scholarly journals A Novel p-Nitrophenol Degradation Gene Cluster from a Gram-Positive Bacterium, Rhodococcus opacus SAO101

2004 ◽  
Vol 186 (15) ◽  
pp. 4894-4902 ◽  
Author(s):  
Wataru Kitagawa ◽  
Nobutada Kimura ◽  
Yoichi Kamagata
Keyword(s):  
Gene Cluster ◽  
Ralstonia Eutropha ◽  
Amino Acid Sequences ◽  
Expression Vectors ◽  
Rhodococcus Opacus ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Crude Cell Extract ◽  
Cell Extracts ◽  
Degrading Bacteria

ABSTRACT p-Nitrophenol (4-NP) is recognized as an environmental contaminant; it is used primarily for manufacturing medicines and pesticides. To date, several 4-NP-degrading bacteria have been isolated; however, the genetic information remains very limited. In this study, a novel 4-NP degradation gene cluster from a gram-positive bacterium, Rhodococcus opacus SAO101, was identified and characterized. The deduced amino acid sequences of npcB, npcA, and npcC showed identity with phenol 2-hydroxylase component B (reductase, PheA2) of Geobacillus thermoglucosidasius A7 (32%), with 2,4,6-trichlorophenol monooxygenase (TcpA) of Ralstonia eutropha JMP134 (44%), and with hydroxyquinol 1,2-dioxygenase (ORF2) of Arthrobacter sp. strain BA-5-17 (76%), respectively. The npcB, npcA, and npcC genes were cloned into pET-17b to construct the respective expression vectors pETnpcB, pETnpcA, and pETnpcC. Conversion of 4-NP was observed when a mixture of crude cell extracts of Escherichia coli containing pETnpcB and pETnpcA was used in the experiment. The mixture converted 4-NP to hydroxyquinol and also converted 4-nitrocatechol (4-NCA) to hydroxyquinol. Furthermore, the crude cell extract of E. coli containing pETnpcC converted hydroxyquinol to maleylacetate. These results suggested that npcB and npcA encode the two-component 4-NP/4-NCA monooxygenase and that npcC encodes hydroxyquinol 1,2-dioxygenase. The npcA and npcC mutant strains, SDA1 and SDC1, completely lost the ability to grow on 4-NP as the sole carbon source. These results clearly indicated that the cloned npc genes play an essential role in 4-NP mineralization in R. opacus SAO101.

Sequencing and Characterization of the xyl Operon of a Gram-Positive Bacterium, Tetragenococcus halophila

1998 ◽  
Vol 64 (7) ◽  
pp. 2513-2519 ◽  
Author(s):  
Yasuo Takeda ◽  
Kazuma Takase ◽  
Ichiro Yamato ◽  
Keietsu Abe
Keyword(s):  
Consensus Sequence ◽  
Regulatory Gene ◽  
Regulation Of Gene Expression ◽  
Regulatory Region ◽  
Gc Content ◽  
Amino Acid Sequences ◽  
Dependent Manner ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Palindromic Structure

ABSTRACT The xyl operon of a gram-positive bacterium,Tetragenococcus halophila (previously calledPediococcus halophilus), was cloned and sequenced. The DNA was about 7.7 kb long and contained genes for a ribose binding protein and part of a ribose transporter, xylR (a putative regulatory gene), and the xyl operon, along with its regulatory region and transcription termination signal, in this order. The DNA was AT rich, the GC content being 35.8%, consistent with the GC content of this gram-positive bacterium. The xyl operon consisted of three genes, xylA, encoding a xylose isomerase, xylB, encoding a xylulose kinase, andxylE, encoding a xylose transporter, with predicted molecular weights of 49,400, 56,400, and 51,600, respectively. The deduced amino acid sequences of the XylR, XylA, XylB, and XylE proteins were similar to those of the corresponding proteins in other gram-positive and -negative bacteria, the similarities being 37 to 64%. Each polypeptide of XylB and XylE was expressed functionally inEscherichia coli. XylE transported d-xylose in a sodium ion-dependent manner, suggesting that it is the first described xylose/Na+ symporter. The XylR protein contained a consensus sequence for binding catabolites of glucose, such as glucose-6-phosphate, which has been discovered in glucose and fructose kinases in bacteria. Correspondingly, the regulatory region of this operon contained a putative binding site of XylR with a palindromic structure. Furthermore, it contained a consensus sequence, CRE (catabolite-responsive element), for binding CcpA (catabolite control protein A). We speculate that the transcriptional regulation of this operon resembles the regulation of catabolite-repressible operons such as the amy, lev, xyl, andgnt operons in various gram-positive bacteria. We discuss the significance of the regulation of gene expression of this operon inT. halophila.

