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Extracts of Ruminococcus albus were not able to convert pyruvate to acetyl phosphate, CO2, and H2 after passage through a diethylaminoethyl (DEAE)-cellulose column. Activity was restored by a brown protein fraction eluted from the column with 0.4 M Cl-. The protein was partially purified and shown to have the spectral and biological characteristics of ferredoxin. R. albus ferredoxin, Clostridium pasteurianum ferredoxin, and methyl viologen restored activity for pyruvate decomposition by DEAE-cellulose-treated R. albus extracts. R. albus or C. pasteurianum ferredoxin restored the ability of DEAE-cellulose-treated C. pasteurianum extracts to form H2 and acetyl phosphate from pyruvate. Ferredoxin-free extracts of R. albus reduced nicotinamide adenine dinucleotide (NAD) when supplemented with R. albus or C. pasteurianum ferredoxin or with methyl viologen. These extracts reduced NADP with H2 poorly unless both ferredoxin and NAD were added, which indicates the presence of an NADH:NADP transhydrogenase. Flavin mononucleotide and flavin adenine dinucleotide were rapidly reduced by H2 by ferredoxin-free extracts in the absence of ferredoxin.

Methanobacterium ruminantium was shown to possess a formate dehydrogenase which is linked to factor 420 (F420) as the first low-molecular-weight or anionic electron transfer coenzyme. Reduced F420 obtained from the formate dehydrogenase can be further linked to the formation of hydrogen via the previously described F420-dependent hydrogenase reaction, thus constituting an apparently simple formate hydrogenlyase system, or to the reduction of nicotinamide adenine dinucleotide phosphate via F420:nicotinamide adenine dinucleotide phosphate oxidoreductase. The results indicate that hydrogen and formate, the only known energy sources for M. ruminantium and many other methanogenic bacteria, should be essentially equivalent as sources of electrons in the metabolism of this organism.

Methanobacterium ruminantium was shown to possess a nicotinamide adenine dinucleotide phosphate (NADP)-linked factor 420 (F420)-dependent hydrogenase system. This system was also shown to be present in Methanobacterium strain MOH. The hydrogenase system of M. ruminantium also links directly to F420, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), methyl viologen, and Fe-3 plus. It has a pH optimum of about 8 and an apparent Km for F420 of about 5 x 10-6 M at pH 8 when NADP is the electron acceptor. The F420-NADP oxidoreductase activity is inactive toward nicotinamide adenine dinucleotide (nad) and no NADPH:NAD or FADH2(FMNH2):NAD transhydrogenase system was detected. Neither crude ferredoxin nor boiled crude extract of Clostridium pasteuranum could replace F420 in the NADP-linked hydrogenase reaction of M. ruminantium. Also, neitther F420 nor a curde "ferredoxin" fraction from M. ruminantium extracts could substitute for ferredoxin in the pyruvate-ferredoxin oxidoreductase reaction of C. pasteurianum.

The ndhF gene of the unicellular marine cyanobacterium Synechococcus sp. strain PCC 7002 was cloned and characterized. NdhF is a subunit of the type 1, multisubunit NADH:plastoquinone oxidoreductase (NADH dehydrogenase). The nucleotide sequence of the gene predicts an extremely hydrophobic protein of 664 amino acids with a calculated mass of 72.9 kDa. The ndhF gene was shown to be single copy and transcribed into a monocistronic mRNA of 2,300 nucleotides. An ndhF null mutation was successfully constructed by interposon mutagenesis, demonstrating that NdhF is not required for cell viability under photoautotrophic growth conditions. The mutant strain exhibited a negligible rate of oxygen uptake in the dark, but its photosynthetic properties (oxygen evolution, chlorophyll/P700 ratio, and chlorophyll/P680 ratio) were generally similar to those of the wild type. Although the ndhF mutant strain grew as rapidly as the wild-type strain at high light intensity, the mutant grew more slowly than the wild type at lower light intensities and did not grow at all under photoheterotrophic conditions. The roles of the NADH:plastoquinone oxidoreductase in photosynthetic and respiratory electron transport are discussed.

