Hybrid short- and long-read assembly of an experimentally evolved Sodalis glossinidius strain SgGmmC1*

Hybrid short- and long-read assembly of an experimentally evolved Sodalis 1 glossinidius strain SgGmmC1* 2 3 Poppy Pes c o d 1, 2* , L ee R . H aines 2,4* , Al istair C D arby 3 , Ian Goodhead 1# 4 5 1 School of Sc ience, En gineering and E nvironmen t, Univ er s it y of S alfo rd, Sal ford, UK 6 2 Depar t ment o f Vector Bio logy, Liverpool School of Tr opic al Medic ine, Live rpool, UK 7 3 Institu te o f Integr ative Biol ogy, U niv ersity of Liv er pool, Li v er pool, U K 8 4 Cur rent a d dr ess : Universi t y of N ot re D a me, N ot re Dame, IN, U SA . 9 10 * These author s contr ibuted equal ly to t his work. 11 12 # Addr es s corresponden ce to Ian Goo dhead, i.b.goodh e ad @sal f ord.ac.u k 13 14 15 ABSTRA CT 16 Bacterial symbionts of inse c t s under g o dramatic genome r educ t ion dur ing t heir 17 evoluti onar y t ransition fr o m fre e- l i vin g to h os t -dependent l if e styles, but th e d y nami c s of 18 this proces s r emain poorly under s to od due to th e d i f f iculty of observing these proce sses in 19 real-time. So dali s gloss inidius, a f ac ul t a t iv e endos ymbion t of tsetse flies , pr ovi d es an 20 exc ep tional opport un ity to study t his tr ans it i o n ex perimental ly: U n l ike high ly s pe cialis ed 21 obligate sy mbionts, S. glo s si nidius ca n be cult ured in vi tro and r e tains a large genome ( 4 22 Mbp ) wi t h extensi ve pseudogene content (49%), s u gge sting a recent evolution a r y 23 tr ans it i o n. He re, we pr es ent a compar ative g en omic analysi s of S. gloss inidius s tr ains 24 is o l at ed from l abo rato r y colony-d erived G los s ina mor s it ans mor s it ans , co mparing one aft e r 25 ten years of serial passa ging i n labor ator y cultur e (SgG mmC 1*) to a counter part isolated at 26 the same time (SgGmmB4). H ybrid genome ass embly u sin g O xfo rd N ano pore and Illumina 27 technologies produced a high-quality 4.29 Mbp genome comprisin g one c ir c u l ar 28 chromo s ome and f our plasmids similar. Comp a r ative analy sis r evea led a si gnific ant deletion 29 (17,209 bp) c ont aining 31 g en es, in cluding thiM (inv olved in thiam ine bios ynthesi s) and 30 genes en coding sulfur t ransport ers. Additional ly, we identified multiple small-sca le 31 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 27, 2025. ; https://doi.org/10.1101/2025.06.26.659281doi: bioRxiv preprint mut a t ions ( 8 delet ions, 39 i n s er tions, 10 S NP s ) resulting in frameshifts in g enes including a 32 hemolysin precur sor ( shlA ). Our f indi ng s demo ns t rate th a t , under stable la borat ory 33 conditions without t he s elective pressures of the host environment, S. gl os sinidius cont i n ues 34 to und ergo genome degradat ion. The los s of thiM s upp orts previous hypot hese s of 35 complement a r y metabol ic pathway s bet w een S. gloss inidius and the pr i ma ry symbion t 36 W ig g le s w o r t h i a g lo s s i n i d ia f or thiami n e b i o s ynth es i s. Th i s stud y provides ins ight s into t he 37 evoluti onar y t rajectory of f ac ult ative symbiont s and has implic at ions fo r parat ransg en i c 38 appro a ches u s ing S. glo ssinidius f or trypano s o me c on trol. 39 40 KEY WO RD S: S od al is glossinidius , t s et se s y mbiont, s y mbios is , ex p erimental evoluti on , who l e-41 genome sequencing 42 43 DA TA SU M MAR Y 44 Raw N ano pore and MiSeq r ea d s are available i n t he European Nucleotide A r chiv e und e r 45 Pro je ct P RJEB323 21 (R e ad ac ce ssions : ER X332 1202 and ERX332 1201 r espe ctively). A full 46 GBK forma t ted annot a tion o f SgGmmC1* has b een depos it ed in F ig share at 47 htt ps : / /doi.org/1 0.178 66/ r d. sa lf ord.8052437.v 1 . 