narrow distribution among actinobacteria being limited to the genus Frankia. Recently, the number of nitrogen fixation (nifH) genes identified in other non-Frankia actinobacteria has dramatically increased and has opened investigation on the origin and emergence of diazotrophy among actinobacteria. During the last decade, Mycobacterium flavum, Corynebacterium autotrophicum and a fluorescent Arthrobacter sp. have been reported to have nitrogenase activity, but these studies have not been further verified. Additional reports of nitrogen fixation by Agromyces, Microbacterium, Corynebacterium and Micromonospora isolated from root nodules of leguminous and actinorhizal plants have increased. For several actinobacteria, nitrogen fixation was demonstrated by the ability to grow on nitrogen-free medium, acetylene reduction activity, 15N isotope dilution analysis and identification of a nifH gene via PCR amplification. Moreover, the analyses of draft genome sequences of actinobacteria including Slackia exigua, Rothia mucilaginosa and Gordonibacter pamelaeae have also revealed the presence of nifH-like sequences. Whether these nifH sequences are associated with effective nitrogen fixation in these actinobacteria taxa has not yet been demonstrated. These genes may be vertically or horizontally transferred and be silent sequences. These ideas merit further investigation. This minireview presents a phylogenetic comparison of nitrogen fixation gene (nifH) with the aim of elucidating the processes underlying the evolutionary history of this catalytic ability among actinobacteria.

Microbial Biscuits of Vaterite in Lake Issyk-Kul (Republic of Kyrgyzstan)--Discussion

Giralt et al. (2001) reported the formation of microbial biscuits of vaterite in Lake Issyk-Kul (Republic of Kyrgyzstan). Although they state that the trigger mechanisms for the natural precipitation of vaterite remain unclear, they propose an explanation of the origin for the vaterite biscuits involving bacterial activity. Our recent research seems to support their hypothesis, although the way we obtain vaterite precipitation is not strictly related to lacustrine or natural aqueous environments. We have used laboratory cultures to demonstrate the ability of some soil bacteria to transform an oxalate carbon source into carbonate, a hypothesis supported by observations (Cromack et al. 1977; Verrecchia et al. 1993) and biogeochemical studies (Verrecchia and Dumont 1996). The bacteria used were isolated by Tamer and Aragno (1980) and identified as Xanthobacter autotrophicus (DSM 432, ATCC 35674, syn.: Corynebacterium autotrophicum ), previously described by Wiegel et al. (1978). The bacteria were grown in a B4 media (Boquet et al. 1973) to which calcium acetate or calcium citrate was added. In this experiment, the only way to produce carbonate …

Occurrence of Phosphatidylcholine in Hydrogen‐Oxidizing Bacteria

15 strains of hydrogen-oxidizing bacteria were grown heterotrophically, harvested during the stationary phase of growth, and analyzed for their principal phospholipids. All strains - with the exception of Corynebacterium autotrophicum strain SA 32 and Pseudomonas pseudoflava - contained phosphatidylcholine as a major constituent. It is concluded that the presence of phosphatidylcholine is neither characteristic of a peculiar bacterial genus or family, nor is it absolutely correlated to the ability to oxidize hydrogen. The phosphatidylcholines of all strains contain C19 cyclopropane acid which is, in some strains, predominantly located at C-2 position of the glycerol moiety.

Enrichment and isolation of new nitrogen-fixing hydrogen bacteria

An enrichment method for nitrogen fixing hydrogen bacteria is described. The procedure invariably resulted in the isolation of yellow-pigmented coryneform bacterial strains assigned to Corynebacterium autotrophicum. The procedure included a serial transfer in an ammonium-free mineral liquid medium under an atmosphere of 10% hydrogen, 5% oxygen, 10% carbon dioxide and 75% nitrogen, followed by a short alkali treatment and by streaking on nutrient broth-succinate agar. The ability to fix nitrogen was confirmed by the acetylene reduction test and by 15N2 incorporation.

Isoprenoid quinones in the classification of coryneform and related bacteria.

