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The prominence of the alpha-subclass of Proteobacteria in the marine bacterioplankton community and their role in dimethylsulfide (DMS) production has prompted a detailed examination of dimethylsulfoniopropionate (DMSP) metabolism in a representative isolate of this phylotype, strain LFR. [1-(13)C]DMSP was synthesized, and its metabolism and that of its cleavage product, [1-(13)C]acrylate, were studied using nuclear magnetic resonance (NMR) spectroscopy. [1-(13)C]DMSP additions resulted in the intracellular accumulation and then disappearance of both [1-(13)C]DMSP and [1-(13)C]beta-hydroxypropionate ([1-(13)C]beta-HP), a degradation product. Acrylate, the immediate product of DMSP cleavage, apparently did not accumulate to high enough levels to be detected, suggesting that it was rapidly beta-hydroxylated upon formation. When [1-(13)C]acrylate was added to cell suspensions of strain LFR it was metabolized to [1-(13)C]beta-HP extracellularly, where it first accumulated and was then taken up in the cytosol where it subsequently disappeared, indicating that it was directly decarboxylated. These results were interpreted to mean that DMSP was taken up and metabolized by an intracellular DMSP lyase and acrylase, while added acrylate was beta-hydroxylated on (or near) the cell surface to beta-HP, which accumulated briefly and was then taken up by cells. Growth on acrylate (versus that on glucose) stimulated the rate of acrylate metabolism eightfold, indicating that it acted as an inducer of acrylase activity. DMSP, acrylate, and beta-HP all induced DMSP lyase activity. A putative model is presented that best fits the experimental data regarding the pathway of DMSP and acrylate metabolism in the alpha-proteobacterium, strain LFR.

Dimethylsulfoniopropionate (DMSP), an abundant osmoprotectant found in marine algae and salt marsh cordgrass, can be metabolized to dimethyl sulfide (DMS) and acrylate by microbes having the enzyme DMSP lyase. A suite of DMS-producing bacteria isolated from a salt marsh and adjacent estuarine water on DMSP agar plates differed markedly from the pelagic strains currently in culture. While many of the salt marsh and estuarine isolates produced DMS and methanethiol from methionine and dimethyl sulfoxide, none appeared to be capable of producing both methanethiol and DMS from DMSP. DMSP, and its degradation products acrylate and beta-hydroxypropionate but not methyl-3-mecaptopropionate or 3-mercaptopropionate, served as a carbon source for the growth of all the alpha- and beta- but only some of the gamma-proteobacterium isolates. Phylogenetic analysis of 16S rRNA gene sequences showed that all of the isolates were in the group Proteobacteria, with most of them belonging to the alpha and gamma subclasses. Only one isolate was identified as a beta-proteobacterium, and it had >98% 16S rRNA sequence homology with a terrestrial species of Alcaligenes faecalis. Although bacterial population analysis based on culturability has its limitations, bacteria from the alpha and gamma subclasses of the Proteobacteria were the dominant DMS producers isolated from salt marsh sediments and estuaries, with the gamma subclass representing 80% of the isolates. The alpha-proteobacterium isolates were all in the Roseobacter subgroup, while many of the gamma-proteobacteria were closely related to the pseudomonads; others were phylogenetically related to Marinomonas, Psychrobacter, or Vibrio species. These data suggest that DMSP cleavage to DMS and acrylate is a characteristic widely distributed among different phylotypes in the salt marsh-estuarine ecosystem.

Dimethylsulfoniopropionate (DMSP) is degraded to dimethylsulfide (DMS) and acrylate by the enzyme DMSP lyase. DMS or acrylate can serve as a carbon source for both free-living and endophytic bacteria in the marine environment. In this study, we report on the mechanism of DMSP-acrylate metabolism by Alcaligenes faecalis M3A. Suspensions of citrate-grown cells expressed a low level of DMSP lyase activity that could be induced to much higher levels in the presence of DMSP, acrylate, and its metabolic product, beta-hydroxypropionate. DMSP was degraded outside the cell, resulting in an extracellular accumulation of acrylate, which in suspensions of citrate-grown cells was then metabolized at a low endogenous rate. The inducible nature of acrylate metabolism was evidenced by both an increase in the rate of its degradation over time and the ability of acrylate-grown cells to metabolize this molecule at about an eight times higher rate than citrate-grown cells. Therefore, acrylate induces both its production (from DMSP) and its degradation by an acrylase enzyme. (1)H and (13)C nuclear magnetic resonance analyses were used to identify the products resulting from [1-(13)C]acrylate metabolism. The results indicated that A. faecalis first metabolized acrylate to beta-hydroxypropionate outside the cell, which was followed by its intracellular accumulation and subsequent induction of DMSP lyase activity. In summary, the mechanism of DMSP degradation to acrylate and the subsequent degradation of acrylate to beta-hydroxypropionate in the aerobic beta-Proteobacterium A. faecalis has been described.

