Although nitrate stimulated the capacity of Clostridium thermoautotrophicum and Clostridium thermoaceticum to oxidize (utilize) substrates under heterotrophic conditions, it inhibited autotrophic H 2 -CO 2 -dependent growth. Under basal medium conditions, nitrate was also inhibitory to the use of one-carbon substrates (i.e., CO, formate, methanol, or the O-methyl groups of vanillate or syringate) as sole carbon energy sources. This inhibitory effect of nitrate was bypassed when both O-methyl groups and CO were provided concomitantly; H 2 -CO 2 did not replace CO. These results indicated that nitrate blocked the reduction of CO 2 to the methyl and carbonyl levels. On the basis of the inability of acetogenic cells (i.e., cells cultivated without nitrate) to consume or reduce nitrate in resting-cell assays, the capacity to dissimilate nitrate was not constitutive. Nitrate had no appreciable effect on the specific activities of enzymes central to the acetyl-coenzyme A (CoA) pathway. However, membranes obtained from cells cultivated under nitrate-dissimilating conditions were deficient in the b-type cytochrome that was typical of membranes from acetogenic cells, i.e., cells dependent upon the synthesis of acetate for the conservation of energy. Collectively, these findings indicated that (i) C. thermoautotrophicum and C. thermoaceticum cannot engage the carbon-fixing capacities of the acetyl-CoA pathway in the presence of nitrate and (ii) the nitrate block on the acetyl-CoA pathway occurs via an alteration in electron transport.Most investigations of the CO 2 -fixing acetyl-coenzyme A (CoA) pathway of acetogenic bacteria have focused on the dissimilatory and energy-conserving features of the pathway (13,16,61). However, the anabolic and catabolic functions of the pathway are conceived to be integrated (Fig. 1). The concept that acetogens use the acetyl-CoA pathway for the autotrophic assimilation of carbon is based on a relatively limited number of observations, mainly the absence of other autotrophic CO 2 -assimilating enzymes in Acetobacterium woodii and carbon-labeling patterns obtained with Clostridium thermoaceticum (22, 37). Under heterotrophic conditions (i.e., conditions which do not require obligatory use of the acetyl-CoA pathway for the assimilation of carbon), acetogens may not be strictly dependent on this process, since a number of alternative energy-conserving electron acceptors can be utilized, including fumarate (14, 42), aromatic acrylates (1, 43, 57), inorganic sulfur compounds (2, 3, 27), pyruvate (44), or nitrate (52). Because the use of alternative electron acceptors by acetogens has been examined primarily under heterotrophic conditions, the potential impact of competing reductant sinks on the autotrophic potentials of acetogens is largely unknown. The acetogens C. thermoaceticum (20) and Clostridium thermoautotrophicum (60) are phylogenically very closely related (55) and dissimilate nitrate preferentially to the reduction of CO 2 under heterotrophic conditions (21, 52). The main objective of ...

The phototrophic consortium "Chlorochromatium aggregatum" was enriched from sediment samples of a eutrophic freshwater lake and was maintained at high numbers in anoxic sulfide-reduced medium. Growth of intact consortia was observed only in the light and in the presence of 2-oxoglutarate as an organic carbon source. Consortia of "C. aggregatum" reached maximum growth rates at light intensities >/= 5 &mgr;mol quanta m-2 s-1. Of ten compounds tested, sulfide, thiosulfate, 2-oxoglutarate, and citrate served as a chemoattractant for "C. aggregatum". When incubated in the presence of sulfide and in the light, epibionts reduced the fluorochrome 5-cyano-2, 3-di-4-tolyl-tetrazolium chloride (CTC). Reduction of CTC was not observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) or in the dark, indicating that sulfide serves as an electron donor for the phototrophic epibiont. Motile consortia accumulated scotophobically in microcuvettes at a wavelength of 740 nm. Since this wavelength corresponds to the position of the absorption maximum of bacteriochlorophylls c or d, the photosynthetic pigments are most likely the photoreceptors of the scotophobic response. It is concluded that, within the consortia, a rapid interspecies signal transfer occurs between the nonmotile, green-colored epibiont and the motile, colorless central bacterium.

