Fundamentals of the fermentative production of hydrogen.

The molecular details behind hydrogen evolution during fermentation are reviewed. Hydrogen is evolved by hydrogenase, a class of enzymes containing complex metallo-centers. In most cases, sugars are degraded to pyruvate which in turn is converted to a variety of fermentation products. Various pathways leading to fermentative hydrogen generation are outlined and discussed. Thermophilic fermentations have higher yields than mesophilic ones. Yields are thought to be limited to 4H2 per glucose under standard conditions. The highlights of some actual studies of fermentations are presented and ways of potentially increasing hydrogen yields are discussed. It may be possible to achieve higher hydrogen yields by carrying out fermentations under microaerobic conditions where limited respiration could provide additional reducing power to drive the nearly complete conversion of sugar substrates to hydrogen.

Regulation of nitrogenase in the photosynthetic bacterium Rhodobacter sphaeroides containing draTG and nifHDK genes from Rhodobacter capsulatus.

The photosynthetic bacteria Rhodobacter capsulatus and Rhodospirillum rubrum regulate their nitrogenase activity by the reversible ADP-ribosylation of nitrogenase Fe-protein in response to ammonium addition or darkness. This regulation is mediated by two enzymes, dinitrogenase reductase ADP-ribosyl transferase (DRAT) and dinitrogenase reductase activating glycohydrolase (DRAG). Recently, we demonstrated that another photosynthetic bacterium, Rhodobacter sphaeroides, appears to have no draTG genes, and no evidence of Fe-protein ADP-ribosylation was found in this bacterium under a variety of growth and incubation conditions. Here we show that four different strains of Rba. sphaeroides are incapable of modifying Fe-protein, whereas four out of five Rba. capsulatus strains possess this ability. Introduction of Rba. capsulatus draTG and nifHDK (structural genes for nitrogenase proteins) into Rba. sphaeroides had no effect on in vivo nitrogenase activity and on nitrogenase switch-off by ammonium. However, transfer of draTG from Rba. capsulatus was sufficient to confer on Rba. sphaeroides the ability to reversibly modify the nitrogenase Fe-protein in response to either ammonium addition or darkness. These data suggest that Rba. sphaeroides, which lacks DRAT and DRAG, possesses all the elements necessary for the transduction of signals generated by ammonium or darkness to these proteins.

Regulation of nitrogenase activity in Rhodobacter capsulatus under dark microoxic conditions.

Rhodobacter capsulatus modulates its in vivo nitrogenase activity in the light in response to the addition of NH4+ in a variety of ways: with ADP-ribosylation of the Fe-protein of nitrogenase, with a switch-off response that is independent of ADP-ribosylation, and with a "magnitude response." In the light, these responses are differentially shown by cultures that differ in the degree of their nitrogen limitation. Here we examined the response of these culture types to the addition of NH4+ under dark, microoxic conditions and found that all three responses can be observed under these conditions. However, the magnitude response was much more sensitive to the ammonium concentration, and the ADP-ribosylation response correlated only poorly with activity changes, similar to results obtained in the light. In contrast to previous reports, Fe-protein was not ADP-ribosylated in response to the presence of oxygen.

Mutations permitting fdhF (formate dehydrogenase H) expression in a selD mutant of Salmonella typhimurium.

Translation of fdhF mRNA, encoding formate dehydrogenase H, requires the context-specific insertion of selenocysteine at an opal codon. We have cloned the Tn10 insertion previously shown to block fdhF transcription in Salmonella typhimurium and shown that it lies in selD, which encodes phosphoselenate synthetase. A spontaneous mutant of the selD::Tn10 strain that expresses anfdhF::lacZ protein fusion (where lacZ is fused after the opal codon) was isolated and characterized. Suppression showed the same context requirement as previously reported for SelB insertion of selenocysteine. The suppressing mutation was mapped by P22 co-transduction to the 74-75 min of the Salmonella typhimurium genome.

The presence of ADP-ribosylated Fe protein of nitrogenase in Rhodobacter capsulatus is correlated with cellular nitrogen status.

