Posted on Authorea 16 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159493101.19143630 — This a preprint and has not been peer reviewed. Data may be preliminary. K,Hle,&Tej,19;Tno ahsn obn 95 n ute eei tde eeldtheir revealed studies genetic herbicides further with and contaminated 1995) soils Holben, from & mainly Matheson, isolated Tonso, been 1994; have Tiedje, They & 2,4-D. Holben, degrade (Ka, to ability the have 2000), 2002), Gr Meer, al., et Kaschabek, der (Kitagawa parts van Thiel, equal & 2001; of Westing, xylosoxidans Zehnder, Lappin-Scott, mixture A. 2003; Leveau, & a Tomasallo, Laemmli, Burton, is as & Vallaeys, 1997; such It Weber, (Smejkal, environments Harker, chemical, different program. Christian, defoliant & from warfare isolated and (Filer Stellman, chemical bacterium herbicide 134 (Stellman, of its types an 2,4,5-T of different is Many and part which 1984). as 2,4-D Orange, army herbicides, Agent military two 1971, U.S. of the to by 1961 used from was War Vietnam the During We these between energy. Introduction events free mating 1. and recent carbon in of recruited sources been sole have the as may of herbicides potential members the genetic other use of corresponding and to those the species pathways of to metabolic some identical with that virtually equipped hypothesize are are in products that those gene species to the terial identical of almost sequences are petrii acid products B. amino their gene the into the with insight pinatubonensis of comprehensive along Cupriavidus sequences a organization acid obtained gene amino We the the prisingly, energy. with with free along cluster and petrii organization gene carbon B. gene A of The strains. sources these 10.2. sole of BT1 as analysis them genomic by used pathways species Vietnam degradation 3 in last base the military only Hoa were Bien They from xylosoxidans (2,4,5-T). mobacter soil 2,4,5-trichlorophenoxyacetate with and inoculated (2,4-D) as 2,4-dichlorophenoxyacetate cultures classified herbicides enrichment the from with isolated contaminated were strains bacterial Four Abstract 2020 16, July 4 3 2 1 Parsons Nguyen Anh Lan Thi 2,4 of degradation in player acid dichlorophenoxyacetic key a as emerges Orange. petrii Agent with of contaminated bioremediation soil in Vietnamese involved pathways metabolic and Species irLf Solutions MicroLife Amsterdam Technology van and Universiteit Science of Academy Vietnam Amsterdam Universiteit Vrije S184 loehr u nihetapoc a ucsflyrsle nbotn ieettpso proteobac- of types different 3 boosting in resulted successfully has approach enrichment our Altogether, DSM12804. S184 The DSM12804. 3 rmBrouwer Bram , suooa aeruginosa Pseudomonas subsp. T 02 epciey l fte eeal odgae24Dad245Tdrn utvto,but cultivation, during 2,4,5-T and 2,4-D degrade to able were them of 4 All respectively. 10.2, BT1 denitrificans .petrii B. .aeruginosa P. M14 eerdt stp I type as to referred JMP134, 1 h a aDang Ha Cam Thi , 1 β- jl eBoer de Tjalf , and T . tanta eioae a ulcmlmn fthe of complement full a has isolated we that strain 9.2 BT1 T 2.2, BT1 γ- S40 Vde,Vhe,&Hiau 2004), Heinaru, & Vahter, (Vedler, EST4002 proteobacteria. Al and PAOlc pigmnshistidinilytica Sphingomonas 4 n ...vnSpanning van R.J.M. and , Halomonas 2 tfd ac Koekkoek Jacco , 1 ee,adcerydffrn rmtoeof those from different clearly and genes, p F3(lisebr M (Kleinsteuber, EF43 sp. T 5.2, BT1 tfdCDEF 1 atnBraster Martin , nn,Mu Schl & Mau, ¨ oning, odtlapetrii Bordetella ee a on in found was genes 1 Sphingomonas Bradyrhizobium Bordetella .pinatubonensis C. tfdABCDEF le,&Bbl 2001) Babel, & uller, ¨ T .,and 9.2, BT1 .xylosoxidans A. 1 John , mn,2005), ¨ omann, .xylosoxidans A. p TFD44 sp. ee.Sur- genes. p HW13 sp. Achro- JMP and Posted on Authorea 16 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159493101.19143630 — This a preprint and has not been peer reviewed. Data may be preliminary. fsciy-o n ctlCA(abrs agnn ta. 96 abrse l,19) nytanother yet In 1995). al., formation et same in Daubaras the resulting 1996; are ultimately al., merge, product et pathways ultimate maleyla- degradation Danganan, and Tfd (Daubaras, a substrate and acetyl-CoA by their Tft and 3-OXA yet the succinyl-CoA proteins, Here to of 2002). TftGH converted al., from is et differ (Kumari latter that proteins The the LinD (2-MA). by semialdehyde clea- and 4-hydroxymuconic maleylacetate encoded ring yield to Subsequent reductase to 1998). reacts cetate (TftH) al., then 1,2-dioxygenase et hydroxyquinone which (Zaborina by (4-HMSA) catalysed (HQ) then hydroquinone is to vage then to and 2,5-DCHQ dechlorinates (CHQ) (TftG) chlorohydroquinone 2,5-dichlorohydroquinone dehydrochlorinase into reductive 2,5-dichlorohydroxyquinone DCBQ converts First, then (3-OXA). H factor 3 unknown 2003; An The Xun, 1998). (2,5-DCHQ). al., & et chlorophenol-4- Gisi Zaborina a 1995; 1998; rickson, Chakrabarty, by & (2,5-DCBQ) Kitano, 2,5-dichloro-p-benzoquinone (2,4,5-TCP). berger, the to 2,4,5-trichlorophenol by oxidized to encoded further 2,4,5-T monooxygenase is converts compound that latter has one oxygenase The first source. The energy acid all 2,4,5-T. free 2005), 2,4,5-trichlorophenoxyacetic of and Sayler, degradation of carbon & in involved Sanseverino, sole Hay, clusters as 1981), Menn, gene 2,4,5-T 3 Rice, utilizing Chakrabarty, has 2007; of al., & capable et were Huong which Chatterjee, 2016; of Itoh, 1990), Kellogg, & re- Suyama, Baskunov, 2007; been Sano, & (Hayashi, have Suyama, degraders Evtushenko, & 2,4,5-T Pertsova, aerobic eva, Itoh, few in genera a Huong, of is only members database 1994; are bacteria, known KEGG 2,4-D-degrading best the of The to of diversity ported. according the sequence enzymes to acetyl- of a contrast list and In in A Succinyl-CoA acetyl-CoA cycle. (TCA) successively. and S1. acid Table (PcaF), Gordon, succinyl-CoA tricarboxylic supplementary thiolase the Perkins, to fuel 3-oxoadipyl-CoA 1984; degraded may a CoA Chapman, then and the & is (PcaIJ) of Liu 2-MA CoA-transferase products 2000; the involving al., 1990). reactions et Lurquin, the (Laemmli and & by successively carboxymethylenebutenolidase, Caceres, encoded a reductase, enzymes cycloisomerase, maleylacetate 2-maleylacetate the dichloromuconate a to by a degraded pathway 1,2-dioxygenase, sequentially ortho-cleavage chlorocatechol then an is via catechol gene 1993a, (2-MA) The substituted by Hausinger, The encoded & of 2000). (Fukumori is (3,5-DCC). (2,4-DCP) copies al., which 3,5-dichlorocatechol 2,4-dichlorophenol 2 et 6-monooxygenase into 2,4-dichlorophenol 2,4-D (Laemmli had Subsequently, of pJP4 even chain 1993b). plasmid side species acetate on this the another of that cleavage one revealed to Vallaeys, was an 1987; adjacent it Zenk, encodes located & Subsequently, Timmis, cluster Streber, gene 1996). 