Posted on Authorea 15 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161339244.46256081/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. Lrgo hthstesgaueo e pn aaoi eecutricuigtetregnsta together that genes the three to the adjacent including segment cluster plasmids. gene unique secondary Ti catabolic a and these opine also Ri new of but of a nature types of catabolism, signature other agropine 724 dynamic the to for a has the compared responsible showed that was revealing TL-region genes chromid plasmid inversion primary the the Ri identified Mbp The but agropine we the K84, 1.8 Thus respectively. of strain large sequence biocontrol (pRi1855), a The avirulent plasmid by chromosomes. the 2,005,144 Ri accompanied of a bp from that deletion chromosome, long-reads to 252,168 bp kbp similar as 3,958,212 a very well a and was as of chromosome) chromosome sequencing consists (secondary genome Illumina chromid The from bp short-reads sequencing. Technology obtained Nanopore we Oxford genome complete the a of assemble sequence genome complete zogenes) the here report We Abstract Netherlands The author Leiden, *Corresponding 72, Sylviusweg plasmid University, Ri Leiden Biology, agropine of the Institute in Hooykaas* system J.J. Paul opine Hooykaas, new J.G. a Marjolein of root presence hairy the of suggests R. of analysis sequence use the genome facilitate purposes. will The biotechnological of sequence was for synthesis available gene and in The this involved laboratory cluster. by be the catabolic encoded may both adjacent protein thus novel in the The and LBA9402 a by plasmids plasmid. especially revealed degraded Ti Ri and be also agropine agropine rhizogenes can the and analysis that for chrysopine for sequence opine gene unique of responsible Our unknown is synthases catabolic genes the which opine succinamopine dehydrogenase. the TL-DNA, new the identified opine the a to we of of an related end Thus signature most encode right the together plasmids. very has the Ti that that at TL-region and genes kbp gene the Ri three 724 to of the a adjacent types segment showed including sequence unique other chromid The cluster a to the chromosomes. also but compared secondary but these was K84, catabolism, of plasmid strain agropine nature Ri dynamic biocontrol the avirulent agropine revealing the inversion the Mbp a of of 1.8 of that respectively. large consists (pRi1855), a to plasmid by genome similar Ri accompanied The bp very deletion short- 252,168 sequencing. was obtained a Technology chromosome we and chromosome) Nanopore genome primary (secondary Oxford complete chromid The from bp a 2,005,144 long-reads assemble a as to chromosome, well bp order 3,958,212 as In sequencing disease. Illumina LBA9402 root strain from hairy rhizogenes) reads Agrobacterium causing (formerly strain rhizogenes pathogenic Rhizobium the a of (NCPPB1855rifR), sequence genome complete the here report We Abstract 2021 15, February 1 Hooykaas plasmid Marjolein Ri new agropine a the of in presence system the opine strain suggests rhizogenes analysis Rhizobium Bioinformatics root LBA9402. hairy of sequence genome The ednUniversity Leiden tanLA42(CP15rf) ahgncsri asn ar otdsae nodrto order In disease. root hairy causing strain pathogenic a (NCPPB1855rifR), LBA9402 strain 1 n alHooykaas Paul and hzbu rhizogenes Rhizobium 1 1 hzbu hzgns( rhizogenes Rhizobium tanLA42 Bioinformatics LBA9402. strain formerly goatru rhi- Agrobacterium Posted on Authorea 15 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161339244.46256081/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. a e nqeaesicuigoeta epeitecdsanwoiectblccutr nldn the synthase including opine cluster, novel TL-DNA. catabolic a this the for opine of that gene new end revealed candidate a right LBA9402 A encodes very showed of dehydrogenase. the predict chromid plasmid opine at we Ri the an identified that agropine defining was that one for the dynamic but LBA9402 including characteristic the of underscoring genes of similar, areas sequence inversion, three unique chromid very the large few a of was and by a Analysis chromosome chromosome accompanied had deletion chromid. the the kbb the that widely comparing 724 of most large found By nature a the we to a of due 2020). K84 present one differences strain we large is al. Here of which et NCPPB1855, those 2009). (Desmet strain with al. wild-type strains et of laboratory for derivative (Slater and available used rifampicin-resistant 84 is NCBI, a Kerr sequence on is agent of genome available strain complete sequence biocontrol are one genomic agrocin-producing strains only complete avirulent several now second the of to of up sequences However, that (Jumas-Bilak genome , 2014). chromid Draft for al. a published 2010). et been called has (Kajala al. sometimes sequence 2. et genome megacircle, biovar draft Harrison plasmid-derived one 1-3) to 1998; a (biovars belonging and groups al. bacteria different chromosome et three name the a species properties for the circles, The physiological used for DNA 1977). their commonly bioreactors Panagopoulos of and the now in basis (Kerr of grown is the ago bacteria long On be various already nature can gall. distinguished in were that crown that or roots years root the obtain over hairy to apparent 2015). industry become expression has al. in gene It et 2014), and (Mehrotra function metabolites al. gene secondary for et of instance (Ron production for roots research in in (Collier application; analysis engineering and genome On research plant biotechnological for 1993). for useful al. vector a et Ri into (Dessaux the mikimopine plasmid and contains which mannopine, Ri which opines, cucumopine, Like the pathogen, These agropine, which on the degraded, produced, T-region and 1983). distinguished. of formed nowadays the are growth opines are al. to opines the specific plasmids et adjacent called the support (Petit region of compounds T-DNA, sugars a basis unusual the the or in roots by usually acids hairy encoded genes keto and enzymes of catabolic by acids cells amino formed the of are In condensates 1982). specific 1987). in Chilton are grow and al. can (Bevan et that genome plant (Jouanin roots the into into develop integrated that bacterium and related cells the plant by of The to used Expression is transferred 1980). which is that Nester T-region) to and (the similar is (White induced determinants is extensive tumefaciens virulence root Agrobacterium inducing essential hairy (Weller which by the by tomatoes contains mechanism greenhouse and molecular