scholarly journals Separate Upper Pathway Ring Cleavage Dioxygenases Are Required for Growth of Sphingomonas wittichii RW1 on Dibenzofuran and Dibenzo-p-dioxin

2021 ◽  
Author(s):  
Thamer Y. Mutter ◽  
Gerben J. Zylstra
Keyword(s):  
Sole Source ◽  
Gene Replacement ◽  
Ring Cleavage ◽  
Promoter Strength ◽  
Double Knockout ◽  
Catabolic Pathways ◽  
Sphingomonas Wittichii ◽  
Physiological Approach ◽  
The Subject ◽  
Related Compounds

Sphingomonas wittichii RW1 is one of a few strains known to grow on the related compounds dibenzofuran (DBF) and dibenzo-p-dioxin (DXN) as the sole source of carbon. Previous work by others (B. Happe, L. D. Eltis, H. Poth, R. Hedderich, and K. N. Timmis, J Bacteriol 175:7313-20, 1993, doi: 10.1128/jb.175.22.7313-7320.1993) showed that purified DbfB had significant ring cleavage activity against the DBF metabolite trihydroxybiphenyl but little activity against the DXN metabolite trihydroxybiphenylether. We took a physiological approach to positively identify ring cleavage enzymes involved in the DBF and DXN pathways. Knockout of dbfB on the RW1 megaplasmid pSWIT02 results in a strain that grows slowly on DBF but normally on DXN confirming that DbfB is not involved in DXN degradation. Knockout of SWIT3046 on the RW1 chromosome results in a strain that grows normally on DBF but that does not grow on DXN demonstrating that SWIT3046 is required for DXN degradation. A double knockout strain does not grow on either DBF or DXN demonstrating that these are the only ring cleavage enzymes involved in RW1 DBF and DXN degradation. Substitution of dbfB by SWIT3046 results in a strain that grows normally (equal to wild type) on both DBF and DXN showing that promoter strength is important for SWIT3046 to take the place of DbfB in DBF degradation. Thus both dbfB and SWIT3046 encoded enzymes are involved in DBF degradation but only the SWIT3046 encoded enzyme is involved in DXN degradation. Importance S. wittichii RW1 has been the subject of numerous investigations due to the fact that it is one of only a few strains known to grow on DXN as the sole carbon and energy source. However, while the genome has been sequenced and several DBF pathway enzymes have been purified, there has been very little research using physiological techniques to precisely identify the genes and enzymes involved in the RW1 DBF and DXN catabolic pathways. Using knockout and gene replacement mutagenesis our work identifies separate upper pathway ring cleavage enzymes involved in the related catabolic pathways for DBF and DXN degradation. The identification of a new enzyme involved in DXN biodegradation explains why the pathway of DBF degradation on the RW1 megaplasmid pSWIT02 is inefficient for DXN degradation. In addition, our work demonstrates that both plasmid and chromosomally encoded enzymes are necessary for DXN degradation suggesting that the DXN pathway has only recently evolved.

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 Novel Pathway for the Degradation of 2-Chloro-4-Nitrobenzoic Acid by Acinetobacter sp. Strain RKJ12

2011 ◽  
Vol 77 (18) ◽  
pp. 6606-6613 ◽  
Author(s):  
Dhan Prakash ◽  
Ravi Kumar ◽  
R. K. Jain ◽  
B. N. Tiwary
Keyword(s):  
Salicylic Acid ◽  
Tricarboxylic Acid Cycle ◽  
Sole Source ◽  
Degradation Pathway ◽  
Ring Cleavage ◽  
Muconic Acid ◽  
Content Type ◽  
Nitrobenzoic Acid ◽  
Catabolic Genes ◽  
Catechol 1,2 Dioxygenase

