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 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.

Aerobic benzoyl-CoA catabolic pathway inAzoarcus evansii: studies on the non-oxygenolytic ring cleavage enzyme

2005 ◽  
Vol 56 (6) ◽  
pp. 1586-1600 ◽  
Author(s):  
Johannes Gescher ◽  
Wolfgang Eisenreich ◽  
Jürgen Wörth ◽  
Adelbert Bacher ◽  
Georg Fuchs
Keyword(s):  
Ring Cleavage ◽  
Catabolic Pathway ◽  
Cleavage Enzyme

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 Cathepsin B and D deficiency in the mouse pancreas induces impaired autophagy and chronic pancreatitis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hideaki Iwama ◽  
Sally Mehanna ◽  
Mai Imasaka ◽  
Shinsuke Hashidume ◽  
Hiroshi Nishiura ◽  
...  
Keyword(s):  
Chronic Pancreatitis ◽  
Cathepsin B ◽  
Gene Knockout ◽  
Cathepsin L ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Double Knockout ◽  
Lysosomal Proteases ◽  
Autophagic Activity

AbstractThe major lysosomal proteases, Cathepsin B (CTSB), Cathepsin D (CTSD) and Cathepsin L (CTSL), are implicated in autophagic activity. To investigate the role of each cathepsin in the exocrine pancreas, we generated mice in which the pancreas was specifically deficient in Ctsb, Ctsd and Ctsl. Each of these gene knockout (KO) and Ctsb;Ctsl and Ctsd;Ctsl double-knockout (DKO) mice were almost normal. However, we found cytoplasmic degeneration in the pancreatic acinar cells of Ctsb;Ctsd DKO mice, similar to autophagy related 5 (Atg5) KO mice. LC3 and p62 (autophagy markers) showed remarkable accumulation and the numbers of autophagosomes and autolysosomes were increased in the pancreatic acinar cells of Ctsb;Ctsd DKO mice. Moreover, these Ctsb;Ctsd DKO mice also developed chronic pancreatitis (CP). Thus, we conclude that both Ctsb and Ctsd deficiency caused impaired autophagy in the pancreatic acinar cells, and induced CP in mice.

scholarly journals Proteasomal Components Required for Cell Growth and Stress Responses in the Haloarchaeon Haloferax volcanii

2008 ◽  
Vol 190 (24) ◽  
pp. 8096-8105 ◽  
Author(s):  
Guangyin Zhou ◽  
David Kowalczyk ◽  
Matthew A. Humbard ◽  
Sunil Rohatgi ◽  
Julie A. Maupin-Furlow
Keyword(s):  
Stress Responses ◽  
Gene Knockout ◽  
Inorganic Nitrogen ◽  
Nitrogen Sources ◽  
Genetic System ◽  
Haloferax Volcanii ◽  
Double Knockout ◽  
Genes Encoding ◽  
Proteasome Gene

ABSTRACT Little is known regarding the biological roles of archaeal proteases. The haloarchaeon Haloferax volcanii is an ideal model for understanding these enzymes, as it is one of few archaea with an established genetic system. In this report, a series of H. volcanii mutant strains with markerless and/or conditional knockouts in each known proteasome gene was systematically generated and characterized. This included single and double knockouts of genes encoding the 20S core α1 (psmA), β (psmB), and α2 (psmC) subunits as well as genes (panA and panB) encoding proteasome-activating nucleotidase (PAN) proteins closely related to the regulatory particle triple-A ATPases (Rpt) of eukaryotic 26S proteasomes. Our results demonstrate that 20S proteasomes are required for growth. Although synthesis of 20S proteasomes containing either α1 or α2 could be separately abolished via gene knockout with little to no impact on growth, conditional depletion of either β alone or α1 and α2 together rendered the cells inviable. In contrast, the PAN proteins were not essential based on the robust growth of the panA panB double knockout strain. Deletion of genes encoding either α1 or PanA did, however, render cells more sensitive to growth on organic versus inorganic nitrogen sources and hypo-osmotic stress and limited growth in the presence of l-canavanine. Abolishment of α1 synthesis also had a severe impact on the ability of cells to withstand thermal stress. This contrasted with what was seen for panA knockouts, which displayed enhanced thermotolerance. Together, these results provide new and important insight into the biological role of proteasomes in archaea.

Towards the Final Legitimation of the Party

2017 ◽  
Author(s):  
Piero Ignazi
Keyword(s):  
Twentieth Century ◽  
Great Britain ◽  
Political Parties ◽  
Political Party ◽  
Sole Source ◽  
The Political ◽  
Party Formation ◽  
Post War ◽  
Dominant Influence

Chapter 3 investigates the process of party formation in France, Germany, Great Britain, and Italy, and demonstrates the important role of cultural and societal premises for the development of political parties in the nineteenth century. Particular attention is paid in this context to the conditions in which the two mass parties, socialists and Christian democrats, were established. A larger set of Western European countries included in this analysis is thoroughly scrutinized. Despite discontent among traditional liberal-conservative elites, full endorsement of the political party was achieved at the beginning of the twentieth century. Particular attention is paid to the emergence of the interwar totalitarian party, especially under the guise of Italian and German fascism, when ‘the party’ attained its most dominant influence as the sole source and locus of power. The chapter concludes by suggesting hidden and unaccounted heritages of that experience in post-war politics.

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.

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