Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 73
Filter
Add more filters










Publication year range
1.
Gan To Kagaku Ryoho ; 42(12): 1718-9, 2015 Nov.
Article in Japanese | MEDLINE | ID: mdl-26805149

ABSTRACT

We report a 3-year survival case of cholangiocarcinoma treated with S-1 monotherapy despite positive margins after palliative bile duct resection. A 66 year-old man visited our hospital for jaundice. Because a smooth round defect was observed in the middle bile duct on ERCP, an impacted stone was suspected. Bile duct incision was performed, but the suspected stone was a tumor that was pathologically diagnosed as cholangiocarcinoma. Although pancreaticoduodenectomy was recommended, the patient decided to undergo palliative bile duct resection. Postoperative pathological examination showed moderately tubular adenocarcinoma with lymph node metastasis. The surgical margins of the hepatic side, duodenal side, and exfoliated surface were all positive. Subsequently, the patient chose to undergo S-1 monotherapy for maintaining his lifestyle. S-1 was orally administered at 100mg/day for 4 weeks, followed by 2 weeks of rest. He has continued S-1 monotherapy and survived for 3 years without evidence of recurrence.


Subject(s)
Antimetabolites, Antineoplastic/therapeutic use , Bile Duct Neoplasms/drug therapy , Bile Duct Neoplasms/surgery , Bile Ducts, Extrahepatic/surgery , Cholangiocarcinoma/drug therapy , Cholangiocarcinoma/surgery , Oxonic Acid/therapeutic use , Tegafur/therapeutic use , Aged , Bile Duct Neoplasms/pathology , Bile Ducts, Extrahepatic/pathology , Drug Combinations , Humans , Male , Pancreaticoduodenectomy , Prognosis
2.
Biosci Biotechnol Biochem ; 78(6): 1094-101, 2014.
Article in English | MEDLINE | ID: mdl-25036142

ABSTRACT

Strain OC11 was isolated from seawater sampled at the coast of Chiba, Japan, in artificial seawater medium with carbazole (CAR) as the sole carbon source. Its 16S ribosomal RNA gene sequence suggested that strain OC11 belongs to the genus Janibacter. The CAR-degradation genes (car genes) of strain OC11 were PCR amplified, using degenerate primers designed based on the car gene sequences of other CAR-degrading bacteria. Complete nucleotide sequences encoding six complete open reading frames were determined, and the first known ferredoxin reductase gene (carAd) was found from a CAR-degrading bacterium isolated from the marine environment. An experiment using a mutant strain suggested that the car genes of strain OC11 are functional in CAR degradation. Southern hybridization indicated that strain OC11 had one car gene cluster in vivo. RT-PCR revealed that transcription of carOC11 constitutes an operon.


Subject(s)
Actinomycetales/genetics , Actinomycetales/metabolism , Aquatic Organisms/genetics , Aquatic Organisms/metabolism , Carbazoles/metabolism , Actinomycetales/isolation & purification , Amino Acid Sequence , Aquatic Organisms/isolation & purification , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA, Bacterial/genetics , Molecular Sequence Data , Sequence Analysis, DNA , Transcription, Genetic
3.
Appl Microbiol Biotechnol ; 97(10): 4485-98, 2013 May.
Article in English | MEDLINE | ID: mdl-23179619

ABSTRACT

A dibenzofuran (DF)-degrader Terrabacter sp. strain DBF63 harbors the dbfA and dbfBC genes for DF degradation and the fln-dbfA, pht, and pca gene clusters for the utilization of fluorene (FN) as a sole carbon source. From this strain, dfdA1, the gene encoding the second DF dioxygenase was detected using degenerate polymerase chain reaction (PCR) and the dfdA1A2A3A4 genes were cloned from a cosmid library of the DBF63 genome. Nucleotide sequencing revealed that the dfdA genes showed considerably high identities with those of other actinobacteria, such as Terrabacter sp. strain YK3 and Rhodococcus sp. strain HA01. In the neighboring region of the dfdA genes, as many as 11 homologs for transposase and integrase genes and the putative extradiol dioxygenase gene disrupted by an insertion sequence (dfdB::ISTesp2) were found, suggesting that repeated gene rearrangement had occurred. Quantitative reverse transcription-PCR analysis revealed that dfdA1 was expressed primarily in the DF-fed strain, whereas dbfA1 was expressed in the FN-cultured strain, apparently indicating that the dfdA genes are induced by DF for the initial hydroxylation of DF in strain DBF63. Furthermore, two polycistronic gene cassettes consisting of either dfdA or dbfA together with the dbfBC gene were constructed and expressed heterologously in Streptomyces lividans, degrading DF to salicylate. Furthermore, the expressed DfdA dioxygenase degraded dibenzo-p-dioxin, carbazole, dibenzothiophene, anthracene, phenanthrene, and biphenyl, thereby exhibiting a broader substrate range than that of the DbfA dioxygenase.