48 49 IMP ACT S TATEME NT 50 In this s t ud y we p rovide new i n s ights into t he ongoing ge n ome degradatio n o f S odali s 51 gloss inidius, a f acult a t iv e end os ymbiont of tsetse flies impl icated in thei r ab ili t y t o t r a n s mit 52 tr ypanos omia si s, d uring prolon g ed la bor ator y cultur e. Through high- quali ty c omparative 53 genomics we reveal si gn i f ic ant gene los s and mutational changes, including disrupt i o n of 54 pathway s i n v olved in nut r ient b iosynt hesis. The se findings en han c e our understanding of 55 s y mbiont genome evolu tion and inf or m t he dev el o pmen t of S. gloss inidius as a poten tial 56 tool for parat ransge n i c strat egi es in c ontro l l ing t rypanosome transmis s ion. 57 58 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 27, 2025. ; https://doi.org/10.1101/2025.06.26.659281doi: bioRxiv preprint IN TRO D U C TI O N 59 Tset s e f l ies (Genu s : Glossina) are unu s ual insect s that , li ke keds and bat flies , r eprodu c e b y 60 givi n g b i r th to l ive young (adenot rop hic viv ipar ity). As obligate haemat ophag es , tset se 61 harbo urs a limit ed bac t er ial microbi o me due t o their r es t ricted diet, f eed i ng solely on blood 62 in the adul t li f e stage and on pr oteina ceous mil k fr om the femal e ’ s spec ialised a c ce ssor y 63 glands d uring the larval s t age (Minchi n, 1905). At l east fo ur spec ies of ba cteria play 64 impor tan t r oles in the nu tri t i o n, fecu ndity and vec t orial ca p a city of t s et s e flies: 65 W ig g le s w o r t h i a g lo s s i n i d ia , an ob l igat e p rima ry s ymbiont is ubiquitous; Sodalis gloss inidius , 66 a facultative commensal symbiont , and Wolbachia and S pi r op lasma, paras i t ic r eproduct i ve 67 manipulato rs, all have va ried in fectio n r a t es acro ss t he wide dis tr ibution o f ts et s e acro s s 68 sub-Saharan Af rica ( Doudou mis et al , 2017) . S. glos s inidius is a G r am-negative sec on dar y 69 bacterial endosymbiont t hat has been l inked t o mod ifying tsetse fly su s c e pt ibil i t y to 70 tr ypanos ome i n fec t ion by parasi t es b elonging to t he genus Tr ypanos om a ( Trappeniers et al , 71 2019; Makhulu e t a l, 2021 ; K al lu et al, 2023) . Trypano somes cau se a fat al d i sease in hu man s 72 and animals called tr y p a n os omi a sis ( Maudlin and Ellis , 1985). 73 74 Oblig at e i n s e ct endos ymbiont geno mes demo ns t rate spe c ialisation to t he ir host 75 environme nt by being non culture- v ia ble and having s ma ll, compact genomes relative to 76 fr ee- liv ing r el a t iv es (Moran e t al., 2008). S. gloss inidius i s notable fo r being the fir s t 77 endosymbiont dis covered to be bo t h amenable to in vitro culture (Dale & Maudlin, 1999) 78 and for hav ing a rela t i vely large geno me: The S. gloss inidius geno me is 4 Mbp , similar in siz e 79 to fr ee -li ving bac t eria, but large in comparison to t he 0.7 Mbp genome of W . glos s inidia ; 80 both o f th ese featur es sug gest a r ec ent s witch to ho s t a s s ociation and s ymbiosis (Toh et a l., 81 2006). 82 83 The tr ans ition o f bac t erial genomes from f ree -liv ing to endosymbiosis i s po orly understood . 84 Evidence su g ges t s endo symbiont ev olut ion i nvolv es inactivation of funct i onal genes via 85 small insert ions or deleti ons to pro du ce “p seudogenes ” . The se p s eud ogen es are 86 sub s equ ently l o s t in lar g e delet ion e v e n ts f ac ilitat ed by f requent populat io n bot tlen eck s 87 (Goodhead and D a r by, 2 015). As mut ation s ac c u mulate in non-esse n tial g en es , fr ames hif ts 88 and large number s of i n s er tions o r de letions (indels) result in los s of gene funct i o n i n a 89 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 27, 2025. ; https://doi.org/10.1101/2025.06.26.659281doi: bioRxiv preprint pro c es s known a s p s eudo g en i sation; pseudogene s may or may not r etain f unction 90 depending on t he l e vel o f degr adatio n (Goodhead et al, 2020). 