Menaquinones were the only isoprenoid quinones found in 85 of the 95 coryneform bacteria examined. Dihydromenaquinones having nine isoprene units were the main components isolated from Corynebacterium bovis, from other glutamic acid-producing strains, and from Arthrobacter globiformis and related species. Dihydromenaquinones with eight isoprene units were found in Brevibacterium linens, the remaining Corynebacterium species and strains probably belonging to the genus Rhodococcus. Tetrahydromenaquinones with eight isoprene units were found in Arthrobacter simplex and Arthrobacter tumescens, and with nine isoprene units in Cellulomonas and Oerskovia. Kurthia and Curtobacterium were characterized by menaquinones with seven and nine isoprene units, respectively, and Microbacterium lacticum and Corynebacterium aquaticum had comparable amounts of menaquinones with 10 and 11 isoprene units. Strains received as Brevibacterium leucinophagum, Corynebacterium autotrophicum, Corynebacterium nephridii, Mycobacterium flavum, Mycoplana rubra and Protaminobacter ruber contained uniquinones as their sole isoprenoid quinones. The isoprenoid quinone data correlate well with major trends in coryneform taxonomy and are of value in the classification of coryneform and related bacteria.

Transfer of the Nitrogen-Fixing Hydrogen Bacterium Corynebacterium autotrophicum Baumgarten et al. to Xanthobacter gen. nov.

Thirty-five nitrogen-fixing, hydrogen-oxidizing bacteria, all members of Cory-nebacterium autotrophicum Baumgarten, Reh, and Schlegel 1974, and including the type strain of this species, were compared with 28 strains of coryneform bacteria by numerical taxonomy methods. The nitrogen-fixing hydrogen bacteria formed a well defined cluster; their similarity to the other strains tested was low, however. Furthermore, the chemotaxonomic characters of these strains excluded them from the coryneform bacteria. Therefore C. autotrophicum was reclassified, following the keys of Bergey's Manual of Determinative Bacteriology (8th ed.) as a species of the family Azotobacteraceae. Although taxonomically close to Beijerinckia and Derxia, the nitrogen-fixing, hydrogen-oxidizing bacteria are regarded as representing a new genus, for which we propose the name Xanthobacter. C. autotrophicum Baumgarten, Reh, and Schlegel is transferred to the new genus as Xanthobacter autotrophicus (Baumgarten et al.) comb. nov.

The Nitrogen‐Fixing System of Corynebacterium autotrophicum

Both nitrogenase components were isolated and purified from sucrose‐grown, nitrogen‐fixing cells of Corynebacterium autotrophicum by DEAE‐cellulose and Sephadex gel chromatography. The MoFe protein (Ca1) and the Fe protein (Ca2) appeared as single bands in sodium dodecylsulfate gel electrophoresis (Serva). Ca1 had an average molecular weight of 232000 with two non‐identical subunits (Mr 61200 and 57500) on Koch‐Light sodium dodecylsulfate gels; the Ca2 protein had an average molecular weight of 72600 with two identical subunits. Ca1 contained 2.2 mol Mo/mol, 23.1 mol Fe/mol and 20.1 mol S2−/mol, the Ca2 protein 3.8 mol Fe/mol and 2.4 mol S2−/mol. The amino acid composition of both nitrogenase proteins was similar to those of other N2‐fixing bacteria except for the occurrence of an unidentified derivative.Electron paramagnetic resonance (EPR) spectroscopy of Ca1 protein gave a rhombic signal with g values of 4.13, 3.72 and 2.03 at pH 6.2 with small features to low field of the g= 4.13 peak and to high field of the g= 3.72 peak. At pH 9.25 the intensity of the EPR signal was transferred to minor features with g‐values of 4.31 and 3.54 and of 4.68 and 3.31. Upon addition of acetylene to Ca1 at pH 9.25 the signal returned to the low‐pH form. The additional features were interpreted as being exhibited by two additional conformations of the protein; the change in form with acetylene confirms that acetylene binds to the MoFe protein. The EPR spectrum of Ca2 had features at g values of 2.04, 1.95 and 1.89 and an integrated intensity of 0.28 electron/molecule of protein. The MoFe protein and Fe protein of C. autotrophicum revealed no cold lability. The pH optima for C2H2, N2 and H3O+ reductions occurred between pH 7.1–7.3 with Bes buffer; in Pipes buffer the reduction of all three substrates was inhibited by 40%. At high molar ratios of Ca1 protein to Ca2 protein C2H2 reduction suffered a lag phase before a linear rate occurred. The rate of N2 reduction was linear but low whereas the rate of H3O+ reduction under argon remained constant. H3O+ reduction under N2 (concomitant H2 evolution) increased with excess Ca1. The results suggest different sites or forms of the enzyme for the three substrates at the nitrogenase protein and a second role for the Fe protein during N2 reduction.Two ferredoxins were isolated with the optical properties of eight‐iron, eight‐sulfur proteins. They exhibited an absorption ratio A390/A280 of 0.71 (Fd I) and 0.76 (Fd II) and peaks at 280 nm (Fd I), 285 nm (Fd II) and 390 nm and shoulders at 305 nm. The molecular weights were 12400 (Fd I) and 12800 (Fd II) in sodium dodecylsulfate gels and 12900 for a mixture of both proteins using thinlayer gel filtration. Fd I contained 10.0 mol Fe/mol and 9.4 mol S2−/mol, Fd II 8.1 mol Fe/mol and 10.4 mol S2−/mol, as determined colourimetrically. Using the atomic absorption method 7.9 mol Fe/mol protein and 8.4 mol Fe/mol protein were measured for Fd I and Fd II, respectively. Both proteins gave identical EPR spectra in the oxidised (g= 2.00) and in the reduced state (g= 2.07, 1.94, 1.88) which integrated to 0.12 electron per molecule of protein in both instances. Since the ferredoxins isolated from obligate aerobic nitrogen‐fixing bacteria such as Azotobacter vinelandii and Mycobacterium flavum exhibit paramagnetic features only in the oxidised state, the two ferredoxins of C. autotrophicum represent a new type of ferredoxin within this group.

