Introduction of H2-Uptake Hydrogenase Genes Into Rhizobial Strains Improves Symbiotic Nitrogen Fixation in Vicia sativa and Lotus corniculatus Forage Legumes

Biological nitrogen fixation by the Rhizobium-legume symbiosis allows the conversion of atmospheric nitrogen into ammonia within root nodules mediated by the nitrogenase enzyme. Nitrogenase activity results in the evolution of hydrogen as a result of a side reaction intrinsic to the activity of this enzyme. Some rhizobia, and also other nitrogen fixers, induce a NiFe uptake hydrogenase (Hup) to recycle hydrogen produced by nitrogenase, thus improving the efficiency of the nitrogen fixation process. In this work we report the generation and symbiotic behavior of hydrogenase-positive Rhizobium leguminosarum and Mesorhizobium loti strains effective in vetch (Vicia sativa) and birsfoot trefoil (Lotus corniculatus) forage crops, respectively. The ability of hydrogen recycling was transferred to these strains through the incorporation of hup minitransposon TnHB100, thus leading to full recycling of hydrogen in nodules. Inoculation of Vicia and Lotus plants with these engineered strains led to significant increases in the levels of nitrogen incorporated into the host legumes. The level of improvement of symbiotic performance was dependent on the recipient strain and also on the legume host. These results indicate that hydrogen recycling has the potential to improve symbiotic nitrogen fixation in forage plants.

Changes of the Proteome and Acetylome during Transition into the Stationary Phase in the Organohalide-Respiring Dehalococcoides mccartyi Strain CBDB1

The strictly anaerobic bactGIerium Dehalococcoides mccartyi obligatorily depends on organohalide respiration for energy conservation and growth. The bacterium also plays an important role in bioremediation. Since there is no guarantee of a continuous supply of halogenated substrates in its natural environment, the question arises of how D. mccartyi maintains the synthesis and activity of dehalogenating enzymes under these conditions. Acetylation is a means by which energy-restricted microorganisms can modulate and maintain protein levels and their functionality. Here, we analyzed the proteome and Nε-lysine acetylome of D. mccartyi strain CBDB1 during growth with 1,2,3-trichlorobenzene as an electron acceptor. The high abundance of the membrane-localized organohalide respiration complex, consisting of the reductive dehalogenases CbrA and CbdbA80, the uptake hydrogenase HupLS, and the organohalide respiration-associated molybdoenzyme OmeA, was shown throughout growth. In addition, the number of acetylated proteins increased from 5% to 11% during the transition from the exponential to the stationary phase. Acetylation of the key proteins of central acetate metabolism and of CbrA, CbdbA80, and TatA, a component of the twin-arginine translocation machinery, suggests that acetylation might contribute to maintenance of the organohalide-respiring capacity of the bacterium during the stationary phase, thus providing a means of ensuring membrane protein integrity and a proton gradient.

Azotobacter vinelandiiNitrogenase Activity, Hydrogen Production, and Response to Oxygen Exposure

ABSTRACTAzotobacter vinelandiiselectively utilizes three types of nitrogenase (molybdenum, vanadium, and iron only) to fix N2, with their expression regulated by the presence or absence of different metal cofactors in its environment. Each alternative nitrogenase isoenzyme is predicted to have different electron flux requirements based onin vitromeasurements, with the molybdenum nitrogenase requiring the lowest flux and the iron-only nitrogenase requiring the highest. Here, prior characterized strains, derepressed in nitrogenase synthesis and also deficient in uptake hydrogenase, were further modified to generate new mutants lacking the ability to produce poly-β-hydroxybutyrate (PHB). PHB is a storage polymer generated under oxygen-limiting conditions and can represent up to 70% of the cells' dry weight. The absence of such granules facilitated the study of relationships between catalytic biomass and product molar yields across different adaptive respiration conditions. The released hydrogen gas observed during growth, due to the inability of the mutants to recapture hydrogen, allowed for direct monitoring ofin vivonitrogenase activity for each isoenzyme. The data presented here show that increasing oxygen exposure limits equally thein vivoactivities of all nitrogenase isoenzymes, while under comparative conditions, the Mo nitrogenase enzyme evolves more hydrogen per unit of biomass than the alternative isoenzymes.IMPORTANCEA. vinelandiihas been a focus of intense research for over 100 years. It has been investigated for a variety of functions, including agricultural fertilization and hydrogen production. All of these endeavors are centered aroundA. vinelandii's ability to fix nitrogen aerobically using three nitrogenase isoenzymes. The majority of research up to this point has targetedin vitromeasurements of the molybdenum nitrogenase, and robust data contrasting how oxygen impacts thein vivoactivity of each nitrogenase isoenzyme are lacking. This article aims to providein vivonitrogenase activity data using a real-time evaluation of hydrogen gas released by derepressed nitrogenase mutants lacking an uptake hydrogenase and PHB accumulation.

Bioenergetics of lactate vs. acetate outside TCA enhanced the hydrogen evolution levels in two newly isolated strains of the photosynthetic bacterium Rhodopseudomonas

Two local hydrogen-evolving strains of purple nonsulfur bacteria have been isolated, characterized, and identified as Rhodopseudomonas sp. TUT (strains Rh1 and Rh2). Lactate followed by succinate and malate supported the highest amounts of H2 production, growth (O.D.660nm, proteins and bacteriochlorphyll contents), nitrogenase activity, and uptake hydrogenase; the least of which was acetate. Alginate-immobilized cells evolved higher hydrogen amounts than free cell counterparts. Rh1 was more productive than Rh2 at all circumstances. Lactate-dependent hydrogen evolution was more than twice that of acetate, due to ATP productivity (2/–1, respectively), which is limiting to the nitrogenase activity. The preference of lactate over other acids indicates the feasibility of using these two strains in hydrogen production from dairy wastewater.

Turning around the electron flow in an uptake hydrogenase. EPR spectroscopy and in vivo activity of a designed mutant in HupSL from Nostoc punctiforme

The uptake hydrogenase HupSL became a H2 producer in N. punctiforme after modifying the proximal FeS cluster with the single point mutation C12P.

Genome Sequence of Bradyrhizobium tropiciagri Strain CNPSo 1112 T , Isolated from a Root Nodule of Neonotonia wightii

CNPSo 1112 T is a nitrogen-fixing symbiont of perennial soybean, a tropical legume forage. Its draft genome indicates a large genome with a circular chromosome and 9,554 coding sequences (CDSs). Operons of nodulation, nitrogen fixation, and uptake hydrogenase were present in the symbiotic island, and the genome encompasses several CDSs of stress tolerance.