1981; this Pemberton, Soulas, & & (Don Wright, 2,4-D of Fulthorpe, degradation the for the enzymes on the studies on based was originally 2,4-D of degradation pinatubonensis in 2018). involved C. Amada, genes of & description Kawakami, first Mita, The (Kijima, TfdA of function the replaces so-called a possesses of a organization of of Their complement members full include a bacteria have and II usually Group They enzymes 2004). tion. of 2,4-D-degrading al., subdivision the et the classified Itoh to express Rhodoferax, been belong to Ralstonia, bacteria have potential I bacteria Pseudomonas, genetic Group These 2019). and Tavares, degradation. phylogeny & γ- 2,4-D Giongo, their Pinheiro, for on Rebelo, enzymes based (Serbent, the groups encoding two genes into key of possession rtoatraadicueseisfo h genera the from species include and proteobacteria Bradyrhizobium laiee eutrophus) Alcaligenes α- eoltrt-eedn ,-ihoohnxaeaedoyeae hc aaye h initial the catalyses which dioxygenase, 2,4-dichlorophenoxyacetate ketoglutarate-dependent sri M 3 M 17 rvosytermed previously 1197, LMG / 134 JMP (strain rd Ktgw ta. 02 hrkv,Iskv hrno ooo,&Mruhv,2018). Markusheva, & Korobov, Zhurenko, Iasakov, Zharikova, 2002; al., et (Kitagawa cadABCD tfd eecluster, gene ee sdffrn rmta fgopIseisad importantly, and, species I group of that from different is genes eecutrecdn h uuiso ighdoyaigdoyeaethat dioxygenase hydroxylating ring a of subunits the encoding cluster gene tftCD hwn hti otie ocalled so a contained it that showing , tftE tftEFGH ee notoou of orthologue an gene, ee nte2 the on genes and tfdF Variovorax α- and noe h nye o ovrigDH o3-oxoadipate to DCHQ converting for enzymes the encodes , Burkholderia rtoatra lsl eae otegenera the to related closely proteobacteria, Sphingomonas pcaIJF 2 nd crmbce,Brhlei,Dlta Halomonas, Delftia, Burkholderia, Achromobacter, Hffan lisebr M Kleinsteuber, (Hoffmann, lse Dnaa ta. 94 abrs Hersh- Daubaras, 1994; al., et (Danganan cluster ee,tels fwihecd 3-oxoadipate a encode which of 3 last the genes, Dnaa,Y,Duaa,Xn Chakrabarty, & Xun, Daubaras, Ye, (Danganan, be,Dnaa,Xn hkaat,&Hend- & Chakrabarty, Xun, Danganan, ubner, ¨ tfdABCDEF tfdF Hoge l,20) and 2007), al., et (Huong . fA genes tftAB .paucimobilis S. .nctr asoi eutropha Ralstonia necator, C. tfdABCDEF tfdCDEF eecutrfr24Ddegrada- 2,4-D for cluster gene ukodracepacia Burkholderia tfdB noigtetosubunits two the encoding ee,wihicuea include which genes, eecutrexpressing cluster gene ovrs24DPinto 2,4-DCP converts Nocardioides le,&Bbl 2003; Babel, & uller, ¨ tanB0hsLinE has B90 strain β- proteobacterium Bradyrhizobium Sphingomonas Sphingomonas (Golovl- AC1100 β- tfdA and and Posted on Authorea 16 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159493101.19143630 — This a preprint and has not been peer reviewed. Data may be preliminary. ehrad) h QM a pliga lcr pa oiaini eaieinmd.Tetransitions The mode. ion the Valkenswaard negative a Biosolve, in (both ionization methanol spray 3 and electro Germany). mm, acid an Bremen 2 applying formic was Bruker, x 0.1% the TQ-MS (both of 50 The for gradient detector a collected Netherlands). a was selective on applying layer mass separated UHPLC top Netherlands), Elute quadrupole were the the an of triple compounds on ml EVOQ measured target MgSO 2 were an The of Then, 2,4,5-T to and rpm. g 2,4-D 10,000 (2 coupled of at salts system concentrations min of remaining 5 The mixture for analysis. A centrifugation LCMS/MS acetonitrile min. by of 15 followed ml for 2 min, which vortexed 5 after C and tubes, of extracted Falcon added g 15-ml was were 0.5 to acid herbicides transferred formic 2015). were The Tuzimski, ml) 1% & 8.2.1. (2 (Rejczak with version cultures method Safe) the Prism and of using Rugged, ana- aliquots Effective, Briefly, performed remaining Data Cheap, Easy, were (LC-MS/MS). the (Quick, QuEChERS tests detecting spectrometry the by using mass biodegradation chromatography assessed the liquid was of tandem communities lyses by bacterial herbicides of of ability using concentration 2,4,5-T-degrading time and LC-MS/MS in 2,4-D by Thermo determined The 2,4,5-T GO, were and (Multiskan samples 2,4-D Series). 1200 of spectrophotometer culture Technologies, Analysis a all (Aligent 2.2 determined (LC-MS/MS) was in using was spectrometry herbicides 2,4,5-T nm same mass growth the and 600 the chromatography Cell with 2,4-D liquid at under triplicate. amended tandem of cultures experiment in cells Concentration of degradation performed dead U.S). the absorbance independently Scientific, with supplemented for the were flask medium experiments BS measuring inoculum A into All by cultures. as transferred control. enrichment dominant 2,4,5-T and the as as mg/l BS isolated for used sterilized 100 were those with that and as times strains 2,4-D 2 conditions Bacterial washed mg/l centrifugation ml. by min, 100 200 harvested 5 were per with cells peptone, for agar The g rpm medium. g 1.0 (NB) 10,000 1.5 contained broth at and medium nutrient in (NA) NaCl, overnight agar g pre-cultured 30 Nutrient were 0.5 CFUs at experiment. extract, the agar meat of nutrient g start on 1.0 the shaker after isolated rotary points were a time on (CFUs) different dark units carbon- the as forming in used Colony incubated and 2 were pure, a 95% Cultures inoculate than assays. more degradation 30 Sigma, during from at source purchased energy were free compounds (10 and Both airbase 2,4,5-T. mg/l Hoa 100 KH Bien NaNO containing and culture) from g/l per 0.5 ml collected 40 was (BS, medium studies salt enrichment for 106 soil contaminated Herbicide species 2.1 of view methods integrative sequences phenoxyacetates. an and genome chlorinated of in their Materials these resulted degradation unravel of 2. efforts the to degradation These monitor iii) the potential. to and in metabolic ii) biomass, involved their defined pathways cellular sources, in under and insight in energy strains obtain increases indigenous free to with the and order along culture carbon in in to time sole potential i) in compounds. as genetic to herbicides these 2,4,5-T and was these of physiological and study breakdown their 2,4-D this in of in with involved sole understanding approach conditions genes as fundamental The had 2,4,5-T more process. with which and a metabolic contaminated 2,4-D of getting that with heavily both at cultures and soil aimed enrichment energy, we in in free Here, dominant present and were originally carbon which of of strains sources all bacterial Vietnam, indigenous in isolated Orange Agent we 2011). study, al., this et Takenaka In 2005; (Tft- al., et chlorophenol-4-monooxygenase Ferraroni enzymes (Ferraroni 1996; Chakrabarty, (ChqB) the 1,2-dioxygenase via hydroxyquinol al and 2-MA et (HbqR) to reductase converted 2-hydroxy-1,4-benzoquinone is CD), 2,5-DCHQ pathway, parallel ° utrn odtosadioaino atra strains bacterial of isolation and conditions Culturing 81.”) ogNiPoic,Venm e rm fhriiecnaiae olwsaddi basal in added was soil contaminated herbicide of grams Ten Vietnam. Province, Nai Dong 48’19.3”E), ° 05.Teeezmsaefudi f.e. in found are enzymes These 2005). . n 0 p te1 (the rpm 200 and C 6 H 5 nd NaO nihetwt 0 gl24Dad10m/ ,,- n ute nuae o 5days. 25 for incubated further and 2,4,5-T mg/l 100 and 2,4-D mg/l 200 with enrichment 3 7 .2H . / vndrZa ta. 02,adte eesplmne ih10m/ ,- and 2,4-D mg/l 100 with supplemented were they and 2012), al., et Zaan der (van g/l 0.4 2 ,ad02 fC of g 0.25 and O, st nihet.Atrcliainfr2 as hs utrswr hnue to used then were cultures these days, 26 for cultivation After enrichment). 