Subject(s)
Actinomycetales/genetics , Dioxygenases/genetics , Genes, Bacterial , Streptomyces lividans/genetics , Chromatography, High Pressure Liquid , DNA, Bacterial/genetics , Reverse Transcriptase Polymerase Chain Reaction
4.
Biosci Biotechnol Biochem ; 76(3): 608-12, 2012.
Article in English | MEDLINE | ID: mdl-22451411

ABSTRACT

The EpsF and EpsG of the methanol-assimilating bacterium Methylobacillus sp. 12S are involved in the synthesis of a high molecular weight exopolysaccharide, methanolan. These proteins share homology with chain-length determiners in other polysaccharide-producing bacteria. The N- and C-termini of EpsF were found to locate to the cytoplasm, and EpsF was predicted to have two transmembrane regions. EpsG showed both ATPase and autophosphorylation activities.


Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Membrane/metabolism , Methylobacillus/cytology , Methylobacillus/genetics , Polysaccharides/biosynthesis , Polysaccharides/chemistry , Amino Acid Sequence , Bacterial Proteins/chemistry , Methylobacillus/metabolism , Molecular Sequence Data
5.
Biotechnol Lett ; 33(9): 1859-64, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21553289

ABSTRACT

The car genes from a carbazole (CAR)-degrading bacterium, Kordiimonas sp. OC9, were functionally and transcriptionally analysed. The enzymatic activity for the protein coded by carBaBb using pBOC93 (carAaAcBa), pBOC93-2 (carAaAcBb), and pBOC94 (carAaAcBaBb) was confirmed. Resting cells using Escherichia coli harbouring pBOC95 (carAaAcBaBbC) revealed the function of the carC gene product in the conversion of CAR to anthranilic acid by expressing it with CarAaAcBaBb. The pathway of CAR metabolism to anthranilic acid in marine CAR-degraders was elucidated. Transcriptional analysis using RT-PCR revealed that car genes are related to CAR degradation in response to CAR exposure in strain OC9. RT-PCR analysis of the operon structure showed that the car gene cluster of strain OC9 has two distinct operons in one car gene cluster. The localisation of the car gene cluster of strain OC9 was also determined.


Subject(s)
Alphaproteobacteria/enzymology , Alphaproteobacteria/genetics , Carbazoles/metabolism , Metabolic Networks and Pathways/genetics , Escherichia coli/genetics , Gene Expression Profiling , Multigene Family , Operon , Plasmids , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic , ortho-Aminobenzoates/metabolism
6.
Biotechnol Lett ; 32(11): 1725-31, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20652619

ABSTRACT

Carbazole (CAR)-degrading genes (carRAaCBaBb) were isolated from marine CAR-degrading isolate strain OC9 (probably Kordiimonas gwangyangensis) using shotgun cloning experiments and showed 35-65% similarity with previously reported CAR-degrading genes. In addition, a ferredoxin-like gene (carAc) was found downstream of carR, although it was not homologous with any reported ferredoxin components of the CAR 1,9a-dioxygenase (CARDO) system. The carAc-deduced amino acid sequence possessed consensus sequences for chloroplast-type iron-sulfur proteins for binding the [2Fe-2S] cluster. These car genes were arranged in the order of carAcRAaCBaBb, but carRAc and carAaCBaBb genes were the opposite orientation. Escherichia coli JM109 cells harboring pBOC91 (carAa) converted CAR to 2'-aminobiphenyl-2,3-diol at a ratio of 12%, and the transformation ratio of CAR increased from 12 to 100% when carAc was added, indicating that CarAc is the ferredoxin component of the CARDO system in strain OC9. This is the first finding of a chloroplast-type ferredoxin component in a CARDO system. Biotransformation tests with aromatic compounds revealed that the strain OC9 CarAaAc showed activity with polycyclic aromatic hydrocarbons and dioxin compounds and exhibited significant activity for fluorene, unlike previously reported CARDOs.