91 92 S. glos sinidius r epresents a un i q ue mid-way point in the ev oluti on a r y transition t owa r ds 93 obligate endosymbiosi s , evidenced b y t he high propor tion o f pseudogenes in its geno me: 94 49% compared to t he usual 1% pr es ent i n f r ee-l iving bac t eria (Goodhead e t al., 2017; Toh et 95 al., 2006). Ho w ever , w h i lst th e o bli gat e en dos ymb i o nt W . glos si nidia is pre sent in all 96 individuals of t h e G los s ina genus , S. glossinidius is found at r ates r anging f rom 1% - 94% in 97 wild tsetse po pulations ( Farikou et al 2011; Den nis et al, 201 4; Mf opit et al , 2023). Thi s lack 98 of consi s t e n cy implies S . glos s inidius retains a facultative re lationship with i ts host and 99 impacts our ability to study ba cter i al genome degr adation in natu r al s ett in g s . Indeed, 100 genomic informati on for S . glossi nidi us ha s lar ge ly been collected fro m is olates derived f r om 101 Gl o s sina u nder labo rat ory conditions (Toh et al., 2006; Belda et al., 2010; G oodhead et al., 102 2017). 103 104 S. glos sinidius ha s s everal att ribut es t h a t make it a p rom ising mic r oorganis m for u s e in 105 tsetse and trypano some contr ol s t rategies – i t is amenable to cultur e in v itro , has a 106 demonstr able a b i l it y t o inf ect the t a rget vector, and can be genetically man i p ula t ed t o 107 express contr ol factors (Bear d et al, 1 993; W elbur n et al, 1987). A r ec o mbinant strain of S. 108 gloss inidius ex pr essing fun ctional anti -t rypanosome Nanobodies ® ha s been used to deliver 109 the Nanobodies to tsetse in an in v i v o lab-based proof o f c o nc ept s t ud y ( De Voogh t et al 110 2012). Whil s t promising, the effector molecule w as only s t able wit hin the t setse ho s t when 111 its native S. glossi n i dius population w as s ignificantly suppre s s ed. Thi s s u g g ests fitnes s 112 differences bet w een nat iv e and lab- reared strains of th e b a ct e r ium, mean i ng fur ther stud i es 113 into t he fitn ess of l ab- derived S. g los s inidi us versu s n atural populat i ons ar e war ranted. A 114 thor ough under s t anding of the pathw ay of genome d eg r adation in lab-ba sed s y stems wi ll 115 als o b e vital fo r paratr ans genic appro a che s, to pr edic t i mpac t s on ba cter ia l f i t nes s or t heir 116 ability to succes s f ull y infect t he tsetse ho s t . 117 118 Her e we p rese n t a high-qua lity whole genome sequence a s se mbly and an n otation o f a 119 strain of S. gloss inidius i solated f rom a lab colony o f G lo s s ina mor s it ans mo r s it ans th a t w as 120 serially pas saged in vitr o f or t en year s. We h av e compar ed i t s genome t o th at o f a 121 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 27, 2025. ; https://doi.org/10.1101/2025.06.26.659281doi: bioRxiv preprint counter part isolated at t he s ame time and f rom t he s ame colony a s t he original s t rain, with 122 the aim o f d i r ectly obs er vi n g genome r eduction under la b o rator y s et tings . We identify both 123 small and lar ge-s cale deletions as well as smaller scale mutat ions , pr oduce a b etter 124 under s t a n ding of pathway s und er s el ection in labor at ory settings , and hyp othesis t he 125 impact on natu ral S . g lossini d i us populat i o ns . 