The recA gene of Synechococcus sp. strain PCC 7002 was detected and cloned from a lambda gtwes genomic library by heterologous hybridization by using a gene-internal fragment of the Escherichia coli recA gene as the probe. The gene encodes a 38-kilodalton polypeptide which is antigenically related to the RecA protein of E. coli. The nucleotide sequence of a portion of the gene was determined. The translation of this region was 55% homologous to the E. coli protein; allowances for conservative amino acid replacements yield a homology value of about 74%. The cyanobacterial recA gene product was proficient in restoring homologous recombination and partial resistance to UV irradiation to recA mutants of E. coli. Heterologous hybridization experiments, in which the Synechococcus sp. strain PCC 7002 recA gene was used as the probe, indicate that a homologous gene is probably present in all cyanobacterial strains.

The pathway for the anaerobic catabolism of gallic acid by Eubacterium oxidoreducans was studied by using both in vivo and cell-free systems. Cells grown with gallate and crotonate, but with no formate or H2, excreted pyrogallol and phloroglucinol into the medium. Gallate was decarboxylated by crude cell extracts, with pyrogallol as the only detectable product. Whole cells converted pyrogallol to phloroglucinol. A phloroglucinol reductase catalyzed the conversion of phloroglucinol to dihydrophloroglucinol when NADPH was used as the source of electrons. Both formate dehydrogenase (EC 1.2.1.43) and hydrogenase (EC 1.18.99.1) were present in cell extracts of gallate-formate-grown cells. These two enzymes were both NADP linked. Since either H2 or formate is required for cell growth with gallate or phloroglucinol, these results suggest that the oxidation of the reduced substrate may be indirectly linked to the reduction of phloroglucinol. A dihydrophloroglucinol hydrolase was present, which hydrolyzed dihydrophloroglucinol to 3-hydroxy-5-oxohexanoate. This six-carbon ring cleavage product then presumably can be broken down by a series of reactions similar to beta-oxidation. These reactions cleaved the six-carbon acid to 3-hydroxybutyryl-coenzyme A yielding acetate and butyrate as end products. A number of key enzymes involved in beta-oxidation and substrate-level phosphorylation were demonstrated in cell extracts.

In Cyanophora paradoxa, the allophycocyanin apoprotein subunits, alpha and beta, are encoded in the cyanelle (plastid) genome. These genes were transferred to the cyanobacterium Synechococcus sp. PCC 7002 on a plasmid replicon. Phycobilisomes isolated from transformed cyanobacteria were found to contain C. paradoxa allophycocyanin subunits. Thus, these plastid genes are expressed in the cyanobacterium as polypeptides which become linked to a chromophore and are incorporated into the light-harvesting apparatus.

The nucleotide sequence and transcript initiation site of the Synechococcus sp. strain PCC 7002 recA gene have been determined. The deduced amino acid sequence of the RecA protein of this cyanobacterium is 56% identical and 73% similar to the Escherichia coli RecA protein. Northern (RNA) blot analysis indicates that the Synechococcus strain PCC 7002 recA gene is transcribed as a monocistronic transcript 1,200 bases in length. The 5' endpoint of the recA mRNA was mapped by primer extension by using synthetic oligonucleotides of 17 and 27 nucleotides as primers. The nucleotide sequence 5' to the mapped endpoint contained sequence motifs bearing a striking resemblance to the heat shock (sigma 32-specific) promoters of E. coli but did not contain sequences similar to the E. coli SOS operator recognized by the LexA repressor. An insertion mutation introduced into the recA locus of Synechococcus strain PCC 7002 via homologous recombination resulted in the formation of diploids carrying both mutant and wild-type recA alleles. A variety of growth regimens and transformation procedures failed to produce a recA Synechococcus strain PCC 7002 mutant. However, introduction into these diploid cells of the E. coli recA gene in trans on a biphasic shuttle vector resulted in segregation of the cyanobacterial recA alleles, indicating that the E. coli recA gene was able to provide a function required for growth of recA Synechococcus strain PCC 7002 cells. This interpretation is supported by the observation that the E. coli recA gene is maintained in these cells when antibiotic selection for the shuttle vector is removed.

