Adaption and Degradation Strategies of Methylotrophic 1,4-Dioxane Degrading Strain Xanthobacter sp. YN2 Revealed by Transcriptome-Scale Analysis.

Biodegradation of 1,4-dioxane (dioxane) contamination has gained much attention for decades. In our previous work, we isolated a highly efficient dioxane degrader, Xanthobacter sp. YN2, but the underlying mechanisms of its extraordinary degradation performance remained unresolved. In this study, we performed a comparative transcriptome analysis of YN2 grown on dioxane and citrate to elucidate its genetic degradation mechanism and investigated the transcriptomes of different dioxane degradation stages (T0, T24, T48). We also analyzed the transcriptional response of YN2 over time during which the carbon source switched from citrate to dioxane. The results indicate that strain YN2 was a methylotroph, which provides YN2 a major advantage as a pollutant degrader. A large number of genes involved in dioxane metabolism were constitutively expressed prior to dioxane exposure. Multiple genes related to the catabolism of each intermediate were upregulated by treatment in response to dioxane. Glyoxylate metabolism was essential during dioxane degradation by YN2, and the key intermediate glyoxylate was metabolized through three routes: glyoxylate carboligase pathway, malate synthase pathway, and anaplerotic ethylmalonyl-CoA pathway. Genes related to quorum sensing and transporters were significantly upregulated during the early stages of degradation (T0, T24) prior to dioxane depletion, while the expression of genes encoding two-component systems was significantly increased at late degradation stages (T48) when total organic carbon in the culture was exhausted. This study is the first to report the participation of genes encoding glyoxalase, as well as methylotrophic genes xoxF and mox, in dioxane metabolism. The present study reveals multiple genetic and transcriptional strategies used by YN2 to rapidly increase biomass during growth on dioxane, achieve high degradation efficiency and tolerance, and adapt to dioxane exposure quickly, which provides useful information regarding the molecular basis for efficient dioxane biodegradation.

A New Catabolic Plasmid in Xanthobacter and Starkeya spp. from a 1,2-Dichloroethane-Contaminated Site.

UNLABELLED: 1,2-Dichloroethane (DCA) is a problematic xenobiotic groundwater pollutant. Bacteria are capable of biodegrading DCA, but the evolution of such bacteria is not well understood. In particular, the mechanisms by which bacteria acquire the key dehalogenase genes dhlA and dhlB have not been well defined. In this study, the genomic context of dhlA and dhlB was determined in three aerobic DCA-degrading bacteria (Starkeya novella strain EL1, Xanthobacter autotrophicus strain EL4, and Xanthobacter flavus strain EL8) isolated from a groundwater treatment plant (GTP). A haloalkane dehalogenase gene (dhlA) identical to the canonical dhlA gene from Xanthobacter sp. strain GJ10 was present in all three isolates, and, in each case, the dhlA gene was carried on a variant of a 37-kb circular plasmid, which was named pDCA. Sequence analysis of the repA replication initiator gene indicated that pDCA was a member of the pTAR plasmid family, related to catabolic plasmids from the Alphaproteobacteria, which enable growth on aromatics, dimethylformamide, and tartrate. Genes for plasmid replication, mobilization, and stabilization were identified, along with two insertion sequences (ISXa1 and ISPme1) which were likely to have mobilized dhlA and dhlB and played a role in the evolution of aerobic DCA-degrading bacteria. Two haloacid dehalogenase genes (dhlB1 and dhlB2) were detected in the GTP isolates; dhlB1 was most likely chromosomal and was similar to the canonical dhlB gene from strain GJ10, while dhlB2 was carried on pDCA and was not closely related to dhlB1 Heterologous expression of the DhlB2 protein confirmed that this plasmid-borne dehalogenase was capable of chloroacetate dechlorination. IMPORTANCE: Earlier studies on the DCA-degrading Xanthobacter sp. strain GJ10 indicated that the key dehalogenases dhlA and dhlB were carried on a 225-kb linear plasmid and on the chromosome, respectively. The present study has found a dramatically different gene organization in more recently isolated DCA-degrading Xanthobacter strains from Australia, in which a relatively small circular plasmid (pDCA) carries both dhlA and dhlB homologs. pDCA represents a true organochlorine-catabolic plasmid, first because its only obvious metabolic phenotype is dehalogenation of organochlorines, and second because acquisition of this plasmid provides both key enzymes required for carbon-chlorine bond cleavage. The discovery of the alternative haloacid dehalogenase dhlB2 in pDCA increases the known genetic diversity of bacterial chloroacetate-hydrolyzing enzymes.

