Mobile Anaerobe Laboratory

A laboratory cart equipped for obtaining clinical samples at bedside or operatory and for inoculating prereduced media for cultivation of medically important anaerobes is described.     

Mobile Anaerobe Laboratory

A laboratory cart equipped for obtaining clinical samples at bedside or operatory and for inoculating prereduced media for cultivation of medically important anaerobes is described.     

Synthetic Medium for Cellulolytic Anaerobe,Ruminococcus albus

Some physico-chemical and heat-induced gelling properties of myosin heavy chains (MHCs) from rabbit skeletal muscle were studied. MHCs were found to be almost devoid of ATPase activity, possibly because of the absence of myosin light chains. MHCs formed precipitates below and ionic strength of 0.3, and above this ionic strength MHCs became soluble. On heating to 65°C at low concentrations of the protein, MHCs lost their solubility and formed aggregates even at high ionic conditions. Partially irreversible change in the conformation of MHC (from helical to random coil) also occurred in heated MHCs.

The heat-induced gel strength of MHCs was found to be almost equal to that of intact myosin molecules with identical protein concentrations. This suggests that the gelling potential of myosin is solely confined in MHC. Although myosin light chains do not seem to contribute to the gelling power of myosin, possibly they provide some stability to the gel if the pH is varied above the optimum value, i.e., 6.0. The addition of actin to a MHC system weakened the gel strength of the latter proportionally to the quantity of added actin.     

Catabolism of Phloroglucinol by the Rumen Anaerobe Coprococcus

A rumen isolate, Coprococcus, sp. Pe₁5, was found to carry phloroglucinol reductase, which catalyzed the initial step in the breakdown of phloroglucinol. The organism uses phloroglucinol as the sole source of carbon and energy when grown in the absence of oxygen. Induced levels of enzyme were detected in cells grown either on phloroglucinol or on other carbon sources in the presence of limiting quantities of phloroglucinol. Although the organism is a strict anaerobe, the enzyme from anaerobically grown cells was insensitive to air. The partially purified enzyme required reduced nicotinamide adenine dinucleotide phosphate as an electron donor and was specific for phloroglucinol. However, partial enzyme activity (14 to 17%) was also detected in the presence of 2-methyl-1,4-naphthoquinone but not in the presence of several other phenolic compounds. The enzyme exhibited a higher affinity for phloroglucinol than for reduced nicotinamide adenine dinucleotide phosphate, with K_m values of 3.0 × 10⁻⁵ M and 29.0 × 10⁻⁵ M, respectively. The optimum pH for maximal enzyme activity was 7.4, and the molecular weight of the native protein was about 130,000, as determined by the Sephadex gel filtration technique.     

L-Asparaginase Production by the Rumen Anaerobe Vibrio succinogenes

The rumen anaerobe Vibrio succinogenes possesses a constitutive L-asparaginase. The amount of enzyme produced is affected by the compound supplied to the organism to generate the fumaric acid it requires as a terminal electron acceptor. When nitrate is provided as the terminal electron acceptor, the amount of enzyme produced is affected by the compound provided to satisfy the nutritional requirement of the organism for succinic acid. Specific activities of up to 8.4 IU/mg of protein in cell-free extracts have been obtained. This specific activity is higher than has been previously reported for any organism. The enzyme has an apparent K(m) of 1.7 x 10(-5) M and low activity towards L-glutamine when assayed at pH 8.5.     