Isolation of lipopolysaccharides and the effect of Polymyxin B on the outer membrane of Corynebacterium autotrophicum

Lipopolysaccharides (LPS) from Corynebacterium autotrophicum were isolated and analyzed. Autotrophically grown cells contained 2--5 mg of partly purified LPS per g dry weight of lyophilized cells. Serological cross reaction with Lipid A antigen of Salmonella minnesota confirmed the presence of LPS in C. autotrophicum. Electron microscopy of negatively stained Polymyxin B-treated cells showed formation of blebs on the Outer Membrane indicating an interaction of Polymyxin B specifically with LPS. Up to now, no Gram-positive organisms are known which contain any LPS. Thus, C. autotrophicum, though giving opposite results when the Gram-staining reaction was applied by several authors, has to be classified into the group of Gram-negative bacteria.

Lipid patterns of various hydrogen oxidizing bacterial species

Strains of hydrogen-oxidizing bacteria, Pseudomonas facilis, Nocardia opaca 1 b, Paracoccus denitrificans and Corynebacterium autotrophicum 7 C, were grown both under chemolithoautotrophic (referred to as autotrophic) and under chemoorganoheterotrophic (referred to as heterotrophic) conditions and harvested during the phase of stationary growth. The lipid patterns of all strains were studied by quantitative chemical analyses, UV spectra, TLC and GLC of fatty acid methyl esters. In general, there were no essential qualitative differences between lipid patterns of autotrophically and those of heterotrophically grown bacteria of the same strain. The conditions of growth apparently influence only the quantitative composition of lipids. In particular, Nocardia opaca is comparatively rich in triglycerides. Ubiquinones were found in Pseudomonas facilis and in Corynebacterium autotrophicum. The latter finding is quite unusual in a Corynebacterium. Carotenoid glycosides are probably present in Corynebacterium autotrophicum, thus explaining the orange color of this organism. The bacteria studied contain a C 19 cyclopropane acid, Pseudomonas facilis and Corneynebacterium autotrophicum also

Micromorphology of Gram-negative hydrogen bacteria

The cell morphology, the arrangement and fine structure of flagella and the piliation of the following Gram-negative aerobic hydrogen bacteria have been studied: Alcaligenes eutrophus, Alcaligenes paradoxus, Alcaligenes ruhlandii, Pseudomonas flava, Pseudomonas pseudoflava, Pseudomonas palleronii, Pseudomonas facilis, Aquaspirillum autotrophicum, Paracoccus denitrificans, Corynebacterium autotrophicum, and strains MA 2 and SA 35. The identity of the bacteria was examined by their substrate spectra and type of flagellation. Three types of flagellar fine structure were differentiated. The presence of pili was noted in strains of Alcaligenes paradoxus, Pseudomonas flava, P.pseudoflava, P.palleronii, and P.facilis.