Mercury Reduction and Methyl Mercury Degradation by the Soil Bacterium Xanthobacter autotrophicus Py2.

Two previously uncharacterized potential broad-spectrum mercury (Hg) resistance operons (mer) are present on the chromosome of the soil Alphaproteobacteria Xanthobacter autotrophicus Py2. These operons, mer1 and mer2, contain two features which are commonly found in mer operons in the genomes of soil and marine Alphaproteobacteria, but are not present in previously characterized mer operons: a gene for the mercuric reductase (MerA) that encodes an alkylmercury lyase domain typical of those found on the MerB protein, and the presence of an additional gene, which we are calling merK, with homology to glutathione reductase. Here, we demonstrate that Py2 is resistant to 0.2 µM inorganic mercury [Hg(II)] and 0.05 µM methylmercury (MeHg). Py2 is capable of converting MeHg and Hg(II) to elemental mercury [Hg(0)], and reduction of Hg(II) is induced by incubation in sub toxic concentrations of Hg(II). Transcription of the merA genes increased with Hg(II) treatment, and in both operons merK resides on the same polycistronic mRNA as merA. We propose the use of Py2 as a model system for studying the contribution of mer to Hg mobility in soil and marine ecosystems.

Valence bond and enzyme catalysis: a time to break down and a time to build up.

Understanding enzyme catalysis and developing ability to control of it are two great challenges in biochemistry. A few successful examples of computational-based enzyme design have proved the fantastic potential of computational approaches in this field, however, relatively modest rate enhancements have been reported and the further development of complementary methods is still required. Herein we propose a conceptually simple scheme to identify the specific role that each residue plays in catalysis. The scheme is based on a breakdown of the total catalytic effect into contributions of individual protein residues, which are further decomposed into chemically interpretable components by using valence bond theory. The scheme is shown to shed light on the origin of catalysis in wild-type haloalkane dehalogenase (wt-DhlA) and its mutants. Furthermore, the understanding gained through our scheme is shown to have great potential in facilitating the selection of non-optimal sites for catalysis and suggesting effective mutations to enhance the enzymatic rate.

Enzyme activity and gene expression profiles of Xanthobacter autotrophicus GJ10 during aerobic biodegradation of 1,2-dichloroethane.

Xanthobacter autotrophicus GJ10 has been widely studied because of its ability to degrade halogenated compounds, especially 1,2-dichloroethane (1,2-DCA), which is achieved through chromosomal as well as plasmid pAUX1 encoded 1,2-DCA degrading genes. This work described the gene expression and enzyme activity profiles as well as the intermediates formed during the 1,2-DCA degradation by this organism. A correlation between gene expression, enzyme activity and metabolic intermediates, after the induction of GJ10 grown culture with 1,2-DCA, was established at different time intervals. Haloalkane dehalogenase (dhlA) and haloacid dehalogenase (dhlB) were constitutively expressed while the expression of alcohol dehydrogenase (max) and aldehyde dehydrogenase (ald) was found to be inducible. The DhlA and DhlB activities were relatively higher compared to that of the inducible enzymes, Max and Ald. To the best of our knowledge, this is the first study to correlate gene expression profiles with enzyme activity and metabolite formation during 1,2-DCA degradation process in GJ10. Findings from this study may assist in fully understanding the mechanism of 1,2-DCA degradation by GJ10. It could also assist in the design and implementation of appropriate bioaugmentation strategies for complete removal of 1,2-DCA from contaminated environment.

Substrate specificity of haloalkane dehalogenases.