which called nurseries, the long cucumbers plasmid, tree in for (Ri) of in was problems inducing problem harvest that a causing bacterium the as a increasingly reducing encountered agent, also thereby originally was is adventitious mats, It of disease formation root the 1930). the by (Riker characterized nowadays is sites range, but wound host wide infected a from with disease roots plant neoplastic a root, Hairy Introduction succinamopine Ri-plasmid; will cyclodeaminase; ornithine sequence opine; synthase available chromid; rhizogenes; The Agrobacterium cluster. words: Key catabolic adjacent synthesis the in involved by be to degraded purposes. related may of be most thus use can was and the gene that the plasmids this facilitate of opine Ti by end unknown encoded agropine right protein and the very The chrysopine the of at plasmid. of gene Ri synthases novel agropine succinamopine a the the revealed for also unique analysis is sequence which Our TL-DNA, dehydrogenase. opine an encode .tmfces .rhizogenes A. tumefaciens, A. rol gnslctdi h -N ed otetasomto fnra el notmrcells tumor into cells normal of transformation the to leads T-DNA the in located -genes .rhizogenes R. oidc rw altmr npat.Drn neto,pr fteR plasmid Ri the of part infection, During plants. in tumors gall crown induce to n seilyLA42i ohtelbrtr n o biotechnological for and laboratory the both in LBA9402 especially and a endsre ydltn h -N ee nodrt ovr it convert to order in genes T-DNA the deleting by disarmed been has .rhizogenes, R. nvitro in goatru rhizogenes, Agrobacterium .rhizogenes R. utr nteasneo de ln rwhregulators growth plant added of absence the in culture 2 hto h ar otidcn tanLA42 This LBA9402. strain inducing root hairy the of that tal et 08.As,tebceima uhi used is such as bacterium the Also, 2018). . tanAC184cnitn f4 scaffolds 43 of consisting ATCC15834 strain .rhizogenes R. otisalre bu 0 b root- kbp 200 about large, a contains Rhizobiaceae tal et tan aetomegabase two have strains 00.Tecausal The 2000). . hzbu rhizogenes Rhizobium aiymycause may family .rhizogenes R. Posted on Authorea 15 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161339244.46256081/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. . h eoi euneof sequence genomic The 3.1 under Archive Read Sequence the in Results deposited SRR10177304. are and reads raw SRR10177303 The numbers accessions CP044124. and CP044123 were CP044122, of 1 bers sequence Table genome in complete shown The as identities version availability Percentage Jalview Data Illustrator. with 2.4 seqinr. visualized Adobe package and and R and 2013) the 2009) pRi-1855 with (Katoh between calculated al. method et and et L-INS-I and (Alikhan (Waterhouse regions 7.471, Ri rings 2.11.1.2 version catabolism concentric other MAFFT erythritol in with with hits between pRi1855 performed the compare comparisons visualize to the the to lusitanum used align For and was bium generate to 1e-10) BRIG to used 2011). cut-off was used 2010). e-value (progressiveMauve) was al. BLASTN, Mauve al. CGView (with et and 2005). plasmids (Darling 2015) Wishart Ti genomes shown. and al. K84 are (Stothard et and repeats, pRi1855 2 (Dhillon LBA9402 inverted figure of islands In including map genomic 2017). circular i.e. Tang predict a sequences, and to 2016). (Xie insertion used ISEScan complete was al. using (Huerta-Cepas only identified IslandViewer employed et S5, were was elements) (Arndt figure (IS eggNOG-Mapper supplementary sequences elements proteins and prophage Insertion encoded annotate 2017). the to al. Annotation of et used Genome characterization Prokaryotic was functional NCBI bacteriophage PHASTER using the there- the was addition, For annotated represented contig was In This assembly This (PGAP). The the preparation. Pipeline library assembly. identified. and Illumina the was chromosomes during from two bp concentration removed low the fore 5386 at was representing spiked-in of assembly is contigs contig which Hybrid three fourth sequence, coverage). Besides a (70-fold plasmid 0.4.7. Cutadapt Ri version using trimmed Unicycler bp. adapter using 3441 and performed quality of were length reads read Illumina mean ( quality a The average with on bp, Epi2me). ( filtered 1,027,720,149 (with and totaling demultiplexed end-trimmed reads, were were 298,712 reads reads was MinION Nanopore LBA9402 the for 2.3.4) yield (version by total Albacore followed with library, basecalling another After with DNA methods ng pooled R9.4.1). processing 200 was (version Data with library cell 2.3 generated flow The was MinION a library Kit. an Leiden on sequencing on Barcoding the sequencing sequenced Nanopore Rapid of in-house were house, Oxford SQK-RBK004 (LGTC) Libraries The in Center the DNA Technology done TruSeq using machine. was Genome where 2000 Netherlands), Leiden sequencing combina- HiSeq The the Nanopore a Illumina (Leiden, at using Center platforms. sequenced performed Medical Technologies was was University Nanopore LBA9402 sequencing Oxford of Illumina genome and but The Illumina of columns. tion gravity-flow QIAGEN using isolation rhizogenes that toothpick R. wooden sterile a bacterium. methods The CaCl with the Sequencing g/l, stems of plants. 2.2 3 plant colony infected extract the a yeast on into puncturing Difco by dipped roots g/l, virulence been hairy 5 for had prolific tryptone tested causes (Difco was medium which bacterium TY The and on 1979), grown (Hooykaas was bacterium laboratory our in isolated rhizogenes R. Organism 2.1 methods and Materials > 00b)wt aoit(4fl oeaeatrfitrn) oa f458119-uloiepaired-end 99-nucleotide 4,518,191 of total A filtering). after coverage (64-fold NanoFilt with bp) 5000 tanLA42i iapcnrssatdrvtv fwl-yesri CP15,wihwas which NCPPB1855, strain wild-type of derivative rifampicin-resistant a is LBA9402 strain tanLA42wsclue nT eim(eigr 94,floe ygnmcDNA genomic by followed 1974), (Beringer, medium TY in cultured was LBA9402 strain tan69 LSnwsrnlclywt LS eso ..+ rti lgmnswere alignments Protein 2.9.0+. version BLAST with locally run was BLASTn 629, strain hzbu rhizogenes Rhizobium .rhizogenes R. B90 a eoie nGnakudracsinnum- accession under GenBank in deposited was LBA9402 3 tanLBA9402 strain > 1)adlength and Q10) 2 Φ .6H 14genome X174 2 . g/l). 