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n-Butane was metabolized in Mycobacterium vaccae (JOB5) via terminal oxidation. This organism metabolized 2-butanone through propionate (or propionyl coenzyme A). Subterminal oxidation in M. vaccae was apparently limited to propane.

The cellular fatty acid composition of Mycobacterium vaccae JOB5 and Mycobacterium convolutum R22 was examined after growth on n-alkanes and compared with the fatty acids of the organisms after growth on 1-chlorohexadecane and 1-chlorooctadecane. Growth on n-alkanes resulted in normal fatty acid profiles. Mass spectral analyses indicated that, after growth on the terminally chlorinated n-alkanes, 75 to 86% of the fatty acids in M. convolutum and ca. 55% of the fatty acids in M. vaccae contained chlorine. Neither organism could utilize chloroacetate or 3-chloropropionate as sole source of carbon and energy. When these compounds were added to a growth medium with n-hexadecane as substrate, there was no evidence that chlorinated fatty acids were produced. Terminally chlorinated n-alkanes can be added to the list of n-alkanes, alkenes, and cyclohexylalkane derivatives that can be directly incorporated into cellular fatty acids of hydrocarbon-utilizing organisms.

The cell wall of Thermomicrobium roseum, a Gram-negative, obligately thermophilic bacterium, has a composition unlike other Gram-negative thermophilic organisms. The purified cell wall was composed predominantly of a protein with a monomeric molecular weight of 75 000. The amino acid composition of this protein revealed high concentrations of proline, glutamic acid, glycine, and alanine.

Ten strains of Gram-negative, aerobic, obligately thermophilic bacteria were examined for their response to oxygen toxicity by comparing static with shaken cultures. All of the organisms tested had measurable levels of superoxide dismutase, catalase, and peroxidase. Aeration generally did not result in an increased level of superoxide dismutase in any of the thermophiles. Aeration of organisms obligate for n-alkane substrate caused an increase in cellular peroxidase levels and a corresponding decrease in catalase. The thermophiles that grew on either n-alkanes or complex media did not grow on the hydrocarbon in aerated culture but on a complex medium, aeration effected an increased level of catalase. With the exception of a pink-pigmented thermophile which, when aerated, did not have an increased level of the three oxygen defense enzymes, most of the thermophiles surveyed had an increased level of catalase or peroxidase when exposed to increased oxygen tension. The activity of the enzymes was determined at temperatures from 25 to 65 degrees C and the former temperature was satisfactory for these experiments.

A manganese-containing catalase has been characterized from Thermoleophilum album NM, a gram-negative aerobic bacterium obligate for thermophily and n-alkane substrates. The level of catalase in cells was increased about ninefold by growth in the presence of paraquat (2.5 microM), a superoxide-generating toxicant. Superoxide dismutase levels were unaffected by this compound. The enzyme was purified from cultures grown in the presence of paraquat to greater than 95% homogeneity and had an Mr of 141,000. The enzyme was composed of four subunits, and each had an Mr of 34,000. There were 1.4 +/- 0.4 atoms of manganese present per subunit. The catalase had a Km for hydrogen peroxide of 15 mM and a Vmax of 11 mM/mg. Peroxidase activity, as measured with p-phenylenediamine, copurified with the catalase. Inhibitors of heme-catalase were weak inhibitors of the T. album enzyme. The optimum pH for catalase activity was 8 to 9. The enzyme was stable from pH 6.5 to 11 and retained activity at assay temperatures from 25 to 80 degrees C. The catalase was stable for 24 h of incubation at 60 degrees C.

Mycobacterium vaccae catabolizes propane through a C2 + C1 cleavage. Radiorespirometric and 14C-substrate incorporation studies were conducted to ascertain the fate of the C1 product. Results presented indicate that it is directly assimilated through the cellular reduced C1 pool.

The level of superoxide dismutase, catalase, and peroxidase was determined in obligately thermophilic bacteria after growth either in the presence of methyl viologen (paraquat) or exposed to an increased atmospheric oxygen concentration. In general, the superoxide dismutase level in these organisms was not altered by paraquat addition and had a varied response to oxygenation. Incorporation of paraquat in the growth medium resulted in minimally three-fold higher levels of catalase. The peroxidase level varied as a response to oxygen stress whereas cellular catalase levels were generally higher.