scholarly journals Rhodococcus opacus expresses the xsc gene to utilize taurine as a carbon source or as a nitrogen source but not as a sulfur source

Microbiology ◽  
2004 ◽  
Vol 150 (6) ◽  
pp. 1859-1867 ◽  
Author(s):  
Karin Denger ◽  
Jürgen Ruff ◽  
David Schleheck ◽  
Alasdair M. Cook
Keyword(s):  
Gene Cluster ◽  
Gene Clusters ◽  
Pcr Primers ◽  
Sole Source ◽  
Amino Acid Sequences ◽  
Rhodococcus Opacus ◽  
Sulfur Source ◽  
Gram Positive Bacteria ◽  
Sulfoacetaldehyde Acetyltransferase ◽  
Different Levels

The Gram-positive bacteria Rhodococcus opacus ISO-5 and Rhodococcus sp. RHA1 utilized taurine (2-aminoethanesulfonate) as the sole source of carbon or of nitrogen or of sulfur for growth. Different gene clusters and enzymes were active under these different metabolic situations. Under carbon- or nitrogen-limited conditions three enzymes were induced, though to different levels: taurine-pyruvate aminotransferase (Tpa), alanine dehydrogenase (Ald) and sulfoacetaldehyde acetyltransferase (Xsc). The specific activities of these enzymes in R. opacus ISO-5 were sufficient to explain the growth rates under the different conditions. These three enzymes were purified and characterized, and the nature of each reaction was confirmed. Analyses of the genome of Rhodococcus sp. RHA1 revealed a gene cluster, tauR-ald-tpa, putatively encoding regulation and oxidation of taurine, located 20 kbp from the xsc gene and separate from two candidate phosphotransacetylase (pta) genes, as well as many candidate ABC transporters (tauBC). PCR primers allowed the amplification and sequencing of the tauR-ald-tpa gene cluster and the xsc gene in R. opacus ISO-5. The N-terminal sequences of the three tested proteins matched the derived amino acid sequences of the corresponding genes. The sequences of the four genes found in each Rhodococcus strain shared high degrees of identity (>95 % identical positions). RT-PCR studies proved transcription of the xsc gene when taurine was the source of carbon or of nitrogen. Under sulfur-limited conditions no xsc mRNA was generated and no Xsc was detected. Taurine dioxygenase (TauD), the enzyme catalysing the anticipated desulfonative reaction when taurine sulfur is assimilated, was presumed to be present because oxygen-dependent taurine disappearance was demonstrated with taurine-grown cells only. A putative tauD gene (with three other candidates) was detected in strain ISO-5. Regulation of the different forms of metabolism of taurine remains to be elucidated.

Identification of a gene cluster encoding an arginine ATP-binding-cassette transporter in the genome of the thermophilic Gram-positive bacterium Geobacillus stearothermophilus strain DSMZ 13240

Microbiology ◽  
2005 ◽  
Vol 151 (3) ◽  
pp. 835-840 ◽  
Author(s):  
Rebecca Fleischer ◽  
Antje Wengner ◽  
Frank Scheffel ◽  
Heidi Landmesser ◽  
Erwin Schneider
Keyword(s):  
Amino Acids ◽  
Gene Cluster ◽  
Abc Transporter ◽  
Atp Hydrolysis ◽  
Atp Binding ◽  
Geobacillus Stearothermophilus ◽  
Positive Bacterium ◽  
Gram Positive ◽  
High Affinity ◽  
Atp Binding Cassette