1.3 O Rhizo- Posted on Authorea 15 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161339244.46256081/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. h R15 lsi opie 5,6 p thsa prxmtl %lwrG otn hnters fthe of rest the than content GC lower 4% approximately an has below. It described is bp. be which 252,168 comprises will plasmid, plasmids plasmid and pRi1855 Ri pRi1855 The K84, the agropine strain of by the carried represents properties plasmid circle General catabolic 3.3 kbp nopaline 252 chromid large characteristics dynamic the the more key the from Finally the on different present of were very chromosome. they one erythritol the that of is remarkable presence was of erythritol The it instead catabolize but 1977). expected, Panagopoulos to thus and ability was (Kerr genes The agrobacteria catabolic 2 2014). biotype and al. 1 biotype et distinguishing Barbier 2012; genes Oresnik and with genes operon of set transport a chromid a a the including in found S4) in We region figure fimbriae/pili. erythritol ‘chromids’ and the polysaccharides coined wall to been cell homologous have of and production the chromosome as for primary chromosomes genes content the secondary GC with (developing) similar genes considered a exchange (Slater in are has time seen megacircles but evolutionary secondary over be system, Such that replication can RepABC as chromosome. present plasmid-like particular primary genes a the in of has genes categories megacircle smaller, the of secondary in is category This differences sequence functional major inversion LBA9402 one analysis. no Mbp affect The eggNOG-mapper are 1.8 large there to not a LBA9402). that according did by demonstrating to deletion accompanied 1, aligns seems large LBA9402 figure which the K84 supplementary The deletion, of of kb 85% 1). 724 64% still approximately (and (fig. but large prophage 1), a K84 a to fig. of of due S2, insertion that mainly figure the to (supplementary to aligns similar due less sequence seems are thus replicons For and second-largest 2016) The elements. al. et mobile (Arndt S3). other PHASTER vB figure tool LBA9402, of (supplementary phage search in two (Rhizobium presence/absence 745,195-784,185 phage by position proteins the to of) from surrounded by phage DNA remains due of is to segment (the mainly and unique homology the be have are integrase in may encoded putative K84 proteins which various primary a and example, bp, genes for LBA9402 LBA9402 15 of gene the (from of set of a formation of chromosomes pair a contains repeat position regions mating with (from island direct replication for unique kbp and genomic a system transfer the 100 The secretion DNA for of about Type4 necessary the a enzymes of 3,302,269-3,307,564). one the and only island also in bacterium not but genomic 3,324,071-3,314,501), the encoding tRNA position putative transfer of recombination, extra a DNA clusters DNA an conjugative contains primary rRNA and can for chromosome annotated 3 repair at the As The We the protein, K84 with including replication of collinear chromosome. 2004). translation DnaA genes. that largely and a to tRNA transcription is for al. aligned 53 for as LBA9402 et be division, such of can (Kurtz cell replication contig sequence (NUCmer) for for largest the genes MUMmer of the the using (96% contains S2 K84 K84 figure of of supplementary those chromosome in to seen aligned be be could of megacircles (Slater genome K84 complete strain other only of The sequences sequence coding the using predicted to annotated to online). Comparison was assigned (3 Material 3.2 were assembly Supplementary genes categories rRNA S1, The COG 9 figure 60%. sequences, annotated. (supplementary were coding was eggNOG-mapper genes 5822 with genome that tRNA total, the 252,168 indicating 53 In assembly, of and and (PGAP). the to content bp operons), Pipeline to used G+C 2,005,144 Annotation align was reads Genome bp, The Prokaryotic Illumina 3,958,212 Unicycler NCBI and complete. of MinION is contigs sequencing. both assembly circular of Nanopore three 99.9% the by than in More sequence reads resulted genome respectively. This long complete bp, and assembly. obtained quality hybrid high additionally a a we obtain obtain to LBA9402, sufficient strain not were of alone data sequencing Illumina As deoR tp euao as called (also regulator -type tal. et 09 Harrison 2009; tal. et 09.Ti atrsri osntcnana ipamd u h w LBA9402 two the but plasmid, Ri an contain not does strain latter This 2009). tal. et 00.Tecrmdo B90 otismn eaoi ee,btalso but genes, metabolic many contains LBA9402 of chromid The 2010). eryR iohzbu meliloti Sinorhizobium .rhizogenes R. olwdb ee called genes by followed ) .rhizogenes R. eryEFG 4 bandtu a sta fteaiuetbiocontrol avirulent the of that is far thus obtained tanK84 strain RleM aaoi prnwt genes with operon catabolic a , and P1adohrtie hgs srevealed as phages) tailed other and PPF1 att hzbu leguminosarum Rhizobium eryH sts agrdffrne ewe the between differences Larger -sites. and eryI > 5 euneiett) It identity). sequence 95% Ys ta.20;Geddes 2006; al. et (Yost (supplementary eryABCD and Posted on Authorea 15 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161339244.46256081/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. ar ot omdb goiesriscnanarpn,arpncai,mnoii cdadmannopine and acid mannopinic acid, agropinic agropine, contain strains agropine by formed roots genes Hairy catabolism agrocinopine and plasmid. Agropine the 3.4 a of above area mentioned as different and entirely plasmids, an Ri in mikimopine and cucumopine the from plasmids Ti the the virH1 to locate the next and to contains identify next plasmid now location different could the We completely from a plasmids. at (Krall Ri sequence 5’-phosphate mikimopine pRi1855 which and riboside plasmid, cucumopine zeatin Ri the cytokinin mannopine the the the outside of in synthesis located present not the is catalyzes but that analysis, the Southern carries by still plasmids of Ri deletion mikimopine the and (Hodges that pRiA4 suggesting for The identical before almost plasmids. Ri is these area of essential divergence this the in contains cells plasmids plant Ri into T-DNA new the a of by transfer for responsible genes pRi1855 virulence of region plasmid virulence (Offringa Ri The acid containing agropine acetic The (TR-region) indole T-region paragraph. auxin next additional the a and an TL- of in (TL-region) The biosynthesis discussed T-region in be conserved involved will ). the as 3 revealed synthase besides this opine has and (fig. unknown 1986) an plasmid al. encode of Ri respectively, et may presence (Slightom mannopine 2001), sequenced the the the previously end al. encode besides in was right These et pRiA4 synthesis plasmid the (Moriguchi mannopine present. Ri synthase at for agropine are mikimopine the necessary genes mikimopine