Blue light promotes zero-valent sulfur production in a deep-sea bacterium.

Increasing evidence has shown that light exists in a diverse range of deep-sea environments. We unexpectedly found that blue light is necessary to produce excess zero-valent sulfur (ZVS) in Erythrobacter flavus 21-3, a bacterium that has been recently isolated from a deep-sea cold seep. E. flavus 21-3 is able to convert thiosulfate to ZVS using a novel thiosulfate oxidation pathway comprising a thiosulfate dehydrogenase (TsdA) and a thiosulfohydrolase (SoxB). Using proteomic, bacterial two-hybrid and heterologous expression assays, we found that the light-oxygen-voltage histidine kinase LOV-1477 responds to blue light and activates the diguanylate cyclase DGC-2902 to produce c-di-GMP. Subsequently, the PilZ domain-containing protein mPilZ-1753 binds to c-di-GMP and activates TsdA through direct interaction. Finally, Raman spectroscopy and gene knockout results verified that TsdA and two SoxB homologs cooperate to regulate ZVS production. As ZVS is an energy source for E. flavus 21-3, we propose that deep-sea blue light provides E. flavus 21-3 with a selective advantage in the cold seep, suggesting a previously unappreciated relationship between light-sensing pathways and sulfur metabolism in a deep-sea microorganism.

YeeD is an essential partner for YeeE-mediated thiosulfate uptake in bacteria and regulates thiosulfate ion decomposition.

Uptake of thiosulfate ions as an inorganic sulfur source from the environment is important for bacterial sulfur assimilation. Recently, a selective thiosulfate uptake pathway involving a membrane protein YeeE (TsuA) in Escherichia coli was characterized. YeeE-like proteins are conserved in some bacteria, archaea, and eukaryotes. However, the precise function of YeeE, along with its potential partner protein in the thiosulfate ion uptake pathway, remained unclear. Here, we assessed selective thiosulfate transport via Spirochaeta thermophila YeeE in vitro and characterized E. coli YeeD (TsuB) as an adjacent and essential protein for YeeE-mediated thiosulfate uptake in vivo. We further showed that S. thermophila YeeD possesses thiosulfate decomposition activity and that a conserved cysteine in YeeD was modified to several forms in the presence of thiosulfate. Finally, the crystal structures of S. thermophila YeeE-YeeD fusion proteins at 3.34-Å and 2.60-Å resolutions revealed their interactions. The association was evaluated by a binding assay using purified S. thermophila YeeE and YeeD. Based on these results, a model of the sophisticated uptake of thiosulfate ions by YeeE and YeeD is proposed.

Sulfur and methane oxidation by a single microorganism.

Natural and anthropogenic wetlands are major sources of the atmospheric greenhouse gas methane. Methane emissions from wetlands are mitigated by methanotrophic bacteria at the oxic-anoxic interface, a zone of intense redox cycling of carbon, sulfur, and nitrogen compounds. Here, we report on the isolation of an aerobic methanotrophic bacterium, 'Methylovirgula thiovorans' strain HY1, which possesses metabolic capabilities never before found in any methanotroph. Most notably, strain HY1 is the first bacterium shown to aerobically oxidize both methane and reduced sulfur compounds for growth. Genomic and proteomic analyses showed that soluble methane monooxygenase and XoxF-type alcohol dehydrogenases are responsible for methane and methanol oxidation, respectively. Various pathways for respiratory sulfur oxidation were present, including the Sox-rDsr pathway and the S4I system. Strain HY1 employed the Calvin-Benson-Bassham cycle for CO2 fixation during chemolithoautotrophic growth on reduced sulfur compounds. Proteomic and microrespirometry analyses showed that the metabolic pathways for methane and thiosulfate oxidation were induced in the presence of the respective substrates. Methane and thiosulfate could therefore be independently or simultaneously oxidized. The discovery of this versatile bacterium demonstrates that methanotrophy and thiotrophy are compatible in a single microorganism and underpins the intimate interactions of methane and sulfur cycles in oxic-anoxic interface environments.

Insights into the sulfur metabolism of Chlorobaculum tepidum by label-free quantitative proteomics.

