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 β-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.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  Flavodoxins also can transfer one electron with reduction potentials: E0′ of the quinone (Q)/semiquinone couple (SQ) is at least 200 mV more positive than E0′ of the SQ/quinol (HQ) couple that is −400 mV or more negative [Flavodoxins are small electronic transfer flavoproteins, highly isofuntional with ferredoxins, expressed under oxidative stress and iron-limitation ,9. Ferrenegative . Ferredonegative . Based onegative . The mainegative ,12.Azotobacter vinelandii and Klebsiella pneumoniae, electron carrier between photosystem I and the ferredoxin-NADP(H) reductase under iron deficit in cyanobacteria Anabaena and Synechocystis, methionine biosynthesis in Escherichia coli, pyruvate oxidation in Helicobacter pylori, biotin synthesis in Bacillus subtilis, the reduction of oxidized metal prosthetic groups and antioxidant response in E. coli and P. aeruginosa [Anabaena sp. PCC7119 in tobacco plants plastids enhances the detoxification of 2,4-dinitrotoluene and confers resistance to the redox cycling herbicide paraquat, and other stress conditions, such as extreme temperatures and high irradiation [Flavodoxins were discovered in cyanobacteria and characterized by their role in photosynthesis. Nevertheless, these proteins are involved in several processes, including nitrate reduction in ruginosa ,3,13. Thadiation .Burkholderiales order possess a high metabolic versatility for the degradation of aromatic compounds [Paraburkholderia xenovorans LB400 is a model bacterium to study the degradation of polychlorobiphenyls (PCBs) and other aromatic compounds. P. xenovorans LB400 was isolated from a PCB-contaminated landfill in New York, United States. Strain LB400 possesses a 9.73 Mpb genome, distributed in the major chromosome , the minor chromosome  and the megaplasmid  [P. xenovorans LB400 to (chloro)biphenyls  and chlorobenzoates  increases the expression of molecular chaperones , alkyl hydroperoxide reductase (AhpC), membrane proteins and enzymes involved in energy production, which indicates a stress response [p-cymene degradation, an increase of the molecular chaperones DnaK, GroEL and ClpB, the organic hydroperoxide resistance protein (Ohr) and AhpC was observed [Betaproteobacteria of the ompounds ,16, whicompounds ,18. Para.47 Mbp) . Strain .47 Mbp) . The expresponse ,21. Duriobserved .P. xenovorans LB400 and the analyses of their potential protective effect during oxidative stress induced by paraquat and H2O2. The effect of these oxidizing agents on the susceptibility and survival of P. xenovorans LB400 was studied, along with macromolecules damage and the expression of antioxidant genes.The aims of this study were the identification and characterization of flavodoxins of Hydrogen peroxide was purchased from Merck  and the herbicide methyl viologen dichloride hydrate (paraquat) was obtained from Sigma-Aldrich .P. xenovorans LB400, a DELTA-BLAST  was performed [Pseudomonas aeruginosa PA14 (Accession No. A0A0H2ZDT7), Escherichia coli K12 substr. MG1655 (Accession No. P61949) and Anabaena sp. PCC 7119 (Accession No. P0A3E0) were used as references. The amino acid sequences of the LB400 flavodoxins was compared to the long-chain flavodoxins of P. aeruginosa PA14 (Accession No. A0A0H2ZDT7) and FldA of E. coli K12 substr. MG1655 (Accession No. P61949), and the short-chain flavodoxins of Clostridium beijerinckii MP (Accession No. P00322), and Paraburkholderia phymatum TM815 (Accession No. B2JVA6) using the pairwise sequence alignment tool EMBOSS Needle (https://www.ebi.ac.uk/Tools/psa/emboss_needle/). The PROSITE tool of Expasy [P. aeruginosa PA14 (Accession No. A0A0H2ZDT7), E. coli K12 substr. MG1655 (Accession No. P61949) and Anabaena sp. PCC 7119 (Accession No. P0A3E0), and the short-chain flavodoxins of C. beijerinckii MP (Accession No. P00322), Desulfovibrio desulfuricans ATCC 29577 (Accession No. P26492) and Bacillus subtilis 168 (Accession No. O34737). The identity between LB400 flavodoxins was determined by the pairwise sequence alignment tool EMBOSS Needle (https://www.ebi.ac.uk/Tools/psa/emboss_needle/). The genomic context was analyzed through the Burkholderia database (http://www.burkholderia.com/).To identify genes encoding flavodoxins in the genome of erformed . The amif Expasy  was usedf Expasy  and PROMf Expasy  tools anP. xenovorans LB400 was grown at 30°C in Luria-Bertani (LB) modified medium  or in M9 minimal medium with 5 mM glucose as sole carbon and energy source. E. coli strains were growth at 37°C in LB medium. Growth was determined by measuring turbidity at 600 nm and/or by counting colony-forming units (CFU).http://tree.bio.ed.ac.uk/software/figtree/). The bootstrap values (percentage) above 50% were shown for each branch point.Thirty-nine flavodoxin amino acid sequences with experimental evidence were retrieved from UniProtKB Swiss-Prot/TrEMBL database . IdentifP. xenovorans LB400 were amplified by PCR using specific primers  restriction enzymes. The DNA fragments were cloned using T4 DNA ligase  into the vector p2, which possesses a kanamycin resistance cassette. The ligation reactions were transformed into E. coli JM109 and cells were cultured overnight with kanamycin 25 μg mL-1 as selective marker. Positive clones were checked by enzymatic digestion with BamHI and HindIII and PCR  were checked by PCR using the M13 primers. The overexpression of the FldX1 and FldX2 proteins was confirmed by SDS-PAGE 15% w v-1. To construct the control strain (hereafter WT-p2), the vector p2 without insert was transformed into E. coli S17λpir and transferred to P. xenovorans LB400 by biparental mating. The recombinant strain was checked by PCR using M13 