Structural insight into G-protein chaperone-mediated maturation of a bacterial adenosylcobalamin-dependent mutase.

G-protein metallochaperones are essential for the proper maturation of numerous metalloenzymes. The G-protein chaperone MMAA in humans (MeaB in bacteria) uses GTP hydrolysis to facilitate the delivery of adenosylcobalamin (AdoCbl) to AdoCbl-dependent methylmalonyl-CoA mutase, an essential metabolic enzyme. This G-protein chaperone also facilitates the removal of damaged cobalamin (Cbl) for repair. Although most chaperones are standalone proteins, isobutyryl-CoA mutase fused (IcmF) has a G-protein domain covalently attached to its target mutase. We previously showed that dimeric MeaB undergoes a 180° rotation to reach a state capable of GTP hydrolysis (an active G-protein state), in which so-called switch III residues of one protomer contact the G-nucleotide of the other protomer. However, it was unclear whether other G-protein chaperones also adopted this conformation. Here, we show that the G-protein domain in a fused system forms a similar active conformation, requiring IcmF oligomerization. IcmF oligomerizes both upon Cbl damage and in the presence of the nonhydrolyzable GTP analog, guanosine-5'-[(ß,γ)-methyleno]triphosphate, forming supramolecular complexes observable by mass photometry and EM. Cryo-EM structural analysis reveals that the second protomer of the G-protein intermolecular dimer props open the mutase active site using residues of switch III as a wedge, allowing for AdoCbl insertion or damaged Cbl removal. With the series of structural snapshots now available, we now describe here the molecular basis of G-protein-assisted AdoCbl-dependent mutase maturation, explaining how GTP binding prepares a mutase for cofactor delivery and how GTP hydrolysis allows the mutase to capture the cofactor.

Observing 3-hydroxyanthranilate-3,4-dioxygenase in action through a crystalline lens.

The synthesis of quinolinic acid from tryptophan is a critical step in the de novo biosynthesis of nicotinamide adenine dinucleotide (NAD+) in mammals. Herein, the nonheme iron-based 3-hydroxyanthranilate-3,4-dioxygenase responsible for quinolinic acid production was studied by performing time-resolved in crystallo reactions monitored by UV-vis microspectroscopy, electron paramagnetic resonance (EPR) spectroscopy, and X-ray crystallography. Seven catalytic intermediates were kinetically and structurally resolved in the crystalline state, and each accompanies protein conformational changes at the active site. Among them, a monooxygenated, seven-membered lactone intermediate as a monodentate ligand of the iron center at 1.59-Å resolution was captured, which presumably corresponds to a substrate-based radical species observed by EPR using a slurry of small-sized single crystals. Other structural snapshots determined at around 2.0-Å resolution include monodentate and subsequently bidentate coordinated substrate, superoxo, alkylperoxo, and two metal-bound enol tautomers of the unstable dioxygenase product. These results reveal a detailed stepwise O-atom transfer dioxygenase mechanism along with potential isomerization activity that fine-tunes product profiling and affects the production of quinolinic acid at a junction of the metabolic pathway.

Kinetic and Spectroscopic Characterization of the Catalytic Ternary Complex of Tryptophan 2,3-Dioxygenase.

The first step of the kynurenine pathway for l-tryptophan (l-Trp) degradation is catalyzed by heme-dependent dioxygenases, tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase. In this work, we employed stopped-flow optical absorption spectroscopy to study the kinetic behavior of the Michaelis complex of Cupriavidus metallidurans TDO (cmTDO) to improve our understanding of oxygen activation and initial oxidation of l-Trp. On the basis of the stopped-flow results, rapid freeze-quench (RFQ) experiments were performed to capture and characterize this intermediate by Mössbauer spectroscopy. By incorporating the chlorite dismutase-chlorite system to produce high concentrations of solubilized O2, we were able to capture the Michaelis complex of cmTDO in a nearly quantitative yield. The RFQ-Mössbauer results confirmed the identity of the Michaelis complex as an O2-bound ferrous species. They revealed remarkable similarities between the electronic properties of the Michaelis complex and those of the O2 adduct of myoglobin. We also found that the decay of this reactive intermediate is the rate-limiting step of the catalytic reaction. An inverse α-secondary substrate kinetic isotope effect was observed with a kH/kD of 0.87 ± 0.03 when (indole-d5)-l-Trp was employed as the substrate. This work provides an important piece of spectroscopic evidence of the chemical identity of the Michaelis complex of bacterial TDO.