ABSTRACTThe organismAcinetobactersp. RKJ12 is capable of utilizing 2-chloro-4-nitrobenzoic acid (2C4NBA) as a sole source of carbon, nitrogen, and energy. In the degradation of 2C4NBA by strain RKJ12, various metabolites were isolated and identified by a combination of chromatographic, spectroscopic, and enzymatic activities, revealing a novel assimilation pathway involving both oxidative and reductive catabolic mechanisms. The metabolism of 2C4NBA was initiated by oxidativeorthodehalogenation, leading to the formation of 2-hydroxy-4-nitrobenzoic acid (2H4NBA), which subsequently was metabolized into 2,4-dihydroxybenzoic acid (2,4-DHBA) by a mono-oxygenase with the concomitant release of chloride and nitrite ions. Stoichiometric analysis indicated the consumption of 1 mol O2per conversion of 2C4NBA to 2,4-DHBA, ruling out the possibility of two oxidative reactions. Experiments with labeled H218O and18O2indicated the involvement of mono-oxygenase-catalyzed initial hydrolytic dechlorination and oxidative denitration mechanisms. The further degradation of 2,4-DHBA then proceeds via reductive dehydroxylation involving the formation of salicylic acid. In the lower pathway, the organism transformed salicylic acid into catechol, which was mineralized by theorthoring cleavage catechol-1,2-dioxygenase tocis, cis-muconic acid, ultimately forming tricarboxylic acid cycle intermediates. Furthermore, the studies carried out on a 2C4NBA−derivative and a 2C4NBA+transconjugant demonstrated that the catabolic genes for the 2C4NBA degradation pathway possibly reside on the ∼55-kb transmissible plasmid present in RKJ12.

Transformation of Alachlor by Microbial Communities

Weed Science ◽  
1990 ◽  
Vol 38 (4-5) ◽  
pp. 416-420 ◽  
Author(s):  
Hone L. Sun ◽  
Thomas J. Sheets ◽  
Frederick T. Corbin
Keyword(s):  
Carbon Source ◽  
Growth Medium ◽  
Basal Medium ◽  
Sole Source ◽  
Ring Cleavage ◽  
Side Chain ◽  
Microbial Culture ◽  
Mixed Microbial Culture ◽  
Alternative Carbon Source ◽  
Thin Layer Chromatographic Analysis

A mixed microbial culture able to transform alachlor at a concentration of 50 μg ml-1was obtained from alachlor-treated soil after an enrichment period of 84 days. The microbial community was composed of seven strains of bacteria. No single isolate was able to utilize alachlor as a sole source of carbon. There was no alachlor left in the enriched culture after a 14-day incubation, but only 12% of the14C-ring-labeled alachlor was converted to14CO2through ring cleavage during 14 days in the basal medium amended with alachlor as a sole carbon source. The presence of sucrose as an alternative carbon source decreased the mineralization potential of the enriched culture, but sucrose increased the mineralizing ability of a three-member mixed culture. Thin-layer chromatographic analysis showed that there were four unidentified metabolites of alachlor produced by the enriched culture. Sucrose decreased the amount of two of the four metabolites. The absence of a noticeable decline in radioactivity beyond the initial 12% suggested that the side chain of alachlor was utilized as carbon source by the enriched culture. Little difference in radioactivity between growth medium and cell-free supernatant of the growth medium suggested that the carbon in the ring was not incorporated into the cells of the degrading microorganisms.

Biochemical pathway and degradation of phthalate ester isomers by bacteria

2005 ◽  
Vol 52 (8) ◽  
pp. 241-248 ◽  
Author(s):  
J.-D. Gu ◽  
J. Li ◽  
Y. Wang
Keyword(s):  
Aromatic Ring ◽  
Enrichment Culture ◽  
Klebsiella Oxytoca ◽  
Sole Source ◽  
Phthalate Ester ◽  
Ring Cleavage ◽  
Mangrove Sediment ◽  
Individual Species ◽  
Dimethyl Phthalate ◽  
Dimethyl Phthalate Ester

