Sulfur Globule Research Articles

Dynamically Quantifying Intracellular Elemental Sulfur and Predicting Pertinent Gene Transcription by Raman Spectroscopy in Living Cells.

The ability to monitor changes in metabolites and corresponding gene transcription within living cells is highly desirable. However, most current assays for quantification of metabolites or for gene transcription are destructive, precluding tracking the real-time dynamics of living cells. Here, we used the intracellular elemental sulfur in a Thiophaeococcus mangrovi cell as a proof-of-concept to link the quantity of metabolites and relevant gene transcription in living cells by a nondestructive Raman approach. Raman spectroscopy was utilized to quantify intracellular elemental sulfur noninvasively, and a computational mRR (mRNA and Raman) model was developed to infer the transcription of genes relevant to elemental sulfur. The results showed a significant linear correlation between the exponentially transformed Raman spectral intensity of intracellular elemental sulfur and the mRNA levels of genes encoding sulfur globule proteins in T. mangrovi. The mRR model was verified independently in two genera of Thiocapsa and Thiorhodococcus, and the mRNA levels predicted by mRR showed high consistency with actual gene expression detected by real-time polymerase chain reaction (PCR). This approach could enable noninvasive assessment of the quantity of metabolites and link the pertinent gene expression profiles in living cells, providing useful baseline data to spectroscopically map various omics in real time.

Comparative Genomics of Beggiatoa leptomitoformis Strains D-401 and D-402T with Contrasting Physiology but Extremely High Level of Genomic Identity.

Representatives of filamentous colorless sulfur-oxidizing bacteria often dominate in sulfide biotopes, preventing the diffusion of toxic sulfide into the water column. One of the most intriguing groups is a recently described Beggiatoa leptomitoformis including strains D-401 and D-402T. Both strains have identical genes encoding enzymes which are involved in the oxidation of hydrogen sulfide and thiosulfate. Surprisingly, the B. leptomitoformis strain D-401 is not capable to grow lithotrophically in the presence of reduced sulfur compounds and to accumulate elemental sulfur inside the cells, in contrast to the D-402T strain. In general, genomes of D-401 and D-402T have an extremely high level of identity and only differ in 1 single-letter substitution, 4 single-letter indels, and 16 long inserts. Among long inserts, 14 are transposons. It was shown that in the D-401 strain, a gene coding for a sulfur globule protein was disrupted by one of the mentioned transposons. Based on comparative genomics, RT-qPCR, and HPLC-MS/MS, we can conclude that this gene plays a crucial role in the formation of the sulfur globules inside the cells, and the disruption of its function prevents lithotrophic growth of B. leptomitoformis in the presence of reduced sulfur compounds.

Metabolomic profiling of the purple sulfur bacterium Allochromatium vinosum during growth on different reduced sulfur compounds and malate.

Environmental fluctuations require rapid adjustment of the physiology of bacteria. Anoxygenic phototrophic purple sulfur bacteria, like Allochromatium vinosum, thrive in environments that are characterized by steep gradients of important nutrients for these organisms, i.e., reduced sulfur compounds, light, oxygen and carbon sources. Changing conditions necessitate changes on every level of the underlying cellular and molecular network. Thus far, two global analyses of A. vinosum responses to changes of nutritional conditions have been performed and these focused on gene expression and protein levels. Here, we provide a study on metabolite composition and relate it with transcriptional and proteomic profiling data to provide a more comprehensive insight on the systems level adjustment to available nutrients. We identified 131 individual metabolites and compared availability and concentration under four different growth conditions (sulfide, thiosulfate, elemental sulfur, and malate) and on sulfide for a ΔdsrJ mutant strain. During growth on malate, cysteine was identified to be the least abundant amino acid. Concentrations of the metabolite classes “amino acids” and “organic acids” (i.e., pyruvate and its derivatives) were higher on malate than on reduced sulfur compounds by at least 20 and 50 %, respectively. Similar observations were made for metabolites assigned to anabolism of glucose. Growth on sulfur compounds led to enhanced concentrations of sulfur containing metabolites, while other cell constituents remained unaffected or decreased. Incapability of sulfur globule oxidation of the mutant strain was reflected by a low energy level of the cell and consequently reduced levels of amino acids (40 %) and sugars (65 %).Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-014-0649-7) contains supplementary material, which is available to authorized users.

