scholarly journals Identification and Characterization of Genes Encoding a Putative ABC-Type Transporter Essential for Utilization of γ-Hexachlorocyclohexane in Sphingobium japonicum UT26

2007 ◽  
Vol 189 (10) ◽  
pp. 3712-3720 ◽  
Author(s):  
Ryo Endo ◽  
Yoshiyuki Ohtsubo ◽  
Masataka Tsuda ◽  
Yuji Nagata
Keyword(s):  
Abc Transporter ◽  
Sole Source ◽  
Bacterial Cells ◽  
New Genes ◽  
Hydrophobic Compounds ◽  
Dead End ◽  
Genes Encoding ◽  
Degradation Activity ◽  
Mutant Cells

ABSTRACT Sphingobium japonicum UT26 utilizes γ-hexachlorocyclohexane (γ-HCH) as its sole source of carbon and energy. In our previous studies, we cloned and characterized genes encoding enzymes for the conversion of γ-HCH to β-ketoadipate in UT26. In this study, we analyzed a mutant obtained by transposon mutagenesis and identified and characterized new genes encoding a putative ABC-type transporter essential for the utilization of γ-HCH in strain UT26. This putative ABC transporter consists of four components, permease, ATPase, periplasmic protein, and lipoprotein, encoded by linK, linL, linM, and linN, respectively. Mutation and complementation analyses indicated that all the linKLMN genes are required, probably as a set, for γ-HCH utilization in UT26. Furthermore, the mutant cells deficient in this putative ABC transporter showed (i) higher γ-HCH degradation activity and greater accumulation of the toxic dead-end product 2,5-dichlorophenol (2,5-DCP), (ii) higher sensitivity to 2,5-DCP itself, and (iii) higher permeability of hydrophobic compounds than the wild-type cells. These results strongly suggested that LinKLMN are involved in γ-HCH utilization by controlling membrane hydrophobicity. This study clearly demonstrated that a cellular factor besides catabolic enzymes and transcriptional regulators is essential for utilization of xenobiotic compounds in bacterial cells.

scholarly journals Characterization of the Erwinia chrysanthemi gan Locus, Involved in Galactan Catabolism

2007 ◽  
Vol 189 (19) ◽  
pp. 7053-7061 ◽  
Author(s):  
Aurélie Delangle ◽  
Anne-France Prouvost ◽  
Virginie Cogez ◽  
Jean-Pierre Bohin ◽  
Jean-Marie Lacroix ◽  
...  
Keyword(s):  
Abc Transporter ◽  
Transport System ◽  
Pathogenic Bacteria ◽  
Erwinia Chrysanthemi ◽  
Potato Tubers ◽  
Inner Membrane ◽  
Saintpaulia Ionantha ◽  
Genes Encoding

ABSTRACT β-1,4-Galactan is a major component of the ramified regions of pectin. Analysis of the genome of the plant pathogenic bacteria Erwinia chrysanthemi revealed the presence of a cluster of eight genes encoding proteins potentially involved in galactan utilization. The predicted transport system would comprise a specific porin GanL and an ABC transporter made of four proteins, GanFGK2. Degradation of galactans would be catalyzed by the periplasmic 1,4-β-endogalactanase GanA, which released oligogalactans from trimer to hexamer. After their transport through the inner membrane, oligogalactans would be degraded into galactose by the cytoplasmic 1,4-β-exogalactanase GanB. Mutants affected for the porin or endogalactanase were unable to grow on galactans, but they grew on galactose and on a mixture of galactotriose, galactotetraose, galactopentaose, and galactohexaose. Mutants affected for the periplasmic galactan binding protein, the transporter ATPase, or the exogalactanase were only able to grow on galactose. Thus, the phenotypes of these mutants confirmed the functionality of the gan locus in transport and catabolism of galactans. These mutations did not affect the virulence of E. chrysanthemi on chicory leaves, potato tubers, or Saintpaulia ionantha, suggesting an accessory role of galactan utilization in the bacterial pathogeny.