An enzyme's substrate specificity is one of its most important characteristics. The quantitative comparison of broad-specificity enzymes requires the selection of a homogenous set of substrates for experimental testing, determination of substrate-specificity data and analysis using multivariate statistics. We describe a systematic analysis of the substrate specificities of nine wild-type and four engineered haloalkane dehalogenases. The enzymes were characterized experimentally using a set of 30 substrates selected using statistical experimental design from a set of nearly 200 halogenated compounds. Analysis of the activity data showed that the most universally useful substrates in the assessment of haloalkane dehalogenase activity are 1-bromobutane, 1-iodopropane, 1-iodobutane, 1,2-dibromoethane and 4-bromobutanenitrile. Functional relationships among the enzymes were explored using principal component analysis. Analysis of the untransformed specific activity data revealed that the overall activity of wild-type haloalkane dehalogenases decreases in the following order: LinB~DbjA>DhlA~DhaA~DbeA~DmbA>DatA~DmbC~DrbA. After transforming the data, we were able to classify haloalkane dehalogenases into four SSGs (substrate-specificity groups). These functional groups are clearly distinct from the evolutionary subfamilies, suggesting that phylogenetic analysis cannot be used to predict the substrate specificity of individual haloalkane dehalogenases. Structural and functional comparisons of wild-type and mutant enzymes revealed that the architecture of the active site and the main access tunnel significantly influences the substrate specificity of these enzymes, but is not its only determinant. The identification of other structural determinants of the substrate specificity remains a challenge for further research on haloalkane dehalogenases.

Roles of the redox-active disulfide and histidine residues forming a catalytic dyad in reactions catalyzed by 2-ketopropyl coenzyme M oxidoreductase/carboxylase.

NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC), an atypical member of the disulfide oxidoreductase (DSOR) family of enzymes, catalyzes the reductive cleavage and carboxylation of 2-ketopropyl-coenzyme M [2-(2-ketopropylthio)ethanesulfonate; 2-KPC] to form acetoacetate and coenzyme M (CoM) in the bacterial pathway of propylene metabolism. Structural studies of 2-KPCC from Xanthobacter autotrophicus strain Py2 have revealed a distinctive active-site architecture that includes a putative catalytic triad consisting of two histidine residues that are hydrogen bonded to an ordered water molecule proposed to stabilize enolacetone formed from dithiol-mediated 2-KPC thioether bond cleavage. Site-directed mutants of 2-KPCC were constructed to test the tenets of the mechanism proposed from studies of the native enzyme. Mutagenesis of the interchange thiol of 2-KPCC (C82A) abolished all redox-dependent reactions of 2-KPCC (2-KPC carboxylation or protonation). The air-oxidized C82A mutant, as well as wild-type 2-KPCC, exhibited the characteristic charge transfer absorbance seen in site-directed variants of other DSOR enzymes but with a pK(a) value for C87 (8.8) four units higher (i.e., four orders of magnitude less acidic) than that for the flavin thiol of canonical DSOR enzymes. The same higher pK(a) value was observed in native 2-KPCC when the interchange thiol was alkylated by the CoM analog 2-bromoethanesulfonate. Mutagenesis of the flavin thiol (C87A) also resulted in an inactive enzyme for steady-state redox-dependent reactions, but this variant catalyzed a single-turnover reaction producing a 0.8:1 ratio of product to enzyme. Mutagenesis of the histidine proximal to the ordered water (H137A) led to nearly complete loss of redox-dependent 2-KPCC reactions, while mutagenesis of the distal histidine (H84A) reduced these activities by 58 to 76%. A redox-independent reaction of 2-KPCC (acetoacetate decarboxylation) was not decreased for any of the aforementioned site-directed mutants. We interpreted and rationalized these results in terms of a mechanism of catalysis for 2-KPCC employing a unique hydrophobic active-site architecture promoting thioether bond cleavage and enolacetone formation not seen for other DSOR enzymes.

Bathy phytochromes in rhizobial soil bacteria.

