Denitrification is a distinct means of energy conservation, making use of N oxides as terminal electron acceptors for cellular bioenergetics under anaerobic, microaerophilic, and occasionally aerobic conditions. The process is an essential branch of the global N cycle, reversing dinitrogen fixation, and is associated with chemolithotrophic, phototrophic, diazotrophic, or organotrophic metabolism but generally not with obligately anaerobic life. Discovered more than a century ago and believed to be exclusively a bacterial trait, denitrification has now been found in halophilic and hyperthermophilic archaea and in the mitochondria of fungi, raising evolutionarily intriguing vistas. Important advances in the biochemical characterization of denitrification and the underlying genetics have been achieved with Pseudomonas stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Ralstonia eutropha, and Rhodobacter sphaeroides. Pseudomonads represent one of the largest assemblies of the denitrifying bacteria within a single genus, favoring their use as model organisms. Around 50 genes are required within a single bacterium to encode the core structures of the denitrification apparatus. Much of the denitrification process of gram-negative bacteria has been found confined to the periplasm, whereas the topology and enzymology of the gram-positive bacteria are less well established. The activation and enzymatic transformation of N oxides is based on the redox chemistry of Fe, Cu, and Mo. Biochemical breakthroughs have included the X-ray structures of the two types of respiratory nitrite reductases and the isolation of the novel enzymes nitric oxide reductase and nitrous oxide reductase, as well as their structural characterization by indirect spectroscopic means. This revealed unexpected relationships among denitrification enzymes and respiratory oxygen reductases. Denitrification is intimately related to fundamental cellular processes that include primary and secondary transport, protein translocation, cytochrome c biogenesis, anaerobic gene regulation, metalloprotein assembly, and the biosynthesis of the cofactors molybdopterin and heme D1. An important class of regulators for the anaerobic expression of the denitrification apparatus are transcription factors of the greater FNR family. Nitrate and nitric oxide, in addition to being respiratory substrates, have been identified as signaling molecules for the induction of distinct N oxide-metabolizing enzymes.

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Cell biology and molecular basis of denitrification.

Fluidity Parameters of Lipid Regions Determined by Fluorescence Polarization

PAS domains are newly recognized signaling domains that are widely distributed in proteins from members of the Archaea and Bacteria and from fungi, plants, insects, and vertebrates. They function as input modules in proteins that sense oxygen, redox potential, light, and some other stimuli. Specificity in sensing arises, in part, from different cofactors that may be associated with the PAS fold. Transduction of redox signals may be a common mechanistic theme in many different PAS domains. PAS proteins are always located intracellularly but may monitor the external as well as the internal environment. One way in which prokaryotic PAS proteins sense the environment is by detecting changes in the electron transport system. This serves as an early warning system for any reduction in cellular energy levels. Human PAS proteins include hypoxia-inducible factors and voltage-sensitive ion channels; other PAS proteins are integral components of circadian clocks. Although PAS domains were only recently identified, the signaling functions with which they are associated have long been recognized as fundamental properties of living cells.

PAS Domains: Internal Sensors of Oxygen, Redox Potential, and Light

Hydrophobicity determined by a fluorescence probe method and its correlation with surface properties of proteins

Some bacteria have the remarkable capacity to fix atmospheric nitrogen to ammonia under ambient conditions, a reaction only mimicked on an industrial scale by a chemical process that requires high temperatures, elevated pressure and special catalysts. The ability of microorganisms to use nitrogen gas as the sole nitrogen source and engage in symbioses with host plants confers many ecological advantages, but also incurs physiological penalties because the process is oxygen sensitive and energy dependent. Consequently, biological nitrogen fixation is highly regulated at the transcriptional level by sophisticated regulatory networks that respond to multiple environmental cues.

Genetic regulation of biological nitrogen fixation

A new locus, prrA, involved in the regulation of photosynthesis gene expression in response to oxygen, has been identified in Rhodobacter sphaeroides. Inactivation of prrA results in the absence of photosynthetic spectral complexes. The prrA gene product has strong homology to response regulators associated with signal transduction in other prokaryotes. When prrA is present in multiple copies, cells produce light-harvesting complexes under aerobic growth conditions, suggesting that prrA affects photosynthesis gene expression positively in response to oxygen deprivation. Analysis of the expression of puc::lacZ fusions in wild-type and PrrA- cells revealed a substantial decrease in LacZ expression in the absence of prrA under all conditions of growth, especially when cells were grown anaerobically in the dark in the presence of dimethyl sulfoxide. Northern (RNA) and slot blot hybridizations confirmed the beta-galactoside results for puc and revealed additional positive regulation of puf, puhA, and cycA by PrrA. The effect of truncated PrrA on photosynthesis gene expression in the presence of low oxygen levels can be explained by assuming that PrrA may be effective as a multimer. PrrA was found to act on the downstream regulatory sequences (J. K. Lee and S. Kaplan, J. Bacteriol. 174:1146-1157, 1992) of the puc operon regulatory region. Finally, two spontaneous prrA mutations that abolish prrA function by changing amino acids in the amino-terminal domain of the protein were isolated.

prrA, a putative response regulator involved in oxygen regulation of photosynthesis gene expression in Rhodobacter sphaeroides.

Intracytoplasmic membrane synthesis in synchronous cell populations of Rhodopseudomonas sphaeroides. Fate of "old" and "new" membrane.