scholarly journals Cloning, Sequencing, and Characterization of the Hexahydro-1,3,5-Trinitro-1,3,5-Triazine Degradation Gene Cluster from Rhodococcus rhodochrous

2002 ◽  
Vol 68 (10) ◽  
pp. 4764-4771 ◽  
Author(s):  
Helena M. B. Seth-Smith ◽  
Susan J. Rosser ◽  
Amrik Basran ◽  
Emma R. Travis ◽  
Eric R. Dabbs ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Gene Cluster ◽  
High Explosive ◽  
Sole Source ◽  
Ring Cleavage ◽  
Rhodococcus Rhodochrous ◽  
Resting Cell ◽  
Dead End ◽  
Adrenodoxin Reductase

ABSTRACT Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a high explosive which presents an environmental hazard as a major land and groundwater contaminant. Rhodococcus rhodochrous strain 11Y was isolated from explosive contaminated land and is capable of degrading RDX when provided as the sole source of nitrogen for growth. Products of RDX degradation in resting-cell incubations were analyzed and found to include nitrite, formaldehyde, and formate. No ammonium was excreted into the medium, and no dead-end metabolites were observed. The gene responsible for the degradation of RDX in strain 11Y is a constitutively expressed cytochrome P450-like gene, xplA, which is found in a gene cluster with an adrenodoxin reductase homologue, xplB. The cytochrome P450 also has a flavodoxin domain at the N terminus. This study is the first to present a gene which has been identified as being responsible for RDX biodegradation. The mechanism of action of XplA on RDX is thought to involve initial denitration followed by spontaneous ring cleavage and mineralization.

scholarly journals A Member of the Second Carbohydrate Uptake Subfamily of ATP-Binding Cassette Transporters Is Responsible for Ribonucleoside Uptake in Streptococcus mutans

2006 ◽  
Vol 188 (23) ◽  
pp. 8005-8012 ◽  
Author(s):  
Alexander J. Webb ◽  
Arthur H. F. Hosie
Keyword(s):  
Streptococcus Mutans ◽  
Abc Transporter ◽  
Atp Binding ◽  
Diverse Range ◽  
Adenosine Uptake ◽  
Uptake Rates ◽  
Genes Encoding ◽  
Uptake Transporters ◽  
Atp Binding Cassette

ABSTRACT Streptococcus mutans has a significant number of transporters of the ATP-binding cassette (ABC) superfamily. Members of this superfamily are involved in the translocation of a diverse range of molecules across membranes. However, the functions of many of these members remain unknown. We have investigated the role of the single S. mutans representative of the second subfamily of carbohydrate uptake transporters (CUT2) of the ABC superfamily. The genetic context of genes encoding this transporter indicates that it may have a role in ribonucleoside scavenging. Inactivation of rnsA (ATPase) or rnsB (solute binding protein) resulted in strains resistant to 5-fluorocytidine and 5-fluorouridine (toxic ribonucleoside analogues). As other ribonucleosides including cytidine, uridine, adenosine, 2-deoxyuridine, and 2-deoxycytidine protected S. mutans from 5-fluorocytidine and 5-fluorouridine toxicity, it is likely that this transporter is involved in the uptake of these molecules. Indeed, the rnsA and rnsB mutants were unable to transport [2-14C]cytidine or [2-14C]uridine and had significantly reduced [8-14C]adenosine uptake rates. Characterization of this transporter in wild-type S. mutans indicates that it is a high-affinity (Km = 1 to 2 μM) transporter of cytidine, uridine, and adenosine. The inhibition of [14C]cytidine uptake by a range of structurally related molecules indicates that the CUT2 transporter is involved in the uptake of most ribonucleosides, including 2-deoxyribonucleosides, but not ribose or nucleobases. The characterization of this permease has directly shown for the first time that an ABC transporter is involved in the uptake of ribonucleosides and extends the range of substrates known to be transported by members of the ABC transporter superfamily.

