scholarly journals Evolutionary Relationship between Chlorocatechol Catabolic Enzymes from Rhodococcus opacus 1CP and Their Counterparts in Proteobacteria: Sequence Divergence and Functional Convergence

1998 ◽  
Vol 180 (5) ◽  
pp. 1082-1094 ◽  
Author(s):  
Dirk Eulberg ◽  
Elena M. Kourbatova ◽  
Ludmila A. Golovleva ◽  
Michael Schlömann
Keyword(s):  
Gene Cluster ◽  
Regulatory Protein ◽  
Evolutionary Relationship ◽  
Sequence Divergence ◽  
Functional Adaptation ◽  
Open Reading Frames ◽  
Rhodococcus Opacus ◽  
Sequence Comparisons ◽  
Catabolic Enzymes ◽  
Chloromuconate Cycloisomerase

Biochemical investigations of the muconate and chloromuconate cycloisomerases from the chlorophenol-utilizing strainRhodococcus opacus (erythropolis) 1CP had previously indicated that the chlorocatechol catabolic pathway of this strain may have developed independently from the corresponding pathways of proteobacteria. To test this hypothesis, we cloned the chlorocatechol catabolic gene cluster of strain 1CP by using PCR with primers derived from sequences of N termini and peptides of purified chlorocatechol 1,2-dioxygenase and chloromuconate cycloisomerase. Sequencing of the clones revealed that they comprise different parts of the same gene cluster in which five open reading frames have been identified. The clcB gene for chloromuconate cycloisomerase is transcribed divergently from a gene which codes for a LysR-type regulatory protein, the presumed ClcR. Downstream of clcRbut separated from it by 222 bp, we detected the clcA andclcD genes, which could unambiguously be assigned to chlorocatechol 1,2-dioxygenase and dienelactone hydrolase. A gene coding for a maleylacetate reductase could not be detected. Instead, the product encoded by the fifth open reading frame turned out to be homologous to transposition-related proteins of IS1031 and Tn4811. Sequence comparisons of ClcA and ClcB to other 1,2-dioxygenases and cycloisomerases, respectively, clearly showed that the chlorocatechol catabolic enzymes of R. opacus 1CP represent different branches in the dendrograms than their proteobacterial counterparts. Thus, while the sequences diverged, the functional adaptation to efficient chlorocatechol metabolization occurred independently in proteobacteria and gram-positive bacteria, that is, by functionally convergent evolution.

A linear megaplasmid, p1CP, carrying the genes for chlorocatechol catabolism of Rhodococcus opacus 1CP

Microbiology ◽  
2004 ◽  
Vol 150 (9) ◽  
pp. 3075-3087 ◽  
Author(s):  
Christina König ◽  
Dirk Eulberg ◽  
Janosch Gröning ◽  
Silvia Lakner ◽  
Volker Seibert ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Gel Electrophoresis ◽  
Restriction Analysis ◽  
Carbon Sources ◽  
Gene Clusters ◽  
Rhodococcus Opacus ◽  
Terminal Inverted Repeat ◽  
Sequence Comparisons ◽  
S1 Nuclease ◽  
Genes Encoding

The Gram-positive actinobacterium Rhodococcus opacus 1CP is able to utilize several (chloro)aromatic compounds as sole carbon sources, and gene clusters for various catabolic enzymes and pathways have previously been identified. Pulsed-field gel electrophoresis indicates the occurrence of a 740 kb megaplasmid, designated p1CP. Linear topology and the presence of covalently bound proteins were shown by the unchanged electrophoretic mobility after S1 nuclease treatment and by the immobility of the native plasmid during non-denaturing agarose gel electrophoresis, respectively. Sequence comparisons of both termini revealed a perfect 13 bp terminal inverted repeat (TIR) as part of an imperfect 583/587 bp TIR, as well as two copies of the highly conserved centre (GCTXCGC) of a palindromic motif. An initial restriction analysis of p1CP was performed. By means of PCR and hybridization techniques, p1CP was screened for several genes encoding enzymes of (chloro)aromatic degradation. A single maleylacetate reductase gene macA, the clc gene cluster for 4-chloro-/3,5-dichlorocatechol degradation, and the clc2 gene cluster for 3-chlorocatechol degradation were found on p1CP whereas the cat and pca gene clusters for the catechol and the protocatechuate pathways, respectively, were not. Prolonged cultivation of the wild-type strain 1CP under non-selective conditions led to the isolation of the clc- and clc2-deficient mutants 1CP.01 and 1CP.02 harbouring the shortened plasmid variants p1CP.01 (500 kb) and p1CP.02 (400 kb).