Nutritional Features of the Intestinal Anaerobe Ruminococcus bromii

Of six strains of Ruminococcus bromii studied, five grew in a minimal chemically defined medium containing minerals, NH(4) (+) as nitrogen source, sulfide or sulfate as sulfur source, fructose as energy and carbon source, isobutyrate or 2-methylbutyrate and carbonic acid-bicarbonate as additional carbon sources, and the vitamins biotin, riboflavin, pyridoxine, vitamin B(12) (replaced by L-methionine), pantethine, and tetrahydrofolate. The strains also could utilize cysteine or thiosulfate but not methionine; and strain Z3 failed to use dithiothreitol, thioglycolate, sulfite, or beta-mercaptoethanol as sole sources of sulfur. Mixtures of amino acids, peptides (Casitone), urea, nitrate, asparagine, or glutamine failed to replace NH(4) (+) as N source. Three strains isolated from Americans were identical in nutritional features, whereas one from a Japanese and one from a South African native differed slightly in having requirements for fewer vitamins. One strain from the cecum of a sow grew well in a rumen fluid-supplemented medium but not in the various chemically defined media plus Casitone. The nutritional features suggest that the environment which selects R. bromii contains relatively little amino acid nitrogen and a relatively large amount of NH(4) (+)-N and indicate that these bacteria must depend upon other bacteria such as those that produce NH(4) (+) from urea or protein and those that produce branched-chain volatile acids to grow.     

Metabolism of Dichloromethane by the Strict Anaerobe Dehalobacterium formicoaceticum

The metabolism of dichloromethane by Dehalobacterium formicoaceticum in cell suspensions and crude cell extracts was investigated. The organism is a strictly anaerobic gram-positive bacterium that utilizes exclusively dichloromethane as a growth substrate and ferments this compound to formate and acetate in a molar ratio of 2:1. When [¹³C]dichloromethane was degraded by cell suspensions, formate, the methyl group of acetate, and minor amounts of methanol were labeled, but there was no nuclear magnetic resonance signal corresponding to the carboxyl group of acetate. This finding and previously established carbon and electron balances suggested that dichloromethane was converted to methylene tetrahydrofolate, of which two-thirds was oxidized to formate while one-third gave rise to acetate by incorporation of CO₂ from the medium in the acetyl coenzyme A synthase reaction. When crude desalted extracts were incubated in the presence of dichloromethane, tetrahydrofolate, ATP, methyl viologen, and molecular hydrogen, dichloromethane and tetrahydrofolate were consumed, with the concomitant formation of stoichiometric amounts of methylene tetrahydrofolate. The in vitro transfer of the methylene group of dichloromethane onto tetrahydrofolate required substoichiometric amounts of ATP. The reaction was inhibited in a light-reversible fashion by 20 μM propyl iodide, thus suggesting involvement of a Co(I) corrinoid in the anoxic dehalogenation of dichloromethane. D. formicoaceticum exhibited normal growth with 0.8 mM sodium in the medium, and crude extracts contained ATPase activity that was partially inhibited by N,N′-dicyclohexylcarbodiimide and azide. During growth with dichloromethane, the organism thus may conserve energy not only by substrate-level phosphorylation but also by a chemiosmotic mechanism involving a sodium-independent F₀F₁-type ATP synthase.     

Electron Transport System of the Protoheme-requiring Anaerobe Bacteroides melaninogenicus

Protoheme is essential for the growth of some strains of Bacteroides melaninogenicus. At low concentrations in the growth medium, protoheme determines the doubling time, total cell yield, and amount of cytochrome per bacterium. At high protoheme concentrations, the doubling time, total cell yield, and amount of enzymatically reducible cytochrome appear to remain nearly constant, and protoheme is accumulated by the cell. The accumulated protoheme can support the growth of the bacterium for at least eight generations in a protoheme-free medium. When growth and cytochrome content are proportional during growth at low protoheme concentrations, the bacteria incorporate 10 to 20% of the total available protoheme into a membrane-bound respiratory system. This respiratory system includes cytochrome c, a carbon monoxide-binding pigment, and possibly flavoproteins. The pigments can be reversibly reduced by reduced nicotinamide adenine dinucleotide or endogenous metabolism and can be oxidized anaerobically by fumarate or by shaking in air. Electron transport is inhibited by 2-n-nonyl-4-hydroxy-quinoline-N-oxide.     