The archaebacterium Pyrococcus furiosus is a strict anaerobe that grows optimally at 100 degrees C by a fermentative-type metabolism in which H2 and CO2 are the only detectable products. A ferredoxin, which functions as the electron donor to the hydrogenase of this organism was purified under anaerobic reducing conditions. It had a molecular weight of approximately 12,000 and contained 8 iron atoms and 8 cysteine residues/mol but lacked histidine or arginine residues. Reduction and oxidation of the ferredoxin each required 2 electrons/mol, which is consistent with the presence of two [4Fe-4S] clusters. The reduced protein gave rise to a broad rhombic electronic paramagnetic resonance spectrum, with gz = 2.10, gy = 1.86, gx = 1.80, and a midpoint potential of -345 mV (at pH 8). However, this spectrum represented a minor species, since it quantitated to only approximately 0.3 spins/mol. P. furiosus ferredoxin is therefore distinct from other ferredoxins in that the bulk of its iron is not present as iron-sulfur clusters with an S = 1/2 ground state. The apoferredoxin was reconstituted with iron and sulfide to give a protein that was indistinguishable from the native ferredoxin by its iron content and electron paramagnetic resonance properties, which showed that the novel iron-sulfur clusters were not artifacts of purification. The reduced ferredoxin also functioned as an electron donor for H2 evolution catalyzed by the hydrogenase of the mesophilic eubacterium Clostridium pasteurianum. P. furiosus ferredoxin was resistant to denaturation by sodium dodecyl sulfate (20%, wt/vol) and was remarkably thermostable. Its UV-visible absorption spectrum and electron carrier activity to P. furiosus hydrogenase were unaffected by a 12-h incubation of 95 degrees C.

Cell extracts of Eubacterium oxidoreducens, in the presence of dimethyl sulfoxide, catalyzed the conversion of pyrogallol to phloroglucinol with methyl sulfide as a product. The isomerization reaction also proceeded when 1,2,3,5-benzenetetrol was present rather than dimethyl sulfoxide. An assay to quantitate this activity was developed. The assay followed the disappearance of 1,2,4-benzenetriol as determined colorimetrically after incubation with sodium molybdate at neutral pH. The products of this reaction were resorcinol and 2,6-dihydroxyquinone. The enzyme(s) catalyzing this reaction was purified fivefold from cells grown on gallate plus H2. The purification procedure involved treatment with 40% acetone, precipitation with ammonium sulfate, DEAE-cellulose chromatography, concentration by ultrafiltration (molecular weight cutoff, greater than 100,000), and hydroxylapatite chromatography. This preparation had a specific activity of 14.7 mumol/min per mg of protein and a pH optimum of about 7.3. It was strongly inhibited by p-chloromercuribenzoate. The mechanism of the reaction involved oxidation of the pyrogallol followed by introduction of water. The benzenetetrol intermediate was then reduced and dehydrated to phloroglucinol.

The genes for the following proteins were localized by hybridization analysis on the cyanelle genome of Cyanophora paradoxa: the alpha and beta subunits of phycocyanin (cpcA and cpcB); the alpha and beta subunits of allophycocyanin (apcA and apcB); the large and small subunits of ribulose-1,5-bisphosphate carboxylase (rbcL and rbcS); the two putative chlorophyll alpha-binding apoproteins of the photosystem I-P700 complex (psaA and psaB); four apoproteins believed to be components of the photosystem II core complex (psbA, psbB, psbC, and psbD); the two apoprotein subunits of cytochrome b-559 which is also found in the core complex of photosystem II (psbE and psbF); three subunits of the ATP synthase complex (atpA and atpBE); and the cytochrome f apoprotein (petA). Eighty-five percent of the genome was cloned as BamHI, BglII, or PstI fragments. These cloned fragments were used to construct a physical map of the cyanelle genome and to localize more precisely some of the genes listed above. The genes for phycocyanin and allophycocyanin were not clustered and were separated by about 25 kilobases. Although the rbcL gene was adjacent to the atpBE genes and the psbC and psbD genes were adjacent, the arrangement of other genes encoding various polypeptide subunits of protein complexes involved in photosynthetic functions was dissimilar to that observed for known chloroplast genomes. These results are consistent with the independent development of this cyanelle from a cyanobacterial endosymbiont.

