Evidence for two pathways of thiosulfate oxidation in Starkeya novella (formerly Thiobacillus novellus).

The pathway of thiosulfate oxidation in the facultatively chemolithotrophic, sulfur-oxidizing bacterium Starkeya novella (formerly Thiobacillus novellus) has not been established beyond doubt. Recently, isolation of the sorAB genes, which encode a soluble sulfite:cytochrome c oxidoreductase, has been reported, indicating that a thiosulfate-oxidizing pathway not involving a multienzyme complex may exist in this organism. Here we report the cloning and sequencing of the soxBCD genes from S. novella, which are closely related to the corresponding genes encoding the thiosulfate-oxidizing multienzyme complex from Paracoccus pantotrophus. These findings suggest two distinct pathways for thiosulfate oxidation in S. novella. The expression of sorAB and soxC in cells grown on thiosulfate- and/or glucose-containing media was studied by Western blot analysis. The results showed that the SorAB protein is synthesized in the presence of thiosulfate irrespective of the presence of glucose. In contrast, the SoxC protein is subject to repression by glucose; the repression, however, appears to be dependent on the relative amounts of glucose and thiosulfate present. The regulatory effects observed for the expression of sorAB are likely to be mediated by an extracytoplasmic function sigma factor encoded by the sigE gene identified upstream of sorAB.

Characterization of the cys gene locus from Allochromatium vinosum indicates an unusual sulfate assimilation pathway.

Homologues of the genes cysB, cysI, cysH, cysD, cysN, and selD were identified in the genome of the phototrophic purple sulfur bacterium Allochromatium vinosum (formerly Chromatium vinosum). On the basis of amino acid comparisons these genes encode a ferredoxin-dependent siroheme-sulfite reductase (CysI), a plant-type assimilatory APS reductase without thioredoxin domain (CysH), the two different subunits of heterodimeric ATP sulfurylase (CysDN), a transcriptional regulator (CysB) and a selenophosphate synthase (SelD). cysIHDN appear to form an operon and are preceded by cysB which is transcribed in the opposite direction. SelD is situated downstream of cysN and transcribed divergently to cysIHDN. The lack of a gene for APS kinase and presence of a gene for an assimilatory APS reductase implies that assimilatory sulfate reduction in A. vinosum proceeds along the pathway suggested for higher plants without intermediary formation of PAPS. Two completely separate pathways involving specialized enzymes are used for assimilatory sulfate reduction and dissimilatory sulfur oxidation in A. vinosum. The presence of cysB indicates that the genes for assimilatory sulfate reduction are expressed only in the absence of reduced sulfur compounds.

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.

Molecular genetic evidence for extracytoplasmic localization of sulfur globules in Chromatium vinosum.

Purple sulfur bacteria store sulfur as intracellular globules enclosed by a protein envelope. We cloned the genes sgpA, sgpB, and sgpC, which encode the three different proteins that constitute the sulfur globule envelope of Chromatium vinosum D (DSMZ 180(T)). Southern hybridization analyses and nucleotide sequencing showed that these three genes are not clustered in the same operon. All three genes are preceded by sequences resembling sigma70-dependent promoters, and hairpin structures typical for rho-independent terminators are found immediately downstream of the translational stop codons of sgpA, sgpB, and sgpC. Insertional inactivation of sgpA in Chr. vinosum showed that the presence of only one of the homologous proteins SgpA and SgpB suffices for formation of intact sulfur globules. All three sgp genes encode translation products which - when compared to the isolated proteins - carry amino-terminal extensions. These extensions meet all requirements for typical signal peptides indicating an extracytoplasmic localization of the sulfur globule proteins. A fusion of the phoA gene to the sequence encoding the proposed signal peptide of sgpA led to high specific alkaline phosphatase activities in Escherichia coli, further supporting the envisaged targeting process. Together with electron microscopic evidence these results provide strong indication for an extracytoplasmic localization of the sulfur globules in Chr. vinosum and probably in other Chromatiaceae. Extracytoplasmic formation of stored sulfur could contribute to the transmembranous Deltap that drives ATP synthesis and reverse electron flow in Chr. vinosum.

Sulfide oxidation in the phototrophic sulfur bacterium Chromatium vinosum.