primers. The expression level of genes encoding flavodoxins in recombinant strains was determined by qRT-PCR (Genes encoding flavodoxins (BxeA0278 and BxeB0391) of  primers . The PCR and PCR , using t qRT-PCR .To study the bacteria growth of the recombinant strains, cells were grown in 5 mM glucose. Recombinant strains were growth overnight in modified LB medium at 30°C. Cells were washed in 0.9% NaCl and used as inoculum for M9 minimal medium with 5 mM glucose as sole carbon source. Recombinant strains were cultivated for 24 h at 30°C and turbidity a 600 nm was measured every 3 h.2O2; 20 mM) or the superoxide-producing agent paraquat (20 mM) were deposited on 6 mm diffusion discs and placed on the plates. Images of the growth inhibition zones were measured using the software ImageJ after 24 h incubation at 30°C [Recombinant strains were grown until exponential phase and 100 μL of each culture was spread on modified LB medium agar plates. 15-μL of hydrogen peroxide  during 1 h. Aliquots were taken to determine colony-forming units. The percentage of survival was determined as the ratio of cells exposed to oxidizing agents and untreated cells. Values were calculated as the mean ± SD of results of, at least, three independent experiments.Exponential grown cells of P. xenovorans recombinant strains were exposed to 1 mM H2O2 or 1 mM paraquat during 1 h at 30°C. Bacterial cells were harvested and disrupted by sonication using five pulses of 30 s at 6 Watts with 1-min intervals. Nucleic acid-free extracts were used to precipitate proteins with 10% v v-1 trichloroacetic acid. 400-μL of protein extracts were incubated with 400 μL of 10 mM 2,4-dinitrophenylhydrazine (DNPH) during 10 min. 200-μL of 6 M NaOH was added to the reaction and incubated for 10 min at room temperature. Carbonyl content was determined spectrophotometrically at 450 nm using a molar absorption coefficient of 22,308 M-1cm-1 [Exponentially-grown cells of  M-1cm-1 . Values P. xenovorans recombinant strains were exposed to H2O2 or paraquat (1 mM) for 1 h. Bacterial cells were harvested and disrupted by sonication. Proteins were precipitated with 10% v v-1 trichloroacetic acid and discarded by centrifugation. Supernatants were mixed with a saturated solution of thiobarbituric acid (TBA) in 0.1 M HCl and 10 mM butylated hydroxytoluene. Samples were incubated at 100°C for 1 h. TBA reacted with malondialdehyde and other byproducts of lipid peroxidation forming a colored product. TBARS content was determined spectrophotometrically at 535 nm using a molar absorption coefficient of 156 mM-1cm-1 [Exponentially-grown cultures of mM-1cm-1 . Values P. xenovorans recombinant strains were grown until mid-exponential phase (turbidity600nm ~0.4) and harvested. For the quantification of the expression of genes associated to antioxidant response, exponentially-grown cells (turbidity600nm ~0.4) were exposed to 1 mM paraquat during 1 h and harvested. RNA was extracted from cells using the TRIzol reagent , following the manufacturer instructions. Residual DNA was removed employing the TURBO DNA-free Kit . RNA quality was verified with the NanoDrop One spectrometer . RNA integrity was checked in a 1% w v-1 agarose gel. cDNA was synthetized with the First Strand cDNA Synthesis Kit . The qRT-PCRs were carried out with the KAPA SYBR FAST qPCR Master Mix Kit , following the manufacturer instructions. The gyrB and ftsZ genes were used as reference genes. The results were analyzed using the Hellemans method [Differential expression of genes was analyzed by qRT-PCR. For the quantification of the expression of flavodoxins genes, s method . UntreatStatistical analyses were performed using one-way ANOVA and Tukey test to assess differences in mean values from each experiment. Differences are significant at p<0.05.P. xenovorans LB400, amino acid sequences of three well-characterized flavodoxins were retrieved from the UniProt database: P. aeruginosa PA14 (Accession No. A0A0H2ZDT7), E. coli K12 substr. MG1655 (Accession No. P61949) and Anabaena sp. PCC 7119 (Accession No. P0A3E0). Two putative genes encoding flavodoxins were identified in the genome of P. xenovorans LB400. The BxeA0278 gene (hereafter fldX1) localized at the major chromosome encodes a protein of 190 amino acids. The BxeA0278 gene product showed 50, 26 and 23% identity with the FldP, FldA and IsiB flavodoxins of P. aeruginosa PA14, E. coli K12 and Anabaena sp. PCC7119, respectively. The BxeB0391 gene (hereafter fldX2), is located at the minor chromosome and encodes a protein of 175 amino acids. The BxeB0391 gene product possesses 44, 21 and 21% identity with FldP, FldA and IsiB, respectively. Genes encoding LB400 flavodoxins was compared with the long-chain flavodoxins of P. aeruginosa PA14 (Accession No. A0A0H2ZDT7) and E. coli K12 substr. MG1655 (Accession No. P61949), and the short-chain flavodoxins of C. beijerinckii MP (Accession No. P00322) and P. phymatum TM815 (Accession No. B2JVA6)  . InteresP. aeruginosa PA14. This FMN-BD domain is located in the same positions than in FldP of P. aeruginosa PA14 and FldA of E. coli K12. FldX2 possess a flavodoxin domain between amino acids 7 and 175, and two FMN-BD domains between amino acids 13–17 and 132–175.To determine conserved domains, the primary structures of strain LB400 flavodoxins were analyzed using the PROSITE tool (Expasy). FldX1 possesses a flavodoxin domain between amino acids 4 and 179, and two FMN-binding domains (FMN-BD) between amino acids 10–14 and 93–150. The amino acid sequence of the first FMN-BD (SGYGH) domain is identical to the FldP flavodoxin of The secondary structures of both flavodoxins were predicted using the Phyre2 and PROMALS3D tools . FldX1 afldX1 and fldX2 genes in strain LB400 genome were analyzed. Genes encoding membrane proteins, transporters, proteins