The range of types of microbes with dimethylsulfoniopropionate (DMSP) lyase capability (enzymatic release of dimethylsulfide [DMS] from DMSP) has recently been expanded from bacteria and eukaryotic algae to include fungi (a species of the genus Fusarium [M. K. Bacic and D. C. Yoch, Appl. Environ. Microbiol. 64:106-111, 1998]). Fungi (especially ascomycetes) are the predominant decomposers of shoots of smooth cordgrass, the principal grass of Atlantic salt marshes of the United States. Since the high rates of release of DMS from smooth cordgrass marshes have a temporal peak that coincides with peak shoot death, we hypothesized that cordgrass fungi were involved in this DMS release. We tested seven species of the known smooth cordgrass ascomycetes and discovered that six of them exhibited DMSP lyase activity. We also tested two species of ascomycetes from other DMSP-containing plants, and both were DMSP lyase competent. For comparison, we tested 11 species of ascomycetes and mitosporic fungi from halophytes that do not contain DMSP; of these 11, only 3 were positive for DMSP lyase. A third group tested, marine oomycotes (four species of the genera Halophytophthora and Pythium, mostly from mangroves), showed no DMSP lyase activity. Two of the strains of fungi found to be positive for DMSP lyase also exhibited uptake of DMS, an apparently rare combination of capabilities. In conclusion, a strong correlation exists between a fungal decomposer's ability to catabolize DMSP via the DMSP lyase pathway and the host plant's production of DMSP as a secondary product.

A fungus, Fusarium lateritium, with dimethylsulfoniopropionate (DMSP) lyase activity was isolated from both seawater and a salt marsh due to its ability to grow on DMSP (with the evolution of dimethyl sulfide) as the sole source of carbon. This is the first reported case of DMSP lyase activity in a fungus. Several other common fungal genera tested did not have DMSP lyase activity. DMSP was taken up more rapidly by F. lateritium than it was utilized, leading to its intracellular accumulation. Inhibitor studies with nystatin and cyanide indicated that DMSP uptake was an energy-dependent process. The lyase was inducible by its substrate, DMSP (K(m), 1.2 mM), and by the substrate analogs choline and glycine betaine. During induction, DMSP lyase activity increased with time and then dropped rapidly. This loss of activity could be prevented by spiking the culture with fresh DMSP or choline. The V(max) for DMSP lyase was 34.7 mU . mg of protein. The inhibitory effects of nystatin, and p-chloromercuriphenylsulfonate on DMSP lyase activity suggested that the enzyme is cytosolic. Because plants like Spartina (a marsh grass) and marine algae contain high concentrations of DMSP, we speculate that DMSP-utilizing fungi may be involved in their decay.

Volume 63, no. 8, p. 3183, column 1, line 3 from the bottom: "0.012 versus 0.730" should read "0.022 versus 0.065." [This corrects the article on p. 3182 in vol. 63.].

In anoxic Spartina alterniflora-dominated sediments along a naturally occuring salinity gradient (the Cooper River estuary, South Carolina, U.S.A.), dimethylsulfoniopropionate (DMSP) was metabolized to dimethyl sulfide (DMS) and acrylate by sediment microbes. The rate of DMSP degradation and acrylate mineralization by sediment microbes was similar at all sites along this 25-km transect. However, sediments amended with acrylate (or DMSP) showed significantly higher rates of N2 fixation (measured as acetylene reduction activity) (ARA) in the saline sediments downstream than brackish sediments. These results are consistent with the fact that acrylate stimulated the rates of both denitrification and CO2 production in the saline sediments at the mouth of the river more than tenfold over rates in brackish sediments. Enrichment experiments indicate that microbes capable of using DMSP or acrylate were not present in upstream sediments despite the fact that microbial biomass, percent organic matter, and both glucose-stimulated ARA and denitrification were highest upstream. It appears that acrylate utilizing, N2 fixing, and denitrifying populations are insignificant in the lower salinity sediments of the estuary. These results may reflect the availability of DMSP, which averaged 10.3 nmol g wet wt-1 of saline sediments and levels less than our detection limit (1 &mgr;M) in brackish sediments.

The complete sequence of amino acids of ferredoxin II (FdII) from Rhodospirillum rubrum was determined by repetitive Edman degradation using pyridylethylated-ferredoxin and oxidized, denatured ferredoxin. Peptides derived from trypsin, pepsin, Glu-C endoproteinase, Arg-C endoproteinase, tryptophan specific cleavage and partial acid hydrolysis and C-terminal sequence from carboxypeptidase digestion were used to construct the total sequence. RrFdII is a polypeptide of 104 amino acids having a calculated molecular weight of 11556 excluding the iron and sulfur atoms. The complete amino acid sequence was: PYVVTENCIKCKYQDCVEVCPVDCFYEGENFLVINPDECIDCGVCNPECPAEAIAGKWLEINRKFADLWPNITRKGPAL ADADDWKDKPDKTGLLSENPGKGTV. Sequence comparisons, EPR characteristics and iron analyses indicate that RrFdII has structural features in common with ferredoxins containing [3Fe-4S], [4Fe-4S] centers. Of 104 amino acids, 60 (58%) including all 9 cysteines, are found in identical locations in the 7Fe ferredoxin prototype, Azotobacter vinelandii FdI.