Studies were conducted on the oxidation and assimilation of n-alkyl-substituted cycloalkane substrates by several hydrocarbon-utilizing microorganisms. These microorganisms utilized heptadecylcyclohexane and dodecylcyclohexane as the sole source of carbon and energy. Neither methylcyclohexane nor ethylcyclohexane was utilized as a growth substrate by any organisms tested. Gas-liquid chromatographic analyses of fatty acids present in cells after growth on dodecylcyclohexane confirm direct incorporation of both alpha- and beta-oxidation products. Growth patterns of these organisms on n-alkyl-substituted cyclohexane fatty acids of varying chain lengths suggest a greater probability of ring cleavage when the side chain contains an odd number of carbons.

Mycobacterium vaccae strain JOB-5 cultured in the presence of propane contained an inducible secondary alcohol dehydrogenase. The enzyme was purified 198-fold using DEAE-cellulose, omega-aminopentyl agarose and NAD-agarose chromatography. The Mr of the enzyme was approximately 136000, with subunits of Mr 37000. The pH optimum for the reaction oxidizing propan-2-ol to propanone was 10-10.5 while the optimum for the reverse reaction was 7.5-8.5. The isoelectric point was 4.9. NAD but not NADP could serve as electron acceptor. The apparent Km values for propan-2-ol and NAD were 4.9 X 10(-5)M and 2.8 X 10(-4)M, respectively. The enzyme was inhibited by thiol reagents and metal chelators. It appears to play an essential role in the metabolism of propane by this bacterium.

Protocatechuate 3,4-dioxygenase (EC 1.13.1.3) was purified to homogeneity from extracts of Azotobacter vinelandii. The molecular weight of the oligomeric protein was estimated to be 510 000 by gel filtration and 480 000 by ultracentrifugation. The oligomer appears to be formed by association of equal amounts of nonidentical subunits which were estimated by sodium dodecyl sulfate gel electrophoresis to have respective molecular weights of 23 300 and 25 250. Ten gram-atoms of iron was associated with each mol of oligomer. Therefore, the enzyme appears to be a decamer with the structure 10(alpha beta Fe). T-HE AMINO ACID COMPOSITION OF Azotobacter protocatechuate oxygenase closely resembles the amino acid compositions of protocatechuate 3,4-dioxygenases from Pseudomonas aeruginosa and Thiobacillus sp. These proteins from P. aeruginosa and P. putida are known to be formed by association of nonidentical subunits of a physical size similar to the subunits of the Azotobacter enzyme. Furthermore, antisera prepared against the Azotobacter oxygenase cross-reacted strongly with the isofunctional enzymes from the two fluorescent Pseudomonas species. A weak immunological cross-reaction was observed when the antisera were tested against protocatechuate 3,4-dioxygenase from Acinetobacter calcoaceticus. The results favor the conclusion that the bacterial protocatechuate 3,4-dioxygenases were derived from a common ancestral protein.

Strain RA-6 is a pink-pigmented organism which can grow on a variety of substrates including methylamine. It can utilize methylamine as sole source of carbon via an isocitrate lyase negative serine pathway. Methylamine grown cells contain an inducible primary amine dehydrogenase [primary amine: (acceptor) oxidoreductase (deaminating)] which is not present in succinate grown cells. The amine dehydrogenase was purified to over 90% homogeneity. It is an acidic protein (isoelectric point of 5.37) with a molecular weight of 118,000 containing subunits with approximate molecular weights of 16,500 and 46,000. It is active on an array of primary terminal amines and is strongly inhibited by carbonyl reagents. Cytochrome c or artificial electron acceptors are required for activity; neither NAD nor NADP can serve as primary electron acceptor.