and of and non-conserved genes cucumopine region 2016) cucumopine two plasmids) the the in Ri the (in al. present one mannopine and et T-region still the inserted Franco the are has (Valdes (in of genes IS630 end two cucumopine these right of or very of with copy the plasmids) remnants a T-region At Ri however, only one plasmids. plasmid, but Ri only Ri present, mikimopine have agropine and is plasmids the synthase Ri In agrocinopine the Other for 2018). contains Otten roots. TL-region 3; hairy plasmids. the Ri between of which, (fig. and formation of genes Ti the one similar other very for T-regions, in sufficient two those to and has similar necessary plasmid very ( Ri are replication agropine and the 2015) The pRiA4, al. plasmid et Ri Wetzel agropine 1987; the related al. in ( closely these transfer the discuss conjugative with shall and we dealing and studies (NC 2 previous figure pRi1724 In in pRi visualized 2020; is plasmid plasmids al. Ri these parts. et in mikimopine following areas (Weisberg and different pRi8196 the Ti 2018), plasmid of (NZ chrysopine conservation pTiEU6 Ri pRi2659 2001), al. plasmid mannopine plasmid al. Ri Ti et Ti 2018), et cucumopine Tong succinamopine nopaline Oger results), al. 2001), 2015), (DQ058764; unpublished et pTiBo542 own al. al. plasmid Shao our et et Ti (KX388536; Wood Huang agropine pTiChry5 2019), 2001; 2014; plasmid plasmid Ti al. al. al. and et et Ri et Shao available Henkel (KX388535; Goodner publicly (CP007228; (supplementary (AE007871; to bp pTiAch5 sequence pTiC58 898 pRi1855 plasmid plasmid of plasmid Ti size Ri average octopine an agropine with the sequences: identified compared were We sequences S5). coding protein figure 236 total, In genome. ags , orf15/rolD virH2 o goieboytei BuhzadTunu 1991). Tourneur and (Bouchez biosynthesis agropine for orf3 virE3 orf virJ trsmlsi hsrsetthe respect this in resembles It . ihsm iiaiyt oaiepTi nopaline to similarity some with and virA-virD5 ee eie h oestof set core the Besides gene. virE2 sasn.Ohrtpso ipamd aesimilar have plasmids Ri of types Other absent. is orf8 nyoelarger one only vir ttevr etedi h goieT-einadtemnoieTrgo gene a T-region mannopine the and TL-region agropine the in end left very the At . rgo,aot6 b lcws rmthe from clockwise kbp 65 about -region, ee(Hodges gene r,trb tra, u although but , tal et virA 04.The 2004). . orf15 ee ntearpn ipamdwr led ecie Nsiuh et (Nishiguchi described already were plasmid Ri agropine the in genes ) eethe gene orf virE2 tal et eenfe called hereinafter , / virE3 rolD eei ucinlyrpae yagn called gene a by replaced functionally is gene 04.Tgte iha with Together 2004). . tzs GALLS vir vir gene, n he smaller three and speetthe present is ee etoe bv,the above, mentioned genes rgo ftenpln ipamdadlk ntenopaline the in like and plasmid Ti nopaline the of -region virF virK 5 eewspeiul loietfidi h cucumopine the in identified also previously was gene h euneo h uuoieadmikimopine and cucumopine the of sequence The . eodnpln Ti-like nopaline second a , orf16 virE1 traG h ucinof function The . virE3 tal et orfs and vir P1733 adsFac ta.2016; al. et Franco Valdes CP019703.3; 055 oiuh ta.20) The 2001). al. et Moriguchi 002575; eeams 0kpcounterclockwise kbp 90 almost gene virE2 tzs norpi85sqec efind we sequence pRi1855 our In . 96 n h genes the and 1986) . rgos u a but -regions, ee eroiectblcgnsin genes catabolic opine near gene, ee hc noe nenzyme an encodes which gene, virE2 tzs ee r isn n replaced and missing are genes vir eei rsn,btlocated but present, is gene eino h goieRi agropine the of region tal et curdoc eoethe before once occurred orf16 virF virH2 GALLS GALLS 2002), . rol sukon but unknown, is ee n finally and gene, gnsta are that -genes eei absent is gene mas1 eei the in gene sreported as aux GALLS repABC , -genes mas2 is ) Posted on Authorea 15 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161339244.46256081/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. fteegnsaeivle ntectbls fa pn,agn o nukonoiesnhs hudbe should synthase opine unknown an for gene a opine, an DNA of this catabolism of the end in the involved bacteria at are in located genes metabolism is acid these amino regulator If control AsnC/Lrp to an metabolic known acid 2003). for is amino such al. regulators gene putative the et transcriptional A encoding of a of (Brinkman pathway 2012). family genes and Lrp catabolic al. dehydrogenase pRi1855 The a et saccharopine segment. of of specific Serrano putative part Mello the area a form (de of this may cell. encoding lysine catabolism which In bacterial genes the dehydrogenase, the including in semialdehyde into involved present dehydrogenase. aminoadipate enzymes opine also opine often transport encoding specific are the are present a of proteins dehydrogenase by are for uptake opine genes released for genes an often encoding by required products vicinity genes sides permease the plasmids the both in Ri conserved Besides and on encoding been Ti genes surrounded fully In have by are regulator. cluster accompanied genes Fe-S LysR-type the three a and and These co-factors system FMN S7-S9). and FAD figures the (supplementary for sites binding characteristic to to related homologous closely gene phosphorylated Regulators a mocA release of the kbp. remnant of 1 truncated catabolism than of a further homolog only in intact between involved genes an in dehydratase probably the while 6-phosphogluconate are plasmid However, and These Ti sugar. dehydrogenase octopine 1996). glucose-6-phosphate Farrand the an to and In encode (Kim homology sugar. genes weak phosphorylated These with a Farrand agropine enzymes and and degradation. for and acid TR- Kim and glutamine genes amino by the deoxyfructosyl uptake an described the to into agropine genes adjacent mannopine identified in catabolism the oxidizing plasmid comprize now involved to for for the also have similarity plasmid genes and of high We Ti segment with permease with octopine a agropine plasmid genes 1983). agropine. the contains in catabolic region degrade on located second, This al. (1996) are to a et which 2). strains contain pRi1855, (Petit (fig. host HRI in region opines or enables catabolism mannityl 1855 plasmid and three not Ri transport other but agropine the , The A4 of as 1983). such strains al. et (Petit sotpn,npln n ucnmpn eyrgns oss ftresbnt dACta r encoded (F3X89 are that pRi1855 OdhABC in subunits genes flavin- three putative of a a consist of for dehydrogenase code catabolism by succinamopine together such and and dehydrogenases that transport nopaline opine