Using the sulfide-oxidizing bacterium Geobacillus thermodenitrificans to restrict H2S release during chicken manure composting.

Hydrogen sulfide (H2S) is a toxic gas massively released during chicken manure composting. Diminishing its release requires efficient and low cost methods. In recent years, heterotrophic bacteria capable of rapid H2S oxidation have been discovered but their applications in environmental improvement are rarely reported. Herein, we investigated H2S oxidation activity of a heterotrophic thermophilic bacterium Geobacillus thermodenitrificans DSM465, which contains a H2S oxidation pathway composed by sulfide:quinone oxidoreductase (SQR) and persulfide dioxygenase (PDO). This strain rapidly oxidized H2S to sulfane sulfur and thiosulfate. The oxidation rate reached 5.73 µmol min-1·g-1 of cell dry weight. We used G. thermodenitrificans DSM465 to restrict H2S release during chicken manure composting. The H2S emission during composting process reduced by 27.5% and sulfate content in the final compost increased by 34.4%. In addition, this strain prolonged the high temperature phase by 7 days. Thus, using G. thermodenitrificans DSM465 to control H2S release was an efficient and economic method. This study provided a new strategy for making waste composting environmental friendly and shed light on perspective applications of heterotrophic H2S oxidation bacteria in environmental improvements.

Rapid Detection of the Anti-Tumor Drug Etoposide in Biological Samples by Using a Nanoporous-Gold-Based Electrochemical Sensor.

Monitoring etoposide is important due to its wide usage in anti-tumor therapy; however, the commonly used HPLC method is expensive and often requires complicated extraction and detection procedures. Electrochemical analysis has great application prospects because of its rapid response and high specificity, sensitivity, and efficiency with low cost and high convenience. In this study, we constructed a nanoporous gold (NPG)-modified GCE for the detection of etoposide. The electrochemical oxidation of etoposide by NPG caused a sensitive current peak at +0.27 V with good reproductivity in 50 mM of phosphate buffer (pH 7.4). The relationship between etoposide concentration and peak current was linear in the range between 0.1 and 20 µM and between 20 and 150 µM, with a detection sensitivity of 681.8 µA mM-1 cm-2 and 197.2 µA mM-1 cm-2, respectively, and a limit of detection (LOD) reaching 20 nM. The electrode had a good anti-interference ability to several common anions and cations. Spiked recovery tests in serum, urine, and fermentation broth verified the excellent performance of the sensor in terms of sensitivity, reproducibility, and specificity. This may provide a promising tool for the detection of etoposide in biological samples.

The pathway of recombining short homologous ends in Escherichia coli revealed by the genetic study.

The recombination of short homologous ends in Escherichia coli has been known for 30 years, and it is often used for both site-directed mutagenesis and in vivo cloning. For cloning, a plasmid and target DNA fragments were converted into linear DNA fragments with short homologous ends, which are joined via recombination inside E. coli after transformation. Here this mechanism of joining homologous ends in E. coli was determined by a linearized plasmid with short homologous ends. Two 3'-5' exonucleases ExoIII and ExoX with nonprocessive activity digested linear dsDNA to generate 5' single-strand overhangs, which annealed with each other. The polymerase activity of DNA polymerase I (Pol I) was exclusively employed to fill in the gaps. The strand displacement activity and the 5'-3' exonuclease activity of Pol I were also required, likely to generate 5' phosphate termini for subsequent ligation. Ligase A (LigA) joined the nicks to finish the process. The model involving 5' single-stranded overhangs is different from established recombination pathways that all generate 3' single-stranded overhangs. This recombination is likely common in bacteria since the involved enzymes are ubiquitous.

The Pathway of Sulfide Oxidation to Octasulfur Globules in the Cytoplasm of Aerobic Bacteria.

