Mutants of Azotobacter chroococcum Defective in Hydrogenase Activity

Yates, M. G.; Robson, Richard M.

doi:10.1099/00221287-131-6-1459

Cited by 14 publications

(14 citation statements)

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“…They had different secondary phenotypes and were presumed not to be siblings. Their frequency of reversion was less than 5 x lo3, the limit of the 'scrying' technique (Yates & Robson, 1985). For these reasons they were considered adequate for use in this study.…”

Section: Discussionmentioning

confidence: 99%

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The Beneficial Effect of Hydrogenase in Azotobacter chroococcum Under Nitrogen-fixing, Carbon-limiting Conditions in Continuous and Batch Cultures

1985

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The growth of three hydrogenase minus (Hup-) mutants of Azotobacter chroococcum was compared with that of the parent Hup+ strain in batch or continuous cultures. All three mutants gave similar yields to the parent under N,-fixing conditions at an optimum dilution rate (D) of 0.1 h-l in sucrose-limited N2-fixing cultures. However, at higher D values the steady-state yields of sucroscllimited mutants were lower than those of the parent and washout occurred at lower D values. These observations were confirmed in carbon-limited mixed cultures where the parent strain outgrew the mutant at high D values. Such marked differences were not obtained in SO$--or O?-limited continuous cultures. In batch culture at low sucrose levels the mutants displayed a long division lag compared with the parent, particularly with dilute inocula. Non-N,-fixing (NH,+-grown) conditions removed these differences. We suggest that one beneficial effect of hydrogenase is on the initiation of diazotrophic growth, particularly with restricted carbon/energy supply. I N T R O D U C T I O NAlthough hydrogen is a normal by-product of nitrogenase function (see Postgate, 1982), aerobic diazotrophs rarely evolve H2 when fixing N 2 . Exceptions are some strains of Rhizobium and Bradyrhizohium (see Evans et al., 198 1) and Azotobacter chroococcum grown under certain physiological conditions (Yates et al., 198 1) or pre-treated with an inactivator of hydrogenase, acetylene . H2 evolution by nitrogenase represents a metabolic drain on the organisms since ATP is consumed and, presumably, wasted. H2 production can use 25 to 80% of the electron flux to nitrogenase in viuo (Evans et al., 1981 ;Yates et al., 1981). Most diazotrophs possess a hydrogenase; in aerobes such as the azotobacters and rhizobia the enzyme is essentially unidirectional, catalysing the uptake of H2. It is believed to contribute to the metabolic efficiency of the diazotrophic system by scavenging H1 produced during the nitrogenase function ; possible benefits (Dixon, 1972) include sparing of electron sources for diazotrophy, recovery of ATP through HJinked respiration, respiratory protection of nitrogenase against 0, and protection of the enzyme from inhibition by H2 (see Robson & Postgate, 1980).Hydrogenase ought therefore to increase the metabolic efficiency of aerobic diazotrophy. This matter has been much studied in the legume symbiosis, in plants inoculated with either Hup+ or Hup-strains of rhizobia with conflicting results. Evans et al. ( 1985) estimated that only 61 :(, of all experiments had shown a positive response (higher yields and nitrogen contents) to inoculation with Hup+ over Hup-strains.These data are no doubt much influenced by the complexities of the legume symbiosis. A nonsymbiotic diazotroph is, in principle, a physiologically simpler system with which to analyse the metabolic role of hydrogenase in diazotrophy. In the present work we compared the growth of Hup-mutants of Azotobacter chroococcum with the Hup+ parent strain in batch and continuous cultures under diffe...

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Section: Discussionmentioning

confidence: 99%

“…A . chroococcum MCD-105, 1 19 and 122 are uptake hydrogenase negative (Hup-) mutants obtained by mutagenesis with N-methyl-"-nitro-N-nitrosoguanidine (MNNG) (Yates & Robson, 1985). They were grown in batch culture on Burk's N-free medium (Newton et al, 1953) with or without NH,Cl (18 mM).…”

Section: Methodsmentioning

confidence: 99%

The Beneficial Effect of Hydrogenase in Azotobacter chroococcum Under Nitrogen-fixing, Carbon-limiting Conditions in Continuous and Batch Cultures

1985

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“…A recent examination of polypeptides in Alcaligenes eutrophus allowed the identification of at least five new peptides under the control of the hydrogenase system (28). Based on the frequency of occurrence of Hupmutants, the Azotobacter chroococcum Hup system has been proposed to comprise a substantial number of genes (46).…”

Section: Methodsmentioning

confidence: 99%

Identification and isolation of genes essential for H2 oxidation in Rhodobacter capsulatus

