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Acidithiobacillus ferrooxidans plays an important role in bioleaching in reproducing the mineral oxidant of ferric iron ( Fe3+) by oxidization of ferrous iron ( Fe2+). The high-molecular-weight c-type cytochrome Cyc2 that is located in the external membrane is postulated as the first electron carrier in the Fe2+ oxidation respiratory pathway of A. ferrooxidans. To increase ferrous iron oxidation activity, a recombinant plasmid p TCYC2 containing cyc2 gene under the control of Ptac promoter was constructed and transferred into A. ferrooxidans ATCC19859. The transcriptional level of cyc2 gene was increased by 2.63-fold and Cyc2 protein expression was observed in the recombinant strain compared with the control. The ferrous iron oxidation activity and the arsenic stressed cell growth of the recombinant strain were also elevated. [ABSTRACT FROM AUTHOR]

Highlights: [•] Gene inactivation by ISAtc2 transposition was firstly discovered in A. caldus. [•] The features of ISAtc2 were identified. [•] The transposition frequency of ISAtc2 was ranged from 4% to 7%. [•] No reverse mutation occurred in the mutants within 50 generations. [•] IS elements may regulate the gene expression and metabolic pathways. [Copyright &y& Elsevier]

Abstract: A mercury-resistant plasmid of pTMJ212 which was able to shuttle between Acidithiobacillus caldus and Escherichia coli was constructed by inserting the mercury resistant determinants, the mer operon of Acidithiobacillus ferrooxidans, into the IncQ plasmid of pJRD215. pTMJ212 was transferred from Escherichia coli into Acidithiobacillus caldus through conjugation. Furthermore, pTMJ212 was transferred back from Acidithiobacillus caldus into Escherichia coli, thereby confirming the initial transfer of pTMJ212 from Escherichia coli to Acidithiobacillus caldus. Compared to the control, the cell growth of the recombinant Acidithiobacillus caldus increased markedly under mercury (Hg2+) stress especially at Hg2+ concentrations ranging from 2.0 to 4.5μg/ml. [Copyright &y& Elsevier]

Abstract: Metallothioneins (MTs) are a family of metal binding proteins. Recombinant Escherichia coli expressing the human MT (hMT-1A) gene was constructed for bioaccumulation of heavy metals. In order to increase protein stability, the glutathione S-transferase (GST) gene was fused with the hMT-1A gene and coexpressed. In order to increase MT expression efficiency and metal binding capacity, two, three or four hMT-1A genes were integrated in series and overexpressed in E. coli. The recombinant E. coli expressing the GST fused trimeric hMT-1A protein exhibited the highest Cd(II) and As(III) bioaccumulation ability, 6.36mgCd/g dry cells and 7.59mgAs/g dry cells, respectively. [ABSTRACT FROM AUTHOR]

Abstract: The response of Leptospirillum ferriphilum ML-04 to arsenic stress was analyzed using two-dimensional electrophoresis (2-DE), matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Thirty-eight of 65 significantly differentially expressed arsenic response proteins were identified, and 25 of them have known functions. Three proteins are arsenic resistance system (ARS) member proteins. Two ars operons appear to be present in this strain. In addition to the ARS system, phosphate regulation and glutathione (GSH) synthesis appear involved in As[V] and As[III] tolerance, respectively. These findings provide information potentially useful for the genetic engineering of arsenic resistant organisms. [ABSTRACT FROM AUTHOR]

Introduction: To describe the clinical and laboratory features of systemic lupus erythematosus (SLE) enteritis and to establish a predictive model of risk and severity of lupus enteritis (LE). Methods: Records of patients with SLE complaining about acute digestive symptoms were reviewed. The predictive nomogram for the diagnosis of LE was constructed by using R. The accuracy of the model was tested with correction curves. The receiver operating characteristic curve (ROC curve) program and a Decision curve analysis (DCA) were used for the verification of LE model. Receiver operating characteristic curve was also employed for evaluation of factors in the prediction of severity of LE. Results: During the eight year period, 46 patients were in the LE group, while 32 were in the non-LE group. Abdominal pain, emesis, D-dimer >5 μg/mL, hypo-C3, and anti-SSA positive remained statistically significant and were included into the prediction model. Area under the curve (AUC) of ROC curve in this model was 0.909. Correction curve indicated consistency between the predicted rate and actual diagnostic rates. The DCA showed that the LE model was of benefit. Forty-four patients were included in developing the prediction model of LE severity. Infection, SLE disease activity index (SLEDAI), CT score, and new CT score were validated as risk factors for LE severity. The AUC of the combined SLEDAI, infection and new CT score were 0.870. Conclusion: The LE model exhibits good predictive ability to assess LE risk in SLE patients with acute digestive symptoms. The combination of SLEDAI, infection, and new CT score could improve the assessment of LE severity. [ABSTRACT FROM AUTHOR]