Sulfur-oxidizing bacteria can oxidize hydrogen sulfide (H2S) to produce sulfur globules. Although the process is common, the pathway is unclear. In recombinant Escherichia coli and wild-type Corynebacterium vitaeruminis DSM 20294 with sulfide:quinone oxidoreductase (SQR) but no enzymes to oxidize zero valence sulfur, SQR oxidized H2S into short-chain inorganic polysulfide (H2Sn, n ≥ 2) and organic polysulfide (RSnH, n ≥ 2), which reacted with each other to form long-chain GSnH (n ≥ 2) and H2Sn before producing octasulfur (S8), the main component of elemental sulfur. GSnH also reacted with glutathione (GSH) to form GSnG (n ≥ 2) and H2S; H2S was again oxidized by SQR. After GSH was depleted, SQR simply oxidized H2S to H2Sn, which spontaneously generated S8. S8 aggregated into sulfur globules in the cytoplasm. The results highlight the process of sulfide oxidation to S8 globules in the bacterial cytoplasm and demonstrate the potential of using heterotrophic bacteria with SQR to convert toxic H2S into relatively benign S8 globules. IMPORTANCE Our results provide evidence of H2S oxidation producing octasulfur globules via sulfide:quinone oxidoreductase (SQR) catalysis and spontaneous reactions in the bacterial cytoplasm. Since the process is an important event in geochemical cycling, a better understanding facilitates further studies and provides theoretical support for using heterotrophic bacteria with SQR to oxidize toxic H2S into sulfur globules for recovery.

Sulfane Sulfur is an intrinsic signal activating MexR-regulated antibiotic resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa PAO1, an opportunistic human pathogen, deploys several strategies to resist antibiotics. It uses multidrug efflux pumps, including the MexAB-OprM pump, for antibiotic resistance, and it also produces hydrogen sulfide (H2 S) that provides some defense against antibiotics. MexR functions as a transcriptional repressor of the mexAB-oprM operon. MexR responds to oxidative stresses caused by antibiotic exposure, and it also displays a growth phase-dependent derepression of the mexAB-oprM operon. However, the intrinsic inducer has not been identified. Here, we report that P. aeruginosa PAO1 produced sulfane sulfur, including glutathione persulfide and inorganic polysulfide, produced from either H2 S oxidation or from L-cysteine metabolism. Sulfane sulfur directly reacted with MexR, forming di- and trisulfide cross-links between two Cys residues, to derepress the mexAB-oprM operon. Levels of cellular sulfane sulfur and mexAB-oprM expression varied during growth, and both reached the maximum during the stationary phase of growth. Thus, self-produced H2 S and sulfane sulfur may facilitate antibiotic resistance via inducing the expression of antibiotic resistance genes.

T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis.

The assembly of DNA fragments with homologous arms is becoming popular in routine cloning. For an in vitro assembly reaction, a DNA polymerase is often used either alone for its 3'-5' exonuclease activity or together with a 5'-3' exonuclease for its DNA polymerase activity. Here, we present a 'T5 exonuclease DNA assembly' (TEDA) method that only uses a 5'-3' exonuclease. DNA fragments with short homologous ends were treated by T5 exonuclease and then transformed into Escherichia coli to produce clone colonies. The cloning efficiency was similar to that of the commercial In-Fusion method employing a proprietary DNA polymerase, but higher than that of the Gibson method utilizing T5 exonuclease, Phusion DNA polymerase, and DNA ligase. It also assembled multiple DNA fragments and did simultaneous site-directed mutagenesis at multiple sites. The reaction mixture was simple, and each reaction used 0.04 U of T5 exonuclease that cost 0.25 US cents. The simplicity, cost effectiveness, and cloning efficiency should promote its routine use, especially for labs with a budget constraint. TEDA may trigger further development of DNA assembly methods that employ single exonucleases.

Structural and biochemical characterization of iminodiacetate oxidase from Chelativorans sp. BNC1.

Ethylenediaminetetraacetate (EDTA) is the most abundant organic pollutant in surface water because of its extensive usage and the recalcitrance of stable metal-EDTA complexes. A few bacteria including Chelativorans sp. BNC1 can degrade EDTA with a monooxygenase to ethylenediaminediacetate (EDDA) and then use iminodiacetate oxidase (IdaA) to further degrade EDDA into ethylenediamine in a two-step oxidation. To alleviate EDTA pollution into the environment, deciphering the mechanisms of the metabolizing enzymes is an imperative prerequisite for informed EDTA bioremediation. Although IdaA cannot oxidize glycine, the crystal structure of IdaA shows its tertiary and quaternary structures similar to those of glycine oxidases. All confirmed substrates, EDDA, ethylenediaminemonoacetate, iminodiacetate and sarcosine are secondary amines with at least one N-acetyl group. Each substrate was bound at the re-side face of the isoalloxazine ring in a solvent-connected cavity. The carboxyl group of the substrate was bound by Arg265 and Arg307 . The catalytic residue, Tyr250 , is under the hydrogen bond network to facilitate its deprotonation acting as a general base, removing an acetate group of secondary amines as glyoxylate. Thus, IdaA is a secondary amine oxidase, and our findings improve understanding of molecular mechanism involved in the bioremediation of EDTA and the metabolism of secondary amines.