Localization and stability of hydrogenases from aerobic hydrogen bacteria.

Alcaligenes eutrophus strains H 16, B 19, G 27 and N9A contained two different hydrogenases. One enzyme catalyzed the reduction of NAD by hydrogen and was strictly localized in the soluble cell fraction. While the second enzyme was found to be particulate and unable to react with NAD. All other tested strains, Alcaligenes paradoxus SA 29, Pseudomonas facilis, P. palleronii RH 2, Pseudomonas sp. strain GA 3, Paracoccus denitrificans, Aquaspirillum autotrophicum SA 32, and Corynebacterium autotrophicum 14g and 7C contained only a single enzyme exclusively bound to membranes. This was established using fractional centrifugation, indicator enzyme systems, gently methods of cell disintegration and discontinuous sucrose density gradient centrifugation. In cell-free extracts obtained by rough disruption (sonication) of cells, hydrogenase was associated to particles of different size and sedimentation velocity. A partial solubilization of hydrogenase caused by sonication was observed with P. facilis. Without exception, the particulate hydrogenases were found (1) to be unable to reduce pyridine nucleotides, and (2) to reduce methylene blue at an extremely high activity. The eminent reaction rate of 34 micronmoles H2 oxidized per min and mg protein has been determined in particle suspensions of Pseudomonas sp. strain GA 3. All hydrogenases were stable during storage under hydrogen atmosphere, except the soluble enzyme for A. eutrophus H 16 which was shown to be more stable under aerobic conditions.

Leucine biosynthesis: effect of branched-chain amino acids and threonine on [formula omitted] synthase activity from aerobic and anaerobic microorganisms

a- Isopropylmalate synthase activity was demonstrated in the Sephadex G 25 gel filtrated crude extracts of one yeast and 43 bacterial strains belonging to 14 families. The enzyme was inhibited by leucine from all strains Bacteroides fragilis, Clostridia and several phototropic bacteria. The enzyme was inhibited by leucine from all strains investigated. In crude extracts of 17 species (8 genera) the leucine-mediated inhibition could be relieved by the addition of valine or isoleucine , but not by the addition of threonine or alanine. The enzymes from 11 species (7 genera) were inhibited by 1 mM valine and isoleucine, whereas the enzyme activity from 5 bacteria (4genera) were not so affected. These results suggest that valine and isoleucine are specifically involved in the regulation of leucine biosynthesis in several bacteria. The affect of valine and isoleucine on the IPM-synthase activity from mycobacteria and Corynebacterium autotrophicum lends support to the reclassification of Mycobacterium flavum 301 to C. autotrophicum. The antagonism between 5′,5′,5′-trifluoroleucine and amino acids and a- ketoisovalerate was a- isopropylmalate synthase in the presence or abssence of leucine and the reversal of the 5′,5′,5′-trifluoroleucine-mediated growth inhibition by these amino acids.

Identification and physiological characterization of the nitrogen fixing bacterium Corynebacterium autotrophicum GZ 29.

The coryneform hydrogen bacterium strain GZ 29, assigned to Corynebacterium autotrophicum fixed molecular nitrogen under autotrophic (H2, CO2) as well as under heterotrophic (sucrose) conditions. Physiological parameters of nitrogen fixation were measured under heterotrophic conditions. The optimal dissolved oxygen concentration for cells grown in a fermenter with N2 was rather low (0.14 mg O2/l) compared with cells grown in the presence of NH+4 (4.45 mg O2/l). C. autotrophicum GZ 29 had a doubling time of 3.7 h at 30 degrees C with N2 as N-source and sucrose as carbon source and at optimal pO2. Acetylene reduction reached values of 12 nmoles of ethylene produced/min X mg protein. Although the oxygen concentration in the growing culture was kept constant, the optimal dissolved oxygen tension for the acetylene reduction assay shifted to higher pO2-values. The overall efficiency of nitrogen fixation amounted to 22 mg N fixed/g sucrose consumed; it reached a maximal value of 65 mg N fixed/g sucrose consumed at the beginning of the exponential growth phase. Intact cells reduced acetylene even under anaerobic test conditions; further anaerobic metabolic activity could not be ascertained so far.

Bacterial carotenoids. L. Absolute configuration of zeaxanthin dirhamnoside.