126 127 METH OD S 128 In vitro cultur e of S . gl ossini d i us 129 S. glos sinidius wa s i solated fr om two male G. m. mor sitans from t he colony at the Liv er pool 130 School of Tropical Medicine in 2007 following previously des cribed met ho ds ( Matt hew et 131 al., 2005). The strains w er e named Sg G mmC1 and Sg GmmB4, and wer e i mmediately s t ored 132 at -80° C. The SgGmmB 4 i solate was s equenced following a s ingle pas s a ge fro m a glycerol 133 stoc k a s previously d es cribed using P ac ific Bios c ience s RSII sequen cing ( Goodhead et al, 134 2020). Beginning in 2009 , cultur es of Sg GmmC1 were incubated at 27°C in a microaeroph i l ic 135 atmospher e a n d pas s aged ev er y 1 0-1 4 day s for ten years, al t ernating bet wee n solid-phase 136 growt h on Columbia agar base supple ment ed with d efibrinated ho r s e bloo d (Oxoid/Thermo 137 Fis her ) and liquid-phase growth in serum-fre e insect media (Sigma Aldr ich). We here ref er 138 to t he “ evo lved” s t rain of Soda lis gloss inidius as “ Sg G mm C 1*” . 139 140 Compar ative genome an alys is 141 DNA wa s e xt racted fro m a cell pel let of SgGmmC 1* usin g the Zymo Q u ick DNA kit and 142 quant i f ie d us ing a Qubit™ 3.0. The S gGmmC1 * geno me w a s s equ enc ed u si ng both Oxford 143 Nanopore MinION and Illumina MiSeq platforms. A library for the MinI O N wa s generat ed 144 using the SQ K- L S K108 1D l igat i o n k it a n d s equenced on a R9.4 flow cell, producing 14 9,84 7 145 reads of mean length 3,47 7.62bp and length N50 of 14,4 68bp. A t otal of 52 1,111 ,234 ba se s 146 were sequenced giv ing an e stimated 118x genome coverage. For Illumina MiSeq 147 sequenci n g , a QIA seq FX D NA library pr ep k it a n d v 2 150b p s equencin g c artr idge wer e us ed 148 pro duc ing a to tal of 755,405 paired- e nd reads. MiSeq reads were a ssessed and adaptor s 149 tr imm ed using F a stQC (v0.1 1.4) (A ndrews, 2010). MiSeq read s were tr imm ed for qual ity 150 using fa s t p w it h default settings (v0.12.5) (Chen et al . , 20 18). 151 152 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 27, 2025. ; https://doi.org/10.1101/2025.06.26.659281doi: bioRxiv preprint A hybr i d geno me for SgGmmC1* wa s built u s ing Unicycler (v0.4.8-bet a) (Wi c k et al., 2017) 153 by a ssembling the Mi Seq r eads de no vo and mapping th e lon g MinIO N rea ds onto t his 154 assembly for s tr uctur a l corr ection. Th e hybrid as s embly wa s ann otated usi ng PR OK KA 155 (v1.13.3) (Seeman n, 2014) u s ing t he referen c e strain SgGmmB4 genome (G enBank 156 Acce ssion LN 8 54557 .1) , whi ch wa s de po s ited in 2016. The annot ated Sg G mmC1* assembly 157 was compar ed to Sg G mmB4 us ing t h e Artemis Comparis o n Tool (ACT) (v13.0.0) (Carver et 158 al., 2005) to ident ify s tr uctur a l chang es and lar ge d el et ions , and Snippy (v3 .1) ( S eemann, 159 2017) t o i d entify s ingle-n uc leot ide po lymorphisms (S NP s) and insertions/d el et ions. A n 160 assembly graph of t he as sembly w as pr oduced us ing Banda ge (v0.8.1) (Wi c k et a l., 2015) to 161 asse s s its quality (Dat a not shown). 162 163 RESUL T S 164 Compar ative genomic s of SgGmmC1* and SgG mm B4 165 The assembled Sg G mmC1* genome was 4 ,28 8,97 5b p i n size wit h 54. 43% GC and no 166 unident i fied bas e pairs (N s ) . Th e a s s e mbly con sis t ed of s ix contig s: one c ir cular c hr omosome 167 (4,149, 326b p), four circular p l asmid s (pSG 1 , 81, 522bp ; pSG 2, 26 ,425bp; p S G3 , 19,5 12bp; 168 and pSG4 / 3, 1 0,81 6bp) and o ne pla s mid fr ag ment (pSG2 pla s mid fr ag, 1,3 74bp) were 169 pro duc ed . Sg GmmC1* w as iden tified with 99.99% si mil ar ity to the r efe rence strain 170 Sg G mmB4 us in g BLAST N (v2.8.1) (Fig ure 1) (Zhang et al., 2000) . One large deletion o f 17, 171 209bp wa s iden tified, containing 13 c oding g en es and 1 8 non-coding