scholarly journals Key Aromatic-Ring-Cleaving Enzyme, Protocatechuate 3,4-Dioxygenase, in the Ecologically Important MarineRoseobacter Lineage

2000 ◽  
Vol 66 (11) ◽  
pp. 4662-4672 ◽  
Author(s):  
Alison Buchan ◽  
Lauren S. Collier ◽  
Ellen L. Neidle ◽  
Mary Ann Moran
Keyword(s):  
Aromatic Compounds ◽  
Southern Hybridization ◽  
Ring Cleavage ◽  
Degenerate Pcr ◽  
Cell Extracts ◽  
Aromatic Compound Degradation ◽  
Genes Encoding ◽  
Cleavage Enzyme ◽  
Key Enzyme ◽  
Dioxygenase Activity

ABSTRACT Aromatic compound degradation in six bacteria representing an ecologically important marine taxon of the α-proteobacteria was investigated. Initial screens suggested that isolates in theRoseobacter lineage can degrade aromatic compounds via the β-ketoadipate pathway, a catabolic route that has been well characterized in soil microbes. Six Roseobacter isolates were screened for the presence of protocatechuate 3,4-dioxygenase, a key enzyme in the β-ketoadipate pathway. All six isolates were capable of growth on at least three of the eight aromatic monomers presented (anthranilate, benzoate, p-hydroxybenzoate, salicylate, vanillate, ferulate, protocatechuate, and coumarate). Four of the Roseobacter group isolates had inducible protocatechuate 3,4-dioxygenase activity in cell extracts when grown onp-hydroxybenzoate. The pcaGH genes encoding this ring cleavage enzyme were cloned and sequenced from two isolates,Sagittula stellata E-37 and isolate Y3F, and in both cases the genes could be expressed in Escherichia coli to yield dioxygenase activity. Additional genes involved in the protocatechuate branch of the β-ketoadipate pathway (pcaC,pcaQ, and pobA) were found to cluster withpcaGH in these two isolates. Pairwise sequence analysis of the pca genes revealed greater similarity between the twoRoseobacter group isolates than between genes from eitherRoseobacter strain and soil bacteria. A degenerate PCR primer set targeting a conserved region within PcaH successfully amplified a fragment of pcaH from two additionalRoseobacter group isolates, and Southern hybridization indicated the presence of pcaH in the remaining two isolates. This evidence of protocatechuate 3,4-dioxygenase and the β-ketoadipate pathway was found in all six Roseobacterisolates, suggesting widespread abilities to degrade aromatic compounds in this marine lineage.

scholarly journals Transcriptomic Analysis Reveals a Bifurcated Terephthalate Degradation Pathway in Rhodococcus sp. Strain RHA1

2006 ◽  
Vol 189 (5) ◽  
pp. 1641-1647 ◽  
Author(s):  
Hirofumi Hara ◽  
Lindsay D. Eltis ◽  
Julian E. Davies ◽  
William W. Mohn
Keyword(s):  
Aromatic Compounds ◽  
Degradation Pathway ◽  
Transcriptomic Analysis ◽  
Ring Cleavage ◽  
Wide Range ◽  
Genes Encoding ◽  
Catechol 1,2 Dioxygenase ◽  
Rhodococcus Sp ◽  
Dioxygenase Activity ◽  
In Cells

ABSTRACT Phthalate isomers and their esters are important pollutants whose biodegradation is not well understood. Rhodococcus sp. strain RHA1 is notable for its ability to degrade a wide range of aromatic compounds. RHA1 was previously shown to degrade phthalate (PTH) and to have genes putatively encoding terephthalate (TPA) degradation. Transcriptomic analysis of 8,213 genes indicated that 150 were up-regulated during growth on PTH and that 521 were up-regulated during growth on TPA. Distinct ring cleavage dioxygenase systems were differentially expressed during growth on PTH and TPA. Genes encoding the protocatechuate (PCA) pathway were induced on both substrates, while genes encoding the catechol branch of the PCA pathway were additionally induced only on TPA. Accordingly, protocatechuate-3,4-dioxygenase activity was induced in cells grown on both substrates, while catechol-1,2-dioxygenase activity was induced only in cells grown on TPA. Knockout analysis indicated that pcaL, encoding 3-oxoadipate enol-lactone hydrolase and 4-carboxymuconolactone decarboxylase, was required for growth on both substrates but that pcaB, encoding β-carboxy-cis,cis-muconate lactonizing enzyme, was required for growth on PTH only. These results indicate that PTH is degraded solely via the PCA pathway, whereas TPA is degraded via a bifurcated pathway that additionally includes the catechol branch of the PCA pathway.