Substrate Diversity of L-Threonic Acid Dehydrogenase Homologs.

Despite physiological importance of aldonic sugar acids for living organisms, little is known about metabolic pathways of these compounds. Here, we investigated the functional diversity of homologs of L-threonic acid dehydrogenase (ThrDH; UniProt ID: Q0KBC7), an enzyme composed of two NAD-binding domains (PF14833 and PF03446). Ten ThrDH homologs with different genomic context were studied; seven new enzymatic activities were identified, such as (R)-pantoate dehydrogenase, L-altronic acid dehydrogenase, 6-deoxy-L-talonate dehydrogenase, L-idonic acid dehydrogenase, D-xylonic acid dehydrogenase, D-gluconic acid dehydrogenase, and 2-hydroxy-3-oxopantoate reductase activities. Two associated metabolic pathways were identified: L-idonic acid dehydrogenase was found to be involved in the degradation of L-idonic acid through oxidation/decarboxylation in Agrobacterium radiobacter K84, while 2-hydroxy-3-oxopantoate reductase was found to participate in D-glucarate catabolism through dehydration/cleavage in Ralstonia metallidurans CH34.

Streamlined production, purification, and characterization of recombinant extracellular polyhydroxybutyrate depolymerases.

Heterologous production of extracellular polyhydroxybutyrate (PHB) depolymerases (PhaZs) has been of interest for over 30 years, but implementation is sometimes difficult and can limit the scope of research. With the constant development of tools to improve recombinant protein production in Escherichia coli, we propose a method that takes characteristics of PhaZs from different bacterial strains into account. Recombinant His-tagged versions of PhaZs (rPhaZ) from Comamonas testosteroni 31A, Cupriavidus sp. T1, Marinobacter algicola DG893, Pseudomonas stutzeri, and Ralstonia sp. were successfully produced with varying expression, solubility, and purity levels. PhaZs from C. testosteroni and P. stutzeri were more amenable to heterologous expression in all aspects; however, using the E. coli Rosetta-gami B(DE3) expression strain and establishing optimal conditions for expression and purification (variation of IPTG concentration and use of size exclusion columns) helped circumvent low expression and purity for the other PhaZs. Degradation activity of the rPhaZs was compared using a simple PHB plate-based method, adapted to test for various pH and temperatures. rPhaZ from M. algicola presented the highest activity at 15°C, and rPhaZs from Cupriavidus sp. T1 and Ralstonia sp. had the highest activity at pH 5.4. The methods proposed herein can be used to test the production of soluble recombinant PhaZs and to perform preliminary evaluation for applications that require PHB degradation.

Expression and cloning of catA encoding a catechol 1,2-dioxygenase from the 2,4-D-degrading strain Cupriavidus campinensis BJ71.

Catechol 1,2-dioxygenases catalyze catechol ring-opening, a critical step in the degradation of aromatic compounds. Cupriavidus campinensis BJ71, an efficient 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterial strain, was previously isolated from an environment contaminated with 2,4-D. In this study, catA encoding a catechol 1,2-dioxygenase was cloned from the BJ71 strain. The gene was 939 bp long and encoded a polypeptide of 312 amino acids with a molecular weight of 34 kDa. To investigate its enzymatic characteristics, CatA was heterologously expressed in Escherichia coli. Optimal reaction conditions for the pure enzyme were 35 °C and pH 8.0. The enzyme remained stable within a range of 25 °C-45 °C and pH 6.0-9.0, thus indicating that CatA has wide temperature and pH adaptability. After incubation at 45 °C, the enzyme activity of CatA decreased to 37.12%, but its activity was not affected by incubation at pH 9.0. The pure enzyme was able to use catechol, 4-methyl-catechol and 4-chlorocatechol as substrates. Enzyme kinetic parameters Km and Vmax were 39.97 µM and 10.68 U/mg, respectively. This is the first report of the cloning of a gene encoding a catechol 1,2-dioxygenase from a 2,4-D-degrading bacterial strain.