In this paper we describe the successful isolation of glyT-derived GAA suppressors. A glyT+ strain containing glyV55, the gene for a GGA/G-reading, methane sulfonate and hydroxylamine. The cells were plated to select for reversal of auxotrophy due to a trpA(GAA211) mutation. With either mutagen, greater than 85% of the prototrophs obtained were due to suppressors of the trpA mutation. Approximately 12% of the ethyl methane sulfonate-induced and 37% of the hydroxylamine-induced suppressors were shown to be about 25% cotranscucible with metB, as is glyT. The transfer ribonucleic acid from four metB-linked suppressor strains (two from each mutagen) was examined by reversed-phase column (RPC-5) chromatography. In all four cases, the glycyl-transfer ribonucleic acid profile displayed an alteration of glyT transfer ribonucleic acid. All four suppressors responded to GAG in addition to GAA but did not suppress the known mutant codons of several other trpA mutants. Other properties are discussed, along with possible reasons for our success in obtaining these suppressors.

Selenomonas ruminantium was found to possess two pathways for NH4+ assimilation that resulted in net glutamate synthesis. One pathway fixed NH4+ through the action of an NADPH-linked glutamate dehydrogenase (GDH). Maximal GDH activity required KCl (about 0.48 M), but a variety of monovalent salts could replace KCl. Complete substrate saturation of the enzyme by NH4+ did not occur, and apparent Km values of 6.7 and 23 mM were estimated. Also, an NADH-linked GDH activity was observed but was not stimulated by KCl. Cells grown in media containing non-growth-rate-limiting concentrations of NH4+ had the highest levels of GDH activity. The second pathway fixed NH4+ into the amide of glutamine by an ATP-dependent glutamine synthetase (GS). The GS did not display gamma-glutamyl transferase activity, and no evidence for an adenylylation/deadenylylation control mechanism was detected. GS activity was highest in cells grown under nitrogen limitation. Net glutamate synthesis from glutamine was effected by glutamate synthase activity (GOGAT). The GOGAT activity was reductant dependent, and maximal activity occurred with dithionite-reduced methyl viologen as the source of electrons, although NADPH or NADH could partially replace this artificial donor system. Flavin adenine dinucleotide, flavin mononucleotide, or ferredoxin could not replace methyl viologen. GOGAT activity was maximal in cells grown with NH4+ as sole nitrogen source and decreased in media containing Casamino Acids.

Four cold-sensitive mutants of Escherichia coli, which have defects in the maturation of the 50Sribosomal subunit, were isolated. Each of the mutations was shown to map at a different locus. The loci were assigned the name rim(ribosome maturation) and were shown to map as follows: rimAis co-transduced with ilvDand with pyrE; rimBis co-transduced with aroD;conjugation experiments limited rimDto a region between ilvand malB, and conjugation experiments limited rimCto the 22 to 30 min region of the chromosome. In merodiploids heterozygous for rimA, rimB, or rimD, the wild-type allele was shown to be dominant to the mutant allele. The observation that the rimloci lie outside the strAregion and separate from each other, as well as the recessive character of the rimloci, suggests that the mutants may be defective in ribosome maturation factors rather than being defective in ribosomal structural proteins.

Allison, M. J. (Dairy Cattle Research Branch, USDA, Beltsville, Md.), M. P. Bryant, I. Katz, and M. Keeney. Metabolic function of branched-chain volatile fatty acids, growth factors for ruminococci. II. Biosynthesis of higher branched-chain fatty acids and aldehydes. J. Bacteriol. 83:1084–1093. 1962.—A number of strains of rumen bacteria require branched-chain volatile fatty acids for growth. A strain of Ruminococcus flavefaciensthat requires either isovalerate or isobutyrate incorporates radioactive carbon from isovalerate-1-C14and isovalerate-3-C14into leucine and into the lipid fraction of the cells. Evidence obtained by both paper and gas chromatography indicated that most of the label in the lipid of cells grown in isovalerate-1-C14was in a branched-chain 15-carbon fatty acid, with some in a 17-carbon acid; about 7.5% of the C14was recovered in a branched-chain 15-carbon aldehyde. The aldehydes were in the phospholipid fraction and were presumably present as plasmalogen.A strain of R. albuswas shown to require isobutyrate, 2-methyl-n-butyrate, or 2-ketoisovalerate for growth. This strain did not incorporate appreciable C14from isovalerate-1-C14or isovalerate-3-C14. When grown in a medium containing isobutyrate-1-C14, most of the cellular C14was found in the lipid fraction. Analysis of the lipid demonstrated that the label was present mainly as branched-chain 14-carbon and 16-carbon fatty acids, with 11% of the C14present in 14- and 16-carbon carbonyl compounds, presumably branched-chain aldehydes.Branched-chain 14-, 15-, and 16-carbon fatty acids are major components of the lipids of these rumen bacteria. The possibility that these acids and aldehydes, which are found in ruminant body and milk lipids, may be of microbial origin is discussed.