scholarly journals Genetic Analysis of Dioxin Dioxygenase ofSphingomonas sp. Strain RW1: Catabolic Genes Dispersed on the Genome

1998 ◽  
Vol 180 (15) ◽  
pp. 3954-3966 ◽  
Author(s):  
Jean Armengaud ◽  
Birgitta Happe ◽  
Kenneth N. Timmis
Keyword(s):  
Supply System ◽  
Ring Cleavage ◽  
Gene Encoding ◽  
Genetic Organization ◽  
Catabolic Genes ◽  
Genes Encoding ◽  
Subsequent Step ◽  
Cleavage Enzyme ◽  
Angular Dioxygenation ◽  
Class Iib

ABSTRACT The dioxin dioxygenase of Sphingomonas sp. strain RW1 activates dibenzo-p-dioxin and dibenzofuran for further metabolism by introducing two atoms of oxygen at a pair of vicinal carbon atoms, one of which is involved in one of the bridges between the two aromatic rings, i.e., an angular dioxygenation. ThedxnA1 and dxnA2 cistrons encoding this dioxygenase have been cloned and shown to be located just upstream of a hydrolase gene which specifies an enzyme involved in the subsequent step of the dibenzofuran biodegradative pathway. Genes encoding the electron supply system of the dioxygenase are not clustered with the dioxygenase gene but rather are located on two other distinct and separate genome segments. Moreover, whereas expression ofdxnA1A2 is modulated according to the available carbon source, expression of the dbfB gene encoding the ring cleavage enzyme of the dibenzofuran pathway, which is located in the neighborhood of dxnA1A2 but oriented in the opposite direction, is constitutive. The scattering of genes for the component proteins of dioxin dioxygenase system around the genome ofSphingomonas sp. strain RW1, and the differential expression of dioxin pathway genes, is unusual and contrasts with the typical genetic organization of catabolic pathways where component cistrons tend to be clustered in multicistronic transcriptional units. The sequences of the α and β subunits of the dioxin dioxygenase exhibit only weak similarity to other three component dioxygenases, but some motifs such as the Fe(II) binding site and the [2Fe-2S] cluster ligands are conserved. Dioxin dioxygenase activity in Escherichia coli cells containing the cloned dxnA1A2 gene was achieved only through coexpression of the cognate electron supply system from RW1. Under these conditions, exclusively angular dioxygenation of dibenzofuran and dibenzo-p-dioxin was obtained. The dioxin dioxygenase was not active in E. colicells coexpressing a class IIB electron supply system. In the course of the isolation of the dxnA1 and dxnA2 cistrons, a number of other catabolic genes dispersed over different genome segments were identified, which may indicate greater catabolic potential than was previously suspected. This finding is consistent with the catabolic versatility of members of the genusSphingomonas, which is becoming increasingly evident, and may indicate a less well evolved and regulated but more dynamic genetic organization in this organism than is the case for better-studied pathways in organisms such as Pseudomonas species.

scholarly journals Aerobic Benzoyl-Coenzyme A (CoA) Catabolic Pathway in Azoarcus evansii: Conversion of Ring Cleavage Product by 3,4-Dehydroadipyl-CoA Semialdehyde Dehydrogenase

2006 ◽  
Vol 188 (8) ◽  
pp. 2919-2927 ◽  
Author(s):  
Johannes Gescher ◽  
Wael Ismail ◽  
Ellen Ölgeschläger ◽  
Wolfgang Eisenreich ◽  
Jürgen Wörth ◽  
...  
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Coenzyme A ◽  
Cleavage Product ◽  
Ring Cleavage ◽  
Resonance Spectroscopy ◽  
Catabolic Pathway ◽  
Semialdehyde Dehydrogenase ◽  
Cell Extracts ◽  
Genes Encoding ◽  
Coa Thioesters