Love

Borghese

et al. 1989

J Bacteriol

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Mutants of Rhodobacter capsulatus unable to grow photoautotrophically with H2 and CO2 were isolated.Those lacking uptake hydrogenase activity as measured by H2-dependent methylene blue reduction were analyzed genetically and used in complementation studies for the isolation of the wild-type genes. Results of further subcloning and transposon TnS mutagenesis suggest the involvement of a minimum of five genes. Hybridization to the 2.2-kilobase-pair SstI fragment that lies within the coding region for the large and small subunits of Bradyrhizobium japonicum uptake hydrogenase showed one region of strong homology among the R. capsulatus fragments isolated, which we interpret to mean that one or both structural genes were among the genes isolated.The nonsulfur purple photosynthetic bacterium Rhodobacter capsulatus produces and consumes molecular hydrogen. In this diazotroph, H2 production occurs primarily as a function of the nitrogenase enzyme complex (26, 43) and its consumption via an uptake hydrogenase (6, 40). Although hydrogenase catalyzes the reversible oxidation of H2 to protons and electrons, the enzyme found in R. capsulatus appears to be physiologically important only in an H2 uptake capacity (5, 40). The directionality of the enzyme underscores its essential role in photoautotrophic (19) and chemoautotrophic (31) growth and in recycling H2 generated during N2 reduction (40).Because H2 metabolism influences the efficiency of nitrogen fixation (14, 15), emphasis has been placed on gaining a clearer understanding of the regulation and function of hydrogenases in the diazotrophs (7,14,21). In addition, the potential application of the phototrophs for H2 production from biomass at the expense of light energy (44) provides a second impetus for analysis of this enzyme system. The enzyme has been purified from R. capsulatus and was initially reported to be a monomer (6). Later purification yielded a preparation with higher specific activity and showed the hydrogenase to be a dimer with subunits of 67,000 and 31,000 daltons (Da) (37). As yet, the number of genes essential for uptake hydrogenase biosynthesis and function has not been determined, although at least two have been isolated (7). Recently, the hydrogenase control system in Alcaligenes eutrophus has been implicated in the regulation of five polypeptides in addition to the hydrogenase peptides (28). Thus, it is possible that a regulon may be necessary for the formation, maturation, and metal metabolism of uptake hydrogenase and electron flux from this enzyme.To obtain a more complete picture of the hydrogenase genetic system in R. capsulatus, we isolated mutants unable to grow photoautotrophically and unable to reduce methylene blue with H2. After transductional analysis, complementing fragments were obtained from a pLAFR1 cosmid library. Subcloning and complementation revealed the presence of a minimum of five genes essential for restoration of * Corresponding author. t Missouri Agricultural Experiment Station journal series no. 10644.the mutant phenotype...

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“…The [NiFe] hydrogenase, as found in Desulfovibrio gigas (6,13), is a heterologous dimer of a small and a large subunit (molecular masses, 26 and 62 kilodaltons [kDa], respectively) and is known to be present in a variety of sulfate-reducing and other gram-negative bacteria, e.g., Desulfovibrio vulgaris (10,18), Desulfovibrio africanus (14), Azotobacter vinelandii (22) and Azotobacter chroococcum (34), Bradyrhizobium japonicum (21), Escherichia coli (1,20), and gram-negative photosynthetic bacteria such as Rhodobacter capsulatus (8). The presence of the [NiFe] hydrogenase in these bacteria has been proven by purification and characterization of the enzymes (1,6,10,13,14,18,20,22) and, more recently, by Southern blotting, cloning, and sequencing of the genes for this hydrogenase (8,9,21).…”

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Organization of the genes encoding [Fe] hydrogenase in Desulfovibrio vulgaris subsp. oxamicus Monticello

1989

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The genes encoding the periplasmic [Fe] hydrogenase from Desulfovibrio vulgaris subsp. oxamicus Monticello were cloned by exploiting their homology with the hydAB genes from D. vulgaris subsp. vulgaris Hildenborough, in which this enzyme is present as a heterologous dimer of a and 0i subunits. Nucleotide sequencing showed that the enzyme is encoded by an operon in which the gene for the 46-kilodalton (kDa) a subunit precedes that of the 13.5-kDa 0i subunit, exactly as in the Hildenborough strain. The pairs of hydA and hydB genes are highly homologous; both a subunits (420 amino acid residues) share 79% sequence identity, while the unprocessed ,I subunits (124 and 123 amino acid residues, respectively) share 71 % sequence identity. In contrast, there appears to be no sequence homology outside these coding regions, with the exception of a possible promoter element, which was (1,20), and gram-negative photosynthetic bacteria such as Rhodobacter capsulatus (8). The presence of the [NiFe] hydrogenase in these bacteria has been proven by purification and characterization of the enzymes (1,6,10,13,14,18,20,22) and, more recently, by Southern blotting, cloning, and sequencing of the genes for this hydrogenase (8,9,21

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Mutants of Azotobacter chroococcum Defective in Hydrogenase Activity

Cited by 14 publications

References 23 publications

The Beneficial Effect of Hydrogenase in Azotobacter chroococcum Under Nitrogen-fixing, Carbon-limiting Conditions in Continuous and Batch Cultures

The Beneficial Effect of Hydrogenase in Azotobacter chroococcum Under Nitrogen-fixing, Carbon-limiting Conditions in Continuous and Batch Cultures

Identification and isolation of genes essential for H2 oxidation in Rhodobacter capsulatus

Organization of the genes encoding [Fe] hydrogenase in Desulfovibrio vulgaris subsp. oxamicus Monticello

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