Sulfide oxidation in the phototrophic purple sulfur bacterium Chromatium vinosum D (DSMZ 180(T)) was studied by insertional inactivation of the fccAB genes, which encode flavocytochrome c, a protein that exhibits sulfide dehydrogenase activity in vitro. Flavocytochrome c is located in the periplasmic space as shown by a PhoA fusion to the signal peptide of the hemoprotein subunit. The genotype of the flavocytochrome-c-deficient Chr. vinosum strain FD1 was verified by Southern hybridization and PCR, and the absence of flavocytochrome c in the mutant was proven at the protein level. The oxidation of thiosulfate and intracellular sulfur by the flavocytochrome-c-deficient mutant was comparable to that of the wild-type. Disruption of the fccAB genes did not have any significant effect on the sulfide-oxidizing ability of the cells, showing that flavocytochrome c is not essential for oxidation of sulfide to intracellular sulfur and indicating the presence of a distinct sulfide-oxidizing system. In accordance with these results, Chr. vinosum extracts catalyzed electron transfer from sulfide to externally added duroquinone, indicating the presence of the enzyme sulfide:quinone oxidoreductase (EC 1.8.5.-). Further investigations showed that the sulfide:quinone oxidoreductase activity was sensitive to heat and to quinone analogue inhibitors. The enzyme is strictly membrane-bound and is constitutively expressed. The presence of sulfide:quinone oxidoreductase points to a connection of sulfide oxidation to the membrane electron transport system at the level of the quinone pool in Chr. vinosum.

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.

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.

Characterization of mucoid Pseudomonas aeruginosa strains isolated from technical water systems.

The occurrence of mucoid Pseudomonas aeruginosa strains was investigated in water samples and surface material from non-clinical aquatic environments. Ten of 81 environmental isolates displayed a mucoid colony type after incubation at 36 degrees C for 24 h on Pseudomonas Isolation Agar. The mucoid strains obtained exclusively from surfaces of technical water systems were characterized in terms of medium-dependent expression of mucoid colonial phenotype, exoenzyme profile, pigment production and O-antigen type. The mucoid strains secreted substantially higher quantities of carbohydrate and uronic acid-containing material compared to non-mucoid environmental isolates. Major slime components of the mucoid strains were identified as O-acetylated alginates that contained higher proportions of mannuronate than guluronate monomer residues and were composed of blocks of poly-mannuronate and poly-mannuronate/guluronate, whereas blocks of poly-guluronate were absent. The results suggest that surfaces in aquatic environments may represent a natural habitat for mucoid (i.e. alginate-overproducing) strains of Ps. aeruginosa with properties similar to clinical mucoid strains.

Adenylylsulphate reductase from the sulphate-reducing archaeon Archaeoglobus fulgidus: cloning and characterization of the genes and comparison of the enzyme with other iron-sulphur flavoproteins.

Adenylylsulphate (adenosine-5'-phosphosulphate, APS) reductase from the extremely thermophilic sulphate-reducing archaeon Archaeoglobus fulgidus is an iron-sulphur flavoprotein containing one non-covalently bound flavin group, eight non-haem iron and six labile sulphide atoms per molecule. Reevaluation of the enzyme structure revealed the presence of two different subunits with molecular masses of 80 and 18.5 kDa. The subunits are arranged in an alpha 2 beta subunit structure. We have cloned and sequenced a 2.7 kb segment of DNA containing the genes for the alpha and beta subunits, which we designate aprA and aprB, respectively. The two genes are separated by 17 bp and localized in the order aprBA. While a putative promoter could not be identified in the vicinity of aprBA a probable termination signal was found just downstream of the translation stop codon of aprA. The codon usage for aprBA shows strong preferences for G and C in the third codon position. aprA encodes a 73.3 kDa polypeptide, which shows significant overall similarities with the flavoprotein subunits of the succinate dehydrogenases from Escherichia coli and Bacillus subtilis and the corresponding flavoprotein of E. coli fumarate reductase. Part of the homologous peptide stretches could be assigned to domains that are involved in the binding of the substrate or of the FAD prosthetic group. aprB encodes a 17.1 kDa polypeptide representing an iron-sulphur protein, seven cysteine residues of which are arranged in two clusters typical of ligands of the iron-sulphur centres in ([Fe3S4][Fe4S4]) 7-Fe ferredoxins.

Dissimilatory sulphite reductase from Archaeoglobus fulgidus: physico-chemical properties of the enzyme and cloning, sequencing and analysis of the reductase genes.

