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CooA, the CO-sensing heme protein from Rhodospirillum rubrum, regulates the expression of genes that encode a CO-oxidation system, allowing R. rubrum to use CO as a sole energy source. To better understand the gas-sensing regulation mechanism used by R. rubrum CooA and its homologs in other organisms, we characterized spectroscopically and functionally the Fe(II), Fe(II)-NO, and Fe(II)-CO forms of CooA from Carboxydothermus hydrogenoformans. Surprisingly, and unlike R. rubrum CooA, C. hydrogenoformans CooA binds NO to form a six-coordinate Fe(II)-NO heme that is active for DNA binding in vitro and in vivo. In contrast, R. rubrum CooA, which is exquisitely specific for CO, forms a five-coordinate Fe(II)-NO adduct that is inactive for DNA binding. Based on analyses of protein variants and temperature studies, NO-dependent DNA binding by C. hydrogenoformans CooA is proposed to result from a greater apparent stability of the six-coordinate Fe(II)-NO adduct at room temperature. Results from the present study strengthen the proposal that CO specificity in the CooA activation mechanism is based on the requirement for a small, neutral distal ligand, which in turn affects the relative positioning of the ligand-bound heme.

The P(II) regulatory protein family is unusually widely distributed, being found in all three domains of life. Three P(II) homologs called GlnB, GlnK, and GlnJ have been identified in the photosynthetic bacterium Rhodospirillum rubrum. These have roles in at least four distinct functions, one of which is activation of the nitrogen fixation-specific regulatory protein NifA. The activation of NifA requires only the covalently modified (uridylylated) form of GlnB. GlnK and GlnJ are not involved. However, the basis of specificity for different P(II) homologs in different processes is poorly understood. We examined this specificity by altering GlnJ to support NifA activation. A small number of amino acid substitutions in GlnJ were important for this ability. Two (affecting residues 45 and 54) are in a loop called the T-loop, which contains the site of uridylylation and is believed to be very important for contacts with other proteins, but other critical residues lie in the C terminus (residues 95-97 and 109-112) and near the N terminus (residues 3-5 and 17). Because many of the residues important for P(II)-NifA interaction lie far from the T-loop in the known x-ray crystal structures of P(II) proteins, our results lead to the hypothesis that the T-loop of GlnB is flexible enough to come into proximity with both the C- and N-terminal regions of the protein to bind NifA. Finally, the results show that the level of P(II) accumulation is also an important factor for NifA activation.

Biological sensing of small molecules such as NO, O2, 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.

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 reflect 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.

The primary product of biological nitrogen fixation, ammonia, reversibly regulates nitrogenase activity in a variety of diazotrophs by a process called "NH4(+)-switch-off/on." Strong correlative evidence from work in Azospirillum lipoferum and Rhodospirillum rubrum indicates that this regulation involves both the inactivation of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase and the reactivation by dinitrogenase reductase activating glycohydrolase. The genes encoding these two enzymes, draT and draG, have been cloned from these two organisms, so that direct genetic evidence can be marshaled to test this model in vivo. The draT/G system has been transferred to and monitored in the enteric nitrogen-fixing bacterium Klebsiella pneumoniae, an organism normally devoid of such a regulatory mechanism. The expressed draT and draG genes allowed K. pneumoniae to respond to NH4Cl with a reversible regulation of nitrogenase activity that was correlated with the reversible ADP-ribosylation of dinitrogenase reductase in vivo. Thus, the expression of draT and draG genes in K. pneumoniae is necessary and sufficient to support NH4(+)-switch-off/on, and ADP-ribosylation serves as a reversible regulatory mechanism for controlling nitrogenase activity in prokaryotes.

The nifN and -E gene products are involved in the synthesis of the iron-molybdenum cofactor of dinitrogenase, the enzyme responsible for the reduction of dinitrogen to ammonia. By using the in vitro iron-molybdenum cofactor biosynthesis assay, we have followed the purification of these gene products 450-fold to greater than 95% purity. An overall recovery of 20% was obtained with the purified protein having a specific activity of 6900 units/mg of protein. The protein (hereafter referred to as NIFNE) was found to contain equimolar amounts of the nifN and -E gene products and have a native molecular mass of 200 +/- 10 kDa, which indicates an alpha 2 beta 2 structure. NIFNE was oxygen labile with a half-life of 1 min in air. A UV-visible spectrum of the dye-oxidized protein showed an absorption maximum at 425 nm that could be bleached by reduction of NIFNE with sodium dithionite, suggesting the presence of an Fe center in NIFNE.

Cell-free extracts of nifF and nifJ mutants of Klebsiella pneumoniae are unable to couple acetylene reduction (N2 fixation) by nitrogenase to the oxidation of organic metabolites. However, nifF and nifJ mutants can complement each other in vitro to establish the coupling. This indicates that the products of the nifF and nifJ genes constitute essential elements of the physiological electron pathway to nitrogenase. The electron-transfer-active product of the nifF gene, a flavoprotein, has been purified.