Here we show that the extent of electron flow through the cbb3 oxidase of Rhodobacter sphaeroides is inversely related to the expression levels of those photosynthesis genes that are under control of the PrrBA two-component activation system: the greater the electron flow, the stronger the inhibitory signal generated by the cbb3 oxidase to repress photosynthesis gene expression. Using site-directed mutagenesis, we show that intramolecular electron transfer within the cbb3 oxidase is involved in signal generation and transduction and this signal does not directly involve the intervention of molecular oxygen. In addition to the cbb3 oxidase, the redox state of the quinone pool controls the transcription rate of the puc operon via the AppA–PpsR antirepressor–repressor system. Together, these interacting regulatory circuits are depicted in a model that permits us to understand the regulation by oxygen and light of photosynthesis gene expression in R.sphaeroides.

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Redox signaling: globalization of gene expression.

Rhodobacter sphaeroides has the capacity to grow by aerobic and anaerobic respiration and photosynthetically in the light under anaerobic conditions, as well as fermentatively. It can fix atmospheric nitrogen and carbon dioxide. It resembles other gram-negative members of the class Proteobacteria when growing aerobically, but a reduction in oxygen tension induces an intracellular differentiation of the inner membrane, leading to the formation of the intracytoplasmic membrane system (ICM). The ICM houses the integral membrane pigment-protein complexes constituting the photosystem (PS), comprised of the reaction center (RC) and two light-harvesting (LH) complexes. For an early review of ICM biosynthesis, see reference 44. The LH complexes are designated B800-850 (LHII) and B875 (LHI), based on their respective absorption maxima. The ratio of LHI to RC is fixed at approximately 15:1, whereas the ratio of LHII to the LHI-RC unit is variable, changing in a manner inverse to the incident light intensity. These three pigment-protein complexes are the spectral complexes (SC) of the R. sphaeroides PS. Detailed structural information about these complexes in several species of Rhodobacter is emerging (72).

The LH complexes capture light energy and direct that energy to the RC, where conversion of the excitation energy takes place and is intrinsically coupled with a cyclic flow of electrons, ultimately to the periplasmically localized cytochrome c2, which serves to rereduce the RC to allow a new cycle of electron flow (for further details, see references 42, 44, and 72).

Bacteriochlorophyll (Bchl) absorbs most of the light energy within the SCs and is critical to the assembly and final structure of the SCs (44, 84, 87). The carotenoids (Crt) have a minor role in absorbing light energy (15), but they function to protect the complexes against photo-oxidative damage, dissipate excess radiant energy, and help to maintain the structure and relative abundance of each SC (35, 50, 53).

A reduction in oxygen tension is both necessary and sufficient to induce synthesis of the ICM (reviewed in references 42 and 44), which is gratuitously produced under anaerobic dark growth conditions, in the presence of an alternate electron acceptor such as dimethyl sulfoxide (DMSO). Oxygen tension is the major environmental stimulus controlling PS induction, with variations in light intensity determining the cellular level of the ICM and the abundance of the different SCs. PS formation is tightly regulated, with checkpoints at all levels of information flow, from transcriptional through posttranslational. In the following sections, we will describe what we currently know about the regulatory processes controlling the formation and abundance of SCs in R. sphaeroides. We will present a working model for the regulation of PS formation in R. sphaeroides 2.4.1 which is based on the critical role of cellular redox carriers. For clarity, we will, throughout this review, define aerobic growth as that which occurs under highly oxygenic conditions, under which there are no detectable SCs present in wild-type membranes.

Control of Photosystem Formation in Rhodobacter sphaeroides

The puc operon of Rhodobacter sphaeroides comprises the pucBA structural genes which encode B800-850 light-harvesting beta and alpha polypeptides, respectively. Northern (RNA) blot hybridization analysis of puc operon expression has identified two pucBA-specific transcripts. The small (0.5-kilobase [kb]) transcript encodes the beta and alpha polypeptides and, under photoheterotrophic growth conditions, was approximately 200-fold more abundant than the large (2.3-kb) transcript. The 5' end of the 0.5-kb transcript was mapped at 117 nucleotides upstream from the start of pucB. The 3' ends of the 0.5-kb transcript were mapped to two adjacent nucleotides, which follow a stem-loop structure immediately 3' to the pucA stop codon. Two mutant strains, PUC705-BA and PUC-Pv, were constructed by replacement of the pucBA genes and adjacent DNA in the former case or by insertional interruption of the DNA downstream of the pucBA genes in the latter case. The two mutant strains were devoid of B800-850 complexes during photosynthetic growth but were otherwise apparently normal. The B800-850 phenotype of both PUC705-BA and PUC-Pv was not complemented in trans with a 2.5-kb PstI restriction endonuclease fragment extending from 0.75 kb upstream of pucBA to 1.3 kb downstream of pucBA, despite the presence of the 0.5-kb pucBA-specific transcript. Both of the mutant strains, however, showed restoration of B800-850 expression with a 10.5-kb EcoRI restriction endonuclease fragment in trans encompassing the 2.5-kb PstI fragment. Western immunoblot analysis revealed no B800-850-beta polypeptide as well as no polypeptide designated 15A in either mutant. Nonetheless, under photoheterotrophic growth conditions, the 0.5-kb pucBA-specific transcript was present in PUC-Pv, although no 2.3-kb transcript was detectable. We suggest that the DNA region immediately downstream of pucBA encodes a gene product(s) essential for translational or posttranslational expression of the B800-850 beta and alpha polypeptides.

Samuel Kaplan

Papers

prrA, a putative response regulator involved in oxygen regulation of photosynthesis gene expression in Rhodobacter sphaeroides.

Intracytoplasmic membrane synthesis in synchronous cell populations of Rhodopseudomonas sphaeroides. Fate of "old" and "new" membrane.

Redox signaling: globalization of gene expression.

Control of Photosystem Formation in Rhodobacter sphaeroides

Posttranscriptional control of puc operon expression of B800-850 light-harvesting complex formation in Rhodobacter sphaeroides.