ABSTRACT Benzoate, a strategic intermediate in aerobic aromatic metabolism, is metabolized in various bacteria via an unorthodox pathway. The intermediates of this pathway are coenzyme A (CoA) thioesters throughout, and ring cleavage is nonoxygenolytic. The fate of the ring cleavage product 3,4-dehydroadipyl-CoA semialdehyde was studied in the β-proteobacterium Azoarcus evansii. Cell extracts contained a benzoate-induced, NADP+-specific aldehyde dehydrogenase, which oxidized this intermediate. A postulated putative long-chain aldehyde dehydrogenase gene, which might encode this new enzyme, is located on a cluster of genes encoding enzymes and a transport system required for aerobic benzoate oxidation. The gene was expressed in Escherichia coli, and the maltose-binding protein-tagged enzyme was purified and studied. It is a homodimer composed of 54 kDa (without tag) subunits and was confirmed to be the desired 3,4-dehydroadipyl-CoA semialdehyde dehydrogenase. The reaction product was identified by nuclear magnetic resonance spectroscopy as the corresponding acid 3,4-dehydroadipyl-CoA. Hence, the intermediates of aerobic benzoyl-CoA catabolic pathway recognized so far are benzoyl-CoA; 2,3-dihydro-2,3-dihydroxybenzoyl-CoA; 3,4-dehydroadipyl-CoA semialdehyde plus formate; and 3,4-dehydroadipyl-CoA. The further metabolism is thought to lead to 3-oxoadipyl-CoA, the intermediate at which the conventional and the unorthodox pathways merge.

scholarly journals Origins of the 2,4-Dinitrotoluene Pathway

2002 ◽  
Vol 184 (15) ◽  
pp. 4219-4232 ◽  
Author(s):  
Glenn R. Johnson ◽  
Rakesh K. Jain ◽  
Jim C. Spain
Keyword(s):  
Burkholderia Cepacia ◽  
Ring Cleavage ◽  
Gene Encoding ◽  
Metabolic Intermediates ◽  
Semialdehyde Dehydrogenase ◽  
Ring Fission ◽  
Genes Encoding ◽  
Cleavage Enzyme ◽  
Transport Complex ◽  
Methylmalonate Semialdehyde Dehydrogenase

ABSTRACT The degradation of synthetic compounds requires bacteria to recruit and adapt enzymes from pathways for naturally occurring compounds. Previous work defined the steps in 2,4-dinitrotoluene (2,4-DNT) metabolism through the ring fission reaction. The results presented here characterize subsequent steps in the pathway that yield the central metabolic intermediates pyruvate and propionyl coenzyme A (CoA). The genes encoding the degradative pathway were identified within a 27-kb region of DNA cloned from Burkholderia cepacia R34, a strain that grows using 2,4-DNT as a sole carbon, energy, and nitrogen source. Genes for the lower pathway in 2,4-DNT degradation were found downstream from dntD, the gene encoding the extradiol ring fission enzyme of the pathway. The region includes genes encoding a CoA-dependent methylmalonate semialdehyde dehydrogenase (dntE), a putative NADH-dependent dehydrogenase (ORF13), and a bifunctional isomerase/hydrolase (dntG). Results from analysis of the gene sequence, reverse transcriptase PCR, and enzyme assays indicated that dntD dntE ORF13 dntG composes an operon that encodes the lower pathway. Additional genes that were uncovered encode the 2,4-DNT dioxygenase (dntAaAbAcAd), methylnitrocatechol monooxygenase (dntB), a putative LysR-type transcriptional (ORF12) regulator, an intradiol ring cleavage enzyme (ORF3), a maleylacetate reductase (ORF10), a complete ABC transport complex (ORF5 to ORF8), a putative methyl-accepting chemoreceptor protein (ORF11), and remnants from two transposable elements. Some of the additional gene products might play as-yet-undefined roles in 2,4-DNT degradation; others appear to remain from recruitment of the neighboring genes. The presence of the transposon remnants and vestigial genes suggests that the pathway for 2,4-DNT degradation evolved relatively recently because the extraneous elements have not been eliminated from the region.

scholarly journals A New Bacterial Steroid Degradation Gene Cluster in Comamonas testosteroni TA441 Which Consists of Aromatic-Compound Degradation Genes for Seco-Steroids and 3-Ketosteroid Dehydrogenase Genes

2003 ◽  
Vol 69 (8) ◽  
pp. 4421-4430 ◽  
Author(s):  
Masae Horinouchi ◽  
Toshiaki Hayashi ◽  
Takako Yamamoto ◽  
Toshiaki Kudo
Keyword(s):  
Gene Cluster ◽  
Cholic Acid ◽  
Northern Blot ◽  
Blot Analysis ◽  
Aromatic Compounds ◽  
Gene Disruption ◽  
Northern Blot Analysis ◽  
Comamonas Testosteroni ◽  
Aromatic Compound Degradation ◽  
Cleavage Enzyme