Subject(s)
Alphaproteobacteria/enzymology , Bacterial Proteins/metabolism , Dioxygenases/metabolism , Ferredoxins/metabolism , Alphaproteobacteria/genetics , Carbazoles/metabolism , Chloroplasts/enzymology , Cloning, Molecular , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Escherichia coli/genetics , Ferredoxins/genetics , Gene Expression , Gene Order , Molecular Sequence Data , Multigene Family , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino Acid
7.
J Biosci Bioeng ; 110(4): 415-8, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20573544

ABSTRACT

This is the first report that Lysobacter fixes nitrogen under free-living conditions, as shown by its ability to grow on nitrogen-free medium and accumulate relatively high amounts of ammonia in the culture broth. Growth of the E4 Lysobacter strain, isolated in a screen for nitrogen-fixing and ammonia-producing bacteria, resulted in higher ammonia accumulation (0.53 mM ammonium ion concentration) in media containing glucose rather than other tested carbon sources. The optimum glucose concentration was 0.30% at an initial medium pH of 7.0 and incubation temperature of 30°C. From time-course experiments, when the glucose in the culture was exhausted, ammonia began to be accumulated, and maximum ammonia accumulation (∼1.60 mM) was reached after 8 days of incubation. Ammonia accumulation by this strain required molybdenum, manganese, and iron.


Subject(s)
Ammonia/metabolism , Lysobacter/metabolism , Nitrogen Fixation , Base Sequence , Carbon/metabolism , Culture Media , DNA Primers , DNA, Ribosomal/genetics , Glucose/metabolism , Hydrogen-Ion Concentration , Lysobacter/genetics , Polymerase Chain Reaction , RNA, Ribosomal, 16S/genetics , Temperature
8.
Curr Microbiol ; 61(1): 50-6, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20039169

ABSTRACT

The marine bacterium Neptuniibacter sp. strain CAR-SF utilizes carbazole as its sole carbon and nitrogen sources. Two sets of clustered genes related to carbazole degradation, the upper and lower pathways, were obtained. The marine bacterium genes responsible for the upper carbazole degradation pathway, carAa, carBa, carBb, and carC, encode the terminal oxygenase component of carbazole 1,9a-dioxygenase, the small and large subunits of the meta-cleavage enzyme, and the meta-cleavage compound hydrolase, respectively. The genes involved in the lower degradation pathway encode the anthranilate dioxygenase large and small subunit AntA and AntB, anthranilate dioxygenase reductase AntC, 4-oxalocrotonate tautomerase, and catechol 2,3-dioxygenase. Reverse transcription-polymerase chain reaction confirmed the involvement of the isolated genes in carbazole degradation. Escherichia coli cells transformed with the CarAa of strain CAR-SF required ferredoxin and ferredoxin reductase for biotransformation of carbazole. Although carAc, which encodes the ferredoxin component of carbazole 1,9a-dioxygenase, was not found immediately downstream of carAaBaBbC, the carAc-like gene may be located elsewhere based on Southern hybridization. This is the first report of genes involved in carbazole degradation isolated from a marine bacterium.


Subject(s)
Carbazoles/metabolism , Genes, Bacterial , Oceanospirillaceae/genetics , Oceanospirillaceae/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , Biotransformation , Catechol 2,3-Dioxygenase/genetics , Catechol 2,3-Dioxygenase/metabolism , Dioxygenases/genetics , Dioxygenases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Ferredoxins/genetics , Ferredoxins/metabolism , Hydrolases/genetics , Hydrolases/metabolism , Isomerases/genetics , Isomerases/metabolism , Metabolic Networks and Pathways/genetics , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , Multigene Family , Oceanospirillaceae/enzymology , Open Reading Frames , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic , Transformation, Bacterial
9.
Biosci Biotechnol Biochem ; 73(7): 1647-52, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19584533