Allison, MiltonJ. (Dairy Cattle Research Branch, U. S. Department of Agriculture, Beltsville, Md.), M. P. Bryant, and R. N. Doetsch. Studies on the metabolic function of branched-chain volatile fatty acids, growth factors for ruminococci. I. Incorporation of isovalerate into leucine. J. Bacteriol. 83:523–532. 1962.—Ruminococcus flavefaciensstrain C94, a cellulolytic rumen bacterium, requires either isobutyrate or isovalerate for growth. The organism was grown in the presence of C14-labeled isovalerate, and the metabolic fate of the labeled carbon was studied to obtain information on the functions of this growth factor. Radioactivity from isovalerate-1-C14and isovalerate-3-C14was found mainly in the protein and lipid fractions of the cells. The C14in protein was all in leucine, indicating that a function of isovalerate was to serve as a carbon skeleton for leucine synthesis.As C14in leucine synthesized from isovalerate-1-C14was entirely in carbon 2, the intact isovalerate molecule was apparently incorporated into leucine. This is evidence that leucine was synthesized by a mechanism different from that previously demonstrated in other microorganisms.R. flavefacienshas a definite but limited ability to incorporate exogenous amino acids, including leucine. It incorporated 2% of the C14during growth in uniformly labeled (UL) C14-Chlorellaprotein hydrolyzate; Escherichia coliincorporated 37% of the label under similar conditions. In another experiment, a limited amount of exogenous leucine-2-C14was incorporated into protein of R. flavefaciens. The requirement for isovalerate was not replaced by dl-leucine or 2-ketoisocaproate. It is suggested that isovalerate or isobutyrate is required because R. flavefacienshas a limited ability to incorporate exogenous branched-chain amino acids and a limited ability to synthesize the isopropyl group found in these amino acids and in other components of the cell.

Bacteroides ruminicoladid not take up 14C from exogenous 14C-labeled l-proline or 14C-labeled l-glutamic acid and took up very little 14C from exogenous 14C-labeled l-valine. Growing cultures of B. ruminicolarapidly took up 14C from 14C-proline-labeled peptides of molecular weights up to 2,000 and incorporated it into trichloroacetic acid-insoluble cell material. Uptake and incorporation did not occur at 0 C and were reduced or eliminated in glucose-starved cells, depending upon the length of time the cells were starved. The initial rate of uptake of peptides seemed to exhibit saturation kinetics, but it was impossible to establish this conclusively. The initial uptake of 14C from peptides was not affected by chloramphenicol but the incorporation of it into trichloroacetic acid-insoluble cell material was virtually eliminated. Only moderate amounts of trichloroacetic acid-extractable, labeled material were present in cells during peptide uptake, whether or not chloramphenicol was present. 14C-proline was rapidly released from labeled peptides during uptake, whether or not chloramphenicol was present. The amount of 14C fixed into trichloroacetic acid-insoluble cell material was directly related to the size of peptides originally supplied in the medium. It is concluded that B. ruminicolapossesses a general system for the uptake of peptides, that peptides are rapidly hydrolyzed during or after uptake, and that oligopeptides function only to supply amino acids in a form available to the organism.

