Role of Bradyrhizobium japonicum cytochrome c550 in nitrite and nitrate respiration.

Bradyrhizobium japonicum cytochrome c(550), encoded by cycA, has been previously suggested to play a role in denitrification, the respiratory reduction of nitrate to dinitrogen. However, the exact role of this cytochrome in the denitrification process is unknown. This study shows that cytochrome c(550) is involved in electron transfer to the copper-containing nitrite reductase of B. japonicum, as revealed by the inability of a cycA mutant strain to consume nitrite and, consequently, to grow under denitrifying conditions with nitrite as the electron acceptor. Mutation of cycA had no apparent effect on methylviologen-dependent nitrite reductase activity. However, succinate-dependent nitrite reduction was largely inhibited, suggesting that c(550) is the in vivo electron donor to copper-containing nitrite reductase. In addition, this study demonstrates that a cytochrome c(550) mutation has a negative effect on expression of the periplasmic nitrate reductase. This phenotype can be rescued by extending the growth period of the cells. A model is proposed whereby a mutation in cycA reduces expression of the cbb(3)-type oxidase, affecting oxygen consumption rate by the cells and consequently preventing maximal expression of the periplasmic nitrate reductase during the first days of the growth period.     

In silico analysis reveals substantial variability in the gene contents of the gamma proteobacteria LexA-regulon

MOTIVATION: Motif-prediction algorithm capabilities for the analysis of bacterial regulatory networks and the prediction of new regulatory sites can be greatly enhanced by the use of comparative genomics approaches. In this study, we make use of a consensus-building algorithm and comparative genomics to conduct an in-depth analysis of the LexA-regulon of gamma proteobacteria, and we use the inferred results to study the evolution of this regulatory network and to examine the usefulness of the control sequences and gene contents of regulons in phylogenetic analysis. RESULTS: We show, for the first time, the substantial heterogeneity that the LexA-regulon of gamma proteobacteria displays in terms of gene content and we analyze possible branching points in its evolution. We also demonstrate the feasibility of using regulon-related information to derive sound phylogenetic inferences. AVAILABILITY: Complementary analysis data and both the source code and the Windows-executable files of the consensus-building software are available at http://www.cnm.es/~ivan/RCGScanner/     

Two nitrate/nitrite transporters are encoded within the mobilizable plasmid for nitrate respiration of Thermus thermophilus HB8

Thermus thermophilus HB8 can grow anaerobically by using a membrane-bound nitrate reductase to catalyze the reduction of nitrate as a final electron acceptor in respiration. In contrast to other denitrifiers, the nitrite produced does not continue the reduction pathway but accumulates in the growth medium after its active extrusion from the cell. We describe the presence of two genes, narK1 and narK2, downstream of the nitrate reductase-encoding gene cluster (nar) that code for two homologues to the major facilitator superfamily of transporters. The sequences of NarK1 and NarK2 are 30% identical to each other, but whereas NarK1 clusters in an average-distance tree with putative nitrate transporters, NarK2 does so with putative nitrite exporters. To analyze whether this differential clustering was actually related to functional differences, we isolated derivatives with mutations of one or both genes. Analysis revealed that single mutations had minor effects on growth by nitrate respiration, whereas a double narK1 narK2 mutation abolished this capability. Further analysis allowed us to confirm that the double mutant is completely unable to excrete nitrite, while single mutants have a limitation in the excretion rates compared with the wild type. These data allow us to propose that both proteins are implicated in the transport of nitrate and nitrite, probably acting as nitrate/nitrite antiporters. The possible differential roles of these proteins in vivo are discussed.     

Role of Bradyrhizobium japonicum cytochrome c550 in nitrite and nitrate respiration

Bradyrhizobium japonicum cytochrome c ₅₅₀, encoded by cycA , has been previously suggested to play a role in denitrification, the respiratory reduction of nitrate to dinitrogen. However, the exact role of this cytochrome in the denitrification process is unknown. This study shows that cytochrome c ₅₅₀ is involved in electron transfer to the copper-containing nitrite reductase of B. japonicum , as revealed by the inability of a cycA mutant strain to consume nitrite and, consequently, to grow under denitrifying conditions with nitrite as the electron acceptor. Mutation of cycA had no apparent effect on methylviologen-dependent nitrite reductase activity. However, succinate-dependent nitrite reduction was largely inhibited, suggesting that c ₅₅₀ is the in vivo electron donor to copper-containing nitrite reductase. In addition, this study demonstrates that a cytochrome c ₅₅₀ mutation has a negative effect on expression of the periplasmic nitrate reductase. This phenotype can be rescued by extending the growth period of the cells. A model is proposed whereby a mutation in cycA reduces expression of the cbb ₃-type oxidase, affecting oxygen consumption rate by the cells and consequently preventing maximal expression of the periplasmic nitrate reductase during the first days of the growth period.     

