1. |
Chadhain SM,
Moritz EM,
Kim E,
Zylstra GJ,
( 2007 ) Identification, cloning, and characterization of a multicomponent biphenyl dioxygenase from Sphingobium yanoikuyae B1. PMID : 17647036 : DOI : 10.1007/s10295-007-0235-3 Abstract >>
Sphingobium yanoikuyae B1 utilizes both polycyclic aromatic hydrocarbons (biphenyl, naphthalene, and phenanthrene) and monocyclic aromatic hydrocarbons (toluene, m- and p-xylene) as its sole source of carbon and energy for growth. The majority of the genes for these intertwined monocyclic and polycyclic aromatic pathways are grouped together on a 39 kb fragment of chromosomal DNA. However, this gene cluster is missing several genes encoding essential enzymatic steps in the aromatic degradation pathway, most notably the genes encoding the oxygenase component of the initial polycyclic aromatic hydrocarbon (PAH) dioxygenase. Transposon mutagenesis of strain B1 yielded a mutant blocked in the initial oxidation of PAHs. The transposon insertion point was sequenced and a partial gene sequence encoding an oxygenase component of a putative PAH dioxygenase identified. A cosmid clone from a genomic library of S. yanoikuyae B1 was identified which contains the complete putative PAH oxygenase gene sequence. Separate clones expressing the genes encoding the electron transport components (ferredoxin and reductase) and the PAH dioxygenase were constructed. Incubation of cells expressing the dioxygenase enzyme system with biphenyl or naphthalene resulted in production of the corresponding cis-dihydrodiol confirming PAH dioxygenase activity. This demonstrates that a single multicomponent dioxygenase enzyme is involved in the initial oxidation of both biphenyl and naphthalene in S. yanoikuyae B1.
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2. |
Ferraro DJ,
Brown EN,
Yu CL,
Parales RE,
Gibson DT,
Ramaswamy S,
( 2007 ) Structural investigations of the ferredoxin and terminal oxygenase components of the biphenyl 2,3-dioxygenase from Sphingobium yanoikuyae B1. PMID : 17349044 : DOI : 10.1186/1472-6807-7-10 PMC : PMC1847435 Abstract >>
The initial step involved in oxidative hydroxylation of monoaromatic and polyaromatic compounds by the microorganism Sphingobium yanoikuyae strain B1 (B1), previously known as Sphingomonas yanoikuyae strain B1 and Beijerinckia sp. strain B1, is performed by a set of multiple terminal Rieske non-heme iron oxygenases. These enzymes share a single electron donor system consisting of a reductase and a ferredoxin (BPDO-FB1). One of the terminal Rieske oxygenases, biphenyl 2,3-dioxygenase (BPDO-OB1), is responsible for B1's ability to dihydroxylate large aromatic compounds, such as chrysene and benzo[a]pyrene. In this study, crystal structures of BPDO-OB1 in both native and biphenyl bound forms are described. Sequence and structural comparisons to other Rieske oxygenases show this enzyme to be most similar, with 43.5 % sequence identity, to naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4. While structurally similar to naphthalene 1,2-dioxygenase, the active site entrance is significantly larger than the entrance for naphthalene 1,2-dioxygenase. Differences in active site residues also allow the binding of large aromatic substrates. There are no major structural changes observed upon binding of the substrate. BPDO-FB1 has large sequence identity to other bacterial Rieske ferredoxins whose structures are known and demonstrates a high structural homology; however, differences in side chain composition and conformation around the Rieske cluster binding site are noted. This is the first structure of a Rieske oxygenase that oxidizes substrates with five aromatic rings to be reported. This ability to catalyze the oxidation of larger substrates is a result of both a larger entrance to the active site as well as the ability of the active site to accommodate larger substrates. While the biphenyl ferredoxin is structurally similar to other Rieske ferredoxins, there are distinct changes in the amino acids near the iron-sulfur cluster. Because this ferredoxin is used by multiple oxygenases present in the B1 organism, this ferredoxin-oxygenase system provides the structural platform to dissect the balance between promiscuity and selectivity in protein-protein electron transport systems.