A Comparative Quantitative Proteomic Study Identifies New Proteins Relevant for Sulfur Oxidation in the Purple Sulfur Bacterium Allochromatium vinosum

In the present study, we compared the proteome response of Allochromatium vinosum when growing photoautotrophically in the presence of sulfide, thiosulfate, and elemental sulfur with the proteome response when the organism was growing photoheterotrophically on malate. Applying tandem mass tag analysis as well as two-dimensional (2D) PAGE, we detected 1,955 of the 3,302 predicted proteins by identification of at least two peptides (59.2%) and quantified 1,848 of the identified proteins. Altered relative protein amounts (≥1.5-fold) were observed for 385 proteins, corresponding to 20.8% of the quantified A. vinosum proteome. A significant number of the proteins exhibiting strongly enhanced relative protein levels in the presence of reduced sulfur compounds are well documented essential players during oxidative sulfur metabolism, e.g., the dissimilatory sulfite reductase DsrAB. Changes in protein levels generally matched those observed for the respective relative mRNA levels in a previous study and allowed identification of new genes/proteins participating in oxidative sulfur metabolism. One gene cluster (hyd; Alvin_2036-Alvin_2040) and one hypothetical protein (Alvin_2107) exhibiting strong responses on both the transcriptome and proteome levels were chosen for gene inactivation and phenotypic analyses of the respective mutant strains, which verified the importance of the so-called Isp hydrogenase supercomplex for efficient oxidation of sulfide and a crucial role of Alvin_2107 for the oxidation of sulfur stored in sulfur globules to sulfite. In addition, we analyzed the sulfur globule proteome and identified a new sulfur globule protein (SgpD; Alvin_2515).

Genome-Wide Transcriptional Profiling of the Purple Sulfur Bacterium Allochromatium vinosum DSM 180T during Growth on Different Reduced Sulfur Compounds

The purple sulfur bacterium Allochromatium vinosum DSM 180(T) is one of the best-studied sulfur-oxidizing anoxygenic phototrophic bacteria, and it has been developed into a model organism for laboratory-based studies of oxidative sulfur metabolism. Here, we took advantage of the organism's high metabolic versatility and performed whole-genome transcriptional profiling to investigate the response of A. vinosum cells upon exposure to sulfide, thiosulfate, elemental sulfur, or sulfite compared to photoorganoheterotrophic growth on malate. Differential expression of 1,178 genes was observed, corresponding to 30% of the A. vinosum genome. Relative transcription of 551 genes increased significantly during growth on one of the different sulfur sources, while the relative transcript abundance of 627 genes decreased. A significant number of genes that revealed strongly enhanced relative transcription levels have documented sulfur metabolism-related functions. Among these are the dsr genes, including dsrAB for dissimilatory sulfite reductase, and the sgp genes for the proteins of the sulfur globule envelope, thus confirming former results. In addition, we identified new genes encoding proteins with appropriate subcellular localization and properties to participate in oxidative dissimilatory sulfur metabolism. Those four genes for hypothetical proteins that exhibited the strongest increases of mRNA levels on sulfide and elemental sulfur, respectively, were chosen for inactivation and phenotypic analyses of the respective mutant strains. This approach verified the importance of the encoded proteins for sulfur globule formation during the oxidation of sulfide and thiosulfate and thereby also documented the suitability of comparative transcriptomics for the identification of new sulfur-related genes in anoxygenic phototrophic sulfur bacteria.

