The long-chain flavodoxin FldX1 improves the biodegradation of 4-hydroxyphenylacetate and 3-hydroxyphenylacetate and counteracts the oxidative stress associated to aromatic catabolism in Paraburkholderia xenovorans.

BACKGROUND: Bacterial aromatic degradation may cause oxidative stress. The long-chain flavodoxin FldX1 of Paraburkholderia xenovorans LB400 counteracts reactive oxygen species (ROS). The aim of this study was to evaluate the protective role of FldX1 in P. xenovorans LB400 during the degradation of 4-hydroxyphenylacetate (4-HPA) and 3-hydroxyphenylacetate (3-HPA). METHODS: The functionality of FldX1 was evaluated in P. xenovorans p2-fldX1 that overexpresses FldX1. The effects of FldX1 on P. xenovorans were studied measuring growth on hydroxyphenylacetates, degradation of 4-HPA and 3-HPA, and ROS formation. The effects of hydroxyphenylacetates (HPAs) on the proteome (LC-MS/MS) and gene expression (qRT-PCR) were quantified. Bioaugmentation with strain p2-fldX1 of 4-HPA-polluted soil was assessed, measuring aromatic degradation (HPLC), 4-HPA-degrading bacteria, and plasmid stability. RESULTS: The exposure of P. xenovorans to 4-HPA increased the formation of ROS compared to 3-HPA or glucose. P. xenovorans p2-fldX1 showed an increased growth on 4-HPA and 3-HPA compared to the control strain WT-p2. Strain p2-fldX1 degraded faster 4-HPA and 3-HPA than strain WT-p2. Both WT-p2 and p2-fldX1 cells grown on 4-HPA displayed more changes in the proteome than cells grown on 3-HPA in comparison to glucose-grown cells. Several enzymes involved in ROS detoxification, including AhpC2, AhpF, AhpD3, KatA, Bcp, CpoF1, Prx1 and Prx2, were upregulated by hydroxyphenylacetates. Downregulation of organic hydroperoxide resistance (Ohr) and DpsA proteins was observed. A downregulation of the genes encoding scavenging enzymes (katE and sodB), and gstA and trxB was observed in p2-fldX1 cells, suggesting that FldX1 prevents the antioxidant response. More than 20 membrane proteins, including porins and transporters, showed changes in expression during the growth of both strains on hydroxyphenylacetates. An increased 4-HPA degradation by recombinant strain p2-fldX1 in soil microcosms was observed. In soil, the strain overexpressing the flavodoxin FldX1 showed a lower plasmid loss, compared to WT-p2 strain, suggesting that FldX1 contributes to bacterial fitness. Overall, these results suggest that recombinant strain p2-fldX1 is an attractive bacterium for its application in bioremediation processes of aromatic compounds. CONCLUSIONS: The long-chain flavodoxin FldX1 improved the capability of P. xenovorans to degrade 4-HPA in liquid culture and soil microcosms by protecting cells against the degradation-associated oxidative stress.

Effect of Microclimate on the Mass Emergence of Hypothenemus hampei in Coffee Grown under Shade of Trees and in Full Sun Exposure.

The rainfall regime has a significant impact on the microclimate and mass emergence of the coffee berry borer, Hypothenemus hampei (Coleoptera: Curculionidae) (CBB). Little is known, however, about the shade tree-microclimate-CBB mass emergence interaction. The objective of the present study was to compare the effect of microclimate on the mass emergence of CBB in a full sun-exposed plot with a plot shaded by trees. The experiment was conducted on a Robusta coffee farm in southern Chiapas, Mexico. In each plot, 18 traps baited with an alcohol mixture were installed to capture flying females, collecting caught individuals every hour from 8:00 to 18:00 h. A meteorological station recorded several microclimatic variables on 13 weekly sampling dates from February to May 2022. Significantly more CBB females were captured in the shaded plot. The largest number of CBB captures was recorded between 14:00 and 16:00 h for the shade plot and between 15:00 and 17:00 h for the sun-exposed plot. The mass emergence of CBB showed a positive association with precipitation, dew point, and wind speed samples and a negative association with maximum air temperature, average relative humidity, ultraviolet radiation, wind speed, and equilibrium moisture content. Our observations show that the relationship between shade trees, microclimate, and mass emergence of CBB is complex and that its study helps us to gain deeper insight into CBB bioecology and advance control techniques against this important pest.

