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W B Whitman

Publications and source records attributed to W B Whitman.

At least 55 records · Page 3Linked to original sources
PubMed1987

Role of Amino Acids and Vitamins in Nutrition of Mesophilic Methanococcus spp.

In this study we found that autotrophic methanococci similar to Methanococcus maripaludis obtained up to 57% of their cellular carbon from exogenous amino acids. About 85% of the incorporation was into protein. Primarily nonpolar and basic amino acids and glycine were incorporated; only small amounts of acidic and some polar amino acids were taken up. An additional 10% of the incorporation was into the nucleic acid fraction. Because little CO(2) was formed from the C-amino acids, little metabolism of the amino acids occurred. Therefore the growth stimulation by amino acids was probably due to the sparing of anabolic energy requirements. Of the amino acids incorporated, only alanine was also a sole nitrogen source for these methanococci. In contrast, Methanococcus vannielii and "Methanococcus aeolicus" are autotrophic methanococci which did not incorporate amino acids and did not utilize alanine as a sole nitrogen source. Although glutamine served as a sole nitrogen source for the autotrophic methanococci and Methanococcus voltae, a heterotrophic methanococcus, growth was due to chemical deamination in the medium. M. voltae requires leucine and isoleucine for growth. However, these amino acids were not significant nitrogen sources, and alanine was not a sole nitrogen source for the growth of M. voltae. The branched-chain amino acids were not extensively metabolized by M. voltae. Pantoyl lactone and pantoic acid were readily incorporated by M. voltae. The intact vitamin pantothenate was neither stimulatory to growth nor incorporated. In conclusion, although amino acids and vitamins are nutritionally important to both autotrophic and heterotrophic methanococci, generally they are not subject to extensive catabolism.

Journal Article↗
PubMed1987

Incorporation of Exogenous Purines and Pyrimidines by Methanococcus voltae and Isolation of Analog-Resistant Mutants.

Methanococcus voltae incorporated exogenous adenine, guanine, hypoxanthine, and uracil, but not thymine. Growth of M. voltae was also sensitive to purine and pyrimidine analogs. Of the 20 analogs tested, 12 were inhibitory at 1 mg/ml. The most effective inhibitors were purine analogs with endocyclic substitutions. Nucleoside analogs and analogs with exocyclic substitutions or additions were less effective. Four purine analogs, 8-aza-2,6-diaminopurine, 8-azaguanine, 8-azahypoxanthine, and 6-mercaptopurine and one pyrimidine analog, 6-azauracil, were especially toxic. The MICs were 20, 0.5, 2.0, 80, and 10 mug/ml, respectively. Spontaneous resistance mutants were isolated for these five analogs. The MICs for these mutants were 20.5, 8.2, >65, >41, and 20.5 mg/ml, respectively. These concentrations far exceeded the solubilities of the analogs and represented an increase in resistance of at least three orders of magnitude. In addition to demonstrating cross resistance to several of the analogs, four of these mutants lost the ability to incorporate exogenous bases. These appeared to be mutations in the salvage pathways for purines and pyrimidines. In contrast, the mutant resistant to 6-mercaptopurine was not defective in purine uptake. Instead, it degraded 6-mercaptopurine. In the presence or absence of high concentrations of the analogs, the growth rates of the resistant mutants were no less than one-half of the growth rate of the wild type in the absence of the analog. The high level of resistance and rapid growth are very desirable properties for the application of the mutants in genetic experiments.

Journal Article↗
PubMed1987

Inhibition by corrins of the ATP-dependent activation and CO2 reduction by the methylreductase system in Methanobacterium bryantii.