Quantitative proteomics of Chlorobaculum tepidum: insights into the sulfur metabolism of a phototrophic green sulfur bacterium

Chlorobaculum (Cba.) tepidum is a green sulfur bacterium that oxidizes sulfide, elemental sulfur, and thiosulfate for photosynthetic growth. To gain insight into the sulfur metabolism, the proteome of Cba. tepidum cells sampled under different growth conditions has been quantified using a rapid gel-free, filter-aided sample preparation (FASP) protocol with an in-solution isotopic labeling strategy. Among the 2245 proteins predicted from the Cba. tepidum genome, approximately 970 proteins were detected in unlabeled samples, whereas approximately 630-640 proteins were detected in labeled samples comparing two different growth conditions. Wild-type cells growing on thiosulfate had an increased abundance of periplasmic cytochrome c-555 and proteins of the periplasmic thiosulfate-oxidizing SOX enzyme system when compared with cells growing on sulfide. A dsrM mutant of Cba. tepidum, which lacks the dissimilatory sulfite reductase DsrM protein and therefore is unable to oxidize sulfur globules to sulfite, was also investigated. When compared with wild type, the dsrM cells exhibited an increased abundance of DSR enzymes involved in the initial steps of sulfur globule oxidation (DsrABCL) and a decreased abundance of enzymes putatively involved in sulfite oxidation (Sat-AprAB-QmoABC). The results show that Cba. tepidum regulates the cellular levels of enzymes involved in sulfur metabolism and other electron-transferring processes in response to the availability of reduced sulfur compounds.

Sulfur globule oxidation in green sulfur bacteria is dependent on the dissimilatory sulfite reductase system

Green sulfur bacteria (GSB) oxidize sulfide and thiosulfate to sulfate, with extracellular globules of elemental sulfur as an intermediate. Here we investigated which genes are involved in the formation and consumption of these sulfur globules in the green sulfur bacterium Chlorobaculum tepidum. We show that sulfur globule oxidation is strictly dependent on the dissimilatory sulfite reductase (DSR) system. Deletion of dsrM/CT2244 or dsrT/CT2245, or the two dsrCABL clusters (CT0851-CT0854, CT2247-2250), abolished sulfur globule oxidation and prevented formation of sulfate from sulfide, whereas deletion of dsrU/CT2246 had no effect. The DSR system also seems to be involved in the formation of thiosulfate, because thiosulfate was released from wild-type cells during sulfide oxidation, but not from the dsr mutants. The dsr mutants incapable of complete substrate oxidation oxidized sulfide and thiosulfate about twice as fast as the wild-type, while having only slightly lower growth rates (70-80 % of wild-type). The increased oxidation rates seem to compensate for the incomplete substrate oxidation to satisfy the requirement for reducing equivalents during growth. A mutant in which two sulfide : quinone oxidoreductases (sqrD/CT0117 and sqrF/CT1087) were deleted exhibited a decreased sulfide oxidation rate (~50 % of wild-type), yet formation and consumption of sulfur globules were not affected. The observation that mutants lacking the DSR system maintain efficient growth suggests that the DSR system is dispensable in environments with sufficiently high sulfide concentrations. Thus, the DSR system in GSB may have been acquired by horizontal gene transfer as a response to a need for enhanced substrate utilization in sulfide-limiting habitats.

Growth of Acidithiobacillus Ferrooxidans ATCC 23270 in Thiosulfate Under Oxygen-Limiting Conditions Generates Extracellular Sulfur Globules by Means of a Secreted Tetrathionate Hydrolase.