scholarly journals Requirement of Staphylococcus aureus ATP-Binding Cassette-ATPase FhuC for Iron-Restricted Growth and Evidence that It Functions with More than One Iron Transporter

2006 ◽  
Vol 188 (6) ◽  
pp. 2048-2055 ◽  
Author(s):  
Craig D. Speziali ◽  
Suzanne E. Dale ◽  
James A. Henderson ◽  
Enrique D. Vinés ◽  
David E. Heinrichs
Keyword(s):  
Staphylococcus Aureus ◽  
Abc Transporter ◽  
Binding Protein ◽  
Sole Source ◽  
Deletion Strain ◽  
Growth Defect ◽  
Atp Binding ◽  
Growth Defects ◽  
Atp Binding Cassette

ABSTRACT In Staphylococcus aureus, fhuCBG encodes an ATP-binding cassette (ABC) transporter that is required for the transport of iron(III)-hydroxamates; mutation of either fhuB or fhuG eliminates transport. In this paper, we describe construction and characterization of an S. aureus fhuCBG deletion strain. The ΔfhuCBG::ermC mutation not only resulted in a strain that was incapable of growth on iron(III)-hydroxamates as a sole source of iron but also resulted in a strain which had a profound growth defect in iron-restricted laboratory media. The growth defect was not a result of the inability to transport iron(III)-hydroxamates since S. aureus fhuG::Tn917 and S. aureus fhuD1::Km fhuD2::Tet mutants, which are also unable to transport iron(III)-hydroxamates, do not have similar iron-restricted growth defects. Complementation experiments demonstrated that the growth defect of the ΔfhuCBG::ermC mutant was the result of the inability to express FhuC and that this was the result of an inability to transport iron complexed to the S. aureus siderophore staphylobactin. Transport of iron(III)-staphylobactin is dependent upon SirA (binding protein), SirB (permease), and SirC (permease). S. aureus expressing FhuC with a Walker A K42N mutation could not utilize iron(III)-hydroxamates or iron(III)-staphylobactin as a sole source of iron, supporting the conclusion that FhuC, as expected, functions with FhuB, FhuG, and FhuD1 or FhuD2 to transport iron(III)-hydroxamates and is the “genetically unlinked” ABC-ATPase that functions with SirA, SirB, and SirC to transport iron(III)-staphylobactin. Finally, we demonstrated that the ΔfhuCBG::ermC strain had decreased virulence in a murine kidney abscess model.

scholarly journals Aerobic Biodegradation of 2,4-Dinitroanisole by Nocardioides sp. Strain JS1661

2014 ◽  
Vol 80 (24) ◽  
pp. 7725-7731 ◽  
Author(s):  
Tekle Tafese Fida ◽  
Shannu Palamuru ◽  
Gunjan Pandey ◽  
Jim C. Spain
Keyword(s):  
Microbial Transformation ◽  
Growth Yield ◽  
Sole Source ◽  
Aerobic Biodegradation ◽  
Enzyme Assays ◽  
Content Type ◽  
Dead End ◽  
End Products ◽  
Subsequent Degradation ◽  
Genes Encoding

ABSTRACT2,4-Dinitroanisole (DNAN) is an insensitive munition ingredient used in explosive formulations as a replacement for 2,4,6-trinitrotoluene (TNT). Little is known about the environmental behavior of DNAN. There are reports of microbial transformation to dead-end products, but no bacteria with complete biodegradation capability have been reported.Nocardioidessp. strain JS1661 was isolated from activated sludge based on its ability to grow on DNAN as the sole source of carbon and energy. Enzyme assays indicated that the first reaction involves hydrolytic release of methanol to form 2,4-dinitrophenol (2,4-DNP). Growth yield and enzyme assays indicated that 2,4-DNP underwent subsequent degradation by a previously established pathway involving formation of a hydride-Meisenheimer complex and release of nitrite. Identification of the genes encoding the key enzymes suggested recent evolution of the pathway by recruitment of a novel hydrolase to extend the well-characterized 2,4-DNP pathway.

