Two monofunctional NiFeS carbon monoxide (CO) dehydrogenases, designated CODH I and CODH II, were purified to homogeneity from the anaerobic CO-utilizing eubacterium Carboxydothermus hydrogenoformans. Both enzymes differ in their subunit molecular masses, N-terminal sequences, peptide maps, and immunological reactivities. Immunogold labeling of ultrathin sections revealed both CODHs in association with the inner aspect of the cytoplasmic membrane. Both enzymes catalyze the reaction CO Electron paramagnetic resonance (EPR) spectroscopy revealed signals indicative of [4Fe-4S] clusters. Ni was EPR silent under any conditions tested. It is proposed that CODH I is involved in energy generation and that CODH II serves in anabolic functions.Bacteria which utilize CO as a growth substrate include the aerobic carboxidotrophs and the anaerobic acetogens, sulfatereducers, methanogens, and phototrophs (16,34,42,44,47). Carboxydothermus hydrogenoformans is a strictly anaerobic, thermophilic, gram-positive eubacterium which was isolated from a volcanic hot spring (55). Phylogenetically, C. hydrogenoformans falls into the group of the low-GϩC subphylum of the gram-positive bacteria and shows highest 16S rRNA gene sequence homology to Thermoterrabacterium (52). C. hydrogenoformans utilizes CO under chemolithoautotrophic conditions (55). The bacterium couples the oxidation of CO to CO 2 (E 0 Ј ϭ Ϫ0.52 V) to the reduction of protons to H 2 (E 0 Ј ϭ Ϫ0.41 V) in the energy-conserving reaction CO ϩ H 2 O 3 CO 2 ϩ H 2 , ⌬G 0 Ј ϭ Ϫ20 kJ mol Ϫ1 (54, 55). The metabolism of C. hydrogenoformans is strictly fermentative because the bacterium is obligately anaerobic and utilizes protons as the characteristic intracellular electron acceptor. On the basis of the formation of H 2 as the ultimate fermentation product by C. hydrogenoformans, we propose the terms "hydrogenogenic," "hydrogenogens," and "hydrogenogenesis" to refer to the type of metabolism, the physiological group, and the process of H 2 formation, respectively. Although C. hydrogenoformans is not a phototroph, it is in many ways similar to the phototrophic bacteria Rhodospirillum rubrum and Rhodocyclus gelatinosus, which utilize CO anaerobically in the dark (34,58,59). C.hydrogenoformans is also able to ferment pyruvate to acetate and H 2 (56).Although carbon monoxide dehydrogenases (CODHs) formally catalyze the same reaction (CO ϩ H 2 O 3 CO 2 ϩ 2 e Ϫ ϩ 2 H ϩ ), different types of enzymes serving different metabolic functions operate in the various groups of CO-oxidizing bacteria (15,16,42,43,44,47 (12,26,43) or Hydrogenophaga pseudoflava (27,43) show a dimer of two heterotrimers in an (LMS) 2 subunit structure. Each heterotrimer is composed of a molybdoprotein (L subunit), a flavoprotein (M subunit), and an iron-sulfur protein (S subunit). The molybdoprotein carries the active site, which contains a 1:1 molar complex of molybdopterin cytosine dinucleotide and a molybdenum atom. The iron-sulfur protein contains the type I and type II [2Fe-2S] centers. The flavoprotein contains...