involved in redox processes, biosynthesis of nucleic acids, transcriptional regulators, metabolism of amino acids, proteins and carbohydrates were identified in neighborhoods of the fldX1 and fldX2 genes in strain LB400 genome and of FldP, FldA and IsiB flavodoxins genes of P. aeruginosa PA14, E. coli K12 (MG1655) and Nostoc sp. PCC 7120 (Anabaena), respectively (The neighborhoods of the ectively . NucleicfldX1 gene neighborhood are located genes encoding the membrane proteins OmpW family porin (ompW), two membrane lipoproteins (BxeA0274 and BxeA0275) and a major facilitator transporter (BxeA0282). The ompW gene located downstream of the fldX1 gene encodes the OmpW porin that may be involved in paraquat efflux in strain LB400. The OmpW porin mediates paraquat efflux in Salmonella enterica serovar typhimurium [fldX1 gene is located a LuxR family transcriptional regulator gene (BxeA0280). It has been proposed that the LysR family regulator of P. aeruginosa PA14 encoded by a gene located close to the fldP gene is the repressor of the transcription of the FldP operon [fldX1 gene, however, experimental assays are required to study the regulation. In addition, the gene argC (BxeA0277) encoding N-acetyl-γ-glutamyl-phosphate reductase is in the context of the fldX1 gene. N-acetyl-γ-glutamyl-phosphate reductase ArgC is involved in the synthesis of arginine and proline from glutamate [argC gene in the neighborhood of the fldX1gene. In addition, in the fldx1 gene neighborhood are located genes encoding an orotate phosphoribosyl-transferase  that is involved in nucleotide biosynthesis, a phospholipid-binding protein  and the malic enzyme .In the lutamate . Argininlutamate , which mfldX2 gene neighborhood are located two genes encoding the membrane proteins OmpC family porins  and a gene encoding an aromatic acid exporter . The presence of porins and aromatic exporter may explain in part the bacterial resistance to antibiotics and toxic compounds such as paraquat [ydjC; BxeB0286) involved in carbohydrate metabolism and a gene encoding the RNA polymerase sigma 70 factor  was identified in the fldX2 gene neighborhood.In the paraquat . In addiA. vinelandii OP and P. stutzeri A1501 are clustered with genes involved in nitrogen fixation [P. xenovorans LB400 flavodoxins genes reveals genes encoding proteins involved in the synthesis of nucleic acids and proteins and efflux of toxic compounds, which might influence bacterial fitness during oxidative stress.The long-chain flavodoxin NifF genes of nitrogen-fixating fixation ,44, whicfixation . The genP. xenovorans LB400 flavodoxins, the phylogeny of FldX1, FldX2 and 71 flavodoxins from 43 different bacterial genera was analyzed. From this dataset, 20 amino acid sequences correspond to FldX1 orthologs, while 12 belong to FldX2 orthologs. A total of 39 flavodoxins with experimental evidence were incorporated to evaluate FldX1 and FldX2 evolutionary position among flavodoxins with an assigned biological function. The phylogenetic tree was rooted using the NrdI flavodoxin family, involved in ribonucleotide reductase function, showing eight distinct clusters  on glucose, while no differences were detected between strains p2-fldx2 and control . These results suggest that overexpression of flavodoxin FldX1 is conferring an advantage during aerobic growth on glucose.To characterize the growth of fldX genes in recombinant strains was studied by qRT-PCR using specific primer sets (600nm ~0.4). The fldX1 gene expression was highly induced (>600-fold) in recombinant strain p2-fldX1 compared to the control WT-p2 strain. In recombinant strain p2-fldX2, the fldX2 gene expression was >4000-fold induced compared to the control WT-p2 strain. Interestingly, despite that the genes encoding both flavodoxins were cloned in the same plasmid, the overexpression of fldX2 gene in strain p2-fldX2 is higher (>6-fold) than the overexpression of fldX1 gene in recombinant strain p2-fldX1. These results suggest a higher expression of the fldX1 gene than the fldX2 in WT-p2 strain grown on glucose.The transcription of the mer sets . RecombiP. xenovorans strains overexpressing fldX1 and fldX2 genes to oxidizing agents was assessed. Recombinant P. xenovorans cells on agar plates were exposed to the superoxide-producing redox-cycling agent paraquat or H2O2 (20 mM)  compared to the control strain (22.8 mm). In contrast, after incubation with paraquat the strain overexpressing the fldX2 gene did not show significant differences with the control strain (22.6 mm). After incubation with 20 mM H2O2, the strains overexpressing the fldX1 (10.1 mm) and fldX2 genes (10.7 mm) did not show significant differences with the control strain (10.3 mm).The susceptibility of the  (20 mM) . InteresP. xenovorans during exposure to oxidizing agents was studied by survival assays in liquid medium. Recombinant P. xenovorans cells grown until exponential phase were exposed to paraquat or H2O2 (1 mM and 20 mM) during 1 h  than the control strain (68%). After incubation with 20 mM paraquat, the strain overexpressing the fldX1 gene showed a higher survival (77%) than the control strain (45%). In contrast, the strain overexpressing the fldX2 gene did not show significant differences in survival in 20mM paraquat (43%) with the control strain. On the other side, during exposure to 1 mM H2O2, the strain overexpressing the fldX2 gene (72%) did not show significant differences with the control strain (75%), while the strain overexpressing the fldX1 gene displayed an unexpected lower viability (52%) than the control strain, suggesting that overexpression of the fldX1 gene may cause a detrimental effect toward H2O2 exposure under these conditions. After exposure of the recombinant strains to 20 mM H2O2, no survival of the recombinant strains was observed  during 1 