Yanoikuyae B1 Research Articles

Identification, characterization, and distribution of novel amidase gene aphA in sphingomonads conferring resistance to amphenicol antibiotics.

Amphenicol antibiotics, such as chloramphenicol (CHL), thiamphenicol (TAP), and florfenicol (Ff), are high-risk emerging pollutants. Their extensive usage in aquaculture, livestock, and poultry farming has led to an increase in bacterial antibiotic resistance and facilitated the spread of resistance genes. Yet, limited research has been conducted on the co-resistance of CHL, TAP, and Ff. Herein, a novel amidase AphA was identified from a pure cultured strain that can concurrently mediate the hydrolytic inactivation of CHL, TAP, and Ff, yielding products p-nitrophenylserinol, thiamphenicol amine (TAP-amine), and florfenicol amine (Ff-amine), respectively. The antibacterial activity of these antibiotic hydrolysates exhibited a significant reduction or complete loss in comparison to the parent compounds. Notably, AphA shared less than 26% amino acid sequence identity with previously reported enzymes and exhibited high conservation within the sphingomonad species. Through enzymatic kinetic analysis, the AphA exhibited markedly superior affinity and catalytic activity toward Ff in comparison to CHL and TAP. Site-directed mutagenesis analysis revealed the indispensability of catalytic triad residues, particularly serine 153 and histidine 277, in forming crucial hydrogen bonds essential for AphA's hydrolytic activity. Comparative genomic analysis showed that aphA genes in some species are closely adjacent to various transposable elements, indicating that there is a high potential risk of horizontal gene transfer (HGT). This study established a hydrolysis resistance mechanism of amphenicol antibiotics in sphingomonads, which offers theoretical guidance and a novel marker gene for assessing the prevalent risk of amphenicol antibiotics in the environment.IMPORTANCEAmphenicol antibiotics are pervasive emerging contaminants that present a substantial threat to ecological systems. Few studies have elucidated resistance genes or mechanisms that can act on CHL, TAP, and Ff simultaneously. The results of this study fill this knowledge gap and identify a novel amidase AphA from the bacterium Sphingobium yanoikuyae B1, which mediates three typical amphenicol antibiotic inactivation, and the molecular mechanism is elucidated. The diverse types of transposable elements were identified in the flanking regions of the aphA gene, indicating the risk of horizontal transfer of this antibiotic resistance genes (ARG). These findings offer new insights into the bacterial resistance to amphenicol antibiotics. The gene reported herein can be utilized as a novel genetic diagnostic marker for monitoring the environmental fate of amphenicol antibiotics, thereby enriching risk assessment efforts within the context of antibiotic resistance.

Role of biochar pyrolysis temperature on intracellular and extracellular biodegradation of biochar-adsorbed organic compounds

Immobilizing organic pollutants by adsorption of biochar in farmland soil is a cost-effective remediation method for contaminated soil. As the adsorption capacity of biochar is limited, biodegradation of biochar-adsorbed organic pollutants was a potential way to regenerate biochars and maintain the adsorption performance of biochars to lower the cost. It could be affected by the biochar pyrolysis temperature, but was not evaluated yet. In this study, biodegradation of adsorbed phenanthrene on a series of biochars with pyrolysis temperatures from 150 to 700 °C by Sphingobium yanoikuyae B1 was investigated using batch experiments of biodegradation kinetics at 30 °C, to explore the role of biochar pyrolysis temperature on biodegradation of biochar-adsorbed organic compounds. It was observed that 37.5–47.9% of adsorbed phenanthrene on moderate temperature-pyrolyzed biochars produced at 400 and 500 °C were biodegraded, less than that on high temperature-pyrolyzed biochars produced at ≥600 °C (48.8–60.8%) and low temperature-pyrolyzed biochars produced at ≤300 °C (63.4–92.5%). Phenanthrene adsorbed largely on the low temperature-pyrolyzed biochars by partition mechanism and thus is easily desorbed to water for a dominated intracellular biodegradation. On the high temperature-pyrolyzed biochars, phenanthrene is adsorbed largely by pore-filling mechanism and thus less desorbed to water for intracellular biodegradation. However, high temperature-pyrolyzed biochars can promote microbes to produce siderophore, H2O2 and thus release extracellular •OH for a dominated degradation of adsorbed phenanthrene by Fenton-like reaction. With the increase of biochar pyrolysis temperature, desorption and consequently the intracellular biodegradation of adsorbed phenanthrene on biochars decreased, while the secretion of siderophore and H2O2 by microbes on biochars increased to produce more extracellular •OH for degradation by Fenton-like reaction. The results could provide deep insights into the role of biochar pyrolysis temperature on biodegradation of biochar-adsorbed organic compounds, and optimize the selection of biochar with higher adsorption performance and easier regeneration for soil remediation.