Chlorobaculum tepidum is an anaerobic green sulfur bacterium which oxidizes sulfide, elemental sulfur, and thiosulfate for photosynthetic growth. It can also oxidize sulfide to produce extracellular S0 globules, which can be further oxidized to sulfate and used as an electron donor. Here, we performed label-free quantitative proteomics on total cell lysates prepared from different metabolic states, including a sulfur production state (10 h post-incubation [PI]), the beginning of sulfur consumption (20 h PI), and the end of sulfur consumption (40 h PI), respectively. We observed an increased abundance of the sulfide:quinone oxidoreductase (Sqr) proteins in 10 h PI indicating a sulfur production state. The periplasmic thiosulfate-oxidizing Sox enzymes and the dissimilatory sulfite reductase (Dsr) subunits showed an increased abundance in 20 h PI, corresponding to the sulfur-consuming state. In addition, we found that the abundance of the heterodisulfide-reductase and the sulfhydrogenase operons was influenced by electron donor availability and may be associated with sulfur metabolism. Further, we isolated and analyzed the extracellular sulfur globules in the different metabolic states to study their morphology and the sulfur cluster composition, yielding 58 previously uncharacterized proteins in purified globules. Our results show that C. tepidum regulates the cellular levels of enzymes involved in sulfur metabolism in response to the availability of reduced sulfur compounds.

Hydrogenimonas cancrithermarum sp. nov., a hydrogen- and thiosulfate-oxidizing mesophilic chemolithoautotroph isolated from diffuse-flow fluids on the East Pacific Rise, and an emended description of the genus Hydrogenimonas.

A novel mesophilic, hydrogen- and thiosulfate-oxidizing bacterium, strain ISO32T, was isolated from diffuse-flow hydrothermal fluids from the Crab Spa vent on the East Pacific Rise. Cells of ISO32T were rods, being motile by means of a single polar flagellum. The isolate grew at a temperature range between 30 and 55 °C (optimum, 43 °C), at a pH range between 5.3 and 7.6 (optimum, pH 5.8) and in the presence of 2.0-4.0 % NaCl (optimum, 2.5 %). The isolate was able to grow chemolithoautotrophically with molecular hydrogen, thiosulfate or elemental sulfur as the sole electron donor. Thiosulfate, elemental sulfur, nitrate and molecular oxygen were each used as a sole electron acceptor. Phylogenetic analysis of 16S rRNA gene sequences placed ISO32T in the genus Hydrogenimonas of the class Epsilonproteobacteria, with Hydrogenimonas thermophila EP1-55-1 %T as its closest relative (95.95 % similarity). On the basis of the phylogenetic, physiological and genomic characteristics, it is proposed that the organism represents a novel species within the genus Hydrogenimonas, Hydrogenimonas cancrithermarum sp. nov. The type strain is ISO32T (=JCM 39185T =KCTC 25252T). Furthermore, the genomic properties of members of the genus Hydrogenimonas are distinguished from those of members of other thermophilic genera in the orders Campylobacterales (Nitratiruptor and Nitrosophilus) and Nautiliales (Caminibacter, Nautilia and Lebetimonas), with larger genome sizes and lower 16S rRNA G+C content values. Comprehensive metabolic comparisons based on genomes revealed that genes responsible for the Pta-AckA pathway were observed exclusively in members of mesophilic genera in the order Campylobacterales and of the genus Hydrogenimonas. Our results indicate that the genus Hydrogenimonas contributes to elucidating the evolutionary history of Epsilonproteobacteria in terms of metabolism and transition from a thermophilic to a mesophilic lifestyle.

Copiotrophy in a Marine-Biofilm-Derived Roseobacteraceae Bacterium Can Be Supported by Amino Acid Metabolism and Thiosulfate Oxidation.

Copiotrophic bacteria that respond rapidly to nutrient availability, particularly high concentrations of carbon sources, play indispensable roles in marine carbon cycling. However, the molecular and metabolic mechanisms governing their response to carbon concentration gradients are not well understood. Here, we focused on a new member of the family Roseobacteraceae isolated from coastal marine biofilms and explored the growth strategy at different carbon concentrations. When cultured in a carbon-rich medium, the bacterium grew to significantly higher cell densities than Ruegeria pomeroyi DSS-3, although there was no difference when cultured in media with reduced carbon. Genomic analysis showed that the bacterium utilized various pathways involved in biofilm formation, amino acid metabolism, and energy production via the oxidation of inorganic sulfur compounds. Transcriptomic analysis indicated that 28.4% of genes were regulated by carbon concentration, with increased carbon concentration inducing the expression of key enzymes in the EMP, ED, PP, and TCA cycles, genes responsible for the transformation of amino acids into TCA intermediates, as well as the sox genes for thiosulfate oxidation. Metabolomics showed that amino acid metabolism was enhanced and preferred in the presence of a high carbon concentration. Mutation of the sox genes decreased cell proton motive force when grown with amino acids and thiosulfate. In conclusion, we propose that copiotrophy in this Roseobacteraceae bacterium can be supported by amino acid metabolism and thiosulfate oxidation.

