Deletions of ttrA and pduA genes in Salmonella enterica affect survival within chicken-derived HD-11 macrophages.

In mammals, enteric salmonellas can use tetrathionate (ttr), formed as a by-product from the inflammatory process in the intestine, as electron acceptor in anaerobic respiration, and it can fuel its energy metabolism by degrading the microbial fermentation product 1,2-propanediol. However, recent studies have shown that this mechanism is not important for Salmonella infection in the intestine of poultry, while it prolongs the persistence of Salmonella at systemic sites in this species. In the current study, we show that ΔttrApduA strains of Salmonella enterica have lower net survival within chicken-derived HD-11 macrophages, as CFU was only 2.3% (S. Enteritidis ΔttrApduA), 2.3% (S. Heidelberg ΔttrApduA), and 3.0% (S. Typhimurium ΔttrApduA) compared to wild-type strains after 24 h inside HD-11 macrophage cells. The difference was not related to increased lysis of macrophages, and deletion of ttrA and pduA did not impair the ability of the strains to grow anaerobically. Further studies are indicated to determine the reason why Salmonella ΔttrApduA strains survive less well inside macrophage cell lines.

A recently isolated human commensal Escherichia coli ST10 clone member mediates enhanced thermotolerance and tetrathionate respiration on a P1 phage-derived IncY plasmid.

The ubiquitous human commensal Escherichia coli has been well investigated through its model representative E. coli K-12. In this work, we initially characterized E. coli Fec10, a recently isolated human commensal strain of phylogroup A/sequence type ST10. Compared to E. coli K-12, the 4.88 Mbp Fec10 genome is characterized by distinct single-nucleotide polymorphisms and acquisition of genomic islands. In addition, E. coli Fec10 possesses a 155.86 kbp IncY plasmid, a composite element based on phage P1. pFec10 harbours multiple cargo genes such as coding for a tetrathionate reductase and its corresponding regulatory two-component system. Among the cargo genes is also the Transmissible Locus of Protein Quality Control (TLPQC), which mediates tolerance to lethal temperatures in bacteria. The disaggregase ClpGGI of TLPQC constitutes a major determinant of the thermotolerance of E. coli Fec10. We confirmed stand-alone disaggregation activity, but observed distinct biochemical characteristics of ClpGGI-Fec10 compared to the nearly identical Pseudomonas aeruginosa ClpGGI-SG17M. Furthermore, we noted a unique contribution of ClpGGI-Fec10 to the exquisite thermotolerance of E. coli Fec10, suggesting functional differences between both disaggregases in vivo. Detection of thermotolerance in 10% of human commensal E. coli isolates hints to the successful establishment of food-borne heat-resistant strains in the human gut.

Salmonella Heidelberg side-step gene loss of respiratory requirements in chicken infection model.

Among the important recent observations involving anaerobic respiration was that an electron acceptor produced as a result of an inflammatory response to Salmonella Typhimurium generates a growth advantage over the competing microbiota in the lumen. In this regard, anaerobically, salmonellae can oxidize thiosulphate (S2O32-) converting it into tetrathionate (S4O62-), the process by which it is encoded by ttr gene cluster (ttrSRttrBCA). Another important pathway under aerobic or anaerobic conditions is the 1,2-propanediol-utilization mediated by the pdu gene cluster that promotes Salmonella expansion during colitis. Therefore, we sought to compare in this study, whether Salmonella Heidelberg strains lacking the ttrA, ttrApduA, and ttrACBSR genes experience a disadvantage during cecal colonization in broiler chicks. In contrast to expectations, we found that the gene loss in S. Heidelberg potentially confers an increase in fitness in the chicken infection model. These data argue that S. Heidelberg may trigger an alternative pathway involving the use of an alternative electron acceptor, conferring a growth advantage for S. Heidelberg in chicks.

Deciphering the role of ttrA and pduA genes for Salmonella enterica serovars in a chicken infection model.