scholarly journals A New Modified ortho Cleavage Pathway of 3-Chlorocatechol Degradation by Rhodococcus opacus 1CP: Genetic and Biochemical Evidence

2002 ◽  
Vol 184 (19) ◽  
pp. 5282-5292 ◽  
Author(s):  
Olga V. Moiseeva ◽  
Inna P. Solyanikova ◽  
Stefan R. Kaschabek ◽  
Janosch Gröning ◽  
Monika Thiel ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Pcr Primers ◽  
Open Reading Frames ◽  
Rhodococcus Opacus ◽  
Catabolic Genes ◽  
Rhodococcus Opacus 1Cp ◽  
Dienelactone Hydrolase ◽  
Related Enzyme ◽  
Reading Frames ◽  
Chloromuconate Cycloisomerase

ABSTRACT The 4-chloro- and 2,4-dichlorophenol-degrading strain Rhodococcus opacus 1CP has previously been shown to acquire, during prolonged adaptation, the ability to mineralize 2-chlorophenol. In addition, homogeneous chlorocatechol 1,2-dioxygenase from 2-chlorophenol-grown biomass has shown relatively high activity towards 3-chlorocatechol. Based on sequences of the N terminus and tryptic peptides of this enzyme, degenerate PCR primers were now designed and used for cloning of the respective gene from genomic DNA of strain 1CP. A 9.5-kb fragment containing nine open reading frames was obtained on pROP1. Besides other genes, a gene cluster consisting of four chlorocatechol catabolic genes was identified. As judged by sequence similarity and correspondence of predicted N termini with those of purified enzymes, the open reading frames correspond to genes for a second chlorocatechol 1,2-dioxygenase (ClcA2), a second chloromuconate cycloisomerase (ClcB2), a second dienelactone hydrolase (ClcD2), and a muconolactone isomerase-related enzyme (ClcF). All enzymes of this new cluster are only distantly related to the known chlorocatechol enzymes and appear to represent new evolutionary lines of these activities. UV overlay spectra as well as high-pressure liquid chromatography analyses confirmed that 2-chloro-cis,cis-muconate is transformed by ClcB2 to 5-chloromuconolactone, which during turnover by ClcF gives cis-dienelactone as the sole product. cis-Dienelactone was further hydrolyzed by ClcD2 to maleylacetate. ClcF, despite its sequence similarity to muconolactone isomerases, no longer showed muconolactone-isomerizing activity and thus represents an enzyme dedicated to its new function as a 5-chloromuconolactone dehalogenase. Thus, during 3-chlorocatechol degradation by R. opacus 1CP, dechlorination is catalyzed by a muconolactone isomerase-related enzyme rather than by a specialized chloromuconate cycloisomerase.

scholarly journals Nitrate reductase from the magnetotactic bacteriumMagnetospirillum magnetotacticumMS-1: purification and sequence analyses