Caldwell, D. R. (U.S. Department of Agriculture, Beltsville, Md.), D. C. White, M. P. Bryant, and R. N. Doetsch. Specificity of the heme requirement for growth of Bacteroides ruminicola. J. Bacteriol. 90:1645–1654. 1965.—Previous studies suggested that most strains of Bacteroides ruminicolasubsp. ruminicolarequire heme for growth. Present studies with heme-requiring strain 23 showed that protoheme was replaced by various porphyrins, uroporphyrinogen, coproporphyrinogen, certain iron-free metalloporphyrins, hemes, and certain heme-proteins containing readily removable hemes. Strain 23 utilized a wider range of tetrapyrroles than hemin-requiring bacteria previously studied. Inactive compounds included porphyrin biosynthesis intermediates preceding the tetrapyrrole stage and related compounds; uroporphyrin, chlorophyll, pheophytin, phycoerythrin, bilirubin, pyrrole, FeSO4with or without chelating agents; and representative ferrichrome compounds. Strain 23, two other strains representing predominant biotypes of B. ruminicolasubsp. ruminicola, and one closely related strain grew in media containing heme-free protoporphyrin, mesoporphyrin, hematoporphyrin, or deuteroporphyrin, apparently inserting iron into several nonvinyl porphyrins. Porphobilinogen and porphyrin synthesis, apparently via the commonly known heme synthesis pathway, occurred during growth of heme-independent B. ruminicolasubsp. brevisstrain GA33 in a tetrapyrrole-free medium containing δ-aminolevulinic acid, but δ-aminolevulinic acid metabolism to porphobilinogen or porphyrins could not be detected in cells of heme-requiring strain 23 grown in the same medium with hemin added. Growth of strain 23 with uroporphyrinogen, coproporphyrinogen, or protoporphyrin IX replacing hemin suggests that part of the commonly known heme-biosynthesis pathway is present in this strain, but nutritional and metabolic evidence indicates that some or all of the enzymes synthesizing the tetrapyrrole nucleus from linear molecules are lacking or inactive.

A method for the mass culture of hydrogen-oxidizing methane bacteria has been developed; yields of 50 to 60 g (wet weight) of cells per 12 to 14 liters of culture medium were obtained. The methanogenic organism from the culture of Methanobacillus omelianskiiwas grown in a complex medium which was aerated with a gas mixture of hydrogen and carbon dioxide. Extracts prepared from hydrogen-grown cells formed methane from methyl cobalamin, 5-methyl tetrahydrofolate, serine, pyruvate, and carbon dioxide; these substrates have been shown to be precursors of methane in extracts of the ethyl alcohol-grown culture of M. omelianskii.

Comparison of the fine structure of the methanogenic organism from the culture known as Methanobacillus omelianskiiwith that of Methanobacterium formicicumrevealed a great similarity. Both organisms exhibited a large number of intracytoplasmic membranous elements when stained with phosphotungstic acid. In contrast, no such elements were observed in Methanobacterium ruminantium.

Pittman, K. A. (Agricultural Research Service, Beltsville, Md.), and M. P. Bryant. Peptides and other nitrogen sources for growth of Bacteroides ruminicola. J. Bacteriol. 88:401–410. 1964.—Representative strains of Bacteroides ruminicolawere found to utilize peptide nitrogen or ammonia nitrogen, but not to utilize significant amounts of free amino acid nitrogen or the nitrogen from a variety of other low molecular weight compounds for growth. All strains grew well in a defined medium containing glucose, minerals, B-vitamins, heme, volatile fatty acids, methionine, and cysteine, with ammonia as the main nitrogen source. Methionine and cysteine were required by some strains. The only compounds found to replace ammonia as the main nitrogen source were a few proteins; tryptic digests of protein; peptide-rich fractions of Sephadex G-25 fractionated tryptic digests of casein; and the octapeptides, oxytocin and vasopressin. Most of the nitrogen present in these compounds was utilized. However, the organism did not utilize nitrogen from any of 12 dipeptides, triglycylglycine, glutathione, or mixtures of free amino acids. Possible reasons for the inability of B. ruminicolato utilize low molecular weight nitrogen compounds are discussed.

