scholarly journals Identification and functional analysis of the genes for naphthalenesulfonate catabolism by Sphingomonas xenophaga BN6

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 1929-1940 ◽  
Author(s):  
Andreas Keck ◽  
Doris Conradt ◽  
Anette Mahler ◽  
Andreas Stolz ◽  
Ralf Mattes ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Functional Analysis ◽  
Degradative Pathway ◽  
Ferredoxin Reductase ◽  
Insertional Inactivation ◽  
Genes Encoding ◽  
Transcriptional Units ◽  
Dna Fragment ◽  
Sphingomonas Aromaticivorans ◽  
Sphingomonas Xenophaga

Sphingomonas xenophaga BN6 degrades various (substituted) naphthalenesulfonates to the corresponding (substituted) salicylates. A gene cluster was identified on the plasmid pBN6 which coded for several enzymes participating in the degradative pathway for naphthalenesulfonates. A DNA fragment of 16 915 bp was sequenced which contained 17 ORFs. The genes encoding the 1,2-dihydroxynaphthalene dioxygenase, 2-hydroxychromene-2-carboxylate isomerase, and 2′-hydroxybenzalpyruvate aldolase of the naphthalenesulfonate pathway were identified on the DNA fragment and the encoded proteins heterologously expressed in Escherichia coli. Also, the genes encoding the ferredoxin and ferredoxin reductase of a multi-component, ring-hydroxylating naphthalenesulfonate dioxygenase were identified by insertional inactivation. The identified genes generally demonstrated the highest degree of homology to enzymes encoded by the phenanthrene-degrading organism Sphingomonas sp. P2, or the megaplasmid pNL1 of the naphthalene- and biphenyl-degrading strain Sphingomonas aromaticivorans F199. The genes of S. xenophaga BN6 participating in the degradation of naphthalenesulfonates also shared the same organization in three different transcriptional units as the genes involved in the degradation of naphthalene, biphenyl, and phenanthrene previously found in Sphingomonas sp. P2 and S. aromaticivorans F199. The genes were flanked in S. xenophaga BN6 by ORFs which specify proteins that show the highest homologies to proteins of mobile genetic elements.

Identification and mapping of the gene translation products involved in the first steps of the Comamonas testosteroni B-356 biphenyl/chlorobiphenyl biodegradation pathway

1994 ◽  
Vol 40 (9) ◽  
pp. 743-753 ◽  
Author(s):  
Janique Bergeron ◽  
Darakhshan Ahmad ◽  
Diane Barriault ◽  
Angèle Larose ◽  
Michel Sylvestre ◽  
...  
Keyword(s):  
Cleavage Product ◽  
Enzymatic Conversion ◽  
Comamonas Testosteroni ◽  
Gene Encoding ◽  
Ferredoxin Reductase ◽  
Oxidized Iron ◽  
Sds Page ◽  
Genes Encoding ◽  
Dna Fragment ◽  
T7 Polymerase

In this study, we have mapped Comamonas testosteroni B-356 genes encoding enzymes for the conversion of biphenyl and 4-chlorobiphenyl into the corresponding meta-cleavage compounds onto a 6.3-kb DNA fragment, and we have determined the subunit composition of the enzymes involved in this pathway. The various proteins encoded by this 6.3-kb DNA fragment and by subclones derived from it were overexpressed and selectively labelled using the T7 polymerase promoter system in Escherichia coli. They were then analyzed using SDS-PAGE, which allowed the encoding locus of each polypeptide to be mapped. Despite apparent dissimilarity in the congener selectivity patterns of the initial oxygenase of strain B-356 with those of Pseudomonas sp. strain LB400, the number and sizes of the polypeptides involved in the enzymatic conversion of biphenyl or 4-chlorobiphenyl into the meta-cleavage product appear to be similar in the two strains. In both strains, the bph operon encodes the following: the large (51-kDa polypeptide encoded by bphA) and the small (22-kDa polypeptide encoded by bphE) subunits of the iron sulphur protein, which is thought to interact directly with the substrate to introduce the oxygen molecule; the ferredoxin (12-kDa polypeptide encoded by bphF) involved in electron transfer from the reduced ferredoxin reductase to the oxidized iron sulphur protein; the 29-kDa polypeptide of the 2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase encoded by bphB; and the 32-kDa polypeptide of the 2,3-dihydroxybiphenyl-1,2-dioxygenase encoded by bphC, which catalyzes meta-1,2 fission of the aromatic ring. A major difference between strain B-356 and strain LB400 is that the bphG gene encoding biphenyl dioxygenase ferredoxin reductase is located outside the bph gene cluster in strain B-356. Several lines of evidence indicate that bphG is absent in clones carrying the bph operon from strain B-356.Key words: PCB, gene expression, biphenyl oxygenase, bph.

