scholarly journals Genes for 2,4,5-Trichlorophenoxyacetic Acid Metabolism in Burkholderia cepacia AC1100: Characterization of the tftC and tftD Genes and Locations of the tft Operons on Multiple Replicons

1998 ◽  
Vol 64 (6) ◽  
pp. 2086-2093 ◽  
Author(s):  
Anette Hübner ◽  
Clyde E. Danganan ◽  
Luying Xun ◽  
A. M. Chakrabarty ◽  
William Hendrickson
Keyword(s):  
High Frequency ◽  
Burkholderia Cepacia ◽  
Acid Metabolism ◽  
Genomic Organization ◽  
Genomic Library ◽  
Sole Source ◽  
Amino Acid Sequences ◽  
Klebsiella Pneumonia ◽  
Trichlorophenoxyacetic Acid

ABSTRACT Burkholderia cepacia AC1100 uses the chlorinated aromatic compound 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy. The enzyme which converts the first intermediate in the pathway, 2,4,5-trichlorophenol, to 5-chlorohydroquinone has been purified and consists of two subunits of 58 and 22 kDa, encoded by the tftC and tftDgenes (48). A degenerate primer was designed from the N terminus of the 58-kDa polypeptide and used to isolate a clone containing the tftC and tftD genes from a genomic library of AC1100. The derived amino acid sequences oftftC and tftD show significant homology to the two-component monooxygenases HadA of Burkholderia pickettii, HpaBC of Escherichia coli, and HpaAH ofKlebsiella pneumonia. Expression of the tftCand tftD genes appeared to be induced when they were grown in the presence of 2,4,5-T, as shown by RNA slot blot and primer extension analyses. Three sets of cloned tft genes were used as probes to explore the genomic organization of the pathway. Pulsed-field gel electrophoresis analyses of whole chromosomes ofB. cepacia AC1100 demonstrated that the genome is comprised of five replicons of 4.0, 2.7, 0.53, 0.34, and 0.15 Mbp, designated I to V, respectively. The tft genes are located on the smaller replicons: the tftAB cluster is on replicon IV, tftEFGH is on replicon III, and copies of thetftC and the tftCD operons are found on both replicons III and IV. When cells were grown in the absence of 2,4,5-T, the genes were lost at high frequency by chromosomal deletions and rearrangements to produce 2,4,5-T-negative mutants. In one mutant, thetftA and tftB genes translocated from one replicon to another, with the concomitant loss of tftEFGHand one copy of tftCD.

scholarly journals Characterization of Chlorophenol 4-Monooxygenase (TftD) and NADH:Flavin Adenine Dinucleotide Oxidoreductase (TftC) of Burkholderia cepacia AC1100

2003 ◽  
Vol 185 (9) ◽  
pp. 2786-2792 ◽  
Author(s):  
Michelle R. Gisi ◽  
Luying Xun
Keyword(s):  
Burkholderia Cepacia ◽  
Active Oxygen Species ◽  
Environmental Pollutant ◽  
Flavin Mononucleotide ◽  
Binding Property ◽  
Aromatic Substrates ◽  
Key Enzyme ◽  
Trichlorophenoxyacetic Acid ◽  
New Discovery

ABSTRACT Burkholderia cepacia AC1100 uses 2,4,5-trichlorophenoxyacetic acid, an environmental pollutant, as a sole carbon and energy source. Chlorophenol 4-monooxygenase is a key enzyme in the degradation of 2,4,5-trichlorophenoxyacetic acid, and it was originally characterized as a two-component enzyme (TftC and TftD). Sequence analysis suggests that they are separate enzymes. The two proteins were separately produced in Escherichia coli, purified, and characterized. TftC was an NADH:flavin adenine dinucleotide (FAD) oxidoreductase. A C-terminally His-tagged fusion TftC used NADH to reduce either FAD or flavin mononucleotide (FMN) but did not use NADPH or riboflavin as a substrate. Kinetic and binding property analysis showed that FAD was a better substrate than FMN. TftD was a reduced FAD (FADH2)-utilizing monooxygenase, and FADH2 was supplied by TftC. It converted 2,4,5-trichlorophenol to 2,5-dichloro-p-quinol and then to 5-chlorohydroxyquinol but converted 2,4,6-trichlorophenol only to 2,6-dichloro-p-quinol as the final product. TftD interacted with FADH2 and retarded its rapid oxidation by O2. A spectrum of possible TftD-bound FAD-peroxide was identified, indicating that the peroxide is likely the active oxygen species attacking the aromatic substrates. The reclassification of the two enzymes further supports the new discovery of FADH2-utilizing enzymes, which have homologues in the domains Bacteria and Archaea.