To evaluate the microbial populations involved in the reduction of Fe(III) in an acidic, iron-rich sediment, the anaerobic flow of supplemental carbon and reductant was evaluated in sediment microcosms at the in situ temperature of 12°C. Supplemental glucose and cellobiose stimulated the formation of Fe(II); 42 and 21% of the reducing equivalents that were theoretically obtained from glucose and cellobiose, respectively, were recovered in Fe(II). Likewise, supplemental H2 was consumed by acidic sediments and yielded additional amounts of Fe(II) in a ratio of approximately 1:2. In contrast, supplemental lactate did not stimulate the formation of Fe(II). Supplemental acetate was not consumed and inhibited the formation of Fe(II). Most-probable-number estimates demonstrated that glucose-utilizing acidophilic Fe(III)-reducing bacteria approximated to 1% of the total direct counts of 4′,6-diamidino-2-phenylindole-stained bacteria. From the highest growth-positive dilution of the most-probable-number series at pH 2.3 supplemented with glucose, an isolate, JF-5, that could dissimilate Fe(III) was obtained. JF-5 was an acidophilic, gram-negative, facultative anaerobe that completely oxidized the following substrates via the dissimilation of Fe(III): glucose, fructose, xylose, ethanol, glycerol, malate, glutamate, fumarate, citrate, succinate, and H2. Growth and the reduction of Fe(III) did not occur in the presence of acetate. Cells of JF-5 grown under Fe(III)-reducing conditions formed blebs, i.e., protrusions that were still in contact with the cytoplasmic membrane. Analysis of the 16S rRNA gene sequence of JF-5 demonstrated that it was closely related to an Australian isolate of Acidiphilium cryptum (99.6% sequence similarity), an organism not previously shown to couple the complete oxidation of sugars to the reduction of Fe(III). These collective results indicate that the in situ reduction of Fe(III) in acidic sediments can be mediated by heterotrophicAcidiphilium species that are capable of coupling the reduction of Fe(III) to the complete oxidation of a large variety of substrates including glucose and H2.

Earthworms emit nitrous oxide (N2O) via the activity of bacteria in their gut. Four N2O-producing facultative aerobes, ED1T, ED5T, MH21T and MH72, were isolated from the gut of the earthworm Aporrectodea caliginosa. The isolates produced N2O under conditions that simulated the microenvironment of the earthworm gut. ED1T and ED5T were Gram-negative, motile rods that carried out complete denitrification (i.e. the reduction of nitrate to N2) and contained membranous c-type cytochromes. ED1T grew optimally at 30 °C and pH 7. ED1T oxidized organic acids and reduced (per)chlorate, sulfate, nitrate and nitrite. The closest phylogenetic relative of ED1T was Dechloromonas agitata. ED5T grew optimally at 25 °C and pH 7. ED5T grew mainly on sugars, and nitrate and nitrite were used as alternative electron acceptors. The closest phylogenetic relatives of ED5T were Flavobacterium johnsoniae and Flavobacterium flevense. MH21T and MH72 were motile, spore-forming, rod-shaped bacteria with a three-layered cell wall. Sugars supported the growth of MH21T and MH72. Cells of MH21T grew in chains, were linked by connecting filaments and contained membranous b-type cytochromes. MH21T grew optimally at 30–35 °C and pH 7·7, grew by fermentation and reduced low amounts of nitrite to N2O. The closest phylogenetic relatives of MH21T were Paenibacillus borealis and Paenibacillus chibensis. Based on morphological, physiological and phylogenetic characteristics, ED1T (=DSM 15892T=ATCC BAA-841T), ED5T (=DSM 15936T=ATCC BAA-842T) and MH21T (=DSM 15890T=ATCC BAA-844T) are proposed as type strains of the novel species Dechloromonas denitrificans sp. nov., Flavobacterium denitrificans sp. nov. and Paenibacillus anaericanus sp. nov., respectively. MH72 is considered a new strain of Paenibacillus terrae.

A strictly anaerobic, H 2 -utilizing bacterium, strain SL1, was isolated from the sediment of an acidic coal mine pond. Cells of strain SL1 were sporulating, motile, long rods with a multilayer cell wall. . These results indicate that (i) sediments of acidic coal mine ponds harbour acetogens and (ii) C. scatologenes is an acetogen that tends to lose its capacity to grow acetogenically under H 2 /CO 2 or CO/CO 2 after prolonged laboratory cultivation.

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