ABSTRACT

Desulfotignum balticum utilizes benzoate coupled to sulfate reduction. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis was conducted to detect proteins that increased more after growth on benzoate than on butyrate. A comparison of proteins on 2D gels showed that at least six proteins were expressed. The N-terminal sequences of three proteins exhibited significant identities with the alpha and beta subunits of electron transfer flavoprotein (ETF) from anaerobic aromatic-degraders. By sequence analysis of the fosmid clone insert (37,590 bp) containing the genes encoding the ETF subunits, we identified three genes, whose deduced amino acid sequences showed 58%, 74%, and 62% identity with those of Gmet_2267 (Fe-S oxidoreductase), Gmet_2266 (ETF beta subunit), and Gmet_2265 (ETF alpha subunit) respectively, which exist within the 300-kb genomic island of aromatic-degradation genes from Geobacter metallireducens GS-15. The genes encoding ETF subunits found in this study were upregulated in benzoate utilization.


Subject(s)
Benzoates/pharmacology , Deltaproteobacteria/enzymology , Deltaproteobacteria/genetics , Electron-Transferring Flavoproteins/genetics , Electron-Transferring Flavoproteins/metabolism , Up-Regulation/drug effects , Amino Acid Sequence , Carbon/chemistry , Cloning, Molecular , DNA, Bacterial/genetics , Deltaproteobacteria/drug effects , Deltaproteobacteria/growth & development , Electrophoresis, Gel, Two-Dimensional , Gene Library , Genes, Bacterial/genetics , Molecular Sequence Data , Oxidation-Reduction , Peptides/chemistry , Peptides/metabolism , Protein Subunits/genetics , Protein Subunits/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , Sequence Homology, Nucleic Acid
10.
J Gen Appl Microbiol ; 55(2): 81-6, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19436124

ABSTRACT

Nine thermophilic denitrification bacteria were isolated from field soil, mud, and spa samples. The alignment of 16S rDNA showed that all were identical to the genus Geobacillus. Two of the bacteria produced N2O and N2 gas and the other seven strains produced N2 gas from nitrate. We examined the growth substrates for Geobacillus TDN01 and determined that sodium succinate, pyruvate, formate, acetate, glycerol, glucose, sucrose, and cellobiose well supported growth of the isolate. Growth occurred under the following concentration of NO3- and phosphate: 10-60 mmol/L, and 0.1-50 mmol/L, respectively. Thermophilic TDN01 grown on sodium succinate accumulated nitrite. A time course of denitrification by Geobacillus TDN01 in a jar fermentor revealed that maintaining a pH of around 7 is important for denitrification without accumulating NO2. The NO3- and NO2- consumption ratios of Geobacillus were 44-75 and 9-41 times higher, respectively, than those of Pseudomonas stutzeri JCM 5965T.


Subject(s)
Bacillaceae/classification , Bacillaceae/metabolism , Hot Temperature , Nitrates/metabolism , Soil Microbiology , Bacillaceae/genetics , Bacillaceae/isolation & purification , Bacterial Typing Techniques , DNA, Bacterial/genetics , Nitrous Oxide/metabolism , RNA, Ribosomal, 16S/genetics , Time Factors
11.
J Gen Appl Microbiol ; 55(2): 87-92, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19436125

ABSTRACT

The thermophilic denitrifying bacterium Geobacillus sp. strain TDN01 was examined to determine the effects of nitrogen and carbon sources and nitrate and nitrite concentrations on denitrification in a batch culture. The specific nitrate removal rate was 12 times higher with ammonia than without ammonia. The consumption rates of nitrate and succinate were proportional. Furthermore, the growth rates with 120 and 150 mM nitrate were only slightly lower than those with 60 mM and did not cause notable growth inhibition. Denitrification ability in continuous culture was analyzed based on the data for batch culture. The maximum specific growth rate micromax and substrate saturation constant KS in the Monod equation were determined by gradually changing the dilution rate. The maximum denitrification rate was six times higher than that of mesophilic bacteria.