2003 ◽  
Vol 49 (3) ◽  
pp. 197-206 ◽  
Author(s):  
Azuma Taoka ◽  
Katsuhiko Yoshimatsu ◽  
Masaaki Kanemori ◽  
Yoshihiro Fukumori
Keyword(s):  
Nitrate Reductase ◽  
Gene Cluster ◽  
Phylogenetic Analyses ◽  
Regulatory Protein ◽  
Heme Iron ◽  
Open Reading Frames ◽  
Non Heme Iron ◽  
Close Relationship ◽  
Paracoccus Pantotrophus ◽  
Magnetospirillum Magnetotacticum

We purified the nitrate reductase from the soluble fraction of Magnetospirillum magnetotacticum MS-1. The enzyme was composed of 86- and 17-kDa subunits and contained molybdenum, non-heme iron, and heme c. These properties are very similar to those of the periplasmic nitrate reductase found in Paracoccus pantotrophus. The M. magnetotacticum nap locus was clustered in seven open reading frames, napFDAGHBC. The phylogenetic analyses of NapA, NapB, and NapC suggested a close relationship between M. magnetotacticum nap genes and Escherichia coli nap genes, which is not consistent with the 16S rDNA data. This is the first finding that the α subclass of Proteobacteria possesses a napFDAGHBC-type nap gene cluster. The nap gene cluster had putative fumarate and nitrate reduction regulatory protein (Fnr) and NarL protein binding sites. Furthermore, we investigated the effect of molybdate deficiency in medium on the total iron content of the magnetosome fraction and discussed the physiological function of nitrate reductase in relation to the magnetite synthesis in M. magnetotacticum.Key words: nitrate reductase, magnetotactic bacteria, denitrification, horizontal gene transfer.

scholarly journals The Mithramycin Gene Cluster of Streptomyces argillaceus Contains a Positive Regulatory Gene and Two Repeated DNA Sequences That Are Located at Both Ends of the Cluster

1999 ◽  
Vol 181 (2) ◽  
pp. 642-647 ◽  
Author(s):  
Felipe Lombó ◽  
Alfredo F. Braña ◽  
Carmen Méndez ◽  
José A. Salas
Keyword(s):  
Gene Cluster ◽  
Dna Sequences ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Fold Increase ◽  
Open Reading Frames ◽  
Antibiotic Biosynthesis ◽  
Positive Regulators ◽  
Actinorhodin Production

ABSTRACT Sequencing of a 4.3-kb DNA region from the chromosome ofStreptomyces argillaceus, a mithramycin producer, revealed the presence of two open reading frames (ORFs). The first one (orfA) codes for a protein that resembles several transport proteins. The second one (mtmR) codes for a protein similar to positive regulators involved in antibiotic biosynthesis (DnrI, SnoA, ActII-orf4, CcaR, and RedD) belonging to the Streptomycesantibiotic regulatory protein (SARP) family. Both ORFs are separated by a 1.9-kb, apparently noncoding region. Replacement of themtmR region by an antibiotic resistance cassette completely abolished mithramycin biosynthesis. Expression of mtmR in a high-copy-number vector in S. argillaceus caused a 16-fold increase in mithramycin production. The mtmR gene restored actinorhodin production in Streptomyces coelicolor JF1 mutant, in which the actinorhodin-specific activator ActII-orf4 is inactive, and also stimulated actinorhodin production byStreptomyces lividans TK21. A 241-bp region located 1.9 kb upstream of mtmR was found to be repeated approximately 50 kb downstream of mtmR at the other end of the mithramycin gene cluster. A model to explain a possible route for the acquisition of the mithramycin gene cluster by S. argillaceus is proposed.

scholarly journals Expanding the Diversity of the IS630-Tc1-mariner Superfamily: Discovery of a Unique DD37E Transposon and Reclassification of the DD37D and DD39D Transposons

Genetics ◽  
2001 ◽  
Vol 159 (3) ◽  
pp. 1103-1115 ◽  
Author(s):  
Hongguang Shao ◽  
Zhijian Tu
Keyword(s):  
Phylogenetic Analyses ◽  
Mosquito Species ◽  
Open Reading Frames ◽  
Inverted Repeats ◽  
Sequence Comparisons ◽  
Wide Range ◽  
Multiple Copies ◽  
Catalytic Motif ◽  
Open The Doors ◽  
Reading Frames