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3. |
Ohnishi K,
Okuta A,
Ju J,
Hamada T,
Misono H,
Harayama S,
( 2004 ) Molecular breeding of 2,3-dihydroxybiphenyl 1,2-dioxygenase for enhanced resistance to 3-chlorocatechol. PMID : 15113829 : DOI : 10.1093/jb/mvh037 Abstract >>
3-Chlorobiphenyl is known to be mineralized by biphenyl-utilizing bacteria to 3-chlorobenzoate, which is further metabolized to 3-chlorocatechol. An extradiol dioxygenase, 2,3-dihydroxybiphenyl 1,2-dioxygenase (DHB12O; EC 1.13.11.39), which is encoded by the bphC gene, catalyzes the third step of the upper pathway of 3-chlorobiphenyl degradation. In this study, two full-length bphCs and nine partial fragments of bphCs fused to the 3' end of bphC in Pseudomonas pseudoalcaligenes KF707 were cloned from different biphenyl-utilizing soil bacteria and expressed in Escherichia coli. The enzyme activities of the expressed DHB12Os were inhibited to varying degrees by 3-chlorocatechol, and the E. coli cells overexpressing DHB12O could not grow or grew very slowly in the presence of 3-chlorocatechol. These sensitivities of enzyme activity and cell growth to 3-chlorocatechol were well correlated, and this phenomenon was employed in screening chimeric BphCs formed by family shuffling of bphC genes isolated from Comamonas testosteroni KF704 and C. testosteroni KF712. The resultant DHB12Os were more resistant by a factor of two to 3-chlorocatechol than one of the best parents, KF707 DHB12O.
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4. |
Ochou M,
Saito M,
Kurusu Y,
( 2008 ) Characterization of two compatible small plasmids from Sphingobium yanoikuyae. PMID : 18391447 : DOI : 10.1271/bbb.70813 Abstract >>
We isolated and characterized two small cryptic indigenous plasmids, pYAN-1 (4,896 bp) and pYAN-2 (4,687 bp), from Sphingobium yanoikuyae, and developed a versatile system that permitted genetic manipulation of the genus Sphingomonas. Nucleotide sequencing of both plasmids revealed that they contained mobA, mobs, and repA genes, which are predicted to encode proteins associated with mobilization and replication, in common. Transformation with each plasmid harboring the antibiotic resistance gene by electroporation was fully successful, using Novosphingobium capsulatum as a host.
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5. |
Unterweger B,
Drinkwater N,
Johanesen P,
Lyras D,
Dumsday GJ,
McGowan S,
( 2017 ) X-ray crystal structure of cytochrome P450 monooxygenase CYP101J2 from Sphingobium yanoikuyae strain B2. PMID : 27936485 : DOI : 10.1002/prot.25227 Abstract >>
The cytochrome P450 monooxygenases (P450s) catalyze a vast array of oxygenation reactions that can be useful in biocatalytic applications. CYP101J2 from Sphingobium yanoikuyae is a P450 that catalyzes the hydroxylation of 1,8-cineole. Here we report the crystallization and X-ray structure elucidation of recombinant CYP101J2 to 1.8 ? resolution. The CYP101J2 structure shows the canonical P450-fold and has an open conformation in the absence of substrate. Analysis of the structure revealed that CYP101J2, in the absence of substrate, forms a well-ordered substrate-binding channel that suggests a unique form of substrate guidance in comparison to other bacterial 1,8-cineole-hydroxylating P450 enzymes. Proteins 2017; 85:945-950. ? 2016 Wiley Periodicals, Inc.