gene s (Table 1). This 172 deleted regi o n i n c luded t he c o ding gene th i M , which enc od es a prot ein inv olv ed in t hia mine 173 biosynthe sis and one of t wo c opies of esar that encode s a prot ei n r e lated t o t he quorum-174 sen s ing pr otein Lux R . Fur ther d e le t ed genes in cluded th ree s ulf ur and one sulfite 175 tr ans membrane tr ans po rter protei n s ; one copy of th e p e p tidas e yxeP (the ot her copy of 176 which i s p seudogeni s ed elsewhere in the genome); and mul tipl e sugar pho sphotr ans f erase s 177 (Figure 1 and Table 1). 178 179 Snippy als o ident ified ei ght s ma ll del e t i o ns (r ange of 1-27bp, mean 11.6b p, s tand a r d 180 deviation 6.08), 39 insert i o ns (1- 8bp, mean 3.1bp, std 4. 72) and 10 S N P s . I n to tal, 12/ 32 181 (37.5%) insert i o ns in a codin g region resulted in a frames hift ; Putati vely pseudogenis ed 182 genes in cluded t he putati ve tr ans por t pr otein gene HsrA , one of fiv e c op i es of th e p r oline 183 tr ans po rter gene pr op and a he molys in pr ec u rsor shl A (Table 2). 184 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 27, 2025. ; https://doi.org/10.1101/2025.06.26.659281doi: bioRxiv preprint 185 Raw N ano pore and MiSeq r eads are available f rom the European Nucleotide Ar c hive under 186 Pro je ct P RJEB323 21 (R e ad ac ce ssions : ER X332 1202 and ERX332 1201 r espe ctively). A full 187 GBK forma t ted annot a tion o f SgGmmC1* has been depos it ed in F ig share at 188 htt ps : / /doi.org/1 0.178 66/ r d. sa lf ord.8052437.v 1 . 189 190 DISCU SSI ON 191 The pseudogeni s at ion and deletion of genes de scribed suppor t the hypo thesi s of s ymbiont 192 genome siz e r educ t ion thr ough ps eu d ogenis ation and sub sequent delet i on of genes 193 unneces s ar y to sur v ival in a s table host enviro nment. In the c ase of Sg G mmC 1*, 10 year s of 194 pas s a ging in a minimal med ia w it h c ar efully cont rolled tempe ratur e and a t mospher e has 195 resulted in inactivati on or de leti on of genes unne cessar y to th i s environment. W it hin the 17 196 kbp deletion most coding genes a n d ps eudo g en es were involved in transmembr ane 197 tr ans po rt of or ganic and inor ganic co mpou nds , mainly of carboh y d rate s, o r were 198 membr ane-b ound t ransferases . In t s et s e, a s i n o ther or ganis ms, thiamine is a n es sential 199 cofactor f or amino acid and c ar bohyd rate metab olis m that is not present i n bloo d meals . 200 The tsetse fly l a ck s t he c apa city for t h iamine biosy n thesi s but carr ies gene s for thia mine 201 tr ans po rter s (Int ernation a l Glossina G en ome Initiative, 2012). W. glossinidia and S. 202 gloss inidius c ont ribut e metabol ical ly complement ary cofac t ors to pr oduce an int ac t 203 thiamine b iosynthesi s pat hwa y : W. glossinidia has t he c apacity for B v it ami n bios ynt he s is 204 and the synthe sis of t hiamine monop ho s phat e from which S. glos sinidius a lso benefits 205 (Belda et a l ., 2010; Sn yder et al. , 201 2; Hall et a l ., 2019). 206 207 Serially passa gi n g S. glos s inidius i n t hi amine-ri ch media in vi tr o, wit h the re peat ed 208 populat i o n bo ttl eneck s cau sed b y pa ssa gi n g , ha s re sulted in the de let i on of the th i M gene, a 209 putat iv e hydoxyethylt hia zole kinas e t h a t is an integr al par t o f t he thiamin s al vage II pathway 210 that a llows S. glossini d i us to s ynthesi se th i azole phosphate carboxylate (TH Z-P; Belda et al 211 2010). ThiM h a d b een retained by th e or iginal S. g loss inidius genome desp ite the loss of 212 other c o fac t ors in the thiam ine- p rod u c t ion pathway. That thiM is deleted in the 213 experimen t all y evolved S. g l ossinidi u s