scholarly journals Functional and transcriptional analyses of the initial oxygenase genes for acenaphthene degradation from Sphingomonas sp. strain A4

Microbiology ◽  
2006 ◽  
Vol 152 (8) ◽  
pp. 2455-2467 ◽  
Author(s):  
Atsushi Kouzuma ◽  
Onruthai Pinyakong ◽  
Hideaki Nojiri ◽  
Toshio Omori ◽  
Hisakazu Yamane ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Gene Disruption ◽  
Pcr Analysis ◽  
Nucleotide Sequence Analysis ◽  
Rt Pcr ◽  
Transcription Start ◽  
Ferredoxin Reductase ◽  
Genes Encoding ◽  
Dna Fragment ◽  
Putative Binding Site

Sphingomonas sp. strain A4 is capable of utilizing acenaphthene as its sole carbon and energy source. To isolate the genes responsible for acenaphthene degradation, transposon mutagenesis was performed on strain A4 and four mini-Tn5-inserted mutants lacking the ability to utilize acenaphthene were isolated. In three of the four mini-Tn5 inserted mutants, the mini-Tn5s were inserted into the same locus (within about 16 kb) as the arhA1A2 genes, which had previously been identified as the genes encoding the terminal oxygenase components for the initial oxygenation of acenaphthene. The nucleotide sequence analysis of the corresponding 16.4 kb DNA fragment revealed the existence of 16 ORFs and a partial ORF. From these ORFs, the genes encoding the ferredoxin (ArhA3) and ferredoxin reductase (ArhA4) complementary to ArhA1A2 were identified. RT-PCR analysis suggested that a 13.5 kb gene cluster, consisting of 13 ORFs and including all the arhA genes, forms an operon, although it includes several ORFs that are apparently unnecessary for acenaphthene degradation. Furthermore, using gene disruption and quantitative RT-PCR analyses, the LysR-type activator, ArhR, required for expression of the 13.5 kb gene cluster was also identified. Transcription of the gene cluster by ArhR was induced in the presence of acenaphthene (or its metabolite), and a putative binding site (T-N11-A motif) for ArhR was found upstream from the transcription start point of arhA3.

Expression of the 4-chlorobenzoate dehalogenase genes from Pseudomonas sp. CBS3 in Escherichia coli and identification of the gene translation products

1992 ◽  
Vol 38 (10) ◽  
pp. 1074-1083 ◽  
Author(s):  
Pierre Savard ◽  
Hugues Charest ◽  
Michel Sylvestre ◽  
François Shareck ◽  
Jeffrey D. Scholten ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Deletion Mutants ◽  
Pseudomonas Sp ◽  
Product Analysis ◽  
Pseudomonas Putida Kt2440 ◽  
Whole Cells ◽  
E Coli ◽  
Selective Expression ◽  
Genes Encoding ◽  
Dna Fragment