Cross-linked enzyme aggregates of arylamidase from Cupriavidus oxalaticus ICTDB921: process optimization, characterization, and application for mitigation of acrylamide in industrial wastewater.

Acrylamidase produced by Cupriavidus oxalaticus ICTDB921 was recovered directly from the fermentation broth by ammonium sulfate (40-50%) precipitation and then stabilized by cross-linking with glutaraldehyde. The optimum conditions for the preparation of cross-linked enzyme aggregates of acrylamidase (acrylamidase-CLEAs) were using 60 mM glutaraldehyde for 10 min at 35 °C and initial broth pH of 7.0. Acrylamidase-CLEAs were characterized by SDS-PAGE, FTIR, particle size analyzer and SEM. Cross-linking shifted the optimal temperature and pH from 70 to 50 °C and 5-7 to 6-8, respectively. It also altered the secondary structure fractions, pH and thermal stability along with the kinetic constants, Km and Vmax, respectively. A complete degradation of acrylamide ~ 1.75 g/L in industrial wastewater was achieved after 60 min in a batch process under optimum operating conditions, and the kinetics was best represented by Edward model (R2 = 0.70). Acrylamidase-CLEAs retained ~ 40% of its initial activity after three cycles for both pure acrylamide and industrial wastewater, and were stable for 15 days at 4 °C, retaining ~ 25% of its original activity.

Rapid Biodegradation of the Organophosphorus Insecticide Chlorpyrifos by Cupriavidus nantongensis X1T.

Chlorpyrifos was one of the most widely used organophosphorus insecticides and the neurotoxicity and genotoxicity of chlorpyrifos to mammals, aquatic organisms and other non-target organisms have caused much public concern. Cupriavidus nantongensis X1T, a type of strain of the genus Cupriavidus, is capable of efficiently degrading 200 mg/L of chlorpyrifos within 48 h. This is ~100 fold faster than Enterobacter B-14, a well-studied chlorpyrifos-degrading bacterial strain. Strain X1T can tolerate high concentrations (500 mg/L) of chlorpyrifos over a wide range of temperatures (30-42 °C) and pH values (5-9). RT-qPCR analysis showed that the organophosphorus hydrolase (OpdB) in strain X1T was an inducible enzyme, and the crude enzyme isolated in vitro could still maintain 75% degradation activity. Strain X1T can simultaneously degrade chlorpyrifos and its main hydrolysate 3,5,6-trichloro-2-pyridinol. TCP could be further metabolized through stepwise oxidative dechlorination and further opening of the benzene ring to be completely degraded by the tricarboxylic acid cycle. The results provide a potential means for the remediation of chlorpyrifos- contaminated soil and water.

Application of an efficient indole oxygenase system from Cupriavidus sp. SHE for indigo production.

Indigo, one of the most widely used dyes, is mainly produced by chemical processes, which generate amounts of pollutants and need high energy consumption. Microbial production of indigo from indole has attracted much attention; however, the indole oxygenase has never been explored and applied for indigo production. In the present study, the indole oxygenase indAB genes were successfully cloned from Cupriavidus sp. SHE and heterologously expressed in Escherichia coli BL21(DE3) (designated as IND_AB). Strain IND_AB produced primarily indigo in tryptophan medium by high-performance liquid chromatography-mass spectroscopy (HPLC-MS) analysis. The preferable conditions for indigo production were pH 6.5 (normal pH), 30 °C, 150 rpm, strain inoculation concentration OD600 0.08, and induction with 1 mM IPTG at the time of inoculation. The optimal culture medium compositions were further determined as tryptophan 1.0 g/L, NaCl 3.55 g/L, and yeast extract 5.12 g/L based on single-factor experiment and response surface methodology. The highest indigo yield was 307 mg/L, which was 4.39-fold higher than the original value. This is the first study investigating indigo production using the indole oxygenase system and the results highlighted its potential in bio-indigo industrial application.