h. As no survival of recombinant cells was observed with H2O2 20 mM, only the concentration 1 mM of both oxidizing agents was used in these assays. To quantify carbonyl group content, protein extracts were derivatized with DNPH and the products were measured spectrophotometrically at 450 nm  compared to the control strain (1.21 μmol/mg of protein). For the cells exposed to 1 mM H2O2, strains p2-fldX1 and p2-fldX2 showed less protein carbonylation  than the control strain (0.86 μmol/mg of protein). After paraquat treatment, no significant differences in protein carbonylation were detected between the recombinant strains.The effects of overexpressing the t 450 nm . In absefldX1 did not showed significant difference with the control strain. However, strain p2-fldX2 showed less TBARS content (99.43 pmol/mg of protein) compared to the control strain (134.57 pmol/mg of protein). After the exposure to 1 mM paraquat, P. xenovorans p2-flX1 (138.20 pmol/mg of protein) and p2-fldX2 (117.49 pmol/mg of protein) showed lower lipid peroxidation compared to the control strain (171.11 pmol/mg of protein). During exposure of cells to 1 mM H2O2, strain p2-fldX1 displayed lower levels of lipid peroxidation (98.49 pmol/mg of protein) than the control strain (151.80 pmol/mg of protein). In contrast, strain p2-fldX2 showed a higher TBARS content (198.08 pmol/mg of protein) compared to the control strain.To quantify TBARS, samples reacted with TBA and the products were measured spectrophotometrically at 535 nm . In abseP. xenovorans recombinant strains were assessed , the katG4 gene (>2-fold), the ohr gene (>2-fold), the trxB1 gene (>2-fold) and the hpf gene (>2-fold) compared to cells incubated in absence of paraquat. The katG4 gene encodes a catalase, whereas the fumC gene encodes a ROS-resistant fumarase. The ohr gene encodes an organic hydroperoxide resistance protein, the trxB1 gene encodes a thioredoxin and the hpf encodes a high potential iron-sulfur protein associated to oxidative stress.The effect of paraquat on the expression of ten specific genes involved in oxidative stress of assessed . These gfldX2 showed an upregulation (>2-fold) of the ahpC1 and oxyR genes compared to cells incubated in absence of paraquat. No significant downregulation of antioxidant genes was observed in strain p2-fldX2. The oxyR gene encodes the H2O2-sensing transcriptional regulator OxyR. Therefore, the induction of the oxyR gene by paraquat suggests increased and threatening intracellular levels of H2O2 in p2-fldX2 cells exposed to this oxidizing compound. The ahpC1 gene encodes the alkyl hydroperoxide reductase involved in detoxification of H2O2 at low concentration, which is induced in E. coli by the transcriptional regulator OxyR [Upon exposure to paraquat, recombinant strain p2-tor OxyR .sodB1, trxB2 and gst genes were observed in both recombinant strains. The sodB1 gene encodes the scavenging enzyme superoxide dismutase, the trxB2 gene encodes a thioredoxin, and the gst gene encodes a glutathione S-transferase.In presence of paraquat no changes in expression levels of the fldX1 gene and BxeB0391: fldX2 gene) were identified in P. xenovorans LB400 genome and characterized. To study their function, the fldX genes were cloned and overexpressed in recombinant strains of P. xenovorans. The protective effects to oxidative stress induced by superoxide-producing herbicide paraquat and H2O2 of the expression of these flavodoxins in the recombinant strains p2-fldX1 and p2-fldX2 were characterized.In this study, two genes encoding flavodoxins (BxeA0278: fldX1 (BxeA0278) and fldX2 (BxeB0391) encoding flavodoxins are located at the major and the minor chromosome, respectively. These flavodoxins share 21.4% identity in amino acid sequence. The FldX1 and FldX2 flavodoxins of strain LB400 possessed two FMN-binding domains (FMN-BD) with similar amino acid length and located at similar positions than flavodoxins of E. coli DHOl [H. pylori [Anabaena sp. PCC 7120 [et al. [P. aeruginosa PA14, which were compared to FldA of E. coli and IsiB of Anabaena sp. PCC 7119, sharing the same protein domains. The genomic analyses allowed us to identify two flavodoxin-encoding genes in strain LB400 possessing high similarity in length, amino acid sequence, and predicted secondary structure with typical long-chain (FldX1) and short-chain (FldX2) flavodoxins.Flavodoxins genes were identified in strain LB400 genome based on the amino acid sequences of previously characterized flavodoxins. The genes oli DHOl , H. pylo. pylori  and Anab1 BxeA027 and fldXP. xenovorans LB400 grouped in two different phylogenetic clusters  within a long-chain flavodoxin cluster (VII) and a short-chain flavodoxin cluster (VI), respectively. The distribution of the short-chain flavodoxin cluster III that includes Bacillus flavodoxins, and the cluster V, including the M. elsdenii and C. beijerinckii flavodoxins, correlate with the unrooted tree constructed by Pérez-Dorado et al. [Desulfovibrio desulfuricans in the same cluster as M. elsdenii and C. beijerinckii. The cluster VI contains short-chain flavodoxins homologous to FldX2 and the flavodoxin-4 of Lactobacillus acidophilus ATCC 700396. An NMR structure have been performed to the flavodoxin-4 of strain ATCC 700396 identifying a long insertion between α2 and β3 [Paraburkholderia and Burkholderia orthologs, and to the flavodoxins FldP of P. aeruginosa PA14 and WrpA of B. abortus. FldP is the closest flavodoxin with an assigned function to FldX1  in growth inhibition zones compared to the control strain  and 1 mM (~140%) in liquid medium than the control strain (fldX1 cells displayed a reduction of lipid peroxidation (>20%) compared to control cells after exposure to paraquat (1 mM), but no significant differences in protein carbonylation were detected. In