The photosynthetic bacterium Rhodobacter capsulatus has been shown to regulate its nitrogenase by covalent modification via the reversible ADP-ribosylation of Fe protein in response to darkness or the addition of external NH4+. Here we demonstrate the presence of ADP-ribosylated Fe protein under a variety of steady-state growth conditions. We examined the modification of Fe protein and nitrogenase activity under three different growth conditions that establish different levels of cellular nitrogen: batch growth with limiting NH4+, where the nitrogen status is externally controlled; batch growth on relatively poor nitrogen sources, where the nitrogen status is internally controlled by assimilatory processes; and continuous culture. When cultures were grown to stationary phase with different limiting concentrations of NH4+, the ADP-ribosylation state of Fe protein was found to correlate with cellular nitrogen status. Additionally, actively growing cultures (grown with N2 or glutamate), which had an intermediate cellular nitrogen status, contained a portion of their Fe protein in the modified state. The correlation between cellular nitrogen status and ADP-ribosylation state was corroborated with continuous cultures grown under various degrees of nitrogen limitation. These results show that in R. capsulatus the modification system that ADP-ribosylates nitrogenase in the short term in response to abrupt changes in the environment is also capable of modifying nitrogenase in accordance with long-term cellular conditions.

Short-term regulation of nitrogenase activity by NH4+ in Rhodobacter capsulatus: multiple in vivo nitrogenase responses to NH4+ addition.

The photosynthetic bacterium Rhodobacter capsulatus has been shown to carry out nitrogenase "switch-off," a rapid, reversible inhibition of in vivo activity. Here, we demonstrate that highly nitrogen-limited cultures of both the wild-type strain and a draT draG mutant are capable of nitrogenase switch-off while moderately nitrogen-limited cultures show instead a "magnitude" response, with a decrease in in vivo nitrogenase activity that is proportional to the amount of added NH4+.

Purification and characterization of pyruvate oxidoreductase from the photosynthetic bacterium Rhodobacter capsulatus.

Pyruvate:ferredoxin (flavodoxin) oxidoreductase (POR) was purified 3050-fold to apparent homogeneity from the photosynthetic bacterium Rhodobacter capsulatus using ion-exchange, Reactive Red, and gel filtration chromatography. The isolated enzyme was sensitive to dilution and oxygen (especially when in dilute solution). The molecular mass of the native enzyme was determined by high performance liquid chromatography gel filtration to be 270+/-20 kDa. Since a subunit molecular mass of 130+/-5 kDa was found by denaturing gel electrophoresis, POR from R. capsulatus thus appears to be a homodimer. Electron paramagnetic resonance analysis showed that a free radical was formed upon the addition of pyruvate. This POR is shown to be an indiscriminate electron donor causing the full reduction of R. capsulatus flavodoxin (Fld), R. capsulatus ferredoxin I (FdI), R. capsulatus ferredoxin II (FdII), as well as the major plant-type ferredoxin (FdI) from Anabaena variabilis. The purified enzyme can couple the oxidation of pyruvate to the reduction of nitrogenase in a coupled system with either R. capsulatus ferredoxins or nif-specific flavodoxin, NifF; (Fld>FdI>FdII). Immunoblot analysis shows that R. capsulatus POR is constitutively synthesized, with synthesis augmented under nitrogen-fixing conditions (34+/-13%) and decreased in acetate and aerobically grown cells.

Stopped-flow kinetic studies of low potential electron carriers of the photosynthetic bacterium, Rhodobacter capsulatus: ferredoxin I and NifF.

The kinetics of electron-transfer reactions involving nif-specific proteins from Rhodobacter capsulatus; ferredoxin I, NifF, Fe-protein of nitrogenase and dithionite were studied using stopped-flow spectrophotometry. Kinetic evidence was obtained for the formation of a tight (0.44 microM) complex between NifF and Fe-protein. Under the same conditions, FdI interacted only weakly (Kd > 325 microM) with Fe-protein. There was no evidence for complex formation between NifF and FdI since the reaction NifFSQ + FdIred had a bimolecular rate constant of 12.5 +/- 1.2 x 10(3) M-1 s-1. These results suggest that NifF, which is present in only small quantities in the cell, can make a significant contribution to the overall rate of nitrogen fixation due its high reactivity with Fe-protein. Moreover, the apparent lack of specific interaction between NifF and FdI suggest that they act in vivo in parallel to reduce Fe-protein and not in series.

Regulation of synthesis of pyruvate carboxylase in the photosynthetic bacterium Rhodobacter capsulatus.