Molecular and biochemical characterization of 2-chloro-4-nitrophenol degradation via the 1,2,4-benzenetriol pathway in a Gram-negative bacterium.

2-Chloro-4-nitrophenol (2C4NP) is the most common chlorinated nitrophenol pollutant, and its environmental fate is of great concern. Cupriavidus sp. CNP-8, a Gram-negative bacterium, has been reported to degrade 2C4NP via the 1,2,4-benzenetriol (BT) pathway, significantly different from the (chloro)hydroquinone pathways reported in all other Gram-negative 2C4NP-utilizers. Herein, the BT pathway of the catabolism of 2C4NP in this strain was characterized at the molecular, biochemical, and genetic levels. The hnp gene cluster was suspected to be involved in the catabolism of 2C4NP because the hnp genes are significantly upregulated in the 2C4NP-induced strain CNP-8 compared to the uninduced strain. HnpAB, a two-component FAD-dependent monooxygenase, catalyzes the conversion of 2C4NP to BT via chloro-1,4-benzoquinone, with a Km of 2.7 ± 1.1 µΜ and a kcat/Km of 0.17 ± 0.03 µΜ-1 min-1. hnpA is necessary for strain CNP-8 to utilize 2C4NP in vivo. HnpC, a BT 1,2-dioxygenase, was proved to catalyze BT ring-cleavage with formation of maleylacetate by HPLC-MS analysis. Phylogenetic analysis indicated that HnpA likely has different evolutionary origin compared to other functionally identified 2C4NP monooxygenases. To our knowledge, this is the first report revealing the catabolic mechanism of 2C4NP via the BT pathway in a Gram-negative bacterium, increasing our knowledge of the catabolic diversity for microbial 2C4NP degradation at the molecular and biochemical level.

Comparative genome analysis of completely sequenced Cupriavidus genomes provides insights into the biosynthetic potential and versatile applications of Cupriavidus alkaliphilus ASC-732.

The genome analysis of microorganisms provides valuable information to endorse more extensive research on their potential applications. In this paper, the genome of Cupriavidus alkaliphilus ASC-732, isolated from agave rhizosphere in northeastern Mexico, was analyzed and compared with the genomes of other Cupriavidus species to gain better insight into the parts in the genetic makeup responsible for essential metabolic pathways and others of biotechnological importance. Here, the key genes related to glycolysis, pentose phosphate, and the Entner-Doudoroff and tricarboxylic acid cycle pathways were predicted. Comparative genome analysis revealed that the key genes for hydrogenotrophic growth and carbon fixation pathway, i.e., those coding for hydrogenase and enzymes Calvin-Benson-Bassham cycle, are absent in C. alkaliphilus ASC-732. Furthermore, capabilities for producing polyhydroxyalkanoates and extracellular polysaccharide matrix and degrading xenobiotics were found, and the related pathways are explained. Moreover, biofilm formation and the production of exopolysaccharides and polyhydroxyalkanoates were corroborated with crystal violet staining, calcofluor, and Nile red fluorochromes, confirming the presence of the products of the active genes in these pathways and their related metabolic routes, respectively. Additionally, a large group of genes essential for the resistance and detoxification of several heavy metals were also found. Thus, the present study demonstrates that this strain can respond to various environmental signals, such as energy source, nutrient limitations, virulence, and extreme metals concentration, indicating the possibility to foster C. alkaliphilus ASC-732 in diverse biotechnological applications.

Kinetics and Catabolic Pathways of the Insecticide Chlorpyrifos, Annotation of the Degradation Genes, and Characterization of Enzymes TcpA and Fre in Cupriavidus nantongensis X1T.