Corrins inhibited the ATP-dependent activation of the methylreductase system and the methyl coenzyme M-dependent reduction of CO2 in extracts of Methanobacterium bryantii resolved from low-molecular-weight factors. The concentrations of cobinamides and cobamides required for one-half of maximal inhibition of the ATP-dependent activation were between 1 and 5 microM. Cobinamides were more inhibitory at lower concentrations than cobamides. Deoxyadenosylcobalamin was not inhibitory at concentrations up to 25 microM. The inhibition of CO2 reduction was competitive with respect to CO2. The concentration of methylcobalamin required for one-half of maximal inhibition was 5 microM. Other cobamides inhibited at similar concentrations, but diaquacobinamide inhibited at lower concentrations. With respect to their affinities and specificities for corrins, inhibition of both the ATP-dependent activation and CO2 reduction closely resembled the corrin-dependent activation of the methylreductase described in similar extracts (W. B. Whitman and R. S. Wolfe, J. Bacteriol. 164:165-172, 1985). However, whether the multiple effects of corrins are due to action at a single site is unknown.

Adenosine Triphosphate↗
PubMed1987

Sulfometuron methyl-sensitive and -resistant acetolactate synthases of the archaebacteria Methanococcus spp.

The herbicide sulfometuron methyl (SM) inhibited growth of some methanococci. Of 28 strains tested, the growth of 7 was completely inhibited by 0.55 mM SM. Growth of an additional 14 strains was partially inhibited, and the growth of 7 strains was unaffected by this concentration of SM. In some cases, the branched-chain amino acids protected growth. Growth inhibition was correlated with the Ki for SM of acetolactate synthase (ALS). For the enzymes from bacteria representative of the sensitive, partially resistant, and resistant methanococci (Methanococcus aeolicus, Methanococcus maripaludis, and Methanococcus voltae, respectively), the Ki for SM was 0.0012, 0.34, and greater than 1.0 mM, respectively. Inhibition was uncompetitive with respect to pyruvate. Based on these observations, ALS appeared to be the major if not the sole site of action of SM in the methanococci. The sensitivity of the ALS from these three methanococci to feedback inhibition by branched-chain amino acids was also quite different. Although all three were sensitive to feedback inhibition by valine, the Ki varied 20-fold, from 0.01 to 0.22 mM. Moreover, only the ALS from M. maripaludis was sensitive to inhibition by leucine, and the Ki was 1.8 mM. The Ki for isoleucine for the ALS from both M. maripaludis and M. voltae was about 0.1 mM. The ALS from M. aeolicus was not inhibited by isoleucine. In other respects, the ALSs from the methanococci were very similar. After dialysis, thiamine pyrophosphate but not FAD and Mg2+ was required for maximal activity, and they were all rapidly inactivated by oxygen. Although the methanococcal ALSs exhibited diverse properties, the range of catalytic and regulatory properties closely resembled those of the eubacterial enzymes.

Acetolactate Synthase↗
PubMed1987

Pathway of acetate assimilation in autotrophic and heterotrophic methanococci.

The autotroph Methanococcus maripaludis contained high levels of acetate-coenzyme A ligase, pyruvate synthase, pyruvate, water dikinase, pyruvate carboxylase, and the enzymes of the incomplete reductive tricarboxylic acid cycle. Phosphoenolpyruvate carboxykinase, citrate synthase, and isocitrate dehydrogenase were not detected. In contrast, the heterotroph Methanococcus sp. strain A3 contained acetate kinase, and acetate coenzyme A ligase was virtually absent.

Acetates↗
PubMed1985

A newly-isolated marine methanogen harbors a small cryptic plasmid.

Of 21 recently isolated strains of methanococci, one was found to harbor a small, cryptic, low copy number plasmid. Reproducible recovery was achieved by alkaline lysis of cells pretreated with proteinase K in an osmotically stabilizing buffer. The plasmid was found to contain a single AvaI site. No homology was detected between the plasmid and DNA from any of the other new strains or from five known species of methanococci.

Base Sequence↗
PubMed1985

Activation of the methylreductase system from Methanobacterium bryantii by corrins.

