Paulette M. Vignais

Sustained Photoevolution of Molecular Hydrogen in a Mutant of Synechocystis sp. Strain PCC 6803 Deficient in the Type I NADPH-Dehydrogenase Complex

The interaction between hydrogen metabolism, respiration, and photosynthesis was studied in vivo in whole cells of Synechocystis sp. strain PCC 6803 by continuously monitoring the changes in gas concentrations (H2, CO2, and O2) with an online mass spectrometer. The in vivo activity of the bidirectional [NiFe]hydrogenase [H2:NAD(P) oxidoreductase], encoded by the hoxEFUYH genes, was also measured independently by the proton-deuterium (H-D) exchange reaction in the presence of D2. This technique allowed us to demonstrate that the hydrogenase was insensitive to light, was reversibly inactivated by O2, and could be quickly reactivated by NADH or NADPH (+H2). H2 was evolved by cells incubated anaerobically in the dark, after an adaptation period. This dark H2 evolution was enhanced by exogenously added glucose and resulted from the oxidation of NAD(P)H produced by fermentation reactions. Upon illumination, a short (less than 30-s) burst of H2 output was observed, followed by rapid H2 uptake and a concomitant decrease in CO2 concentration in the cyanobacterial cell suspension. Uptake of both H2 and CO2 was linked to photosynthetic electron transport in the thylakoids. In the ndhB mutant M55, which is defective in the type I NADPH-dehydrogenase complex (NDH-1) and produces only low amounts of O2 in the light, H2 uptake was negligible during dark-to-light transitions, allowing several minutes of continuous H2 production. A sustained rate of photoevolution of H2 corresponding to 6 micro mol of H2 mg of chlorophyll(-1) h(-1) or 2 ml of H2 liter(-1) h(-1) was observed over a longer time period in the presence of glucose and was slightly enhanced by the addition of the O2 scavenger glucose oxidase. By the use of the inhibitors DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] and DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), it was shown that two pathways of electron supply for H2 production operate in M55, namely photolysis of water at the level of photosystem II and carbohydrate-mediated reduction of the plastoquinone pool.

Hydrogenase, Nitrogenase, and Hydrogen Metabolism in the Photosynthetic Bacteria

Publisher Summary This chapter illustrates the H 2 metabolism in photosynthetic bacteria. The developments in the context of the biochemistry and physiology of the photosynthetic bacteria, and their biotechnological applications are presented. Photosynthetic bacteria possess a diverse and evolutionarily ancient metabolism, which is reflected in the different ways in which they can metabolize H 2 . Three enzymes have been implicated in H 2 metabolism in these organisms: (1) nitrogenase, which catalyses unidirectional, ATP-dependent H 2 evolution, and can function either in the light, or in the dark under anaerobic or micro-aerobic conditions; (2) uptake hydrogenase, which is membrane-bound and, although capable of both H 2 evolution and uptake, functions physiologically in the direction of H 2 oxidation; and (3) “classical” or reversible hydrogenase, which may be either soluble or membrane bound and functions mainly during dark anaerobic fermentation. The photosynthetic bacteria represent a tool of great potential in various fields of biotechnology. Their rapid growth rate and their metabolic versatility enable them to survive and proliferate in a wide variety of environments.

Cloning and sequencing of the genes encoding the large and the small subunits of the H2 uptake hydrogenase (hup) of Rhodobacter capsulatus

The structural genes (hup) of the H2 uptake hydrogenase of Rhodobacter capsulatus were isolated from a cosmid gene library of R. capsulatus DNA by hybridization of Bradyrhizobium japonicum. The R. capsulatus genes were localized on a 3.5 kb HindIII fragment. The fragment, cloned onto plasmid pAC76, restored hydrogenase activity and autotrophic growth of the R. capsulatus mutant JP91, deficient in hydrogenase activity (Hup-). The nucleotide sequence, determined by the dideoxy chain termination method, revealed the presence of two open reading frames. The gene encoding the large subunit of hydrogenase (hupL) was identified from the size of its protein product (68,108 dalton) and by alignment with the NH2 amino acid protein sequence determined by Edman degradation. Upstream and separated from the large subunit by only three nucleotides was a gene encoding a 34,256 dalton polypeptide. Its amino acid sequence showed 80% identity with the small subunit of the hydrogenase of B. japonicum. The gene was identified as the structural gene of the small subunit of R. capsulatus hydrogenase (hupS). The R. capsulatus hydrogenase also showed homology of Desulfovibrio baculatus and D. gigas. In the R. capsulatus hydrogenase the Cys residues (13 in the small subunit and 12 in the large subunit) were not arranged in the typical configuration found in [4Fe-4S] feredoxins.

