Walter G. Zumft

Phylogeny of the bacterial superfamily of Crp-Fnr transcription regulators: exploiting the metabolic spectrum by controlling alternative gene programs

The Crp-Fnr regulators, named after the first two identified members, are DNA-binding proteins which predominantly function as positive transcription factors, though roles of repressors are also important. Among over 1200 proteins with an N-terminally located nucleotide-binding domain similar to the cyclic adenosine monophosphate (cAMP) receptor protein, the distinctive additional trait of the Crp-Fnr superfamily is a C-terminally located helix-turn-helix motif for DNA binding. From a curated database of 369 family members exhibiting both features, we provide a protein tree of Crp-Fnr proteins according to their phylogenetic relationships. This results in the assembly of the regulators ArcR, CooA, CprK, Crp, Dnr, FixK, Flp, Fnr, FnrN, MalR, NnrR, NtcA, PrfA, and YeiL and their homologs in distinct clusters. Lead members and representatives of these groups are described, placing emphasis on the less well-known regulators and target processes. Several more groups consist of sequence-derived proteins of unknown physiological roles; some of them are tight clusters of highly similar members. The Crp-Fnr regulators stand out in responding to a broad spectrum of intracellular and exogenous signals such as cAMP, anoxia, the redox state, oxidative and nitrosative stress, nitric oxide, carbon monoxide, 2-oxoglutarate, or temperature. To accomplish their roles, Crp-Fnr members have intrinsic sensory modules allowing the binding of allosteric effector molecules, or have prosthetic groups for the interaction with the signal. The regulatory adaptability and structural flexibility represented in the Crp-Fnr scaffold has led to the evolution of an important group of physiologically versatile transcription factors.

Respiratory transformation of nitrous oxide (N2O) to dinitrogen by Bacteria and Archaea.

N2O is a potent greenhouse gas and stratospheric reactant that has been steadily on the rise since the beginning of industrialization. It is an obligatory inorganic metabolite of denitrifying bacteria, and some production of N2O is also found in nitrifying and methanotrophic bacteria. We focus this review on the respiratory aspect of N2O transformation catalysed by the multicopper enzyme nitrous oxide reductase (N2OR) that provides the bacterial cell with an electron sink for anaerobic growth. Two types of Cu centres discovered in N2OR were both novel structures among the Cu proteins: the mixed-valent dinuclear Cu(A) species at the electron entry site of the enzyme, and the tetranuclear Cu(Z) centre as the first catalytically active Cu-sulfur complex known. Several accessory proteins function as Cu chaperone and ABC transporter systems for the biogenesis of the catalytic centre. We describe here the paradigm of Z-type N2OR, whose characteristics have been studied in most detail in the genera Pseudomonas and Paracoccus. Sequenced bacterial genomes now provide an invaluable additional source of information. New strains harbouring nos genes and capability of N2O utilization are being uncovered. This reveals previously unknown relationships and allows pattern recognition and predictions. The core nos genes, nosZDFYL, share a common phylogeny. Most principal taxonomic lineages follow the same biochemical and genetic pattern and share the Z-type enzyme. A modified N2OR is found in Wolinella succinogenes, and circumstantial evidence also indicates for certain Archaea another type of N2OR. The current picture supports the view of evolution of N2O respiration prior to the separation of the domains Bacteria and Archaea. Lateral nos gene transfer from an epsilon-proteobacterium as donor is suggested for Magnetospirillum magnetotacticum and Dechloromonas aromatica. In a few cases, nos gene clusters are plasmid borne. Inorganic N2O metabolism is associated with a diversity of physiological traits and biochemically challenging metabolic modes or habitats, including halorespiration, diazotrophy, symbiosis, pathogenicity, psychrophily, thermophily, extreme halophily and the marine habitat down to the greatest depth. Components for N2O respiration cover topologically the periplasm and the inner and outer membranes. The Sec and Tat translocons share the task of exporting Nos components to their functional sites. Electron donation to N2OR follows pathways with modifications depending on the host organism. A short chronology of the field is also presented.

The cupric site in nitrous oxide reductase contains a mixed‐valence [Cu(II),Cu(I)] binuclear center: A multifrequency electron paramagnetic resonance investigation

Multifrequency electron paramagnetic resonance (EPR) spectra of the Cu(II) site in nitrous oxide reductase (N2OR) from Pseudomonas stutzeri confirm the assignment of the low field g value at 2.18 consistent with the seven line pattern observed at 9.31 GHz, 10 K. S‐band spectra at 20 K are better resolved than the X‐band spectra recorded at 10 K. The features observed at 2.4, 3.4, 9.31 and 35 GHz are explained by a mixed‐valence [Cu(1.5)..Cu(1.5)] S= 1/2 species with the unpaired electron delocalized between two equivalent Cu nuclei. The resemblance of the N2OR S‐band spectra to the spectra for the EPR‐detectable Cu of cytochrome c oxidase suggests that the S‐band spectrum for cytochrome c oxidase measured below 30 K may also contain hyperfine splittings from two approximately equivalent Cu nuclei.