The synthesis of pyruvate carboxylase (PC) was studied by using quantitative immunoblot analysis with an antibody raised against PC purified from Rhodobacter capsulatus and was found to vary 20-fold depending on the growth conditions. The PC content was high in cells grown on pyruvate or on carbon substrates metabolized via pyruvate (lactate, D-malate, glucose, or fructose) and low in cells grown on tricarboxylic acid (TCA) cycle intermediates or substrates metabolized without intermediate formation of pyruvate (acetate or glutamate). Under dark aerobic growth conditions with lactate as a carbon source, the PC content was approximately twofold higher than that found under light anaerobic growth conditions. The results of incubation experiments demonstrate that PC synthesis is induced by pyruvate and repressed by TCA cycle intermediates, with negative control dominating over positive control. The content of PC in R. capsulatus cells was also directly related to the growth rate in continuous cultures. The analysis of intracellular levels of pyruvate and TCA cycle intermediates in cells grown under different conditions demonstrated that the content of PC is directly proportional to the ratio between pyruvate and C4 dicarboxylates. These results suggest that the regulation of PC synthesis by oxygen and its direct correlation with growth rate may reflect effects on the balance of intracellular pyruvate and C4 dicarboxylates. Thus, this important enzyme is potentially regulated both allosterically and at the level of synthesis.

Cloning, characterization, and regulation of nifF from Rhodobacter capsulatus.

The Rhodobacter capsulatus nifF gene and upstream sequence were cloned by using a probe based on the N-terminal sequence of NifF. nifF was found to not be contained in the previously described nif regions I, II, and III. Comparison of the deduced amino acid sequence showed that it is highly similar to NifF from Azotobacter vinelandii and NifF from Klebsiella pneumoniae. Analysis of translational fusions demonstrated that the regulation of transcription was the same as previously reported at the protein level. Insertional mutagen esis showed that NifF contributes significantly to nitrogenase activity under normal nitrogen-fixing conditions and that it is absolutely required for nitrogen fixation under iron limitation.

Cloning and characterization of a gene cluster, phsBCDEF, necessary for the production of hydrogen sulfide from thiosulfate by Salmonella typhimurium.

We cloned, by complementation of an H2S- mutant, a cluster of Salmonella typhimurium genes, phsBCDEF, that appears to be essential for the anaerobic production of hydrogen sulfide from thiosulfate. Tn5 mutagenesis and ExoIII deletion analysis showed that approx. the entire region of a 3.3-kb subclone was necessary for H2S production. Subsequent sequencing revealed the presence of five potential translationally coupled open reading frames (ORFs). Their putative protein products were confirmed by synthesis from a phage T7 expression system. Comparison of the encoded sequences with previously determined sequences suggests that these genes constitute part of a thiosulfate-reducing operon coding for a membrane-associated electron transport chain which contains proteins potentially capable of ligating iron-sulfur clusters and heme. Immediately upstream from these genes, a region encoding the C-terminal portion of an ORF (OrfA) was identified that showed a high degree of similarity to some other anaerobic terminal reductases, polysulfide reductase (PsrA) of Wolinella succinogenes and dimethylsulfoxide reductase (DmsA), formate dehydrogenase (formate-hydrogene-lyase linked) (FdhF) and nitrate reductase (NarG) of Escherichia coli.

Purification and properties of a nif-specific flavodoxin from the photosynthetic bacterium Rhodobacter capsulatus.

A flavodoxin was isolated from iron-sufficient, nitrogen-limited cultures of the photosynthetic bacterium Rhodobacter capsulatus. Its molecular properties, molecular weight, UV-visible absorption spectrum, and amino acid composition suggest that it is similar to the nif-specific flavodoxin, NifF, of Klebsiella pneumoniae. The results of immunoblotting showed that R. capsulatus flavodoxin is nif specific, since it is absent from ammonia-replete cultures and is not synthesized by the mutant strain J61, which lacks a nif-specific regulator (NifR1). Growth of cultures under iron-deficient conditions causes a small amount of flavodoxin to be synthesized under ammonia-replete conditions and increases its synthesis under N2-fixing conditions, suggesting that its synthesis is under a dual system of control with respect to iron and fixed nitrogen availability. Here we show that flavodoxin, when supplemented with catalytic amounts of methyl viologen, is capable of efficiently reducing nitrogenase in an illuminated chloroplast system. Thus, this nif-specific flavodoxin is a potential in vivo electron carrier to nitrogenase; however, its role in the nitrogen fixation process remains to be established.

Purification and properties of a flavodoxin from the heterocystous cyanobacterium Anabaena sphaerica.