ABSTRACT In Comamonas testosteroni TA441, testosterone is degraded via aromatization of the A ring, which is cleaved by the meta-cleavage enzyme TesB, and further degraded by TesD, the hydrolase for the product of TesB. TesEFG, encoded downstream of TesD, are probably hydratase, aldolase, and dehydrogenase for degradation of 2-oxohex-4-enoicacid, one of the products of TesD. Here we present a new and unique steroid degradation gene cluster in TA441, which consists of ORF18, ORF17, tesI, tesH, ORF11, ORF12, and tesDEFG. TesH and TesI are 3-ketosteroid-Δ1-dehydrogenase and 3-ketosteroid-Δ4(5α)-dehydrogenase, respectively, which work in the early steps of steroid degradation. ORF17 probably encodes the reductase component of 9α-hydroxylase for 1,4-androstadiene-3,17-dione, which is the product of TesH in testosterone degradation. Gene disruption experiments showed that these genes are necessary for steroid degradation and do not have any isozymes in TA441. By Northern blot analysis, these genes were shown to be induced when TA441 was incubated with steroids (testosterone and cholic acid) but not with aromatic compounds [phenol, biphenyl, and 3-(3-hydroxyphenyl)propionic acid], indicating that these genes function exclusively in steroid degradation.

scholarly journals Polyhydroxyalkanoate (PHA) Production in Pseudomonas sp. phDV1 Strain Grown on Phenol as Carbon Sources

2021 ◽  
Vol 9 (8) ◽  
pp. 1636
Author(s):  
Iliana Kanavaki ◽  
Athina Drakonaki ◽  
Ermis Dionisios Geladas ◽  
Apostolos Spyros ◽  
Hao Xie ◽  
...  
Keyword(s):  
Aromatic Compounds ◽  
Carbon Sources ◽  
Biodegradable Plastics ◽  
Pha Synthase ◽  
Pseudomonas Sp ◽  
Degrading Bacterium ◽  
Cell Extracts ◽  
Transmission Electron ◽  
Key Enzyme ◽  
Pha Production

Pseudomonas strains have a variety of potential uses in bioremediation and biosynthesis of biodegradable plastics. Pseudomonas sp. strain phDV1, a Gram-negative phenol degrading bacterium, has been found to utilize monocyclic aromatic compounds as sole carbon source via the meta-cleavage pathway. The degradation of aromatic compounds comprises an important step in the removal of pollutants. The present study aimed to investigate the ability of the Pseudomonas sp. strain phDV1 to produce polyhydroxyalkanoates (PHAs) and examining the effect of phenol concentration on PHA production. The bacterium was cultivated in minimal medium supplemented with different concentrations of phenol ranging from 200–600 mg/L. The activity of the PHA synthase, the key enzyme which produces PHA, was monitored spectroscopically in cells extracts. Furthermore, the PHA synthase was identified by mass spectrometry in cell extracts analyzed by SDS-PAGE. Transmission electron micrographs revealed abundant electron-transparent intracellular granules. The isolated biopolymer was confirmed to be polyhydroxybutyrate (PHB) by FTIR, NMR and MALDI-TOF/TOF analyses. The ability of strain Pseudomonas sp. phDV1 to remove phenol and to produce PHB makes the strain a promising biocatalyst in bioremediation and biosynthesis of biodegradable plastics.

scholarly journals Isolating an active and inactive CACTA transposon from lettuce color mutants and characterizing their family

2021 ◽  
Author(s):  
Csanad Gurdon ◽  
Alexander Kozik ◽  
Rong Tao ◽  
Alexander Poulev ◽  
Isabel Armas ◽  
...  
Keyword(s):  
Anthocyanin Biosynthesis ◽  
Mobile Element ◽  
Bioinformatic Analysis ◽  
Relative Expression Level ◽  
Gene Coding ◽  
Genes Encoding ◽  
Key Enzyme ◽  
Natural Derivative ◽  
Dark Green ◽  
Transposon Insertions