Objective Idiopathic inflammatory myositis-associated interstitial lung disease (IIM-ILD) significantly increases morbidity and mortality. Lung ultrasound B-lines and Krebs von den Lungen-6 (KL-6) are identified as new sonographic and serum markers of ILD, respectively. The aim of our work was to assess the role of B-lines and KL-6 as markers of the severity of IIM-ILD. For this purpose, the correlation among B-lines score, serum KL-6 levels, high-resolution CT (HRCT) score, and pulmonary function tests were investigated in IIM-ILD patients. Methods Thirty-eight patients with IIM-ILD underwent chest HRCT scans, lung ultrasound and pulmonary function tests (independently performed within 1 week) examination. To assess severity and extent of ILD at HRCT, the Warrick score was used. The B-lines score denoting the extension of ILD was calculated by summing the number of B-lines on a total of 50 scanning sites. Serum KL-6 levels (U/ml) was measured by chemiluminescent enzyme immunoassay. Results A significant correlation was found between the B-lines score and serum KL-6 levels (r  = 0.43, P  < 0.01), and between the Warrick score and serum KL-6 levels (r  = 0.45, P  < 0.01). A positive correlation between B-lines score and the Warrick score (r  = 0.87, P  < 0.0001) was also confirmed. Both B-lines score and KL-6 levels inversely correlated to diffusion capacity for carbon monoxide (r  = −0.77, P  < 0.0001 and r  = −0.42, P  < 0.05, respectively) and total lung capacity (r  = −0.73, P  < 0.0001 and r  = −0.36, P  < 0.05, respectively). Moreover, B-lines correlated inversely with forced vital capacity (r  = −0.73, P  < 0.0001), forced expiratory volume in 1 s (r  = −0.69, P  < 0.0001). Conclusion B-lines score and serum KL-6 levels correlate with HRCT findings and pulmonary function tests, supporting their use as measures of IIM-ILD severity. [ABSTRACT FROM AUTHOR]

(D) Contrast-enhanced chest computed tomography scan showing an irregular and poorly defined mass (asterisk) in the right superior mediastina with deformation and partial occlusion of the superior vena cava. A 58-year-old woman was hospitalized in our rheumatology department with a 1-month history of myalgia, edema on bilateral hands and feet, and puffy face, without systemic symptoms such as fever, weight loss, or anorexia. Subsequent contrast-enhanced chest computed tomography scan showed an irregular and poorly defined mass (Figure 1D, asterisk) in the right superior mediastina with deformation and partial occlusion of the superior vena cava. [Extracted from the article]

d -Allulose (or d -Psicose), a C-3 epimer of d -fructose, is a low-calorie rare sugar with excellent physiological functions. The recombinant Escherichia coli expressing d -allulose 3-epimerase for d -allulose conversion from d -fructose was constructed. Under the optimal conditions, 139.3 g/L d -allulose was produced from 500 g/L of d -fructose. In order to decrease the cost for mass production, one-pot reaction method by using immobilized glucose isomerase and recombinant E. coli expressing d -allulose 3-epimerase to produce d -allulose from d -glucose was developed. The immobilized glucose isomerase (200 g/L) and the recombinant E. coli cells (OD 600 2) were mixed and used to transform d -glucose into d -allulose, and 228.5 g/L d -glucose, 216.3 g/L d -fructose and 90.7 g/L d -allulose were obtained from 550 g/L d -glucose after 3 h reaction. After that, d -allulose was separated from the reaction mixture by using simulated moving bed chromatography (SMBC) with the purity of 99.6%. Finally, crystallization was made to obtain the d -allulose crystals with 99.8% purity. The combination of enzyme and catalytic cells in biotransformation greatly expand the flexibility and capability of the catalytic reactions. This method developed in this study can be easily scaled up for mass production of highly purified d -allulose. [Display omitted] • Biotransformation of d -allulose from low cost d -glucose was realized. • Separation of d -allulose were achieved by simulated moving bed chromatography. • The practical process for mass production of d -allulose was established. • Mixed biocatalysts increase the flexibility and capability of the catalyzation. [ABSTRACT FROM AUTHOR]