A flavodoxin was purified to homogeneity from the nitrogen-fixing heterocystous cyanobacterium Anabaena sphaerica grown under iron-limited conditions. The protein has a molecular mass of 21 kDa, and its spectral properties and amino-acid composition are very close to that of flavodoxins from other cyanobacteria. A. sphaerica flavodoxin supported the activities of A. sphaerica NADP reductase and Clostridium butyricum hydrogenase in reconstituted systems with illuminated plant chloroplasts as reductant. With the use of polyclonal anti-flavodoxin antiserum it was found that nitrogen-fixing cultures of A. sphaerica grown under iron-sufficient conditions contain low but significant amounts of flavodoxin (0.2-0.6 micrograms/mg crude extract protein) which increased dramatically (to 8-15 micrograms/mg crude extract protein) after the iron concentration in the medium was decreased to below 1 microM Fe. The flavodoxin content of both iron-limited and iron-sufficient. A. sphaerica was also shown to depend upon the growth phase of the (batch) cultures with a maximum at early exponential phase, coinciding with maximal in-vivo nitrogenase activity. These results suggest that A. sphaerica flavodoxin not only substitutes for ferredoxin under iron-limiting conditions, but also fulfills some specific role under iron-sufficient conditions.

Purification and properties of a bacterial-type ferredoxin from the nitrogen-fixing cyanobacterium Anabaena variabilis ATCC29413.

Three soluble ferredoxins were purified to homogeneity from nitrogen-fixing cultures of Anabaena variabilis (ATCC 29413) and characterized. The purified proteins have different absorption spectra, molecular mass, iron content, amino-acid composition and resistance to O2 inactivation. Two were plant-type ferredoxins FdI and FdxH, corresponding to the previously reported ferredoxins II and I (Böhme, H. and Schrautemeier, B. (1987) Biochim. Biophys. Acta 891, 1-7). The third ferredoxin (ferredoxin III) (previously not described in cyanobacteria) was a bacterial-type ferredoxin. Ferredoxin III has a molecular mass of about 6 kDa and contains 3-4 atoms Fe/mol. Native (oxidized) ferredoxin III shows an EPR-signal at g = 2.014 that disappears after reduction by dithionite, characteristic of ferredoxins containing three-iron clusters. Ferredoxin III, like ferredoxin FdxH, is inactivated by oxygen. Ferredoxin III supports higher rates of C2H2 reduction by Rhodobacter capsulatus nitrogenase than FdI and higher rates of H2 evolution by clostridial hydrogenase than FdI and FdxH. Combined nitrogen suppresses the synthesis of both nitrogenase and ferredoxin III. These data suggest a possible role of ferredoxin III (bacterial-type) in nitrogen fixation by A. variabilis.

Mutations that affect the regulation of phs in Salmonella typhimurium.

The regulation of phs [production of hydrogen sulphide (H2S)] in Salmonella typhimurium is complex. Previous studies have shown that expression is dependent upon the presence of reduced sulphur and anaerobiosis and is modulated by carbon source and growth stage. Transposon mutagenesis failed to find any potential trans-acting factors effective in the regulation of phs in relation to oxygen. Spontaneous mutants capable of expressing phs-lac aerobically were isolated and characterized. These mutations are closely linked to phs and affect not only oxygen regulation but also the requirement for cyclic AMP and reduced sulphur. Analysis of merodiploid strains indicates that these mutations cis-acting and that phs is not subject to autoregulation.

The role of ntrA in the anaerobic metabolism of Salmonella typhimurium.

The possible involvement of NtrA in the expression of several anaerobically induced genes in Salmonella typhimurium was investigated. Unlike Escherichia coli, where hydrogenase 3 is ntrA dependent, the introduction of a mutation in ntrA had virtually no effect on the hydrogenase activity, thought to be hydrogenase 3, of S. typhimurium LT7. Fumarate reductase and alcohol dehydrogenase activities were found to be diminished in ntrA mutant strains, but this may very well be indirect since fdhF mutant strains showed the same effect. These results suggest that in S. typhimurium NtrA is highly specific for the anaerobic expression of fdhF.

Mutations affecting nitrogenase switch-off in Rhodobacter capsulatus.

