Cultivation methods have contributed to our present knowledge about the presence and diversity of microbes in naturally occurring communities. However, it is well established that only a small fraction of prokaryotes have been cultivated by standard methods and, therefore, the prokaryotes that are cultivated may not ref lect the composition and diversity within those communities. Of the two prokaryotic phylogenetic domains, Bacteria and Archaea, members of the former have been shown to be ubiquitous in nature, with ample evidence of vast assemblages of uncultured organisms. There is also now increasingly compelling evidence that the Archaea, which were once thought to occupy a limited number of environments, are also globally widespread. Here we report the use of molecular phylogenetic techniques, which are independent of microbial cultivation, to conduct an assessment of Archaea in a soil microbial community. Small subunit ribosomal RNA genes of Archaea were amplified from soil and cloned. Phylogenetic and nucleotide signature analyses of these cloned small subunit ribosomal RNA gene sequences revealed a cluster of Archaea from a soil microbial community that diverge deeply from the crenarchaeotal line of descent and has the closest affiliation to the lineage of planktonic Archaea. The identification and phylogenetic classification of this archaeal lineage from soil contributes to our understanding of the ecological significance of Archaea as a component of microbial communities in non-extreme environments.

Biological sensing of small molecules such as NO, O 2 , and CO is an important area of research; however, little is know about how CO is sensed biologically. The photosynthetic bacterium Rhodospirillum rubrum responds to CO by activating transcription of two operons that encode a CO-oxidizing system. A protein, CooA, has been identified as necessary for this response. CooA is a member of a family of transcriptional regulators similar to the cAMP receptor protein and fumavate nitrate reduction from Escherichia coli. In this study we report the purification of wild-type CooA from its native organism, R. rubrum, to greater than 95% purity. The purified protein is active in sequence-specific DNA binding in the presence of CO, but not in the absence of CO. Gel filtration experiments reveal the protein to be a dimer in the absence of CO. Purified CooA contains 1.6 mol heme per mol of dimer. Upon interacting with CO, the electronic spectrum of CooA is perturbed, indicating the direct binding of CO to the heme of CooA. A hypothesis for the mechanism of the protein's response to CO is proposed.The sensing of small molecules such as NO, CO, and O 2 is a highly active area of research in biology. Although NO-sensing (soluble guanylyl cyclase) (1, 2) and O 2 -sensing (FixL) (3, 4) proteins have been studied, little data on CO-sensing proteins exists. The CO binding and CO inhibition of metalloproteins such as cytochromes has been extensively researched, but little is known of regulatory factors that have a clear biological role in the recognition of CO. In this paper, we report the isolation and characterization of a bacterial protein that binds CO and elicits a physiologically important response upon activation with CO.

The CO-sensing mechanism of the transcription factor CooA from Rhodospirillum rubrum was studied through a systematic mutational analysis of potential heme ligands. Previous electron paramagnetic resonance (EPR) spectroscopic studies on wild-type CooA suggested that oxidized (FeIII) CooA contains a low-spin heme with a thiolate ligand, presumably a cysteine, bound to its heme iron. In the present report, electronic absorption and EPR analysis of various substitutions at Cys residues establish that Cys75 is a heme ligand in FeIII CooA. However, characterization of heme stability and electronic properties of purified C75S CooA suggest that Cys75 is not a ligand in FeII CooA. Mutational analysis of all CooA His residues showed that His77 is critical for CO-stimulated transcription. On the basis of findings that H77Y CooA is perturbed in its FeII electronic properties and is unable to bind DNA in a site-specific manner in response to CO, His77 appears to be an axial ligand to FeII CooA. These results imply a ligand switch from Cys75 to His77 upon reduction of CooA. In addition, an interaction has been identified between Cys75 and His77 in FeIII CooA that may be involved in the CO-sensing mechanism. Finally, His77 is necessary for the proper conformational change of CooA upon CO binding.

