Purification of Thiobacillus denitrificans siroheme sulfite reductase and investigation of some molecular and catalytic properties.

A siroheme-containing sulfite reductase was isolated from Thiobacillus denitrificans, purified to an electrophoretically homogenous state, and investigated with regard to some of its molecular and catalytic properties. The enzyme was a tetramer with a molecular weight of 160 000, consisting of two types of subunits arranged to an alpha 2 beta 2-structure. The molecular weight of the alpha-subunit was 38 000, that of the beta-subunit 43 000. As prosthetic groups siroheme and Fe/S groupings could be detected. The absorption spectrum showed maxima at 273 nm, 393 nm, and 594 nm; the molar extinction coefficient at these wavelengths were 280, 181, and 60 . 10(3) cm2 . mmol-1, respectively. With reduced viologen dyes the enzyme reduced sulfite to sulfide, thiosulfate and trithionate. In many properties T. denitrificans sulfite reductase closely resembled desulfoviridin, the dissimilatory sulfite reductase of Dssulfovibrio species. It is proposed that the physiological function of this enzyme is not to reduce but rather to form sulfite from reduced sulfur compounds in the course of dissimilatory sulfur oxidation in T. denitrificans.

Cloning and sequencing of the gene encoding the high potential iron-sulfur protein (HiPIP) from the purple sulfur bacterium Chromatium vinosum.

The gene encoding the high potential iron-sulfur protein (HiPIP) of Chromatium vinosum strain D (DSM 180T) was cloned from an EcoRI-HindIII digest of genomic DNA. A nucleotide sequence of 648 bp length was determined which contained the coding region and putative promoter and termination sites. The gene codes for a 122 residue 12761 Da protein. The C-terminal 85 residues are those of the previously biochemically determined sequence, whereas the N-terminal 37 residues constitute a leader peptide which shows characteristics of the double arginine signal sequences of complex cofactor containing periplasmic proteins.

In situ analysis of sulfur in the sulfur globules of phototrophic sulfur bacteria by X-ray absorption near edge spectroscopy.

During the oxidation of sulfide and thiosulfate purple and green sulfur bacteria accumulate globules of 'elemental' sulfur. Although essential for a thorough understanding of sulfur metabolism in these organisms, the exact chemical nature of the stored sulfur is still unclear. We applied sulfur K-edge X-ray absorption near edge spectroscopy (XANES) to probe the forms of sulfur in intact cells. Comparing XANES spectra of Allochromatium vinosum, Thiocapsa roseopersicina, Marichromatium purpuratum, Halorhodospira halophila and Chlorobium vibrioforme grown photolithoautotrophically on sulfide with reference probes (fingerprint method), we found sulfur chains with the structure R-S(n)-R. Evidence for the presence of sulfur rings, polythionates and anionic polysulfides in the sulfur globules of these bacteria was not obtained.

Dissimilatory ATP sulfurylase from the hyperthermophilic sulfate reducer Archaeoglobus fulgidus belongs to the group of homo-oligomeric ATP sulfurylases.

In the hyperthermophilic sulfate reducer Archaeoglobus fulgidus DSM 4304T, two open reading frames (sat and ORF2) are located upstream of the aprBA genes encoding adenosine-5'-phosphosulfate (APS) reductase. sat-ORF2-aprBA probably form a transcriptional unit, since sat is preceded by putative promoter sequences and termination signals are found downstream of aprA. While the 117-residue ORF2 product does not show significant similarity to known proteins, the 456-residue, 52.78-kDa, sat-encoded polypeptide exhibits similarity to the homo-oligomeric adenosine triphosphate (ATP) sulfurylases from sulfur-oxidizing bacteria and from sulfate-assimilating bacteria and eukaryotes. Functional overexpression of sat in Escherichia coli proved that the encoded protein acts as an ATP sulfurylase. The recombinant protein was purified to homogeneity and found to be a homo-dimer. Comparison of sulfate and thiosulfate grown A. fulgidus revealed that ATP sulfurylase and APS reductase are constitutive enzymes. Distance matrix analyses allowed insights into the evolution of prokaryotic ATP sulfurylases.