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6. |
Unterweger B,
Bulach DM,
Scoble J,
Midgley DJ,
Greenfield P,
Lyras D,
Johanesen P,
Dumsday GJ,
( 2016 ) CYP101J2, CYP101J3, and CYP101J4, 1,8-Cineole-Hydroxylating Cytochrome P450 Monooxygenases from Sphingobium yanoikuyae Strain B2. PMID : 27590809 : DOI : 10.1128/AEM.02067-16 PMC : PMC5086545 Abstract >>
We report the isolation and characterization of three new cytochrome P450 monooxygenases: CYP101J2, CYP101J3, and CYP101J4. These P450s were derived from Sphingobium yanoikuyae B2, a strain that was isolated from activated sludge based on its ability to fully mineralize 1,8-cineole. Genome sequencing of this strain in combination with purification of native 1,8-cineole-binding proteins enabled identification of 1,8-cineole-binding P450s. The P450 enzymes were cloned, heterologously expressed (N-terminally His6 tagged) in Escherichia coli BL21(DE3), purified, and spectroscopically characterized. Recombinant whole-cell biotransformation in E. coli demonstrated that all three P450s hydroxylate 1,8-cineole using electron transport partners from E. coli to yield a product putatively identified as (1S)-2�\-hydroxy-1,8-cineole or (1R)-6�\-hydroxy-1,8-cineole. The new P450s belong to the CYP101 family and share 47% and 44% identity with other 1,8-cineole-hydroxylating members found in Novosphingobium aromaticivorans and Pseudomonas putida Compared to P450cin (CYP176A1), a 1,8-cineole-hydroxylating P450 from Citrobacter braakii, these enzymes share less than 30% amino acid sequence identity and hydroxylate 1,8-cineole in a different orientation. Expansion of the enzyme toolbox for modification of 1,8-cineole creates a starting point for use of hydroxylated derivatives in a range of industrial applications. CYP101J2, CYP101J3, and CYP101J4 are cytochrome P450 monooxygenases from S. yanoikuyae B2 that hydroxylate the monoterpenoid 1,8-cineole. These enzymes not only play an important role in microbial degradation of this plant-based chemical but also provide an interesting route to synthesize oxygenated 1,8-cineole derivatives for applications as natural flavor and fragrance precursors or incorporation into polymers. The P450 cytochromes also provide an interesting basis from which to compare other enzymes with a similar function and expand the CYP101 family. This could eventually provide enough bacterial parental enzymes with similar amino acid sequences to enable in vitro evolution via DNA shuffling.
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7. |
Oliveira V,
Gomes NC,
Almeida A,
Silva AM,
Simões MM,
Smalla K,
Cunha ?,
( 2014 ) Hydrocarbon contamination and plant species determine the phylogenetic and functional diversity of endophytic degrading bacteria. PMID : 24765659 : Abstract >>
Salt marsh sediments are sinks for various anthropogenic contaminants, giving rise to significant environmental concern. The process of salt marsh plant survival in such environment is very intriguing and at the same time poorly understood. The plant�Vmicrobe interactions may play a key role in the process of environment and in planta detoxification.In this study, a combination of culture-dependent and culture-independent molecular approaches [enrichment cultures, polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), DNA sequencing] were used to investigate the effect of petroleum hydrocarbons (PH) contamination on the structure and function[polycyclic aromatic hydrocarbon (PAH) dioxygenase genes] of endophytic bacterial communities of salt marsh plant species (Halimione portulacoides and Sarcocornia perennis)in the estuarine system Ria de Aveiro (Portugal). Pseudomonads dominated the cultivable fraction of the endophytic communities in the enrichment cultures. In a set of fifty isolates tested, nine were positive for genes encoding for PAH dioxygenases (nahAc)and four were positive for plasmid carrying genes encoding PAH degradation enzymes(nahAc). Interestingly, these plasmids were only detected in isolates from most severely PH-polluted sites. The results revealed site-specific effects on endophytic communities,related to the level of PH contamination in the sediment, and plant-species-specific ��imprints�� in community structure and in genes encoding for PAH dioxygenases. These results suggest a potential ecological role of bacterial plant symbiosis in the process of plant colonization in urban estuarine areas exposed to PH contamination.