Production of sulfur globules during sulfide or thiosulfate oxidation is a characteristic feature of some sulfur bacteria. Although their generation has been reported in Acidithiobacillus ferrooxidans, its mechanism of formation and deposition, as well as the physiological significance of these globules during sulfur compounds oxidation, are currently unknown. Under oxygen-sufficient conditions (OSC), A. ferrooxidans oxidizes thiosulfate to tetrathionate, which accumulates in the culture medium. Tetrathionate is then oxidized by a tetrathionate hydrolase (TTH) generating thiosulfate, elemental sulfur, and sulfate as final products. We report here a massive production of extracellular conspicuous sulfur globules in thiosulfate-grown A. ferrooxidans cultures shifted to oxygen-limiting conditions (OLC). Concomitantly with sulfur globule deposition, the extracellular concentration of tetrathionate greatly diminished and sulfite accumulated in the culture supernatant. A. ferrooxidans cellular TTH activity was negligible in OLC-incubated cells, indicating that this enzymatic activity was not responsible for tetrathionate disappearance. On the other hand, supernatants from both OSC- and OLC-incubated cells showed extracellular TTH activity, which most likely accounted for tetrathionate consumption in the culture medium. The extracellular TTH activity described here: (i) gives experimental support to the TTH-driven model for hydrophilic sulfur globule generation, (ii) explains the extracellular location of A. ferrooxidans sulfur deposits, and (iii) strongly suggests that the generation of sulfur globules in A. ferrooxidans corresponds to an early step during its adaptation to an anaerobic lifestyle.

Cultivation‐independent characterization of ‘Candidatus Magnetobacterium bavaricum’ via ultrastructural, geochemical, ecological and metagenomic methods

'Candidatus Magnetobacterium bavaricum' is unusual among magnetotactic bacteria (MTB) in terms of cell size (8-10 µm long, 1.5-2 µm in diameter), cell architecture, magnetotactic behaviour and its distinct phylogenetic position in the deep-branching Nitrospira phylum. In the present study, improved magnetic enrichment techniques permitted high-resolution scanning electron microscopy and energy dispersive X-ray analysis, which revealed the intracellular organization of the magnetosome chains. Sulfur globule accumulation in the cytoplasm point towards a sulfur-oxidizing metabolism of 'Candidatus M. bavaricum'. Detailed analysis of 'Candidatus M. bavaricum' microhabitats revealed more complex distribution patterns than previously reported, with cells predominantly found in low oxygen concentration. No correlation to other geochemical parameters could be observed. In addition, the analysis of a metagenomic fosmid library revealed a 34 kb genomic fragment, which contains 33 genes, among them the complete rRNA gene operon of 'Candidatus M. bavaricum' as well as a gene encoding a putative type IV RubisCO large subunit.

In situ analysis of sulfur species in sulfur globules produced from thiosulfate by Thermoanaerobacter sulfurigignens and Thermoanaerobacterium thermosulfurigenes.

The Firmicutes Thermoanaerobacter sulfurigignens and Thermoanaerobacterium thermosulfurigenes convert thiosulfate, forming sulfur globules inside and outside cells. X-ray absorption near-edge structure analysis revealed that the sulfur consisted mainly of sulfur chains with organic end groups similar to sulfur formed in purple sulfur bacteria, suggesting the possibility that the process of sulfur globule formation by bacteria is an ancient feature.

The role of the sulfur globule proteins of Allochromatium vinosum: mutagenesis of the sulfur globule protein genes and expression studies by real-time RT-PCR.

During oxidation of reduced sulfur compounds, the purple sulfur bacterium Allochromatium vinosum stores sulfur in the periplasm in the form of intracellular sulfur globules. The sulfur in the globules is enclosed by a protein envelope that consists of the homologous 10.5-kDa proteins SgpA and SgpB and the smaller 8.5-kDa SgpC. Reporter gene fusions of sgpA and alkaline phosphatase showed the constitutive expression of sgpA in A. vinosum and yielded additional evidence for the periplasmic localization of the sulfur globules. Expression analysis of the wild-type sgp genes by quantitative RT-PCR using the LightCycler system showed the constitutive expression of all three sgp genes. The expression of sgpB and sgpC is significantly enhanced under photolithotrophic conditions. Interestingly, sgpB is expressed ten times less than sgpA and sgpC implying that SgpA and SgpC are the "main proteins" of the sulfur globule envelope. Mutants with inactivated sgpA or sgpB did not show any differences in comparison with the wild-type, i.e., the encoded proteins can replace each other, whereas inactivation of sgpC leads to the formation of considerably smaller sulfur globules. This indicates a role of SgpC for globule expansion. A sgpBC double mutant was unable to grow on sulfide and could not form sulfur globules, showing that the protein envelope is indispensible for the formation and deposition of intracellular sulfur.