strain SgCmmC 1*, i n t he abs ence of W. glossinidia but 214 in the pr es ence of th i amine- rich medi a, support s t he h y po thesi s that thiM i s maintained in 215 S. glos sinidius despite ongoing genome degr adation due to p os itive selec tiv e pr es sure, likely 216 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 27, 2025. ; https://doi.org/10.1101/2025.06.26.659281doi: bioRxiv preprint due to t hia mine b iosy nt hes i s being e ssential for t s et s e survival a n d a chieved thr ough co-217 oper a t ion wi th the wide r mic r obio me (H all , 2019) . 218 219 S. glos sinidius also maintains a f unc t i onal a cylated homos er ine l acto ne ( A HL)- bas ed quo rum 220 sen s ing s ystem t hat puta t iv ely modu l at es gene expres s ion ac cording t o dens it y t hat is 221 hypoth es i sed to aid c o ordination bet ween symbiot ic bacter ia d uring host tis s ue invasion 222 (Pont es et al, 2008; R enoz et al 2023) . Sg GmmB4 cont a ins two fun ct i o nal copies of es a R, 223 which enc o de s an ohlL-responsive t rans c r iptional regulat o r, with the de leted c o py in 224 Sg G mmC2* being proximal to th iM . It is t herefor e lik ely th a t the e volved s train mainta ins 225 quor um s en s ing fun ctions due to t he presence of the function al copy, how ever whether 226 density-dependent gene ex pr es sion h as b een a f fected will r equi re further s tu dy. 227 228 In Sg G mmC1* multiple sulfur- a ssoci a t ed transmembr ane p rot ei n s were de let ed; s ulfu r is 229 used in the biosy n thesis and modification of s ulfur-containing amino acids , me t hion ine and 230 c ysteine (Mbaye et al , 2019). Met hio nine and cysteine are bot h pro d uc ed by W . glossinidia 231 for the tsetse hos t (Bing, et al , 2017) and th e d e gr adation o f t he ir pro duc t i on pathway s in S. 232 gloss inidius hav e been pr edic t ed as it mov es towar ds a sy mbiotic l if es tyle (Belda , et al, 233 2010). A ssuming the deleted sulfur a nd s ulfit e transmembr ane p rot e ins facilitated sulfur 234 compound up take, their deleti o n a d d s evidence to su ggest degr adation of met hion ine and 235 c ysteine biosynthesi s is o ccur ring. 236 237 Carb ohydrate t ransmembrane t ransporter s wer e t he m ost common gr oup of genes 238 containing small mutations between th e evolv ed strain and t he a n c estor s t rain fro m w h i ch 239 it was d e r ived, i n c luding t hos e involv ed in tran s por t o f pr ol ine, mannose, a n d a Glc N A c 240 repr es sor . Fur ther gene f unc t ions p otentially impacted by va r iation included prot ein 241 modification, D N A transcription and regulation, membr ane p rodu c t ion, two phage lytic 242 phase as soc iat ed ge n e s , energ y pr oduction and haemolysis ( Table 2). One notewor thy 243 pseudogeni s at ion identi fied i n SgG m mC1* oc c ur red in shlA , a haemolys is precursor ( Ta b l e 244 2). D uring axenic cultur e it was not ed that when grown on C olumbia blo od agar plates, 245 Sg G mmC1* did not exhibit th e same l evel of hae molys is a s t he original S . gl os si nidius is olat e 246 counter part ( data no t shown), su g ge s ting pheno typic conf i r mation of an effect of this 247 pseudogeni s at ion ev en t. This implies either t hat haemo lysis is an imp ortant trai t in t he 248 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 27, 2025. ; https://doi.org/10.1101/2025.06.26.659281doi: bioRxiv preprint tsetse ho s t , but not one to tho s e grown ou ts ide of i t s nat iv e habi t at, or tha t the conditions 249 for haemo lys i s gene ex pr ession are not met in vi t ro , r e sult ing in a lack of pos it i ve selec t iv e 250 pressure for maintaining the activi ty of this gene enabling i t s delet i on. 251 252 The serial