Subject(s)
Bacillus/growth & development , Bacillus/metabolism , Nitrates/metabolism , Ammonia/metabolism , Cell Culture Techniques , Culture Media/chemistry , Kinetics , Succinic Acid/metabolism , Time Factors
12.
Curr Microbiol ; 59(2): 154-9, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19452212

ABSTRACT

The novel carbazole (CAR)-degrading bacterium Lysobacter sp. strain OC7 has been isolated from seawater and can also utilize naphthalene and phenanthrene as its sole carbon and energy source. The CAR-degradative gene cluster was isolated and encoded five complete open reading frames (ORFs) and two truncated ORFs. Among them, four ORFs showed 40-50% similarity with previously reported CAR-degradative genes. Ferredoxin (carAc) and ferredoxin reductase (carAd) genes, which are necessary for the CAR 1,9a-dioxygenase system, were not found in this car gene cluster. The car (OC7) gene transcripts were strongly detected when CAR was provided. However, these transcripts were also detected when naphthalene was provided. The resting cell reaction with Escherichia coli revealed that CarAa(OC7) can use CarAc and CarAd of Pseudomonas resinovorans CA10 as ferredoxin and ferredoxin reductase, respectively, and converted CAR to 2'-aminobiphenyl-2,3-diol. In 13 marine CAR-degrading isolates, only Caulobacter sp. strain OC6 hybridized with the car (OC7) gene cluster probe. This is the first report showing CAR-degradative genes from the genus Lysobacter.


Subject(s)
Carbazoles/metabolism , Lysobacter/classification , Lysobacter/metabolism , Multigene Family , Naphthalenes/metabolism , Phenanthrenes/metabolism , Seawater/microbiology , Caulobacter/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Escherichia coli/genetics , Gene Expression Profiling , Genes, Bacterial , Lysobacter/genetics , Lysobacter/isolation & purification , Metabolic Networks and Pathways/genetics , Molecular Sequence Data , Open Reading Frames , Phylogeny , Pseudomonas/genetics , Sequence Analysis, DNA
13.
FEMS Microbiol Lett ; 292(2): 203-9, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19187209

ABSTRACT

Eleven carbazole (CAR)-degrading bacterial strains were isolated from seawater collected off the coast of Japan using two different media. Seven isolates were shown to be most closely related to the genera Erythrobacter, Hyphomonas, Sphingosinicella, Caulobacter, and Lysobacter. Meanwhile, strains OC3, OC6S, OC9, and OC11S showed low similarity to known bacteria, the closest relative being Kordiimonas gwangyangensis GW14-5 (90% similarity). Southern hybridization analysis revealed that only five isolates carried car genes similar to those reported in Pseudomonas resinovorans CA10 (car(CA10)) or Sphingomonas sp. strain KA1 (car(KA1)). The isolates were subjected to GC-MS and the results indicated that these strains degrade CAR to anthranilic acid.


Subject(s)
Bacteria/classification , Bacteria/metabolism , Carbazoles/metabolism , Seawater/microbiology , Bacteria/isolation & purification , Bacterial Proteins/genetics , Biotransformation , Blotting, Southern , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Genes, rRNA , Japan , Molecular Sequence Data , Oxidoreductases/genetics , Phylogeny , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Sequence Homology, Nucleic Acid , ortho-Aminobenzoates/metabolism
14.
Biosci Biotechnol Biochem ; 72(12): 3237-48, 2008 Dec.
Article in English | MEDLINE | ID: mdl-19060398