Dimethylsulfoniopropionate (DMSP) lyase enzymatically cleaves DMSP, an algal metabolite, to produce acrylate, a proton, and dimethyl sulfide (DMS), the most abundant volatile sulfur compound emitted from oceans. The physiology of DMS production by DMSP lyase was studied in vivo in an Alcaligenes-like organism, strain M3A, a salt marsh bacterial isolate, and in a marine strain, Pseudomonas doudoroffii. Enzymes from both strains were induced at optimum rates by 1 mM DMSP and vigorous aeration. P. doudoroffii was very sensitive to continued aeration and lost activity rapidly; the enzyme was more stable when aeration ceased. In addition to DMSP, acrylate and several of its analogs acted as inducers of DMSP lyase in Alcaligenes sp. strain M3A but not in P. doudoroffii. Turnover of DMSP by P. doudoroffii was enhanced by 3.5% NaCl or seawater, whereas the Alcaligenes sp. strain M3A enzyme was not salt dependent and salt did not greatly affect its activity. The pH profile showed two peaks of DMSP lyase activity (6.5 and 8.8) for Alcaligenes sp. strain M3A and a single peak at pH 8 for P. doudoroffii. Enzyme activity in both organisms was inhibited by methyl-3-mercaptopropionate and homocysteine. Cyanide, azide and p-chloromercuribenzoate inhibited only the P. doudoroffii DMSP lyase. The apparent K(infm) values for DMSP for cell cultures of Alcaligenes sp. strain M3A and P. doudoroffii were ca. 2 mM and <20 (mu)M, respectively. The differences in the physiology of DMSP metabolism in these two bacterial isolates may enable them to exist in diverse ecological niches.

Dimethyl sulfide (DMS) is quantitatively the most important biogenic sulfur compound emitted from oceans and salt marshes. It is formed primarily by the action of dimethylsulfoniopropionate (DMSP) lyase which cleaves DMSP, an algal osmolyte, to equimolar amounts of DMS and acrylate. This report is the first to describe the isolation and purification of DMSP lyase. The soluble enzyme was purified to electrophoretic homogeneity from a facultatively anaerobic gram-negative rod-shaped marine bacterium identified as an Alcaligenes species by the Vitek gram-negative identification method. The key to successful purification of the enzyme was its binding to, and hydrophobic chromatography on, a phenyl-Sepharose CL-4B column. DMSP lyase biosynthesis was induced by its substrate, DMSP; its product, acrylate; and also by acrylamide. The relative effectivenesses of the inducers were 100, 90, and 204%, respectively. DMSP lyase is a 48-kDa monomer with a Michaelis-Menten constant (K(infm)) for DMSP of 1.4 mM and a V(infmax) of 408 (mu)mol/min/mg of protein. It converted DMSP to DMS and acrylate stoichiometrically. The similar K(infm) values measured for pure DMSP lyase and the axenic culture, seawater, and surface marsh sediment suggest that the microbes in these ecosystems must have enzymes similar to the one purified from our marine isolate. Anoxic sediment populations, however, have a 40-fold-lower K(infm) for this enzyme (30 (mu)M), possibly giving them the capability to metabolize much lower levels of DMSP than the aerobes.

Using an oligonucleotide probe derived from the amino acid sequence of Rhodospirillum rubrum ferredoxin I, the gene (fdxN) was identified, cloned and sequenced. The FdxN coding region is 183 nucleotides which codes for a 61 amino acid (7267 Da) protein. Phylogenetic comparisons between the R. rubrum FdI and other 8Fe-8S nif-coupled ferredoxins showed only moderate degrees of similarity between the amino acid sequences. R. rubrum FdI synthesis was stimulated by nif derepressing conditions, but was not completely repressed by nif repression. Previous reports of an extracellular clostridial-type ferredoxin in R. rubrum could not be confirmed.

Formate dehydrogenase (NAD+ dependent) was isolated from the obligate methanotroph Methylosinus trichosporium OB3b. When the enzyme was isolated anaerobically, two forms of the enzyme were seen on native polyacrylamide gels, DE-52 cellulose and Sephacryl S-300 columns; they were approximately 315,000 and 155,000 daltons. The enzyme showed two subunits on sodium dodecyl sulfate-polyacrylamide gels. The Mr of the alpha-subunit was 53,800 +/- 2,800, and that of the beta-subunit was 102,600 +/- 3,900. The enzyme (Mr 315,000) was composed of these subunits in an apparent alpha 2 beta 2 arrangement. Nonheme iron was present at a concentration ranging from 11 to 18 g-atoms per mol of enzyme (Mr 315,000). Similar levels of acid-labile sulfide were detected. No other metals were found in stoichiometric amounts. When the enzyme was isolated aerobically, there was no cofactor requirement for NAD reduction; however, when isolated anaerobically, activity was 80 to 90% dependent on the addition of flavin mononucleotide (FMN) to the reaction mixture. Furthermore, the addition of formate to an active, anoxic solution of formate dehydrogenase rapidly inactivated it in the absence of an electron acceptor; this activity could be reconstituted approximately 85% by 50 nM FMN. Flavin adenine dinucleotide could not replace FMN in reconstituting enzyme activity. The Kms of formate dehydrogenase for formate, NAD, and FMN were 146, 200, and 0.02 microM, respectively. "Pseudomonas oxalaticus" formate dehydrogenase, which has physical characteristics nearly identical to those of the M. trichosporium enzyme, was also shown to be inactivated under anoxic conditions by formate and reactivated by FMN. The evolutionary significance of this similarity is discussed.