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8. |
Furukawa K,
Hayase N,
Taira K,
Tomizuka N,
( 1989 ) Molecular relationship of chromosomal genes encoding biphenyl/polychlorinated biphenyl catabolism: some soil bacteria possess a highly conserved bph operon. PMID : 2507526 : DOI : 10.1128/jb.171.10.5467-5472.1989 PMC : PMC210385 Abstract >>
All the genes we examined that encoded biphenyl/polychlorinated biphenyl (PCB) degradation were chromosomal, unlike many other degradation-encoding genes, which are plasmid borne. The molecular relationship of genes coding for biphenyl/PCB catabolism in various biphenyl/PCB-degrading Pseudomonas, Achromobacter, Alcaligenes, Moraxella, and Arthrobacter strains was investigated. Among 15 strains tested, 5 Pseudomonas strains and one Alcaligenes strain possessed the bphABC gene cluster on the XhoI 7.2-kilobase fragment corresponding to that of Pseudomonas pseudoalcaligenes KF707. More importantly, the restriction profiles of these XhoI 7.2-kilobase fragments containing bphABC genes were very similar, if not identical, despite the dissimilarity of the flanking chromosomal regions. Three other strains also possessed bphABC genes homologous with those of KF707, and five other strains showed weak or no significant genetic homology with bphABC of KF707. The immunological cross-reactivity of 2,3-dihydroxybiphenyl dioxygenases from various strains corresponded well to the DNA homology. On the other hand, the bphC gene of another PCB-degrading strain, Pseudomonas paucimobilis Q1, lacked genetic as well as immunological homology with any of the other 15 biphenyl/PCB degraders tested. The existence of the nearly identical chromosomal genes among various strains may suggest that a segment containing the bphABC genes has a mechanism for transferring the gene from one strain to another.
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9. |
Jin D,
Kong X,
Cui B,
Bai Z,
Zhang H,
( 2013 ) Biodegradation of di-n-butyl phthalate by a newly isolated halotolerant Sphingobium sp. PMID : 24336064 : DOI : 10.3390/ijms141224046 PMC : PMC3876093 Abstract >>
A Gram-negative strain (TJ) capable of growing aerobically on mixed phthalate esters (PAEs) as the sole carbon and energy source was isolated from the Haihe estuary, Tianjin, China. It was identified as belonging to the Sphingobium genus on the basis of morphological and physiological characteristics and 16S rRNA and gyrb gene sequencing. The batch tests for biodegradation of di-n-butyl phthalate (DBP) by the Sphingobium sp. TJ showed that the optimum conditions were 30 �XC, pH 7.0, and the absence of NaCl. Stain TJ could tolerate up to 4% NaCl in minimal salt medium supplemented with DBP, although the DBP degradation rates slowed as NaCl concentration increased. In addition, substrate tests showed that strain TJ could utilize shorter side-chained PAEs, such as dimethyl phthalate and diethyl phthalate, but could not metabolize long-chained PAEs, such as di-n-octyl phthalate, diisooctyl phthalate, and di-(2-ethyl-hexyl) phthalate. To our knowledge, this is the first report on the biodegradation characteristics of DBP by a member of the Sphingobium genus.