Degradation of dimethyl isophthalate (DMI) and dimethyl phthalate ester (DMPE) was investigated using microorganisms isolated from mangrove sediment of Hong Kong Mai Po Nature Reserve. One enrichment culture was capable of utilizing DMI as the sole source of carbon and energy, but none of the bacteria in the enrichment culture was capable of degrading DMI alone. In co-culture of two bacteria, degradation was observed proceeding through monomethyl isophthalate (MMI) ester and isophthalic acid (IPA) before the aromatic ring opening. Using DMI as the sole carbon and energy source, Klebsiella oxytoca Sc and Methylobacterium mesophilicum Sr degraded DMI through the biochemical cooperation. The initial hydrolytic reaction of the ester bond was by K. oxytoca Sc and the next step of transformation was by M. mesophilicum Sr, and IPA was degraded by both of them. In another investigation, a novel bacterium, strain MPsc, was isolated for degradation of dimethyl phthalate ester (DMPE) also from the mangrove sediment. On the basis of phenotypic, biochemical and 16S rDNA gene sequence analyses, the strain MPsc should be considered as a new bacterium at the genus level (8% differences). This strain, together with a Rhodococcus zopfii isolated from the same mangrove sediment, was able to degrade DMPE aerobically. The consortium consisting of the two species degraded 450mg/l DMPE within 3 days as the sole source of carbon and energy, but none of the individual species alone was able to transform DMPE. Furthermore, the biochemical degradation pathway proceeded through monomethyl phthalate (MMP), phthalic acid (PA) and then protocatechuate before aromatic ring cleavage. Our results suggest that degradation of complex organic compounds including DMI and DMPE may be carried out by several members of microorganisms working together in the natural environments.

The neural crest and the acoustic ganglion

Development ◽  
1967 ◽  
Vol 17 (3) ◽  
pp. 533-541
Author(s):  
M. S. Deol
Keyword(s):  
Neural Crest ◽  
Sole Source ◽  
New Approach ◽  
Present Purpose ◽  
Difference Of Opinion ◽  
Mammalian Embryos ◽  
The Subject ◽  
Otic Placode ◽  
The Difference ◽  
Mutant Genes

Although the origin of the acoustic ganglion has been the subject of numerous studies there is no unanimity of opinion about it. Most of the earlier investigators (Bartelmez, 1922; Adelman, 1925), using mammalian embryos, believed that it arose from the neural crest, but the experiments of Campenhout (1935) and Yntema (1937) on amphibian embryos led them to the view that it was largely, if not wholly, of placodal origin. This view was supported by Halley (1955), who worked on the cat, and later by Batten (1958), who worked on the sheep. In fact Batten stated categorically that the otic placode was the sole source of the acoustic ganglion. It was thought that an entirely new approach to the problem, namely the use of mutant genes, might help to resolve the difference of opinion. The most suitable mutant for the present purpose seemed to be piebald-lethal (symbol s1; Lane, 1966).

The degradation of p-toluenesulfonate by a Pseudomonas

1970 ◽  
Vol 16 (5) ◽  
pp. 309-316 ◽  
Author(s):  
D. D. Focht ◽  
F. D. Williams
Keyword(s):  
Carbon Dioxide ◽  
Oxygen Uptake ◽  
Aromatic Compounds ◽  
Carbon Sources ◽  
Sole Source ◽  
Carbon Chain ◽  
Ring Cleavage ◽  
Resting Cells ◽  
Resting Cell ◽  
Complete Mineralization

A Pseudomonas isolated from sewage was adapted to use p-toluenesulfonate as the sole source of both carbon and sulfur. Very few of over 30 aromatic compounds tested were used for growth as sole carbon sources. Significantly, sulfobenzoate, phenolsulfonates, and isomers of cresolsulfonates did not support growth. Respirometry studies with washed, resting cells showed similar results. In both studies, benzenesulfonate was always used more rapidly than p-toluenesulfonate. The degradation of p-toluenesulfonate was shown to be over 90% of the theoretical value required for complete mineralization to carbon dioxide, water, and sulfate. When resting cells were incubated with 35S-p-toluenesulfonate, the ratio of oxygen uptake to 35S-sulfate liberation remained constant during the complete degradation period. Radiochromatographic analysis showed no 35S-aromatic intermediates in resting-cell supernatants at any time. Resting cells previously incubated with 35S-p-toluenesulfonate liberated two 35S-labeled aromatic intermediates upon disruption. Resting cells incubated with 1-14C-p-toluenesulfonate produced labeled 3-methylcatechol, labeled acetate, and unlabeled pyruvate. The labeled intermediate, 3-methylcatechol, was degraded by cell-free extracts to labeled acetate. Hydroxylation, desulfonation, ring cleavage, and subsequent fissions of the carbon chain occurred in that order; all steps but the first were catalyzed by cell-free extracts.