Aerobic biotransformation of 6:2 fluorotelomer sulfonate by Dietzia aurantiaca J3 under sulfur-limiting conditions.

The polyfluorinated alkyl substance 6:2 fluorotelomer sulfonate (6:2 FTS) has been detected in diverse environments impacted by aqueous film-forming foams used for firefighting. In this study, a bacterial strain (J3) using 6:2 FTS as a sulfur source was isolated from landfill leachate previously exposed to polyfluoroalkyl substances in New South Wales, Australia. Strain J3 shares 99.9% similarity with the 16S rRNA gene of Dietzia aurantiaca CCUG 35676T. Genome sequencing yielded a draft genome sequence of 37 contigs with a G + C content of 69.7%. A gene cluster related to organic sulfur utilisation and assimilation was identified, that included an alkanesulfonate monooxygenase component B (ssuD), an alkanesulfonate permease protein (ssuC), an ABC transporter (ssuB), and an alkanesulfonate-binding protein (ssuA). Proteomic analyses comparing strain J3 cultures using sulfate and 6:2 FTS as sulfur source indicated that the ssu gene cluster was involved in 6:2 FTS biodegradation. Upregulated proteins included the SsuD monooxygenase, the SsuB transporter, the ABC transporter permease (SsuC), an alkanesulfonate-binding protein (SsuA), and a nitrilotriacetate monooxygenase component B. 6:2 Fluorotelomer carboxylic acid (6:2 FTCA) and 6:2 fluorotelomer unsaturated acid (6:2 FTUA) were detected as early degradation products in cultures (after 72 h) while 5:3 fluorotelomer acid (5:3 FTCA), perfluorohexanoic acid (PFHxA) and perfluoropentanoic acid (PFPeA) were detected as later degradation products (after 168 h). This work provides biochemical and metabolic insights into 6:2 FTS biodegradation by the Actinobacterium D. aurantiaca J3, informing the fate of PFAS in the environment.

The OxyR and SoxR transcriptional regulators are involved in a broad oxidative stress response in Paraburkholderia xenovorans LB400.

BACKGROUND: Aerobic metabolism generates reactive oxygen species that may cause critical harm to the cell. The aim of this study is the characterization of the stress responses in the model aromatic-degrading bacterium Paraburkholderia xenovorans LB400 to the oxidizing agents paraquat and H2O2. METHODS: Antioxidant genes were identified by bioinformatic methods in the genome of P. xenovorans LB400, and the phylogeny of its OxyR and SoxR transcriptional regulators were studied. Functionality of the transcriptional regulators from strain LB400 was assessed by complementation with LB400 SoxR of null mutant P. aeruginosa ΔsoxR, and the construction of P. xenovorans pIZoxyR that overexpresses OxyR. The effects of oxidizing agents on P. xenovorans were studied measuring bacterial susceptibility, survival and ROS formation after exposure to paraquat and H2O2. The effects of these oxidants on gene expression (qRT-PCR) and the proteome (LC-MS/MS) were quantified. RESULTS: P. xenovorans LB400 possesses a wide repertoire of genes for the antioxidant defense including the oxyR, ahpC, ahpF, kat, trxB, dpsA and gorA genes, whose orthologous genes are regulated by the transcriptional regulator OxyR in E. coli. The LB400 genome also harbors the soxR, fumC, acnA, sodB, fpr and fldX genes, whose orthologous genes are regulated by the transcriptional regulator SoxR in E. coli. The functionality of the LB400 soxR gene was confirmed by complementation of null mutant P. aeruginosa ΔsoxR. Growth, susceptibility, and ROS formation assays revealed that LB400 cells were more susceptible to paraquat than H2O2. Transcriptional analyses indicated the upregulation of the oxyR, ahpC1, katE and ohrB genes in LB400 cells after exposure to H2O2, whereas the oxyR, fumC, ahpC1, sodB1 and ohrB genes were induced in presence of paraquat. Proteome analysis revealed that paraquat induced the oxidative stress response proteins AhpCF and DpsA, the universal stress protein UspA and the RNA chaperone CspA. Both oxidizing agents induced the Ohr protein, which is involved in organic peroxide resistance. Notably, the overexpression of the LB400 oxyR gene in P. xenovorans significantly decreased the ROS formation and the susceptibility to paraquat, suggesting a broad OxyR-regulated antioxidant response. CONCLUSIONS: This study showed that P. xenovorans LB400 possess a broad range oxidative stress response, which explain the high resistance of this strain to the oxidizing compounds paraquat and H2O2.