Abstract A novel transposon named ITmD37E was discovered in a wide range of mosquito species. Sequence analysis of multiple copies in three Aedes species showed similar terminal inverted repeats and common putative TA target site duplications. The ITmD37E transposases contain a conserved DD37E catalytic motif, which is unique among reported transposons of the IS630-Tc1-mariner superfamily. Sequence comparisons and phylogenetic analyses suggest that ITmD37E forms a novel family distinct from the widely distributed Tc1 (DD34E), mariner (DD34D), and pogo (DDxD) families in the IS630-Tc1-mariner superfamily. The inclusion in the phylogenetic analysis of recently reported transposons and transposons uncovered in our database survey provided revisions to previous classifications and identified two additional families, ITmD37D and ITmD39D, which contain DD37D and DD39D motifs, respectively. The above expansion and reorganization may open the doors to the discovery of related transposons in a broad range of organisms and help illustrate the evolution and structure-function relationships among these distinct transposases in the IS630-Tc1-mariner superfamily. The presence of intact open reading frames and highly similar copies in some of the newly characterized transposons suggests recent transposition. Studies of these novel families may add to the limited repertoire of transgenesis and mutagenesis tools for a wide range of organisms, including the medically important mosquitoes.

scholarly journals Mitochondrial Intronic Open Reading Frames in Podospora: Mobility and Consecutive Exonic Sequence Variations

Genetics ◽  
1996 ◽  
Vol 143 (2) ◽  
pp. 777-788 ◽  
Author(s):  
Carole H Sellem ◽  
Yves d'Aubenton-Carafa ◽  
Michèle Rossignol ◽  
Léon Belcour
Keyword(s):  
Mitochondrial Genome ◽  
Open Reading Frames ◽  
Nucleotide Substitutions ◽  
Sequence Comparisons ◽  
Modular Evolution ◽  
Group I ◽  
Sequence Variations ◽  
Type Strains ◽  
Reading Frames ◽  
Adjacent Exon

Abstract The mitochondrial genome of 23 wild-type strains belonging to three different species of The mitochondrial genome the filamentous fungus Podospora was examined. Among the 15 optional sequences identified are two intronic reading frames, nad1-i4-orf1 and cox1-i7-orf2. We show that the presence of these sequences was strictly correlated with tightly clustered nucleotide substitutions in the adjacent exon. This correlation applies to the presence or absence of closely related open reading frames (ORFs), found at the same genetic locations, in all the Pyrenomycete genera examined. The recent gain of these optional ORFs in the evolution of the genus Podospora probably account for such sequence differences. In the homoplasmic progeny from heteroplasmons constructed between Podospora strains differing by the presence of these optional ORFs, nad1-i4-orf1 and cox1-i7-orf2 appeared highly invasive. Sequence comparisons in the nad1-i4 intron of various strains of the Pyrenomycete family led us to propose a scenario of its evolution that includes several events of loss and gain of intronic ORFs. These results strongly reinforce the idea that group I intronic ORFs are mobile elements and that their transfer, and comcomitant modification of the adjacent exon, could participate in the modular evolution of mitochondrial genomes.