Acidithiobacillus spp. are the most active bacteria in bioleaching and bioremediation, because of their remarkable extreme environmental adaptabilities and unique metabolic characteristics. The researches on regulatory mechanisms of energy metabolism and stress resistance are critical for the understanding and application of Acidithiobacillus spp. However, the lack of an ideal reporter gene has become an obstacle for studying genes expression and regulatory mechanism in these chemoautotrophic bacteria. In this study, we reported the firefly luciferase as a reporter gene for Acidithiobacillus caldus (A. caldus) and created a firefly luciferase (Luc) reporter system. The Luc system was applied for the quantitative analysis of the transcription strength of the promoters of tetH gene and the feoA gene in A. caldus. Moreover, the regulating effect of ferric uptake regulator (Fur) on the feoP gene in A. caldus was determined using the Luc system. The Luc reporter system is not only used in the study of regulatory mechanism of A. caldus, but also applied in the researches of other Acidithiobacillus species. Therefore, this study provides a new useful tool for the studies on the molecular biological mechanism and synthetic biological modification of these chemoautotrophic bacteria, which would promote the industrial application of Acidithiobacillus spp. [ABSTRACT FROM AUTHOR]

A strain SDU10 was isolated from swine manure compost and identified as Pseudomonas stutzeri SDU10. It demonstrated excellent capability in NH4+-N removal. Optimal conditions of NH4+-N removal were determined, which were sodium acetate as the optimal carbon source, carbon to nitrogen (C/N) ratio of 10, temperature of 30 °C, pH of 7.0. Especially, P. stutzeri SDU10 could remove high concentration NH4+-N of 1500.0 and 2000.0 mg/l in 120 h with the NH4+-N removal rates of 91.1% and 61.6%, respectively. In batch experiments, the highest NH4+-N removal rate of 97.6% and chemical oxygen demand (COD) removal rate of 94.2% were obtained at initial C/N ratio 10 during piggery wastewater treatment using P. stutzeri SDU10. Results showed that P. stutzeri SDU10 had the potential for treatment of wastewater of high NH4+-N concentration. [ABSTRACT FROM AUTHOR]

In the past decade, lung ultrasound (LUS) B-lines and serum Krebs von den Lungen-6 (KL-6) antigen have been recognized as biomarkers of the connective tissue disease-associated interstitial lung diseases (CTD-ILDs). Robust data have demonstrated that B-lines total numbers and KL-6 levels are correlated with high-resolution computed tomography findings, pulmonary function test, and some clinical parameters in CTD-ILDs. However, limited data are available regarding the use of these two biomarkers to follow CTD-ILDs. Herein, we report a case with anti-melanoma differentiation-associated gene 5 antibody-positive clinically amyopathic dermatomyositis-associated ILD, successfully treated with high-dose methylprednisolone, cyclophosphamide, intravenous immunoglobulin, pirfenidone, and followed using lung ultrasound and KL-6. [ABSTRACT FROM AUTHOR]

Acute fibrinous and organizing pneumonia (AFOP) is a new histopathological pattern of acute lung injury first described by Beasley et al. in 2002. Hallmarks of pathological findings are characterized by the presence of intra-alveolar fibrin in the form of fibrin “balls” within the alveolar spaces and organizing pneumonia with a patchy distribution. Patients with AFOP may have an acute or subacute clinical presentation. Although the pathogenesis of AFOP is not fully elucidated, it may be associated with autoimmune diseases. Reported herein is a patient diagnosed of acute AFOP associated with primary Sjögren’s syndrome. The patient’s condition promptly improved after treatment with corticosteroid. [ABSTRACT FROM AUTHOR]