Growth Yields in Bacterial Denitrification and Nitrate Ammonification

ABSTRACT Denitrification and nitrate ammonification are considered the highest-energy-yielding respiration systems in anoxic environments after oxygen has been consumed. The corresponding free energy changes are 7 and 35% lower than that of aerobic respiration, respectively. Growth yield determinations with pure cultures of Paracoccus denitrificans and Pseudomonas stutzeri revealed that far less energy is converted via ATP into cell mass than expected from the above calculations. Denitrification with formate or hydrogen as electron donor yielded about 2.4 to 3.0 g dry matter per mol formate or hydrogen and 15 to 18 g dry matter per mol acetate. Similar yields with acetate were obtained with Pseudomonas stutzeri. Wolinella succinogenes and Sulfurospirillum deleyianum, which reduce nitrate to ammonia, both exhibited similar yield values with formate or H2 plus nitrate. The results indicate that ATP synthesis in denitrification is far lower than expected from the free energy changes and even lower than in nitrate ammonification. The results are discussed against the background of our present understanding of electron flow in denitrification and with respect to the importance of denitrification and nitrate ammonification in the environment.

The nirSTBM region coding for cytochrome cd1-dependent nitrite respiration of Pseudomonas stutzeri consists of a cluster of mono-, di-, and tetraheme proteins

Genes for respiratory nitrite reduction (denitrification) of Pseudomonas stutzeri are clustered within 7 kbp. A 4.6‐kbp Hind III‐Kpn I fragment carrying nirS, the structural gene for cytochrome cd 1, was sequenced. An open reading frame immediately downstream of nirScodes for a 22.8‐kDa protein with four heme c‐binding motifs. Mutagenesis of this gene causes an apparent defect in electron donation to cytochrome cd 1. Following this ORF are the structural genes for cytochrome c 552, cytochrome c 551, and ORF5 that codes for a 11.9‐kDa monoheme protein. All cytochromes have a signal sequence for protein export.

Nitric oxide signaling and transcriptional control of denitrification genes in Pseudomonas stutzeri.

The expression of denitrification by a facultatively anaerobic bacterium requires as exogenous signals a low oxygen tension concomitant with an N oxide. We have studied the role of nitric oxide (NO), nitrous oxide (N2O), and nitrite as signal molecules for the expression of the denitrification apparatus of Pseudomonas stutzeri. Transcriptional kinetics of structural genes were monitored by Northern blot analysis in a 60-min time frame after cells were exposed to an N oxide signal. To differentiate the inducer role of NO from that of nitrite, mRNA kinetics were monitored under anoxic conditions in a nirF strain, where NO generation from nitrite is prevented because of a defect in heme D(1) biosynthesis. NO-triggered responses were monitored from the nirSTB operon (encoding cytochrome cd(1) nitrite reductase), the norCB operon (encoding NO reductase), nosZ (encoding nitrous oxide reductase), and nosR (encoding a putative regulator). Transcription of nirSTB and norCB was activated by 5 to 50 nM NO, whereas the nosZ promoter required about 250 nM. Nitrite at 5 to 50 nM elicited no response. At a threshold concentration of 650 nM N2O, we observed in the anoxic cell the transient appearance of nosZ and nosR transcripts. Constant levels of transcripts of both genes were observed in an anoxic cell sparged with N2O. NO at 250 nM stimulated in this cell type the expression of nos genes severalfold. The transcription factor DnrD, a member of the FNR-CRP family, was found to be part of the NO-triggered signal transduction pathway. However, overexpression of dnrD in an engineered strain did not result in NirS synthesis, indicating a need for activation of DnrD. NO modified the transcriptional pattern of the dnrD operon by inducing the transcription of dnrN and dnrO, located upstream of dnrD. Insertional mutagenesis of dnrN altered the kinetic response of the nirSTB operon towards nitrite. Our data establish NO and DnrD as key elements in the regulatory network of denitrification in P. stutzeri. The NO response adds to the previously identified nitrate-nitrite response mediated by the NarXL two-component system for the expression of respiratory nitrate reductase encoded by the narGHJI operon.

Multifrequency EPR evidence for a bimetallic center at the CuA site in cytochrome c oxidase.

Multifrequency electron paramagnetic resonance (EPR) spectra of the Cu(II) site in bovine heart cytochrome c oxidase (COX) and nitrous oxide reductase (N2OR) from Pseudomonas stutzeri confirm the existence of Cu‐Cu interaction in both enzymes. C‐band (4.5 GHz) proves to be a particularly good frequency complementing the spectra of COX and N2OR recorded at 2.4 and 3.5 GHz. Both the high and low field region of the EPR spectra show the presence of a well‐resolved 7‐line pattern consistent with the idea of a binuclear Cu center in COX and N2OR. Based on this assumption consistent g‐values are calculated for gz and gx at four frequencies. No consistent g‐values are obtained with the assumption of a 4‐line pattern indicative for a mononuclear Cu site.

N2O binding at a [4Cu:2S] copper-sulphur cluster in nitrous oxide reductase.