Salmonella enterica serovars use self-induced intestinal inflammation to increase electron acceptor availability and to obtain a growth advantage in the host gut. There is evidence suggesting that the ability of Salmonella to use tetrathionate and 1,2-propanediol provides an advantage in murine infection. Thus, we present here the first study to evaluate both systemic infection and faecal excretion in commercial poultry challenged by Salmonella Enteritidis (SE) and S. Typhimurium (STM) harbouring deletions in ttrA and pduA genes, which are crucial to the metabolism of tetrathionate and 1,2-propanediol, respectively. Mutant strains were excreted at higher rates when compared to the wild-type strains. The highest rates were observed with white egg-layer and brown egg-layer chicks (67.5%), and broiler chicks (56.7%) challenged by SEΔttrAΔpduA, and brown egg-layer chicks (64.8%) challenged by STMΔttrAΔpduA. SEΔttrAΔpduA presented higher bacterial counts in the liver and spleen of the three chicken lineages and caecal contents from the broiler chickens, whereas STMΔttrAΔpduA presented higher counts in the liver and spleen of the broiler and brown-egg chickens for 28 days post-infection (P < 0.05). The ttrA and pduA genes do not appear to be major virulence determinants in faecal excretion or invasiveness for SE and STM in chickens. RESEARCH HIGHLIGHTSttrA and pudA do not impair gut colonization or systemic infection in chicks.Mutant strains were present in higher numbers in broilers than in laying chicks.Mutants of SE and STM showed greater pathogenicity in broiler chicks than layers.

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).

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 .

Possible Involvement of a Tetrathionate Reductase Homolog in Dissimilatory Arsenate Reduction by Anaeromyxobacter sp. Strain PSR-1.

Anaeromyxobacter sp. strain PSR-1, a dissimilatory arsenate [As(V)]-reducing bacterium, can utilize As(V) as a terminal electron acceptor for anaerobic respiration. A previous draft genome analysis revealed that strain PSR-1 lacks typical respiratory As(V) reductase genes (arrAB), which suggested the involvement of another protein in As(V) respiration. Dissimilatory As(V) reductase activity of strain PSR-1 was induced under As(V)-respiring conditions and was localized predominantly in the periplasmic fraction. The activity was visualized by partially denaturing gel electrophoresis, and liquid chromatography-tandem mass spectrometry analysis identified proteins involved in the active band. Among these proteins, a protein annotated as molybdopterin-dependent oxidoreductase (PSR1_00330) exhibited the highest sequence coverage, 76%. Phylogenetic analysis revealed that this protein was a homolog of tetrathionate reductase catalytic subunit TtrA. However, the crude extract of strain PSR-1 did not show significant tetrathionate reductase enzyme activity. Comparative proteomic analysis revealed that the protein PSR1_00330 and a homolog of tetrathionate reductase electron transfer subunit TtrB (PSR1_00329) were expressed abundantly and specifically under As(V)-respiring conditions, respectively. The genes encoding PSR1_00330 and PSR1_00329 formed an operon-like structure along with a gene encoding a c-type cytochrome (cyt c), and their transcription was upregulated under As(V)-respiring conditions. These results suggest that the protein PSR1_00330, which lacks tetrathionate reductase activity, functions as a dissimilatory As(V) reductase in strain PSR-1. Considering the wide distribution of TtrA homologs among bacteria and archaea, they may play a hitherto unknown role along with conventional respiratory As(V) reductase (Arr) in the biogeochemical cycling of arsenic in nature.IMPORTANCE Dissimilatory As(V)-reducing prokaryotes play significant roles in arsenic release and contamination in groundwater and threaten the health of people worldwide. Generally, such prokaryotes reduce As(V) by means of a respiratory As(V) reductase designated Arr. However, some dissimilatory As(V)-reducing prokaryotes such as Anaeromyxobacter sp. strain PSR-1 lack genes encoding Arr, suggesting the involvement of other protein in As(V) reduction. In this study, using multiple proteomic and transcriptional analyses, it was found that the dissimilatory As(V) reductase of strain PSR-1 was a protein closely related to the tetrathionate reductase catalytic subunit (TtrA). Tetrathionate reductase is known to play a role in anaerobic respiration of Salmonella on tetrathionate, but strain PSR-1 showed neither growth on tetrathionate nor significant tetrathionate reductase enzyme activity. These results suggest the possibility that TtrA homologs encoded in a wide variety of archaeal and bacterial genomes might function as dissimilatory As(V) reductases.

Metagenomes and metatranscriptomes shed new light on the microbial-mediated sulfur cycle in a Siberian soda lake.