Phytochromes are biliprotein photoreceptors that are found in plants, bacteria, and fungi. Prototypical phytochromes have a Pr ground state that absorbs in the red spectral range and is converted by light into the Pfr form, which absorbs longer-wavelength, far-red light. Recently, some bacterial phytochromes have been described that undergo dark conversion of Pr to Pfr and thus have a Pfr ground state. We show here that such so-called bathy phytochromes are widely distributed among bacteria that belong to the order Rhizobiales. We measured in vivo spectral properties and the direction of dark conversion for species which have either one or two phytochrome genes. Agrobacterium tumefaciens C58 contains one bathy phytochrome and a second phytochrome which undergoes dark conversion of Pfr to Pr in vivo. The related species Agrobacterium vitis S4 contains also one bathy phytochrome and another phytochrome with novel spectral properties. Rhizobium leguminosarum 3841, Rhizobium etli CIAT652, and Azorhizobium caulinodans ORS571 contain a single phytochrome of the bathy type, whereas Xanthobacter autotrophicus Py2 contains a single phytochrome with dark conversion of Pfr to Pr. We propose that bathy phytochromes are adaptations to the light regime in the soil. Most bacterial phytochromes are light-regulated histidine kinases, some of which have a C-terminal response regulator subunit on the same protein. According to our phylogenetic studies, the group of phytochromes with this domain arrangement has evolved from a bathy phytochrome progenitor.

Isolation of a Xanthobacter sp. degrading dichloromethane and characterization of the gene involved in the degradation.

A bacterial strain able to degrade dichloromethane (DCM) as the sole carbon source was isolated from a wastewater treatment plant receiving domestic and pharmaceutical effluent. 16S rDNA studies revealed the strain to be a Xanthobacter sp. (strain TM1). The new isolated strain when grown aerobically on DCM showed Luong type growth kinetics, with 1(max) of 0.094 h(-1) and S (m) of 1,435 mg l(-1). Strain TM1 was able to degrade other aromatic and aliphatic halogenated compounds, such as halobenzoates, 2-chloroethanol and dichloroethane. The gene for DCM dehalogenase, which is the key enzyme in DCM degradation, was amplified through PCR reactions. Strain TM1 contains type A DCM dehalogenase (dcmAa), while no product could be obtained for type B dehalogense (dcmAb). The sequence was compared against 12 dcmAa from other DCM degrading strains and 98% or 99% similarity was observed with all other previously isolated DCM dehalogenase genes. This is the first time a Xanthobacter sp. is reported to degrade DCM.

The genome of the versatile nitrogen fixer Azorhizobium caulinodans ORS571.

BACKGROUND: Biological nitrogen fixation is a prokaryotic process that plays an essential role in the global nitrogen cycle. Azorhizobium caulinodans ORS571 has the dual capacity to fix nitrogen both as free-living organism and in a symbiotic interaction with Sesbania rostrata. The host is a fast-growing, submergence-tolerant tropical legume on which A. caulinodans can efficiently induce nodule formation on the root system and on adventitious rootlets located on the stem. RESULTS: The 5.37-Mb genome consists of a single circular chromosome with an overall average GC of 67% and numerous islands with varying GC contents. Most nodulation functions as well as a putative type-IV secretion system are found in a distinct symbiosis region. The genome contains a plethora of regulatory and transporter genes and many functions possibly involved in contacting a host. It potentially encodes 4717 proteins of which 96.3% have homologs and 3.7% are unique for A. caulinodans. Phylogenetic analyses show that the diazotroph Xanthobacter autotrophicus is the closest relative among the sequenced genomes, but the synteny between both genomes is very poor. CONCLUSION: The genome analysis reveals that A. caulinodans is a diazotroph that acquired the capacity to nodulate most probably through horizontal gene transfer of a complex symbiosis island. The genome contains numerous genes that reflect a strong adaptive and metabolic potential. These combined features and the availability of the annotated genome make A. caulinodans an attractive organism to explore symbiotic biological nitrogen fixation beyond leguminous plants.

Aerobic Dechlorination of Dichloromethane Using Biostimulation Agent BD-C in Continuous and Batch Cultures of Xanthobacter autotrophicus GJ10.