Microbial degradation of nondesorbable organic compounds on biochars by extracellular reactive oxygen species

Knowledge of microbial degradation of biochar-adsorbed organic pollutants is essential for recovering adsorption performance of biochars and reducing secondary pollution in soil remediation. In previous study, desorption of organic compounds from biochars was perceived as a prerequisite for the microbial degradation. However, microbial degradation of the nondesorbable organic compounds on biochars has not been studied. Therefore, degradation of nondesorbable naphthalene (NAPH), phenanthrene (PHEN) and pyrene (PYR) on a wood chip-derived biochar (WBC700) by Sphingobium yanoikuyae B1 was investigated. Significant microbial degradations of nondesorbable organic compounds were observed and followed the order of NAPH < PHEN < PYR. It was newly observed in this study that the microbial degradation of nondesorbable organic compounds on WBC700 was mainly attributed to the •OH in extracellular fluid of Sphingobium yanoikuyae B1. The extracellular •OH was produced through a Fenton-like reaction involved siderophore, H2O2 and iron ions, which could be significantly enhanced by WBC700. Microbial degradation was higher for larger organic compound (e.g., PYR), because larger molecules were adsorbed in relatively larger micropores of WBC700 and thus could be accessible to more extracellular •OH for degradation. The obtained results could provide a new insight into the microbial degradation of biochar-adsorbed organic pollutants in soil remediation.

Characterization of a novel pathway for xanthene degradation by the engineered strain Sphingobium yanoikuyae B1DR

Polyaromatic hydrocarbons (PAHs) are a group of aromatic compounds that contain at least two rings. These compounds are found naturally in petroleum products and are considered the most prevalent pollutants in the environment. The lack of microorganism capable of degrading some PAHs led to their accumulation in the environment which usually causes major health problems as many of these compounds are known carcinogens. Xanthene is one of the small PAHs which has three rings. Many xanthene derivatives are useful dyes that are used for dyeing wood and cosmetic articles. However, several studies have illustrated that these compounds have toxic and carcinogenic effects. The first step of the bacterial degradation of xanthene is conducted by dioxygenase enzymes that introduces two oxygen atoms in the structure of the aromatic rings. In this study we focused on the bacterial bioremediation of xanthene via Sphingobium yanoikuyae B1DR, an engineered strain carrying the dioxin angular dioxygenase from Sphingomonas wittichii RW1. HPLC analysis of supernatant from resting cells of S. yanoikuyae B1DR grown on xanthene and succinate showed the ability of this strain to transform xanthene to 2-hydroxyphenylacetate that was not produced by the wild type of Sphingobium yanoikuyae B1. Production of 2-hydroxyphenylacetate was confirmed by GC-MS. Our results show the importance of this strain in reducing the toxic effects of xanthene in the environment and showed for the first time that ring-hydroxylation enzymes and hydrolases for biphenyl degradation in S. yanoikuyae B1 may function on metabolites generated from the degradation pathway of xanthene. By analyzing our results we were able to draw a novel pathway for xanthene degradation in S. yanoikuyae B1DR.

Identification of biphenyl 2, 3-dioxygenase and its catabolic role for phenazine degradation in Sphingobium yanoikuyae B1

Phenazines are important nitrogen-containing secondary metabolites that display a range of biological functionalities. However, these compounds have shown lethal effects on humans and, the fate of phenazine in the ecosystem remains uncertain. In this study, we investigated that Sphingobium yanoikuyae B1 could utilize phenazine as a sole carbon source for growth. Intermediate produced during phenazine degradation was purified and identified as 1, 2-dihydrogen 1, 2-dihydroxy phenazine. Biphenyl 2, 3-dioxygenase was determined to be the initial dioxygenase for phenazine degradation through gene cloning and whole cell transformation techniques. Phenazine was converted to 1, 2-dihydrogen 1, 2-dihydroxy phenazine through hydrogenation and hydroxylation, which then transformed to 2-hydroxy phenazine through spontaneous dehydration. ThebphA1fA2f, were evidenced to be the only genes encoding the initial dioxygenase for phenazine degradation. BphB (dihydrodiol dehydrogenase) and BphC (2,3-dihydroxybiphenyl 1,2-dioxygenase) did not exhibit any 1, 2-dihydrogen 1, 2-dihydroxy phenazine and 1, 2-dihydroxy phenazine degradation capability, suggesting no contribution in phenazine degradation. Phylogenetic analysis of the dioxygenases demonstrated enormous biodegradation potential in strain B1. In conclusion, this study opens up new possibilities in better understanding the phenazine degradation in the environment.