scholarly journals Characterization of a Novel Glucosamine-6-Phosphate Deaminase from a Hyperthermophilic Archaeon

2005 ◽  
Vol 187 (20) ◽  
pp. 7038-7044 ◽  
Author(s):  
Takeshi Tanaka ◽  
Fumikazu Takahashi ◽  
Toshiaki Fukui ◽  
Shinsuke Fujiwara ◽  
Haruyuki Atomi ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Abc Transporter ◽  
Catalytic Properties ◽  
Sugar Metabolism ◽  
Amino Sugar ◽  
Hyperthermophilic Archaeon ◽  
Chitin Degradation ◽  
Genes Encoding ◽  
Activity Dependent

ABSTRACT A key step in amino sugar metabolism is the interconversion between fructose-6-phosphate (Fru6P) and glucosamine-6-phosphate (GlcN6P). This conversion is catalyzed in the catabolic and anabolic directions by GlcN6P deaminase and GlcN6P synthase, respectively, two enzymes that show no relationship with one another in terms of primary structure. In this study, we examined the catalytic properties and regulatory features of the glmD gene product (GlmD Tk ) present within a chitin degradation gene cluster in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Although the protein GlmD Tk was predicted as a probable sugar isomerase related to the C-terminal sugar isomerase domain of GlcN6P synthase, the recombinant GlmD Tk clearly exhibited GlcN6P deaminase activity, generating Fru6P and ammonia from GlcN6P. This enzyme also catalyzed the reverse reaction, the ammonia-dependent amination/isomerization of Fru6P to GlcN6P, whereas no GlcN6P synthase activity dependent on glutamine was observed. Kinetic analyses clarified the preference of this enzyme for the deaminase reaction rather than the reverse one, consistent with the catabolic function of GlmD Tk . In T. kodakaraensis cells, glmDTk was polycistronically transcribed together with upstream genes encoding an ABC transporter and a downstream exo-β-glucosaminidase gene (glmATk ) within the gene cluster, and their expression was induced by the chitin degradation intermediate, diacetylchitobiose. The results presented here indicate that GlmD Tk is actually a GlcN6P deaminase functioning in the entry of chitin-derived monosaccharides to glycolysis in this hyperthermophile. This enzyme is the first example of an archaeal GlcN6P deaminase and is a structurally novel type distinct from any previously known GlcN6P deaminase.

Characterization of three genes encoding enzymes of the folate biosynthetic pathway in Plasmodium falciparum

Parasitology ◽  
2001 ◽  
Vol 122 (1) ◽  
pp. 1-13 ◽  
Author(s):  
C.-S. LEE ◽  
E. SALCEDO ◽  
Q. WANG ◽  
P. WANG ◽  
P.F.G. SIMS ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Serine Hydroxymethyltransferase ◽  
Gtp Cyclohydrolase I ◽  
Gene Encoding ◽  
New Genes ◽  
Isolation And Characterization ◽  
Genes Encoding ◽  
Antifolate Drugs ◽  
Dihydroneopterin Aldolase

Although the folate metabolic pathway in malaria parasites is a major chemotherapeutic target, resistance to currently available antifolate drugs is an increasing problem. This pathway, however, includes a number of enzymes that, to date, have not been characterized despite their potential for clinical exploitation. As a step towards evaluation of additional targets in this pathway, we report the isolation and characterization of 3 new genes that encode homologues of GTP cyclohydrolase I (GTP-CH), dihydrofolate synthase/folylpolyglutamate synthase (DHFS/FPGS) and serine hydroxymethyltransferase (SHMT). The genes encoding GTP-CH and SHMT are unambiguously assigned to chromosome 12, while that for DHFS/FPGS is tentatively assigned to chromosome 13. All 3 genes are expressed in blood-stage parasites, yielding transcripts of which only ca 60–70% is accounted for by coding sequence. All 3 of the proteins predicted to be encoded by these genes display sequence differences compared to the human host homologues that may be of functional significance. These data bring the complement of cloned genes that encode activities in the pathway to seven, leaving only the gene encoding dihydroneopterin aldolase (DHNA) to be identified in the route from GTP to folate synthesis and folate turnover in the thymidylate cycle.