A single gene cluster encoding components of a putative ATP-binding cassette (ABC) transporter for basic amino acids was identified in the incomplete genome sequence of the thermophilic Gram-positive bacterium Geobacillus stearothermophilus by blast searches. The cluster comprises three genes, and these were amplified from chromosomal DNA of G. stearothermophilus, ligated into plasmid vectors and expressed in Escherichia coli. The purified solute-binding protein (designated ArtJ) was demonstrated to bind l-arginine with high affinity (K d=0·39±0·06 μM). Competition experiments revealed only partial inhibition by excess l-lysine (38 %) and l-ornithine (46 %), while no inhibition was observed with l-histidine or other amino acids tested. The membrane-associated transport complex, composed of a permease (designated ArtM) and an ATPase component (designated ArtP), was solubilized from E. coli membranes by decanoylsucrose and purified by metal-affinity chromatography. The ArtMP complex, when incorporated into liposomes formed from a crude extract of G. stearothermophilus lipids, displayed ATPase activity in the presence of ArtJ only. Addition of l-arginine further stimulated the activity twofold. ATP hydrolysis was optimal at 60 °C and sensitive to the specific inhibitor vanadate. Analysis of kinetic parameters revealed a maximal velocity of ATP hydrolysis of 0·71 μmol Pi min−1 (mg protein)−1 and a K m (ATP) of 1·59 mM. Together, these results identify the ArtJMP complex as a high-affinity arginine ABC transporter.

scholarly journals Identification and Heterologous Expression of Genes Involved in Anaerobic Dissimilatory Phosphite Oxidation by Desulfotignum phosphitoxidans

2010 ◽  
Vol 192 (19) ◽  
pp. 5237-5244 ◽  
Author(s):  
Diliana Dancheva Simeonova ◽  
Marlena Marie Wilson ◽  
William W. Metcalf ◽  
Bernhard Schink
Keyword(s):  
Sequence Analysis ◽  
Gene Cluster ◽  
Electron Donor ◽  
Genomic Library ◽  
Amino Acid Sequences ◽  
Strictly Anaerobic ◽  
Oxidation Activity ◽  
Cell Extracts ◽  
Expression Of Genes ◽  
Phosphorus Source

ABSTRACT Desulfotignum phosphitoxidans is a strictly anaerobic, Gram-negative bacterium that utilizes phosphite as the sole electron source for homoacetogenic CO2 reduction or sulfate reduction. A genomic library of D. phosphitoxidans, constructed using the fosmid vector pJK050, was screened for clones harboring the genes involved in phosphite oxidation via PCR using primers developed based on the amino acid sequences of phosphite-induced proteins. Sequence analysis of two positive clones revealed a putative operon of seven genes predicted to be involved in phosphite oxidation. Four of these genes (ptxD-ptdFCG) were cloned and heterologously expressed in Desulfotignum balticum, a related strain that cannot use phosphite as either an electron donor or as a phosphorus source. The ptxD-ptdFCG gene cluster was sufficient to confer phosphite uptake and oxidation ability to the D. balticum host strain but did not allow use of phosphite as an electron donor for chemolithotrophic growth. Phosphite oxidation activity was measured in cell extracts of D. balticum transconjugants, suggesting that all genes required for phosphite oxidation were cloned. Genes of the phosphite gene cluster were assigned putative functions on the basis of sequence analysis and enzyme assays.

scholarly journals A Pathway Closely Related to the d-Tagatose Pathway of Gram-Negative Enterobacteria Identified in the Gram-Positive Bacterium Bacillus licheniformis

2013 ◽  
Vol 79 (11) ◽  
pp. 3511-3515 ◽  
Author(s):  
Edwige Van der Heiden ◽  
Michaël Delmarcelle ◽  
Sarah Lebrun ◽  
Régine Freichels ◽  
Alain Brans ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Gene Cluster ◽  
Bacillus Licheniformis ◽  
Klebsiella Oxytoca ◽  
Negative Bacterium ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type ◽  
Bacterium Bacillus

ABSTRACTWe report the first identification of a gene cluster involved ind-tagatose catabolism inBacillus licheniformis. The pathway is closely related to thed-tagatose pathway of the Gram-negative bacteriumKlebsiella oxytoca, in contrast to thed-tagatose 6-phosphate pathway described in the Gram-positive bacteriumStaphylococcus aureus.

scholarly journals Regulation of the Alkane Hydroxylase CYP153 Gene in a Gram-Positive Alkane-Degrading Bacterium, Dietzia sp. Strain DQ12-45-1b

2015 ◽  
Vol 82 (2) ◽  
pp. 608-619 ◽  
Author(s):  
Jie-Liang Liang ◽  
Jing-Hong JiangYang ◽  
Yong Nie ◽  
Xiao-Lei Wu
Keyword(s):  
Gene Cluster ◽  
Mutant Strain ◽  
Gene Transcription ◽  
Gene Promoter ◽  
Gene Clusters ◽  
Gram Positive ◽  
Content Type ◽  
Degrading Bacterium ◽  
Degrading Bacteria ◽  
Arac Family