Zeaxanthin dirhamnoside is the major carotenoid of Corynebacterium autotrophicum. The absolute configuration 3R,3'R followed from CD-properties of its hexaacetate, alpha-L-assignment and 1C4 conformation were concluded from 1H NMR data by comparison with model compounds.

Enrichment and isolation of nitrogen fixing hydrogen bacteria.

An enrichment method for nitrogen fixing hydrogen bacteria is described. The procedure invariably resulted in the isolation of yellow-pigmented coryneform bacterial strains assigned to Corynebacterium autotrophicum. The procedure included a serial transfer in an ammonium-free mineral liquid medium under an atmosphere of 10% hydrogen, 5% oxygen, 10% carbon dioxide and 75% nitrogen, followed by a short alkali treatment and by streaking on nutrient broth-succinate agar. The ability to fix nitrogen was confirmed by the acetylene reduction test and by 15N2 incorporation.

Aromatic amino acid biosynthesis in Alcaligenes eutrophus H16

Properties and regulation of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHP-synthase), EC4.1.2.15, from Alcaligenes eutrophus H16 were investigated. DAHP synthase was unstable during manipulations such as dialysis, dilution, ammonium sulfate fractionation, chromatography on DEAE-cellulose or Sephadex G-200. For kinetic measurements Sephadex G-25 treated crude extracts were used. The enzyme was not affected by thiol reagents, EDTA or divalent metal ions. The activation energy, deltaH, amounted to 16100 cal/mole. Between pH 7.2 and pH 8.2 there was little change of enzyme activity. The Km-values for the two substrates were found to be 0.043 mM phosphoenolpyruvate and 0.055 mM erythrose-4-phosphate. DAHP-synthase was inhibited by 0.5 mM phenylalanine for 60% and by 0.5 mM tyrosine for 20%. In the presence of both amino acids cumulative inhibition occurred amounting to about 70%. No other amino acid exerted inhibitory effects. A repression of DAHP-synthase by the aromatic amino acids was not observed. Some other strains of hydrogen bacteria were included in this study. The DAHP synthase from strain 12/60/X and Corynebacterium autotrophicum 7C was unregulated. The enzyme from strain 33/X was subject to retro-tyrosine inhibition and from strain 3/2, H1 and H20 were subject to cumulative inhibition.

Taxonomic studies on some gram-positive coryneform hydrogen bacteria

Recently isolated coryneform hydrogen bacteria were investigated under taxonomical aspects. Strains 7 C, RH 10, and 14 g are characterized by the snapping type of cell division, 68.5 to 69.7% GC content, dl-diaminopimelic acid in the cell wall, content of metachromatic granules, weak utilization of sugars and inhibitory effect of citrate. The strains are placed to the group 1—genus Corynebacterium—of the classification of coryneform bacteria of Yamada and Komagata (1972) and the name Corynebacterium autotrophicum sp.nov. is proposed. Strains 11 X and RH 12 are characterized by the bending type of cell division, a GC content of 70.2 and 70.5%, ll-diaminopimelic acid in the cell wall, absence of metachromatic granules, utilization of several sugars and no changes in cell morphology by citrate. The strains have to be placed to group 6 of coryneform bacteria.

A new coryneform hydrogen bacterium: Corynebacterium autotrophicum strain 7 C

1. Corynebacterium autotrophicum strain 7 C was isolated from an enrichment culture designed for propane oxidizing bacteria. The cells are Grampositive, immotile, short, irregularly formed rods. The colonies are yellow-pigmented and slimy. The yellow pigmentation is due to carotenoids. 2. Growth occurs either autotrophically in mineral medium under an atmosphere of 70% H2+20% O2+10% CO2 or heterotrophically with fructose or many organic acids as substrates. 3. The hexoses and gluconate are degraded via the Entner-Doudoroff pathway. 6-Phosphogluconate dehydrogenase is not detectable. 4. A NAD reducing hydrogenase has not been detected; the hydrogenase is localized in the particle fraction of the crude extract and reduces methylene blue. The specific activity of hydrogenase in the crude extract of autotrophically grown cells is 2400 μl H2/mg protein · hr. During growth on fructose the enzyme is constitutively formed (1200 μl H2/mg protein · hr). 5. The utilization of fructose was suppressed by hydrogen. The inhibitory effect was significant, when either fully adapted or autotrophically grown cells were exposed to a hydrogen containing atmosphere.