The fatty acid pattern in Mycobacterium vaccae strain JOB5 was examined after growth on n-alkanes (C14-C18), 1-alkenes (C14-C18), 2- or 3-methyl octadecane, and 8-heptadecene. It was evident that monoterminal oxidation of n-alkanes was followed by beta-oxidation and that both parent fatty acid and products of beta-oxidation were incorporated into cellular lipids. Radioactive experiments demonstrated that there was desaturation of long-chain fatty acids. There was no evidence of chain elongation. Growth on 1-alkenes resulted in the incorporation of fatty acids that were products of two primary modes of oxidation: (1) methyl group attack resulting in omega-unsaturated fatty acids and (2) double-bond attack resulting in the removal of one carbon from the substrate. Cells of strain JOB5 grown on 2- and 3-methyl octadecane contained the corresponding iso- or anteiso-fatty acids in significant quantity. Cells cultured on 8-heptadecene contained 8- and 9-heptadecenoic acids, 6- and 7-pentadecenoic acids, 9- and 10-methyl heptadecanoic acids, and 7- and 8-methyl pentadecanoic acids. Fatty acid composition (C13 to C19) was affected by substrate chain length and was additionally modified by cellular control mechanisms.

The fatty acid compositions of two filamentous fungi (Cunninghamella elegans and Penicillium zonatum) and a yeast (Candida lipolytica) were determined after the organisms were grown on 1-chlorohexadecane or 1-chlorooctadecane. These organisms utilized the chlorinated alkanes as sole sources of carbon and energy. Analyses of the fatty acids present after growth on the chlorinated alkanes indicated that 60 to 70% of the total fatty acids in C. elegans were chlorinated. Approximately 50% of the fatty acids in C. lipolytica were also chlorinated. P. zonatum contained 20% 1-chlorohexadecanoic acid after growth on either substrate but did not incorporate C18 chlorinated fatty acids.

Mycobacterium convolutum strain NPA-1 can utilize n-propylamine (NPA), isopropylamine (IPA), and 1,3-propane diamine (PD) as sole source of carbon, nitrogen, and energy. Enzyme assays, fatty acid profiles, and 14CO2 incorporation experiments indicate that NPA is deaminated to propionate and further metabolized via the methylmalonyl succinate pathway, and IPA and PD were metabolized (after deamination) through a C2 + C1 cleavage. An inducible amine dehydrogenase was present in cell extracts after growth on the three amines. Polyacrylamide gel electrophoresis of cell extracts from NPA- and IPA-grown cells yielded one major band of amine dehydrogenase activity. When extracts of NPA-grown cells were assayed with NPA, IPA, or PD as substrate, the relative position of the major band on gel electrophoresis was equivalent. Similar results were obtained with extracts prepared from IPA-grown cells. Sephadex G-100 chromatography also indicated one major peak of activity. This suggests that one enzyme of broad specificity is involved in deamination of IPA, NPA, and PD. IPA-grown cells utilized NPA readily, whereas NPA-grown cells could not utilize IPA without lag. Since amine dehydrogenase activity was present in extracts of cells after growth on either substrate, this lag was probably due to the inability to transport IPA without an induction period. The molecular weight of the amine dehydrogenase was approximately 38,500 as determined by gel filtration.

There was approximately five times more hemoprotein (amine dehydrogenase) in crude extracts obtained from Pseudomonas putida grown on benzylamine than present in extracts from succinate-grown cells. The difference (reduced minus oxidized) spectrum of the purified enzyme possessed alpha,beta, and gamma bands at 550, 523, and 416 nm, respectively. The difference spectrum of the pyridine hemochrome derivative had absorption maxima at 416, 520, and 550 nm. These results, together with the fact that the heme group was covalently bound to the enzyme, indicated that the amine dehydrogenase from P. putida was a hemoprotein which contained heme c. The heme content was calculated at 2.01 mol/mol of enzyme. The enzyme was composed of two nonidentical subunits, but heme was present solely in the heavier unit. Carbon monoxide did not inhibit enzymatic activity, nor would it combine with the reduced or oxidized form of the enzyme. Amine dehydrogenase activity was inhibited by carbonyl agents with semicarbazide and cuprizone acting noncompetitively, whereas KCN and isoniazid inhibited by competitive and uncompetitive mechanisms, respectively. Spectral observations suggested that inhibition by these reagents was not due to an interaction with the heme moiety.

After growth of Mycobacterium vaccae strain JOB5 on acetate or propane, the cellular fatty acids were isolated and identified by a combination of gas-chromatographic, mass-spectral, and chemical means. The fatty acids ranged from C12 to C19 and were a mixture of saturated, monounsaturated, and methyl-branched components. The double bond was in the delta9 position in the C15 to C18 unsaturated acids. The single methyl branch was located on the C10 position of Br-C17, Br-C18, and Br-C19 fatty acids. Branched-chain synthesis occurs at the expense of an unsaturated precursor fatty acid; the double bond serves as the site of methylation. Results suggest that S-adenosylmethionine is the methyl donor involved.