Whole genome sequencing and analysis reveal insights into the genetic structure, diversity and evolutionary relatedness of luxI and luxR homologs in bacteria belonging to the Sphingomonadaceae family.

Here we report the draft genomes and annotation of four N-acyl homoserine lactone (AHL)-producing members from the family Sphingomonadaceae. Comparative genomic analyses of 62 Sphingomonadaceae genomes were performed to gain insights into the distribution of the canonical luxI/R-type quorum sensing (QS) network within this family. Forty genomes contained at least one luxR homolog while the genome of Sphingobium yanoikuyae B1 contained seven Open Reading Frames (ORFs) that have significant homology to that of luxR. Thirty-three genomes contained at least one luxI homolog while the genomes of Sphingobium sp. SYK6, Sphingobium japonicum, and Sphingobium lactosutens contained four luxI. Using phylogenetic analysis, the sphingomonad LuxR homologs formed five distinct clades with two minor clades located near the plant associated bacteria (PAB) LuxR solo clade. This work for the first time shows that 13 Sphingobium and one Sphingomonas genome(s) contain three convergently oriented genes composed of two tandem luxR genes proximal to one luxI (luxR-luxR-luxI). Interestingly, luxI solos were identified in two Sphingobium species and may represent species that contribute to AHL-based QS system by contributing AHL molecules but are unable to perceive AHLs as signals. This work provides the most comprehensive description of the luxI/R circuitry and genome-based taxonomical description of the available sphingomonad genomes to date indicating that the presence of luxR solos and luxI solos are not an uncommon feature in members of the Sphingomonadaceae family.

Effect of high pressure on hydrocarbon-degrading bacteria.

The blowout of the Deepwater Horizon in the Gulf of Mexico in 2010 occurred at a depth of 1500 m, corresponding to a hydrostatic pressure of 15 MPa. Up to now, knowledge about the impact of high pressure on oil-degrading bacteria has been scarce. To investigate how the biodegradation of crude oil and its components is influenced by high pressures, like those in deep-sea environments, hydrocarbon degradation and growth of two model strains were studied in high-pressure reactors. The alkane-degrading strain Rhodococcus qingshengii TUHH-12 grew well on n-hexadecane at 15 MPa at a rate of 0.16 h−1, although slightly slower than at ambient pressure (0.36 h−1). In contrast, the growth of the aromatic hydrocarbon degrading strain Sphingobium yanoikuyae B1 was highly affected by elevated pressures. Pressures of up to 8.8 MPa had little effect on growth of this strain. However, above this pressure growth decreased and at 12 MPa or more no more growth was observed. Nevertheless, S. yanoikuyae continued to convert naphthalene at pressure >12 MPa, although at a lower rate than at 0.1 MPa. This suggests that certain metabolic functions of this bacterium were inhibited by pressure to a greater extent than the enzymes responsible for naphthalene degradation. These results show that high pressure has a strong influence on the biodegradation of crude oil components and that, contrary to previous assumptions, the role of pressure cannot be discounted when estimating the biodegradation and ultimate fate of deep-sea oil releases such as the Deepwater Horizon event.

Experimental results and integrated modeling of bacterial growth on an insoluble hydrophobic substrate (phenanthrene).

Metabolism of a low-solubility substrate is limited by dissolution and availability and can hardly be determined. We developed a numerical model for simultaneously calculating dissolution kinetics of such substrates and their metabolism and microbial growth (Monod kinetics with decay) and tested it with three aerobic phenanthrene (PHE) degraders: Novosphingobium pentaromativorans US6-1, Sphingomonas sp. EPA505, and Sphingobium yanoikuyae B1. PHE was present as microcrystals, providing non-limiting conditions for growth. Total PHE and protein concentration were tracked over 6-12 days. The model was fitted to the test results for the rates of dissolution, metabolism, and growth. The strains showed similar efficiency, with vmax values of 12-18 g dw g(-1) d(-1), yields of 0.21 g g(-1), maximum growth rates of 2.5-3.8 d(-1), and decay rates of 0.04-0.05 d(-1). Sensitivity analysis with the model shows that (i) retention in crystals or NAPLs or by sequestration competes with biodegradation, (ii) bacterial growth conditions (dissolution flux and resulting chemical activity of substrate) are more relevant for the final state of the system than the initial biomass, and (iii) the desorption flux regulates the turnover in the presence of solid-state, sequestered (aged), or NAPL substrate sources.