A spontaneous pleiotropic Nif- mutation in Rhodospirillum rubrum has been partially characterized biochemically and by complementation analysis with recombinant plasmids carrying Azotobacter vinelandii DNA in the vicinity of ORF12 [Jacobson et al. (1989) J. Bacteriol 171: 1017-1027]. In addition to being unable to grow on N2 as a nitrogen source the phenotypic characterization of this and other metronidazole enriched spontaneous mutants showed (a) no nitrogenase activity, (b) the absence of NifHDK polypeptides, (c) a slower growth rate on NH4+, (d) approximately 50% higher glutamine synthetase (GS) activity than the wild-type, which was repressible, (e) an inability to switch-off GS activity in response to an NH4+ up-shift, and (f) an inability to modify (32P-label) the GS polypeptide. The apparent relationship between the absence of nifHDK expression and the absence of GS adenylylation cannot be explained in terms of the current model for nif gene regulation. However, R. rubrum transconjugants receiving A. vinelandii DNA which originated immediately upstream from nifH, restored all aspects of the wild-type phenotype. These data suggest a here-to-fore unrecognized relationship between nif expression and GS switch-off (adenylylation) activity, and the existence of a previously unidentified regulatory locus in Azotobacter that complements this mutation.

Nitrogen-starved purple non-sulphur bacteria have an active unregulated form of nitrogenase (nitrogenase A); however, the nitrogenase of a glutamine synthetase-negative mutant of Rhodopseudomonas capsulata, when nitrogen-starved, was predominantly inactive and required activation by Mn2+ and activating-factor protein. This regulatory form of nitrogenase has been called nitrogenase R. Treatment of wild-type cells (containing nitrogenase A) with methionine sulphoximine, an inhibitor of glutamine synthetase, converted the enzyme into nitrogenase R. Glutamine synthetase thus appears to control the intracellular concentrations of nitrogenase A and R and in this way regulates nitrogenase activity in the photosynthetic bacterium.

In vivo switch-off of nitrogenase activity by NH4+ is a reversible process in Rhodobacter sphaeroides and Methylosinus trichosporium OB3b. The same pattern of switch-off in Rhodospirillum rubrum is explained by ADP-ribosylation of one of the Fe protein subunits, however, no evidence of covalent modification could be found in the subunits from either R. sphaeroides or M. trichosporium. Fe protein subunits from these organisms showed no variant behaviour on SDS-PAGE, nor were they 32P-labeled following switch-off. These observations suggest either that the attachment of the modifying group to the Fe protein in these organisms is quite labile and does not survive in vitro manipulation, or that the mechanism of switch-off is different than that seen in Rhodospirillum.

Ethyl methanesulfonate rendered a high percentage of Rhodospirillum rubrum cells plasmidless and photosynthetically incompetent (Kuhl et al., J. Bacteriol. 156:737-742, 1983). By probing restriction endonuclease-digested chromosomal DNA from these plasmidless strains with 32P-labeled R. rubrum plasmid DNA, we showed that no homology exists between the plasmid and the chromosomal DNA of the mutant. Loss of the plasmid in all the nonphotosynthetic isolates was accompanied by the synthesis of spirilloxanthin under aerobic growth conditions, resistance to cycloserine and HgCl2, and loss of ability to grow fermentatively on fructose. Changes in both the protein and lipid composition of the membranes and the impaired uptake of 203HgCl2 in the plasmidless strains (compared with the wild type) suggest either that membrane modification occurs as a result of plasmid loss, accounting for several of the acquired phenotype characteristics of the cured strains, or that both membrane modification and plasmid loss are part of the same pleiotropic mutation.

A single plasmid of 55 kilobases was found in crude cell lysates of each of nine strains of Rhodospirillum rubrum. Restriction endonuclease analysis showed identical fragment patterns with a given nuclease for all plasmids except one, for which an additional EcoRI site was observed. Elimination of the plasmid required that the cells be passaged several times in 25 mM calcium-containing medium, followed by at least two passages under photosynthetic growth conditions in low-calcium medium before treatment with ethyl methanesulfonate. The resulting plasmidless mutants only grew aerobically and were all incapable of pigment formation and photosynthetic growth, suggesting that plasmid DNA is required for photosynthetic competence in R. rubrum.