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.

The Structural Basis of the Binding of Various Aminopolycarboxylates by the Periplasmic EDTA-Binding Protein EppA from Chelativorans sp. BNC1.

The widespread use of synthetic aminopolycarboxylates, such as ethylenediaminetetraacetate (EDTA), as chelating agents has led to their contamination in the environment as stable metal-chelate complexes. Microorganisms can transport free EDTA, but not metal-EDTA complexes, into cells for metabolism. An ABC-type transporter for free EDTA uptake in Chelativorans sp. BNC1 was investigated to understand the mechanism of the ligand selectivity. We solved the X-ray crystal structure of the periplasmic EDTA-binding protein (EppA) and analyzed its structure-function relations through isothermal titration calorimetry, site-directed mutagenesis, molecular docking, and quantum chemical analysis. EppA had high affinities for EDTA and other aminopolycarboxylates, which agrees with structural analysis, showing that its binding pocket could accommodate free aminopolycarboxylates. Further, key amino acid residues involved in the binding were identified. Our results suggest that EppA is a general binding protein for the uptake of free aminopolycarboxylates. This finding suggests that bacterial cells import free aminopolycarboxylates, explaining why stable metal-chelate complexes are resistant to degradation, as they are not transported into the cells for degradation.

Sensitive Method for Reliable Quantification of Sulfane Sulfur in Biological Samples.

Sulfane sulfur has been recognized as a common cellular component, participating in regulating enzyme activities and signaling pathways. However, the quantification of total sulfane sulfur in biological samples is still a challenge. Here, we developed a method to address the need. All tested sulfane sulfur reacted with sulfite and quantitatively converted to thiosulfate when heated at 95 °C in a solution of pH 9.5 for 10 min. The assay condition was also sufficient to convert total sulfane sulfur in biological samples to thiosulfate for further derivatization and quantification. We applied the method to detect sulfane sulfur contents at different growth phases of bacteria, yeast, mammalian cells, and zebrafish. Total sulfane sulfur contents in all of them increased in the early stage, kept at a steady state for a period, and declined sharply in the late stage of the growth. Sulfane sulfur contents varied in different species. For Escherichia coli, growth media also affected the sulfane sulfur contents. Total sulfane sulfur contents from different organs of mouse and shrimp were also detected, varying from 1 to 10 nmol/(mg of protein). Thus, the new method is suitable for the quantification of total sulfane sulfur in biological samples.

Developing Polysulfide-Sensitive GFPs for Real-Time Analysis of Polysulfides in Live Cells and Subcellular Organelles.

Polysulfides are newly discovered cellular contents, and they are involved in multiple intracellular processes, including redox homeostasis and protein sulfhydration. The dynamic changes of polysulfides inside the cell are directly related to these processes. To monitor the intracellular dynamics and subcellular levels of polysulfides, we developed green-fluorescent-protein (GFP)-based probes that are polysulfide-specific. A pair of cysteine residues was introduced near the GFP chromophore with the spatial distance between the cysteine residues designed to allow the formation of internal -S n- ( n ≥ 3) bonds but not -S2- (disulfide) bonds. We tested these probes in model microorganisms and found that they displayed ratiometric changes to intracellular polysulfides that had clear variations associated with the growth phases. The distribution of polysulfides in subcellular organelles is heterogeneous, suggesting that polysulfides have multiple origins and functions in cells. These probes provided long-desired tools for polysulfide in vivo studies.

FisR activates σ54 -dependent transcription of sulfide-oxidizing genes in Cupriavidus pinatubonensis JMP134.

Some heterotrophic bacteria are able to oxidize sulfide (H2 S, HS- and S2- ) to sulfite and thiosulfate via polysulfide. The genes coding for the oxidation enzymes in Cupriavidus pinatubonensis JMP134 have recently been identified; however, their regulation is unknown. A regulator gene is adjacent to the operon of the sulfide-oxidizing genes, encoding a σ54 -dependent transcription factor (FisR) with three domains: an R domain, an AAA+ domain and a DNA-binding domain. Here it is reported that the regulator responds to the presence of sulfide and activates the sulfide-oxidizing genes. FisR binds to its cognate operator at -114 to -135 bp of the transcription start of the operon. When polysulfide reacts with the R domain of FisR through the three conserved cysteine residues (C53, C64 and C71), FisR activates the expression of the operon. FisR is highly sensitive to polysulfide, activating σ54 -dependent transcription of sulfide-oxidizing genes for sulfide removal. Further, sequence analysis indicates that FisR-type regulators are relatively common for controlling sulfide-oxidizing genes under sulfide stress in the Proteobacteria.