Respiratory Physiology of a Facultative Anaerobe, Romanomermis culicivorax

The respiratory physiology of postparasitic larval and adult Romanornermis culicivorax was studied by manometric and polarographic methods. Endogenous respiration rates were relatively low and unaffected by postemergent development. The respiratory quotient (RQ) of larvae and young adults was 0.4 but increased to 0.7 about 3 wk after emergence. Exogenous glucose (0.02 mM) had no effect on QO₂ or RQ. Respiration of adults and larvae was completely inhibited by KCN (l mM) but not by NaN₃ (l mM) or 2,4-DNP (0.1 mM). The nematodes survived exposure to cyanide (l mM) for 18 h. Carbon dioxide (10%) inhibited respiration. The postparasitic stages of the nematode were mixed respiratory regulators-conformers. Exposure to anaerohic conditions resulted in an increased postanaerobic oxygen consumption which persisted for 3.5 h. The experiments confirmed that the postemergent stages of the nematode are facultative anaerobes.     

Chemically Defined Medium for Growth of Clostridium thermocellum, a Cellulolytic Thermophilic Anaerobe

Nutritional requirements of Clostridium thermocellum were examined, and a defined medium was formulated which supported reproducible growth through 10 serial subcultures.     

Leucine Dehydrogenase of a Thermophilic Anaerobe,Clostridium thermoaceticum: Gene Cloning,Purification and Characterization

The leucine dehydrogenase (l-leucine: NAD⁺ oxidoreductase, deaminating, EC 1.4.1.9) gene of Clostridium thermoaceticum was cloned and expressed in Escherichia coli C600 with a vector plasmid, pICD242, which was constructed from pBR322 and the leucine dehydrogenase gene derived from C. thermoaceticum. The enzyme overproduced in the clone was purified about 12 fold to homogeneity by heat treatment and another two steps with a yield of 46%. The enzyme of E. coli- pICD242 was immunochemically identical with that of C. thermoaceticum. The enzyme has a molecular weight of about 350,000 and consists of six subunits identical in molecular weight (56,000). The enzyme is not inactivated by heat treatment: at pH 7.2 and 75°C for 15 min; at 55°C and various pH’s between 6.0 and 10.0 for 10 min. The enzyme catalyzes the oxidative deamination of branched-chain l-amino acids and the reductive amination of their 2-oxo analogues in the presence of NAD⁺ and NADH, respectively. The pro-S hydrogen at C-4 of the dihydronicotin- amide ring of NADH is exclusively transferred to the substrate; the enzyme is B stereospecific. The enzymological properties are very similar to those of the Bacillus stearothermophilus enzyme [T. Ohshima, S. Nagata and K. Soda, Arch. Microbiol., 141, 407 (1985)].     

Ultrastructure of Putrefactive Anaerobe 3679h During Sporulation

Sporulation of putrefactive anaerobe 3679h was studied by observation of ultra-thin sections in the electron microscope. The customary stages were observed during forespore formation, spore maturation, and liberation of the free spore. The exosporium was partially laminated and devoid of the hairlike surface projections observed on spores of some species. Atypical membrane configurations occurred in some cells, and, occasionally, cells with bipolar forespores were found.     

Nitrite-induced Germination of Putrefactive Anaerobe 3679h Spores

Sodium nitrite alone has been shown to stimulate germination of PA 3679h spores. The process was accelerated by using increased concentrations of sodium nitrite, a low pH, and a high temperature of incubation. At low concentrations of nitrite (0.01 to 0.2%), the delay of 36 to 48 hr occurred before germination commenced at 37 C. However, with 3.45% nitrite at 45 C and pH 6.0, most of the spores germinated within 1 hr. At pH 7.0, the germination rate decreased markedly, and at pH 8.0 it was nil. The greatest acceleration in germination rate occurred near 60 C. Hydroxylamine was completely inhibitory to nitrite-induced germination. Sodium nitrite, in turn, inhibited germination by l-alanine, the degree of inhibition being influenced by nitrite concentration and pH.     

Cellulase and Sugar Formation by Bacteroides cellulosolvens, a Newly Isolated Cellulolytic Anaerobe

A newly isolated mesophilic anaerobe, Bacteroides cellulosolvens, has the ability to produce cellulase and to degrade cellulose to cellobiose and glucose. It does not utilize glucose, and it lacks β-glucosidase activity. This anaerobe appears to degrade cellulose to cellobiose by cellulase action, and the presence of cells appears necessary for the formation of glucose.