It is important to construct microbiological treatment systems for organic solvent-contaminated water. We developed a continuous culture supplemented with a biostimulation agent named BD-C, which is formulated from canola oil, and Xanthobacter autotrophicus strain GJ10 for an aerobic dichloromethane (DCM)-dechlorinating microorganism. The continuous culture was a chemostat constructed using a 1 L screw-capped bottle containing artificial wastewater medium with 2.0 mM DCM and 1.0% (v/v) BD-C. The expression of genes for DCM metabolism in the dechlorinating aerobe was monitored and analyzed by reverse transcription-quantitative PCR. Strain GJ10 was able to dechlorinate approximately 74% of the DCM in medium supplemented with BD-C during 12 days of incubation. The DCM dechlorination rate was calculated to be 0.11 mM/day. The ΔΔCT method showed that expression of haloalkane dehalogenase increased 5.4 times in the presence of BD-C. Based on batch culture growth tests conducted with mineral salt medium containing three DCM concentrations (0.07, 0.20, 0.43 and 0.65 mM) with BD-C, the apparent maximum specific consumption rate (νmax) and the saturation constant (Ks) determined for DCM degradation in this test were 19.0 nmol/h/CFU and 0.44 mM, respectively. In conclusion, BD-C enhanced the aerobic degradation of DCM by strain GJ10.

Engineering Bacteria to Catabolize the Carbonaceous Component of Sarin: Teaching E. coli to Eat Isopropanol.

We report an engineered strain of Escherichia coli that catabolizes the carbonaceous component of the extremely toxic chemical warfare agent sarin. Enzymatic decomposition of sarin generates isopropanol waste that, with this engineered strain, is then transformed into acetyl-CoA by enzymatic conversion with a key reaction performed by the acetone carboxylase complex (ACX). We engineered the heterologous expression of the ACX complex from Xanthobacter autotrophicus PY2 to match the naturally occurring subunit stoichiometry and purified the recombinant complex from E. coli for biochemical analysis. Incorporating this ACX complex and enzymes from diverse organisms, we introduced an isopropanol degradation pathway in E. coli, optimized induction conditions, and decoupled enzyme expression to probe pathway bottlenecks. Our engineered E. coli consumed 65% of isopropanol compared to no-cell controls and was able to grow on isopropanol as a sole carbon source. In the process, reconstitution of this large ACX complex (370 kDa) in a system naïve to its structural and mechanistic requirements allowed us to study this otherwise cryptic enzyme in more detail than would have been possible in the less genetically tractable native Xanthobacter system.

Heterologous expression of alkene monooxygenase components from Xanthobacter autotrophicus Py2 and reconstitution of the active complex.

The coupling protein and ferredoxin from Xanthobacter autotrophicus Py2 alkene monooxygenase (Xamo) have been functionally expressed in both N-terminal affinity tagged fusion and native forms in Escherichia coli. However, attempts to express the NADH-oxidoreductase and oxygenase, always resulted in the production of inactive, insoluble proteins. Nevertheless, the recombinant reductase from the toluene 4-monooxygenase of Pseudomonas mendocina KR1 was found to functionally complement the Xamo system. In vitro reconstitution, using the recombinant coupling protein and other components purified from the wild type, showed that steady-state epoxidation rate and coupling efficiency were dependent on the relative concentration of Xamo components in the reaction. The optimal molar stoichiometric ratio of Xamo components was determined to be approximately 1:0.25-0.3:2:2 (oxygenase hexamer:reductase:ferredoxin:coupling protein), suggesting the formation of an efficient catalytic complex at the minimal stoichiometric ratio to saturate the probable two-fold symmetry binding sites on the oxygenase.

Genetic organization of the dhlA gene encoding 1,2-dichloroethane dechlorinase from Xanthobacter flavus UE15.