Amino acid and nucleotide pools were measured in nitrogenase-containing Rhodospirillum rubrum cultures during NH4+- or dark-induced inactivation (switch-off) of the Fe protein. A big increase in the glutamine pool size preceded NH4+ switch-off of nitrogenase activity, but the glutamine pool remained unchanged during dark switch-off. Furthermore, methionine sulfoximine had no effect on the rate of dark switch-off, suggesting that glutamine plays no role in this process. In the absence of NH4+ azaserine, an inhibitor of glutamate synthate, raised glutamine pool levels sufficiently to initiate switch-off in vivo. While added NH4+ substantially increased the size of the nucleotide pools in N-limited cells, the kinetics of nucleotide synthesis were all similar and followed (rather than preceded) Fe protein inactivation. Darkness had little effect on nucleotide pool sizes. Glutamate pool sizes were also found to be important in NH4+ switch-off because of the role of this molecule as a glutamine precursor. Much of the diversity reported in the observations on NH4+ switch-off appears to be due to variations in glutamate pool sizes prior to the NH4+ shock. The nitrogen nutritional background is an important factor in determining whether darkness initiates nitrogenase switch-off; however, no link has yet been established between this and NH4+ (glutamine) switch-off.

A combination of inhibitors and carbon substrates was used to determine the relative contribution of sulfate-reducing bacteria (SRB) and fermenting bacteria to nitrogen fixation in a salt marsh sediment and on the roots of Spartina alterniflora. Because a lag period precedes acetylene-reducing activity (ARA) in amended sediments, an extensive analysis was done to be sure that this activity was due to the activation of dormant cells, not simply to cell proliferation. Since ARA was not affected by metabolic inhibitors such as rifampin, nalidixic acid, or methionine sulfoximine, it appeared that cell growth was not responsible for this activity. Instead, dormant cells were being activated by the added energy source. Molybdate inhibition studies with glucose-amended sediment slurries indicated that ARA in the upper 5 cm of the salt marsh was due primarily (70%) to SRB and that below that level (5 to 10 cm) it was due primarily (greater than 90%) to fermenting bacteria. ARA associated with washed roots of intact S. alterniflora plants was not inhibited by molybdate, which indicates that bacteria other than SRB were responsible. However, when the roots were excised from the plant, the activity (per unit of root mass) was 10-fold higher and was severely inhibited by molybdate. While this high activity is probably an artifact, due to the release of oxidizable substrates from the excised roots, it indicates that SRB are present in high numbers on Spartina roots.

The characteristics of soluble and membrane-bound glutamine synthetase (GS) from Rhodospirillum rubrum were compared with those of the enzyme located in situ (measured in detergent-treated cells). The results suggest that in vivo GS may be associated with, or bound to, the chromatophore membranes. GS was found to reversibly associate and dissociate from purified chromatophores as a function of the ionic strength of the buffer or the Mg2+ concentration. Solubilized GS was purified to homogeneity and found to be similar to the GS of enteric bacteria in that its molecular weight was about 600,000 and it had one type of subunit of 51,000 molecular weight. Removal of GS from the membrane had no effect on the Km values for the substrates of the biosynthetic reaction, but it did have a substantial effect on both its Mg2+ requirement (the Km increased 10-fold) and the sensitivity of the gamma-glutamyl transferase reaction to the inhibitor methionine sulfoximine (the I0.5 decreased from 1,500 to 60 microM). Both observations suggest that the active site of GS is influenced by its association with the membrane. GS activity was shown to respond to NH4+, phosphodiesterase, Mg2+, and adenylylation cofactors in a manner identical to that of the GS of the coliform bacteria, suggesting that the former may also respond to adenylylation and deadenylylation. Finally, R. rubrum GS was also inhibited by NH4+ by a newly observed, as yet undefined, system.

The high-potential 4Fe-4S* center ofAzotobacter vinelandii ferredoxin I has been titrated potentiometrically by a reductive procedure. The absorbance decrease at 510 nm accompanying the reduction of the high-potential center titrated with an Em of 320 mV (n = 1). The low-potential 4Fe-4S* center was titrated by using the absorbance decrease at 410 nm to monitor its reduction. This center exhibited an Em of -424 mV (n = 1).

The purification to homogeneity of the non-heme iron protein, sometimes referred to as either "red protein" or "paramagnetic protein", from Clostridium pasteurianum W5 extracts is described and its physicochemical properties studied. This paramagnetic protein (g= 1.94) has a molecular weight of about 25000 and contains two iron and two acid-labile sulfur atoms per mol of protein. Its midpoint potential at pH 7.5, as determined by electron paramagnetic resonance titration, is -300 mV. Optical circular dichroism and electron paramagnetic resonance spectra of the paramagnetic protein are similar to those of two iron-two acid-labile sulfur ferredoxins. The biochemical reduction of the purified protein was also studied.