response to paraquat (1 mM), strain p2-fldX1 downregulated (>2-fold) the fumC, katG4, hpf, trxB1 and ohr genes, but do not significantly upregulated any of the other genes associated to oxidative stress including the key oxyR and ahpC genes that were studied, suggesting that the oxidative stress response was not activated under these conditions. Overall, these results indicate the strain p2-fldX1 that overexpress the long-chain flavodoxin FldX1 showed a remarkably resistance to paraquat. Flavodoxins such as FldX1 may replace ferredoxins and scavenge ROS during oxidative stress, but also consume electron donors during paraquat redox cycling [The long-chain flavodoxin FldX1 of c growth . It has oli K-12 ,50. The pathways . Exposurconitase . After el strain . Interesl strain . Strain  20 mM ~10% and 1  cycling .P. xenovorans showed only low protection to oxidative stress induced by the presence of hydrogen peroxide in the medium. A decrease in cell susceptibility on plates and increase in the survival in liquid medium of strain p2-fldX1 in presence of H2O2 was not observed. However, strain p2-fldX1 showed a decrease in protein carbonylation (~30%) and lipid peroxidation (~35%) after exposure to H2O2 (1 mM). E. coli strains BW25113 pBAD and KS400 incubated with H2O2 (0.1 mM) showed 28- and 50-fold higher protein carbonylation, respectively, and similar lipid peroxidation than strain p2-fldX1 [P. putida KT2440 to H2O2 (1.5 mM), the lipid peroxidation was 4-fold higher than the lipoperoxidation observed in strain p2-fldX1 [E. coli growth has been reported to occur at low H2O2 concentrations (0.03 mM), inactivating key enzymes containing active-site sulfhydryl residues [P. xenovorans strains, at a higher concentration of H2O2 (1 mM), exhibited a high survival and lower protein and membrane damage.In contrast, the overexpression of long-chain flavodoxin FldX1 of p2-fldX1 ,53. Aftep2-fldX1 . Inhibitresidues . P. xenofldX2 do not displayed significant differences in growth inhibition compared to the control strain. Strain p2-fldX2 showed a higher survival (~150%) than control strain only at the lowest paraquat concentration (1 mM), but not at 20 mM paraquat. Recombinant strain p2-fldX2 displayed a reduction of lipid peroxidation (~30%) after exposure to paraquat (1 mM), but protein carbonylation did not changed. P. xenovorans strain p2-fldX2 showed a significant upregulation by paraquat (1 mM) of the antioxidant oxyR and ahpC1 genes, suggesting increased intracellular levels of ROS (H2O2) by the superoxide-producing paraquat. H2O2 activates the expression of the antioxidant oxyR and ahpC1 genes in P. xenovorans LB400 [2O2. High expression of FldX2 did not influence susceptibility to H2O2 (20 mM) on agar plates and survival in liquid medium in presence of H2O2 (1 and 20 mM). Interestingly, strain p2-fldX2 showed a decrease in protein carbonylation (~30%) after exposure to H2O2 (1 mM).The short-chain flavodoxin FldX2 only protected partially to oxidative stress induced by paraquat compared to FldX1, in spite of its higher expression than FldX 1 in recombinant strains. After exposure to paraquat (20 mM) on agar plates, strain p2-ns LB400 . On the P. aeruginosa and P. putida [P. aeruginosa fldP mutant showed enhanced sensitivity toward H2O2 toxicity. In contrast, P. aeruginosa mioC mutant is more tolerant than the wild type strain to oxidative stress induced by paraquat or H2O2 [P. aeruginosa enhances survival to oxidizing conditions, such as the exposure to H2O2 and its intracellular presence in mammalian macrophages [E. coli, P. fluorescens and Ensifer meliloti strains overexpressing the long-chain flavodoxin IsiB from Anabaena sp. PCC7119 showed a higher survival in presence of paraquat and H2O2 [Both long-chain flavodoxin FldP, which is closely related to FldX1, and short-chain flavodoxin MioC have been reported in . putida . P. aeru or H2O2 . Long-chrophages . The lonand H2O2 .2O2 detoxification, it has been demonstrated in E. coli that Ahp scavenges H2O2 at low concentrations, while catalase detoxify higher concentrations of hydrogen peroxide [fldX1 and p2-fldx2, no changes were observed in the katG4 gene expression levels. This may suggest that the intracellular H2O2 concentration is not high enough to induce the expression of the gene encoding the catalase KatG4. Therefore, hydrogen peroxide may be scavenged by AhpC under these conditions. The ahpC1 gene is upregulated by paraquat in strain p2-fldx2.Although alkyl hydroperoxide reductase (AhpC) and catalase are responsible for Hperoxide . In strafldX1 and p2-fldX2 did not exhibit upregulation of the sodB1 gene. However, P. xenovorans LB400 genome possess other three sodB homologues , that could be counteracting the superoxide generated by paraquat. The higher viability of strains p2-fldX1 and p2-fldX2 in presence of the superoxide-forming paraquat, suggests that FldX1 and FldX2 may be involved in an alternative ROS scavenging-mechanism to thrive oxidative stress. The organic hydroperoxide resistance (Ohr) protein is a thiol-dependent peroxidase induced by organic hydroperoxides [P. xenovorans strains overexpressing flavodoxins, compared to the control strain, which correlates with no upregulation of the ohr gene during the incubation with paraquat. The antioxidant fumC, hpf, trxB1, trxB2 and gst genes were not induced in P. xenovorans recombinant strains exposed to paraquat. Glutathione S-transferases catalyze the conjugation of reduced glutathione to xenobiotic substrates for detoxification [E. coli, the upregulation of the fumC gene encoding a class II fumarate hydratase (FumC), occurs during oxidative stress damage, to replace class I iron-sulfur cluster-depending fumarases (FumA and FumB) [Alcaligenes aquatilis QD168 displayed an upregulation of two gene copies of ahpC (ahpC1 