Structural mechanism for bacterial oxidation of oceanic trimethylamine into trimethylamine N-oxide.

Trimethylamine (TMA) and trimethylamine N-oxide (TMAO) are widespread in the ocean and are important nitrogen source for bacteria. TMA monooxygenase (Tmm), a bacterial flavin-containing monooxygenase (FMO), is found widespread in marine bacteria and is responsible for converting TMA to TMAO. However, the molecular mechanism of TMA oxygenation by Tmm has not been explained. Here, we determined the crystal structures of two reaction intermediates of a marine bacterial Tmm (RnTmm) and elucidated the catalytic mechanism of TMA oxidation by RnTmm. The catalytic process of Tmm consists of a reductive half-reaction and an oxidative half-reaction. In the reductive half-reaction, FAD is reduced and a C4a-hydroperoxyflavin intermediate forms. In the oxidative half-reaction, this intermediate attracts TMA through electronic interactions. After TMA binding, NADP+ bends and interacts with D317, shutting off the entrance to create a protected micro-environment for catalysis and exposing C4a-hydroperoxyflavin to TMA for oxidation. Sequence analysis suggests that the proposed catalytic mechanism is common for bacterial Tmms. These findings reveal the catalytic process of TMA oxidation by marine bacterial Tmm and first show that NADP+ undergoes a conformational change in the oxidative half-reaction of FMOs.

The Complete Pathway for Thiosulfate Utilization in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is known to grow with thiosulfate as a sulfur source, and it produces more ethanol when using thiosulfate than using sulfate. Here, we report how it assimilates thiosulfate. S. cerevisiae absorbed thiosulfate into the cell through two sulfate permeases, Sul1 and Sul2. Two rhodaneses, Rdl1 and Rdl2, converted thiosulfate to a persulfide and sulfite. The persulfide was reduced by cellular thiols to H2S, and sulfite was reduced by sulfite reductase to H2S. Cysteine synthase incorporated H2S into O-acetyl-l-homoserine to produce l-homocysteine, which is the precursor for cysteine and methionine in S. cerevisiae Several other rhodaneses replaced Rdl1 and Rdl2 for thiosulfate utilization in the yeast. Thus, any organisms with the sulfate assimilation system potentially could use thiosulfate as a sulfur source, since rhodaneses are common in most organisms.IMPORTANCE The complete pathway of thiosulfate assimilation in baker's yeast is determined. The finding reveals the extensive overlap between sulfate and thiosulfate assimilation. Rhodanese is the only additional enzyme for thiosulfate utilization. The common presence of rhodanese in most organisms, including Bacteria, Archaea, and Eukarya, suggests that most organisms with the sulfate assimilation system also use thiosulfate. Since it takes less energy to reduce thiosulfate than sulfate for assimilation, thiosulfate has the potential to become a choice of sulfur in optimized media for industrial fermentation.

H2S biotreatment with sulfide-oxidizing heterotrophic bacteria.

Many industrial activities produce H2S, which is toxic at high levels and odorous at even very low levels. Chemolithotrophic sulfur-oxidizing bacteria are often used in its remediation. Recently, we have reported that many heterotrophic bacteria can use sulfide:quinone oxidoreductase and persulfide dioxygenase to oxidize H2S to thiosulfate and sulfite. These bacteria may also potentially be used in H2S biotreatment. Here we report how various heterotrophic bacteria with these enzymes were cultured with organic compounds and the cells were able to rapidly oxidize H2S to zero-valence sulfur and thiosulfate, causing no apparent acidification. Some also converted the produced thiosulfate to tetrathionate. The rates of sulfide oxidation by some of the tested bacteria in suspension, ranging from 8 to 50 µmol min-1 g-1 of cell dry weight at pH 7.4, sufficient for H2S biotreatment. The immobilized bacteria removed H2S as efficiently as the bacteria in suspension, and the inclusion of Fe3O4 nanoparticles during immobilization resulted in increased efficiency for sulfide removal, in part due to chemical oxidation H2S by Fe3O4. Thus, heterotrophic bacteria may be used for H2S biotreatment under aerobic conditions.