6 H 6 Na Burkholderia 2 O 2 7 PO 15H .1.5 3 4 . /,(NH g/l, 0.5 2 )wsaddt h ooeaeadmxdfor mixed and homogenate the to added was O) μ and uaC8clm Peoee,Utrecht) (Phenomenex, column C18 Luna m Nocardioides o ysra iuino h utrsat cultures the of dilution serial by C 4 ) 2 SO 4 .5gl MgSO g/l, 0.25 pce Duaa,Sio & Saido, (Daubaras, species 4 . fNaCl, of g 0.5 , 4 . /,CaCl g/l, 0.2 ° 58’14.3”N 2 Posted on Authorea 16 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159493101.19143630 — This a preprint and has not been peer reviewed. Data may be preliminary. Fg )wsvsaie sn h EAvrin7sfwr yteniho-onn ehdwt 1,000 with method neighbor-joining the by software tree 7 phylogenetic reference version a with and MEGA compared aligned the were were using sequences bacteria The isolated visualized database. 4 was KEGG the 3) and from NCBI (Fig. homologues the TftA in available of sequences sequences acid GenBank amino The in (Tatusov, 5.2 deposited species BT1 were closest brackets. (PRJNA586721), (PRJNA587043) between as 2.2 2.5 in 10.2 hits BT1 listed BT1 most numbers of and the Bioproject genomes their with (PRJNA586752), for NCBI-annotated with database species The 9.2 (COG) the 2000). BT1 Groups taking homologues Koonin, (PRJNA586745), Orthologues closest by & of The and Clusters Natale, 2015). the separately Galperin, al., using gene et analysis Weber predicted Ramulu, metabolite BLAST 2019; each secondary Golaconda by CAZy al., and determined Lombard, SignalP et the was 2014; with Armenteros and species predicted (Almagro al., were databases) AntiSMASH et peptides hamap using Signal (Jones clusters 2014). and families) Henrissat, (Pfam & 2014). 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(the by Scientific, checked B.V (ThermoFischer were BaseClear DNA kit genomic Assay extracted HS PowerSoil the dsDNA MoBio of quantity the and using quality extracted annotation was and Center DNA assembly, National Genomic sequencing, the genome at extraction, search DNA BLAST Genomic the 2.4 using 16S database known (http://www.ncbi.nlm.gov.BLAST/). GenBank with Information compared the were Biotechnology in PCR sequences for Germany). deposited These Jena, sequences Europe. Analytik Macrogen gene (Biometra, by rRNA Thermocycler sequenced a and Pelle- in purified performed Barns, were were (Weisburg, products Reactions previously 1991). described Lane, AGAGTTTGATYMTGGCTCAG & as ACGGYTACCTTGTTACGACTT-3’) (5’- tier, (5’- 8F primers 1512R universal and extrac- with was -3’) out bacteria carried of was DNA Amplification analysis. PowerSoil sequence instructions. MoBio gene rRNA the strains 16S using on isolated ted based of identified were sequencing isolates gene bacterial The rRNA 16S and isolation DNA 2.3 4 software. at review store Data and water deionized in 253 were 221 were 2,4-D for Phylogeny > 9 n 255 and 195 > 6 n 219 and 163 > 9 qatfiradqaie,rsetvl) eoeaayi,smlswr diluted were samples analysis, Before respectively). qualifier, and (quantifier 197 ® ° ni neto.Dt custo n nlsswr efre ihMS with performed were analyses and acquisition Data injection. until C N slto i Crsa,C,UA codn otemanufacturer’s the to according USA) CA, (Carlsbad, kit Isolation DNA > 6 qatfiradqaie,rsetvl) o ,,- h transitions the 2,4,5-T For respectively). qualifier, and (quantifier 161 4 ® N slto i Crsa,C,UA.The USA). CA, (Carlsbad, kit Isolation DNA Posted on Authorea 16 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159493101.19143630 — This a preprint and has not been peer reviewed. Data may be preliminary. r itda el h hehl o eeto fteotooospoen a e tamnmmo 45% of minimum a least at at set should they was so proteins 2,4,5-T orthologous and the 2,4-D and of of genomes degradation materials selection selected (initial) the showed for the A-D. in species from S3 threshold described Table 4 expressed The Supplementary all is those in Importantly, well. analyses identity. and listed as these sequences are protein organism listed for selected each KEGG sequences script are for our similar the The analyses for between of isolates. these look identity S1. of to list 4 Percentages Table used results our A Supplementary then The were of in pathways. section. genes shown sequences methods enzymes selected metabolic is genome the the these parameters of the all sequences additional in of in acid some involved sequences amino and database acid being KEGG properties amino further The as their and the with benzoate database gathered to along we KEGG 2,4,5-T enzymes end, and the this 2,4-D To degrade in acetyl-CoA. to identified potential and genetic succinyl-CoA their to for on genomes 4 the days. 2,4,5-T inspected of 10 We Degradation required mixture. this the degradation mixture with herbicide those the in for in within days Pathways removed 10 while was cultivation, took the compound it of and carbon former while properties days The 2,4-D, compounds, growth of 4 only 2,4,5-T. These 2 with and source only. took the cultures 2,4-D 2,4,5-T herbicides. sole of the of with in mixture rates both as observed days degradation the was of 2 2,4-D the in yield mixture with only and less part a rates slightly with in with to were coincided cultures regard yields culture the responses with and the performance in the rates from growth poorest observed studied those Both were we energy. with degrade capacities, yields free C) completely and its highest and to on and 2B ability view growth (Figs its Fastest comprehensive herbicides with more the along a of growth removal get of biomass of to yields maximum that order and rate, to of In rates growth comparable slower 2,4,5-T. to was apparent and respect yield its 2,4-D with final of to the performer that Due but best than days, yield. cultivation. the less 7 growth of slightly after days maximum was obtained 4 its was biomass after reach reached final 3 to was other longer its growth and The bit Also, maximum 2,4-D formed. growing. cases, The of were cultivation. started both removal that of it In days complete products before 10 show the 10 within days however, on herbicides after 2 did, data the again It of no were removal decreased have growers lysis. complete subsequently we fastest and and but which growth death cultivation, substantial cultivation, cell showed of 1A) of strains of days (Fig. days result 10 curves 7 within a C). growth after 2,4,5-T and as their biomass 1B with likely determined (Figs We supplemented in most time energy. medium increase days, in free salt small 2,4,5-T and basal a and carbon in 2,4-D with cultured of of were sources removal isolates the sole the and as where 2,4,5-T cultures experiment and enrichment growth 2,4-D from a isolated out strains set four We by 2,4,5-T and 2,4-D of Biodegradation herbicide ( their 16S similar study the highly respectively. were that to 10.2 showed BT1 selected and as results 9.2, were them BT1 BLAST enrichments 5.2, that. BT1 the achieve 2.2, BT1 in to to isolates make-up the species of genetic dominant sequences their rRNA most and the were capacities isolates as varied biodegradation dominant strains up the These that popped energy. indicated that free results and The communities Staphylococcus plates. carbon soil agar of Sphingopyxis, nutrient contaminated sources on herbicide sole colony genera as of from from colour 2,4,5-T isolated and and shape were 2,4-D in with colony cultures bacterial in of enriched types bacteria different 2,4,5-T-degrading Fourteen and 2,4-D- of classification and Isolation Results 2016). 