Reaction of Thiosulfate Dehydrogenase with a Substrate Mimic Induces Dissociation of the Cysteine Heme Ligand Giving Insights into the Mechanism of Oxidative Catalysis.

Thiosulfate dehydrogenases are bacterial cytochromes that contribute to the oxidation of inorganic sulfur. The active sites of these enzymes contain low-spin c-type heme with Cys-/His axial ligation. However, the reduction potentials of these hemes are several hundred mV more negative than that of the thiosulfate/tetrathionate couple (Em, +198 mV), making it difficult to rationalize the thiosulfate oxidizing capability. Here, we describe the reaction of Campylobacter jejuni thiosulfate dehydrogenase (TsdA) with sulfite, an analogue of thiosulfate. The reaction leads to stoichiometric conversion of the active site Cys to cysteinyl sulfonate (Cα-CH2-S-SO3-) such that the protein exists in a form closely resembling a proposed intermediate in the pathway for thiosulfate oxidation that carries a cysteinyl thiosulfate (Cα-CH2-S-SSO3-). The active site heme in the stable sulfonated protein displays an Em approximately 200 mV more positive than the Cys-/His-ligated state. This can explain the thiosulfate oxidizing activity of the enzyme and allows us to propose a catalytic mechanism for thiosulfate oxidation. Substrate-driven release of the Cys heme ligand allows that side chain to provide the site of substrate binding and redox transformation; the neighboring heme then simply provides a site for electron relay to an appropriate partner. This chemistry is distinct from that displayed by the Cys-ligated hemes found in gas-sensing hemoproteins and in enzymes such as the cytochromes P450. Thus, a further class of thiolate-ligated hemes is proposed, as exemplified by the TsdA centers that have evolved to catalyze the controlled redox transformations of inorganic oxo anions of sulfur.

Improved microvascular reactivity after aged garlic extract intake is not mediated by hydrogen sulfide in older adults at risk for cardiovascular disease: a randomized clinical trial.

PURPOSE: This study aimed to investigate the effects of AGE on microvascular reactivity, systolic blood pressure (SBP), and diastolic blood pressure (DBP) in older individuals at high risk for cardiovascular disease (CVD). Urinary thiosulfate was also investigated as an indirect marker of endogenous hydrogen sulfide (H2S) synthesis. The study was conducted in a randomized, double-blind, crossover, and placebo-controlled way. METHODS: Twenty-eight participants (14 male), 67 ± 6 years old with CVD risk factors, ingested 2.4 g of AGE or placebo (PLA). Near-infrared spectroscopy evaluated tissue oxygen saturation (StO2) during a vascular occlusion test (30 s baseline, 5 min occlusion, and 2 min reperfusion). The upslope of StO2 signal after cuff release was calculated to measure microvascular reactivity. Urinary thiosulfate levels were measured using a high-performance liquid chromatography system. RESULTS: The upslope of StO2 was significantly faster after AGE (1.01 ± 0.37% s-1) intake compared to PLA (0.83 ± 0.35% s-1; P < 0.001; d = 0.50). Relative changes in Δ% SBP from pre- to post-AGE intake (- 5.17 ± 5.77%) was significantly different compared to Δ% PLA (0.32 ± 5.99%; P = 0.001; d = 0.93). No significant changes in urinary thiosulfate concentrations were observed between interventions. Moreover, no significant gender effect in any parameter assessed was found. CONCLUSION: This study demonstrated that a single dose of AGE improved microvascular reactivity in older adults at risk of CVD despite such an effect was not linked with urinary thiosulfate levels. This trial was registered at clinicaltrials.gov as NCT04008693 (May 19, 2020).

Thiosulfate-Cyanide Sulfurtransferase a Mitochondrial Essential Enzyme: From Cell Metabolism to the Biotechnological Applications.