Nitrogenase activity in Rhodospirillum rubrum was inhibited by NH4+ more rapidly in low light than in high light. Furthermore, the nitrogenase of cells exposed to phosphorylation uncouplers was inhibited by NH4+ more rapidly than was the nitrogenase of controls without an uncoupler. These observations suggest that high levels of photosynthate inhibit the nitrogenase inactivation system. L-Methionine-DL-sulfoximine, a glutamine synthetase inhibitor, prevented NH4+ from inhibiting nitrogenase activity, which suggests that NH4+ must be processed at least to glutamine for inhibition to occur. An inhibitor of glutamate synthase activity, 6-diazo-5-oxo-L-norleucine, inhibited nitrogenase activity in the absence of NH4+, but only in cells exposed to low light. The mechanism of 6-diazo-5-oxo-L-norleucine inhibition appeared to be the same as that induced by NH4+, because nitrogenase activity could be restored in vitro by activating enzyme and Mn2+. The inhibitor data suggest that the glutamine pool or a molecule that responds to it activates the Fe protein-modifying (or protein-inactivating) system and that the accumulation of this (unidentified) molecule is retarded when the cells are exposed to high light. It was confirmed here that Anabaena nitrogenase is also inhibited by NH4+, but only when the cells are incubated under low light. This inhibition, however, unlike that in R. rubrum, could be completely reversed in high light, suggesting that the mechanisms of nitrogenase inhibition by NH4+ in these two phototrophs are different.

Nitrogenase in Chromatium vinosum was rapidly, but reversibly inhibited by NH4+. Activity of the Fe protein component of nitrogenase required both Mn2+ and activating enzyme. Activating enzyme from Rhodospirillum rubrum could replace Chromatium chromatophores in activating the Chromatium Fe protein, and conversely, a protein fraction prepared from Chromatium chromatophores was effective in activating R. rubrum Fe protein. Inactive Chromatium Fe protein contained a peptide covalently modified by a phosphate-containing molecule, which migrated the same in SDS-polyacrylamide gels as the modified subunit of R. rubrum Fe protein. In sum, these observations suggest that Chromatium nitrogenase activity is regulated by a covalent modification of the Fe protein in a manner similar to that of R. rubrum.

In coupling the reducing power of illuminated chloroplasts to the nitrogenase from photosynthetically grown Rhodospirillum rubrum cells, one of the native ferredoxins. FdI, was found to be three times more effective than FdII.

Nitrogenase (N(2)ase) from the photosynthetic bacterium Rhodospirillum rubrum can exist in two forms, an unregulated form (N(2)ase A) and a regulatory form (N(2)ase R), the latter being identified in vitro by its need for activation by a Mn(2+)-dependent N(2)ase activating system. The physiological significance of this Mn(2+)-dependent N(2)ase activating system was suggested here by observations that growth of R. rubrum and Rhodopseudomonas capsulata on N(2) gas (a condition that produces active N(2)ase R) required Mn(2+), but growth on ammonia or glutamate did not. Manganese could not be shown to be required for the biosynthesis of either nitrogenase or glutamine synthetase or for glutamine synthetase turnover, but it was required for the in vitro activation of N(2)ases from N(2) and glutamate-grown R. rubrum and R. capsulata cells. Chromatium N(2)ase, in contrast, was always fully active and did not require Mn(2+) activation, suggesting that only the purple nonsulfur bacteria are capable of controlling their N(2)ase activity by this new type of regulatory system. Although R. rubrum could not substitute Fe(2+) for Mn(2+) in the in vivo N(2) fixation process, Fe(2+) and, to a lesser extent, Co(2+) could substitute for Mn(2+) in the in vitro activation of N(2)ase. Electron paramagnetic resonance spectroscopy of buffer-washed R. rubrum chromatophores showed lines characteristic of Mn(2+). Removal of the Mn(2+)-dependent N(2)ase activating factor by a salt wash of the chromatophores removed 90% of the Mn(2+), which suggested a specific coupling of this metal to the activating factor. The data presented here all indicate that Mn(2+) plays an important physiological role in regulating the N(2) fixation process by these photosynthetic bacteria.

Other investigators have isolated soluble, low-potential, c-type cytochromes (cytochrome c3) from a few photosynthetic procaryotes, i.e., a cyanobacterium and two species of purple nonsulfur bacteria. However, such cytochromes appeared to be absent from other purple bacteria, including Rhodospirillum rubrum and Chromatium vinosum. We now report evidence for the presence of low-potential c-type cytochromes in these two species, in which they were found to be bound to the photosynthetic membranes. Evidence for a membrane-bound, low-potential c-type cytochrome was also found in Rhodopseudomonas sphaeoides. The low-potential c-type cytochrome of R. rubrum was solubilized by a Triton X-100 treatment of chromatophores and was partly purified. It was found to have a molecular weight of about 17,000, a midpoint oxidation-reduction potential of -192 mV, and an alpha-absorption peak at 552 nm. It appears that low-potential c-type cytochromes may be present in all purple photosynthetic bacteria, of both the sulfur and the nonsulfur types.

In the photosynthetic bacterium Rhodopseudomonas capsulata, NH4+ switch-off of nitrogenase activity can be mimicked by its analog, methylamine. Like NH4+, methylamine appeared to require processing by glutamine synthetase (GS) before it was effective; gamma-glutamylmethylamide was shown to be the product of this reaction. Evidence that this glutamine analog functioned directly to initiate nitrogenase inactivation was suggested first by the fact that it was a poor substrate for glutamate synthase (i.e., it was not further metabolized by this pathway) and secondly, azaserine which blocks the transfer of the glutamine amide group had no effect on CH3NH3+ (or NH4+) switch-off. These observations are taken as preliminary evidence to suggest that when NH4+ inhibits nitrogenase activity, inactivation is initiated by glutamine itself, and not a molecule derived from it. Finally, evidence was presented that R. capsulata would use CH3NH3+ as a nitrogen substrate, but lag periods and generation times increased with subsequent passages.