genes Flavin-containing the characteristic octopine, three in as S7-S9). the involved figures all (supplementary be identified dehydrogenase may we opine originated TL-region containing area have this the In of may border opine. It new right the the the to sequences. to to adjacent insertion introduced adjacent adjacent map segment IS5-like been of the map have by chromosome on may the surrounded another sequenced kbp and on recently is of 64-88 genes chromosome it kbp from metabolic the as (area 20-40 mainly from kbp transposition from contains 24 transposable by area (area include about latter pRi1855 kbp These and The into with 20 fig.2) described. genes). areas plasmids in about catabolic Ti unique TL-region of and agropine several the Ri areas has of of larger plasmid border types two other right pRi1855 genes the and contains the of region (fig.2) Besides, none This in elements and about plasmids. found 2001). regulators Ri of are transcription other region that al. several the large genes encodes et with a and 2) (Moriguchi shares metabolism figure chemoreceptors It glycerol of two map transport, function. the sugar unknown on in of involved kbp putatively 88-153 genes from with (area regions kbp several Ri pRi1855 65 has mikimopine in plasmid and system pRi1855 cucumopine opine The the novel from a absent for has but Genes plasmid plasmid, 3.5 Ri Ri agropine mannopine The the of in 2005). set plasmids. present a al. an also pRi1855 et of are in (Baek presence genes TL-region plasmid the the Ri of matches agropine end which the left from the to absent adjacent be to thought was noxABC/ooxABC n between and mocC lk ee ragdi admi h eoe(aaaee l 05.Tethree The 2015). al. et (Watanabe genome the in tandem in arranged genes -like and 84,F3X89 28345, mocD Rhizobium hzbu lusitanum Rhizobium acs u eeto fa of detection Our . agcA eefrteboytei farcnpn nteT-ein These TL-region. the in agrocinopine of biosynthesis the for gene 85,F3X89 28350, mocS o h eatns ovrigarpn nomannopine, into agropine converting delactonase the for pce,a eysmlrsrtho N a eetdi the in detected was DNA of stretch similar very a as species, mocA and a rsn npi85 swl sahmlgof homolog a as well as pRi1855, in present was 6 mocR 85)ecd lsl eae rtisi hc the which in proteins related closely encode 28355) mocD tan69(upeetr gr 6.Te2 kbp 20 The S6). figure (supplementary 629 strain mocDE r rsn na dnia oiini rn of front in position identical an in present are acc eei R15 a eakbea uhgene such as remarkable was pRi1855 in gene mocB ee o goioiectbls fi.2), (fig. catabolism agrocinopine for genes eemnn h eojgs liberating deconjugase the determining a rsn u oadlto fmore of deletion a to due present was mocA and .rhizogenes R. mocB mocC encode mocC , Posted on Authorea 15 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161339244.46256081/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. aaaee l 04.Aprnl,i a vleas na pn sciaoie ytae hrfr,it Therefore, synthase. (succinamopine) the opine of an both in or also dehydrogenase either evolve alanine that can an it possible into Apparently, evolve seem to would 2014). reported al. been has et function Watanabe its instance For in time. Ti some nopaline for the by (encoded known encoded ( is proteins cyclodeaminase cyclodeaminase ornithine these ornithine the That with All identity plasmid. 19-21% about 1). instance, al. (table for et share, identity They (Oger 93% pTiBo542 share agropine by themselves the encoded by and which proteins 2018) The synthases, al. succinamopine proteins. et related two (Shao most pTiChry5 Using the chrysopine as 2018). of 2001) al. 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All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161339244.46256081/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. abe ,e l 04 rtrtlfestepnoepopaeptwyvatrenwioeae edn to in leading transfer isomerases 17815-17820. factor new R three 111: via USA 1974. pathway Sci JE. phosphate Acad Beringer are pentose Natl the compound, Proc feeds amadori Brucella. Erythritol 221-233. an in 2014. (DFG), 53: D-erythrose-4-phosphate glutamine Ecol al. deoxyfructosyl Microbiol et Acids FEMS of T, Nucleic Barbier utilization Rhizobiaceae. tool. for family search the Genes phage in 2005. PHAST dispersed the widely al. of et version C, faster Baek simple better, (BRIG): a PHASTER: Generator W16-W21. Image 2016. 44(W1): Ring Res al. BLAST et D, 2011. Arndt 12:402 SA. Genomics Beatson BMC NLB, comparisons. Zakour genome prokaryote NK, Petty NF, Alikhan under Archive Read Sequence cited the in Literature deposited SRR10177304. are and reads raw SRR10177303 The numbers accessions CP044124. and CP044123 CP044122, of bers sequence genome complete The Academy Netherlands Verk Royal van the statement by Marcel availability by supported Dr was Data Professor P.J.J.H. isolation, Academy data. DNA as Illumina with appointment the help of Sciences. the analyses for of with initial Shao the associated Shuai to fellowship contributions and the for Dulk-Ras Henkel den Chris Dr Amke and article. to this grateful for are tab We information supporting the in online Acknowledgements found be may Information Supporting Additional purposes. biotechnological Material for of Supplementary and use the laboratory facilitate the will sequence both octopine available in needed the the dehydrogenase application, LBA9402 encode frequent opine the its to the of on known view encode opine. genes are but In may unknown the that gene, thus novel of genes and Three synthase the of 2015), of the plasmids. trios Ri al. catabolism to the and et for of close Ti (Watanabe signature different dehydrogenase located many the nopaline often in have and seen are indeed arrangement together genes an here border, catabolic located right Opine the of synthase. side other opine an new that a hypothesize for We in 2014). activity dehydrogenase al. alanine as ( such activities encoding enzymatic fulgidus new cyclodeaminase both still encoding Ornithine that which genes discovered into and evolved plasmids. we (ocd) Ti Here the cyclodeaminase chrysopine with 2001). ornithine 44-47% and of to al. identity related larger significant et (Trovato one evolutionary also the have has activity is at plasmid cyclodeaminase that synthase Ri ornithine opine protein agropine an has the a for also encodes coding that gene shows gene now bacteria. This a have sequence the Our sofar of described source T-region. nutritional plasmids ( the a Ri of a as and end for serve Ti right import not that very of formed types but cannot