and ahpC2) and two copies of the transcriptional regulator soxR (soxR1 and soxR2) [P. putida KT2440 to paraquat induced expression of a ferredoxin reductase (fpr), a superoxide dismutase (sodA) and a fumarate hydratase (fumC-1) [Although paraquat is a source of superoxide radical, strains p2-eroxides , such asfication . High pofication . Thioredfication . In E. cd soxR2) . Exposur(fumC-1) .oxyR and ahpC genes were only significantly induced by paraquat in strain p2-fldX2 and not in strain p2-fldX1, indicating that flavodoxin FldX1 exerts a higher protective effect against oxidative stress induced by paraquat. These results also support our hypothesis that FldX1 may be related to an alternative ROS scavenging-mechanism to thrive oxidative stress, which partially involves the OxyR regulon.The fldX1 cells than in control strain cells grown on glucose and not exposed to oxidizing agents  are induced in the presence of paraquat, as part of the SoxRS regulon, which controls the response to redox-cycling agents [P. xenovorans, flavodoxin FldX1 exert a higher effect against paraquat than H2O2.This may suggest that the gene fldX1 is regulated by SoxR.In g agents . In P. xP. xenovorans during oxidative stress induced by paraquat and H2O2, indicating the role of these proteins in the reestablishment of the cell redox balance. Interestingly, FldX1 conferred higher protection than FldX2 to paraquat and H2O2. This resulted in more resistant cells to paraquat, an increase in viability and a decrease in the macromolecule damage in presence of oxidizing agents.Overall, the overexpression long-chain flavodoxin FldX1 and short-chain flavodoxin FdlX2 exerted a protective role to P. xenovorans strain LB400 were identified and characterized. A phylogenetic analysis of LB400 flavodoxins indicates that they belong to different evolutionary clusters of long-chain and short-chain flavodoxins. Assays of bacterial susceptibility, survival, macromolecules damage and gene expression indicate that the flavodoxins FldX1 and FdlX2 are involved in the resistance of strain LB400 to oxidative stress caused by paraquat and H2O2. Several assays indicate that FldX1 and FldX2 are involved in protection against oxidative stress, including (i) the partial protection conferred by FldX1 overexpression to P. xenovorans susceptibility to 20 mM paraquat on agar plates  thed medium , (iii) tand H2O2  and (iv)paraquat . P. xeno


UGT1A1) and/or solute carrier organic anion transporter family member 1B (SLCO1B) polymorphic variants and their combined effects on mild unconjugated hyperbilirubinemia in Chinese adults. Fourteen genetic variants in the UGT1A1 or SLCO1B gene were genotyped through sequencing in 148 adults with unconjugated hyperbilirubinemia and 158 healthy controls. Variants c.-3275T > G, (TA)6>(TA)7, c.211G > A or c.1091C > T within the UGT1A1 gene as well as c.521T > C within the SLCO1B1 gene appear to be genetic risk factors for inherited unconjugated hyperbilirubinemia. After adjusting for covariates, the results of multivariate logistic regressions revealed that odds ratios (ORs)  of these five variants were 2.35 , 2.38 , 2.99 , 7.60 , and 2.54 , respectively. The OR for unconjugated hyperbilirubinemia is positively correlated with the cumulative number of these five variants in adults. And the greater the number of genetic variations, the higher the total bilirubin level. Adults carrying diplotype 3/4 (homozygous c.-3275T > G and heterozygous (TA)6>(TA)7) had higher bilirubin levels than those with diplotypes 1/3 (heterozygous c.-3275T > G and (TA)6>(TA)7)) or 1/4 (heterozygous c.-3275T > G) (P < 0.05). Similarly, bilirubin levels in individuals with diplotype 2/4 (heterozygous c.-3275T > G and c.211G > A) were higher than adults carrying diplotypes 1/2 (heterozygous c.211G > A) or 1/4 (P < 0.001). For subjects with heterozygous or homozygous variant c.211G> A, as the number of c.521T > C alleles variation increased, the incidence of unconjugated hyperbilirubinemia increased, but it was not statistically significant. Our results indicate that variants of UGT1A1 and/or SLCO1B1 have combined effects on Chinese adult mild unconjugated hyperbilirubinemia.The potential for genetic variation to cause adult unconjugated hyperbilirubinemia is increasingly being recognized. However, the cumulative effects of genetic variants have not been fully illuminated. The current study aimed to investigate the effects of uridine diphospho-glucuronosyl transferase 1A1 ( In the (SLCO1B) .UGT1A1 gene is considered to be a major factor controlling serum bilirubin levels (UGT1A1 gene often underline Gilbert Syndrome (GS), a mild unconjugated hyperbilirubinemia condition 7TAA (35.7–41.5%)  , while t7–41.5%) . An exomubinemia . Moreoveed in GS , suggestSLCO1B, which is closely related to the transportation of bilirubin and organic anions into hepatocytes, has also been found to play a role in bilirubin homeostasis  and c.521T > C  were also found to be risk factors for neonatal unconjugated hyperbilirubinemia , were enrolled in this study. The 158 control subjects were enrolled from the same health examination people with bilirubin values of less than 17.1 µmol/l. Blood samples were taken in 10–12 h fasting state in the morning and the subjects were kept from strenuous exercise. And clinical records including the gender, age as well as biochemical test results were collected. Exclusion criteria were: (1) a history of chronic liver disease ; (2) abnormal liver functions  or aspartate aminotransferase (AST) > 50 U/L); (3) hemolysis signs ; (4) evidence of infection or biliary obstruction or pregnancy; (5) a history of drug use within 6 months.This Study Was Approved by the Ethics Committee of Beijing You an Hospital, Capital Medical University and a Written Informed Consent Form