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10. |
Narciso-da-Rocha C,
Vaz-Moreira I,
Manaia CM,
( 2014 ) Genotypic diversity and antibiotic resistance in Sphingomonadaceae isolated from hospital tap water. PMID : 23892027 : DOI : 10.1016/j.scitotenv.2013.06.109 Abstract >>
The aim of this study was to infer about the modes and extent of dispersion of Sphingomonadaceae via tap water. Sphingomonadaceae isolated from tap water samples in different places of a hospital were compared, based on intra-species genetic variability and antibiotic resistance phenotypes. These isolates were also compared with others isolated before from houses and dental chairs, served by the same municipal water supply system. Sphingomonadaceae from hospital tap water comprised members of the genera Sphingomonas, Sphingobium, Novosphingobium and Blastomonas. In general, distinct genotypes of Sphingomonadaceae were detected in different hospital areas and in tap water outside the hospital, suggesting these bacteria are not persistent or widespread in the urban water distribution system. Possible intrinsic antibiotic resistance, observed in most or all members of the family or of a genus, was observed for colistin in Sphingomonadaceae, aminoglycosides in the genus Blastomonas and beta-lactams in the genus Sphingobium. Possible acquired resistance phenotypes, not common to all members of a given species, comprised fluoroquinolones, cephalosporins and sulphonamides. Although the potential of Sphingomonadaceae as opportunistic pathogens may be low, the capacity of these bacteria to thrive in water supply systems, combined with the intrinsic or acquired antibiotic resistance, may raise the risk associated with their occurrence in hospital tap water.
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11. |
Hotta Y,
Sato H,
Hosoda A,
Tamura H,
( 2012 ) MALDI-TOF MS analysis of ribosomal proteins coded in S10 and spc operons rapidly classified the Sphingomonadaceae as alkylphenol polyethoxylate-degrading bacteria from the environment. PMID : 22324315 : DOI : 10.1111/j.1574-6968.2012.02525.x Abstract >>
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) using ribosomal subunit proteins coded in the S10-spc-alpha operon as biomarkers was applied for the classification of the Sphingomonadaceae from the environment. To construct a ribosomal protein database, S10-spc-alpha operon of type strains of the Sphingomonadaceae and their related alkylphenol polyethoxylate (APEO(n))-degrading bacteria were sequenced using specific primers designed based on nucleotide sequences of genome-sequenced strains. The observed MALDI mass spectra of intact cells were compared with the theoretical mass of the constructed ribosomal protein database. The nine selected biomarkers coded in the S10-spc-alpha operon, L18, L22, L24, L29, L30, S08, S14, S17, and S19, could successfully distinguish the Sphingopyxis terrae NBRC 15098(T) and APEO(n) -degrading bacteria strain BSN20, despite only one base difference in the 16S rRNA gene sequence. This method, named the S10-GERMS (S10-spc-alpha operon gene-encoded ribosomal protein mass spectrum) method, is a significantly useful tool for bacterial discrimination of the Sphingomonadaceae at the strain level and can detect and monitor the main APEO(n) -degrading bacteria in the environment.
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12. |
Vaz-Moreira I,
Nunes OC,
Manaia CM,
( 2011 ) Diversity and antibiotic resistance patterns of Sphingomonadaceae isolates from drinking water. PMID : 21705522 : DOI : 10.1128/AEM.00579-11 PMC : PMC3165245 Abstract >>
Sphingomonadaceae (n = 86) were isolated from a drinking water treatment plant (n = 6), tap water (n = 55), cup fillers for dental chairs (n = 21), and a water demineralization filter (n = 4). The bacterial isolates were identified based on analysis of the 16S rRNA gene sequence, and intraspecies variation was assessed on the basis of atpD gene sequence analysis. The isolates were identified as members of the genera Sphingomonas (n = 27), Sphingobium (n = 28), Novosphingobium (n = 12), Sphingopyxis (n = 7), and Blastomonas (n = 12). The patterns of susceptibility to five classes of antibiotics were analyzed and compared for the different sites of isolation and taxonomic groups. Colistin resistance was observed to be intrinsic (92%). The highest antibiotic resistance prevalence values were observed in members of the genera Sphingomonas and Sphingobium and for beta-lactams, ciprofloxacin, and cotrimoxazole. In tap water and in water from dental chairs, antibiotic resistance was more prevalent than in the other samples, mainly due to the predominance of isolates of the genera Sphingomonas and Sphingobium. These two genera presented distinct patterns of association with antibiotic resistance, suggesting different paths of resistance development. Antibiotic resistance patterns were often related to the species rather than to the site or strain, suggesting the importance of vertical resistance transmission in these bacteria. This is the first study demonstrating that members of the family Sphingomonadaceae are potential reservoirs of antibiotic resistance in drinking water.