scholarly journals Characterization of bacterium types isolated from commercial laying hen farms in Ogun State Nigeria

2018 ◽  
Vol 71 (3) ◽  
pp. 137
Author(s):  
Oluwawemimo Adebowale ◽  
Olanike Adeyemo
Keyword(s):  
Burkholderia Cepacia ◽  
Vibrio Vulnificus ◽  
Klebsiella Oxytoca ◽  
Klebsiella Pneumonia ◽  
Morganella Morganii ◽  
Ogun State ◽  
Vibrio Mimicus ◽  
Citrobacter Diversus ◽  
Providencia Stuartii

L’étude a porté sur la répartition des types de bactéries isolées à partir de fèces et de litières de volaille dans les élevages commerciaux de poules pondeuses des agglomérations de Remo et d’Egba, Etat d’Ogun, Nigeria. Au total, 29 espèces de fermenteurs du lactose et de non-fermenteurs du lactose ont été récoltées. Les bactéries isolées des fèces comprenaient Aeromonas hydrophila (27,5 %), Providencia stuartii (15,5 %), Actinobacillus sp. (9,1 %), Burkholderia cepacia (7,7 %), Serratia marcescens (4,9 %), Citrobacter diversus (4,9 %), Klebsiella oxytoca (4,2 %) et Enterobacter gergoviae (4,2 %). Les autres espèces étaient Escherichia coli (2,1 %), Plesiomonas shigelloides (2,1 %), Vibrio alginolyticus (2,1 %), Morganella morganii (2,1 %), Pantoea agglomerans (1,4 %), Vibrio mimicus (1,4 %), Pseudomonas aeruginosa (1,4 %), Burkholderia pseudomallei (1,4 %), Salmonella arizonae (0,7 %), Klebsiella pneumonia (0,7 %), Acinetobacter iwoffii (0,7 %), Vibrio vulnificus (0,7 %), Shewanella putrefaciens (0,7 %), Proteus mirabilis (0,7 %) et Proteus vulgaris (0,7 %). Il y avait 66,7 % de similarité entre le profil bactérien des litières et celui des fèces ; quelques souches supplémentaires ont été identifiées dans les litières. Aucune variation (p = 0,64) n’a été observée dans le nombre de types de bactéries isolés provenant d’échantillons de fèces et de litières. Cependant, le nombre de types de bactéries isolés à partir des matières fécales a été significativement différent (p = 0,002) entre les deux zones étudiées. Les résultats suggèrent qu’il existe un risque potentiel de transmission bactérienne à grande échelle au sein des populations de volailles, et aux humains qui y sont en contact étroit.

scholarly journals Molecular and Functional Characterization of a Unique Sucrose Hydrolase from Xanthomonas axonopodis pv. glycines

2004 ◽  
Vol 186 (2) ◽  
pp. 411-418 ◽  
Author(s):  
Hong-Suk Kim ◽  
Hyoung-Joon Park ◽  
Sunggi Heu ◽  
Jin Jung
Keyword(s):  
Insertional Mutagenesis ◽  
Catalytic Mechanism ◽  
Genomic Library ◽  
Functional Characterization ◽  
Amino Acid Sequences ◽  
Protein Coding ◽  
Reading Frame ◽  
Xanthomonas Axonopodis ◽  
Sucrose Induction

ABSTRACT A novel sucrose hydrolase (SUH) from Xanthomonas axonopodis pv. glycines, a causative agent of bacterial pustule disease on soybeans, was studied at the functional and molecular levels. SUH was shown to act rather specifically on sucrose (Km = 2.5 mM) but not on sucrose-6-phosphate. Protein analysis of purified SUH revealed that, in this monomeric enzyme with an estimated molecular mass of 70,223 ± 12 Da, amino acid sequences determined for several segments have corresponding nucleotide sequences in XAC3490, a protein-coding gene found in the genome of X. axonopodis pv. citri. Based on this information, the SUH gene, consisting of an open reading frame of 1,935 bp, was cloned by screening a genomic library of X. axonopodis pv. glycines 8ra. Database searches and sequence comparison revealed that SUH has significant homology to some family 13 enzymes, with all of the crucial invariant residues involved in the catalytic mechanism conserved, but it shows no similarity to known invertases belonging to family 32. suh expression in X. axonopodis pv. glycines requires sucrose induction, and insertional mutagenesis resulted in an absence of sucrose-inducible sucrose hydrolase activity in crude protein extracts and a sucrose-negative phenotype. Recombinant SUH, overproduced in Escherichia coli and purified, was shown to have the same enzymatic characteristics in terms of kinetic parameters.