Two uptake hydrogenases were found in the obligate methanotroph Methylosinus trichosporium OB3b; one was constitutive, and a second was induced by H2. Both hydrogenases could be assayed by measuring methylene blue reduction anaerobically or by coupling their activity to nitrogenase acetylene reduction activity in vivo in an O2-dependent reaction. The H2 concentration for half-maximal activity of the inducible and constitutive hydrogenases in both assays was 0.01 and 0.5 bar (1 and 50 kPa), respectively, making it easy to distinguish these enzymes from one another both in vivo and in vitro. Hydrogen uptake was shown to be coupled to ATP synthesis in methane-starved cells. Methane, methanol, formate, succinate, and glucose all repressed the H2-mediated synthesis of the inducible hydrogenase. Furthermore, this enzyme was only expressed in N-starved cultures and was repressed by NH4+ and NO3-; synthesis of the constitutive hydrogenase was not affected by excess N in the growth medium. In nickel-free, EDTA-containing medium, the activities of these two enzymes were negligible; however, both enzyme activities appeared rapidly following the addition of nickel to the culture. Chloramphenicol, when added along with nickel, had no effect on the rapid appearance of either the constitutive or inducible activity, indicating that nickel is not required for synthesis of the hydrogenase apoproteins. These observations all suggest that these hydrogenases are nickel-containing enzymes. Finally, both hydrogenases were soluble and could be fractionated by 20% ammonium sulfate; the constitutive enzyme remained in the supernatant solution, while the inducible enzyme was precipitated under these conditions.

The regulatory effect of NH4+ on nitrogen fixation in a Spartina alterniflora salt marsh was examined. Acetylene reduction activity (ARA) measured in situ was only partially inhibited by NH4+ in both the light and dark after 2 h. In vitro analysis of bulk sediment divided into sediment particles, live and dead roots, and rhizomes showed that microbes associated with sediment and dead roots have a great potential for anaerobic C2H2 reduction, but only if amended with a carbon source such as mannose. Only live roots had significant rates of ARA without an added carbon source. In sediment, N2-fixing mannose enrichment cultures could be distinguished from those enriched by lactate in that only the latter were rapidly inhibited by NH4+. Ammonia also inhibited ARA in dead and live roots and in surface-sterilized roots. The rate of this inhibition appeared to be too rapid to be attributed to the repression and subsequent dilution of nitrogenase. The kinetic characteristics of this inhibition and its prevention in root-associated microbes by methionine sulfoximine are consistent with the NH4+ switch-off-switch-on mechanism of nitrogenase regulation.

The photosynthetic bacterium Rhodospirillum rubrum regulates the activity of its nitrogenase (N2ase) by interconverting the enzyme into three distinct enzymatic species: N2ase A (a fully active form) and two regulatory forms, N2ase Ractive and N2ase Rinactive. N2ase R is distinguished from N2ase A in vitro by the requirement of its Fe protein for activation by a Mn2+-dependent activating factor. N2ase is converted from the A to the R form in response to certain environmental factors such as carbon starvation, depletion of intracellular adenosine triphosphate, or the addition of NH4+ (or glutamate) to a culture of N-starved cells. The rapid inhibition of R. rubrum N2ase in vivo by NH4+ was shown to result from the conversion of N2ase A to N2ase Rinactive. On depletion of NH4+ from the culture, whole-cell N2ase activity returned; however, the enzyme remained in the R form. Unlike the effect of NH4+, adding glutamate to cells containing N2ase A did not inhibit in vivo activity, but converted the enzyme to the R form (N2ase Ractive). Although glutamate-induced N2ase R formation was much slower than the NH4+-induced reaction, it occurred in the presence of rifampin, indicating that de novo protein synthesis was not involved. This suggested that N2ase R was formed by a modification of N2ase A. Although glutamine synthetase in involved in the conversion of N2ase A to R, the adenylylation state of glutamine synthetase appears not to be involved in regulating this nitrogenase reaction.