Acidithiobacillus caldus is a chemolithoautotrophic sulfur-oxidizing bacterium that is widely used for bioleaching processes. Acidithiobacillus spp. are suggested to contain sulfur dioxygenases (SDOs) that facilitate sulfur oxidation. In this study, two putative sdo genes (A5904_0421 and A5904_1112) were detected in the genome of A. caldus MTH-04 by BLASTP searching with the previously identified SDO (A5904_0790). We cloned and expressed these genes, and detected the SDO activity of recombinant protein A5904_0421 by a GSH-dependent in vitro assay. Phylogenetic analysis indicated that A5904_0421and its homologous SDOs, mainly found in autotrophic bacteria, were distantly related to known SDOs and were categorized as a new subgroup of SDOs. The potential functions of genes A5904_0421 (termed sdo1) and A5904_0790 (termed sdo2) were investigated by generating three knockout mutants (Δsdo1, Δsdo2 and Δsdo1&2), two sdo overexpression strains (OE-sdo1 and OE-sdo2) and two sdo complemented strains (Δsdo1/sdo1’ and Δsdo2/sdo2’) of A. caldus MTH-04. Deletion or overexpression of the sdo genes did not obviously affect growth of the bacteria on S0, indicating that the SDOs did not play an essential role in the oxidation of extracellular elemental sulfur in A. caldus. The deletion of sdo1 resulted in complete inhibition of growth on tetrathionate, slight inhibition of growth on thiosulfate and increased GSH-dependent sulfur oxidation activity on S0. Transcriptional analysis revealed a strong correlation between sdo1 and the tetrathionate intermediate pathway. The deletion of sdo2 promoted bacterial growth on tetrathionate and thiosulfate, and overexpression of sdo2 altered gene expression patterns of sulfide:quinone oxidoreductase and rhodanese. Taken together, the results suggest that sdo1 is essential for the survival of A. caldus when tetrathionate is used as the sole energy resource, and sdo2 may also play a role in sulfur metabolism. [ABSTRACT FROM AUTHOR]

Background: Acidithiobacillus caldus, a Gram-negative, chemolithotrophic sulfur-oxidizing bacterium, is widely applied in bioleaching. The absence of an ideal selection marker has become a major obstacle to achieve high efficiency of the gene transfer system for A. caldus. Plasmid pJRD215, widely used in Acidithiobacillus spp., has severe drawbacks in molecular manipulations and potential biosafety issues due to its mobility. Therefore, finding a new selection marker and constructing new plasmids have become an urgent and fundamental work for A. caldus. Results: Effective inhibitory effect of chloramphenicol on the growth of A. caldus was elucidated for the first time. The P2-cat gene cassette, including a chloramphenicol acetyltransferase gene (cat) from plasmid pACBSR and a promoter (P2) upstream of the tetracycline resistance gene on pBR322, was designed, chloramphenicol acetyltransferase was expressed in A. caldus, and the enzyme activity was assessed. A new vector pSDU1 carrying the replication and mobilization regions derived from pJRD215, the P2-cat gene cassette and a multiple cloning site from pUC19 was successfully constructed. Compared with pJRD215, pSDU1 had a 27-fold increase in electrotransformation efficiency (30.43±0.88×104 CFU/μg DNA for pSDU1 and 1.09±0.11×104 CFU/μg DNA for pJRD215), better carrying capacity and could offer more convenience for the restriction enzyme digestion. In addition, the generated plasmid pSDU1Δmob, a novel non-mobilizable derivative of pSDU1 lacking some DNA sequences involved in the mobilization process, had increased copy number in A. caldus and lost its mobility for biosafety considerations. Both pSDU1 and pSDU1Δmob exhibited stable maintenance in A. caldus within 50 passages. However, further deletion of orfEF region involved in regulating repAC operon resulted in a negative effect on transformation efficiency, copy number and stability of plasmid pSDU1ΔmobΔorfEF in A. caldus. Conclusion: Chloramphenicol was proved to be an ideal selection marker for A. caldus. Novel plasmids carrying cat gene were constructed. The utilization of these vectors will undoubtedly facilitate efficient genetic manipulations and accelerate the research progress in A. caldus. [ABSTRACT FROM AUTHOR]