White, D. C. (Rockefeller Institute, New York, N.Y.), M. P. Bryant, and D. R. Caldwell. Cytochrome-linked fermentation in Bacteroides ruminicola. J. Bacteriol. 84:822–828. 1962—Previous studies showed that Bacteroides ruminicola, an anaerobic, saccharolytic, ruminal bacterium, ferments glucose with the production of succinic, acetic, and formic acids, requires a large amount of CO2, and most strains require heme for growth. Difference spectra of cell suspensions of both heme-requiring strain 23, B. ruminicolasubsp. ruminicola, and heme-independent strain GA33, B. ruminicolasubsp. brevis, showed the presence of a cytochrome (absorption maxima at 560 mμ, near 530 mμ, and 428 mμ) similar to cytochrome b. This cytochrome and flavoprotein (trough at 450 mμ) in the cells, reduced by endogenous metabolism, were oxidized on addition of air, CO2, oxalacetate, malate, or fumarate but no oxidation occurred in the presence of succinate, malonate, lactate, pyruvate, aspartate, citrate, NO3−, SO4=, 2-n-heptyl or hydroxyquinoline-N-oxide (HOQNO), amytal or azide. The oxidation of these cellular pigments by fumarate was not inhibited by CN−, CO, malonate, succinate, amytal, or HOQNO. Glucose and reduced diphosphopyridine nucleotide (DPNH), but not succinate, reduced the pigments in frozen-thawed cells previously exposed to air for 4 hr at room temperature. The results suggest that this cytochrome and flavoprotein form an electron transport system for fumarate reduction to succinate by DPNH generated by glycolysis, and that succinate is produced via CO2condensation with pyruvate or phosphoenolpyruvate and with oxalacetate, malate, and fumarate as intermediates. A pigment similar to cytochrome o(absorption maxima at 570, 555, and 416 mμ) was observed when reduced cells were treated with CO and compared to reduced cells, but there was no detectable cytochrome oxidase activity. The function of this pigment is obscure. No peroxidase or catalase activity was detected in either strain. Pyridine hemochromogens of both strains indicate one major heme, a protoheme-like pigment, with absorption in the α region maximum at 556 mμ. As B. ruminicolais one of the most numerous of rumen bacteria and ferments a wide variety of carbohydrates of importance in ruminant rations, cytochrome must be of importance in electron transport in rumen contents, a highly anaerobic environment.

Bryant, M. P. (U.S. Department of Agriculture, Beltsville, Md.) and I. M. Robinson. Some nutritional characteristics of predominant culturable ruminal bacteria. J. Bacteriol. 84:605–614. 1962.—The effect of enzymatic hydrolysate of casein, NH4+, a mixture of volatile fatty acids (acetic, n-valeric, isovaleric, 2-methylbutyric, and isobutyric), hemin, and ruminal fluid on growth of 89 freshly isolated strains of predominant culturable ruminal bacteria was studied, using basal media containing glucose, cellobiose, or maltose as energy source, minerals, cysteine, and S=as reducing agents, and H2CO3-HCO3−buffer. Of these strains, 13% (four morphological groups) grew poorly or not at all in defined medium plus casein hydrolysate; 6% (one morphological group) required casein hydrolysate; 56% (four morphological groups) grew with either NH4+or casein hydrolysate as the main source of nitrogen; and NH4+, but not casein hydrolysate, was essential for 25% of the strains (five morphological groups). The volatile fatty acid mixture excluding acetate was essential for 19% of the strains (five morphological groups), and this mixture and acetate were necessary for good growth of 23% of the strains (one morphological group) when casein hydrolysate was excluded from the medium; 30% of the strains (one morphological group) required hemin. Similar studies are reported on 35 old laboratory strains of ruminal bacteria, most of which were previously identified. The results indicate that most strains of ruminal bacteria can be grown in defined media, and suggest the relative importance of NH4+and volatile fatty acids and the relative lack of importance of organic nitrogen compounds such as amino acids in the nutrition of these bacteria.