scholarly journals Cloning, Sequencing, and Characterization of the Hexahydro-1,3,5-Trinitro-1,3,5-Triazine Degradation Gene Cluster from Rhodococcus rhodochrous

2002 ◽  
Vol 68 (10) ◽  
pp. 4764-4771 ◽  
Author(s):  
Helena M. B. Seth-Smith ◽  
Susan J. Rosser ◽  
Amrik Basran ◽  
Emma R. Travis ◽  
Eric R. Dabbs ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Gene Cluster ◽  
High Explosive ◽  
Sole Source ◽  
Ring Cleavage ◽  
Rhodococcus Rhodochrous ◽  
Resting Cell ◽  
Dead End ◽  
Adrenodoxin Reductase

ABSTRACT Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a high explosive which presents an environmental hazard as a major land and groundwater contaminant. Rhodococcus rhodochrous strain 11Y was isolated from explosive contaminated land and is capable of degrading RDX when provided as the sole source of nitrogen for growth. Products of RDX degradation in resting-cell incubations were analyzed and found to include nitrite, formaldehyde, and formate. No ammonium was excreted into the medium, and no dead-end metabolites were observed. The gene responsible for the degradation of RDX in strain 11Y is a constitutively expressed cytochrome P450-like gene, xplA, which is found in a gene cluster with an adrenodoxin reductase homologue, xplB. The cytochrome P450 also has a flavodoxin domain at the N terminus. This study is the first to present a gene which has been identified as being responsible for RDX biodegradation. The mechanism of action of XplA on RDX is thought to involve initial denitration followed by spontaneous ring cleavage and mineralization.

scholarly journals Cloning and Sequencing of a Novel meta-Cleavage Dioxygenase Gene Whose Product Is Involved in Degradation of γ-Hexachlorocyclohexane in Sphingomonas paucimobilis

1999 ◽  
Vol 181 (21) ◽  
pp. 6712-6719 ◽  
Author(s):  
Keisuke Miyauchi ◽  
Yugo Adachi ◽  
Yuji Nagata ◽  
Masamichi Takagi
Keyword(s):  
Amino Acids ◽  
Sole Source ◽  
Ring Cleavage ◽  
Hydroxyl Groups ◽  
Aromatic Rings ◽  
Cloning And Sequencing ◽  
Direct Demonstration ◽  
Rate Of Reaction ◽  
Dioxygenase Gene ◽  
New Type

ABSTRACT Sphingomonas (formerly Pseudomonas)paucimobilis UT26 utilizes γ-hexachlorocyclohexane (γ-HCH), a halogenated organic insecticide, as a sole source of carbon and energy. In a previous study, we showed that γ-HCH is degraded to chlorohydroquinone (CHQ) and then to hydroquinone (HQ), although the rate of reaction from CHQ to HQ was slow (K. Miyauchi, S. K. Suh, Y. Nagata, and M. Takagi, J. Bacteriol. 180:1354–1359, 1998). In this study, we cloned and characterized a gene, designated linE, which is located upstream oflinD and is directly involved in the degradation of CHQ. The LinE protein consists of 321 amino acids, and all of the amino acids which are reported to be essential for the activity ofmeta-cleavage dioxygenases are conserved in LinE.Escherichia coli overproducing LinE could convert both CHQ and HQ, producing γ-hydroxymuconic semialdehyde and maleylacetate, respectively, with consumption of O2 but could not convert catechol, which is one of the major substrates formeta-cleavage dioxygenases. LinE seems to be resistant to the acylchloride, which is the ring cleavage product of CHQ and which seems to react with water to be converted to maleylacetate. These results indicated that LinE is a novel type ofmeta-cleavage dioxygenase, designated (chloro)hydroquinone 1,2-dioxygenase, which cleaves aromatic rings with two hydroxyl groups at para positions preferably. This study represents a direct demonstration of a new type of ring cleavage pathway for aromatic compounds, the hydroquinone pathway.

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