The fatty acid pattern in hydrocarbon-utilizing filamentous fungi was determined after growth on acetate, propionate, n-alkanes (C(13) to C(15)), and alk-1-enes (C(14) to C(18)). The fatty acid profile of Cunninghamella elegans and Penicillium zonatum after growth on acetate shows a predominance of even-carbon fatty acids (C(16), C(18:1), C(18:2)), whereas cells grown on propionate showed significantly higher levels of odd-carbon fatty acids (C(15), C(17), C(17:1)). Growth on n-alkanes resulted in the incorporation of fatty acids homologous to the growth substrate. Cunninghamella elegans grown on the alk-1-enes from C(14) to C(18) incorporated the unsaturated substrate into cellular fatty acid after oxidation at the saturated end of the molecule. Regardless of substrate these fungi contain, predominantly, fatty acids 18 carbons in length.

Hydrocarbon-utilizing microorganisms in our culture collection oxidized propylene but could not utilize it as the sole source of carbon and energy. When propane-grown cells of Mycobacterium convulutum were placed on propylene, acrylate, the terminally oxidized, three-carbon unsaturated acid, accumulated. A mixed culture and an axenic culture (strain PL-1) that utilized propylene as the sole source of carbon and energy were isolated from soil. Respiration rates, enzyme assays, fatty acid profiles, and 14CO2 incorporation experiments suggest that both the mixed culture and strain PL-1 oxidize propylene via attack at the double bond, resulting in a C2+C1 cleavage of the molecule.

The metabolism of propane and propionate by a soil isolate (Brevibacterium sp. strain JOB5) was investigated. The presence of isocitrate lyase in cells grown on isopropanol, acetate, or propane and the absence of this inducible enzyme in n-propanol- and propionate-grown cells suggested that propane is not metabolized via C-terminal oxidation. Methylmalonyl coenzyme A mutase and malate synthase are constitutive in this organism. The incorporation of (14)CO(2) into pyruvate accumulated during propionate utilization suggests that propionate is metabolized via the methyl-malonyl-succinate pathway. These results were further substantiated by radiorespirometric studies with propionate-1-(14)C, -2-(14)C, and -3-(14)C as substrate. Propane -2-(14)C was shown, by unlabeled competitor experiments, to be oxidized to acetone; acetone and isopropanol are oxidized in this organism to acetol. Cleavage of acetol to acetate and CO(2) would yield the inducer for the isocitrate lyase present in propane-grown cells.

Studies were conducted on the oxidation and assimilation of various three-carbon compounds by a gram-positive rod isolated from soil and designated strain R-22. This organism can utilize propane, propionate, or n-propylamine as sole source of carbon and energy. Respiration rates, enzyme assays, and (14)CO(2) incorporation experiments suggest that propane is metabolized via methyl ketone formation; propionate and n-propylamine are metabolized via the methylmalonyl-succinate pathway. Isocitrate lyase activity was found in cells grown on acetate and was not present in cells grown on propionate or n-propylamine. (14)CO(2) was incorporated into pyruvate when propionate and n-propylamine were oxidized in the presence of NaAsO(2), but insignificant radioactivity was found in pyruvate produced during the oxidation of propane and acetone. The n-propylamine dissimilatory mechanism was inducible in strain R-22, and amine dehydrogenase activity was detected in cells grown on n-propylamine. Radiorespirometer and (14)CO(2) incorporation studies with several propane-utilizing organisms indicate that the methylmalonyl-succinate pathway is the predominant one for the metabolism of propionate.