The influence of a H2-utilizing organism, Vibrio succinogenes, on the fermentation of limiting amounts of glucose by a carbohydrate-fermenting, H2-producing organism, Ruminococcus albus, was studied in continuous cultures. Growth of V. succinogenesdepended on the production of H2from glucose by R. albus. V. succinogenesused the H2produced by R. albusto obtain energy for growth by reducing fumarate in the medium. Fumarate was not metabolized by R. albusalone. The only products detected in continuous cultures of R. albusalone were acetate, ethanol, and H2. CO2was not measured. The only products detected in the mixed cultures were acetate and succinate. No free H2was produced. No formate or any other volatile fatty acid, no succinate or other dicarboxylic acids, lactate, alcohols other than ethanol, pyruvate, or other keto-acids, acetoin, or diacetyl were detected in cultures of R. albusalone or in mixed cultures. The moles of product per 100 mol of glucose fermented were approximately 69 for ethanol, 74 for acetate, 237 for H2for R. albusalone and 147 for acetate and 384 for succinate for the mixed culture. Each mole of succinate is equivalent to the production of 1 mol of H2by R. albus. Thus, in the mixed cultures, ethanol production by R. albusis eliminated with a corresponding increase in acetate and H2formation. The mixed-culture pattern is consistent with the hypothesis that nicotinamide adenine dinucleotide (reduced form), formed during glycolysis by R. albus, is reoxidized during ethanol formation when R. albusis grown alone and is reoxidized by conversion to nicotinamide adenine dinucleotide and H2when R. albusis grown with V. succinogenes. The ecological significance of this interspecies transfer of H2gas and the theoretical basis for its causing changes in fermentation patterns of R. albusare discussed.

S organism, an anaerobic gram-negative rod, which is one of two bacterial species isolated from the culture known as “Methanobacillus omelianskii,” ferments ethanol to acetate and H2. The present study shows that extracts of this organism contain ferredoxin and produce acetate from acetaldehyde via aldehyde: ferredoxin oxidoreductase activity. Electrons generated in the reaction are given off as H2by a previously demonstrated ferredoxin-linked hydrogenase system. Extracts were shown to contain good phosphotransacetylase and acetokinase activities, but no mechanism of adenosine triphosphate generation during acetaldehyde conversion to acetate could be detected. No evidence could be obtained for coenzyme A (CoASH) or phosphate requirement or for formation of acetyl CoA or acetyl phosphate.

S organism ferments ethanol to acetate and H2but grows poorly on ethanol unless the partial pressure of H2is kept low, as when it is grown in combination with an H2-utilizing methanogenic bacterium. The present study shows that S organism contains an alcohol dehydrogenase and a formate dehydrogenase, both of which require nicotinamide adenine dinucleotide (NAD) for activity. Hydrogen is evolved from NADH generated by these activities via a ferredoxin-dependent oxidation of NADH to NAD and H2. NADH:NADP oxido-reductase activity was also demonstrated. The relationship of these activities to the growth of S organism is discussed.

Previous work showed that Methanobacillus omelianskiiwas a mixed culture of an ethanol-oxidizing organism called S organism and a hydrogen-utilizing methane bacterium, strain MOH. S organism grows poorly on ethanol unless a hydrogen-utilizing methanogenic bacterium is included to utilize the H2produced during growth. Further studies have shown that, among many substrates tested, only ethanol, n-propanol, n-butanol, isobutanol, n-pentanol, acetaldehyde, oxalacetate, and pyruvate are fermented by S organism, either alone or in combination with Methanobacterium ruminantium. It grew better in pure culture with pyruvate than with alcohols. H2gas phase inhibited growth on pyruvate as well as on alcohol. When grown alone on pyruvate, S organism produced mainly acetate, ethanol, and CO2, in addition to a small amount of H2. When combined with M. ruminantium, no H2and very little ethanol were produced and acetate production was increased. When M. ruminantiumwas present, electrons from pyruvate oxidation by S organism were channeled almost entirely to H2and hence to methane formation rather than ethanol. Also, S organism utilized more pyruvate when grown with M. ruminantium. Attempts to obtain better growth of S organism on ethanol by addition of many possible electron acceptors were unsuccessful. It grew best between 32 and 45 C, had a per cent guanine plus cytosine content of deoxyribonucleic acid bases of 47.27 ± 0.1, contained no cytochrome, and could be grown on a defined medium with pyruvate as the energy and carbon source and with (NH4)2SO4as the main nitrogen source. These and other results suggest that S organism belongs in a new genus, but assignment of a definite taxonomic status should await isolation and characterization of more strains.