Was Obtained From All Participants.UGT1A1 were PCR amplified and sequenced, as reported before  were investigated. The PCR products were purified from agarose gel and sequenced via an ABI3730XL sequencer .Genomic DNA was extracted from whole blood using a QIAamp DNA Blood Mini Kit 51106  according to the manufacturer's recommended protocol. The promoter, all 5 exons, exon–intron boundaries, and a region in the distal promoter  of d before . SimilarUGT1A1 and 4 of SLCO1B) were selected. Analysis of linkage disequilibrium (LD) and haplotype were performed by using SNPStats website (https://www.snpstats.net/start.htm). Strong LD was defined as both |D′| and r2 > 0.8.Selection of SNPs was done based on their association with serum bilirubin levels as well as their prevalence in Asia population . Finally2) test. Logistic regression models were performed to evaluate the effects of SNPs on hyperbilirubinemia. The odds ratios (ORs) with corresponding 95% confidence interval (CI) were estimated between the case and the control groups by using binary logistic regression models after adjusting for clinical factors . The relation between log-transformed total bilirubin (TB) and different genotypes was also assessed. All statistical analyses were performed by using SPSS version 23.0 . A P-value < 0.05 was considered to be statistically significant.Continuous variables were expressed as mean and standard deviation (SD) and then statistically evaluated by using Student's t-test. Categorical variables were compared by using the chi-squared (χP = 0.006) and albumin (ALB) , but lower platelet (PLT) values . Besides, there were no obvious differences between the case and the control groups concerning age, gender, blood routine, and other plasma biochemical results.A total of 146 adults with hyperbilirubinemia were enrolled in the case group and 158 adults with bilirubin values of less than 17.1µmol/L in the control group. The clinical features of the case and control groups are shown in UGT1A1 variations are presented in 6>(TA)7: 41.78% vs 22.78%, P < 0.001; c.211G > A: 50.68% vs 29.11%, P < 0.001]. In addition, 8.22% of cases featured homozygous (TA)7 variant, while no controls carried this homozygous variation. The incidence of other six UGT1A1 variations in the case group was also significantly higher for c.-3275T > G , c.-3152G > A , c.686C > A , IVS1+2842G > T , IVS1+2925T > G , and c.1091C > T , but lower for IVS2+15T > C . The proportion of case subjects with c.1456T > G variant was not statistically different from that of control subjects . Moreover, the three variants c.686C > A, c.1091C > T, and c.1456T > G presented only heterozygous forms or wild-type in cases and controls. The LD pattern across the multiple SNPs of UGT1A1 is shown in UGT1A1 , where each |D′| > 0.8 and r2 > 0.8. Moderate LD was present between (TA)n repeat polymorphism and c.-3275T > G (|D′| > 0.8 and r2= 0.4320).The frequencies of SLCO1B gene observed among the cases and controls are also shown in SLCO1B1, IVS8+2087T > C and g.21074122C > T within SLCO1B3) were investigated in this study. However, there was only a statistical difference of the frequency of 521T > C variation between the case and the control groups . A low pairwise LD was found between the four polymorphisms of SLCO1B (r2 < 0.3) . There were also strong associations between other common variations in UGT1A1 and hyperbilirubinemia, including c.-3275T > G , (TA)6>(TA)7 , and c.211G > A . Variant IVS2+15T > C appeared to reduce the risk of hyperbilirubinemia . Specifically, variant c.521T > C within SLCO1B1 was found to have a significantly increased risk of hyperbilirubinemia . The log10-transferred TB of different variants is shown in UGT1A1 or variant c.521T > C of SLCO1B1 have higher bilirubin values than those with wild type. However, in the cases of variants IVS2+15T > C or IVS7+2087T > C, the opposite is true.Initially, the OR with 95% CI of single suspected variant for hyperbilirubinemia was assessed by using separate binary logistic regression , 10.92 , 18.43  and 34.38 , respectively. Besides, the greater the number of genetic variations, the higher the total bilirubin level , representing wild type. Compared with haplotype 1, haplotypes 2 [–3275T–(TA)6–211A–1091C], 3 [–3275G–(TA)7–211G–1091C], 4 [–3275G–(TA)6–211G–1091C] and 5 [–3275G–(TA)6–211G–1091A] all increased the risk of unconjugated hyperbilirubinemia (P < 0.05).To further analyze the combined effects of UGT1A1 diplotypes are shown in UGT1A1 diplotypes, 3/3 (homozygous variation of c.-3275T > G and (TA)6>(TA)7) featured the highest levels of bilirubin. The diplotypes 1/4 and 4/4 represent heterozygous or homozygous c.-3275T > G variant, respectively, and their bilirubin levels are lower than normal. Moreover, adults carrying diplotype 3/4 (homozygous c.-3275T > G and heterozygous (TA)6>(TA)7) had higher bilirubin levels than those with diplotypes 1/3 (heterozygous c.-3275T > G and (TA)6>(TA)7)) or 1/4 (heterozygous c.-3275T > G) (P < 0.05) (P < 0.001). However, there was no significant difference in bilirubin levels among adults with diplotype 1/4, 1/5 or 4/5. These data demonstrated an additive effect of variants c.