scholarly journals Cloning and Characterization of a Cryptic Haloacid Dehalogenase from Burkholderia cepacia MBA4

1999 ◽  
Vol 181 (19) ◽  
pp. 6003-6009 ◽  
Author(s):  
Jimmy S. H. Tsang ◽  
Laiju Sam
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Burkholderia Cepacia ◽  
Amino Acid Sequences ◽  
Predicted Amino Acid Sequence ◽  
Haloacid Dehalogenase ◽  
Reading Frame ◽  
Relative Molecular Weight

ABSTRACT Burkholderia cepacia MBA4 has been shown to produce a single dehalogenase batch culture. Moreover, other cryptic dehalogenases were also detected when the cells were grown in continuous culture. In this paper, we report the cloning and characterization of one of the cryptic dehalogenases in MBA4. This cryptic haloacid dehalogenase, designated Chd1, was expressed constitutively in Escherichia coli. This recombinant Chd1 had a relative molecular weight of 58,000 and existed predominantly as a dimer. The subunits had a relative molecular weight of 27,000. Chd1 exhibited isomer specificity, being active towards thel-isomer of 2-monochloropropionic acid only. The structural gene, chd1, was isolated on a 1.7-kb PstI fragment. This fragment contains a functional promoter, because expression of chd1 in E. coli is orientation independent. The nucleotide sequence of this fragment was determined and characterized. An open reading frame of 840 bp encoding a putative peptide of 280 amino acids was identified. This corresponds closely with the size of the subunit. The nucleotide sequence of chd1 did not show any homology with those of other dehalogenase genes. Comparison of the predicted amino acid sequence, however, shows significant homology, ranging from 42 to 50%, with the amino acid sequences of many other dehalogenases. Chd1 is unusual in having a long leader sequence, a property of periplasmic enzymes.

Molecular Characterization of the GenespcaG and pcaH, Encoding Protocatechuate 3,4-Dioxygenase, Which Are Essential for Vanillin Catabolism inPseudomonas sp. Strain HR199

1999 ◽  
Vol 65 (3) ◽  
pp. 951-960 ◽  
Author(s):  
Jörg Overhage ◽  
Andreas U. Kresse ◽  
Horst Priefert ◽  
Horst Sommer ◽  
Gerhard Krammer ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Sole Carbon Source ◽  
Genomic Library ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Regulatory Proteins ◽  
Β Subunit ◽  
Catabolic Pathway ◽  
Reading Frames

ABSTRACT Pseudomonas sp. strain HR199 is able to utilize eugenol (4-allyl-2-methoxyphenol), vanillin (4-hydroxy-3-methoxybenzaldehyde), or protocatechuate as the sole carbon source for growth. Mutants of this strain which were impaired in the catabolism of vanillin but retained the ability to utilize eugenol or protocatechuate were obtained after nitrosoguanidine mutagenesis. One mutant (SK6169) was used as recipient of a Pseudomonas sp. strain HR199 genomic library in cosmid pVK100, and phenotypic complementation was achieved with a 5.8-kbp EcoRI fragment (E58). The amino acid sequences deduced from two corresponding open reading frames (ORF) identified on E58 revealed high degrees of homology to pcaGand pcaH, encoding the two subunits of protocatechuate 3,4-dioxygenase. Three additional ORF most probably encoded a 4-hydroxybenzoate 3-hydroxylase (PobA) and two putative regulatory proteins, which exhibited homology to PcaQ of Agrobacterium tumefaciens and PobR of Pseudomonas aeruginosa, respectively. Since mutant SK6169 was also complemented by a subfragment of E58 that harbored only pcaH, this mutant was most probably lacking a functional β subunit of the protocatechuate 3,4-dioxygenase. Since this mutant was still able to grow on protocatechuate and lacked protocatechuate 4,5-dioxygenase and protocatechuate 2,3-dioxygenase, the degradation had to be catalyzed by different enzymes. Two other mutants (SK6184 and SK6190), which were also impaired in the catabolism of vanillin, were not complemented by fragment E58. Since these mutants accumulated 3-carboxy muconolactone during cultivation on eugenol, they most probably exhibited a defect in a step of the catabolic pathway following the orthocleavage. Moreover, in these mutants cyclization of 3-carboxymuconic acid seems to occur by a syn absolute stereochemical course, which is normally only observed forcis,cis-muconate lactonization in pseudomonads. In conclusion, vanillin is degraded through theortho-cleavage pathway in Pseudomonas sp. strain HR199 whereas protocatechuate could also be metabolized via a different pathway in the mutants.