Xanthobacter flavus strain UE15 was isolated in wastewater obtained from the Ulsan industrial complex, Korea. This strain functions as a 1,2-dichloroethane (1,2-DCA) degrader, via a mechanism of hydrolytic dechlorination, under aerobic conditions. The UE15 strain was also capable of dechlorinating other chloroaliphatics, such as 2-chloroacetic acid and 2-chloropropionic acid. The dhlA gene encoding 1,2-DCA dechlorinase was cloned from the genomic DNA of the UE15 strain, and its nucleotide sequence was determined to consist of 933 base pairs. The deduced amino acid sequence of the DhlA dechlorinase exhibited 100% homology with the corresponding enzyme from X. autotrophicus GJ10, but only 27 to 29% homology with the corresponding enzymes from Rhodococcus rhodochrous, Pseudomonas pavonaceae, and Mycobacterium sp. strain GP1, which all dechlorinate haloalkane compounds. The UE15 strain has an ORF1 (1,356 bp) downstream from the dhlA gene. The OFR1 shows 99% amino acid sequence homology with the transposase reported from X. autotrophicus GJ10. The transposase gene was not found in the vicinity of the dhlA in the GJ10 strain, but rather beside the dhlB gene coding for haloacid dechlorinase. The dhlA and dhlB genes were confirmed to be located at separate chromosomal loci in the Xanthobacter flavus UE15 strain as well as in X. autotrophicus GJ10. The dhlA and transposase genes of the UE15 strain were found to be parenthesized by a pair of insertion sequences, IS1247, which were also found on both sides of the transposase gene in the GJ10 strain. This unique structure of the dhlA gene organization in X. flavus strain UE15 suggested that the dechlorinase gene, dhlA, is transferred with the help of the transposase gene.

Getting a handle on the role of coenzyme M in alkene metabolism.

Coenzyme M (2-mercaptoethanesulfonate; CoM) is one of several atypical cofactors discovered in methanogenic archaea which participate in the biological reduction of CO(2) to methane. Elegantly simple, CoM, so named for its role as a methyl carrier in all methanogenic archaea, is the smallest known organic cofactor. It was thought that this cofactor was used exclusively in methanogenesis until it was recently discovered that CoM is a key cofactor in the pathway of propylene metabolism in the gram-negative soil microorganism Xanthobacter autotrophicus Py2. A four-step pathway requiring CoM converts propylene and CO(2) to acetoacetate, which feeds into central metabolism. In this process, CoM is used to activate and convert highly electrophilic epoxypropane, formed from propylene epoxidation, into a nucleophilic species that undergoes carboxylation. The unique properties of CoM provide a chemical handle for orienting compounds for site-specific redox chemistry and stereospecific catalysis. The three-dimensional structures of several of the enzymes in the pathway of propylene metabolism in defined states have been determined, providing significant insights into both the enzyme mechanisms and the role of CoM in this pathway. These studies provide the structural basis for understanding the efficacy of CoM as a handle to direct organic substrate transformations at the active sites of enzymes.

Engineering a catabolic pathway in plants for the degradation of 1,2-dichloroethane.

Plants are increasingly being employed to clean up environmental pollutants such as heavy metals; however, a major limitation of phytoremediation is the inability of plants to mineralize most organic pollutants. A key component of organic pollutants is halogenated aliphatic compounds that include 1,2-dichloroethane (1,2-DCA). Although plants lack the enzymatic activity required to metabolize this compound, two bacterial enzymes, haloalkane dehalogenase (DhlA) and haloacid dehalogenase (DhlB) from the bacterium Xanthobacter autotrophicus GJ10, have the ability to dehalogenate a range of halogenated aliphatics, including 1,2-DCA. We have engineered the dhlA and dhlB genes into tobacco (Nicotiana tabacum 'Xanthi') plants and used 1,2-DCA as a model substrate to demonstrate the ability of the transgenic tobacco to remediate a range of halogenated, aliphatic hydrocarbons. DhlA converts 1,2-DCA to 2-chloroethanol, which is then metabolized to the phytotoxic 2-chloroacetaldehyde, then chloroacetic acid, by endogenous plant alcohol dehydrogenase and aldehyde dehydrogenase activities, respectively. Chloroacetic acid is dehalogenated by DhlB to produce the glyoxylate cycle intermediate glycolate. Plants expressing only DhlA produced phytotoxic levels of chlorinated intermediates and died, while plants expressing DhlA together with DhlB thrived at levels of 1,2-DCA that were toxic to DhlA-expressing plants. This represents a significant advance in the development of a low-cost phytoremediation approach toward the clean-up of halogenated organic pollutants from contaminated soil and groundwater.