Nitrous oxide (N2O) is generated by natural and anthropogenic processes and has a critical role in environmental chemistry. It has an ozone-depleting potential similar to that of hydrochlorofluorocarbons as well as a global warming potential exceeding that of CO2 300-fold. In bacterial denitrification, N2O is reduced to N2 by the copper-dependent nitrous oxide reductase (N2OR). This enzyme carries the mixed-valent CuA centre and the unique, tetranuclear CuZ site. Previous structural data were obtained with enzyme isolated in the presence of air that is catalytically inactive without prior reduction. Its CuZ site was described as a [4Cu:S] centre, and the substrate-binding mode and reduction mechanism remained elusive. Here we report the structure of purple N2OR from Pseudomonas stutzeri, handled under the exclusion of dioxygen, and locate the substrate in N2O-pressurized crystals. The active CuZ cluster contains two sulphur atoms, yielding a [4Cu:2S] stoichiometry; and N2O bound side-on at CuZ, in close proximity to CuA. With the substrate located between the two clusters, electrons are transferred directly from CuA to N2O, which is activated by side-on binding in a specific binding pocket on the face of the [4Cu:2S] centre. These results reconcile a multitude of available biochemical data on N2OR that could not be explained by earlier structures, and outline a mechanistic pathway in which both metal centres and the intervening protein act in concert to achieve catalysis. This structure represents the first direct observation, to our knowledge, of N2O bound to its reductase, and sheds light on the functionality of metalloenzymes that activate inert small-molecule substrates. The principle of using distinct clusters for substrate activation and for reduction may be relevant for similar systems, in particular nitrogen-fixing nitrogenase.

Copper-containing nitrite reductase from Pseudomonas aureofaciens is functional in a mutationally cytochrome cd1-free background (NirS−) of Pseudomonas stutzeri

The structural gene, nirK, for the respiratory Cu-containing nitrite reductase from denitrifying Pseudomonas aureofaciens was isolated and sequenced. It encodes a polypeptide of 363 amino acids including a signal peptide of 24 amino acids for protein export. The sequence showed 63.8% positional identity with the amino acid sequence of “Achromobacter cycloclastes” nitrite reductase. Ligands for the blue, type I Cu-binding site and for a putative type-II site were identified. The nirK gene was transferred to the mutant MK202 of P. stutzeri which lacks cytochrome cd1 nitrite reductase due to a transposon Tn5 insertion in its structural gene, nirS. The heterologous enzyme was active in vitro and in vivo in this background and restored the mutationally interrupted denitrification pathway. Transfer of nirK to Escherichia coli resulted in an active nitrite reductase in vitro. Expression of the nirS gene from P. stutzeri in P. aureofaciens and E. coli led to nonfunctional gene products. Nitrite reductase activity of cell extract from either bacterium could be reconstituted by addition of heme d1, indicating that both heterologous hosts synthesized a cytochrome cd1 without the d1-group.

Multiple transcription factors of the FNR family in denitrifying Pseudomonas stutzeri: characterization of four fnr‐like genes, regulatory responses and cognate metabolic processes

Pseudomonas stutzeri is a facultative anaerobic bacterium with the capability of denitrification. In searching for regulators that control the expression of this trait in response to oxygen withdrawal, we have found an unprecedented multiplicity of four genes encoding transcription factors of the FNR family. The fnrA gene encodes a genuine FNR‐type regulator, which is expressed constitutively and controls the cytochrome cbb3‐type terminal oxidase (the cco operon), cytochrome c peroxidase (the ccp gene) and the oxygen‐independent coproporphyrinogen III oxidase (the hemN gene), in addition to its previously demonstrated role in arginine catabolism (the arc operon). The fnr homologues dnrD, dnrE and dnrS encode regulators of a new subgroup within the FNR family. Their main distinctive feature is the lack of cysteine residues for complexing the [4Fe–4S] centre of redox‐active FNR‐type regulators. However, they form a phylogenetic lineage separate from the FixK branch of FNR proteins, which also lack this cysteine signature. We have studied the expression of the dnr genes under aerobic, oxygen‐limited and denitrifying conditions. DnrD is a key regulator of denitrification by selective activation of the genes for cytochrome cd1 nitrite reductase and NO reductase. The dnrD gene is part of the 30 kb region carrying denitrification genes of P. stutzeri. Transcription of dnrD was activated in O2‐limited cells and particularly strongly in denitrifying cells, but was not under the control of FnrA. In response to denitrifying growth conditions, dnrD was transcribed as part of an operon together with genes downstream and upstream of dnrD. dnrS was found about 9 kb upstream of dnrD, next to the nrdD gene for anaerobic ribonucleotide reductase. The transcription of dnrS required FnrA in O2‐limited cells. Mutation of dnrS affected nrdD and the expression of ferredoxin I as an element of the oxidative stress response. The dnrE gene is part of the nar region encoding functions for respiratory nitrate reduction. We found the highest amount of dnrE transcripts in aerobically nitrate‐challenged cells. The gene was transcribed from two promoters, P1 and P2, of which promoter P1 was under the control of the nitrate response regulator NarL. The multiplicity of FNR factors in P. stutzeri underlines the versatility of the FNR scaffold to serve for transcriptional regulation directed at anaerobic or nitrate‐activated metabolic processes.