Characterization of A Phenanthrene-degrading Strain and Cloning of Degradation-related Gene*

A bacterial strain 2F5-2 capable of degrading phenanthrene was isolated from petroleum-contaminated soil.It was preliminarily identified as Sphingobium sp.according to its physiological biochemical characteristics and the analysis of its 16S rRNA gene sequence.Strain 2F5-2 could degrade 100% of 100 mg/L phenanthrene within 10 h.The optimal pH and temperature for the degradation were 7 and 30 ℃,respectively.The analysis of its phenanthrene-degrading pathway revealed that strain 2F5-2 degraded phenanthrene via salicylate pathway.The aromatic-ring-hydroxylating dioxygenases α-subunit encoding gene phdA was cloned and analyzed.It had 97.9%,98%,100% identity to the phdA gene from Sphingomonas sp.P2,Sphingobium yanoikuyae B1 and Sphingomonas sp.ZP1,respectively,indicating the conservation of this gene.Fig 6,Ref 16

Characterization of a ring-hydroxylating dioxygenase from phenanthrene-degrading Sphingomonas sp. strain LH128 able to oxidize benz[a]anthracene

Sphingomonas sp. strain LH128 was isolated from a polycyclic aromatic hydrocarbon (PAH)-contaminated soil using phenanthrene as the sole source of carbon and energy. A dioxygenase complex, phnA1fA2f, encoding the alpha and beta subunit of a terminal dioxygenase responsible for the initial attack on PAHs, was identified and isolated from this strain. PhnA1f showed 98%, 78%, and 78% identity to the alpha subunit of PAH dioxygenase from Novosphingobium aromaticivorans strain F199, Sphingomonas sp. strain CHY-1, and Sphingobium yanoikuyae strain B1, respectively. When overexpressed in Escherichia coli, PhnA1fA2f was able to oxidize low-molecular-weight PAHs, chlorinated biphenyls, dibenzo-p-dioxin, and the high-molecular-weight PAHs benz[a]anthracene, chrysene, and pyrene. The action of PhnA1fA2f on benz[a]anthracene produced two benz[a]anthracene dihydrodiols.

Identification, cloning, and characterization of a multicomponent biphenyl dioxygenase from Sphingobium yanoikuyae B1

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.

Enhanced degradation of benzo[ a]pyrene by Mycobacterium sp. in conjunction with green alga

Previous work in this laboratory has confirmed that the bacteria Mycobacterium sp. strain RJGII.135 and Sphingomonas yanoikuyae strain B1 and the green alga Selanastrum capricornutum strain UTEX 1648 degrade benzo[ a]pyrene (BaP) to various BaP intermediates. S. capricornutum was first grown with BaP for 4 days. The organic extract of this media was then introduced into separate cultures of strain RJGII.135 and strain B1; separate cultures were grown with BaP for comparison. Cultures grown with BaP and those grown with the algal/BaP extract showed similar mineralization patterns. The quantity of total metabolites formed was greater in bacterial cultures grown with the algal/BaP extract than those grown with BaP alone. For strain RJGII.135, only 27% of the original BaP remained in cultures grown with the algal/BaP extract; 59% remained in cultures grown with BaP. For strain B1, only 6% of the original BaP remained in cultures grown with the algal/BaP extract; 38% remained in cultures grown with BaP. These results indicate that strategies utilizing organisms together may be necessary in being able to degrade large, recalcitrant polycyclic aromatic hydrocarbons (PAHs) such as BaP.

Structural investigations of the ferredoxin and terminal oxygenase components of the biphenyl 2,3-dioxygenase from Sphingobium yanoikuyae B1

BackgroundThe 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.ResultsIn 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.ConclusionThis 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.