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.

Determination of the thickness of the boundary layer surrounding bacterial PHA inclusion bodies, and implications for models describing the molecular architecture of this layer

AbstractThe thickness of the boundary layer surrounding polyhydroxyalkanoic acid (PHA) inclusion bodies in bacteria was determined from electron micrographs of ultrathin cell sections. The obtained values were compared with the thickness of boundary layers of other kinds of prokaryotic inclusion bodies known to be monolayers (sulfur globule, hydrocarbon‐containing inclusion, carboxysome, gas vesicle, chiorobium vesicle) and the thickness of typical bilayer membranes (cytoplasmic membrane, photosynthetic vesicle). The data are discussed in view of contradicting models published recently for the molecular architecture of the boundary layer of PHA inclusion bodies.

Isolation and characterization of sulfur globule proteins from Chromatium vinosum and Thiocapsa roseopersicina.

Purple sulfur bacteria store sulfur as intracellular globules enclosed by a protein envelope. The proteins associated with sulfur globules of Chromatium vinosum and Thiocapsa roseopersicina were isolated by extraction into 50% aqueous acetonitrile containing 1% trifluoroacetic acid and 10 mM dithiothreitol. The extracted proteins were separated by reversed-phase HPLC, revealing three major proteins from C. vinosum and two from T. roseopersicina. All of these proteins have similar, rather unusual amino acid compositions, being rich in glycine and aromatic amino acids, particularly tyrosine. The molecular masses of the C. vinosum proteins were determined to be 10,498, 10,651, and 8,479 Da, while those from T. roseopersicina were found to be 10,661 and 8,759 Da by laser desorption time-of-flight mass spectrometry. The larger T. roseopersicina protein is N-terminally blocked, probably by acetylation, but small amounts of the unblocked form (mass = 10,619) were also isolated by HPLC. Protein sequencing showed that the two larger C. vinosum proteins are homologous to each other and to the large T. roseopersicina protein. The 8,479 Da C. vinosum and 8,759 Da T. roseopersicina proteins are also homologous, indicating that sulfur globule proteins are conserved between different species of purple sulfur bacteria.

Isolation and characterization of sulfur globule proteins from Chromatium vinosum and Thiocapsa roseopersicina

Isolation and characterization of sulfur globule proteins from Chromatium vinosum and Thiocapsa roseopersicina

PH-Dependent metabolism of thiosulfate and sulfur globules in the chemolithotrophic marine bacterium Thiomicrospira crunogena

Respiring cells of the chemolithotrophic bacterium Thiomicrospira crunogena produced sulfur globules from the sulfane sulfur of thiosulfate below pH 7, and consumed the globules above pH 7. The switch in metabolism was immediate and reversible upon titration of the culture. The consumed sulfur globules remained in a membrane-bound form and were not oxidized unless the medium was depleted of thiosulfate. Sulfur globule production but not uptake was blocked by azide. Anoxia, thiol-binding agents, and inhibitors of protein synthesis blocked globule uptake. Transitory accumulations of sulfite and polythionates appeared to be reaction products of thiosulfate and sulfur globules. A model depicting the pH sensitivity and biochemistry of sulfur globule production and consumption is proposed.

Influence of sulfur accumulation and composition of sulfur globule on cell volume and buoyant density of Chromatium vinosum

Average cell volume and cell buoyant density of Chromatium vinosum DSM 185 growing in sulfide limited continuous cultures, were found to increase with increasing dilution rate. It was found that the increase in buoyant density was mainly a consequence of the accumulation of elemental sulfur. The contribution of other compounds such as protein, bacteriochlorophyll a and glycogen, was almost negligible. It was concluded that the sulfur globule is constituted by at least two fractions, sulfur and an unidentified moiety with a density lower than that of sulfur, probably water.