different are permease, bacterium the opines agropine which All the an in for neoplasias because induce genes or Agrobacteria contains genes plasmid This pRi1855 catabolic mannopine. the system. the degrade Indeed transport cannot mannopine induce cell. pRi1855 cannot the carrying in bacteria mannopine mannopine the because that known be is may it However, mannopine. and orf16 ttevr ih n fteT-ein hc hrs5%iett ihteneighboring the with identity 55% shares which TL-region, the of end right very the at ) n arpn eyrgns ciiyin activity dehydrogenase tauropine and .rhizogenes R. ocd hzbu leguminosarum Rhizobium lk eemyhv vle ntearpn ipamdit gene a into plasmid Ri agropine the in evolved have may gene -like susL B90 a eoie nGnakudracsinnum- accession under GenBank in deposited was LBA9402 aihnrajaponica Halichondria ee noigsciaoiesnhs nteagropine the in synthase succinamopine encoding genes 8 e irbo 84:188-198. Microbiol Gen J . ocd ee aebe eotdt have to reported been have genes ) Sam ta.21;Wtnb et Watanabe 2013; al. et (Sharma .rhizogenes R. rolD Archaeoglobus n especially and rolD and orf16 gene. orf Posted on Authorea 15 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161339244.46256081/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. ao .21.MFTmlil euneainetsfwr eso :ipoeet npromneand performance in improvements 7: version 30:772-780. software Evol alignment Biol sequence Mol multiple usability. of MAFFT sequence 2013. genome S. Katoh Draft Unconventional 2749–2755. 2014. e01108-14. 180: SM. 2: 1998. Bacteriol Brady Announcements J A. DA, Genome Proteobacteria. Coil Allardet-Servent the K, M, of Kajala Ramuz subgroup alpha G, the 53-63. Bourg in of 53: organization S, TL-DNA Science genomic the Michaux-Charachon Plant of tobacco. E, fragment regenerated kb Jumas-Bilak by to 4.3 phenotype assignment a transformed of orthology pRi Transfer through the 1987. confers annotation al. functional et L, genome-wide 2115-2122. Jouanin Fast 34: Evol 2017. Biol Mol al. eggNOG-mapper. et J, Huerta-Cepas of sequence genome Complete e00570-15. Netherlands. 2015. 3: plant The al. University, with et Rhizobiaceae Leiden YY, of Thesis. interactions Huang the PhD in approach. functions genetic determined plasmid A of 3065-3077. Agrobac- cells. role 186: for The Bacteriol substitutes 1979. J protein PJJ. Hooykaas GALLS VirE2. rhizogenes protein Agrobacterium DNA-binding 2004. single-stranded W. tumefaciens Ream octopine-type terium J, the of Cuperus sequence LD, Genome Hodges 2014. chro- PJ. a Hooykaas X, not tumefaciens Zhang ’chromid’: terium A, bacterial Dulk-Ras 141-148. den the CV, 18: Introducing Henkel Microbiol 2010. Trends agent JPW. plasmid. biotechnology Young a and NKD, not pathogen Kim mosome, plant RPJ, Lower the PW, of Harrison sequence and Genome transport 2001. the for al. tumefaciens necessary et locus B, complex in Goodner a l-arabitol of and characterization visualization adonitol discovery, Genetic erythritol, island of 2012. genomic catabolism IJ. interactive Oresnik flexible, W104–W108. BA, more Geddes 43: Res 3: Acids IslandViewer Nucl 2015. analysis. and al. parasitism. et of BK, mediators chemical Dhillon opines, of biochemistry 31-38. and Chemistry : 34 1993. Phytochem J. achievements Tempe current A, Petit breeding: 2435-2451. Y, plant Dessaux 104: for tool Biotechnol innovative Microbiol an as Appl agrobacteria animal Rhizogenic limitations. and and 2020. plant the al. to et through relationship S, degradation its Desmet Lysine 905-911. and 2012. bacteria 586: P. in Arruda Lett SDH N, and FEBS Zanata loss LKR E, enzymes. gain, bacteria: Kiyota in TR, gene pathway Figueira saccharopine with Silva the e alignment GC, genome Serrano multiple Mello e11147. de progressiveMauve: 5: ONE 2010. PLOS NT. rearrangement. Perna 573–583. and B, system 95: stacking Mau J gene AE, Plant Agrobacterium-based Darling robust plants. and Arabidopsis versatile regulators. transgenic A transcriptional quality of 2018. high R. family generates Thilmony Lrp J, The Thomson 2003. R, J. Collier Oost der 287-94. van 48: WM, Vos Microbiol de Mol TJ, Ettema AB, the Brinkman of T-DNA 1982. MD. 357-384. Chilton MW, Bevan 5.Sine24 2323-2328. 294: Science C58. tanAh.Gnm non.2 e00225-14. 2: Announc. Genome Ach5. strain Agrobacterium S.meliloti. 9 goatru tumefaciens Agrobacterium irbooy18 2180-2191. 158: Microbiology iadR lsis n e ee 16: Genet Rev Ann Plasmids. Ri and Ti hzbu rhizogenes Rhizobium goatru rhizogenes Agrobacterium c5 eoeAnnounc. Genome Ach5. tanATCC15834. strain Agrobacterium tanA4 strain Agrobac- Posted on Authorea 15 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161339244.46256081/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. lgtmJ,Drn-adfM oai ,Tpe .18.Ncetd euneaayi fT-N of TL-DNA of analysis sequence Nucleotide 1986. D. Tepfer 261(1):108-21. L, Jouanin rhizogenes Agrobacterium M, three Durand-Tardif 2501-2511. of JL, 191: sequences Slightom Bacteriol Genome J cyclodeaminase-like bacteria. 2009. ornithine in an genomes al. multichromosome of et characterization SC, Functional Slater 2013. 11: PE. Evolution Verslues of and S, protein Biology Shinde Genome S, pTiEU6 Sharma Ti-plasmid Ti-plasmids. succinamopine nopaline-type of sequence with Complete relatedness 2480–2491. 2019. evolutionary PJJ. Hooykaas its GPH, 96-97:1-6. reveals Heusden Plasmid plasmid van Chry5. tumor-inducing S, the strain of Shao tumefaciens sequence 455-469. Complete Agrobacterium 166: hypervirulent 2018. Physiol the PJJ. Plant Hooykaas from GPH, model. pTiChry5 Heusden van a 507-540. X, as Zhang 41: using S, tomato Res Shao transformation using Agr function root J and Hairy trees. expression 2014. apple gene nursery type-specific al. of et root M, hairy Ron infectious on Studies 204-214. 1930. in 190: AJ. Plasmids Riker Genet concept: gen opine Mol the of degradation. extension Further opine for 1983. al. et A, Petit 375-420. The family 418: pTiBo542: the 2018. of in L. analysis plasmids and Otten of sequence evolution nucleotide 83: and Complete USA structure 2001. Sci about 169-170. SK. Acad us Farrand Natl tells GJ, genomics Proc mutants Olsen what rhizogenes aux C, Agrobacterium Reich tumor-inducing of PM, tumefaciens Oger plasmid Agrobacterium Ri the of of Complementation TR-region replicator 6935-6939. the 1986. 8. the from 1– of genes al. 206: determination by Genet et sequence Gen IA, and Mol Offringa Characterization pRiA4b. plasmid 1987. hairy-root-inducing the A. in Oka region M, Takanami its 771-784. R, indicates 307: Nishiguchi plasmid plasmids Biol: (Ri) (Sym) symbiotic Mol root-inducing and plant J (Ti) a Rhizobiaceae. tumor-inducing of between in sequence relationship nucleotide evolutionary complete and to The structure chimeric timeline 1189-1201. 