ABSTRACT

Carbazole 1,9a-dioxygenase (CARDO) consists of terminal oxygenase (CARDO-O) and electron transport components. CARDO can catalyze specific oxygenation for various substrates: angular dioxygenation for carbazole and dibenzo-p-dioxin, lateral dioxygenation for anthracene, and monooxygenation for methylene carbon of fluorene and sulfide sulfur of dibenzothiophene. To elucidate the molecular mechanism determining its unique substrate specificity, 17 CARDO-O site-directed mutants at amino acid residues I262, F275, Q282, and F329, which form the substrate-interacting wall around the iron active site by CARDO-O crystal structure, were generated and characterized. F329 replacement dramatically reduced oxygenation activity. However, several mutants produced different products from the wild-type enzyme to a large extent: I262V and Q282Y (1-hydroxycarbazole), F275W (4-hydroxyfluorene), F275A (unidentified cis-dihydrodiol of fluoranthene), and I262A and I262W (monohydroxydibenzothiophenes). These results suggest the possibility that the respective substrates bind to the active sites of CARDO-O mutants in a different orientation from that of the wild-type enzyme.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Dioxygenases/chemistry , Dioxygenases/metabolism , Anthracenes/metabolism , Bacterial Proteins/genetics , Carbazoles/metabolism , Catalytic Domain , Dioxins/metabolism , Dioxygenases/genetics , Escherichia coli/cytology , Escherichia coli/metabolism , Fluorenes/metabolism , Models, Molecular , Mutagenesis, Site-Directed , Mutation , Oxidation-Reduction , Protein Conformation , Substrate Specificity , Thiophenes/metabolism
15.
J Bacteriol ; 190(13): 4521-31, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18456803

ABSTRACT

Pseudomonas putida DS1 is able to utilize dimethyl sulfone as a sulfur source. Expression of the sfnFG operon responsible for dimethyl sulfone oxygenation is directly regulated by a sigma(54)-dependent transcriptional activator, SfnR, which is encoded within the sfnECR operon. We investigated the transcription mechanism for the sulfate starvation-induced expression of these sfn operons. Using an in vivo transcription assay and in vitro DNA-binding experiments, we revealed that SfnR negatively regulates the expression of sfnECR by binding to the downstream region of the transcription start point. Additionally, we demonstrated that a LysR-type transcriptional regulator, CysB, directly activates the expression of sfnECR by binding to its upstream region. CysB is a master regulator that controls the sulfate starvation response of the sfn operons, as is the case for the sulfonate utilization genes of Escherichia coli, although CysB(DS1) appeared to differ from that of E. coli CysB in terms of the effect of O-acetylserine on DNA-binding ability. Furthermore, we investigated what effector molecules repress the expression of sfnFG and sfnECR in vivo by using the disruptants of the sulfate assimilatory genes cysNC and cysI. The measurements of mRNA levels of the sfn operons in these gene disruptants suggested that the expression of sfnFG is repressed by sulfate itself while the expression of sfnECR is repressed by the downstream metabolites in the sulfate assimilatory pathway, such as sulfide and cysteine. These results indicate that SfnR plays a role independent of CysB in the sulfate starvation-induced expression of the sfn operons.


Subject(s)
Bacterial Proteins/metabolism , Pseudomonas putida/metabolism , Sulfates/pharmacology , Trans-Activators/metabolism , Transcription Factors/metabolism , Amino Acid Motifs/genetics , Bacterial Proteins/genetics , Base Sequence , Dimethyl Sulfoxide/metabolism , Electrophoretic Mobility Shift Assay , Gene Expression Regulation, Bacterial/drug effects , Genes, Bacterial/genetics , Models, Biological , Molecular Sequence Data , Operon/genetics , Promoter Regions, Genetic/genetics , Protein Binding , Pseudomonas putida/drug effects , Pseudomonas putida/genetics , RNA Polymerase Sigma 54/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sulfates/metabolism , Sulfones/metabolism , Trans-Activators/genetics , Transcription Factors/genetics , Transcription Initiation Site
16.
Appl Microbiol Biotechnol ; 79(1): 87-95, 2008 May.
Article in English | MEDLINE | ID: mdl-18324396

ABSTRACT

Subtractive hybridization (SH) and random arbitrarily primed PCR (RAP-PCR) were used to detect genes involved in anaerobic benzoate degradation by Desulfotignum balticum. Through SH, we obtained 121 DNA sequences specific for D. balticum but not for D. phosphitoxidans (a non-benzoate-assimilating species). Furthermore, RAP-PCR analysis showed that a 651-bp DNA fragment, having 55% homology with the solute-binding protein of the ABC transporter system in Methanosarcina barkeri, was expressed when D. balticum was grown on benzoate, but not on pyruvate. By shotgun sequencing of the fosmid clone (38,071 bp) containing the DNA fragment, 33 open reading frames (ORFs) and two incomplete ORFs were annotated, and several genes within this region corresponded to the DNA fragments obtained by SH. An 11.3-kb gene cluster (ORF10-17) revealed through reverse transcription-PCR showed homology with the ABC transporter system and TonB-dependent receptors, both of which are presumably involved in the uptake of siderophore/heme/vitamin B(12), and was expressed in response to growth on benzoate.