The OxyR and SoxR transcriptional regulators are involved in a broad oxidative stress response in Paraburkholderia xenovorans LB400

BACKGROUND: Aerobic metabolism generates reactive oxygen species that may cause critical harm to the cell. The aim of this study is the characterization of the stress responses in the model aromatic degrading bacterium Paraburkholderia xenovorans LB400 to the oxidizing agents paraquat and H 2 O2. METHODS: Antioxidant genes were identified by bioinformatic methods in the genome of P. xenovorans LB400, and the phylogeny of its OxyR and SoxR transcriptional regulators were studied. Functionality of the transcriptional regulators from strain LB400 was assessed by complementation with LB400 SoxR of null mutant P. aeruginosa ΔsoxR, and the construction of P. xenovorans pIZ oxyR that overexpresses OxyR. The effects of oxidizing agents on P. xenovorans were studied measuring bacterial susceptibility, survival and ROS formation after exposure to paraquat and H 2 O2. The effects of these oxidants on gene expression (qRT PCR) and the proteome (LC-MS/MS) were quantified. RESULTS: P. xenovorans LB400 possesses a wide repertoire of genes for the antioxidant defense including the oxyR , ahpC , ahpF , kat , trxB , dpsA and gorA genes, whose orthologous genes are regulated by the transcriptional regulator OxyR in E. coli . The LB400 genome also harbors the soxR, fumC , acnA , sodB , fpr and fldX genes, whose orthologous genes are regulated by the transcriptional regulator SoxR in E. coli . The functionality of the LB400 soxR gene was confirmed by complementation of null mutant P. aeruginosa Δ soxR . Growth, susceptibility, and ROS formation assays revealed that LB400 cells were more susceptible to paraquat than H2O2. Transcriptional analyses indicated the upregulation of the oxyR , ahpC1 , katE and ohrB genes in LB400 cells after exposure to H2O2, whereas the oxyR , fumC , ahpC1 , sodB1 and ohrB genes were induced in presence of paraquat. Proteome analysis revealed that paraquat induced the oxidative stress response proteins AhpCF and DpsA, the universal stress protein UspA and the RNA chaperone CspA. Both oxidizing agents induced the Ohr protein, which is involved in organic peroxide resistance. Notably, the overexpression of the LB400 oxyR gene in P. xenovorans significantly decreased the ROS formation and the susceptibility to paraquat, suggesting a broad OxyR regulated antioxidant response. CONCLUSIONS: This study showed that P. xenovorans LB400 possess a broad range oxidative stress response, which explain the high resistance of this strain to the oxidizing compounds paraquat and H2O2.

Comparative Genomics of Pathogenic Clavibacter michiganensis subsp. michiganensis Strains from Chile Reveals Potential Virulence Features for Tomato Plants.