2001. biotechnology-indicative 252: al. root 5:R12. Protoplasma et Hairy Biology Moriguchi, outlook. Genome 2015. future AK. genomes. and Kukreja, large links LU, comparing missing for Rahman C58 understand software V, tumefaciens Srivastav open S, and Agrobacterium Versatile Mehrotra from 2004. protein al. Tzs et S, The Kurtz FEBS 2002. AMP. and 315-318. C. diphosphate 4-hydroxy-3-methyl-2-(E)-butenyl 527: Baron from Lett MH, 5’-phosphate of opine riboside Zenk synthesis zeatin gall M, for produces crown 178:3275-3284. Raschke responsible the Bacteriol genes L, of J T-region Krall catabolism tumor. the for plant of the responsible homologs by genes are opine plasmid-encoded this tumefaciens Ti Agrobacterium by 1996. mannopine SK. Farrand KS, Kim 172-179. 90: Z. Phytopath. of Biotypes control. 1977. CG. Panagopoulos A, Kerr rbdpi thaliana Arabidopsis Agrobacterium goietp lsi.Ietfiaino pnraigfae.JBo Chem Biol J frames. reading open of Identification plasmid. type agropine M ln il1:182. 13: Biol Plant BMC . hntpcpatct ( plasticity phenotypic goatru radiobacter Agrobacterium 10 Agrobacterium goatru rhizogenes Agrobacterium Plast ee.CretTpMcoilImmunol Microbiol Top Current genes. ) ivr epeuiaeteeouinof evolution the elucidate help biovars var. goatru rhizogenes Agrobacterium tumefaciens Rhizobiaceae sato o xlrn cell exploring for tool a as n hi biological their and lsi 45: Plasmid . cooperate Posted on Authorea 15 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161339244.46256081/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. eolt.Rdaesaergoswt h aeoinain hra leaesaini nes orientation. inverse in package align R areas the blue with whereas visualized orientation, were 2 same blocks the aligned Fig. with The regions progressiveMauve. are using areas K84 Red of genoplotR. that of to chromid aligned was the in rearrangements Large 1 Fig. catabolism erythritol in involved prokaryotic genes in of Legends Characterization elements 2006. sequence MF. insertion Hynes TC, of in Noel identification AM, Rath automated CK, Yost ISEScan: 3340-3347. 33: 2017. Bioinformatics H. genomes. Tang Z, Xie engineer genetic natural the of genome 2317-2323. The 294: in 2001. traits al.. virulence et DW, encodes Wood plasmid root: 1134-1141. Hairy 141: groups. 1980. Bacteriol evolving EW. separately Nester and FF, structurally 3337–3357. White two 7: into The divide Evolution. Rhizobiales and 2015. the Biology SK. of Genome Farrand members 43-50. in V, Chakravartty systems 49: transfer Pathol GJ, Plant Rhizogenic Olsen UK. 2000. ME, the GM. Wetzel in McPherson mat D, root Hargreaves cucumber TM, and O’Neill 1 plasmids. DE, multiple virulence Stead 2-a agrobacterial SA, of Version Weller transmission Jalview global and eaba5256. conservation 2009. 368: Unexpected Science GJ. 1189-1191. 2020. Barton al. 25: et Bioinformatics M, Weisberg workbench. Clamp analysis DMA, and dehy- editor Martin opine alignment JB, flavin-containing sequence Procter of Characterization e0138434. AM, Waterhouse 10: 2015. One Y. PLOS Watanabe bacteria. F, from Fukumori drogenase R, Sueda S, Watanabe cyclodeaminase/ putative Ornithine in involved 2014. Y. archaeon Watanabe perthermophilic Y, Tozawa Genome S, NCPPB2659. Watanabe strain rhizogenes Agrobacterium of sequence e00746-16. genome 4: Draft Announcements 2016. al. et Franco Valdes 7:108. From Plants 2018. development. P. plant Costantino control R, 13449-13453. Mattioli 98: M, 1167-1174. USA Trovato oncogene Sci 20: plant Acad Biol The Natl Agric 2001. Proc J P. cucumopine-type Costantino Int cyclodeaminase. F, of Linhares roots. sequence B, hairy Maras genome inducing M, Trovato engineer complete genetic The nature’s a 2018. of (NCPPB2659), K599 sequence al. et genome X, Draft Tong 2020. e00506-20. 9: Announcements al. Resource et MG, Bioinformatics Thompson CGView. using exploration and visualization 537-539. genome Circular 21: 2005. DS, Wishart P, Stothard hzbu leguminosarum Rhizobium trans hroocslitoralis Thermococcus 3hdoylpoiemtbls.FB pnBo4 2211-5463 4: Bio Open FEBS metabolism. -3-hydroxy-l-proline bv. Viciae irbooy12 2061-2074. 152: Microbiology . R.rhizogenes ucin sanovel a as functions .rhizogenes A. 11 h hoi/eodr hoooeo LBA9402 of chromosome chromid/secondary The . goatru fabrum Agrobacterium oDt ln 5S xliigpoieto proline exploiting P5CS: plant to RolD repABC goatru tumefaciens Agrobacterium Δ -yrln--abxlt reductase 1-pyrroline-2-carboxylate rolD μ cytli ooo rmtehy- the from homolog -crystallin lsiswt quorum-regulated with plasmids goatru rhizogenes Agrobacterium noe ucinlornithine functional a encodes goatru rhizogenes Agrobacterium Ru1 Microbiology ARqua1. Agrobacterium 5.Science. C58. biovar strain J . Posted on Authorea 15 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161339244.46256081/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. itnl oontieccoemns.Mlil euneaineto rtisecddby encoded proteins of alignment sequence pRi-1855, Multiple of cyclodeaminase. ornithine to by distantly genes encoded of proteins overview pRi8196: The color. Schematic type, same cucumopine the plasmids. have pRi2659: Ri 4 genes type, of Homologous Fig. mikimopine T-regions type. (pRi1724: the agropine plasmids of pRi1855: Ri end type, different right mannopine of very T-regions the the the on at in indicated located are genes genes Unique and regions of the number indicates 3 A intensity Fig. Color rings legend. inner the plasmids. The in Ti shown plasmids.. and ring. as Ti Ri outermost similarity, and various sequence Ri and of other Ri1855 degree to between analysis comparisons comparative BLASTn and show pRi1855 of representation Circular suooa putida Pseudomonas susL from oosaeacrigt h lsa oo scheme. color X Clustal the to according are Colors . .tumefaciens A. orf15 and orf16 tan hy n o4,and Bo542, and Chry5 strains r vltoayrltdt ucnmpn ytaeadmore and synthase succinamopine to related evolutionary are 12 ocd from .tumefaciens A. orf15 and 5 and C58 orf16 Posted on Authorea 15 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161339244.46256081/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. LBA9402 chromid K84 chromid Figure 1 0 Mb 0 Mb 0.2 Mb 0.2 Mb 0.4 Mb 0.4 Mb 0.6 Mb 0.6 Mb 0.8 Mb 0.8 Mb 1 Mb 1 Mb 13 1.2 Mb 1.2 Mb 1.4 Mb 1.4 Mb 1.6 Mb 1.6 Mb 1.8 Mb 1.8 Mb 2 Mb 2 Mb 2.2 Mb 2.4 Mb 500 kb 2.6 Mb Posted on Authorea 15 