Thiosulfate: cyanide sulfurtransferase (TST), also named rhodanese, is an enzyme widely distributed in both prokaryotes and eukaryotes, where it plays a relevant role in mitochondrial function. TST enzyme is involved in several biochemical processes such as: cyanide detoxification, the transport of sulfur and selenium in biologically available forms, the restoration of iron-sulfur clusters, redox system maintenance and the mitochondrial import of 5S rRNA. Recently, the relevance of TST in metabolic diseases, such as diabetes, has been highlighted, opening the way for research on important aspects of sulfur metabolism in diabetes. This review underlines the structural and functional characteristics of TST, describing the physiological role and biomedical and biotechnological applications of this essential enzyme.

Thiosulfate oxidation in sulfur-reducing Shewanella oneidensis and its unexpected influences on the cytochrome c content.

Thiosulfate, an important form of sulfur compounds, can serve as both electron donor and acceptor in various microorganisms. In Shewanella oneidensis, a bacterium renowned for respiratory versatility, thiosulfate reduction has long been recognized but whether it can catalyse thiosulfate oxidation remains elusive. In this study, we discovered that S. oneidensis is capable of thiosulfate oxidation, a process specifically catalysed by two periplasmic cytochrome c (cyt c) proteins, TsdA and TsdB, which act as the catalytic subunit and the electron transfer subunit respectively. In the presence of oxygen, oxidation of thiosulfate has priority over reduction. Intriguingly, thiosulfate oxidation negatively regulates the cyt c content in S. oneidensis cells, largely by reducing intracellular levels of cAMP, which as the cofactor modulates activity of global regulator Crp required for transcription of many cyt c genes. This unexpected finding provides an additional dimension to interplays between the respiration regulator and the respiratory pathways in S. oneidensis. Moreover, the data presented here identified S. oneidensis as the first bacterium known to date owning both functional thiosulfate reductase and dehydrogenase, and importantly, genomics analyses suggested that the number of bacterial species possessing this feature is rather limited.

Insights into growth kinetics and roles of enzymes of Krebs' cycle and sulfur oxidation during exochemolithoheterotrophic growth of Achromobacter aegrifaciens NCCB 38021 on succinate with thiosulfate as the auxiliary electron donor.

Achromobacter aegrifaciens NCCB 38021 was grown heterotrophically on succinate versus exochemolithoheterotrophically on succinate with thiosulfate as auxiliary electron donor. In batch culture, no significant differences in specific molar growth yield or specific growth rate were found for the two growth conditions, but in continuous culture in the succinate-limited chemostat, the maximum specific growth yield coefficient increased by 23.3% with thiosulfate present, consistent with previous studies of endo- and exochemolithoheterotrophs and thermodynamic predictions. Thiosulfate oxidation was coupled to respiration at cytochrome c551, and thiosulfate-dependent ATP biosynthesis occurred. Specific activities of cytochrome c-linked thiosulfate dehydrogenase (E.C. 1.8.2.2) and two other enzymes of sulfur metabolism were significantly higher in exochemolithoheterotrophically grown cell extracts, while those of succinyl-transferring 2-oxoglutarate dehydrogenase (E.C. 1.2.4.2), fumarate hydratase (E.C. 4.2.1.2) and malate dehydrogenase (NAD+, E.C. 1.1.1.37) were significantly lower-presumably owing to less need to generate reducing equivalents during Krebs' cycle, since they could be produced from thiosulfate oxidation.

Hydrogen Sulfide Metabolite, Sodium Thiosulfate: Clinical Applications and Underlying Molecular Mechanisms.

Thiosulfate in the form of sodium thiosulfate (STS) is a major oxidation product of hydrogen sulfide (H2S), an endogenous signaling molecule and the third member of the gasotransmitter family. STS is currently used in the clinical treatment of acute cyanide poisoning, cisplatin toxicities in cancer therapy, and calciphylaxis in dialysis patients. Burgeoning evidence show that STS has antioxidant and anti-inflammatory properties, making it a potential therapeutic candidate molecule that can target multiple molecular pathways in various diseases and drug-induced toxicities. This review discusses the biochemical and molecular pathways in the generation of STS from H2S, its clinical usefulness, and potential clinical applications, as well as the molecular mechanisms underlying these clinical applications and a future perspective in kidney transplantation.