The genus Clavibacter has been associated largely with plant diseases. The aims of this study were to characterize the genomes and the virulence factors of Chilean C. michiganensis subsp. michiganensis strains VL527, MSF322 and OP3, and to define their phylogenomic positions within the species, Clavibacter michiganensis. VL527 and MSF322 genomes possess 3,396,632 and 3,399,199 bp, respectively, with a pCM2-like plasmid in strain VL527, with pCM1- and pCM2-like plasmids in strain MSF322. OP3 genome is composed of a chromosome and three plasmids (including pCM1- and pCM2-like plasmids) of 3,466,104 bp. Genomic analyses confirmed the phylogenetic relationships of the Chilean strains among C.michiganensis subsp. michiganensis and showed their low genomic diversity. Different virulence levels in tomato plants were observable. Phylogenetic analyses of the virulence factors revealed that the pelA1 gene (chp/tomA region)-that grouped Chilean strains in three distinct clusters-and proteases and hydrolases encoding genes, exclusive for each of the Chilean strains, may be involved in these observed virulence levels. Based on genomic similarity (ANIm) analyses, a proposal to combine and reclassify C. michiganensis subsp. phaseoli and subsp. chilensis at the species level, as C. phaseoli sp. nov., as well as to reclassify C. michiganensis subsp. californiensis as the species C. californiensis sp. nov. may be justified.

Complete Genome Sequence of Hydrocarbon-Degrading Halotolerant Acinetobacter radioresistens DD78, Isolated from the Aconcagua River Mouth in Central Chile.

Acinetobacter radioresistens strain DD78 (= CCUG 69565) is a soil hydrocarbon-degrading and biosurfactant-producing bacterium isolated from chronically crude oil-polluted soil of the Aconcagua River mouth in Chile. The 3.25-Mb A. radioresistens DD78 genome (41.8% GC content) was completely sequenced, with 4 replicons, 2,970 coding sequences, and 77 tRNAs.

Long-chain flavodoxin FldX1 improves Paraburkholderia xenovorans LB400 tolerance to oxidative stress caused by paraquat and H2O2.

Flavodoxins are small electron transfer proteins containing flavin mononucleotide (FMN) as a prosthetic group, which play an important role during oxidative stress or iron limitation. The aims of this study were the identification and characterization of flavodoxins in the model aromatic-degrader Paraburkholderia xenovorans LB400 and the analyses of their protective effects during oxidative stress induced by paraquat and H2O2. Two genes (BxeA0278 and BxeB0391) encoding flavodoxins (hereafter referred to as fldX for flavodoxin from P. xenovorans), were identified at the LB400 major and minor chromosome. Genomic context of the flavodoxin-encoding genes showed genes encoding membrane proteins, transporters, and proteins involved in redox processes and biosynthesis of macromolecules. A secondary structure prediction of both LB400 flavodoxins showed the characteristic flavodoxin structure of five ß-sheets intercalated with five α-helices. FldX1 contains a loop intercalated in the fifth ß-strand, which indicates that it belongs to the long-chain flavodoxins, whereas FldX2 is a short-chain flavodoxin. A phylogenetic analysis of 73 flavodoxins from 43 bacterial genera revealed eight clusters (I-VIII), while FldX1 and FldX2 grouped separately within a long-chain and a short-chain flavodoxin clades. FldX1 and FldX2 were overexpressed in P. xenovorans. Interestingly, the strain overexpressing the long-chain flavodoxin FldX1 (p2-fldX1) showed a faster growth in glucose than the control strain. The recombinant strain overexpressing the long-chain flavodoxin FldX1 (p2-fldx1) exposed to paraquat (20 mM) possessed lower susceptibility to growth inhibition on plates and higher survival in liquid medium than the control strain. The strains overexpressing the flavodoxins FldX1 and FldX2 showed higher survival during exposure to 1 mM paraquat (>95%) than the control strain (68%). Compared to the control strain, strains overexpressing FldX1 and FldX2 showed lower lipid peroxidation (>20%) after exposure to 1 mM paraquat and a lower protein carbonylation (~30%) after exposure to 1 mM H2O2 was observed. During exposure to paraquat, strain p2-fldx1 downregulated the katG4, hpf, trxB1 and ohr genes (> 2-fold), whereas strain p2-fldx2 upregulated the oxyR and ahpC1 genes (> 2-fold). In conclusion, the flavodoxins FldX1 and FldX2 of P. xenovorans LB400 conferred protection to cells exposed to the oxidizing agents paraquat and H2O2.