Acidithiobacillus ferrooxidans is a model organism for investigating metal sulfide bioleaching. The regulatory mechanism of gene expression by metabolizing various substrates is critical for understanding its role in bioleaching processes. However, no reporter has been successfully employed to study gene expression in A. ferrooxidans to date. In this study, a sensitive and robust reporter system based on β-glucuronidase (GusA) was described for feasible application in A. ferrooxidans. A set of vectors, which contained the transcriptional and translational fusions of gusA, were constructed and employed to analyze promoter activity and efficiency of translation initiation in A. ferrooxidans. Ptac and P2811 were screened out from ten tested promoters and could be used as strong promoters for gene overexpression in A. ferrooxidans. Among the four translational fusions of gusA with different start codons, ATG was most active, followed by TTG and GTG, while CTG showed the least activity. The transcriptional inhibition effect of an IclR-like transcription factor was also observed on its own encoding gene AFE_1668 as well as its neighboring AFE_1667. In addition, the specific chromogenic reaction of GusA could be detected and visualized by colonies of A. ferrooxidans containing gusA expression plasmids. Generally, the established GusA reporter system would be applied not only for quantitative analysis of promoter strength and its transcriptional regulation but also for qualitative colony screening in A. ferrooxidans in the future. [ABSTRACT FROM AUTHOR]

The acidophilic bioleaching bacteria can usually survive in high concentrations of copper ions because of their special living environment. However, little is known about the copper homeostatic mechanisms of A cidithiobacillus thiooxidans, an important member of bioleaching bacteria. Here, a putative multicopper oxidase gene ( cueO) was detected from the draft genome of A . thiooxidans ATCC 19377. The transcriptional level of cueO in response to 10 mM CuSO4 was upregulated 25.01 ± 2.59 folds. The response of PcueO to copper was also detected and might be stimulated by a putative CueR protein. Then, by using the counter-selectable marker lacZ and enhancing the expression of endonuclease I- SceI with tac promoter, a modified markerless gene disruption system was developed and the cueO gene disruption mutant (Δ cueO) of A . thiooxidans was successfully constructed with a markedly improved second homologous recombination frequency of 0.28 ± 0.048. The Δ cueO mutant was more sensitive to external copper and nearly completely lost the phenoloxidase activity; however, the activity could be restored after complementing the cueO gene. All results suggest the close relation of cueO gene to copper tolerance in A . thiooxidans. In addition, the developed efficient markerless gene knockout method can also be introduced into other A cidithiobacillus strains. [ABSTRACT FROM AUTHOR]

Elemental sulfur (S) oxidation in Acidithiobacillus spp. is an important process in metal sulfide bioleaching. However, the gene that encodes the sulfur dioxygenase (SDO) for S oxidation has remained unclarified in Acidithiobacillus spp. By BLASTP with the eukaryotic mitochondrial sulfur dioxygenases (ETHE1s), the putative sdo genes (AFE_0269 and ACAL_0790) were recovered from the genomes of Acidithiobacillus ferrooxidans ATCC 23270 and Acidithiobacillus caldus MTH-04. The purified recombinant proteins of AFE_0269 and ACAL_0790 exhibited remarkable SDO activity at optimal mildly alkaline pH by using the GSH-dependent in vitro assay. Then, a sdo knockout mutant and a sdo overexpression strain of A. ferrooxidans ATCC 23270 were constructed and characterized. By overexpressing sdo in A. ferrooxidans ATCC 23270, a significantly increased transcriptional level of sdo (91-fold) and a 2.5-fold increase in SDO activity were observed when S was used as sole energy source. The sdo knockout mutant of A. ferrooxidans displayed a slightly reduced growth capacity in S-medium compared with the wild type but still maintained high S-oxidizing activity, suggesting that there is at least one other S-oxidizing enzyme besides SDO in A. ferrooxidans ATCC 23270 cells. In addition, no obvious changes in transcriptional levels of selected genes related to sulfur oxidation was observed in response to the sdo overexpression or knockout in A. ferrooxidans when cultivated in S-medium. All the results might suggest that SDO is involved in sulfide detoxification rather than bioenergetic S oxidation in chemolithotrophic bacteria. [ABSTRACT FROM AUTHOR]