Abstract Dietary flavonoids play an important role in human nutrition and health. Flavonoid biosynthesis genes have recently been identified in lettuce (Lactuca sativa); however, few mutants have been characterized. We now report the causative mutations in Green Super Lettuce (GSL), a natural light green mutant derived from red cultivar NAR; and GSL-Dark Green (GSL-DG), an olive-green natural derivative of GSL. GSL harbors CACTA 1 (LsC1), a 3.9-kb active nonautonomous CACTA superfamily transposon inserted in the 5′ untranslated region of anthocyanidin synthase (ANS), a gene coding for a key enzyme in anthocyanin biosynthesis. Both terminal inverted repeats (TIRs) of this transposon were intact, enabling somatic excision of the mobile element, which led to the restoration of ANS expression and the accumulation of red anthocyanins in sectors on otherwise green leaves. GSL-DG harbors CACTA 2 (LsC2), a 1.1-kb truncated copy of LsC1 that lacks one of the TIRs, rendering the transposon inactive. RNA-sequencing and reverse transcription quantitative PCR of NAR, GSL, and GSL-DG indicated the relative expression level of ANS was strongly influenced by the transposon insertions. Analysis of flavonoid content indicated leaf cyanidin levels correlated positively with ANS expression. Bioinformatic analysis of the cv Salinas lettuce reference genome led to the discovery and characterization of an LsC1 transposon family with a putative transposon copy number greater than 1,700. Homologs of tnpA and tnpD, the genes encoding two proteins necessary for activation of transposition of CACTA elements, were also identified in the lettuce genome.

scholarly journals Genetic Investigation of the Catabolic Pathway for Degradation of Abietane Diterpenoids by Pseudomonas abietaniphilaBKME-9

2000 ◽  
Vol 182 (13) ◽  
pp. 3784-3793 ◽  
Author(s):  
Vincent J. J. Martin ◽  
William W. Mohn
Keyword(s):  
Gene Cluster ◽  
Sequence Similarity ◽  
Open Reading Frames ◽  
Ring Cleavage ◽  
Catabolic Pathway ◽  
Transcriptional Fusion ◽  
Abietane Diterpenoids ◽  
Genes Encoding ◽  
Dna Fragment ◽  
Reading Frames

ABSTRACT We have cloned and sequenced the dit gene cluster encoding enzymes of the catabolic pathway for abietane diterpenoid degradation by Pseudomonas abietaniphila BKME-9. Thedit gene cluster is located on a 16.7-kb DNA fragment containing 13 complete open reading frames (ORFs) and 1 partial ORF. The genes ditA1A2A3 encode the α and β subunits and the ferredoxin of the dioxygenase which hydroxylates 7-oxodehydroabietic acid to 7-oxo-11,12-dihydroxy-8,13-abietadien acid. The dioxygenase mutant strain BKME-941 (ditA1::Tn5) did not grow on nonaromatic abietanes, and transformed palustric and abietic acids to 7-oxodehydroabietic acid in cell suspension assays. Thus, nonaromatic abietanes are aromatized prior to further degradation. Catechol 2,3-dioxygenase activity of xylEtranscriptional fusion strains showed induction of ditA1and ditA3 by abietic, dehydroabietic, and 7-oxodehydroabietic acids, which support the growth of strain BKME-9, as well as by isopimaric and 12,14-dichlorodehydroabietic acids, which are diterpenoids that do not support the growth of strain BKME-9. In addition to the aromatic-ring-hydroxylating dioxygenase genes, thedit cluster includes ditC, encoding an extradiol ring cleavage dioxygenase, and ditR, encoding an IclR-type transcriptional regulator. Although ditR is not strictly required for the growth of strain BKME-9 on abietanes, aditR::Kmr mutation in aditA3::xylE reporter strain demonstrated that it encodes an inducer-dependent transcriptional activator of ditA3. An ORF with sequence similarity to genes encoding permeases (ditE) is linked with genes involved in abietane degradation.

Differential Roles of Three Different Upper Pathway Meta Ring Cleavage Product Hydrolases in the Degradation of Dibenzo- p -dioxin and Dibenzofuran by Sphingomonas wittichii RW1

2021 ◽  
Author(s):  
Thamer Y. Mutter ◽  
Gerben J. Zylstra
Keyword(s):  
Gene Knockout ◽  
Sole Source ◽  
Cleavage Product ◽  
Ring Cleavage ◽  
Catabolic Pathway ◽  
Double Knockout ◽  
Cleavage Enzyme ◽  
Sphingomonas Wittichii ◽  
Physiological Approach