3. Tamura, & Stecher, (Kumar, replicates bootstrap .petrii B. .aeruginosa P. .aeruginosa P. T . nclue ihol ,- rol ,,- n oprdtegot aa(i.2)and 2A) (Fig. data growth the compared and 2,4,5-T only or 2,4-D only with cultures in 9.2 BT1 hdnbce,Atrbce,Pedmns crmbce,Shnooa,Bsa Pedobacter, Bosea, Sphingomonas, Achromobacter, Pseudomonas, Arthrobacter, Rhodanobacter, , .histidinilytica S. .petrii B. T 2.2, BT1 and T . and 9.2 BT1 S.histidinilytica Ancylobacter ., .petrii B. .xylosoxidans A. and , Tbe2) (Table T 5.2, BT1 .xylosoxidans A. 5 . .petrii B. mn hs slts rmngtv strains Gram-negative 4 isolates, these Among T 02 u h atroehdalgtm of time lag a had one latter the but 10.2, BT1 .aeruginosa P. T .,and 9.2, BT1 epciey hrfr,w classified we Therefore, respectively. , .histidinilytica S. .petrii B. T . hwdpo growth poor showed 2.2 BT1 .xylosoxidans A. T ..Telte was latter The 9.2. BT1 .petrii B. T . edda needed 5.2 BT1 > 9 identity) 99% T 10.2, BT1 T 9.2. BT1 Posted on Authorea 16 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159493101.19143630 — This a preprint and has not been peer reviewed. Data may be preliminary. o n ctlCA motnl,i osnthv napparent an are have genes not Their does 3859. it and Importantly, 2507 acetyl-CoA. 1633, TftA, and 524, of CoA ORFs role are the histidinilytica over 30% S. take to to of close the candidates homologues TftA to Likely with reference related accompanied proteins the reaction. all that to this assume identities for to we with responsible identity step, all are dioxygenase high produce hydroxylating proteins with to ring TftAB genes enzymes initial reference the the of find the also homologues Also, perform not reasonable has to did lacks It product. potential We it the However, degradation 3407). BT. consume 2,4,5-T (ORF to to in genome 2-HBQ or the convert on to substrate able elsewhere the be found may was that it TfdA, show so reference comparisons the pairwise species to the between databases, identity Vietnamese Also, those the the isolated of 5A). than in copy lower the (Fig. second much deposited of different are sequence are organization are identities organizations genome 2 organizations the genetic gene these gene its the Their of the with compared ancestors Also, AM902716.1). one also between 100%. We transfer to the gene 99 with horizontal the from evolution. of ranging of recent kind which identity during some of The of all acetyl-CoA. suggestive high, identical remarkably and is succinyl- the M35097.1) is into no. genes 3-oxoadipate-CoA structural of other cleavage the tfdBCDEF the to for by opposed sandwiched order responsible genes direction the four enzyme transcription in the the of transcribed with One and and gene. clustered genes 4 are by They separated 5A). HBQ into (Fig. 2,4,5-T 2-MA a convert into to 2,4-D enzymes of conversion the encoding petrii genes B. the species. Yet, this 2-MA. in to identified not BT were via are 2-HBQ products of breakdown conversion 2,4,5-T from of counterparts degradation KEGG further their from with identities for protein 32% candidate reference obvious (around likely KEGG an A the Its find respectively. with not species. 2,4,5-T, tity did these and We of 2,4-D amino S3). ancestors of Table corresponding between conversion (ORF2511; and event genome organization transfer the gene gene on recent This a of a AbsAc. those of of flavodoxin, to subunits result identical the almost the is are donor sequences encoding acid electron genes the in are described and first Downstream AbsAb 2,3-dioxygenase 5A). 2-aminobenzenesulfonate a (Fig. to a it similar on most from dioxygenase emphasis hydroxylating transcribed with ring isolates divergently 4 and the tream of to description orthologous 2,4,5-T. further xylosoxidans sequences and a A. their 2,4-D 4. is convert identifying Fig. Below to by in S1). potential species chlorobenzene shown (Fig. genetic isolated pathways and is their 4 reference pathways ‘chlorocyclohexane our the their these maps in of and of pathways those 2,4,5-T database representation the and KEGG graphical reconstructed 2,4-D A the then degrade We degradation’. pathways using to to ‘benzoate reference herbicides species As the and reason group. 4 constructed the degradation’ TftA had the first of the We we of to KEGG acetyl-CoA. degradation potential than as and the proteins minimum succinyl-CoA for AntA from these to the enzymes and to ultimately judge closer reference metabolites of BenA to group further the tree found CadA, able with we phylogenetic were include that along A we homologues also 3 such role. the enzymes Fig. of that some reference in over that shown take These believe is could databases. isolates that NCBI 4 enzymes and the unknown from yet homologues as TftA or 2,4,5-TCP, into 2,4,5-T a have Alcaligenes lysR tfdA -type T . a ulcmlmn of complement full a has 9.2 BT1 noe rtiswt h EGrfrnepoen from proteins reference KEGG the with proteins encoded eet erd ,- no24DP n a and 2,4-DCP, into 2,4-D degrade to gene pce Mme,Rff ukr ok 99.Tectltcpr scmoe fAsaand AbsAa of composed is part catalytic The 1999). Cook, & Junker, Ruff, (Mampel, species tfdS T 02hsits has 10.2 BT1 T . a h eei oeta oepesalezmsfrdgaaino ,- osuccinyl- to 2,4-D of degradation for enzymes all express to potential genetic the has 5.2 BT1 tfdA ooou ptemaddvretytasrbdfo ht i oedownstream more bit A that. from transcribed divergently and upstream homologue ee o lsee ihother with clustered not gene, tfdCDEF tftB . .pinatubonensis C. ee lsee ln iharegulatory a with along clustered genes tfdABCDEF .petrii B. tfdA .petrii B. n remaining and 6 tftAB hbqR tfd S 20.Ti ihdge fiett a ethe be may identity of degree high This 12804. DSM hbqR ee n ihteepesdpoenhvn 45% having protein expressed the with and genes eestt noetedoyeaefrconverting for dioxygenase the encode to set gene .cepacia B. eei o lsee ihohrgnsinvolved genes other with clustered not is gene ee noigteezmsfrtesequential the for enzymes the encoding genes T . and 9.2 BT1 and .Toefor Those ). chqB tfdA tfdA tfd tfdA tftAB .pinatubonensis C. .petrii B. .Ohrgnsptnilyivle in involved potentially genes Other ). ee sagn noigaPcaF-type a encoding gene a is genes ee h ucino the of function The gene. xrsigHq n hBfrthe for ChqB and HbqR expressing eenor gene sOF36 TbeS,3%iden- 35% S3, (Table 3265 ORF is .pinatubonensis C. ee u sti tanhsthe has strain this as but genes tftAB tfdB S 20 acsinno. (accession 12804 DSM tftAB eei oae elsewhere located is gene r Rs21 n 2314 and 2313 ORFs are tfdS ee o h initial the for genes M14(accession JMP134 eelctdups- located gene β- tfdCDEFB TbeS4 (Table proteobacteria hbqR tfdA ). gene gene with tfdA A Posted on Authorea 16 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159493101.19143630 — This a preprint and has not been peer reviewed. Data may be preliminary. ovr ,- tlatit ,-C,adw pclt htti sahee ytepouto R30,which ORF3804, of product the by to achieved reference able for found is KEGG is set this are the It that with PcaF acetyl-CoA. speculate identity and we and 38% succinyl-CoA and PcaIJ has 2,4-DCP, to into 3-oxoadipate encoding has least of Genes at breakdown it 2,4-D further 3-oxoadipate. convert as for to genome compounds catechol the on via related