The genes encoding the 4-chlorobenzoate dehalogenase of Pseudomonas sp. strain CBS3 were, in an earlier study, cloned in Escherichia coli DH1 with the cosmid vector pPSA843 and then mobilized to the 4-chlorobenzoate dehalogenase minus strain Pseudomonas putida KT2440. In this paper we report on the expression of 4-chlorobenzoate dehalogenase in these clones and on the polypeptide composition of the active enzyme. The dehalogenase activity in whole cells suspended in 3.2 mM 4-chlorobenzoate (30 °C) was determined to be approximately 27 units (micromoles 4-hydroxybenzoate produced per minute) per 100 g of E. coli - pPSA843 cells and approximately 28 units per 100 g of P. putida-pPSA843 cells. Dehalogenase activity in fresh cellular extracts (pH 7.4, 30 °C) prepared from the is. E. coli and P. putida clones was unstable and at least 20-fold lower than that observed with the whole cells. The polypeptide components of the dehalogenase were identified by selective expression of the cloned dehalogenase genes and analysis of the gene translation products. Analysis of dehalogenase activity in Ω insertion mutants and deletion mutants circumscribed the dehalogenase genes to a 4.8-kilobase (4.8 kb) stretch of the 9.5-kb DNA fragment. Selective expression of the dehalogenase genes from a cloned 4.8-kb DNA fragment in a maxicell system revealed a 30-kDa polypeptide as one of the components of the dehalogenase system. Selective expression of the dehalogenase genes using the T7 polymerase promoter system revealed the 30-kDa polypeptide and 57- and 16-kDa polypeptide products. Determination of which of the three polypeptides were translated in deletion mutants provided the relative positions of the encoding genes on a single DNA strand and the direction in which they are transcribed. Key words: peptide expression, bacteria, dehalogenation, gene product analysis.

The clp1 Gene of the Mushroom Coprinus cinereus Is Essential for A-Regulated Sexual Development

Genetics ◽  
2001 ◽  
Vol 157 (1) ◽  
pp. 133-140
Author(s):  
Kazumi Inada ◽  
Yoshinori Morimoto ◽  
Toshihide Arima ◽  
Yukio Murata ◽  
Takashi Kamada
Keyword(s):  
Sexual Development ◽  
Genomic Dna ◽  
Mutant Allele ◽  
Coprinus Cinereus ◽  
Mating Partner ◽  
Essential Step ◽  
Genes Encoding ◽  
Dna Fragment ◽  
Necessary And Sufficient ◽  
Novel Protein

Abstract Sexual development in the mushroom Coprinus cinereus is under the control of the A and B mating-type loci, both of which must be different for a compatible, dikaryotic mycelium to form between two parents. The A genes, encoding proteins with homeodomain motifs, regulate conjugate division of the two nuclei from each mating partner and promote the formation of clamp connections. The latter are hyphal configurations required for the maintenance of the nuclear status in the dikaryotic phase of basidiomycetes. The B genes encode pheromones and pheromone receptors. They regulate the cellular fusions that complete clamp connections during growth, as well as the nuclear migration required for dikaryosis. The AmutBmut strain (326) of C. cinereus, in which both A- and B-regulated pathways are constitutively activated by mutations, produces, without mating, dikaryon-like, fertile hyphae with clamp connections. In this study we isolated and characterized clampless1-1 (clp1-1), a mutation that blocks clamp formation, an essential step in A-regulated sexual development, in the AmutBmut background. A genomic DNA fragment that rescues the clp1-1 mutation was identified by transformations. Sequencing of the genomic DNA, together with RACE experiments, identified an ORF interrupted by one intron, encoding a novel protein of 365 amino acids. The clp1-1 mutant allele carries a deletion of four nucleotides, which is predicted to cause elimination of codon 128 and frameshifts thereafter. The clp1 transcript was normally detected only in the presence of the A protein heterodimer formed when homokaryons with compatible A genes were mated. Forced expression of clp1 by promoter replacements induced clamp development without the need for a compatible A gene combination. These results indicate that expression of clp1 is necessary and sufficient for induction of the A-regulated pathway that leads to clamp development.

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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