Chlorpyrifos is one of the most used organophosphorus insecticides. It is commonly degraded to 3,5,6-trichloro-2-pyridinol (TCP), which is water-soluble and toxic. Bacteria can degrade chlorpyrifos and TCP, but the biodegradation mechanism has not been well-characterized. Recently isolated Cupriavidus nantongensis X1T can completely degrade 100 mg/L chlorpyrifos and 20 mg/L TCP with half-lives of 6 and 8 h, respectively. We annotated a complete gene cluster responsible for TCP degradation in recently sequenced strain X1T. Two key genes, tcpA and fre, were cloned from X1T and transferred and expressed in Escherichia coli BL21(DE3). Degradation of TCP by X1T whole cell was compared with that by the enzymes 2,4,6-trichlorophenol monooxygenase and NAD(P)H:flavin reductase expressed and purified from E. coli BL21(DE3). Novel metabolites of TCP were isolated and characterized, indicating stepwise dechlorination of TCP, which was confirmed by TCP disappearance, mass balance, and detection and formation kinetics of chloride ion from TCP.

Magnetic cross-linked enzyme aggregates of acrylamidase from Cupriavidus oxalaticus ICTDB921 for biodegradation of acrylamide from industrial waste water.

Acrylamidase from Cupriavidus oxalaticus ICTDB921 was immobilized on magnetic nanoparticles (MNPs) for degradation of acrylamide (a group 2A carcinogen and an environmental contaminant) from industrial waste water. Acrylamidase-MNPs were prepared (maximum recovery ∼94%) at optimized process parameters viz. 1.5:1 (v/v) of acetone: crude acrylamidase/5 mM of glutaraldehyde/90 min/1.5:1 of enzyme: MNP ratio. MNPs and acrylamidase-MNPs were characterized by particle size analysis, FTIR, XRD, SEM and vibrating sample magnetometer. Acrylamidase-MNPs showed a shift in optimum pH (8-8.5) and temperature (60-65 °C) with higher pH/thermal stability vis-à-vis free enzyme. A significant increase in kinetic constants, thermal inactivation constants and thermodynamic parameters were noted for acrylamidase-MNPs. A complete degradation of acrylamide ∼2100 mg/L was achieved in industrial waste water under optimized conditions for batch process and the kinetics was best represented by Haldane model. Acrylamidase-MNPs retained >80% of its initial activity after 4 cycles for both pure acrylamide and industrial waste water.

High PHA density fed-batch cultivation strategies for 4HB-rich P(3HB-co-4HB) copolymer production by transformant Cupriavidus malaysiensis USMAA1020.

P(3HB-co-4HB) with a high 4HB monomer composition was previously successfully produced using the transformant Cupriavidus malaysiensis USMAA1020 containing an additional copy of the PHA synthase gene. In this study, high PHA density fed-batch cultivation strategies were developed for such 4HB-rich P(3HB-co-4HB). The pulse, constant and mixed feeding strategies resulted in high PHA accumulation, with a PHA content of 74-92 wt% and 4HB monomer composition of 92-99 mol%. The pulse-feed of carbon and nitrogen resulted in higher PHA concentration (30.7 g/L) than carbon alone (22.3 g/L), suggesting that a trace amount of nitrogen is essential to support cell density for PHA accumulation. Constant feeding was found to be a more feasible strategy than mixed feeding, since the latter caused a drastic fluctuation in the C/N ratio, as evidenced by higher biomass formation indicating more carbon flux towards the competitive TCA pathway. A two-times carbon and nitrogen pulse feeding was the most optimal strategy achieving 92 wt% accommodation of the total biomass, with the highest PHA concentration (46 g/L) and yield (Yp/x) of 11.5 g/g. The strategy has kept the C/N at optimal ratio during the active PHA-producing phase. This is the first report of the production of high PHA density for 4HB-rich P(3HB-co-4HB).