Purification, characterization, and crystallization of the components of a biphenyl dioxygenase system from Sphingobium yanoikuyae B1

Sphingobium yanoikuyae B1 initiates the catabolism of biphenyl by adding dioxygen to the aromatic nucleus to form (+)-cis-(2R, 3S)-dihydroxy-1-phenylcyclohexa-4,6-diene. The present study focuses on the biphenyl 2,3-dioxygenase system, which catalyzes the dioxygenation reaction. This enzyme has been shown to have a broad substrate range, catalyzing the dioxygenation of not only biphenyl, but also three- and four-ring polycyclic aromatic hydrocarbons. Extracts prepared from biphenyl-grown B1 cells contained three protein components that were required for the oxidation of biphenyl. The genes encoding the three components (bphA4, bphA3 and bphA1f,A2f) were expressed in Escherichia coli. Biotransformations of biphenyl, naphthalene, phenanthrene, and benzo[a]pyrene as substrates using the recombinant E. coli strain resulted in the formation of the expected cis-dihydrodiol products previously shown to be produced by biphenyl-induced strain B1. The three protein components were purified to apparent homogeneity and characterized in detail. The reductase component (bphA4), designated reductase(BPH-B1), was a 43 kD monomer containing one mol FAD/mol reductase(BPH-B1). The ferredoxin component (bphA3), designated ferredoxin(BPH-B1), was a 12 kD monomer containing approximately 2 g-atoms each of iron and acid-labile sulfur. The oxygenase component (bphA1f,A2f), designated oxygenase(BPH-B1), was a 217 kD heterotrimer consisting of alpha and beta subunits (approximately 51 and 21 kD, respectively). The iron and acid-labile sulfur contents of oxygenase(BPH-B1) per alphabeta were 2.4 and 1.8 g-atom per mol, respectively. Reduced ferredoxin(BPH-B1) and oxygenase(BPH-B1) each gave EPR signals typical of Rieske [2Fe-2S] proteins. Crystals of reductase(BPH-B1), ferredoxin(BPH-B1) and oxygenase(BPH-B1 )diffracted to 2.5 A, 2.0 A and 1.75 A, respectively. The structures of the three proteins are currently being determined.

Effect of inoculum pretreatment on survival, activity and catabolic gene expression of Sphingobium yanoikuyae B1 in an aged polycyclic aromatic hydrocarbon-contaminated soil

The survival and effectiveness of a bioaugmentation strain in its target environment depend not only on physicochemical parameters in the soil but also on the physiological state of the inoculated organism. This study examined the effect of variations in inoculum pretreatment on the survival, metabolic activity (measured as rRNA content) and polycyclic aromatic hydrocarbon (PAH)-catabolic gene expression of Sphingobium yanoikuyae B1 in an aged PAH-contaminated soil. RNA denaturing gradient gel electrophoresis analysis showed stable colonization of PAH-contaminated soil by S. yanoikuyae B1 after four pretreatments (growth in complex or minimal medium, starvation, or acclimation to phenanthrene). By contrast, extractable CFUs decreased with time for all four treatments, and significantly faster for Luria Bertani-grown inocula, suggesting that these cells adhered strongly to soil particles while remaining metabolically active. Pretreatment of the inoculum had a dramatic effect on the expression of genes specific to the PAH-degradation pathway. The highest levels of bphC and xylE expression were seen for inocula that had been precultivated on complex medium, and degradation of PAHs was significantly enhanced in soils treated with these inocula. The results suggest that using complex media instead of minimal media for cultivating bioaugmentation inocula may improve the subsequent efficiency of contaminant biodegradation in the soil.

Autecological properties of soil sphingomonads involved in the degradation of polycyclic aromatic hydrocarbons

Autecological properties that are thought to be important for polycyclic aromatic hydrocarbon (PAH)-degradation by bacteria in contaminated soils include the ability to utilize a broad range of carbon sources, efficient biofilm formation, cell-surface hydrophobicity, surfactant production, motility, and chemotaxis. Sphingomonas species are common PAH-degraders, and a selection of PAH-degrading sphingomonad strains isolated from contaminated soils was therefore characterized in terms of these properties. All the sphingomonads tested were relatively hydrophilic and were able to grow as biofilms on a phenanthrene-coated surface, though biofilm formation under other conditions was variable. Sphingobium yanoikuyae B1 was able to utilize the greatest range of carbon sources, though it was not chemotaxic towards any of the substrates tested. Other sphingomonad strains were considerably less flexible in their catabolic range. None of the strains produced detectable surfactant and swimming motility varied between the strains. Examination of the total Sphingomonas community in the soils tested showed that one of the isolates studied was present at significant levels, suggesting that it can thrive under PAH-contaminated conditions despite the lack of many of the tested characteristics. We conclude that these properties are not essential for survival and persistence of Sphingomonas in PAH-contaminated soils.