that elsewhere benzoate and suggest of findings benzoate conversion these anthranilate of sequential substrates, together, degradation their their All in convert supports catechol. which BenABCD, specialized to sub-groups, and respectively, the AntABC benzoate, in systems, and position enzyme their Both shows functions. proteins suggested electron BenA and flavodoxin-type AntA (BenD). including the of homologues dehydrogenase as set carboxylate AntC a a cyclohexadiene likely and most dihydroxy also most core one a is The and catalytic downstream clusters. (BenABC) more the gene one as which 2 The AntAB of donor. these both 1,2-dioxygenase, flanking oxygenases, anthranilate genes ring-hydroxylating an their of the regulators, subunits to AraC-type the close of encoding control clusters apparent gene under 2 are upstream More of composed in clusters erythropolis Gene found the . is by 2014) organization sandwiched Lorenzo, the gene and encompassing to direction similar cluster equivalent same A gene the possibly the in gene at Transcribed regulatory typical look 3-OXA. a closer to of A degradation part catB 5C. catechol make in Fig. they involved in as are is features interesting cluster some gene shows this have of which map 5532, A and 5533 aeruginosa dioxygenases. ORFs from P. hydroxylating of respectively, ring TftB, products of proteins. and gene family reference TftA the counterparts same database by their KEGG the out with to identity carried belong 30% be they around may as by reaction that encoded that obvious do be Alternatively, have can must not proteins enzyme does CadABCD that another degradation It the speculate Hence further 2,4,5-TCP. to for proteins. catechol tempting to Cat into is least substrates the It aromatic by unknown TftAB. yet maleylacetate and as to BenAB their convert AntAB, systems like enzyme dioxygenases these hydroxylating ring of family the a of Upstream has it S4). that observation as the aeruginosa them by a regard of exemplified we control complementary further apparent Nevertheless, the with is S3). clusters Table aromatics gene see the chlorinated 42%, in occurrence and their 32 to of between because addition (in In proteins reference intermediates. KEGG TCA-cycle to suggested way hence the the acetyl-CoA, of all and on 2,4-D set succinyl-CoA and from contain into another with pathway organization proteins, clusters cluster cluster full Tfd both gene gene a the Also, the that of making of level indicates product of S4). the strain the potential (Table on isolated 2-MA, the sequences both our of acid conversion 95%) from amino for than those enzymes (more their with identity latter of of the level degree of the high products a gene share the they of comparison pairwise A AY598949.1), a by Sphingomonas no. encoded (accession dioxygenase TFD44 hydroxylating other sp. ring the a between by sandwiched over is taken likely is product and tftAB catA T ..Piws dniiso h aRB rtisfo ohseisaeaon 0 (Table 50% around are species both from proteins CatRABC the of identities Pairwise 2.2. BT1 C29 acsinn.F953.)and FM995530.1) no. (accession CCM2595 cat benABCD tfdD ee ih eOF 00ad13.Tepoenfo R 00hs3%iett ihthe with identity 30% has 1030 ORF from protein The 1031. and 1030 ORFs be might genes T . has 2.2 BT1 p R5(ceso o F9271 Fg C Nesne l,21;Nesne l,2013). al., et Nielsen 2017; al., et (Nielsen 4C) (Fig. KF494257.1) no. (accession ERG5 sp. pcaIJF ee sagn oooosto homologous gene a is genes eecutrhs2srcua ee ihhgetsmlrt with similarity highest with genes structural 2 has cluster gene and catR cat tfd ee.Ti n a esice nfrgot nclrctcos Importantly, chlorocatechols. on growth for on switched be may one This genes. tfdE ee daett its to adjacent genes and eecutrae2st fgnsta eebetoeecdn h uuiso the of subunits 2 the encoding those resemble that genes of sets 2 are cluster gene euaoygn Fg C.Isgn raiainlosqiesmlrt h n of one the to similar quite looks organization gene Its 5C). (Fig. gene regulatory catAB ee nbt lsessaealwiett ihtercutrat rmthe from counterparts their with identity low a share clusters both in genes cad tfd ee,i a nte n ihcpe of copies with one another has it genes, ee oetal novdi ,- radw.Tergnsaeclustered. are genes Their breakdown. 2,4-D in involved potentially genes ee Fg B.Asmlrgn raiaini enin seen is organization gene similar A 5B). (Fig. genes catR pigbu herbicidovorans Sphingobium .putida P. tfdA MFl,Cuai hkaat,19;Njr ta. 2002). al., et Nojiri 1998; Chakrabarty, & Chugani, (McFall, catRABC cat catC rmspecies from eecutr(i.5) h hlgntcte ihTftA with tree phylogenetic The 5C). (Fig. cluster gene benABCD n ptembtdvretytasrbda transcribed divergently but upstream and t2(i´nz ´rzPnoa hvr´a ´a,&de (Jim´enez, P´erez-Pantoja, Chavarr´ıa, D´ıaz, & mt-2 7 ee u raie ieetyaeas on in found also are differently organized but genes Burkholderia .pinatubonensis C. eecutrecdn ezaedioxygenase benzoate a encoding cluster gene .histidinilytica S. catABC tftAB p H acsinn.AB035483.1). no. (accession TH2 sp. H(ceso o J2811 and AJ628861.1) no. (accession MH tfd ee,yti sal ocneti at it convert to able is it yet genes, eecutr h rtiso which of proteins the cluster, gene ben ee.Isvraiiyi degrading in versatility Its genes. tfdCDEF and catABCD M14 natraiegene alternative An JMP134. cadABCD .cepacia B. T ..I ih ethat be might It 5.2. BT1 Burkholderia pcaIJF .aeruginosa P. cat antABC ee ni,aogwith along it, on genes eecutr o the for clusters gene eecutrunder cluster gene ee omk the make to genes eecutrthat cluster gene htsrea the as serve that ee omake to genes Sphingomonas catAB p H has TH2 sp. T . is 2.2 BT1 lysR tfd genes genes -type R. P. Posted on Authorea 16 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159493101.19143630 — This a preprint and has not been peer reviewed. Data may be preliminary. ar u h aerato.Ti spriual refrteTd n fEpoen.I n ok tthe at looks one If proteins. to TfdE able and of are TfdD proteins yet identity, the II low type for such and true that have I member particularly show type that is a we the species those This by Indeed, of one identify identities reaction. counterpart. from catalysed not pairwise same differ KEGG may is substantially the proteins the 2,4,5-T cases out KEGG with some of carry the identity in degradation Hence, enzymes low in orthologous another. relatively enzymes the step to a key its that breakdown of initial with indicate the of 2,4,5-T biomass the but identities encoding fate for in family, pairwise least genes the recruited increase TftA at of of some be the that unaware suite may see of however, are full likely, enzymes did we a we unknown more yet species yet as is cultures, the growth as the Perhaps of bacterial makes of either degradation. and it all sequential in that in its recognize or 2,4,5-T for not enzyme of alternative did We an disappearance encoding metabolites. the gene also a identity. noticed lower carries We of with it growth homologue that and TfdA 2,4-D assume a of we have of disappearance culture, use explain does the to it in order as 10.2 In clear BT1 database. KEGG less the is in however, homologue like potential, Just genetic 1999). Its (Kohler, with energy. 