Nitrogen fixation and hydrogen metabolism in photosynthetic bacteria

The photosynthetic bacteria are found in a wide range of specialized aquatic environments. These bacteria represent important members of the microbial community since they are capable of carrying out two of the most important processes on earth, namely, photosynthesis and nitrogen fixation, at the expense of solar energy. Since the discovery that these bacteria could fix atmospheric nitrogen, there has been an intensification of studies relating to both the biochemistry and physiology of this process. The practical importance of this field is emphasized by a consideration of the tremendous energy input required for the production of artificial nitrogenous fertilizer. The present communication aims to briefly review the current state of knowledge relating to certain aspects of nitrogen fixation by the photosynthetic bacteria. The topics that will be discussed include a general survey of the nitrogenase system in the various photosynthetic bacteria, the regulation of both nitrogenase biosynthesis and activity, recent advances in the genetics of the nitrogen fixing system, and the hydrogen cycle in these bacteria. In addition, a brief discussion of some of some of the possible practical applications provided by the photosynthetic bacteria will be presented.

Organization of the genes necessary for hydrogenase expression in Rhodobacter capsulatus. Sequence analysis and identification of two hyp regulatory mutants

A 25kbp DNA fragment from the chromosome of Rhodobacter capsulatus B10 carrying hydrogenase (hup) determinants was completely sequenced. Coding regions corresponding to 20 open reading frames were identified. The R. capsulatus hydrogenase‐specific gene (hup and hyp) products bear significant structural identity to hydrogenase gene products from Escherichia coli (13), from Rhizobium liguminosarum (16), from Azotobacter vinelandii (10) and from Alcaligenes eutrophus (11). The sequential arrangement of the R. capsulatus genes is: hupR2‐hupU‐hypF‐hupS‐hupL‐hupM‐hupD‐hupF‐hupG‐hupH‐huoJ‐hupK‐hypA‐hypB‐hupR1‐hypC‐hypD‐hypE‐ORF19‐ORF20, all contiguous and transcribed from the same DNA strand. The last two potential genes do not encode products that are related to identified hydrogenase‐specific gene products in other species. The sequence of the 12 R. capsulatus genes underlined above is presented. The mutation site in two of the Hup− mutants used in this study, RS13 and RCC12, was identified in the hypF gene (deletion of one G) and in the hypD qene (deletion of 54 bp), respectively. The hypF gene product shares 45% identity with the product of hydA from E. coli and the product of hypF from R. leguminosarum. Those products present at their N‐terminus a Cys arrangement typical of zinc‐finger proteins. The G deletion in the C‐terminal region of hypF in the RS13 mutant

Binding of adenosine diphosphate and of antagonist ligands to the mitochondrial ADP carrier

Summary The properties of adenine nucleotide translocation in mitochondria are reviewed. The inhibitory properties of specific inhibitors: (35S)-atractyloside, (35S)carboxyatractyloside (or (35S)gummiferin), (14C)bongkrekic acid, are analysed and compared to their binding properties. In contrast to atractyloside, carboxyatractyloside is not a competitive inhibitor of ADP binding and ADP translocation. Double labelling experiments using (35S)-atractyloside and (3H)ADP confirm that ADP and ATR compete for binding to the inner mitochondrial membrane. Binding of ADP to its translocase brings about membrane conformation changes with the exposure of a small number of -SH groups. Some models illustrating possible modes of translocation which incorporate these findings are discussed.