Effect of Sphingobium yanoikuyae B1 inoculation on bacterial community dynamics and polycyclic aromatic hydrocarbon degradation in aged and freshly PAH-contaminated soils

Sphingobium yanoikuyae B1 is able to degrade a range of polycyclic aromatic hydrocarbons (PAHs) and as a sphingomonad belongs to one of the dominant genera found in PAH-contaminated soils. We examined the ecological effect that soil inoculation with S. yanoikuyae B1 has on the native bacterial community in three different soils: aged PAH-contaminated soil from an industrial site, compost freshly contaminated with PAHs and un-contaminated compost. Survival of S. yanoikuyae B1 was dependent on the presence of PAHs, and the strain was unable to colonize un-contaminated compost. Inoculation with S. yanoikuyae B1 did not cause extensive changes in the native bacterial community of either soil, as assessed by denaturing gel electrophoresis, but its presence led to an increase in the population level of two other species in the aged contaminated soil community and appeared to have an antagonistic affect on several members of the contaminated compost community, indicating niche competition.

Evidence for the role of 2-hydroxychromene-2-carboxylate isomerase in the degradation of anthracene by Sphingomonas yanoikuyae B1.

Sphingomonas yanoikuyae B1 is extremely versatile in its catabolic ability. An insertional mutant strain, S. yamoikuyae EK504, which is unable to grow on naphthalene due to the loss of 2-hydroxychromene-2-carboxylate isomerase activity, was utilized to investigate the role of this enzyme in the degradation of anthracene by S. yanoikuyae B1. Although EK504 is unable to grow on anthracene, this strain could transform anthracene to some extent. A metabolite in the degradation of anthracene by EK504 was isolated by high-pressure liquid chromatography (HPLC) and was identified as 6,7-benzocoumarin by UV-visible, gas-chromatographic, HPLC/mass-spectrometric, and 1H nuclear magnetic resonance spectral techniques. The identification of 6,7-benzocoumarin provides direct chemical and genetic evidence for the involvement of nahD in the degradation of anthracene by S. yanoikuyae B1.

Catabolic role of a three-component salicylate oxygenase from Sphingomonas yanoikuyae B1 in polycyclic aromatic hydrocarbon degradation

Sphingomonas yanoikuyae B1 possesses several different multicomponent oxygenases involved in metabolizing aromatic compounds. Six different pairs of genes encoding large and small subunits of oxygenase iron–sulfur protein components have previously been identified in a gene cluster involved in the degradation of both monocyclic and polycyclic aromatic hydrocarbons. Insertional inactivation of one of the oxygenase large subunit genes, bphA1c, results in a mutant strain unable to grow on naphthalene, phenanthrene, or salicylate. The knockout mutant accumulates salicylate from naphthalene and 1-hydroxy-2-naphthoic acid from phenanthrene indicating the loss of salicylate oxygenase activity. Complementation experiments verify that the salicylate oxygenase in S. yanoikuyae B1 is a three-component enzyme consisting of an oxygenase encoded by bphA2cA1c, a ferredoxin encoded by the adjacent bphA3, and a ferredoxin reductase encoded by bphA4 located over 25 kb away. Expression of bphA3-bphA2c-bphA1c genes in Escherichia coli demonstrated the ability of salicylate oxygenase to convert salicylate to catechol and 3-, 4-, and 5-methylsalicylate to methylcatechols.

Xylene monooxygenase, a membrane-spanning non-heme diiron enzyme that hydroxylates hydrocarbons via a substrate radical intermediate.

The non-heme diiron enzyme xylene monooxygenase (XylM) has been shown to hydroxylate hydrocarbons via a hydrogen abstraction-carbon radical recombination mechanism (oxygen rebound). Using the radical clock bicyclo[4.1.0]heptane (norcarane) in a whole-cell assay, and observing the ratio of rearranged 3-(hydroxymethyl)cyclohexene and unrearranged 2-norcaranol products, the lifetime of the substrate radical was determined to be approximately 0.2 ns. The wild-type organism Pseudomonas putida mt-2 and two separate Escherichia coli clones expressing xylMA genes gave similar results. One clone produced the Pseudomonas putida mt-2 XylMA hydroxylase and the other produced Sphingomonas yanoikuyae B1 XylMA hydroxylase. Clones were constructed by inserting genes for xylene monooxygenase and xylene monooxygenase reductase downstream from an IPTG-inducible T7 promoter. Mechanistic investigations using whole-cell assays will facilitate more rapid screening of structure-function relationships and the identification of novel oxygenases. This approach should enable the construction of a picture of the key metalloenzymes and the mechanisms they use in selected parts of the global carbon cycle without requiring the isolation of every protein involved.