BACKGROUND: The planetary sulfur cycle is a complex web of chemical reactions that can be microbial-mediated or can occur spontaneously in the environment, depending on the temperature and pH. Inorganic sulfur compounds can serve as energy sources for specialized prokaryotes and are important substrates for microbial growth in general. Here, we investigate dissimilatory sulfur cycling in the brine and sediments of a southwestern Siberian soda lake characterized by an extremely high pH and salinity, combining meta-omics analyses of its uniquely adapted highly diverse prokaryote communities with biogeochemical profiling to identify key microbial players and expand our understanding of sulfur cycling under haloalkaline conditions. RESULTS: Peak microbial activity was found in the top 4 cm of the sediments, a layer with a steep drop in oxygen concentration and redox potential. The majority of sulfur was present as sulfate or iron sulfide. Thiosulfate was readily oxidized by microbes in the presence of oxygen, but oxidation was partially inhibited by light. We obtained 1032 metagenome-assembled genomes, including novel population genomes of characterized colorless sulfur-oxidizing bacteria (SOB), anoxygenic purple sulfur bacteria, heterotrophic SOB, and highly active lithoautotrophic sulfate reducers. Surprisingly, we discovered the potential for nitrogen fixation in a new genus of colorless SOB, carbon fixation in a new species of phototrophic Gemmatimonadetes, and elemental sulfur/sulfite reduction in the "Candidatus Woesearchaeota." Polysulfide/thiosulfate and tetrathionate reductases were actively transcribed by various (facultative) anaerobes. CONCLUSIONS: The recovery of over 200 genomes that encoded enzymes capable of catalyzing key reactions in the inorganic sulfur cycle indicates complete cycling between sulfate and sulfide at moderately hypersaline and extreme alkaline conditions. Our results suggest that more taxonomic groups are involved in sulfur dissimilation than previously assumed.

Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation.

There is a groundswell of interest in using genetically engineered sensor bacteria to study gut microbiota pathways, and diagnose or treat associated diseases. Here, we computationally identify the first biological thiosulfate sensor and an improved tetrathionate sensor, both two-component systems from marine Shewanella species, and validate them in laboratory Escherichia coli Then, we port these sensors into a gut-adapted probiotic E. coli strain, and develop a method based upon oral gavage and flow cytometry of colon and fecal samples to demonstrate that colon inflammation (colitis) activates the thiosulfate sensor in mice harboring native gut microbiota. Our thiosulfate sensor may have applications in bacterial diagnostics or therapeutics. Finally, our approach can be replicated for a wide range of bacterial sensors and should thus enable a new class of minimally invasive studies of gut microbiota pathways.

Characterization of tetrathionate hydrolase from the marine acidophilic sulfur-oxidizing bacterium, Acidithiobacillus thiooxidans strain SH.

Tetrathionate hydrolase (4THase), a key enzyme of the S4-intermediate (S4I) pathway, was partially purified from marine acidophilic bacterium, Acidithiobacillus thiooxidans strain SH, and the gene encoding this enzyme (SH-tth) was identified. SH-Tth is a homodimer with a molecular mass of 97 ± 3 kDa, and contains a subunit 52 kDa in size. Enzyme activity was stimulated in the presence of 1 M NaCl, and showed the maximum at pH 3.0. Although 4THases from A. thiooxidans and the closely related Acidithiobacillus caldus strain have been reported to be periplasmic enzymes, SH-Tth seems to be localized on the outer membrane of the cell, and acts as a peripheral protein. Furthermore, both 4THase activity and SH-Tth proteins were detected in sulfur-grown cells of strain SH. These results suggested that SH-Tth is involved in elemental sulfur-oxidation, which is distinct from sulfur-oxidation in other sulfur-oxidizing strains such as A. thiooxidans and A. caldus.

Thiosulfate dehydrogenase (TsdA) from Allochromatium vinosum: structural and functional insights into thiosulfate oxidation.

Although the oxidative condensation of two thiosulfate anions to tetrathionate constitutes a well documented and significant part of the natural sulfur cycle, little is known about the enzymes catalyzing this reaction. In the purple sulfur bacterium Allochromatium vinosum, the reaction is catalyzed by the periplasmic diheme c-type cytochrome thiosulfate dehydrogenase (TsdA). Here, we report the crystal structure of the "as isolated" form of A. vinosum TsdA to 1.98 Šresolution and those of several redox states of the enzyme to different resolutions. The protein contains two typical class I c-type cytochrome domains wrapped around two hemes axially coordinated by His(53)/Cys(96) and His(164)/Lys(208). These domains are very similar, suggesting a gene duplication event during evolution. A ligand switch from Lys(208) to Met(209) is observed upon reduction of the enzyme. Cys(96) is an essential residue for catalysis, with the specific activity of the enzyme being completely abolished in several TsdA-Cys(96) variants. TsdA-K208N, K208G, and M209G variants were catalytically active in thiosulfate oxidation as well as in tetrathionate reduction, pointing to heme 2 as the electron exit point. In this study, we provide spectroscopic and structural evidence that the TsdA reaction cycle involves the transient presence of heme 1 in the high-spin state caused by movement of the Sγ atom of Cys(96) out of the iron coordination sphere. Based on the presented data, we draw important conclusions about the enzyme and propose a possible reaction mechanism for TsdA.