Genomic and Physiological Traits of the Marine Bacterium Alcaligenes aquatilis QD168 Isolated From Quintero Bay, Central Chile, Reveal a Robust Adaptive Response to Environmental Stressors.

Alcaligenes aquatilis QD168 is a marine, aromatic hydrocarbon-degrading bacterium, isolated from an oil-polluted sediment of Quintero Bay, an industrial-coastal zone that has been chronically impacted by diverse pollutants. The aims of this study were to characterize the phylogenomic positions of Alcaligenes spp. and to characterize the genetic determinants and the physiological response of A. aquatilis QD168 to model environmental stressors (benzene, oxidizing agents, and salt). Phylogenomic analyses, using 35 housekeeping genes, clustered A. aquatilis QD168 with four other strains of Alcaligenes spp. (A. aquatilis BU33N, A. faecalis JQ135, A. faecalis UBA3227, and A. faecalis UBA7629). Genomic sequence analyses of A. aquatilis QD168 with 25 Alcaligenes spp., using ANIb, indicated that A. aquatilis BU33N is the closest related strain, with 96.8% ANIb similarity. Strain QD168 harbors 95 genes encoding proteins of seven central catabolic pathways, as well as sixteen peripheral catabolic pathways/reactions for aromatic compounds. A. aquatilis QD168 was able to grow on 3-hydroxybenzoate, 4-hydroxybenzoate, benzoate, benzene, 3-hydroxycinnamate, cinnamate, anthranilate, benzamide, 4-aminobenzoate, nicotinate, toluene, biphenyl and tryptophan, as sole carbon or nitrogen source. Benzene degradation was further analyzed by growth, metabolite identification and gene expression analyses. Benzene strongly induced the expression of the genes encoding phenol hydroxylase (dmpP) and catechol 1,2-dioxygenase (catA). Additionally, 30 genes encoding transcriptional regulators, scavenging enzymes, oxidative damage repair systems and isozymes involved in oxidative stress response were identified. Oxidative stress response of strain QD168 to hydrogen peroxide and paraquat was characterized, demonstrating that A. aquatilis QD168 is notably more resistant to paraquat than to H2O2. Genetic determinants (47 genes) for osmoprotective responses were identified, correlating with observed high halotolerance by strain QD168. The physiological adaptation of A. aquatilis QD168 to environmental stressors such as pollutants, oxidative stress and salinity may be exploited for bioremediation of oil-polluted saline sites.

Complete Genome Sequence of the Marine Hydrocarbon Degrader Alcaligenes aquatilis QD168, Isolated from Crude Oil-Polluted Sediment of Quintero Bay, Central Chile.

Alcaligenes aquatilis strain QD168 (= CCUG 69566) is a marine hydrocarbon-degrading bacterium isolated from crude oil-polluted sediment from Quintero Bay, Central Chile. Here, we present the 4.32-Mb complete genome sequence of strain QD168, with 3,892 coding sequences, 58 tRNAs, and a 56.3% G+C content.

Complete Genome Sequence of the Hydrocarbon-Degrading Strain Achromobacter sp. B7, Isolated during Petroleum Hydrocarbon Bioremediation in the Valparaiso Region, Chile.