Acetylene reduction by nitrogenase from Rhodospirillum rubrum, unlike that by other nitrogenases, was recently found by other investigators to require an activation of the iron protein of nitrogenase by an activating system comprising a chromatophore membrane component, adenosine 5'-triphosphate (ATP), and divalent metal ions. In an extension of this work, we observed that the same activating system was also required for nitrogenase-linked H(2) evolution. However, we found that, depending on their nitrogen nutrition regime, R. rubrum cells produced two forms of nitrogenase that differed in their Fe protein components. Cells whose nitrogen supply was totally exhausted before harvest yielded predominantly a form of nitrogenase (A) whose enzymatic activity was not governed by the activating system, whereas cells supplied up to harvest time with N(2) or glutamate yielded predominantly a form of nitrogenase (R) whose enzymatic activity was regulated by the activating system. An unexpected finding was the rapid (less than 10 min in some cases) intracellular conversion of nitrogenase A to nitrogenase R brought about by the addition to nitrogen-starved cells of glutamine, asparagine, or, particularly, ammonia. This finding suggests that mechanisms other than de novo protein synthesis were involved in the conversion of nitrogenase A to the R form. The molecular weights of the Fe protein and Mo-Fe protein components from nitrogenases A and R were the same. However, nitrogenase A appeared to be larger in size, because it had more Fe protein units per Mo-Fe protein than did nitrogenase R. A distinguishing property of the Fe protein from nitrogenase R was its ATP requirement. When combined with the Mo-Fe protein (from either nitrogenase A or nitrogenase R), the R form of Fe protein required a lower ATP concentration but bound or utilized more ATP molecules during acetylene reduction than did the A form of Fe protein. No differences between the Fe proteins from the two forms of nitrogenase were found in the electron paramagnetic resonance spectrum, midpoint oxidation-reduction potential, or sensitivity to iron chelators.

The coupling of root-associated nitrogen fixation and plant photosynthesis was examined in the salt marsh grass Spartina alterniflora. In both field experiments and hydroponic assay chambers, nitrogen fixation associated with the roots was rapidly enhanced by stimulating plant photosynthesis. A kinetic analysis of acetylene reduction activity (ARA) showed that a five-to sixfold stimulation occurred within 10 to 60 min after the plant leaves were exposed to light or increased CO2 concentrations (with the light held constant). In field experiments, CO2 enrichment increased plant-associated ARA by 27%. Further evidence of the dependence of ARA on plant photosynthate was obtained when activity in excised roots was shown to decrease after young greenhouse plants were placed in the dark. Seasonal variation in the ARA of excised plant roots from field cores appears to be related to the annual cycle of net photosynthesis in S. alterniflora.

Rhodospirillum rubrum was shown to grow fermentatively on fructose with N2 as a nitrogen source. The nitrogenase activity of these cells was regulated by the NH4+ switch-off/switch-on mechanism in a manner identical to that for photosynthetically grown cells. In vitro, the inactive nitrogenase Fe protein from fermenting cells was reactivated by an endogenous membrane-bound, Mn2+-dependent activating enzyme that was interchangeable with the activating enzyme isolated from photosynthetic membranes.

The Fe protein activating enzyme for Rhodospirillum rubrum nitrogenase was purified to approximately 90% homogeneity, using DE52-cellulose chromatography and sucrose density gradient centrifugation. Activating enzyme consists of a single polypeptide of molecular weight approximately 24,000. ATP was required for catalytic activity, but was relatively ineffective in the absence of Mg2+. When the concentration of MgATP2- was held in excess, there was an additional requirement for a free divalent metal ion (Mn2+) for enzyme activity. Kinetic experiments showed that the presence of Mg2+ influenced the apparent binding of Mn2+ by the enzyme, resulting in a lowering of the concentration of Mn2+ required to give half-maximum activity (K alpha) as the free Mg2+ concentration was increased. A low concentration of Mn2+ had a sparing effect on the requirement for free Mg2+. There is apparently a single metal-binding site on activating enzyme which preferentially binds Mn2+ as a positive effector, and free Mg2+ can compete for this site.

A ferredoxin-NAD+ oxidoreductase (EC 1.18.1.3) has been isolated from extracts of the obligate methanotroph Methylosinus trichosporium OB3b. This enzyme was shown to couple electron flow from formate dehydrogenase (NAD+ requiring) to ferredoxin. Ferredoxin-NAD+ reductase was purified to homogeneity by conventional chromatography techniques and was shown to be a flavoprotein with a molecular weight of 36,000 +/- 1,000. This ferredoxin reductase was specific for NADH (Km, 125 microM) and coupled electron flow to the native ferredoxin and to ferredoxins from spinach, Clostridium pasteurianum, and Rhodospirillum rubrum (ferredoxin II). M. trichosporium ferredoxin saturated the ferredoxin-NAD+ reductase at a concentration 2 orders of magnitude lower (3 nM) than did spinach ferredoxin (0.4 microM). Ferredoxin-NAD+ reductase also had transhydrogenase activity which transferred electrons and protons from NADH to thionicotinamide adenine dinucleotide phosphate (Km, 9 microM) and from NADPH to 3-acetylpyridine adenine dinucleotide (Km, 16 microM). Reconstitution of a soluble electron transport pathway that coupled formate oxidation to ferredoxin reduction required formate dehydrogenase, NAD+, and ferredoxin-NAD+ reductase.

A flavoprotein, named azotoflavin, was isolated from an extract of Azotobacter vinelandii cells, which linked the reducing power generated by illuminated spinach chloroplasts to the Azotobacter nitrogen-fixing enzyme complex. The photoreduction of the yellow azotoflavin by chloroplasts produced a stable, free-radical semiquinone, blue in color, with properties similar to those described by other investigators for an Azotobacter flavoprotein of unknown biological function.