Thiosulfate transfer mediated by DsrE/TusA homologs from acidothermophilic sulfur-oxidizing archaeon Metallosphaera cuprina.

Conserved clusters of genes encoding DsrE and TusA homologs occur in many archaeal and bacterial sulfur oxidizers. TusA has a well documented function as a sulfurtransferase in tRNA modification and molybdenum cofactor biosynthesis in Escherichia coli, and DsrE is an active site subunit of the DsrEFH complex that is essential for sulfur trafficking in the phototrophic sulfur-oxidizing Allochromatium vinosum. In the acidothermophilic sulfur (S(0))- and tetrathionate (S4O6(2-))-oxidizing Metallosphaera cuprina Ar-4, a dsrE3A-dsrE2B-tusA arrangement is situated immediately between genes encoding dihydrolipoamide dehydrogenase and a heterodisulfide reductase-like complex. In this study, the biochemical features and sulfur transferring abilities of the DsrE2B, DsrE3A, and TusA proteins were investigated. DsrE3A and TusA proved to react with tetrathionate but not with NaSH, glutathione persulfide, polysulfide, thiosulfate, or sulfite. The products were identified as protein-Cys-S-thiosulfonates. DsrE3A was also able to cleave the thiosulfate group from TusA-Cys(18)-S-thiosulfonate. DsrE2B did not react with any of the sulfur compounds tested. DsrE3A and TusA interacted physically with each other and formed a heterocomplex. The cysteine residue (Cys(18)) of TusA is crucial for this interaction. The single cysteine mutants DsrE3A-C(93)S and DsrE3A-C(101)S retained the ability to transfer the thiosulfonate group to TusA. TusA-C(18)S neither reacted with tetrathionate nor was it loaded with thiosulfate with DsrE3A-Cys-S-thiosulfonate as the donor. The transfer of thiosulfate, mediated by a DsrE-like protein and TusA, is unprecedented not only in M. cuprina but also in other sulfur-oxidizing prokaryotes. The results of this study provide new knowledge on oxidative microbial sulfur metabolism.

The sole cysteine residue (Cys301) of tetrathionate hydrolase from Acidithiobacillus ferrooxidans does not play a role in enzyme activity.

Cysteine residues are absolutely indispensable for the reactions of almost all enzymes involved in the dissimilatory oxidation pathways of reduced inorganic sulfur compounds. Tetrathionate hydrolase from the acidophilic iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans (Af-Tth) catalyzes tetrathionate hydrolysis to generate elemental sulfur, thiosulfate, and sulfate. Af-Tth is a key enzyme in the dissimilatory sulfur oxidation pathway in this bacterium. Only one cysteine residue (Cys301) has been identified in the deduced amino acid sequence of the Af-Tth gene. In order to clarify the role of the sole cysteine residue, a site-specific mutant enzyme (C301A) was generated. No difference was observed in the retention volumes of the wild-type and mutant Af-Tth enzymes by gel-filtration column chromatography, and surprisingly the enzyme activities measured in the cysteine-deficient and wild-type enzymes were the same. These results suggest that the sole cysteine residue (Cys301) in Af-Tth is involved in neither the tetrathionate hydrolysis reaction nor the subunit assembly. Af-Tth may thus have a novel cysteine-independent reaction mechanism.

Multifarious characteristics of sulfur-oxidizing bacteria residing in rice rhizosphere.

Sulfur-oxidizing bacteria (SOB) are versatile microorganisms known for their ability to oxidize various reduced sulfur compounds, namely, elemental sulfur (S0), hydrogen sulfide (H2S), tetrathionate (S4O62-), and trithionate (S3O62-) to sulfate (SO42-). In this study, out of twelve SOB isolates from rice rhizosphere, five were screened based on their sulfur oxidation potential, viz., SOB1, SOB2, SOB3, SOB4, and SOB5, and were identified as Ochrobactrum soli SOB1, Achromobacter xylosoxidans SOB2, Stenotrophomonas maltophilia SOB3, Brucella tritici SOB4, and Stenotrophomonas pavanii SOB5, respectively. All the isolates displayed chemolithotrophic nutritional mode by consuming thiosulfate and accumulating trithionate and tetrathionate in the growth medium which is ultimately oxidized to sulfate. The strains were authenticated with the production of thiosulfate oxidizing enzymes such as rhodanese and sulfite oxidase. Despite their tendency to oxidize reduced sulfur compounds, B. tritici SOB4 and S. pavanii SOB5 were also found to possess phosphate and zinc solubilization potential, acetic acid, and indole acetic acid (IAA) production and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. The presence of sulfanyl (R-SH) groups was noticed in the A. xylosoxidans SOB2. Elemental sulfur conversion into sulfate was noted in the S. maltophilia SOB3, and hydrogen sulfide conversion into sulfate was observed in the Ochromobacter soli SOB1. Sulfur oxidation potential coupled with beneficial properties of the isolates widen the knowledge on SOB.