A dense accumulation of the phototrophic consortium "Pelochromatium roseum" in a small, eutrophic, freshwater lake (Dagowsee, Brandenburg, Germany) was investigated. Within the chemocline, the number of epibionts of the consortia represented up to 19% of the total number of bacteria. Per "P. roseum" a mean value of 20 epibionts was determined. Similar to other aquatic habitats, consortia in the Dagowsee were found only at low light intensities (< 7 &mgr;mol quanta m-2 s-1) and low sulfide concentrations (0-100 &mgr;M). In dialysis cultures of "P. roseum", bacterial cells remained in a stable association only when incubated at light intensities between 5 and 10 &mgr;mol quanta m-2 s-1. Intact consortia from natural samples had a buoyant density of 1046.8 kg m-3, which was much higher than that of ambient chemocline water (995.8 kg m-3). Under environmental conditions and without motility, this density difference would result in rapid sedimentation of consortia toward the lake bottom. Our results indicate that (1) consortia are adapted to a very narrow regime of light intensities and sulfide concentrations, (2) motility and tactic responses must be of ecological significance for the colonization of the free water column of lakes, and (3) phototrophic growth of consortia can be explained only by a cycling of sulfur species in the chemocline, possibly within the consortia themselves.

The phylogenetic affiliation of epibionts occurring in three morphologically distinct types of green-colored phototrophic consortia was investigated. Intact consortia of the types "Chlorochromatium aggregatum", "C. glebulum", and a third previously undescribed type, tentatively named "C. magnum" were mechanically separated from accompanying bacteria by either micromanipulation or by chemotactic accumulation in sulfide-containing capillaries. A 540-base-pair-long fragment of the 16S rRNA gene of the epibionts was amplified employing PCR primers specific for green sulfur bacteria. DNA fragments were separated by denaturing gradient gel electrophoresis and subsequently sequenced. The results of this phylogenetic analysis indicated that the symbiotic epibionts, together with only a few free-living strains, form a cluster within the green sulfur bacterial radiation which is only distantly related to the majority of known representatives of this phylum. Consortia with identical morphology but different origin exhibited significant differences in their partial 16S rRNA gene sequences, which could be confirmed by analysis of the 16S rRNA secondary structure. The phylogenetic affiliation of the chemotrophic central rod-shaped bacterium of "C. aggregatum" and "C. magnum" was analyzed by fluorescent in situ hybridization. According to our results and contrary to earlier assumptions, the central bacterium is a member of the beta-subgroup of the Proteobacteria.

Oxalate and glyoxylate supported growth and acetate synthesis by Moorella thermoacetica in the presence of nitrate under basal (without yeast extract) culture conditions. In oxalate cultures, acetate formation occurred concomitant with growth and nitrate was reduced in the stationary phase. Growth in the presence of [(14)C]bicarbonate or [(14)C]oxalate showed that CO(2) reduction to acetate and biomass or oxalate oxidation to CO(2) was not affected by nitrate. However, cells engaged in oxalate-dependent acetogenesis in the presence of nitrate lacked a membranous b-type cytochrome, which was present in cells grown in the absence of nitrate. In glyoxylate cultures, growth was coupled to nitrate reduction and acetate was formed in the stationary phase after nitrate was totally consumed. In the absence of nitrate, glyoxylate-grown cells incorporated less CO(2) into biomass than oxalate-grown cells. CO(2) conversion to biomass by glyoxylate-grown cells decreased when cells were grown in the presence of nitrate. These results suggest that: (1) oxalate-grown cells prefer CO(2) as an electron sink and bypass the nitrate block on the acetyl-CoA pathway at the level of reductant flow and (2) glyoxylate-grown cells prefer nitrate as an electron sink and bypass the nitrate block of the acetyl-CoA pathway by assimilating carbon via an unknown process that supplements or replaces the acetyl-CoA pathway. In this regard, enzymes of known pathways for the assimilation of two-carbon compounds were not detected in glyoxylate- or oxalate-grown cells.