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161339244.46256081/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. Figure 2 virH1,2 repABC trb genes IS3 virF virK IS66 traI IS66 traR tzs virB genes virA tra genes virG virC genes GALLS virD genes virE3 andmetabolism agrocinopine transport 252,168 bp pRi1855 IS630 14 TL-DNA anddegradation opine transport IS5 TR-DNA anddegradation agropine transport IS5 Posted on Authorea 15 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161339244.46256081/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. pRi1855 TR-DNA pRi1855 TL-DNA pRi8196 pRi2659 pRi1724 Figure 3 acs acs acs acs iaaH 40 kb orf2 orf2 135 kb orf2 orf2 orf3n orf3n iaaM orf3n orf3n 15 kb rolB−li orf8 orf8 IS630 5 kb 5 kb k e orf8 45 kb 140 kb rolA rolA orf8 mas2 rolB rolB rolA 20 kb rolB rolA mas1 15 rolC rolC 10 kb 10 kb rolB rolC orf13 orf13 50 kb ags 145 kb orf13 orf13a orf13a rolC orf13a orf14 orf14 orf13 25 kb orf14 cus orf13a mis mas2 15 kb 15 kb orf14 5 kb orf15 mas1 orf16 Posted on Authorea 15 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161339244.46256081/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. P.putida_ocd C58_ocd Chry5_susL Bo542_susL LBA9402_orf16 LBA9402_orf15 P.putida_ocd C58_ocd Chry5_susL Bo542_susL LBA9402_orf16 LBA9402_orf15 P.putida_ocd C58_ocd Chry5_susL Bo542_susL LBA9402_orf16 LBA9402_orf15 P.putida_ocd C58_ocd Chry5_susL Bo542_susL LBA9402_orf16 LBA9402_orf15 Figure 4 P.putida_ocd C58_ocd Chry5_susL Bo542_susL LBA9402_orf16 LBA9402_orf15 P.putida_ocd C58_ocd Chry5_susL Bo542_susL LBA9402_orf16 LBA9402_orf15 P.putida_ocd C58_ocd Chry5_susL Bo542_susL LBA9402_orf16 LBA9402_orf15 P.putida_ocd C58_ocd Chry5_susL Bo542_susL LBA9402_orf16 LBA9402_orf15 318 342 337 339 280 304 281 281 283 285 226 250 233 233 235 237 174 199 180 180 181 183 123 148 127 127 127 129 69 94 73 73 73 75 30 55 31 31 31 31 1 1 1 1 1 1 G G M M M M M M T S K E V V V N D P P P P L L L L L L A Y L L L L K K K K K K L L L L L - - - I G G G G G G G G Q Q Q Q Q Q S D R E K K K P Y P A P K A A R L L L L L L Y Y N N - - - I - - - - M G D E D D D S L V R R V V D D R R D D E R D D P D A A L L L L V V I I - - - - - I - - I I I I Q Q Q H H D D A D D D E K K K R K C C Y F T T E C R R K N N N H H ------G G G G H H S S H H N N P P S V V V V P K A A F F S F F F R ------G G G G G A D D Y Y Y Y K K K T N D K K R R H H S S N A K R P ------A H S K S S S T K K L F F A S D D S E P P P P P P R R Y Y Y Y A ------I I I - - - - - Q Q Q Q W W W W W W S T A A T A A S A A E E T T T P P S S R R K K D A K S S ------Q M N N E E D H E Y T D K V V V E E L L L L N N N N - - - - - I I I I - - - - I I - - I I I I - Q Q Q M L L L R R L L L Y Y Y Y R E A A K E K T E E N D T T E E A A L V E E ------M M Q V L L A A R D H H L L F R A A A A A V V V A K R R A F F T L - - - - - I I - - - - - I I I P A A D D D D A R E E D E A C V V V V A A F F F F R S K N Y T S S A A N N S H C ------G Q Q W W W G G K K P E D D S S H H S T N L L L L L L N N N K E A K E E E E ------G G G Q V D S S V A V L L V V S S E E N D F F F F L R L L E K F F L R V ------I I I - - - G G G G G G Q G E P L L L L L L L L V A T T R R H P P P P D E R K H H C H ------I I - - - M M Q W W W W D D F F R L E E K S A E S S A S N N A A A A T T L L T L K K L A ------Q Q G G G G G G Q G G D E D D N N R A A L A E A A Y L V V R R S R R K S S T T ------M Q Q M K K S D A A L L L L A A E E K K T H H H H H H E E E E P P A A R D A S ------G G Q Q Q Q A A V V S S S P L L V V A A S V V N V P E S K R L L R D I I I ------I I G G G G P T L K E K Y S S A A F F D D D D K K K K A K K S A D D S S K V I ------I I G G G G V A A S S E E R R E E V K K V A L L L L L L L A T S A F F I I I I I I ------W W Q Q V V V A D D K F F E E A A V V L L V F Y Y F Y A K S S F F R R V V I I ------G G G Q Q C C A A D D L L F F V L L L L L L L L V L N E E A N S N K E E ------I I I - - - M G G G G Q Q D D D D V V V A A V V A S R R R R A S P T L L L F L H Y F ------I I I I I - T L P P P P E P D D D D L L R R L L D D R R N D F F F F L F N ------I I I I I I - - - - - M M M M R L T T A A A A P P L T T T T A A A A A S V V K K R K D D D D D N - - - - I - I - - - - 16 G Q Q R V L H H N N L L D V T T A A A A F F L L L L D D S S F V K K S S R K T V ------R R F F F F F F T T A A A A V A T T T T H K N N N N S S T T T S S E N Y P I I I ------M M M M G G G G G G Q A D D T T S S R R T T N N K A E E S Y A P P P V F F ------G G G A S S T T A P E E E E A A S S S Y H L N N Y Y E E R T R R - - - - I I ------I I G E E T A D D S S N L T A A P P R R R L V D S K F V V V V V V E E I I I ------G G G G G G G G G G R R R R K K P P K K S S S A V L L L L L A A V V R R ------G M N N N N P P P P S C K L L L L F S S P E R R F F L L L L E E ------V V V A A V V R R R R L L S S S S H H T N L A A A A A A I ------I - - - - I I G Q Q M M N N D D A H F F F F V S S L L V V S S R L S S S S I I I I I ------I M M Q Q Q M A A S A Y N N N E E E D K K R E E E E D E E R S R E E Y ------I M M G G G G L L L V A A P P A E R K K R K L V D N N P D D V D D D D D D L L ------G G G G G G G G G A A A A T T K A V T T K K V V L L A D Y F L V P A I I I I I ------I I W W W W W T S A A A A A P L L L L D D E E V R A A A A A A V V C V A I I I I - - - - V A V V V V D D L L H Y L L L A L V A L R R T T H V I I I I I I I I I I I I - - - - G G G L L A A A A F H F F Y Y T S A A T A Y Y L L L L T T S S S S E E E E E E D S D G G G G R R A A V A P P S S S S P D P P D P D D S S S S A A F F E N L L T V V I I I G G G M M G G Q Y Y H H H H V V R R R R D N K K N N T R R R R L L R V V A A A A Y I M M G G G K K R R A A A A D D C R R R R R R P P L L L L V V V V L V V T I I I I I I G G G H L V V V V P P N K T T T T T T V T V A E T T T T L R Y Y Y Y D E I - - I I I Q Q M G Q G G G G G G Q L E T T T T L E T S K A A A A A S P N P P P P D E E E E L L W W W W Q W W K K K K P P L L L L A A S S S S A A D D C C F Y F F T A E A R K K K T G G G M M L L L L S S T S T T N N N N T T F F L F K F F V F L L L L F A V L L R R I M G G G S S T T V L L L L A A E E H F S S A A A S S T K K V E E A L V V - - - - I I Q G G Q G M E E K H H S S K R R R E E L A P P L L R L D D D R E K A A K N L - - - - M M M V H H L L C L L L L A A A A Y Y Y Y Y Y L L V V V V R S L R T K R T I I I I I G G G G Q Q G G G N N D N D D S V V V V A A C C C C S S S S A A A D S P P V L I - - G Q G G G G G G M Q P P R K A D D E D A R R R E A A V L V A A D D D E H N N V A E I M Q G Q G S S D R V V V V L N N E E R K K D D N N F F V V V T A Y S S A A S T D - - 280 280 282 284 225 249 232 232 234 236 173 198 179 179 180 182 122 147 126 126 126 128 350 371 349 344 317 341 333 333 336 338 279 303 68 93 72 72 72 74 29 54 30 30 30 30