In vivo 'switch-off' and subsequent reactivation of nitrogenase activity in Rhodobacter capsulatus or Rhodospirillum rubrum in response to a variety of environmental stimuli, including the addition of fixed nitrogen, is thought to be due to the action of two nitrogenase Fe protein modifying activities; DRAT (dinitrogenase reductase ADP-ribosyl transferase) and DRAG (dinitrogenase reductase-activating glycohydrolase). Here it is demonstrated that strains, including one mutated in glnB, that constitutively express nif in the presence of fixed nitrogen are never-the-less capable of Fe protein modification. Thus the regulation of Fe protein modification is separate from that of its expression. The observations that Mn-deficient cultures are unable to fix nitrogen and that DRAG activity requires a divalent metal cation, most notably Mn2+, prompted the search for mutants (pseudo-prototrophs) capable of in vivo nitrogen fixation under Mn-deficient conditions. In the present study the isolation and partial characterization of several putative mutants is described. One, AF1, was shown to be altered in the in vivo regulation of N2ase activity in response to fixed nitrogen and to have an altered in vitro activity in glutamate grown cells. However, this strain was shown to possess in vitro DRAT activity and to have a modifiable Fe protein. Two-dimensional gel analysis indicates that this strain is altered in the synthesis of a 48 kDa protein of as yet unknown function. Thus, the mutation in this strain must affect, in an as yet undetermined manner, the response of the modifying system to fixed nitrogen.

Mutations in trans that affect formate dehydrogenase (fdhF) gene expression in Salmonella typhimurium.

Expression of the fdhF locus of Salmonella typhimurium is shown to be dependent upon ntrA and oxrB. However, the oxrB8 mutation is pleiotropic and also affects the expression of hyd, pepT, and chlC, whereas a mutation in ntrA does not. Insertional inactivation with Tn10 and localized mutagenesis permitted the definition and partial characterization of two new genes, fdhS and fdhR, which appear to be involved in the positive regulation of fdhF expression. Both genes were mapped to the 71- to 72-min region of the S. typhimurium chromosome with the gene order fdhS-crp-fdhR-rpsL. Mutations in fdhS specifically affect fdhF expression without affecting the expression of the other anaerobically induced genes or enzymes that were tested, including hyd, pepT, chlC, nitrite reductase, sulfite reductase, and trimethylamine-N-oxide reductase. Both fdhR and fdhS may be involved in fdhF regulation vis-à-vis oxygen, since localized mutagenesis produced alleles of both genes that permitted the aerobic expression of fdhF. However, fdhR may more directly interact with fdhF, since insertional inactivation of fdhS does not abolish aerobic expression of fdhF in fdhR mutant strains. Taken together, these results suggest that fdhS and fdhR act in concert under anaerobic conditions to activate fdhF transcription.

Characterization of anaerobic sulfite reduction by Salmonella typhimurium and purification of the anaerobically induced sulfite reductase.

Mutants of Salmonella typhimurium that lack the biosynthetic sulfite reductase (cysI and cysJ mutants) retain the ability to reduce sulfite for growth under anaerobic conditions (E. L. Barrett and G. W. Chang, J. Gen. Microbiol., 115:513-516, 1979). Here we report studies of sulfite reduction by a cysI mutant of S. typhimurium and purification of the associated anaerobic sulfite reductase. Sulfite reduction for anaerobic growth did not require a reducing atmosphere but was prevented by an argon atmosphere contaminated with air (less than 0.33%). It was also prevented by the presence of 0.1 mM nitrate, which argues against a strictly biosynthetic role for anaerobic sulfite reduction. Anaerobic growth in liquid minimal medium, but not on agar, was found to require additions of trace amounts (10(-7)M) of cysteine. Spontaneous mutants that grew under the argon contaminated with air also lost the requirement for 10(-7)M cysteine for anaerobic growth in liquid. A role for sulfite reduction in anaerobic energy generation was contraindicated by the findings that sulfite reduction did not improve cell yields, and anaerobic sulfite reductase activity was greatest during the stationary phase of growth. Sulfite reductase was purified from the cytoplasmic fraction of the anaerobically grown cysI mutant and was purified 190-fold. The most effective donor in crude extracts was NADH. NADPH and methyl viologen were, respectively, 40 and 30% as effective as NADH. Oxygen reversibly inhibited the enzyme. Two high-molecular-weight proteins separated by gel filtration (Mr 360,000 and 490,000, respectively) were required for maximal activity with NADH. Indirect evidence, including in vitro complementation experiments with a cysG mutant extract, suggested that the 360,000-Mr component contains siroheme and is the terminal reductase. This component was further purified to near homogeneity and was found to consist of a single subunit of molecular weight 67,500. The anaerobic sulfite reductase showed some resemblance to the biosynthetic sulfite reductase, but apparently it has a unique, as yet unidentified function.