Sphingomonas wittichii RW1 grows on the two related compounds dibenzofuran (DBF) and dibenzo- p -dioxin (DXN) as the sole source of carbon. Previous work by others (P.V. Bunz, R. Falchetto, and A.M. Cook. Biodegradation 4:171-8, 1993, doi: 10.1007/BF00695119) identified two upper pathway meta cleavage product hydrolases (DxnB1 and DxnB2) active on the DBF upper pathway metabolite 2-hydroxy-6-oxo-6-(2-hydroxyphenyl)-hexa-2,4-dienoate. We took a physiological approach to determine the role of these two enzymes in the degradation of DBF and DXN by RW1. Single knockouts of either plasmid located dbfB1 or chromosome located dbfB2 had no effect on RW1 growth on either DBF or DXN. However, a double knockout lost the ability to grow on DBF but still grew normally on DXN demonstrating that DbfB1 and DbfB2 are the only hydrolases involved in the DBF upper pathway. Using a transcriptomic-guided approach we identified a constitutively expressed third hydrolase encoded by the chromosomally located SWIT0910 gene. Knockout of SWIT0910 resulted in a strain that no longer grows on DXN but still grows normally on DBF. Thus the DbfB1 and DbfB2 hydrolases function in the DBF but not the DXN catabolic pathway and the SWIT0190 hydrolase functions in the DXN but not the DBF catabolic pathway. Importance S. wittichii RW1 is one of only a few strains known to grow on DXN as the sole course of carbon. Much of the work deciphering the related RW1 DXN and DBF catabolic pathways has involved genome gazing, transcriptomics, proteomics, heterologous expression, and enzyme purification and characterization. Very little research has utilized physiological techniques to precisely dissect the genes and enzymes involved in DBF and DXN degradation. Previous work by others identified and extensively characterized two RW1 upper pathway hydrolases. Our present work demonstrates that these two enzymes are involved in DBF but not DXN degradation. In addition, our work identified a third constitutively expressed hydrolase that is involved in DXN but not DBF degradation. Combined with our previous work, this means that the RW1 DXN upper pathway involves genes from three very different locations in the genome: an initial plasmid-encoded dioxygenase and a ring cleavage enzyme and hydrolase encoded on opposite sides of the chromosome.

scholarly journals Evidence that a Linear Megaplasmid Encodes Enzymes of Aliphatic Alkene and Epoxide Metabolism and Coenzyme M (2-Mercaptoethanesulfonate) Biosynthesis in XanthobacterStrain Py2

2001 ◽  
Vol 183 (7) ◽  
pp. 2172-2177 ◽  
Author(s):  
Jonathan G. Krum ◽  
Scott A. Ensign
Keyword(s):  
Ring Opening ◽  
Methanogenic Archaea ◽  
Hypothetical Protein ◽  
Epoxide Ring ◽  
Coenzyme M ◽  
Epoxide Ring Opening ◽  
Propylene Oxidation ◽  
Key Enzymes ◽  
Genes Encoding ◽  
Key Enzyme

ABSTRACT The bacterial metabolism of propylene proceeds by epoxidation to epoxypropane followed by a sequence of three reactions resulting in epoxide ring opening and carboxylation to form acetoacetate. Coenzyme M (2-mercaptoethanesulfonic acid) (CoM) plays a central role in epoxide carboxylation by serving as the nucleophile for epoxide ring opening and the carrier of the C3 unit that is ultimately carboxylated to acetoacetate, releasing CoM. In the present work, a 320-kb linear megaplasmid has been identified in the gram-negative bacterium Xanthobacter strain Py2, which contains the genes encoding the key enzymes of propylene oxidation and epoxide carboxylation. Repeated subculturing of Xanthobacter strain Py2 under nonselective conditions, i.e., with glucose or acetate as the carbon source in the absence of propylene, resulted in the loss of the propylene-positive phenotype. The propylene-negative phenotype correlated with the loss of the 320-kb linear megaplasmid, loss of induction and expression of alkene monooxgenase and epoxide carboxylation enzyme activities, and the loss of CoM biosynthetic capability. Sequence analysis of a hypothetical protein (XecG), encoded by a gene located downstream of the genes for the four enzymes of epoxide carboxylation, revealed a high degree of sequence identity with proteins of as-yet unassigned functions in the methanogenic archaeaMethanobacterium thermoautotrophicum andMethanococcus jannaschii and in Bacillus subtilis. The M. jannaschii homolog of XecG, MJ0255, is located next to a gene, MJ0256, that has been shown to encode a key enzyme of CoM biosynthesis (M. Graupner, H. Xu, and R. H. White, J. Bacteriol. 182: 4862–4867, 2000). We propose that the propylene-positive phenotype of Xanthobacter strain Py2 is dependent on the selective maintenance of a linear megaplasmid containing the genes for the key enzymes of alkene oxidation, epoxide carboxylation, and CoM biosynthesis.

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