Campbell, J. N. (American Meat Institute Foundation, Chicago, Ill.), James B. Evans Jerome J. Perry, and C. F. Niven, Jr. An extracellular material elaborated by Micrococcus sodonensis. J. Bacteriol. 82:828-837. 1961.-When actively growing in a yeast extract-tryptone broth, Micrococcus sodonensis produced rather large quantities of an extracellular material. On a dry weight basis, this material consisted of approximately 80% deoxyribonucleic acid (DNA), 6% ribonucleic acid, 13% protein, and a small quantity of a cell pigment which contained an associated iron porphyrinlike compound. The extracellular material was not produced in other complex or synthetic media. Removal of cells from the appropriate growth medium and suspension in distilled water resulted in the prevention of formation of the material if it had not already begun, or its immediate cessation in those cells which had been actively producing the material. Cells producing the extracellular material exhibited an increasingly higher concentration of intracellular DNA as incubation proceeded, whereas cells growing under conditions not supporting such production showed a decline in intracellular DNA as the culture aged. A similar increase of intracellular DNA, but not release of extracellular material, could be effected by addition of yeast extract or high levels of purine compounds to a medium which did not support the production of extracellular material. The base composition and ratios of both extracellular and intracellular DNA produced under all conditions tested were compared and found to be similar or identical, and to correspond to the classical Watson-Crick structure for DNA.

Perry, Jerome J. (North Carolina State University, Raleigh), and James B. Evans. Oxidation and assimilation of carbohydrates by Micrococcus sodonensis. J. Bacteriol. 91:33-38. 1966.-Micrococcus sodonensis is a biotin-requiring strict aerobe that cannot utilize carbohydrates as sole sources of carbon and energy. However, addition of mannose, glucose, sucrose, or maltose to a medium on which the organism can grow resulted in an increase in total growth. M. sodonensis oxidized these sugars without induction, thus indicating the presence of constitutive enzymes for their transport, activation, and metabolism. Under appropriate nonproliferating cell conditions, glucose was readily incorporated into essential constituents of the cell. When glucose-1-C(14) and glucose-6-C(14) were oxidized by nonproliferating cells, the label was found in both the protein and nucleic acid fractions of the cell. The respiratory quotients of cells oxidizing glucose in saline and in phosphate buffer indicated assimilation of sugar carbon in buffer and virtually no assimilation in saline. The ability of M. sodonensis to completely oxidize glucose and to grow on intermediates of glucose oxidation but not on glucose suggests that glucose may suppress or repress some reaction(s) necessary for growth, and that growth substrates either derepress or circumvent this block.

Staphylococcus epidermidis has been isolated in significant numbers from growing tobacco leaves. The organism is also present on cured and aged tobacco.

Sugars can serve as the germinant for basidiospores of the wood-rotting fungus Lenzites saepiaria. Hexoses sterilized by autoclaving were better germinants than hexoses that were sterilized by filtration. The degradation products in heated hexose which were responsible for the stimulation of germination were levulinic and formic acid. Another product of hexose degradation, hydroxymethyl furfural, had a marked effect on outgrowth of L. saepiaria basidiospores and on the development of mycelia. Basidiospores that germinated in the presence of hydroxymethyl furfural yielded large rounded bodies that, in some cases, developed as a chain of yeastlike cells. Addition of hydroxymethyl furfural to developing mycelia resulted in the production of chains of round yeastlike structures. Similar results were obtained by treating basidiospores or mycelia with phenethyl alcohol.

Washed vegetative cells of Bacillus mycoides obtained and treated under specified conditions have been found to sporulate when shaken in distilled water under specified conditions. Within limitations of the methods, a heat-resistant cell (spore) is produced for each heat-sensitive vegetative cell present initially. Several different experiments designed to detect massive lysis and cell growth during sporulation in distilled water yielded uniformly negative results. Evidence is furnished for the conclusion that a freshly formed spore (heat-resistant cell) weighs considerably less than its progenitor vegetative cell. The observed results are most satisfactorily explained as a direct conversion of a vegetative cell to a spore.

The fatty acid pattern in hydrocarbon- and ketone-utilizing bacteria after growth on various substrates was examined. The fatty acid composition of one hydrocarbon-utilizing organism (Mycobacterium sp. strain OFS) was investigated in detail after growth on n-alkanes, 1-alkenes, ketones, and n-alcohols. n-Alkanes shorter than C(13) or longer than C(17) were not incorporated into cellular fatty acids without some degradation. Strain OFS incorporated C(14) to C(17) 1-alkenes into cellular fatty acids as the omega-monoenoic fatty acid. Methyl ketones were incorporated into strain OFS after removal of one- or two-carbon fragments from the carbonyl end of the molecule. An organism isolated by enrichment on methyl ketones was incapable of n-alkane utilization but could grow on, although not incorporate, ketones or long chain n-alcohols into cellular fatty acids.