ABSTRACTCYP153, one of the most common medium-chainn-alkane hydroxylases belonging to the cytochrome P450 superfamily, is widely expressed inn-alkane-degrading bacteria. CYP153 is also thought to cooperate with AlkB in degrading variousn-alkanes. However, the mechanisms regulating the expression of the protein remain largely unknown. In this paper, we studied CYP153 gene transcription regulation by the potential AraC family regulator (CypR) located upstream of the CYP153 gene cluster in a broad-spectrumn-alkane-degrading Gram-positive bacterium,Dietziasp. strain DQ12-45-1b. We first identified the transcriptional start site and the promoter of the CYP153 gene cluster. Sequence alignment of upstream regions of CYP153 gene clusters revealed high conservation in the −10 and −35 regions inActinobacteria. Further analysis of the β-galactosidase activity in the CYP153 gene promoter-lacZfusion cell indicated that the CYP153 gene promoter was induced byn-alkanes comprised of 8 to 14 carbon atoms, but not by derived decanol and decanic acid. Moreover, we constructed acypRmutant strain and found that the CYP153 gene promoter activities and CYP153 gene transcriptional levels in the mutant strain were depressed compared with those in the wild-type strain in the presence ofn-alkanes, suggesting that CypR served as an activator for the CYP153 gene promoter. By comparing CYP153 gene arrangements inActinobacteriaandProteobacteria, we found that the AraC family regulator is ubiquitously located upstream of the CYP153 gene, suggesting its universal regulatory role in CYP153 gene transcription. We further hypothesize that the observed mode of CYP153 gene regulation is shared by manyActinobacteria.

scholarly journals Novel 2,4-Dichlorophenoxyacetic Acid Degradation Genes from Oligotrophic Bradyrhizobium sp. Strain HW13 Isolated from a Pristine Environment

2002 ◽  
Vol 184 (2) ◽  
pp. 509-518 ◽  
Author(s):  
Wataru Kitagawa ◽  
Sachiko Takami ◽  
Keisuke Miyauchi ◽  
Eiji Masai ◽  
Yoichi Kamagata ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Burkholderia Cepacia ◽  
Ralstonia Eutropha ◽  
Rhodococcus Erythropolis ◽  
Amino Acid Sequences ◽  
Dichlorophenoxyacetic Acid ◽  
Phenol Derivatives ◽  
Degrading Bacteria ◽  
2,4 Dichlorophenoxyacetic Acid

ABSTRACT The tfd genes of Ralstonia eutropha JMP134 are the only well-characterized set of genes responsible for 2,4-dichlorophenoxyacetic acid (2,4-D) degradation among 2,4-D-degrading bacteria. A new family of 2,4-D degradation genes, cadRABKC, was cloned and characterized from Bradyrhizobium sp. strain HW13, a strain that was isolated from a buried Hawaiian soil that has never experienced anthropogenic chemicals. The cadR gene was inferred to encode an AraC/XylS type of transcriptional regulator from its deduced amino acid sequence. The cadABC genes were predicted to encode 2,4-D oxygenase subunits from their deduced amino acid sequences that showed 46, 44, and 37% identities with the TftA and TftB subunits of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) oxygenase of Burkholderia cepacia AC1100 and with a putative ferredoxin, ThcC, of Rhodococcus erythropolis NI86/21, respectively. They are thoroughly different from the 2,4-D dioxygenase gene, tfdA, of R. eutropha JMP134. The cadK gene was presumed to encode a 2,4-D transport protein from its deduced amino acid sequence that showed 60% identity with the 2,4-D transporter, TfdK, of strain JMP134. Sinorhizobium meliloti Rm1021 cells containing cadRABKC transformed several phenoxyacetic acids, including 2,4-D and 2,4,5-T, to corresponding phenol derivatives. Frameshift mutations indicated that each of the cadRABC genes was essential for 2,4-D conversion in strain Rm1021 but that cadK was not. Five 2,4-D degraders, including Bradyrhizobium and Sphingomonas strains, were found to have cadA gene homologs, suggesting that these 2,4-D degraders share 2,4-D degradation genes similar to those of strain HW13 cadABC.

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