Adapting to oxygen: 3-Hydroxyanthrinilate 3,4-dioxygenase employs loop dynamics to accommodate two substrates with disparate polarities.

3-Hydroxyanthranilate 3,4-dioxygenase (HAO) is an iron-dependent protein that activates O2 and inserts both oxygen atoms into 3-hydroxyanthranilate (3-HAA). An intriguing question is how HAO can rapidly bind O2, even though local O2 concentrations and diffusion rates are relatively low. Here, a close inspection of the HAO structures revealed that substrate- and inhibitor-bound structures exhibit a closed conformation with three hydrophobic loop regions moving toward the catalytic iron center, whereas the ligand-free structure is open. We hypothesized that these loop movements enhance O2 binding to the binary complex of HAO and 3-HAA. We found that the carboxyl end of 3-HAA triggers changes in two loop regions and that the third loop movement appears to be driven by an H-bond interaction between Asn27 and Ile142 Mutational analyses revealed that N27A, I142A, and I142P variants cannot form a closed conformation, and steady-state kinetic assays indicated that these variants have a substantially higher Km for O2 than WT HAO. This observation suggested enhanced hydrophobicity at the iron center resulting from the concerted loop movements after the binding of the primary substrate, which is hydrophilic. Given that O2 is nonpolar, the increased hydrophobicity at the iron center of the binary complex appears to be essential for rapid O2 binding and activation, explaining the reason for the 3-HAA-induced loop movements. Because substrate binding-induced open-to-closed conformational changes are common, the results reported here may help further our understanding of how oxygen is enriched in nonheme iron-dependent dioxygenases.

Characterization of d-succinylase from Cupriavidus sp. P4-10-C and its application in d-amino acid synthesis.

d-Amino acids are important building blocks for various compounds, such as pharmaceuticals and agrochemicals. A more cost-effective enzymatic method for d-amino acid production is needed in the industry. We improved a one-pot enzymatic method for d-amino acid production by the dynamic kinetic resolution of N-succinyl amino acids using two enzymes: d-succinylase (DSA) from Cupriavidus sp. P4-10-C, which hydrolyzes N-succinyl-d-amino acids enantioselectively to their corresponding d-amino acid, and N-succinyl amino acid racemase (NSAR, EC.4.2.1.113) from Geobacillus stearothermophilus NCA1503. In this study, DSA and NSAR were purified and their properties were investigated. The optimum temperature of DSA was 50°C and it was stable up to 55°C. The optimum pH of DSA and NSAR was around 7.5. In d-phenylalanine production, the optical purity of product was improved to 91.6% ee from the examination about enzyme concentration. Moreover, 100 mM N-succinyl-dl-tryptophan was converted to d-tryptophan at 81.8% yield with 94.7% ee. This enzymatic method could be useful for the industrial production of various d-amino acids.

Cupriavidus pinatubonensis AEO106 deals with copper-induced oxidative stress before engaging in biodegradation of the herbicide 4-chloro-2-methylphenoxyacetic acid.

BACKGROUND: Microbial degradation of phenoxy acid (PA) herbicides in agricultural soils is important to minimize herbicide leaching to groundwater reservoirs. Degradation may, however, be hampered by exposure of the degrader bacteria to toxic metals as copper (Cu) in the soil environment. Exposure to Cu leads to accumulation of intracellular reactive oxygen species (ROS) in some bacteria, but it is not known how Cu-derived ROS and an ensuing oxidative stress affect the degradation of PA herbicides. Based on the previously proposed paradigm that bacteria deal with environmental stress before they engage in biodegradation, we studied how the degradation of the PA herbicide 2-methyl-4-chlorophenoxyacetic acid (MCPA) by the model PA degrader Cupriavidus pinatubonensis AEO106 was affected by Cu exposure. RESULTS: Exposure of C. pinatubonensis in batch culture to sublethal concentrations of Cu increased accumulation of ROS measured by the oxidant sensing probe 2,7-dichlorodihydrofluorescein diacetate and flow cytometry, and resulted in upregulation of a gene encoding a protein belong to the Ohr/OsmC protein family. The ohr/osmC gene was also highly induced by H2O2 exposure suggesting that it is involved in the oxidative stress response in C. pinatubonensis. The increased ROS accumulation and increased expression of the oxidative stress defense coincided with a delay in the catabolic performance, since both expression of the catabolic tfdA gene and MCPA mineralization were delayed compared to unexposed control cells. CONCLUSIONS: The current study suggests that Cu-induced ROS accumulation in C. pinatubonensis activates a stress response involving the product of the ohr/osmC gene. Further, the stress response is launched before induction of the catabolic tfdA gene and mineralization occurs.