Isolation and characterization of a 9-fluorenone-degrading bacterial strain and its role in synergistic degradation of fluorene by a consortium

1998 ◽  
Vol 44 (8) ◽  
pp. 734-742
Author(s):  
Mercè Casellas ◽  
Magdalena Grifoll ◽  
Jordi Sabaté ◽  
Anna Maria Solanas
Keyword(s):  
Carbon Assimilation ◽  
Sole Source ◽  
Growth Media ◽  
Pseudomonas Mendocina ◽  
Cell Biomass ◽  
Isolation And Characterization ◽  
Dead End ◽  
Angular Dioxygenation ◽  
Synergistic Degradation

Pseudomonas mendocina MC2, able to use 9-fluorenone but not fluorene as its sole source of carbon and energy, was isolated. Identification of metabolites in growth media and washed cell suspensions indicated that strain MC2 metabolizes 9-fluorenone via angular dioxygenation of the ketone, to give 1,1a-dihydroxy-1-hydro-9-fluorenone, followed by the opening of the five-membered ring and further degradation of the resulting biphenyl derivative by reactions akin to those of biphenyl metabolism, which produce phthalate as an intermediate. The aim of this research was to study the biodegradation of fluorene by a co-culture of strain MC2 and Arthrobacter sp.strain F101, which grows on fluorene and simultaneously transforms a fraction of the substrate to 9-fluorenone, which accumulates as a dead-end product. Growing with 0.1 g fluorene/L, Arthrobacter sp. strain F101 caused the total removal of this compound from the cultures, but when this strain was grown with 1g fluorene/L, only 16% of the fluorene was used. The addition of 9-fluorenone to cultures growing on fluorene showed that 9-fluorenone inhibits fluorene degradation. Finally, when Pseudomonas mendocina MC2 and Arthrobacter sp. strain F101 were co-cultured with 1g fluorene/L as a sole source of carbon and energy, the growth of the strains completely removed fluorene in 2 days. 9-Fluorenone did not accumulate and the carbon assimilation into cell biomass was estimated as approximately 46%. Key words: microbial consortium, fluorene, 9-fluorenone, biodegradation.

scholarly journals l-Rhamnose Transport Is Sugar Kinase (RhaK) Dependent in Rhizobium leguminosarum bv. trifolii

2007 ◽  
Vol 189 (23) ◽  
pp. 8437-8446 ◽  
Author(s):  
Jason S. Richardson ◽  
Ivan J. Oresnik
Keyword(s):  
Abc Transporter ◽  
Rhizobium Leguminosarum ◽  
Nodule Occupancy ◽  
Vitro Assay ◽  
Catabolic Genes ◽  
Genes Encoding ◽  
Pentose Sugar ◽  
Sugar Kinase

ABSTRACT Strains of Rhizobium leguminosarum which are unable to catabolize l-rhamnose, a methyl-pentose sugar, are compromised in the ability to compete for nodule occupancy versus wild-type strains. Previous characterization of the 11-kb region necessary for the utilization of rhamnose identified a locus carrying catabolic genes and genes encoding the components of an ABC transporter. Genetic evidence suggested that the putative kinase RhaK carried out the first step in the catabolism of rhamnose. Characterization of this kinase led to the observation that strains carrying rhamnose kinase mutations were unable to transport rhamnose into the cell. The absence of a functional rhamnose kinase did not stop the transcription and translation of the ABC transporter components. By developing an in vitro assay for RhaK activity, we have been able to show that (i) RhaK activity is consistent with RhaK phosphorylating rhamnose and (ii) biochemical activity of RhaK is necessary for rhamnose transport.

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