Subject(s)
Benzoates/metabolism , Deltaproteobacteria/growth & development , Deltaproteobacteria/genetics , Genome, Bacterial , ATP-Binding Cassette Transporters/genetics , Bacterial Proteins/genetics , Base Sequence , Culture Media , DNA, Bacterial/analysis , Genomic Library , Membrane Proteins/genetics , Methyltransferases/genetics , Molecular Sequence Data , Molybdenum/metabolism , Nucleic Acid Hybridization , Open Reading Frames , Polymerase Chain Reaction , Pyruvic Acid/metabolism , Sodium Selenite/metabolism
17.
Microbiology (Reading) ; 153(Pt 9): 3091-3098, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17768252

ABSTRACT

The sigma(54)-dependent transcriptional regulator SfnR is essential for the use of dimethyl sulfone (DMSO(2)) as a sulfur source by Pseudomonas putida DS1. SfnR binds three SfnR-binding sites (sites 1, 2 and 3) within an intergenic region of the divergently transcribed sfnAB and sfnFG gene clusters. The site 1 region, proximal to the sfnF gene, is indispensable for the expression of the sfnFG operon, which encodes components of DMSO(2) monooxygenase. We investigated the transcriptional regulation of the sfnAB operon and possible functions of the sfnA gene. RT-PCR analysis revealed that the sfnAB gene cluster, which is similar to homologues of the acyl-CoA dehydrogenase family, was transcribed as an operon, and its expression was regulated by SfnR under conditions of sulfate starvation. Deletion analyses using lacZ as a reporter demonstrated that the region up to at least -138 bp from the transcription start point of sfnA (containing sites 2 and 3) was necessary for the expression of the sfnAB operon. A growth test of the sfnA-disrupted mutant revealed the possibility that sfnA may be involved in the use of methanethiol as a sulfur source.


Subject(s)
Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Pseudomonas putida/physiology , RNA Polymerase Sigma 54/metabolism , Sulfates/pharmacology , Trans-Activators/metabolism , Transcription, Genetic , Bacterial Proteins/genetics , Base Sequence , Binding Sites/genetics , Gene Deletion , Heat-Shock Response , Molecular Sequence Data , Operon , Pseudomonas putida/genetics , Pseudomonas putida/growth & development , Pseudomonas putida/metabolism , Sulfates/metabolism , Sulfhydryl Compounds/metabolism , Sulfides/metabolism , Trans-Activators/genetics
18.
FEMS Microbiol Lett ; 275(1): 175-81, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17711452

ABSTRACT

Many bacteria living in soil have developed the ability to use a wide variety of organosulfur compounds. Pseudomonas putida strain DS1 is able to utilize dimethyl sulfide as a sulfur source via a series of oxidation reactions that sequentially produce dimethyl sulfoxide, dimethyl sulfone (DMSO2), methanesulfonate, and sulfite. To isolate novel genes involved in DMSO2 utilization, a transposon-based mutagenesis of DS1 was performed. Of c. 10,000 strains containing mini-Tn5 inserts, 11 mutants lacked the ability to utilize DMSO2, and their insertion sites were determined. In addition to the cysNC, cysH, and cysM genes involved in sulfate assimilation, the ptsP gene encoding the phosphoenolpyruvate:sugar phosphotransferase system (PTS) family protein EI(Ntr) was identified, which is necessary for DMSO2 utilization. Using quantitative reverse transcriptase-polymerase chain reaction analysis, it was demonstrated that the expression of the sfn genes, necessary for DMSO2 utilization, was impaired in the ptsP disruptant. To the authors' knowledge, this is the first report of a PTS protein that is involved in bacterial assimilation of organosulfur compounds.