Heme ligation and redox chemistry in two bacterial thiosulfate dehydrogenase (TsdA) enzymes.

Thiosulfate dehydrogenases (TsdAs) are bidirectional bacterial di-heme enzymes that catalyze the interconversion of tetrathionate and thiosulfate at measurable rates in both directions. In contrast to our knowledge of TsdA activities, information on the redox properties in the absence of substrates is rather scant. To address this deficit, we combined magnetic CD (MCD) spectroscopy and protein film electrochemistry (PFE) in a study to resolve heme ligation and redox chemistry in two representative TsdAs. We examined the TsdAs from Campylobacter jejuni, a microaerobic human pathogen, and from the purple sulfur bacterium Allochromatium vinosum In these organisms, the enzyme functions as a tetrathionate reductase and a thiosulfate oxidase, respectively. The active site Heme 1 in both enzymes has His/Cys ligation in the ferric and ferrous states and the midpoint potentials (Em ) of the corresponding redox transformations are similar, -185 mV versus standard hydrogen electrode (SHE). However, fundamental differences are observed in the properties of the second, electron transferring, Heme 2. In C. jejuni, TsdA Heme 2 has His/Met ligation and an Em of +172 mV. In A. vinosum TsdA, Heme 2 reduction triggers a switch from His/Lys ligation (Em , -129 mV) to His/Met (Em , +266 mV), but the rates of interconversion are such that His/Lys ligation would be retained during turnover. In summary, our findings have unambiguously assigned Em values to defined axial ligand sets in TsdAs, specified the rates of Heme 2 ligand exchange in the A. vinosum enzyme, and provided information relevant to describing their catalytic mechanism(s).

Comparison of sulfide-oxidizing Sulfurimonas strains reveals a new mode of thiosulfate formation in subsurface environments.

Sulfur-oxidizing Sulfurimonas spp. are widespread in sediments, hydrothermal vent fields, aquifers and subsurface environments such as oil reservoirs where they play an important role in the sulfur cycle. We determined the genome sequence of the oil field isolate Sulfurimonas sp. strain CVO and compared its gene expression during nitrate-dependent sulfide oxidation to the coastal sediment isolate Sulfurimonas denitrificans. Formation of elemental sulfur (S0 ) and high expression of sulfide quinone oxidoreductase (SQR) genes indicates that sulfide oxidation in both strains is mediated by SQR. Subsequent oxidation of S0 was achieved by the sulfur oxidation enzyme complex (SOX). In the coastal S. denitrificans, the genes are arranged and expressed as two clusters: soxXY1 Z1 AB and soxCDY2 Z2 H, and sulfate was the sole metabolic end product. By contrast, the oil field strain CVO has only the soxCDY2 Z2 H cluster and not soxXY1 Z1 AB. Despite the absence of the soxXY1 Z1 AB cluster, strain CVO oxidized S0 to thiosulfate and sulfate, demonstrating that soxCDY2 Z2 H genes alone are sufficient for S0 oxidation in Sulfurimonas spp. and that thiosulfate is an additional metabolic end product. Screening of publicly available metagenomes revealed that Sulfurimonas spp. with only the soxCDY2 Z2 H cluster are widespread suggesting this mechanism of thiosulfate formation is environmentally significant.

Molecular mechanism of sulfur chemolithotrophy in the betaproteobacterium Pusillimonas ginsengisoli SBSA.

Chemolithotrophic sulfur oxidation represents a significant part of the biogeochemical cycling of this element. Due to its long evolutionary history, this ancient metabolism is well known for its extensive mechanistic and phylogenetic diversification across a diverse taxonomic spectrum. Here we carried out whole-genome sequencing and analysis of a new betaproteobacterial isolate, Pusillimonas ginsengisoli SBSA, which is found to oxidize thiosulfate via the formation of tetrathionate as an intermediate. The 4.7 Mb SBSA genome was found to encompass a soxCDYZAXOB operon, plus single thiosulfate dehydrogenase (tsdA) and sulfite : acceptor oxidoreductase (sorAB) genes. Recombination-based knockout of tsdA revealed that the entire thiosulfate is first converted to tetrathionate by the activity of thiosulfate dehydrogenase (TsdA) and the Sox pathway is not functional in this bacterium despite the presence of all necessary sox genes. The ∆soxYZ and ∆soxXA knockout mutants exhibited a wild-type-like phenotype for thiosulfate/tetrathionate oxidation, whereas ∆soxB, ∆soxCD and soxO::KanR mutants only oxidized thiosulfate up to tetrathionate intermediate and had complete impairment in tetrathionate oxidation. The substrate-dependent O2 consumption rate of whole cells and the sulfur-oxidizing enzyme activities of cell-free extracts, measured in the presence/absence of thiol inhibitors/glutathione, indicated that glutathione plays a key role in SBSA tetrathionate oxidation. The present findings collectively indicate that the potential glutathione : tetrathionate coupling in P. ginsengisoli involves a novel enzymatic component, which is different from the dual-functional thiol dehydrotransferase (ThdT), while subsequent oxidation of the sulfur intermediates produced (e.g. glutathione : sulfodisulfane molecules) may proceed via the iterative action of soxBCD .