Gentisate 1,2-dioxygenase from Xanthobacter polyaromaticivorans 127W.

A putative gentisate 1,2-dioxygenase was encoded in the dibenzothiophene degradation gene cluster (dbd) from Xanthobacter polyaromaticivorans 127W. The deduced amino acid sequence showed high sequence similarity with gentisate dioxygenases from Pseudomonas alcaligenes (AAD49427, 65% identical), Bradyrhizobium japonicum (NP_766750, 64%), and P. aeruginosa (ZP_00135722, 54%), and moderate similarity with 1-hydroxy-2-naphthoate dioxygenase from Nocardioides sp. KP7 (BAA31235, 33%) and salicylate dioxygenase from Pseudaminobacter salicylatoxidans (AAQ91293, 33%). The enzyme, GDOxp, was heterologously produced in Escherichia coli and purified to homogeneity. GDOxp formed a tetramer and exhibited high dioxygenase activity against 1,4-dihydroxy 2-naphthoate as well as gentisate, suggesting unusually broad substrate specificity. GDOxp easily released ferrous ion under unfavorable temperature and pH conditions to become an inactive monomer protein. An inactive monomer protein can reconstitute a tetramer structure and restore enzyme activity in a cooperative manner upon the addition of ferrous ion. Chymotryptic digestion and protein truncation experiments suggested that the N-terminal region is important for the tetramerization of GDOxp.

Biodegradation of bis(2-chloroethyl) ether by Xanthobacter sp. strain ENV481.

Degradation of bis(2-chloroethyl) ether (BCEE) was observed to occur in two bacterial strains. Strain ENV481, a Xanthobacter sp. strain, was isolated by enrichment culturing of samples from a Superfund site located in the northeastern United States. The strain was able to grow on BCEE or 2-chloroethylethyl ether as the sole source of carbon and energy. BCEE degradation in strain ENV481 was facilitated by sequential dehalogenation reactions resulting in the formation of 2-(2-chloroethoxy)ethanol and diethylene glycol (DEG), respectively. 2-Hydroxyethoxyacetic acid was detected as a product of DEG catabolism by the strain. Degradation of BCEE by strain ENV481 was independent of oxygen, and the strain was not able to grow on a mixture of benzene, ethylbenzene, toluene, and xylenes, other prevalent contaminants at the site. Another bacterial isolate, Pseudonocardia sp. strain ENV478 (S. Vainberg et al., Appl. Environ. Microbiol. 72:5218-5224, 2006), degraded BCEE after growth on tetrahydrofuran or propane but was not able to grow on BCEE as a sole carbon source. BCEE degradation by strain ENV478 appeared to be facilitated by a monooxygenase-mediated O-dealkylation mechanism, and it resulted in the accumulation of 2-chloroacetic acid that was not readily degraded by the strain.

Stability and performance of Xanthobacter autotrophicus GJ10 during 1,2-dichloroethane biodegradation.

A nucleic acid-based approach was used to investigate the dynamics of a microbial community dominated by Xanthobacter autotrophicus GJ10 in the degradation of synthetic wastewater containing 1,2-dichloroethane (DCE). This study was performed over a 140-day period in a nonsterile continuous stirred-tank bioreactor (CSTB) subjected to different operational regimens: nutrient-limiting conditions, baseline operation, and the introduction of glucose as a cosubstrate. The microbial community was analyzed by a combination of fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE). Under nutrient-limiting conditions, DCE degradation was restricted, but this did not affect the dominance of strain GJ10, determined by FISH to comprise 85% of the active population. During baseline operation, DCE degradation improved significantly to over 99.5% and then remained constant throughout the subsequent experimental period. DGGE profiles revealed a stable, complex community, while FISH indicated that strain GJ10 remained the dominant species. During the addition of glucose as a cosubstrate, DGGE profiles showed a proliferation of other species in the CSTB. The percentage of strain GJ10 dropped to 8% of the active population in just 5 days, although this did not affect the DCE biodegradation performance. The return to baseline conditions was accompanied by the reestablishment of strain GJ10 as the dominant species, suggesting that this system responds robustly to external perturbations, both at the functional biodegradation level and at the individual strain level.