Subject(s)
Bacterial Proteins/physiology , Dimethyl Sulfoxide/metabolism , Genes, Bacterial/physiology , Phosphoenolpyruvate Sugar Phosphotransferase System/physiology , Pseudomonas putida/enzymology , Sulfones/metabolism , Bacterial Proteins/genetics , DNA Transposable Elements/genetics , Mutagenesis, Insertional , Mutation , Phosphoenolpyruvate Sugar Phosphotransferase System/genetics , Pseudomonas putida/genetics
19.
J Bacteriol ; 189(5): 2007-20, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17172338

ABSTRACT

We determined the complete 254,797-bp nucleotide sequence of the plasmid pCAR3, a carbazole-degradative plasmid from Sphingomonas sp. strain KA1. A region of about 65 kb involved in replication and conjugative transfer showed similarity to a region of plasmid pNL1 isolated from the aromatic-degrading Novosphingobium aromaticivorans strain F199. The presence of many insertion sequences, transposons, repeat sequences, and their remnants suggest plasticity of this plasmid in genetic structure. Although pCAR3 is thought to carry clustered genes for conjugative transfer, a filter-mating assay between KA1 and a pCAR3-cured strain (KA1W) was unsuccessful, indicating that pCAR3 might be deficient in conjugative transfer. Several degradative genes were found on pCAR3, including two kinds of carbazole-degradative gene clusters (car-I and car-II), and genes for electron transfer components of initial oxygenase for carbazole (fdxI, fdrI, and fdrII). Putative genes were identified for the degradation of anthranilate (and), catechol (cat), 2-hydroxypenta-2,4-dienoate (carDFE), dibenzofuran/fluorene (dbf/fln), protocatechuate (lig), and phthalate (oph). It appears that pCAR3 may carry clustered genes (car-I, car-II, fdxI, fdrI, fdrII, and, and cat) for the degradation of carbazole into tricarboxylic acid cycle intermediates; KA1W completely lost the ability to grow on carbazole, and the carbazole-degradative genes listed above were all expressed when KA1 was grown on carbazole. Reverse transcription-PCR analysis also revealed that the transcription of car-I, car-II, and cat genes was induced by carbazole or its metabolic intermediate. Southern hybridization analyses with probes prepared from car-I, car-II, repA, parA, traI, and traD genes indicated that several Sphingomonas carbazole degraders have DNA regions similar to parts of pCAR3.


Subject(s)
Carbazoles/metabolism , Plasmids , Sphingomonas/genetics , Base Sequence , Biodegradation, Environmental , Conjugation, Genetic , DNA Transposable Elements , Molecular Sequence Data , Open Reading Frames , Sphingomonas/metabolism
20.
Structure ; 14(12): 1779-89, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17161368

ABSTRACT

Carbazole 1,9a-dioxygenase (CARDO), a member of the Rieske nonheme iron oxygenase system (ROS), consists of a terminal oxygenase (CARDO-O) and electron transfer components (ferredoxin [CARDO-F] and ferredoxin reductase [CARDO-R]). We determined the crystal structures of the nonreduced, reduced, and substrate-bound binary complexes of CARDO-O with its electron donor, CARDO-F, at 1.9, 1.8, and 2.0 A resolutions, respectively. These structures provide the first structure-based interpretation of intercomponent electron transfer between two Rieske [2Fe-2S] clusters of ferredoxin and oxygenase in ROS. Three molecules of CARDO-F bind to the subunit boundary of one CARDO-O trimeric molecule, and specific binding created by electrostatic and hydrophobic interactions with conformational changes suitably aligns the two Rieske clusters for electron transfer. Additionally, conformational changes upon binding carbazole resulted in the closure of a lid over the substrate-binding pocket, thereby seemingly trapping carbazole at the substrate-binding site.


Subject(s)
Bacterial Proteins/chemistry , Dioxygenases/chemistry , Electron Transport Complex III/chemistry , Ferredoxins/chemistry , Iron-Sulfur Proteins/chemistry , Oxygenases/chemistry , Bacterial Proteins/metabolism , Carbazoles/chemistry , Dimerization , Dioxygenases/metabolism , Electrons , Models, Molecular , Molecular Conformation , Oxygen/chemistry , Pseudomonas/enzymology , Reactive Oxygen Species , Spectrophotometry, Ultraviolet , Static Electricity , Substrate Specificity
SELECTION OF CITATIONS
SEARCH DETAIL
...