The Heterotrophic Bacterium Cupriavidus pinatubonensis JMP134 Oxidizes Sulfide to Sulfate with Thiosulfate as a Key Intermediate.

Heterotrophic bacteria actively participate in the biogeochemical cycle of sulfur on Earth. The heterotrophic bacterium Cupriavidus pinatubonensis JMP134 contains several enzymes involved in sulfur oxidation, but how these enzymes work together to oxidize sulfide in the bacterium has not been studied. Using gene-deletion and whole-cell assays, we determined that the bacterium uses sulfide:quinone oxidoreductase to oxidize sulfide to polysulfide, which is further oxidized to sulfite by persulfide dioxygenase. Sulfite spontaneously reacts with polysulfide to produce thiosulfate. The sulfur-oxidizing (Sox) system oxidizes thiosulfate to sulfate. Flavocytochrome c sulfide dehydrogenase enhances thiosulfate oxidation by the Sox system but couples with the Sox system for sulfide oxidation to sulfate in the absence of sulfide:quinone oxidoreductase. Thus, C. pinatubonensis JMP134 contains a main pathway and a contingent pathway for sulfide oxidation.IMPORTANCE We establish a new pathway of sulfide oxidation with thiosulfate as a key intermediate in Cupriavidus pinatubonensis JMP134. The bacterium mainly oxidizes sulfide by using sulfide:quinone oxidoreductase, persulfide dioxygenase, and the Sox system with thiosulfate as a key intermediate. Although the purified and reconstituted Sox system oxidizes sulfide, its rate of sulfide oxidation in C. pinatubonensis JMP134 is too low to be physiologically relevant. The findings reveal how these sulfur-oxidizing enzymes participate in sulfide oxidation in a single bacterium.

Metabolism of Sulfur-Containing Amino Acids: How the Body Copes with Excess Methionine, Cysteine, and Sulfide.

Metabolism of excess methionine (Met) to homocysteine (Hcy) by transmethylation is facilitated by the expression of methionine adenosyltransferase (MAT) I/III and glycine N-methyltransferase (GNMT) in liver, and a lack of either enzyme results in hypermethioninemia despite normal concentrations of MATII and methyltransferases other than GNMT. The further metabolism of Hcy by the transsulfuration pathway is facilitated by activation of cystathionine ß-synthase (CBS) by S-adenosylmethionine (SAM) as well as the relatively high KM of CBS for Hcy. Transmethylation plus transsulfuration effects catabolism of the Met molecule along with transfer of the sulfur atom of Met to serine to synthesize cysteine (Cys). Oxidation and excretion of Met sulfur depend upon Cys catabolism and sulfur oxidation pathways. Excess Cys is oxidized by cysteine dioxygenase 1 (CDO1) and further metabolized to taurine or sulfate. Some Cys is normally metabolized by desulfhydration pathways, and the hydrogen sulfide (H2S) produced is further oxidized to sulfate. If Cys or Hcy concentrations are elevated, Cys or Hcy desulfhydration can result in excess H2S and thiosulfate production. Excess Cys or Met may also promote their limited metabolism by transamination pathways.

Limnobacter alexandrii sp. nov., a thiosulfate-oxidizing, heterotrophic and EPS-bearing Burkholderiaceae isolated from cultivable phycosphere microbiota of toxic Alexandrium catenella LZT09.