Corrins activated the methylreductase system from Methanobacterium bryantii three- to fivefold in extracts resolved from low-molecular-weight factors. Corrins did not substitute for ATP and component B, which were also required for maximal activity. The concentration of diaquacobinamides required for one-half maximal activity was 1 microM. The concentrations of cyanocobalamin, methylcobalamin, Co alpha-(5-hydroxybenzimidazoyl)-Co beta-cyanocobamide, and 5'-deoxyadenosylcobinamide required for one-half maximal activity were between 4 and 7 microM. Deoxyadenosylcobalamin was nearly inactive. Activation was independent of thiols, coenzyme M, and ATP. Activation was also observed after partial purification of the methylreductase system by agarose column chromatography. Corrins were required in catalytic concentrations, methylcobalamin was not required, and methanogenesis was enzymatic. Corrin activation of the methylreductase is a novel effect on methanogenesis. However, the physiological significance of the corrin activation is uncertain.

Cobamides↗
PubMed1984

Purification and analysis of cobamides of Methanobacterium bryantii by high-performance liquid chromatography.

Cobamides from Methanobacterium bryantii were purified by isocratic reverse-phase high-performance liquid chromatography at neutral pH in readily extractable buffers. A gradient adaptation of this chromatography system on C-18 muBondapak columns in buffers containing aqueous methanol and 100 mM LiCl readily separated Co alpha-(5-hydroxybenzimidazoyl)-Co beta-cyanocobamide, Co alpha-(5-hydroxybenzimidazoyl)-Co beta-adenosylcobamide, cyanocobalamin, adenosylcobalamin, and methylcobalamin. Aquacobalamin was not resolved from cyanocobalamin by this procedure. The gradient procedure was also useful in following the fate of tritiated cyanocobalamin in cell-free extracts of M. bryantii.

Chromatography, High Pressure Liquid↗
PubMed1983

Growth and plating efficiency of methanococci on agar media.

Plating techniques for cultivation of methanogenic bacteria have been optimized for two members of the genus Methanococcus. Methanococcus maripaludis and Methanococcus voltae were cultivated on aerobically and anaerobically prepared agar media. Maintenance of O(2) levels below 5 mul/liter within an anaerobic glove box was necessary during plating and incubation for 90% recovery of plated cells. Under an atmosphere of H(2), CO(2), and H(2)S (79:20:1), 2 to 3 days of incubation at 37 degrees C were sufficient for the formation of visible colonies. The viability of plated cells was significantly affected by the growth phase of the culture, H(2)S concentration, and the volume of medium per plate. In addition, colony size of methanococci was affected by agar type, as well as by the concentrations of agar, H(2)S, and bicarbonate.

Journal Article↗
PubMed1983

Activation of the methylreductase system from Methanobacterium bryantii by ATP.

The methylreductase of Methanobacterium bryantii required ATP for activity. There was sufficient ATP synthesis in extracts to account for the observed activity. Hexokinase inhibited the methylreductase by competing for endogenously synthesized ATP. The uncoupler, carbonyl cyanide p-trifluoromethyoxyphenyl hydrazone, inhibited only at concentrations greater than 0.5 mM, and detergents and non-halogenated membrane-permeable-ions did not inhibit. Thus, membrane proton gradients are not important in activation. In addition, maximal activation was obtained with less than 0.25 mM ATP, was inhibited by beta, gamma-imido ATP, and was strongly temperature dependent. The activated state was very unstable, having a half-life of 5 to 15 min. After gel filtration at 5 degrees C, the methylreductase retained partial activity for a short time in the absence of ATP. These observations indicate that activation involves the modification of a protein or protein-bound cofactor of the methylreductase system.

Adenosine Triphosphate↗
PubMed1983

Complementation of argG and hisA mutations of Escherichia coli by DNA cloned from the archaebacterium Methanococcus voltae.