Phototrophic bacteria (an incoherent group of prokaryotes). A taxonomic versus phylogenetic survey.

In spite of their apparently consistent classical systematic scheme the phototropic bacteria are, as 16S-rRNA oligonucleotide cataloguing and sequencing have shown, deeply split into phylogenetic divisions of very little relationships between one another. Phototrophy as a mode of energy metabolism occurs in the phylogenetic divisions of a) "Gram-positive eubacteria", b) "Cyanobacteria/Chloroplasts", c) "Green Sulfur Bacteria", d) "Chloroflexus and related taxa", and e) "Purple Bacteria and related taxa", i.e. in five of the nine phylogenetic divisions of eubacteria. The arising disagreements are discussed and an attempt is made towards a stepwise reconciliation of taxonomy with phylogeny. The strong and the weak points in the taxonomy of phototrophic eubacteria are pointed out within the existing families. Emphasis is given to areas where taxonomic studies are urgently needed.

Ectothiorhodospira mobilis Pelsh, a photosynthetic sulfur bacterium depositing sulfur outside the cells.

From salt flats on the Galapagos Islands, two strains of a red photosynthetic bacterium were isolated and identified as Ectothiorhodospira mobilis, an organism first described by Pelsh in 1937. The cells are curved in a short spiral, 0.7 to 1.0 mu wide and 2.0 to 4.8 mu long. They are motile by a polar tuft of flagella. Cells contain several large stacks of lamellar membranes, carrying the pigments bacteriochlorophyll a and carotenoids of the spirillo xanthin series. Cell division occurs by binary fission, not budding. The organism is strictly anaerobic and obligately photosynthetic. Its ability to grow well with sulfide, sulfur, thiosulfate, or sulfite as photosynthetic H donors puts it taxonomically in the Thiorhodaceae. During growth with sulfide, elementary sulfur is deposited outside the cells in the medium and disappears during further growth. A limited number of organic carbon compounds can be utilized as hydrogen donors in place of inorganic sulfur compounds. Under these conditions, sulfate can serve as the sulfur source. The enzymes catalase and hydrogenase are present. The newly isolated strains require vitamin B(12). They also require a salinity of 2 to 3% NaCl, but they are not extreme halophiles. The organism is not identical with any of the species listed in Bergey's Manual.

Marine sponges as habitats of anaerobic phototrophic bacteria.

Enrichment cultures were prepared with different media for phototrophic bacteria from four species of marine sponges, collected from oxic coastal waters near Split (Yugoslavia). We obtained pure cultures of six strains ofChromatiaceae and two strains ofRhodospirillaceae by agar shake dilution. TheRhodospirillaceae were identified asRhodopseudomonas sulfidophila and a marine form ofRhodopseudomonas palustris. TheChromatiaceae were identified asChromatium vinosum, Chromatium gracile, Chromatium minutissimum. Ectothiorhodospira mobilis, and a Chromatium species, which in some respects resemblesChromatium minus. The occurrence of strictly anaerobic phototrophic bacteria in aerobic sponges is discussed with respect to nutrition and possible syntrophism.

Cysteine and S-sulfocysteine biosynthesis in phototrophic bacteria.

Forteen species (17 strains) of phototrophic bacteria as well as one strain of Thiobacillus denitrificans were tested for cysteine synthase and S-sulfocysteine synthase. All strains contain cysteine sythase active with O-acetylserine; only the Chromatiaceae, two species of the Rhodospirillaceae and T. denitrificans contain S-sulfocysteine synthase. In six species repression by different sulfur compounds in the medium was studied. In Chromatium vinosum, cysteine synthase was found to be constitutive, while in the Rhodospirillaceae tested the enzyme is repressed by sulfide. Thiosulfate had a derepressive effect in Rhodopseudomonas globiformis but strongly repressed cysteine synthase in R. sulfidophila and R. palustris. Cysteine had only moderate effects with the species tested.

Improved synthesis and rapid isolation millimole quantities of adenylysulfate.