A novel Gram-negative, aerobic, motile and short rod-shaped bacterium with exopolysaccharides production, designated as LZ-4T, was isolated from cultivable phycosphere microbiota of harmful algal blooms-causing marine dinoflagellate Alexandrium catenella LZT09 which produces paralytic shellfish poisoning toxins. Strain LZ-4T was able to use thiosulfate (optimum concentration 10 mM) as energy source for bacterial growth. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain LZ-4T belonged to the genus Limnobacter, showing high 16S rRNA gene sequences similarities with L. thiooxidans DSM 13612T (99.4%), L. humi NBRC 11650T (98.2%) and L. litoralis NBRC 105857T (97.2%), respectively. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between LZ-4T and L. thiooxidans DSM 13612T were 78.9 and 21.9%, respectively. Both values were far lower than the thresholds (95-96% for ANI and 70% for dDDH) generally accepted for new species delineation. The respiratory quinone of strain LZ-4T was Q-8. The dominant cellular fatty acids were determined as summed feature 3 (C16:1 ω6c/ω7c), summed feature 8 (C18:1 ω6c/ω7c) and C16:0. Polar lipids profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unidentified aminolipids and three unidentified polar lipids. The genomic DNA G+C content of strain LZ-4T was 52.5 mol%. Based on polyphasic characterization, strain LZ-4T represents a novel species of the genus Limnobacter, for which the name Limnobacter alexandrii sp. nov. is proposed. The type strain is LZ-4T (=CCTCC AB 2019004T =KCTC 72281T).

Role of cAMP receptor protein in phenotype and stress tolerance in Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is exposed to biotic and abiotic stresses. The survival of Salmonella in nature depends on the global regulators like cAMP receptor protein (CRP). The role of CRP in the phenotypic characteristics and stress tolerance was elucidated in S. Typhimurium using a crp gene null mutant (Δcrp). A 1.6-fold decrease in the cell size, a two-fold reduction in the colony size, and a 3.5-fold decrease in motility were observed in the Δcrp compared with the S. Typhimurium wild-type (WT). H2 S production on selective media was affected in the Δcrp. The utilisation of d-mannose, d-glycerol and d-mannitol was completely affected, whereas that of d-galactose and d-fructose was partially affected. The utilisation of d-arabinose was induced in the Δcrp. The growth rate of the Δcrp in Luria Bertani medium was unaffected. However, in the glucose-containing minimal medium, the growth rate of the Δcrp was reduced by 1.5-fold compared with the WT. The Δcrp was able to utilise ethanolamine as the sole carbon source similar to the WT. The Δcrp was more tolerant to heat and oxidative stress. Overexpression of heat and oxidative stress-related genes was observed in the Δcrp in the stationary phase. The Δcrp was less tolerant to radiation stress compared with the WT. The current findings decisively establish the CRP protein as a global regulator. The CRP affects multiple phenotypes, carbon metabolism and stress physiology of S. Typhimurium.

Operation performance and microbial community of sulfur-based autotrophic denitrification sludge with different sulfur sources.

Operation performance and bacterial community structure of sulfur-based autotrophic denitrification (SAD) based on different sulfur sources served as electron donor was first parallelly compared among three sequencing batch reactors. Sulfur and sodium thiosulfate systems achieved similar operation performance and were superior to that of sodium sulfide. When the influent NO3--N concentration ranged from 50 to 150 mg/L, the effluent NO3--N concentrations of the sulfur and sodium thiosulfate systems were 0-5.99 mg/L and 0-4.52 mg/L, respectively, without NO2--N accumulation. However, when the effluent concentration of NO3--N in the sodium sulfide system was 0-10.38 mg/L, that of NO2--N in the effluent was 0-39.85 mg/L. In addition, participation of sulfur sources presented obvious pressure on the bacterial community structure based on the high-throughput sequencing. Microbial diversity results indicated that sludge with elemental sulfur as electron donor had the richest microbial diversity, followed by sodium thiosulfate and sodium sulfide. Moreover, sludge with elemental sulfur and sodium thiosulfate as electron donor demonstrated more similar community structure compared with the sludge that denitrified with sodium sulfide according to the microbial similarity analysis. The 9.34%, 24.3% and 29.6% of sequences could be assigned to potential SAD organisms from sludge denitrifying with elemental sulfur, sodium thiosulfate and sodium sulfide, respectively. Furthermore, all sludge denitrifying with different sulfur sources showed an enrichment of separate core functional microorganisms. This study could provide an insight into improving the understanding of SAD in engineering applications.