Under dark, anaerobic conditions in the presence of sufficient nickel, Rhodospirillum rubrum grows with a doubling time of under 5 h by coupling the oxidation of CO to the reduction of H ؉ to H 2 . CO-dependent growth of R. rubrum UR294, bearing a kanamycin resistance cassette in cooC, depends on a medium nickel level ninefold higher than that required for optimal growth of coo ؉ strains.Numerous microorganisms oxidize CO to CO 2 : in aerobes the oxidation is catalyzed by an inducible molybdenum-containing oxidase and is coupled to the reduction of oxygen; in strictly anaerobic bacteria and archaea, CO oxidation occurs on a constitutively expressed nickel-containing carbon monoxide dehydrogenase (CODH) and is linked to a variety of reductions. These CODH enzymes are the key components of metabolic processes that interconvert single carbon units and acetyl coenzyme A, leading ultimately to the generation of acetate or methane or the reduction of sulfate. Aerobic and anaerobic CO oxidation and the fundamental role of CODH in anaerobic pathways of carbon metabolism have been reviewed previously (5,13,20,23,24,29,31,33,34).The oxidation of CO to CO 2 (E 0 Ј ϭ Ϫ0.52 V) coupled to the reduction of protons to H 2 (E 0 Ј ϭ Ϫ0.41 V) under anaerobic conditions has been shown to support the growth of a few organisms and may be a component in the energetics of others. CO-tolerant photosynthetic growth of a bacterium was reported by Hirsch in 1968 (15), and dark CO-dependent growth and H 2 production by Rhodocyclus gelatinosus (formerly Rhodopseudomonas gelatinosa) was established by Uffen and coworkers (4, 6, 30-32). Dashekvicz and Uffen also suggested that Rhodospirillum rubrum was capable of slow growth under similar conditions (6, 31). More recently Svetlichny et al. demonstrated the CO-oxidizing and H 2 -generating metabolism and rapid growth of a nonphotosynthetic thermophilic anaerobe, Carboxydothermus hydrogenoformans (27). Methanosarcina barkeri cultures (25) as well as cell suspensions of methanogenic (3, 28), acetogenic (8), and sulfate-reducing (21) organisms also catalyze this reaction with some evidence for coupling to ATP generation (2, 8) and formation of a transmembrane proton gradient (3,8). Hence, CO-dependent H 2 production may be essential to a variety of anaerobic energy generation mechanisms (2,8,13,25,28).Efforts in our laboratories have elaborated the biochemistry and molecular biology of the CO-oxidizing and H 2 -producing system of R. rubrum. Under anaerobic conditions, regardless of the presence of light or other carbon sources, CO induces the synthesis of several proteins, including CODH, an associated Fe-S protein, and a CO-tolerant hydrogenase. The 67-kDa Ni-CODH and the 21-kDa Fe-S protein have been purified and characterized biochemically. In vivo as well as in vitro, electrons derived from CO oxidation, evidently at a Ni-Fe center of CODH (10), are conveyed via the Fe-S protein (and probably other intermediates) to the hydrogenase (11). The genes for these enzymes and additional components h...

(38,61). The enzymes generally have high affinity for CO and couple its oxidation in vitro with the reduction of acceptors such as methylene blue; most do not reduce low-potential acceptors. The purified enzymes contain a molybdenum cofactor plus Fe and perhaps Se, Zn, and Cu (38). The structural genes from Pseudomonas thermocarboxydovorans have been cloned (5).In anaerobic organisms, including acetogens, dissimilatory sulfate reducers, and methanogens, the CODHs are constitutively synthesized metalloproteins, usually 02 labile, that catalyze the interconversion of single-carbon moieties with acetyl coenzyme A (acetyl-CoA); the readily assayed oxidation of CO to CO2 (coupled with the reduction of low-potential electron acceptors) is but one catalytic function. The interconversion is a fundamental anaerobic metabolic process, and the physiology and biochemistry of these bacteria have been the subjects of several reviews (29,33,43,54,62