Achromobacter sp. strain B7 (= CCUG 72081) was isolated from a diesel-polluted soil from the Valparaiso Region, Chile, subjected to bioremediation with a hydrocarbon-degrading enrichment. The complete genome sequence of Achromobacter sp. B7 has been determined to have a size of 6.24 Mb, 5,578 coding sequences, 57 tRNAs, and a G+C content of 64.8%.

An aryl dioxygenase shows remarkable double dioxygenation capacity for diverse bis-aryl compounds, provided they are carbocyclic.

The bacterial dioxygenation of mono- or polycyclic aromatic compounds is an intensely studied field. However, only in a few cases has the repeated dioxygenation of a substrate possessing more than a single aromatic ring been described. We previously characterized the aryl-hydroxylating dioxygenase BphA-B4h, an artificial hybrid of the dioxygenases of the biphenyl degraders Burkholderia xenovorans LB400 and Pseudomonas sp. strain B4-Magdeburg, which contains the active site of the latter enzyme, as an exceptionally powerful biocatalyst. We now show that this dioxygenase possesses a remarkable capacity for the double dioxygenation of various bicyclic aromatic compounds, provided that they are carbocyclic. Two groups of biphenyl analogues were examined: series A compounds containing one heterocyclic aromatic ring and series B compounds containing two homocyclic aromatic rings. Whereas all of the seven partially heterocyclic biphenyl analogues were solely dioxygenated in the homocyclic ring, four of the six carbocyclic bis-aryls were converted into ortho,meta-hydroxylated bis-dihydrodiols. Potential reasons for failure of heterocyclic dioxygenations are discussed. The obtained bis-dihydrodiols may, as we also show here, be enzymatically re-aromatized to yield the corresponding tetraphenols. This opens a way to a range of new polyphenolic products, a class of compounds known to exert multiple biological activities. Several of the obtained compounds are novel molecules.

Stepwise conversion of flavonoids by engineered dioxygenases and dehydrogenase: Characterization of novel biotransformation products.

Flavonoids are a large group of plant secondary metabolites that exert various biological and pharmacological effects. In this context, the generation of derivatives is of considerable interest. The introduction of hydroxy groups is of particular relevance, as they are known to be involved in many of the biological interactions and furthermore enable additional modifications, such as glycosylations. Bacterial aryl-hydroxylating dioxygenases (ARHDOs) have proven to be very useful for the conversion of aromatic structures into versatile building blocks for different kinds of derivatizations. Such enzymes have been used with varying success for the oxidation of flavonoids. In order to find better ARHDOs for the hydroxylation of such substrates, we carried out biotransformation trials with a collection of hybrid ARHDOs of different origin, using resting cells of recombinant strains. This identified enzymes able to transform all of the flavonoids examined, typically in yields above 50%. It also showed that moderately reactive substituents of flavonoids, such as hydroxy or amino groups, can lead to spontaneous follow-up reactions with the dienediol structures generated by dioxygenation. A report of flavanone epoxidation, a reaction never before observed to be catalyzed by an ARHDO, is challenged by our results. All ARHDOs examined converted this substrate into a dehydrogenase-transformable dihydrodiol. All dihydrodiols obtained by dioxygenation of the examined flavonoids were successfully re-aromatized into catechols by a bacterial dehydrogenase. These metabolites were usually stable. However, the catechols formed from flavanone and 2'-hydroxy-chalcone, respectively, were interconvertible under mild conditions. Altogether, we isolated and characterized 13 compounds that have not previously been described. The biotransformations reported here give access to novel flavonoid derivatives that may be applied for biological screens as well as for further modification, such as glycodiversification.

Permissivity of the biphenyl-specific aerobic bacterial metabolic pathway towards analogues with various steric requirements.