Tetrathionate hydrolase from the acidophilic microorganisms.

Tetrathionate hydrolase (TTH) is a unique enzyme found in acidophilic sulfur-oxidizing microorganisms, such as bacteria and archaea. This enzyme catalyzes the hydrolysis of tetrathionate to thiosulfate, elemental sulfur, and sulfate. It is also involved in dissimilatory sulfur oxidation metabolism, the S4-intermediate pathway. TTHs have been purified and characterized from acidophilic autotrophic sulfur-oxidizing microorganisms. All purified TTHs show an optimum pH in the acidic range, suggesting that they are localized in the periplasmic space or outer membrane. In particular, the gene encoding TTH from Acidithiobacillus ferrooxidans (Af-tth) was identified and recombinantly expressed in Escherichia coli cells. TTH activity could be recovered from the recombinant inclusion bodies by acid refolding treatment for crystallization. The mechanism of tetrathionate hydrolysis was then elucidated by X-ray crystal structure analysis. Af-tth is highly expressed in tetrathionate-grown cells but not in iron-grown cells. These unique structural properties, reaction mechanisms, gene expression, and regulatory mechanisms are discussed in this review.

Crystallization and preliminary X-ray diffraction analysis of tetrathionate hydrolase from Acidithiobacillus ferrooxidans.

Tetrathionate hydrolase (4THase) from the iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans catalyses the disproportionate hydrolysis of tetrathionate to elemental sulfur, thiosulfate and sulfate. The gene encoding 4THase (Af-tth) was expressed as inclusion bodies in recombinant Escherichia coli. Recombinant Af-Tth was activated by refolding under acidic conditions and was then purified to homogeneity. The recombinant protein was crystallized in 20 mM glycine buffer pH 10 containing 50 mM sodium chloride and 33%(v/v) PEG 1000 using the hanging-drop vapour-diffusion method. The crystal was a hexagonal cylinder with dimensions of 0.2 × 0.05 × 0.05 mm. X-ray crystallographic analysis showed that the crystal diffracted to 2.15 Å resolution and belongs to space group P3(1) or P3(2), with unit-cell parameters a = b = 92.1, c = 232.6 Å.

Recombinant expression using the tetrathionate hydrolase promoter in Acidithiobacillus ferrooxidans.

In the iron- and sulfur-oxidizing acidophilic chemolithoautotrophic bacterium, Acidithiobacillus ferrooxidans, tetrathionate hydrolase gene (Af-tth) is highly expressed during tetrathionate growth. The expression levels of Af-tth were specifically determined by quantitative reverse transcription-polymerase chain reaction and the expression ratios of S0/Fe2+ and S4O62-/Fe2+ were found to be 68 ± 21 and 181 ± 5, respectively. The transcriptional start site was identified by primer extension. Promoter regions of Af-tth were cloned into the expression shuttle vector pMPJC and GFP gene was under the direction of the regions. Green fluorescence was observed by UV irradiation in recombinant A. ferrooxidans harboring the plasmid colonies grown on tetrathionate. Furthermore, His-tagged Af-Tth was synthesized in the recombinant cells grown on tetrathionate. Recombinant, His-tagged Af-Tth in an active form, was rapidly purified through metal-affinity column chromatography, although recombinant Af-Tth was synthesized in the inclusion bodies of Escherichia coli and acid-refolding treatment was necessary to recover the activity. The specific activity of purified Af-Tth from recombinant A. ferrooxidans (2.2 ± 0.37 U mg-1) was similar to that of acid-refolded Af-Tth from recombinant E. coli (2.5 ± 0.18 U mg-1). This method can be applied not only to heterologous expression but also to homologous expression of target genes for modification or specific mutation in A. ferrooxidans cells.