Structural and biochemical characterization of EDTA monooxygenase and its physical interaction with a partner flavin reductase.

Ethylenediaminetetraacetate (EDTA) is currently the most abundant organic pollutant due to its recalcitrance and extensive use. Only a few bacteria can degrade it, using EDTA monooxygenase (EmoA) to initiate the degradation. EmoA is an FMNH2 -dependent monooxygenase that requires an NADH:FMN oxidoreductase (EmoB) to provide FMNH2 as a cosubstrate. Although EmoA has been identified from Chelativorans (ex. Mesorhizobium) sp. BNC1, its catalytic mechanism is unknown. Crystal structures of EmoA revealed a domain-like insertion into a TIM-barrel, which might serve as a flexible lid for the active site. Docking of MgEDTA(2-) into EmoA identified an intricate hydrogen bond network connected to Tyr(71) , which should potentially lower its pKa. Tyr(71) , along with nearby Glu(70) and a peroxy flavin, facilitates a keto-enol transition of the leaving acetyl group of EDTA. Further, for the first time, the physical interaction between EmoA and EmoB was observed by ITC, molecular docking and enzyme kinetic assay, which enhanced both EmoA and EmoB activities probably through coupled channelling of FMNH2 .

Cytoplasmic Localization of Sulfide:Quinone Oxidoreductase and Persulfide Dioxygenase of Cupriavidus pinatubonensis JMP134.

Heterotrophic bacteria have recently been reported to oxidize sulfide to sulfite and thiosulfate by using sulfide:quinone oxidoreductase (SQR) and persulfide dioxygenase (PDO). In chemolithotrophic bacteria, both SQR and PDO have been reported to function in the periplasmic space, with SQR as a peripheral membrane protein whose C terminus inserts into the cytoplasmic membrane and PDO as a soluble protein. Cupriavidus pinatubonensis JMP134, best known for its ability to degrade 2,4-dichlorophenoxyacetic acid and other aromatic pollutants, has a gene cluster of sqr and pdo encoding C. pinatubonensis SQR (CpSQR) and CpPDO2. When cloned in Escherichia coli, the enzymes are functional. Here we investigated whether they function in the periplasmic space or in the cytoplasm in heterotrophic bacteria. By using sequence analysis, biochemical detection, and green fluorescent protein (GFP)/PhoA fusion proteins, we found that CpSQR was located on the cytoplasmic side of the membrane and CpPDO2 was a soluble protein in the cytoplasm with a tendency to be peripherally located near the membrane. The location proximity of these proteins near the membrane in the cytoplasm may facilitate sulfide oxidation in heterotrophic bacteria. The information may guide the use of heterotrophic bacteria in bioremediation of organic pollutants as well as H2S.IMPORTANCE Sulfide (H2S, HS-, and S2-), which is common in natural gas and wastewater, causes a serious malodor at low levels and is deadly at high levels. Microbial oxidation of sulfide is a valid bioremediation method, in which chemolithotrophic bacteria that use sulfide as the energy source are often used to remove sulfide. Heterotrophic bacteria with SQR and PDO have recently been reported to oxidize sulfide to sulfite and thiosulfate. Cupriavidus pinatubonensis JMP134 has been extensively characterized for its ability to degrade organic pollutants, and it also contains SQR and PDO. This paper shows the localization of SQR and PDO inside the cytoplasm in the vicinity of the membrane. The information may provide guidance for using heterotrophic bacteria in sulfide bioremediation.

Cupriavidus necator H16 Uses Flavocytochrome c Sulfide Dehydrogenase To Oxidize Self-Produced and Added Sulfide.