It has repeatedly been shown that aryl-hydroxylating dioxygenases do not possess a very high substrate specificity. To gain more insight into this phenomenon, we examined two powerful biphenyl dioxygenases, the well-known wild-type enzyme from Burkholderia xenovorans LB400 (BphA-LB400) and a hybrid enzyme, based on a dioxygenase from Pseudomonas sp. B4-Magdeburg (BphA-B4h), for their abilities to dioxygenate a selection of eight biphenyl analogues in which the second aromatic ring was replaced by aliphatic as well as aliphatic/aromatic moieties, reflecting a variety of steric requirements. Interestingly, both enzymes were able to catalyse transformation of almost all of these compounds. While the products formed were identical, major differences were observed in transformation rates. In most cases, BphA-B4h proved to be a significantly more powerful catalyst than BphA-LB400. NMR characterization of the reaction products showed that the metabolite obtained from biphenylene underwent angular dioxygenation, whereas all other compounds were subject to lateral dioxygenation at ortho and meta carbons. Subsequent growth studies revealed that both dioxygenase source strains were able to utilize several of the biphenyl analogues as sole sources of carbon and energy. Therefore, prototype BphBCD enzymes of the biphenyl degradative pathway were examined for their ability to further catabolize the lateral dioxygenation products. All of the ortho- and meta-hydroxylated compounds were converted to acids, showing that this pathway is quite permissive, enabling catalysis of the turnover of a fairly wide variety of metabolites.

Bioremediation of petroleum hydrocarbons: catabolic genes, microbial communities, and applications.

Bioremediation is an environmental sustainable and cost-effective technology for the cleanup of hydrocarbon-polluted soils and coasts. In spite of that longer times are usually required compared with physicochemical strategies, complete degradation of the pollutant can be achieved, and no further confinement of polluted matrix is needed. Microbial aerobic degradation is achieved by the incorporation of molecular oxygen into the inert hydrocarbon molecule and funneling intermediates into central catabolic pathways. Several families of alkane monooxygenases and ring hydroxylating dioxygenases are distributed mainly among Proteobacteria, Actinobacteria, Firmicutes and Fungi strains. Catabolic routes, regulatory networks, and tolerance/resistance mechanisms have been characterized in model hydrocarbon-degrading bacteria to understand and optimize their metabolic capabilities, providing the basis to enhance microbial fitness in order to improve hydrocarbon removal. However, microbial communities taken as a whole play a key role in hydrocarbon pollution events. Microbial community dynamics during biodegradation is crucial for understanding how they respond and adapt to pollution and remediation. Several strategies have been applied worldwide for the recovery of sites contaminated with persistent organic pollutants, such as polycyclic aromatic hydrocarbons and petroleum derivatives. Common strategies include controlling environmental variables (e.g., oxygen availability, hydrocarbon solubility, nutrient balance) and managing hydrocarbon-degrading microorganisms, in order to overcome the rate-limiting factors that slow down hydrocarbon biodegradation.

Genomic and functional analyses of the gentisate and protocatechuate ring-cleavage pathways and related 3-hydroxybenzoate and 4-hydroxybenzoate peripheral pathways in Burkholderia xenovorans LB400.