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13. |
( 1996 ) Sequence and expression of an isocitrate dehydrogenase-encoding gene from a polycyclic aromatic hydrocarbon oxidizer, Sphingomonas yanoikuyae B1. PMID : 8626059 : DOI : 10.1016/0378-1119(95)00732-6 Abstract >>
An 18.5-kb DNA fragment was cloned from Sphingomonas yanoikuyae (Sy) B1 (previously Beijerinckia B1). Analysis of a 4.3-kb sequence revealed an isocitrate dehydrogenase (Idh)-encoding gene (idhA), an unidentified open reading frame (ORF) and a partial glucosamine synthetase-encoding ORF (glmS). As in a number of bacteria, Tn7 insertion was found specifically at a site past the stop codon of glmS. The predicted 406-amino-acid sequence of IdhA shows, for the first time, an extensive sequence identity (66%) with an eukaryotic NADP+-specific Idh. The idhA gene was expressed in Escherichia coli. Identical restriction fragments carrying idhA were found in B1, Sy IFO15102 and Sy Q1 (formerly S. paucimobilis Q1), indicating a well-conserved idh gene.
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14. |
Kim E,
Zylstra GJ,
( 1995 ) Molecular and biochemical characterization of two meta-cleavage dioxygenases involved in biphenyl and m-xylene degradation by Beijerinckia sp. strain B1. PMID : 7768806 : DOI : 10.1128/jb.177.11.3095-3103.1995 PMC : PMC176998 Abstract >>
Beijerinckia sp. strain B1 is able to grow on either biphenyl or m-xylene as the sole source of carbon and is capable of cooxidizing many polycyclic aromatic hydrocarbons. The catabolic pathways for biphenyl and m-xylene degradation are coinduced and share common downstream enzymatic reactions. The catabolic pathway for biphenyl degradation involves two meta-cleavage steps, one for 2,3-dihydroxybiphenyl and a second for catechol. The catabolic pathway for m-xylene involves one m-cleavage step for 3-methylcatechol. The genes for two meta-cleavage dioxygenases were cloned from Beijerinckia sp. strain B1 on a single fragment of genomic DNA. The two genes are located approximately 5.5 kb away from one another. Expression of each gene separately in Escherichia coli and analysis of the meta-cleavage dioxygenase produced showed that one enzyme was more specific for 2,3-dihydroxybiphenyl while the second was more specific for catechol. The genes for the two meta-cleavage enzymes were thus labeled bphC and xylE for 2,3-dihydroxybiphenyl 1,2-dioxygenase and catechol 2,3-dioxygenase, respectively. Nondenaturing polyacrylamide gel electrophoresis followed by enzyme activity staining showed that the two meta-cleavage dioxygenases could be easily separated from each other. Similar analyses of Beijerinckia sp. strain B1 grown on succinate, biphenyl, or m-xylene indicate that both meta-cleavage enzymes are induced when cells are grown on either biphenyl or m-xylene. The nucleotide sequence was determined for both bphC and xylE. The two genes are transcribed in opposite directions, demonstrating that at least two operons must be involved in biphenyl degradation by Beijerinckia sp. strain B1. Analysis of the deduced amino acid sequence indicates that 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC) falls into the class of meta-cleavage dioxygenases acting on dihydroxylated polycyclic aromatic hydrocarbons and is somewhat distinct from the main group of meta-cleavage dioxygenases acting on 2,3-dihydroxybiphenyl. Catechol 2,3-dioxygenase (XyIE) falls into the class of meta-cleavage enzymes acting on dihydroxylated monocyclic aromatic hydrocarbons but shows little similarity to the canonical TOL plasmid-encoded catechol 2,3-dioxygenase.
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