The genetic improvement of biomining bacteria including Acidithiobacillus caldus could facilitate the bioleaching process of sulfur-containing minerals. However, the available vectors for use in A. caldus are very scanty and limited to relatively large broad-host-range IncQ plasmids. In this study, a set of small, mobilizable plasmid vectors (pBBR1MCS-6, pMSD1 and pMSD2) were constructed based on plasmid pBBR1MCS-2, which does not belong to the IncQ, IncW, or IncP groups. The function of the tac promoter on 5.8-kb pMSD2 was determined by inserting a kanamycin-resistant reporter gene. The resulting recombinant pMSD2-Km was successfully transferred by conjugation into A. caldus MTH-04 with transfer frequency of 1.38±0.64×10−5. The stability and plasmid copy number of pMSD2-Km in A. caldus MTH-04 were 75±2.7% and 5–6 copies per cell, respectively. By inserting an arsABC operon into pMSD2, an arsenic-resistant recombinant pMSD2-As was constructed and transferred into A. caldus MTH-04 by conjugation. The arsenic tolerance of A. caldus MTH-04 containing pMSD2-As was obviously increased up to 45mM of NaAsO2. These vectors could be applied in genetic improvement of A. caldus as well as other bioleaching bacteria. [ABSTRACT FROM AUTHOR]

L-Xylulose is a potentially valuable rare sugar used as starting material for antiviral and anticancer drug development in pharmaceutical industries. In this study, cofactor engineering was applied to improve the efficiency of L -xylulose production from xylitol. A water-forming NAD+ regeneration enzyme (NADH oxidase) from Streptococcus mutans ATCC 25175 was introduced into E. coli with xylitol-4-dehydrogenase (XDH) of Pantoea ananatis resulting in recombinant cells harboring the vector pETDuet -xdh-SmNox. The co-expression system exhibited optimal activity at a temperature of 37 °C and pH 8.5, and the addition of Mg2+ enhanced the catalytic activity by 1.19 fold. Co-expression of NADH oxidase with XDH enzyme resulted in increased L -xylulose concentration and productivity from xylitol as well as the intracellular NAD+ concentration. In a 1 L bioconversion system the final concentration and productivity of L -xylulose from 50 g/L of xylitol reached 48.45 g/L, and 2.42 g/L.h respectively. Overall, this study is a suitable approach for large-scale production of L -xylulose from xylitol. • XDH from P. ananatis catalyzes NAD+-dependent interconversion between xylitol and L -xylulose. • A water-forming NADH oxidase from S. mutans was co-expressed with XDH in E. coli. • The NAD+ regeneration has been enhanced by the co-expression system. • The system resulted in an increased L -xylulose production at relatively higher substrate concentration. [ABSTRACT FROM AUTHOR]

Abstract: An efficient genetic system for introducing genes into biomining microorganisms is essential not only to experimentally determine the functions of genes predicted based on bioinformatic analysis, but also for their genetic breeding. In this study, a small broad-host-range vector named pBBR1MCS-2, which does not belong to the IncQ, IncW, or IncP groups, was studied for the feasibility of its use in conjugative gene transfer into extremely acidophilic strains of Acidithiobacillus. To do this, a recombinant plasmid pBBR-tac-Sm, a derivative of pBBR1MCS-2, was constructed and the streptomycin resistant gene (Sm r ) was used as the reporter gene. Using conjugation, pBBR-tac-Sm was successfully transferred into three tested strains of Acidithiobacillus. Then we measured its transfer frequency, its stability in Acidithiobacillus cells, and the level of resistance to streptomycin of the transconjugants and compared this with the IncQ plasmid pJRD215 control. Our results indicate that pBBR1MCS-2 provides a new and useful tool in the genetic manipulation of Acidithiobacillus strains. [Copyright &y& Elsevier]

Abstract: Respirometry was used to study the growth and activity of six pure cultures of acidophilic bioleaching strains grown on a concentration series of low-grade chalcopyrite ores under various pH conditions. Sulfolobus metallicus, Acidithiobacillus ferrooxidans, Acidianus brierleyi and Leptospirillum ferriphilum were able to grow on a very low-grade ore (equivalent to 0.1% Cu content). However, the two sulphur-oxidising bacteria Acidithiobacillus caldus and Acidithiobacillus thiooxidans grew poorly on the low-grade ore. Growth rates of all strains, except for perhaps S. metallicus at highest ore grades, displayed growth that was limited by substrate availability on this low-grade ore (0.5% Cu content in the ore). The decrease in solution pH from 3.0 to 1.0 enhanced both the cell growth and Cu dissolution. [Copyright &y& Elsevier]