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.

scholarly journals Bias between the Left and Right Inverted Repeats during IS911 Targeted Insertion

2008 ◽  
Vol 190 (18) ◽  
pp. 6111-6118 ◽  
Author(s):  
P. Rousseau ◽  
C. Loot ◽  
C. Turlan ◽  
S. Nolivos ◽  
M. Chandler
Keyword(s):  
Insertion Sequence ◽  
Regulatory Protein ◽  
Open Reading Frames ◽  
Inverted Repeats ◽  
Functional Differences ◽  
Left And Right ◽  
Ordered Assembly ◽  
Overlapping Open Reading Frames ◽  
Reading Frames

ABSTRACT IS911 is a bacterial insertion sequence composed of two consecutive overlapping open reading frames (ORFs [orfA and orfB]) encoding the transposase (OrfAB) as well as a regulatory protein (OrfA). These ORFs are bordered by terminal left and right inverted repeats (IRL and IRR, respectively) with several differences in nucleotide sequence. IS911 transposition is asymmetric: each end is cleaved on one strand to generate a free 3′-OH, which is then used as the nucleophile in attacking the opposite insertion sequence (IS) end to generate a free IS circle. This will be inserted into a new target site. We show here that the ends exhibit functional differences which, in vivo, may favor the use of one compared to the other during transposition. Electromobility shift assays showed that a truncated form of the transposase [OrfAB(1-149)] exhibits higher affinity for IRR than for IRL. While there was no detectable difference in IR activities during the early steps of transposition, IRR was more efficient during the final insertion steps. We show here that the differential activities between the two IRs correlate with the different affinities of OrfAB(1-149) for the IRs during assembly of the nucleoprotein complexes leading to transposition. We conclude that the two inverted repeats are not equivalent during IS911 transposition and that this asymmetry may intervene to determine the ordered assembly of the different protein-DNA complexes involved in the reaction.

scholarly journals Establishment of anIn Vitrod-Cycloserine-Synthesizing System by UsingO-Ureido-l-Serine Synthase and d-Cycloserine Synthetase Found in the Biosynthetic Pathway

2013 ◽  
Vol 57 (6) ◽  
pp. 2603-2612 ◽  
Author(s):  
Narutoshi Uda ◽  
Yasuyuki Matoba ◽  
Takanori Kumagai ◽  
Kosuke Oda ◽  
Masafumi Noda ◽  
...  
Keyword(s):  
Gene Cluster ◽  
High Performance ◽  
Sequence Similarity ◽  
Open Reading Frames ◽  
Homocysteine Thiolactone ◽  
Putative Gene ◽  
Content Type ◽  
Dna Fragment ◽  
Layer Chromatography

ABSTRACTWe have recently cloned a DNA fragment containing a gene cluster that is responsible for the biosynthesis of an antituberculosis antibiotic,d-cycloserine. The gene cluster is composed of 10 open reading frames, designateddcsAtodcsJ. Judging from the sequence similarity between each putative gene product and known proteins, DcsC, which displays high homology to diaminopimelate epimerase, may catalyze the racemization ofO-ureidoserine. DcsD is similar toO-acetylserine sulfhydrylase, which generatesl-cysteine usingO-acetyl-l-serine with sulfide, and therefore, DcsD may be a synthase to generateO-ureido-l-serine usingO-acetyl-l-serine and hydroxyurea. DcsG, which exhibits similarity to a family of enzymes with an ATP-grasp fold, may be an ATP-dependent synthetase convertingO-ureido-d-serine intod-cycloserine. In the present study, to characterize the enzymatic functions of DcsC, DcsD, and DcsG, each protein was overexpressed inEscherichia coliand purified to near homogeneity. The biochemical function of each of the reactions catalyzed by these three proteins was verified by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and, in some cases, mass spectrometry. The results from this study demonstrate that by using a mixture of the three purified enzymes and the two commercially available substratesO-acetyl-l-serine and hydroxyurea, synthesis ofd-cycloserine was successfully attained. Thesein vitrostudies yield the conclusion that DcsD and DcsG are necessary for the syntheses ofO-ureido-l-serine andd-cycloserine, respectively. DcsD was also able to catalyze the synthesis ofl-cysteine when sulfide was added instead of hydroxyurea. Furthermore, the present study shows that DcsG can also form other cyclicd-amino acid analogs, such asd-homocysteine thiolactone.

Sign in / Sign up

Export Citation Format

Share Document