Production of sulfide (H2S, HS-, and S2-) by heterotrophic bacteria during aerobic growth is a common phenomenon. Some bacteria with sulfide:quinone oxidoreductase (SQR) and persulfide dioxygenase (PDO) can oxidize self-produced sulfide to sulfite and thiosulfate, but other bacteria without these enzymes release sulfide into the medium, from which H2S can volatilize into the gas phase. Here, we report that Cupriavidus necator H16, with the fccA and fccB genes encoding flavocytochrome c sulfide dehydrogenases (FCSDs), also oxidized self-produced H2S. A mutant in which fccA and fccB were deleted accumulated and released H2S. When fccA and fccB were expressed in Pseudomonas aeruginosa strain Pa3K with deletions of its sqr and pdo genes, the recombinant rapidly oxidized sulfide to sulfane sulfur. When PDO was also cloned into the recombinant, the recombinant with both FCSD and PDO oxidized sulfide to sulfite and thiosulfate. Thus, the proposed pathway is similar to the pathway catalyzed by SQR and PDO, in which FCSD oxidizes sulfide to polysulfide, polysulfide spontaneously reacts with reduced glutathione (GSH) to produce glutathione persulfide (GSSH), and PDO oxidizes GSSH to sulfite, which chemically reacts with polysulfide to produce thiosulfate. About 20.6% of sequenced bacterial genomes contain SQR, and only 3.9% contain FCSD. This is not a surprise, since SQR is more efficient in conserving energy because it passes electrons from sulfide oxidation into the electron transport chain at the quinone level, while FCSD passes electrons to cytochrome c The transport of electrons from the latter to O2 conserves less energy. FCSDs are grouped into three subgroups, well conserved at the taxonomic level. Thus, our data show the diversity in sulfide oxidation by heterotrophic bacteria.IMPORTANCE Heterotrophic bacteria with SQR and PDO can oxidize self-produced sulfide and do not release H2S into the gas phase. C. necator H16 has FCSD but not SQR, and it does not release H2S. We confirmed that the bacterium used FCSD for the oxidation of self-produced sulfide. The bacterium also oxidized added sulfide. The common presence of SQRs, FCSDs, and PDOs in heterotrophic bacteria suggests the significant role of heterotrophic bacteria in sulfide oxidation, participating in sulfur biogeochemical cycling. Further, FCSDs have been identified in anaerobic photosynthetic bacteria and chemolithotrophic bacteria, but their physiological roles are unknown. We showed that heterotrophic bacteria use FCSDs to oxidize self-produced sulfide and extraneous sulfide, and they may be used for H2S bioremediation.

New insights into the QuikChange™ process guide the use of Phusion DNA polymerase for site-directed mutagenesis.

The QuikChange™ site-directed mutagenesis method is popular but imperfect. An improvement by using partially overlapping primers has been reported several times; however, it is incompatible with the proposed mechanism. The QuikChange™ method using complementary primers is proposed to linearly amplify a target plasmid with the products annealing to produce double-stranded DNA molecules with 5'-overhangs. The overhang annealing is supposed to form circular plasmids with staggered breaks, which can be repaired in Escherichia coli after transformation. Here, we demonstrated that the PCR enzyme fills the 5'-overhangs in the early cycles, and the product is then used as the template for exponential amplification. The linear DNA molecules with homologous ends are joined to generate the plasmid with the desired mutations through homologous recombination in E. coli. The correct understanding is important to method improvements, guiding us to use partially overlapping primers and Phusion DNA polymerase for site-directed mutagenesis. Phusion did not amplify a plasmid with complementary primers but used partially overlapping primers to amplify the plasmid, producing linear DNA molecules with homologous ends for site-directed mutagenesis.

Characterizations of Two Bacterial Persulfide Dioxygenases of the Metallo-ß-lactamase Superfamily.

Persulfide dioxygenases (PDOs), also known as sulfur dioxygenases (SDOs), oxidize glutathione persulfide (GSSH) to sulfite and GSH. PDOs belong to the metallo-ß-lactamase superfamily and play critical roles in animals, plants, and microorganisms, including sulfide detoxification. The structures of two PDOs from human and Arabidopsis thaliana have been reported; however, little is known about the substrate binding and catalytic mechanism. The crystal structures of two bacterial PDOs from Pseudomonas putida and Myxococcus xanthus were determined at 1.5- and 2.5-Å resolution, respectively. The structures of both PDOs were homodimers, and their metal centers and ß-lactamase folds were superimposable with those of related enzymes, especially the glyoxalases II. The PDOs share similar Fe(II) coordination and a secondary coordination sphere-based hydrogen bond network that is absent in glyoxalases II, in which the corresponding residues are involved instead in coordinating a second metal ion. The crystal structure of the complex between the Pseudomonas PDO and GSH also reveals the similarity of substrate binding between it and glyoxalases II. Further analysis implicates an identical mode of substrate binding by known PDOs. Thus, the data not only reveal the differences in metal binding and coordination between the dioxygenases and the hydrolytic enzymes in the metallo-ß-lactamase superfamily, but also provide detailed information on substrate binding by PDOs.