DNA derived from the methanogenic archaebacterium Methanococcus voltae was digested with PstI restriction endonuclease and cloned into the PstI site of pBR322. The recombinant plasmids generated were used to transform a multiply auxotrophic strain of Escherichia coli with selection for tetracycline resistance. Plasmids complementing the argG(pAW1) or hisA(pAW2) mutations were isolated and characterized. Nick-translated pAW1 and pAW2 hybridized to the predicted M. voltae PstI fragments but not to digested E. coli DNA. A novel 55,000-dalton protein was synthesized in UV-irradiated cells by pAW1, whereas pAW2 synthesized a novel 26,000-dalton protein. Derivatives of pAW1 carrying insertion elements no longer complemented the argG mutation and failed to produce the 55,000-dalton protein. When an AccI fragment was deleted from pAW2, complementation of hisA did not occur and no 26,000-dalton protein was synthesized. The effect of orientation of the cloned DNA within the vector on complementation and polypeptide synthesis was examined.

Arginine↗
PubMed1982

Nickel-containing factor F430: chromophore of the methylreductase of Methanobacterium.

The yellow chromophore of the methyl-coenzyme M methylreductase of Methanobacterium thermoautotrophicum has been found to be the nickel-containing factor F430. Treatment of 63Ni-labeled methylreductase with 80% aqueous methanol released the radiolabel as well as the yellow chromophore; both properties were associated with a single compound that was found to be identical to F430, the stoichiometry being 1 mol of nickel per mol of F430 and 2 mol of F430 per mol of methylreductase.

Chromatography, High Pressure Liquid↗
PubMed1982

Nutrition and carbon metabolism of Methanococcus voltae.

Methanococcus voltae is a heterotrophic, H2-oxidizing methanogenic bacterium. In complex medium, this bacterium has a doubling time of 1.2 h at its temperature optimum of 38 degrees C. In defined medium, optimal growth is obtained with 0.75 mM isoleucine, 0.75 mM leucine, 2.5 mM acetate, 5 mM NH4Cl, 84 mM MgSO4, 0.4 M NaCl, 1 mM CaCl2, 10 microM Fe2O3, and 0.2 microM NiCl2. In addition, pantothenate, sodium selenate, and cobalt stimulate growth. Optimal growth is obtained between pH 6.0 and 7.0 with either H2 or formate as the electron donor. The volatile fatty acids 2-methylbutyrate and isovalerate can substitute for isoleucine and leucine, respectively. Cellular carbon is derived from acetate (31%), isoleucine (22%), leucine (25%), and carbon dioxide (23%). The amino acids and fatty acids are incorporated almost exclusively into protein. A comparison of the incorporation of U-14C-amino acids and 1-14C-fatty acids indicated that the fatty acids are degraded during incorporation into cell protein. The distribution of carbon from the amino acids suggests that acetyl coenzyme A is not a major intermediate in the degradation of these compounds. Thus, M. voltae may convert isoleucine and leucine to other amino acids by a unique mechanism. The lipid carbon is derived largely from acetate. Thus, the isoprenoid lipids are synthesized de novo from acetate rather than by degradation of leucine. The carbon in the nucleic acids is derived from carbon dioxide (45%), the C-1 of acetate (25%), the C-2 of acetate (22%), and isoleucine and leucine (7%). This labeling pattern is consistent with known biochemical pathways.

Acetates↗
PubMed1980

Isolation and sequence of the pyridoxal 5'-phosphate active-site peptide from Rhodospirillum rubrum ribulose-1,5-bisphosphate carboxylase/oxygenase.

Ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum was modified with pyridoxal 5'-phosphate and then reduced with sodium borohydride. Both carboxylase and oxygenase activities were lost when one molecule of pyridoxal 5'-phosphate was bound per enzyme dimer. Peptide maps of modified enzyme showed one N6-(phosphopyridoxal)lysine-containing peptide. This peptide was isolated by gel filtration and cation-exchange chromatography and its sequence determined as Ala-Leu-Gly-Arg-Pro-Glu-Val-Asp-(PLP-Lys)-Gly-Thr-Leu-Val-Ile-Lys. Since activation of the enzyme with Mg2+/CO2 enhances pyridoxal 5'-phosphate modification and subsequent inactivation and the substrate ribulose bisphosphate protects against modification, the modified lysyl group is most certainly at the catalytic site and not at the activation site of the enzyme.

Amino Acid Sequence↗