2,4,5-T free degrade and not carbon could strain M this with 1999; S but studies (Kohler, 2004), herbicides previous Kohler, acid phenoxyalkanoic in Previously,& of 2007). reported degradation al., the also et for Huong were 2011; findings Piotrowska-Seget, & These Zmijowska, compounds. aromatic catR T . ihrgr otebekono h ebcdsi ute mhszdb h bevto htit that observation the by emphasized further one is to herbicides similar the of quite of versatility are breakdown with The CadA, pathways. the 2 and and to a BenA reactions regard has different TftA, with in as of tasks 5.2 such set specific BT1 dioxygenases, obvious have hydroxylating an they have ring yet from not another of fact does family it in the that is of know fragment now a We PCR detected data). the we unpublished study, al., the previous within et a common (Anh In quite be 2013). to al., seems This function. TfdA the so-called Instead, 4 dioxygenase. of 2,4-D genus 2008). dependent the al., acid et of a-ketoglutaric (Gross species studies by physiological to 2,4,5-T of correlated or not 2,4-D were these of Like but degradation degradation, show herbicide that in involved studies no knowledge, Bordetella our to by are, species 2.4-D 3 there these of in 2,4,5-T breakdown of The degradation cultures detailed for sequential pathways a batch the The with for in enzymes dioxide. along clear. key grew carbon studies the less to and Physiological express way are to energy. the data) potential all free the unpublished 2,4-D have isolated and of they al., were carbon degradation that et herbicides, revealed of genomes (Nguyen sources these their sole on culturing of in as enrichments inspection of 2,4,5-T during 2,4,5-T end and numbers and the 2,4-D their with at 2,4-D increase and species of grow dominant degradation to as the shown were in 3 players All key as identified and as were 9.2 They proteobacteria BT1 Orange. Agent of with contaminated species soil Vietnamese 3 identified have We Discussion 4. from TftA reference KEGG ˙ . tfd histidinilytica pcaIJF .petrii B. pcaIJF ee hc ol lo tt rwo aehl swl n mhszsisvraiiyi rwhon growth in versatility its emphasizes and well as catechols on grow to it allow would which gene, ee.Adsiciefaue oee,i h bevto hti osnthv a have not does it that observation the is however, feature, distinctive A genes. nd eecutrwith cluster gene hr sol n eotpbihdta naesisgnm eune hc ipasgenes displays which sequence, genome its unravels that published report one only is There . ee.As oal stefidn fa3 a of finding the is notable Also genes. ee,btw ol o dniya identify not could we but genes, cadABCD .xylosoxidans A. T .,also 9.2, BT1 T .,also 5.2, BT1 ee noigtesbnt farn yrxltn ixgns htrpae the replaces that dioxygenase hydroxylating ring a of subunits the encoding genes tfdCDEF .cepacia B. .histidinilytica S. .petrii B. T 02 h rti an is first the 10.2, BT1 A.xylosoxidans cadA ee ln iharegulatory a with along genes n t s scro n reeeg orei oe nigas finding novel a is source energy free and carbon as use its and AC1100. hsrsl ute nesoe h vrl iwta members that view overall the underscores further result This . T . a rwo h ebcdsadhsacmaal set comparable a has and herbicides the on grow can 5.2 BT1 tfdA tftA T 02cngo n24Daduei ssl oreof source sole as it use and 2,4-D on grow can 10.2 BT1 eefamn sn C ihdegenerate with PCR using fragment gene ooou ihmr hn4%iett otereference the to identity 45% than more with homologue rd 8 α- .pinatubonensis C. eecutrwith cluster gene rtoatru,telte are 2 latter the proteobacterium, α- rtoatra(ile ta. 07 ile et Nielsen 2017; al., et (Nielsen proteobacteria tfdBCDEF .petrii B. lysR .herbicidovorans S. le,Bre eln a e Meer, der van Werlen, Byrde, uller, ¨ .histidinilytica S. ee niscutrsnwc set a sandwich cluster its in genes catABCD o xml hni snoteworthy is it than example for tp eejs as just gene -type T . rwn n245T It 2,4,5-T. on growing 9.2 BT1 tfdBCDEF Sphingomonas ee n regulatory a and genes .petrii B. tfdA tftAB T 5.2 BT1 Hwsreported was MH β- S eet aean make to gene ee,adthat and genes, proteobacteria. tfdS A.xylosoxidans . histidinilytica T . and 9.2 BT1 tftA ee along genes n again and .petrii B. , (Cyco´n, primers Posted on Authorea 16 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159493101.19143630 — This a preprint and has not been peer reviewed. Data may be preliminary. adi,I . tsa-oisi . aisn .(94.U nsoe .Soedrvdgriaincues germination Watson. Smoke-derived Ex. I. smoke: torr. networks. in attenuata . Up neural Nicotiana (1994). S., deep annual, R. Brunak, Davidson, postfire using & O., for predictions L., Winther, Staszak-Kozinski, peptide T., N., I. signal T. Baldwin, improves Petersen, 5.0 K., SignalP C. (2019). Sønderby, 37 H. D., Biotechnology, K. Nielsen, . Tsirigos, . J., J. Armenteros, 826/QD-BKHCN). Almagro management. code data project in (MOST, assistance Vietnam Affairs References for Technology Economic Melkonian & 6. of Chrats Science Ministry and of Dutch Olivier Ministry the Brett the from thank grant of We Foundation-FES grant BE-Basic a a by by and supported approach is an work Such This use. well. they as that bioremediation pathway of of Acknowledgements: degradation types types in combined5. the other insight and we for fundamental that applied where in more be involved approach a easily species boost ecology in the may to resulted 2,4,5-T, activity systems and TfdB analyses integrative 2,4-D and downstream our of TfdA and some Altogether, genomics have already with pathway. that aromatics physiology clusters other species degrading these of in Hence 2,4-D breakdown transfer 2004). and genetic the Wood, fires via & Bentley, useful wood Fishman, during become Tao, might formed 1994; also Davidson, are & Staszak-Kozinski, they (Baldwin, as phenoxyacetates the than allows older now it the consequence, by tfdCDEF a produced As were which the 1940’s. of 2,4,5-T, these the and that after and 2,4-D and recently, only as quite 9.2 Company such occurred Paint events aromatics exchange Chemical chlorinated gene American that on suggest growth we supported high, events so the are of identities the location Since genetic ( database the the about in from information deposited clusters members already gene was between The that between transfer events. one exchange the gene Apparently, from horizontal 12804). different of DSM is acid soils events amino Vietnamese recent from of the suggests isolated those with observation and That these along hand hand. of organizations one other the cluster of the on those a gene between on I) as 95%) of cluster regarded than similarities (gene (more be JMP134 products may of gene culture degrees scavengers. their enrichment to of manages high the sequences consumers it such, noticed primary Alternatively, As from cultures. further cells. ranging enrichment We levels lysed cultures the trophic enrichment of in different contents the herbicides with the in the web on of dominance food feeding metabolites its by on for survive feeds reason to it possible that A be catechol. by may to preceded respectively, is anthranilate, cluster and same the as catC 2017). al., of that showed sources et which Yang as studies, Ju´arez-Ram´ırez, herbicides other Rubio-Granados, (Marr´on-Montiel, with the Gal´ındez-Mayer, Ruiz-Ordaz, contrast 2,4-D use 2006; & in biodegrade to is able This herbicides. were the cultures on enrichment energy. the free from and isolated carbon species dominant all Not we Hence, that 2000). argument al., valid et a 45%. (Laemmli as respectively identity the identities, low of (TfdD) of counterparts 35% observation and this (TfdE) use 15% could only share they that eeadaregulatory a and gene tfdABCDEF .pinatubonensis C. β- ee,hneterpout ev ocnet(hoiae)ctco no2M.Ctcosaemuch are Catechols 2-MA. into catechol (chlorinated) convert to serve products their hence genes, rtoatra ti upiigta h lse n t eepout from products gene its and cluster the that surprising is It proteobacteria. 