Construction of an Engineered Escherichia coli with Efficient Chemotactic and Metabolizing Ability toward Tetrathionate.

The emergence of genetically engineered bacteria has provided a new means for the diagnosis and treatment of diseases. However, in vivo applications of these engineered bacteria are hindered by their inefficient accumulation in areas of inflammation. In this study, we constructed an engineered Escherichia coli (E. coli) for directional migration toward tetrathionate (a biomarker of gut inflammation), which is regulated by the TtrSR two-component system (TCS) from Shewanella baltica OS195 (S. baltica). Specifically, we removed endogenous cheZ to control the motility of E. coli. Moreover, we introduced the reductase gene cluster (ttrBCA) from Salmonella enterica serotype typhimurium (S. typhimurium), a major pathogen causing gut inflammation, into E. coli to metabolize tetrathionate. The resulting strain was tested for its motility along the gradients of tetrathionate; the engineered strain exhibits tropism to tetrathionate compared with the original strain. Furthermore, the engineered E. coli could only restore its smooth swimming ability when tetrathionate existed. With these modifications enabling tetrathionate-mediated chemotactic and metabolizing activity, this strategy with therapeutic elements will provide a great potential opportunity for target treatment of various diseases by swapping the corresponding genetic circuits.

Improvement of Mueller-Kauffman Tetrathionate-Novobiocin (MKTTn) enrichment medium for the detection of Salmonella enterica by the addition of ex situ-generated tetrathionate.

The detection of Salmonella in food is based on the use of a selective enrichment broth such as Muller-Kauffman Tetrathionate-Novobiocin (MKTTn), in which tetrathionate plays a key role by providing Salmonella with a growth advantage. As sodium tetrathionate is unstable, it is generated in situ by the addition of iodine (Lugol's solution) before seeding. This step is cumbersome as the solution is easily spilled, compromising the performance of the medium and hindering the work of technicians. The aim of this study was to optimize MKTTn broth by generating tetrathionate ex situ through an external reaction between iodine and thiosulphate followed by lyophilization. Quality control procedures were performed to compare the modified and original media, testing pure productivity (enrichment with 50-120 CFU of Salmonella Thyphimurium ATCC 14028 and Salmonella Enteritidis ATCC 13076 and plating on Xylose Lysine Deoxycholate agar, XLD), mixed productivity (50-120 CFU of Salmonella strains and Pseudomonas aeruginosa and Escherichia coli at ≥104 CFU and XLD plating) and selectivity (≥104 CFU of P. aeruginosa and Enterococcus faecalis and plating on Tryptone Casein Soy agar, TSA). The modified MKTTn medium (S/L) performed comparably with the original medium in terms of growth of both Salmonella strains (>300 colonies in XLD), alone or with P. aeruginosa and E. coli. Quantitative assays showed no statistically significant differences in the number of colonies grown on XLD after 10-5 dilution (p = 0.7015 with S. Thyphimurium ATCC 14028 and p = 0.2387 with S. Enteritidis ATCC 13076; ANOVA test). MKTTn medium (S/L) was also selective against E. coli (≤100 colonies) and E. faecalis (<10 colonies). These results suggest that adding tetrathionate as a lyophilisate (S/L) is a feasible alternative to the use of Lugol's solution for the preparation of MKTTn enrichment broth and does not affect the properties of the medium.

The emergence of the tetrathionate reductase operon in the Escherichia coli/Shigella pan-genome.

Escherichia coli pathogenic variants (pathovars) are generally characterized by defined virulence traits and are susceptible to the evolution of hybridized identities due to the considerable plasticity of the E. coli genome. We have isolated a strain from a purified diet intended for research animals that further demonstrates the ability of E. coli to acquire novel genetic elements leading potentially to emergent new pathovars. Utilizing next generation sequencing to obtain a whole genome profile, we report an atypical strain of E. coli, EcoFA807-17, possessing a tetrathionate reductase (ttr) operon, which enables the utilization of tetrathionate as an electron acceptor, thus facilitating respiration in anaerobic environments such as the mammalian gut. The ttr operon is a potent virulence factor for several enteric pathogens, most prominently Salmonella enterica. However, the presence of chromosomally integrated tetrathionate reductase genes does not appear to have been previously reported in wild-type E. coli or Shigella. Accordingly, it is possible that the appearance of this virulence factor may signal the evolution of new mechanisms of pathogenicity in E. coli and Shigella and may potentially alter the effectiveness of existing assays using tetrathionate reductase as a unique marker for the detection of Salmonella enterica.