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.

Native and Foreign Proteins Secreted by the Cupriavidus metallidurans Type II System and an Alternative Mechanism.

The type II secretion system (T2SS), which transports selected periplasmic proteins across the outer membrane, has rarely been studied in nonpathogens or in organisms classified as Betaproteobacteria. Therefore, we studied Cupriavidus metallidurans (Cme), a facultative chemilithoautotroph. Gel analysis of extracellular proteins revealed no remarkable differences between the wild type and the T2SS mutants. However, enzyme assays revealed that native extracellular alkaline phosphatase is a T2SS substrate, because activity was 10-fold greater for the wild type than a T2SS mutant. In Cme engineered to produce three Ralstonia solanacearum (Rso) exoenzymes, at least 95% of their total activities were extracellular, but unexpectedly high percentages of these exoenzymes remained extracellular in T2SS mutants cultured in rich broth. These conditions appear to permit an alternative secretion process, because neither cell lysis nor periplasmic leakage was observed when Cme produced a Pectobacterium carotovorum exoenzyme, and wild-type Cme cultured in minimal medium secreted 98% of Rso polygalacturonase, but 92% of this exoenzyme remained intracellular in T2SS mutants. We concluded that Cme has a functional T2SS despite lacking any abundant native T2SS substrates. The efficient secretion of three foreign exoenzymes by Cme is remarkable, but so too is the indication of an alternative secretion process in rich culture conditions. When not transiting the T2SS, we suggest that Rso exoenzymes are probably selectively packaged into outer membrane vesicles. Phylogenetic analysis of T2SS proteins supports the existence of at least three T2SS subfamilies, and we propose that Cme, as a representative of the Betaproteobacteria, could become a new useful model system for studying T2SS substrate specificity.

Recombinant Escherichia coli with sulfide:quinone oxidoreductase and persulfide dioxygenase rapidly oxidises sulfide to sulfite and thiosulfate via a new pathway.

Many heterotrophic bacteria contain sulfide:quinone oxidoreductase (SQR) and persulfide dioxygenase (PDO) genes. It is unclear how these enzymes cooperate to oxidise sulfide in bacteria. Cupriavidus pinatubonensis JMP134 contains a gene cluster of sqr and pdo, and their functions were analysed in Escherichia coli. Recombinant E. coli cells with SQR and PDO rapidly oxidised sulfide to thiosulfate and sulfite. The SQR also contains a DUF442 domain that was shown to have rhodanese activities. E. coli cells with PDO and SQR-C94S, an active site mutant of the rhodanese domain, oxidised sulfide to thiosulfate with transitory accumulation of polysulfides. Cellular and enzymatic evidence showed that DUF442 speeds up the reaction of polysulfides with glutathione to produce glutathione persulfide (GSSH). Thus, SQR oxidises sulfide to polysulfides; rhodanese enhances the reaction of polysulfides with glutathione to produce GSSH; PDO oxidises GSSH to sulfite; sulfite spontaneously reacts with polysulfides to generate thiosulfate. The pathway is different from the proposed mitochondrial pathway because it has polysulfides, that is, disulfide and trisulfide, as intermediates. The data demonstrated that heterotrophic bacteria with SQR and PDO can rapidly oxidise sulfide to thiosulfate and sulfite, providing the foundation for using heterotrophic bacteria with SQR and PDO for sulfide bioremediation.