4,4043 doi:10.1038/s41587-019-0036-z 420-423. (4), tfdD ee.Tecutr from clusters The genes. .aeruginosa P. .aeruginosa P. and M14t erd ,- l h a o2M ste aeafl complement full a have they as 2-MA to way the all 2,4-D degrade to JMP134 catR tfdE .pinatubonensis C. ee ugsiefrarl nctco eaoim htwudmk sense make would That metabolism. catechol in role a for suggestive gene, ee from genes T . oshave does 2.2 BT1 benABCD T . a oovosstof set obvious no has 2.2 BT1 .xylosoxidans A. .petrii B. .pinatubonensis C. tfd and lsesfo u sltsa ehv nyasto contigs. of set a only have we as isolates our from clusters and antABC 9 .petrii B. catAB pce ihtermtn ateswr independent were partners mating their with species .petrii B. ora fCeia clg,20 Ecology, Chemical of Journal eecutr,alwn tt ovr benzoate convert to it allowing clusters, gene T 02and 10.2 BT1 ee,tog,btteeaecutrdwt a with clustered are these but though, genes, S 20 r npamd,btw aeno have we but plasmids, on are 12804 DSM lhuhte hr niett esthan less identity an share they although , S184and DSM12804 S tfd .petrii B. . histidinilytica ee,wihepan t orgrowth poor its explains which genes, Pseudomonas .petrii B. T . and 9.2 BT1 .xylosoxidans A. T . a elhave well may 5.2 BT1 S 20 aeonly have 12804 DSM pce eeal to able were species .pinatubonensis C. .petrii B. 9,2345-2371. (9), .petrii B. T 10.2 BT1 .petrii B. htwe that Nature BT1 Posted on Authorea 16 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159493101.19143630 — This a preprint and has not been peer reviewed. Data may be preliminary. rs,R,Gza,C . eaha . o ats .A,Pee,D . obi,R,...Martinez-Arias, of . . Degradation . R., (1990). Koebnik, H., P. Bordetellae. D. pathogenic Pieper, of A., B. traits virulence V. and Santos, Baskunov, bacteria link: dos missing M., & The Sebaihia, (2008). 2,4- A., R. I., C. Guzman, L. R., of Gross, Evtushenko, N., a characterization R. by doi:10.1007/BF00119763 Pertsova, and acid A., 2,4,5-Trichlorophenoxyacetic L. Purification Golovleva, NADH:flavin and (TftD) (1993b). 4-monooxygenase doi:10.1128/jb.185.9.2786-2792.2003 of chlorophenol 2786-2792. of (TftC) (9), Characterization P. oxidoreductase (2003). dinucleotide L. Xun, adenine & R. R., M. Gisi, Hausinger, & 24311-24317. dioxygenase. F., dichlorophenoxyacetate/alpha-ketoglutarate Fukumori, doi:10.1128/jb.175.7.2083-2086.1993 dioxygenase. (1993a). alpha-ketoglutarate-dependent acid P. an 2,4-dichlorophenoxyacetic is R. the nooxygenase” Hausinger, of & agent F., inducing Fukumori, the of pJP4. Identification plasmid (1997). by R. encoded A. pathway Harker, & K., (2005). Filer, F. Briganti, . . biodegradation. from . aromatics A., isolated doi:10.1074/jbc.M500666200 Scozzafava, polychlorinated from plasmids S., 1,2-dioxygenase in Kaschabek, degradation hydroxyquinol M., ved the pesticide Thiel, of M., six structure V. of Travkin, Crystal J., Properties Seifert, (1981). M., Ferraroni, M. J. Pemberton, paradoxus & Alcaligenes H., R. Don, by and 1,2-dioxygenase metabolism hydroxyquinol acid of 2,4,5-trichlorophenoxyacetic Purification of (1996). cepacia pathway M. lower A. the Chakrabarty, reductase: & maleylacetate K., gene Saido, a L., of D. analysis Daubaras, Sequence (1995). M. 61 by A. Microbiology, Chakrabarty, acid Environmental 2,4,5-trichlorophenoxyacetic & and of K., metabolism Kitano, in D., involved C. cluster Hershberger, L., D. Daubaras, by acid insight. 2,4,5-trichlorophenoxyacetic H of E., C. Biodegradation Danganan, acceptance. GenBank L., after sequences; D. Genome added Daubaras, 2020; be Spanning; to van J.M. identifier Rob Persistent and NCBI; Boer sequence at de Nucleotide in Tjalf Nguyen, (1994). oxygenase Anh M. Lan acid [dataset]Thi A. 2,4,5-trichlorophenoxyacetic Chakrabarty, encoding & L., genes Xun, the L., of cepacia D. analysis Daubaras, functional W., R. and Ye, E., C. 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Ye, A., ubner, 4,1279-1289. (4), ¨ oadodssimplex Nocardioides laiee eutrophus Alcaligenes sedwdwt ohtemtblcvraiiyo environmental of versatility metabolic the both with endowed is ukodracepacia Burkholderia . ora fBceilg,145 Bacteriology, of Journal ora fBooia hmsr,280 Chemistry, Biological of Journal 10 M eois 9 Genomics, BMC ukodracepacia Burkholderia ora fBooia hmsr,268 Chemistry, Biological of Journal oadodssimplex Nocardioides 1) 4276-4279. (11), 1) 4100-4106. (11), ora fBceilg,175 Bacteriology, of Journal culture. M14”,-ihoohnxaeaemo- ”2,4-dichlorophenoxyacetate JMP134 2,1818 doi:10.2478/s11535-011- 188-198. (2), AC1100. ukodracepacia Burkholderia 4.doi:10.1186/1471-2164-9-449 449. , idgaain 1 Biodegradation, ora fBceilg,185 Bacteriology, of Journal tanA10:evolutionary AC1100: strain 2,681-686. (2), 1,317-320. (1), E e nyeinvol- enzyme key a 3E, 2) 21144-21154. (22), AC1100. 7,2083-2086. (7), Pseudomonas 4,263-271. (4), Burkholderia 5,455-477. 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No reuse without permission. — https://doi.org/10.22541/au.159493101.19143630 — This a preprint and has not been peer reviewed. Data may be preliminary. al :Gnm sebydt fte4bceilisolates. bacterial 4 the of data assembly Genome 1: Table Tables authored tables, draft. and final Figures figures the and prepared approved Tables data, paper, the the analysed of final experiments, drafts the the reviewed draft. designed or approved final Spanning the draft, van approved J.M. the paper, Rob reviewed the isolates, of drafts the reviewed of Brouwer Abraham genome the annotated and draft. final analyzed draft. the Boer approved draft. de draft, final the the Tjalf reviewed approved suggestions, draft, gave the Parsons reviewed R. experiments, draft. John laboratory final the draft. the in final approved assisted the herbicides, approved Braster the paper, Martin of the concentration of the drafts determined reviewed Koekkoek tables, material, Jacco and soil draft. figures delivered final prepared Dang the data, Ha approved the Cam paper, analysed Thi the research, of the drafts performed reviewed and or designed authored Anh Lan Thi Nguyen Contributions Author xylosoxidans Achromobacter petrii Bordetella along available publicly become histidinilytica Sphingomonas will in data deposited the review were manuscript, for aeruginosa (PR- study initially the Pseudomonas data, of this 9.2 number. these acceptance DOI of to BT1 assigned After Links findings an (PRJNA586745), brackets. with below. the between 5.2 in listed support listed BT1 are numbers that only, Bioproject (PRJNA586721), (PRJNA587043) their 2.2 10.2 with GenBank BT1 BT1 and of Bac- JNA586752), genomes (2018). NCBI-annotated V. The T. 