Endonuclease V of Escherichia coli prevents mutations from nitrosative deamination during nitrate/nitrite respiration

Endonuclease V (Endo V) of Escherichia coli participates in the excision repair of hypoxanthine and xanthine (deaminated adenine and guanine) in DNA. It thereby reduces the mutagenic effects of nitrous acid by attacking lesions caused by nitrosative deamination. Nitrosating agents may be produced endogenously when E. coli is grown in oxygen-poor cultures, during which nitrate and nitrite replace oxygen as preferred electron acceptors. In this study, the protective effect of Endo V was observed under such conditions. During micro-aerobic growth, an nfi (Endo V) mutation enhanced the frequency of nitrate- and nitrite-induced A:T-->G:C and G:C-->A:T transition mutations, which are consistent with a defect in the removal of DNA hypoxanthine and xanthine, respectively. Similar effects were observed in saturated, aerobic cultures but not in well-aerated, logarithmically growing ones. A narG (nitrate reductase) mutation blocked the mutagenesis of the nfi mutant by nitrate but not by nitrite. These results differed from those of previous studies in which cell suspensions generated an exogenous nitrosating agent from nitrite, but not from nitrate, in a reaction that was narG-dependent. Nitrate/nitrite metabolism is also known to generate endogenous alkylating agents through N-nitrosation. However, an nfi mutation did not appreciably enhance mutagenesis by N-methyl-N-nitrosourea, suggesting that the mutator effect of nfi is not due to a defect in alkylation repair. The overall results indicate that Endo V functions during normal growth by helping to repair nitrosatively deaminated bases in DNA, which are by-products of anaerobic nitrate/nitrite respiration.     

Comparative genomics: genome-wide analysis in metazoan eukaryotes

The increasing number of complete and nearly complete metazoan genome sequences provides a significant amount of material for large-scale comparative genomic analysis. Finding new effective methods to analyse such enormous datasets has been the object of intense research. Three main areas in comparative genomics have recently shown important developments: whole-genome alignment, gene prediction and regulatory-region prediction. Each of these areas improves the methods of deciphering long genomic sequences and uncovering what lies hidden in them.     

Comparative genomics analysis of metallothioneins in twelve Drosophila species

Metallothioneins (MTs) are a superfamily of Cys-rich polypeptides that bind heavy metal ions, both for physiological and detoxification purposes. They are present in all organisms, but their origin is probably polyphyletic, so that MT evolutionary studies are rather scarce. We present a thorough search and analysis of the MT coding sequences in the 12 Drosophila genomes completely sequenced, taking as reference the features reported for D. melanogaster, where four isogenes (MtnA to MtnD) are known and deeply characterized. We include a fifth isoform in this study, named MtnE, and recently annotated. The MTs polymorphism pattern is essentially the same for the 12 Drosophila species. Invariably, a MtnA form and an MtnB-cluster, comprising the MtnB-to-MtnE forms in tandem array, are observed. The whole set of genes are kept in the same synteny element (Muller E), but implicated in rearrangement events (mainly inversions), encompassing all or some of the isogenes. Gene exon/intron architecture, and cDNA and protein sequences appear highly conserved through Drosophila speciation, concordantly with an essential function for MT isoforms in flies, even for those previously considered as minor products. Data presented here will be comprehensively analyzed to provide a valuable guide for future MT evolutionary, structure and function studies.     