A dissimilatory sulphite reductase was isolated from the extremely thermophilic dissimilatory sulphate-reducing archaeon Archaeoglobus fulgidus. In common with other dissimilatory sulphite reductases thus far characterized, the enzyme has an alpha 2 beta 2-structure and contains sirohaem, non-haem iron atoms and acid labile sulphide. The oxidized enzyme exhibited absorption maxima at 281, 394, 545 and 593 nm with a weak band around 715 nm. We have cloned and sequenced the genes for the alpha and beta subunits of this enzyme, which we designate dsrA and dsrB, respectively. They are contiguous in the order dsrA dsrB and probably comprise an operon, since dsrA is preceded by sequences characteristic of promoters in methanogenic archaea, and dsrB is followed by a sequence resembling termination signals in extremely thermophilic sulphur-dependent archaea. dsrA and dsrB encode 47.4 kDa and 41.7 kDa peptides, which have 25.6% amino acid sequence identity, indicating that they may have arisen by duplication of an ancestral gene. Each deduced peptide contains cysteine clusters resembling those postulated to bind sirohaem-[Fe4S4] complexes in sulphite reductases and nitrite reductases from other species. The dsrB encoded peptide lacks a single cysteine residue in one of the two clusters, suggesting that only the alpha subunit binds a sirohaem-[Fe4S4] complex, and chemical analyses showed the presence of only two sirohaems per alpha 2 beta 2 enzyme molecule. Both deduced peptides also contain an arrangement of cysteine residues characteristic of [Fe4S4] ferredoxins, and chemical analyses were consistent with the presence of six [Fe4S4] clusters per alpha 2 beta 2 enzyme molecule, two of which would be expected to be associated with sirohaem while the other four could bind to the ferredoxin-like sites.

Biosynthesis and function of trehalose in Ectothiorhodospira halochloris.

Trehalose 6-phosphate synthase, catalyzing the reaction between UDP-glucose and glucose 6-phosphate and forming trehalose 6-phosphate, was isolated and partially purified (30-fold) from the phototrophic, halo-alkaliphilic bacterium Ectothiorhodospira halochloris. The activity is stabilized by 20 mM MgCl2, 50 mM NaCe and 2M glycine betaine. The molecular weight was 63000. The enriched enzyme had a MgCl2 optimum at 3-6mM, a pH optimum at 7.5 (in Tris-HCl buffer) and a temperature optimum at 50 degrees C. The Km-values were 1.5 x 10(-3) M for UDP-glucose and 2 x 10(-3) M for glucose 6-phosphate. The enzyme showed a salinity dependence with optimal concentrations between 100 and 300mM salt. Higher concentrations of salt resulted in a decrease in activity. In the presence of inhibitory salt concentrations the compatible solute glycine betaine had a protective effect with a maximum between 0.5 and 2.0M.

Degradation of diphenylether by Pseudomonas cepacia Et4: enzymatic release of phenol from 2,3-dihydroxydiphenylether.

2,3-Dihydroxybiphenyl dioxygenase from Pseudomonas cepacia Et 4 was found to catalyze the ring fission of 2,3-dihydroxydiphenylether in the course of diphenylether degradation. The enzyme was purified and characterized. It had a molecular mass of 240 kDa and is dissociated by SDS into eight subunits of equal mass (31 kDa). The purified enzyme was found to be most active with 2,3-dihydroxybiphenyl as substrate and showed moderate activity with 2,3-dihydroxydiphenylether, catechol and some 3-substituted catechols. The Km-value of 1 microM for 2,3-dihydroxydiphenylether indicated a high affinity of the enzyme towards this substrate. The cleavage of 2,3-dihydroxydiphenylether by 2,3-dihydroxybiphenyl dioxygenase lead to the formation of phenol and 2-pyrone-6-carboxylate as products of ring fission and ether cleavage without participation of free intermediates. Isotope labeling experiments carried out with 18O2 and H2(18)O indicated the incorporation of 18O from the atmosphere into the carboxyl residue as well as into the carbonyl oxygen of the lactone moiety of 2-pyrone-6-carboxylate. Based on these experimental findings the reaction mechanism for the formation of phenol and 2-pyrone-6-carboxylate is proposed in accordance with the mechanism suggested by Kersten et al. (1982).

Spectroscopic studies on APS reductase isolated from the hyperthermophilic sulfate-reducing archaebacterium Archaeglobus fulgidus.

Adenylyl sulfate (APS) reductase, the key enzyme of the dissimilatory sulfate respiration, catalyzes the reduction of APS (the activated form of sulfate) to sulfite with release of AMP. A spectroscopic study was carried out with the APS reductase purified from the extremely thermophilic sulfate-reducing archaebacterium Archaeoglobus fulgidus DSM 4304. Combined ultraviolet/visible spectroscopy and low temperature electron paramagnetic resonance (EPR) studies were used in order to characterize the active centers and the reactivity towards AMP and sulfite of this enzyme. The A. fulgidus APS reductase is an iron-sulfur flavoprotein containing two distinct [4Fe-4S] clusters (Centers I and II) very similar to the homologous enzyme from Desulfovibrio gigas. Center I, which has a high redox potential, is reduced by AMP and sulfite, and Center II has a very negative redox potential.