-3275T > G and (TA)6>(TA)7 or c.211G > A on unconjugated hyperbilirubinemia.All subjects were divided into 14 diplotypes, covering a total of 303 (99.7%) adults. All bilirubin values (log10-transformed) among 6/(TA)7 and c.211G > A in UGT1A1 and variation c.521T > C in SLCO1B was further explored. In UGT1A1-normal subjects, homozygous variation c.521T > C increased total bilirubin level and the incidence of unconjugated hyperbilirubinemia compared to wild-type or heterozygous forms (P = 0.046); as the number of variation c.521T > C alleles increased, the incidence of unconjugated hyperbilirubinemia increased, but it was not statistically significant (P = 0.186). When carried homozygous variation c.211G > A, the incidence of hyperbilirubinemia for individuals featured c.521T > C was higher, but it did not reach statistically significant . In addition, among the 21 adults with the homozygous c.211G > A, all 5 subjected with the heterozygous variation c.521T > C suffered from hyperbilirubinemia. But for (TA)6>(TA)7, we did not observe similar results. These data indicated a cumulative effect of variants c.521T > C and c.211G > A on unconjugated hyperbilirubinemia.The association between the two common variations (TA)UGT1A1 and SLCO1B on mild unconjugated hyperbilirubinemia in adults. Our results indicated that variations of UGT1A1 and SLCO1B genes had a substantial impact on unconjugated bilirubin levels in Chinese adults. Notably, variant c.521T > C in the SLCO1B may be independent genetic risk factors for inherited unconjugated hyperbilirubinemia. Furthermore, the risk of unconjugated hyperbilirubinemia is positively correlated with the cumulative number of variants in adults. To our knowledge, this is the first report that showed the combined effect of UGT1A1 and SLCO1B variants on mild unconjugated hyperbilirubinemia in adults.GS, a benign condition that occurs in 5–10% of the population , is char6>(TA)7 and c.211G > A of UGT1A1 both reduce UGT1A1 enzyme activity and increase unconjugated bilirubin level (6>(TA)7 and c.211G > A with the incidence of unconjugated hyperbilirubinemia. Additionally, the frequencies of variants (TA)6>(TA)7 and c.211G > A in the case group were significantly higher than those in the control group. And subjects with these two heterozygous or homozygous variants had higher mean total serum bilirubin levels than adults carrying the wild type.Two common variants (TA)in level . Our resUGT1A1 c.686C > A, c.1091C > T and c.1456T > G can also lead to Gilbert syndrome amongst the Asian population 7 and c.-3275T > G mutations together may be essential to the Gilbert syndrome (6>(TA)7 or c.211G > A had higher bilirubin levels than those carrying any of the above heterozygous variants. These data indicate a combined effect on unconjugated hyperbilirubinemia for different variants in UGT1A1.Earlier studies suggested the decrease in transcription caused by both (TA)syndrome . In the syndrome . In addiSLCO1B1 and SCLO1B3 . Besides, the variant c.521T > C showed an additive effect on UGT1A1 gene variants, particularly the variant c.211G > A. These results indicated that the change of amino acid  at codon 174 of SLCO1B1 may reduce the function in unconjugated bilirubin transportation. However, the function of the variants should be validated in cell and animal model experiments. Interestingly, simultaneous homozygous mutations in SLCO1B1 and SLCO1B3, resulting in disruption of hepatic reuptake of bilirubin, may account for the predominantly conjugated hyperbilirubinemia of Rotor syndrome  and indocyanine green (ICG) was markedly impaired, indicating a role of impaired hepatic bilirubin uptake . Genetic SCLO1B3 . In the detected . A novelsyndrome . These fUGT1A6 , heme oxygenase (HMOX), biliverdin reductase A (BLVRA), nucleoporin 153 (NUP153) and UGT1A6 . HoweverUGT1A1 and SLCO1B genetic variation of Chinese adults with hyperbilirubinemia and emphatically explored the combined effects of multiple variants. Consistent with the GWAS, our study confirmed the role of the single genetic variation. Moreover, we found that the variants of UGT1A1 and SLCO1B1 have combined effects on the level of bilirubin. The function of the variants should be validated in cell and animal model experiments.This study has some limitation. First, the level of bilirubin in GS is fluctuating, especially in particular conditions such as prolonged fasting, fever, and strenuous exercise . Althoug6>(TA)7, c.211G > A, and c.1091C > T within UGT1A1 as well as c.521T > C within SLCO1B1 are independent risk factors of mild unconjugated hyperbilirubinemia. Our results also reveal that there is a combined effect on unconjugated hyperbilirubinemia for c.-3275T > G as well as (TA)6>(TA)7 or c.211G > A in UGT1A1. Further, co-expression of c.211G > A of UGT1A1 and c.521T > C of SLCO1B1 has a cumulative effect on unconjugated hyperbilirubinemia.In conclusion, this study demonstrates the impact of genetic factors on non- hemolytic unconjugated hyperbilirubinemia in Chinese population. Variations c.-3275T > G, (TA)The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.The studies involving human participants were reviewed and approved by Ethics committee of Beijing Youan Hospital, Capital Medical University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.JB, LL, SZ, and ZD: Conception and design. LL: Patient recruitment. JB and CL: Collection of data. SL and LB: Analysing data. JB: Manuscript draft. JB, LL, YC, SZ and ZD: Editing and revision. All authors read and approved the final manuscript.This study was supported by Beijing Municipal Science and Technology Project (No.Z171100002217070), National Key R&D Program of China (No.2017YFA0103000), National Science and Technology Key Project on “Major Infectious Diseases such as HIV/AIDS, Viral Hepatitis Prevention and Treatment” , “Beijing Municipal Administration of Hospitals” Ascent Plan (No. DFL20151601), Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support (No.ZYLX201806), the Digestive Medical Coordinated Development Center of Beijing Municipal Administration of Hospitals (No.XXZ0503), the You An fund for liver diseases and AIDS (YNKTTS201801189), and the Basic-Clinical Cooperation Project of Capital Medical University (17JL47). Science and technology innovation service capacity building - high-precision discipline construction project (11920703).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.