An improved enzymatic method for the synthesis of adenylysulfate (APS) from adenosine 5'-phosphate using APS-reductase from Thiobacillus denitrificans is described. Isolation of millimole quantitities of this sulfur nucleotide is achieved rapidly by means of ion exchange chromatography on a strongly basic ion exchange resin. A facile and reproducible desalting procedure is described.

Cystine catabolism in mycelia of Microsporum gypseum, a dermatophytic fungus.

The fate of 35S label was studied during cystine degradation by mycelia of the dermatophytic fungus Microsporum gypseum. Excess free cystine in the medium was readily taken up and its sulfur moiety excreted as inorganic sulfate and sulfite. At intervals after 3-60 min of incubation with 35S cystine the products of cystine catabolism were extracted from the mycelia by boiling water and separated by thin layer chromatography and electrophoresis. A total of 10 sulfur-containing compounds were identified, and their relative radioactivity was assessed. After 3 min the mycelia contained, in addition to cystine, labeled cysteine and particularly cysteine sulfinic acid which was accompanied by a smaller amount of cysteic acid. Later on, oxidized and reduced glutathione, inorganic sulfate and taurine appeared consecutively. In all extracts, small amounts of labeled S-sulfocysteine were found, not, however, sulfite. The results suggest that the intermediates of cysteine degradation in the fungal mycelia are cysteine, cysteine sulfinate, unstable sulfinylpyruvate, sulfite and sulfate, i.e., that the catabolic pattern is similar to that of higher organisms. The formation and the role of S-sulfocysteine, cysteic acid, and of taurine is not yet completely understood, although certainly autoxidative processes are involved in the formation of the latter two compounds, and sulfitolysis in that of the former compound.

Purification and properties of adenylyl sulfate reductase from the phototrophic sulfur bacterium, Thiocapsa roseopersicina.

Adenylyl sulfate reductase was purified from Thiocapsa roseopersicina 60- to 80- fold, and the properties were studied. The molecular weight is 180,000. The enzyme contains, per molecule; one flavine group, two heme groups of cytochrome c character, four atoms of nonheme iron, and six labile sulfide groups. Cytochrome c and ferricyanide serve as electron acceptors. With ferricyanide as the electron acceptor, the pH optimum of the enzyme is at 8.0; with cytochrome c, the pH optimum is at 9.0. Of the nucleotides studied, adenosine 5'-monophosphate is most effective. The influence of substrate concentrations on the activity of the enzyme was studied, and the K(m) values for sulfite, adenosine 5'-monophosphate, ferricyanide, and cytochrome c were determined. The properties of the enzyme are compared with those of adenylyl sulfate reductases purified from sulfate-reducing bacteria and thiobacilli.

Reduction of adenylylsulfate and 3'-phosphoadenylylsulfate in phototrophic bacteria.

Extracts of 14 species of phototrophic bacteria, partly grown with different sulfur compounds, were tested for their ability to form volatile sulfur compounds from adenylylsulfate (APS) and 3'-phosphoadenylylsulfate (PAPS). The Rhodospirillum species showed marked activities with both APS and PAPS while the Rhodopseudomonas species seem to prefer PAPS. The Chromatiaceae exhibited the strongest activities with APS, whereas Chlorobium limicola had equally high activity with PAPS.

Taxonomy of phototrophic green and purple bacteria: a review.