scholarly journals Aerobic Degradation of Dinitrotoluenes and Pathway for Bacterial Degradation of 2,6-Dinitrotoluene

2000 ◽  
Vol 66 (5) ◽  
pp. 2139-2147 ◽  
Author(s):  
Shirley F. Nishino ◽  
George C. Paoli ◽  
Jim C. Spain
Keyword(s):  
Burkholderia Cepacia ◽  
Dienoic Acid ◽  
Sole Source ◽  
Bacterial Degradation ◽  
Ring Cleavage ◽  
Bacterial Strains ◽  
Carbon And Nitrogen ◽  
Isolation And Characterization ◽  
Oxidative Pathway

ABSTRACT An oxidative pathway for the mineralization of 2,4-dinitrotoluene (2,4-DNT) by Burkholderia sp. strain DNT has been reported previously. We report here the isolation of additional strains with the ability to mineralize 2,4-DNT by the same pathway and the isolation and characterization of bacterial strains that mineralize 2,6-dinitrotoluene (2,6-DNT) by a different pathway.Burkholderia cepacia strain JS850 andHydrogenophaga palleronii strain JS863 grew on 2,6-DNT as the sole source of carbon and nitrogen. The initial steps in the pathway for degradation of 2,6-DNT were determined by simultaneous induction, enzyme assays, and identification of metabolites through mass spectroscopy and nuclear magnetic resonance. 2,6-DNT was converted to 3-methyl-4-nitrocatechol by a dioxygenation reaction accompanied by the release of nitrite. 3-Methyl-4-nitrocatechol was the substrate for extradiol ring cleavage yielding 2-hydroxy-5-nitro-6-oxohepta-2,4-dienoic acid, which was converted to 2-hydroxy-5-nitropenta-2,4-dienoic acid. 2,4-DNT-degrading strains also converted 2,6-DNT to 3-methyl-4-nitrocatechol but did not metabolize the 3-methyl-4-nitrocatechol. Although 2,6-DNT prevented the degradation of 2,4-DNT by 2,4-DNT-degrading strains, the effect was not the result of inhibition of 2,4-DNT dioxygenase by 2,6-DNT or of 4-methyl-5-nitrocatechol monooxygenase by 3-methyl-4-nitrocatechol.

Characterization of a cDNA for Rhesus Monkey Protein C Inhibitor – Evidence for N-Terminal Involvement in Heparin Stimulation

1995 ◽  
Vol 74 (04) ◽  
pp. 1079-1087 ◽  
Author(s):  
Klaus-P Radtke ◽  
José A Fernández ◽  
Bruno O Villoutreix ◽  
Judith S Greengard ◽  
John H Griffin
Keyword(s):  
Rhesus Monkey ◽  
Protein C ◽  
Activated Protein C ◽  
Pcr Amplification ◽  
Amino Acid Sequences ◽  
Heparin Binding ◽  
Reactive Center ◽  
Protein C Inhibitor ◽  
Polymerase Chain

SummarycDNAs for protein C inhibitor (PCI) were cloned from human and rhesus monkey 1 liver RNAs by reverse transcription and polymerase chain reaction (PCR) amplification. Sequencing showed that rhesus monkey and human PCI cDNAs were 93% identical. Predicted amino acid sequences differed at 26 of 387 residues. Pour of these differences (T352M, N359S, R362K, L3631) were in the reactive center loop that is important for inhibitory specificity, and two were in the N-terminal helix (M8T, E13K) that is implicated in glycosaminoglycan binding. PCI in human or rhesus monkey plasma showed comparable inhibitory activity towards human activated protein C in the presence of 10 U/ml heparin. However, maximal acceleration of the inhibition of activated protein C required 5-fold lower heparin concentration for rhesus monkey than for human plasma, consistent with the interpretation that the additional positive charge (E13K) in a putative-heparin binding region increased the affinity for heparin.

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