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Weisburg, 43 Research, Acids 4-5.doi:10.1016/j.biortech.2017.01.069 146-151. , ilg ultnRves 8 Reviews, Bulletin Biology tanB .:https://dataview.ncbi.nlm.nih.gov/object/PRJNA586752?reviewer=anls0i9ag2l5ehv9f3q7clktoi 9.2: 1 strain:BT 1) 4667-4675. (17), W) 2723 doi:10.1093/nar/gkv437 W237-243. (W1), ora fBceilg,173 Bacteriology, of Journal tanB122 https://dataview.ncbi.nlm.nih.gov/object/PRJNA586721?reviewer=q7ko9k52t4a6uqlcrjdl3sdci2 2.2: strain:BT1 tanB11.:https://dataview.ncbi.nlm.nih.gov/object/PRJNA587043?reviewer=ca8ottdc5qkfr9gdon77qj5ffi 10.2: strain:BT1 tanB152 https://dataview.ncbi.nlm.nih.gov/object/PRJNA586745?reviewer=i6f5dcie0oc5aamc0lu4h8g3ea 5.2: strain:BT1 155-167. , 2,6773 doi:10.1128/jb.173.2.697-703.1991 697-703. (2), 14 ukodracepacia Burkholderia α- eoltrt-eedn dioxygenase ketoglutarate-dependent irsuc Technology, Bioresource AC1100. ora of Journal Posted on Authorea 16 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159493101.19143630 — This a preprint and has not been peer reviewed. Data may be preliminary. Figures as ‘2’ and herbicides. with ‘1’ the medium by on of (denoted communities enrichments half soil second means, T1 and of It first respectively) the names, genome. from isolate size. the derived the contig of isolates in N50 The 50% bacterial digit the last cover to of kbps. equal List to 5 or than needed 2: than smaller Table length larger contigs contigs species. contig of in removal homologous minimum is after closest the sequence determined and as genome are isolate defined contigs the genes is of predicted between number N50 of identity and Nr of N50 length, percentage Total overall an indicate Percentages N50 (Mbps) length Total 10.2 Contigs BT1 9.2 BT1 % 5.2 BT1 species 2.2 homologous BT1 Closest type Species crmbce xylosoxidans Achromobacter petrii Bordetella histidinilytica Sphingomonas aeruginosa Pseudomonas 4B111.2 BT1 BT110.2 14 9.2 BT1 13 8.2 BT1 .2 12 5 BT1 2.2 BT1 11 .2 1 BT1 10 10.1 BT1 9 .1 7 BT1 8 .1 Class 6 BT1 7 5.1 BT1 6 3.1 BT1 5 2.1 BT1 4 1.1 BT1 3 species related Closest 2 1 Isolates S/N nyoatrdichloromethanicus Ancylobacter xylosoxidans Achromobacter petrii Bordetella Staphylococcus histidinilytica Sphingomonas aeruginosa Pseudomonas ginsengisoli Sphingopyxis tournemirensis Pedobacter Bosea Sphingomonas Achromobacter nitroreducens Pseudomonas oxydans Arthrobacter denitrificans Rhodanobacter sp. 56 .9 0225732 5577 208222 5797 141283 6369 169073 144405 6.098 6.098 6.049 60 6.930 68 75 73 60 99 71 88 sp. p Bacilli sp. 15 sp. α- α- α- β- β- α- γ- α- Sphingobacteriia β- γ- Actinobacteria γ- Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Posted on Authorea 16 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159493101.19143630 — This a preprint and has not been peer reviewed. Data may be preliminary. ahgot uv a utatdfo hs ute ni hs uvs ro asrpeettestandard the represent bars Error curves. mean. these salt the in mineral of on 5% in further within (C) those inoculated was herbicide 2,4,5-T from which single subtracted deviation of as 2,4,5-T was degradation or curve 2,4-D and growth only each with (B) or 2,4-D 2,4,5-T and of 2,4-D with of degradation mixture a (A), either growth with medium Bacterial 2. Figure curves. within these was in which on The deviation further standard listed. those the species represent from isolated bars salt subtracted 4 Error mineral was the in curve replicates. of mean. (C) three growth the either 2,4,5-T of of each with 5% of means of inoculated degradation the start 2,4,5-T are and the and Symbols (B) at 2,4-D 2,4-D of density of mixture optical degradation a (A), with growth medium Bacterial 1. Figure .petrii B. T ..Dt onsaetemaso he elcts h pia est ttesatof start the at density optical The replicates. three of means the are points Data 9.2. BT1 16 Posted on Authorea 16 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159493101.19143630 — This a preprint and has not been peer reviewed. Data may be preliminary. 1404: 4898: ORF 4892, ORF 1030, ADP1, (WP AntA bactercalcoaceticus CLUSTAL by 64-7, calculated alignments bacterium, sequence options. Gammaproteobacterial on databases along default based NCBI database 7 the Burkholderia KEGG and MEGA the KEGG using using isolated from in from method sequences 4 W counterparts AntA sequences neighbour-joining the and matching the protein of BenA by sequences reference best CadA, homologues with their include TftA Homologues along with relevant blue) black). of in light relatedness (names in the (names showing Dendrogram species 3. Figure 5061: Prolinoborus .xylosoxidans A. .histidinilytica S. Sphingomonas 058357594.1): fA(AAB39767.1): TftA eA(SPJ20396.1): BenA , Niveispirillum T 10.2. BT1 aA(YP CadA T 5.2, BT1 iroclamassiliensis Vitreoscilla .aeruginosa P. Bordetella 008494438.1): Bradyrhizobium ukodracepacian Burkholderia Rhodospirillales nA(WP AntA rlnbrsfasciculus Prolinoborus T 2.2, BT1 - β- α-, R 2507: ORF Sphingomonas 153203307.1): fA(WP TftA and 17 eA(OYV39246.1): BenA Sphingomonas , Acinetobacter .petrii B. γ- rtoatra h edormwsobtained was dendrogram The proteobacteria. ampoebceilTt (MSQ67289.1): TftA Gammaproteobacterial , Niveispirillum 130222982.1): p R5adisolates and ERG5 sp. T 9.2, BT1 , Acinetobacter R 53 R 54 R 39 ORF 5379, ORF 1504, ORF 5533, ORF nA(O85673): AntA Achromobacter Rhodospirillales Bradyrhizobium p SYP-B3756, sp. eA(P07769): BenA cntbce baylyi Acinetobacter Pseudomonas R 33 ORF 2313, ORF atru 20- bacterium p Leo121, sp. Vitreoscilla Acineto- ORF Posted on Authorea 16 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159493101.19143630 — This a preprint and has not been peer reviewed. Data may be preliminary. 18 Posted on Authorea 16 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159493101.19143630 — This a preprint and has not been peer reviewed. Data may be preliminary. R5(ceso o KF494257.1). no. (accession ERG5 mt-2, AM902716.1), the for bp. in length A.x their reflecting sizes their P.a with genes the of direction transcription from the of and (B), the (A), the of Organization S.histidinilytica 5. Figure the in enzymes the encoding Genes 2,4,5-T. and 2,4-D S1. of Table degradation Supplementary the in listed for are pathways pathways KEGG 4. Figure T 2.2 BT1 T 10.2: BT1 .erythropolis R. B. tfd tfd sp I . lse n 1721frthe for U16782.1 and cluster / .aeruginosa P. : cad TH2 R.e .xylosoxidans A. cat T .,aogwt hs from those with along 5.2, BT1 eecutrof cluster gene Burkholderia : CCM2595: eecutr of clusters gene CCM2595, T 2.2, BT1 .erythropolis R. T 10.2, BT1 tfd .putida P. .histidinilytica S. p H acsinn.AB035483.1), no. (accession TH2 sp. P eecutr of clusters gene . T 5.2 BT1 S.h . aeruginosa tfd C.p t2 and mt-2, II JMP134: cluster), C29 acsinn.FM995530.1), no. (accession CCM2595 .pinatubonensis C. T . ln ihta from that with along 5.2 BT1 : .histidinilytica S. T . and 2.2 BT1 19 .petrii B. Burkholderia B.p .pinatubonensis C. S 12804: DSM .histidinilytica S. T 9.2, BT1 p H C.Tearw ersn the represent arrows The (C). TH2 sp. T 5.2, BT1 M14and JMP134 .petrii B. S. M14(ceso o M35097.1 no. (accession JMP134 .xylosoxidans A. B.p p ERG5 sp. T 9.2: BT1 S 20 acsinno. (accession 12804 DSM pigmnssp. Sphingomonas T . ln ihthose with along 5.2 BT1 .pertussis B. P.p pigmnssp. Sphingomonas : .petrii B. mt-2 T 02 and 10.2, BT1 S 12804 DSM .putida P. : T 9.2, BT1 ERG5