The presently existing classification for the green and purple bacteria comprises physiological-ecological assemblages of phototrophic bacteria with anoxygenic photosynthesis. The taxonomic units of the different levels were based entirely on common phenotypic traits, including morphological, cytological, physiological and biochemical characteristics. Since degrees of resemblance form the basis of the grouping, this classification cannot reflect the genetic or evolutionary relatedness of these bacteria, neither among themselves nor with other bacteria. The advantage of the artificial system, however, is the use of features which can be established in most laboratories and which allow the comparison and identification of newly isolated strains with those already studied and described. The four existing families correspond to the four major recognized, ecophysiological groups, the Chlorobiaceae and Chloroflexaceae among the green bacteria, and the Chromatiaceae and Rhodospirillaceae among the purple bacteria. Our knowledge of all these groups is incomplete; this is reflected by the fact that seven new species have been described during the past three years (6th Newsletter on phot. bacteria, Trüper and Hansen, 1982). The description of the new genus and species Erythrobacter longus (Shiba and Simidu, 1982) is also interesting, as it comprises aerobic chemoorganotrophic marine bacteria which form bacteriochlorophyll a and carotenoids; however, no strains were able to grow phototrophilcally. Significant success is currently being obtained in the different approaches toward elucidating the genetic relationships within and outside of the purple and green bacteria. Detailed studies of the lipopolysaccharides of several species and genera of the Rhodospirillaceae (Weckesser et al., 1979, and more recent paper) have proven to be very useful for the recognition of relationships or dissimilarities between the species of a genus or between different genera. Amino acid sequence studies of cytochromes c from Rhodospirillaceae, other bacteria and eukaryotic organisms (Dickerson, 1980) have led to the recognition of four different groups of cytochrome c molecules (long, medium and two groups of short protein chains). The subdivision of the Rhodospirillaceae into three species groups, each possessing one of the three types of cytochrome c, proved to be in total agreement with the results of oligonucleotide cataloging of the 16 S ribosomal RNA of these bacteria (Gibson et al., 1979). The latter method also revealed that several chemotropic bacteria, including the nitrifying bacteria, are more closely related to certain purple bacteria than different species of the purple bacteria among themselves (Seewaldt et al., 1982).(ABSTRACT TRUNCATED AT 400 WORDS)

O-Acetylserine sulfhydrylase and S-sulfocysteine synthase activities of Rhodospirillum tenue.

O-Acetylserine sulfhydrylase in cell-free extracts of Rhodospirillum tenue was markedly repressed after growth in the presence of sulfide or thiosulfate, whereas S-sulfocysteine synthase activity remained almost unchanged. Purification on DE52 cellulose resulted in the separation of two proteins: Protein I with a molecular weight of 57000 had O-acetylserine sulfhydrylase activity only, while protein II with a molecular weight of 46000 had S-sulfocysteine synthase activity in addition. The activity of protein II with O-acetylserine plus sulfide was about 1.5 of that with O-acetylserine plus thiosulfate. Protein I from sulfate-grown cells possessed 74% of the total O-acetylserine sulfhydrylase, protein II 26%. Growth with sulfide repressed only the synthesis of protein I, which after separation showed only 19% of the measurable O-acetylserine sulfhydrylase, whereas protein II now possessed 81%. Regulatory and kinetic phenomena of the two activities were studied. In addition to the phototrophic bacteria studied earlier, also Rhodomicrobium vannielii, Rhodopseudomonas acidophila, Rhodocyclus purpureus and Thiocystis violacea were found to contain O-acetylserine sulfhydrylase activities; the latter two species contained S-sulfocysteine synthase activities in addition.

Malic enzyme of chromatium vinosum.

Malic enzyme of the phototropic bacterium Chromatium vinosum strain D that lacks malate dehydrogenase was partially purified yielding a specific activity of 55 units/mg protein. The constitutive enzyme with a molecular weight of 110,000 and a pH optimum of 8.0 was absolutely dependent on the presence of a monovalent cation (NH4+, K+, Cs+, or Rb+) as well as a divalent cation (Mn2+, or Mg2+). The enzyme was inhibited by oxaloacetate, glyoxyate, and NADPH. The K0.5 value for L-malate and the inhibition constants for oxaloacetate and glyoxylate are dependent on the concentration of the monovalent cation, whereas the Km value for NADP (18 microM) and the KI value for NADPH (42 microM) are independent. Throughout all kinetic measurements hyperbolic saturation curves and linear double reciprocal plots were obtained.

Partial purification and characterization of ADP sulfurylase from the purple sulfur bacterium Thiocapsa roseopersicina.

High activities of ADP and ATP sulfurylase were found in the soluble protein fraction of Thiocapsa roseopersicina strain 6311 (DSM 219). ADP sulfurylase was partially purified and characterized. It was a very labile soluble enzyme with a molecular weight of 250,000. The optimum pH was 7.5 and the optimal temperature 35 degrees C. Under test conditions the apparent Km values were determined to be 0.33 mM for adenylylsulfate and 13 mM for phosphate.