In this study, the gentisate and protocatechuate pathways in Burkholderia xenovorans LB400 were analyzed by genomic and functional approaches, and their role in 3-hydroxybenzoate (3-HBA) and 4-hydroxybenzoate (4-HBA) degradation was proposed. The LB400 genome possesses two identical mhbRTDHI gene clusters encoding the gentisate pathway and one mhbM gene encoding a 3-HBA 6-hydroxylase that converts 3-HBA into gentisate. The pca genes encoding the protocatechuate pathway and the pobA gene encoding the 4-HBA 3-monooxygenase that oxidizes 4-HBA into protocatechuate are arranged in gene clusters and single genes mainly at the minor chromosome, but also at the major chromosome and the megaplasmid. Strain LB400 was able to grow on gentisate, protocatechuate, 3-HBA and 4-HBA. Transcriptional analyses showed that the mhbD gene encoding the gentisate 1,2-dioxygenase was expressed during growth on 3-HBA, 4-HBA and gentisate, whereas the pcaG gene encoding the protocatechuate 3,4-dioxygenase was expressed only during growth on 4-HBA and protocatechuate. The mhbM gene encoding the 3-HBA 6-hydroxylase was transcribed in strain LB400 during growth on HBAs, gentisate, protocatechuate and glucose. The pobA gene encoding the 4-HBA 3-monooxygenase was expressed during growth on HBAs and glucose. 3-HBA- and 4-HBA-grown LB400 cells showed gentisate 1,2-dioxygenase activity, whereas protocatechuate 3,4-dioxygenase activity was observed only in 4-HBA-grown cells. The mhbR gene encoding a MarR-type transcriptional regulator that probably regulates the expression of the MhbT transporter, and the pcaQ and pcaR genes encoding LysR-type transcriptional regulators that regulate pcaHG and pcaIJBDC genes, respectively, were transcribed during growth on both HBAs, gentisate, protocatechuate and glucose, suggesting a basal constitutive expression. The results indicate active gentisate, protocatechuate, 3-HBA and 4-HBA catabolic pathways in B. xenovorans LB400 and suggest that 3-HBA is channeled exclusively through the gentisate route, whereas 4-HBA is funneled into the protocatechuate central pathway and potentially into the gentisate pathway.

Dioxygenation of the biphenyl dioxygenation product.

Two biphenyl dioxygenases (BphAs) were shown to catalyze dioxygenation of biphenyldienediol in the nonoxidized ring to form the respective symmetrical biphenyl-bis-dienediol. This novel metabolite served as a growth substrate for both BphA source strains. Its catabolism through the upper bph pathway of Burkholderia xenovorans LB400 was analyzed.

The homogentisate and homoprotocatechuate central pathways are involved in 3- and 4-hydroxyphenylacetate degradation by Burkholderia xenovorans LB400.

BACKGROUND: Genome characterization of the model PCB-degrading bacterium Burkholderia xenovorans LB400 revealed the presence of eleven central pathways for aromatic compounds degradation, among them, the homogentisate and the homoprotocatechuate pathways. However, the functionality of these central pathways in strain LB400 has not been assessed and related peripheral pathways has not been described. METHODOLOGY/PRINCIPAL FINDINGS: The aims of this study were to determine the functionality of the homogentisate and homoprotocatechuate central pathways in B. xenovorans LB400 and to establish their role in 3-hydroxyphenylacetate (3-HPA) and 4-hydroxyphenylacetate (4-HPA) catabolism. Strain LB400 was able to grow using 3-HPA and 4-HPA as sole carbon source. A genomic search in LB400 suggested the presence of mhaAB and hpaBC genes clusters encoding proteins of the 3-hydroxyphenylacetate and 4-hydroxyphenylacetate peripheral pathways. LB400 cells grown with 3-HPA and 4-HPA degraded homogentisate and homoprotocatechuate and showed homogentisate 1,2-dioxygenase and homoprotocatechuate 2,3-dioxygenase activities. Transcriptional analyses by RT-PCR showed the expression of two chromosomally-encoded homogentisate dioxygenases (BxeA2725 and BxeA3900) and the hpaD gene encoding the homoprotocatechuate 2,3-dioxygenase during 3-HPA and 4-HPA degradation. The proteome analyses by two-dimensional polyacrilamide gel electrophoresis of B. xenovorans LB400 grown in 3-HPA and 4-HPA showed the induction of fumarylacetoacetate hydrolase HmgB (BxeA3899). CONCLUSIONS/SIGNIFICANCE: This study revealed that strain LB400 used both homogentisate and homoprotocatechuate ring-cleavage pathways for 3- hydroxyphenylacetate and 4-hydroxyphenylacetate catabolism and that these four catabolic routes are functional, confirming the metabolic versatility of B. xenovorans LB400.