Biochemistry of nitrate respiration in Pseudomonas stutzeri. I. Aerobic and nitrate respiration routes of carbohydrate catabolism

Spangler, W. J. (Oregon State University, Corvallis), and C. M. Gilmour. Biochemistry of nitrate respiration in Pseudomonas stutzeri. I. Aerobic and nitrate respiration routes of carbohydrate catabolism. J. Bacteriol. 91:245-250. 1966.-The metabolic pathways of glucose catabolism were studied in Pseudomonas stutzeri under aerobic conditions and under conditions of nitrate respiration. Studies on both glucose and gluconate catabolism, by the radiorespirometric method, indicated that these substrates are degraded in the same manner, i.e., the Entner-Doudoroff and pentose phosphate pathways. There appeared to be no major shift in primary metabolic pathways when nitrate was used as the terminal hydrogen acceptor in nitrate respiration as opposed to aerobic respiration with free molecular oxygen. It was shown that glucose is not degraded to any appreciable extent under anaerobic conditions in the absence of nitrate. Tentative evidence suggests that the tricarboxylic acid cycle functions under both conditions of oxygen relationships and that the rate of carbon oxidation via the tricarboxylic acid cycle is slower with nitrate respiration than under aerobic conditions.     

Induction of nitrate and nitrite reductases in Anabaena cylindrica

Induction of nitrate and nitrite reductases in Anabaena cylindrica(FOGG strain) was investigated. At various stages of algal growthin the presence of nitrate or nitrite, the levels of these enzymeswere determined using cell-free preparations. Nitrate and nitritereductases were induced by the respective substrates. Nitratedid not act either as an inducer or as a repressor of nitritereductase. ¹This work was supported by grant No. 8814 from the Ministryof Education²Department of Biology, Faculty of Science, Tokyo MetropolitanUniversitySetagaya-ku, Tokyo, Japan (Received June 18, 1970; )     

Regulation of nitrate and nitrite reduction in barley leaves

Reduction of nitrate and accumulation of nitrite were studied in barley (Hordeum vulgare L. cv. Gars Clipper ex Napier) leaf sections in the dark and in the light, under aerobic (air and mixtures of O₂ and N₂) or anaerobic (N₂) conditions. Oxygen prevented nitrite accumulation but had no effect on accumulated or infiltrated nitrite. Most of the nitrite accumulated under dark-anaerobic conditions was in the "cytoplasmic" (the cell section between the plasma lemma and the tonoplast) fraction of the tissue. Reduction of nitrate was stimulated by 2, 4-dinitrophenol in tissue under dark-air and by 3-(3', 4'-dichlorophenyl)-l, l-dimethyl urea (DCMU) and carbonyl cyanide m -chlorophenylhydrazone (CCCP) in tissue under all environmental conditions studied. Nitrite accumulated in the light in DCMU-treated tissue under N₂ or under aerobic conditions in the presence of CCCP. On its own, CCCP did not promote accumulation of nitrite in leaf sections under light-air. A model for the reduction of nitrate and nitrite is proposed.     

Effects of electron donors on nitrate removal by nitrate and nitrite reductases

Effects of artificial electron donors to deliver reducing power on enzymic denitrification were investigated using nitrate reductase and nitrite reductase obtained fromOchrobactrum antropi. The activity of nitrite reductase in the soluble portion was almost the same as that in the precipitated portion of the cell extract. Nitrate removal efficiency was higher with benzyl viologen than with methyl viologen or NADH as an artificial electron donor. The turn-over numbers of nitrate and nitrite reductase were 14.1 and 1.9 μmol of nitrogen reduced/min·mg cell extracts, respectively when benzyl viologen was used as an electron donor.     

The position of nitrate respiration in evolution

Egami's hypothesis that oxygen respiration evolved from nitrate respiration, and this from nitrate fermentation, is not accepted. The reasons are: (1) Presumably there was no nitrate before O₂ in the biosphere. (2) On mechanistic grounds, respiration (oxidative phosphorylation) is to be derived directly from photosynthesis (photosynthetic phosphorylation) rather than from any form of fermentation.     

The estimation of nitrate and nitrite in saliva and urine

A method for the estimation of nitrate and nitrite is described in which nitrate is converted to nitrite by Klebsiella pneumoniae (UNF 9232) and nitrite is estimated by the Griess reaction before and after incubation. The method is suitable for the estimation of 1–25 nmol of each ion in body fluids, many samples can be handled simultaneously, and special apparatus is not required.