The hydrogenase structural operon in Rhodobacter capsulatus contains a third gene, hupM, necessary for the formation of a physiologically competent hydrogenase

The hupM gene, previously called ORFX, found downstream from and contiguous with the structural hydrogenase genes hupS and hupL in Rhodobacter capsulatus, is shown here to form a single hupSLM transcription unit with the two other genes. The hupM gene was Inactivated by interposon mutagenesis. The two selected mutants, BCX1 and BCX2, which contained the kanamycin‐resistance gene in opposite orientation, still exhibited hydrogenase activity when assayed with the artificial electron acceptors benzyl‐viologen and methylene blue. However, the hydrogenase was not physiologically active in these mutants, which could not grow autotrophically and were unable to recycle electrons to nitrogenase or to respire on H2. The hupM gene starts nine base pairs downstream from the TGA stop codon of hupL gene, which encodes the large subunit of the [NiFe]hydrogenase of Rhodobacter capsulatus. The three contiguous genes hupS, hupL and hupM were subcloned downstream from the promoter of hupSL, either with the promoter in the correct orientation (plasmid pBC8) or with the promoter in the opposite orientation (plasmid pBC9), then the constructs were introduced into the mutant strains. Only plasmid pBC8 could restore the formation of a competent hydrogenase in mutants BCX1 and BCX2, indicating that the hupM gene is expressed only from the hupSL promoter.

Regulation of nitrogenase activity through iron protein interconversion into an active and an inactive form in Rhodopseudomonas capsulata

Abstract The iron protein of nitrogenase from Rhodopseudomonas capsulata has been isolated in two forms depending on the nitrogen source used for growth. When isolated from cells grown on a limiting amount of ammonia, the Fe protein was composed of two equal subunits of apparent molecular weight 33 500. In contrast, the Fe protein purified from glutamate-grown cells was mainly inactive and composed of two different subunits of apparent M r 33 500 and 38 000. A two-dimensional immunoelectrophoresis technique was devised to allow direct determination, in cell-free extracts, of the subunit composition of Fe protein. By this method it is demonstrated that the Fe protein shifted from an active form (single type of subunit) to an inactive form (two distinct subunits) in cells submitted to an ammonia shock (15 mM NH 4 Cl). Upon incubation of inactivated cells in a nitrogen-free medium, in vivo nitrogenase activity was recovered and, at the same time, the Fe protein was shifted back to the active form. This activation process was antagonized by chloramphenicol. In cells grown in the presence of [ 32 P]phosphate, radioactivity was incorporated in the 38 kDa subunit of the inactive Fe protein. Upon treatment with the activating enzyme from Rhodospirillum rubrum , removal of radioactive phosphate was concomitant with the disappearance of the 38 kDa subunit. Altogether, these results suggest that nitrogenase activity in Rps. capsulata is controlled by the interconversion of the Fe protein between an active and an inactive form as in R. rubrum . This system of regulation appeared to turn off nitrogenase activity with more or less efficiency, depending on factors such as the nature and availability of the nitrogen source and the culture age.

Cloning and sequences of the structural (hupSLC) and accessory (hupDHI) genes for hydrogenase biosynthesis in Thiocapsa roseopersicina

The first molecular biology study on the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina is reported, namely, the construction of cosmid libraries and isolation of a hydrogenase gene cluster by hybridization with hydrogenase structural genes from the purple non-sulfur bacterium, Rhodobacter capsulatus. The sequenced gene cluster contains six open reading frames, the products of which show significant degrees of identity (from 40 to 78%) with hydrogenase gene products necessary for biosynthesis of the group-I of [NiFe]hydrogenases. The structural hupSLC genes encode the small and large hydrogenase subunits and a hydrophobic protein shown to accept electrons from hydrogenase in R. capsulatus. They are followed downstream by three genes, hupDHI, which are similar to hydrogenase accessory genes found in other bacteria.

Genetic Mapping of the Rhodopseudomonas capsulata Chromosome Shows Non-clustering of Genes Involved in Nitrogen Fixation

Summary: The mutant R plasmid pTH10 was used to construct a circular linkage map of the Rhodopseudomonas capsulata B10 chromosome. Mutations affecting nitrogen fixation (nif mutations) were dispersed in several groups on the chromosome. Biochemical analysis of nif mutants allowed identification of the structural gene for the nitrogenase component II or Fe protein (nifH) and a putative regulatory gene, possibly nifA. These two genes appeared closely linked in conjugation experiments, but represented two distinct linkage groups in crosses mediated by gene transfer agent. Other mutants were affected in the synthesis and/or stability of the nitrogenase component I or MoFe protein; synthesis of component II was also affected, but to a lesser extent. In two of these mutants, nitrogenase activity and the content of component I was increased five- to sixfold by the addition of 1 mM-molybdate to the growth medium.