passa g ing of S . glos s inidius imitat es the r epeated p opulati on b ot tlenecks in 253 natur al hos t -s ymbiont sy stems, whi ch ar e k n own to contribu te t o symbiont genome 254 redu c t i o n. The in v i t r o pas sage s were r epeated ev ery 10-14 day s , wher eas t he l ab- rear ed 255 tsetse life c y cle take s mor e than four weeks. The higher fr equency of pa ssa ging i n vitr o than 256 tr ans mis si o n of S. gl ossinidius between t s et s e in viv o may have increas ed t he bott l e n ec king 257 effect on genome degradatio n. 258 259 The Black Queen H y p othesi s de s c r ibes t he c o- dependencies bet w een a sso c iated o rganis ms 260 in s ymbiotic r elationships ; t hes e co-depen denc ies are for med by reductive genomic 261 evoluti on dr iv en b y r epeated popula tion bottl en e cks in a r e s t ricted enviro nmen t (Mo r ris et 262 al., 2012). The large deletion in SgGmmC1*, ac c ompanied by ina ctivation a nd 263 pseudogeni s at ion of genes via indels , suppor ts the con cept the S. glos sinidus i s capable o f 264 ongoing genome r educ t ion by s t eps a s de s cribed pr evi o us ly ( Goodhead and Dar by, 201 5). 265 Futur e w o rk wil l addr es s the eff e ct o f additional s election p r ess ur e s mor e c losel y replica t ing 266 those found in experimenta l systems fro m is olates fro m tsetse h os t s to examine such 267 pro c es s e s in real-wor l d sy s t ems and t hereby examine the tr ajec t ory of S. gl os s inidius 268 towar ds obligate s ymbiosis. 269 270 Long-ter m ex p er imen t a l evo lution of S. gl ossinidius isolates un der varyin g selection 271 pressure s, s u ch a s increa sed oxidative s tr ess or nu trient d e privat ion, may alter t h e 272 evoluti onar y t rajectory of S. gloss inidius and eluc idat e the roles of gene s y s tems and 273 sel ect i ve pr essures thereo n. S imi lar ly , long- term passaging of S. glos s inidiu s thro ugh tse t s e 274 expose d t o tr y pano s omes ma y result in genetic, epigenetic or tr ans cript ion c h a n g e s in the 275 pro duc ed S. gloss inidius s t rain, increasi n g ou r understand i n g of the tr i p a r t i t e int e raction 276 between symbiont, ho s t, and par a s ite. Similarly, studyin g the ability of the ev olved lab strain 277 Sg G mmC1* to infect t s etse flies wou l d allow for ex am inat i on of t he r ol e S . glossini d i us 278 genes h a ve on t rypano some vec t or c ompetence. 279 280 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 27, 2025. ; https://doi.org/10.1101/2025.06.26.659281doi: bioRxiv preprint ACK NO W LED G EME N TS 281 The author s ac knowledge s upp ort fr o m the University of Salfo rd f or genom ics and 282 comput a t ional i nfrastru c t ure and the Liverpool School of Tr opic al Med i cine for s upp orting 283 the t s et s e colony work. 284 285 FUNDI N G I NF ORMATI O N 286 PP wa s suppor ted by a U niversit y of Salfor d a n d U nive rsity Alliance Doctoral Tr aining 287 Scholarship. This work wa s f unded by a Wellcome Trust S EED award t o IG : 200690 /Z / 16/ Z. 288 289 AUTHOR C O N TR IBUTI O N S 290 Sodalis gloss inidius is olat e s were coll ec t ed by ACD and mai n tained by L RH . G en ome 291 sequenci n g and analy sis wa s per formed by P P and I G. Furt her analy s is and manuscr i p t 292 prep a r ation and r ev iew was per formed by P P, L RH , ACD and IG. 293 294 CO NFLIC TS O F IN TER ES T 295 The author s declare t hat ther e are no conflicts o f interest.296 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 27, 2025. ; https://doi.org/10.1101/2025.06.26.659281doi: bioRxiv preprint FI G URES 297 Figure 1: Large deletion in the SgGmmC1* genome. Top: Full geno me al ig nmen t of Soda lis 298 gloss inidius ev olved strain Sg GmmC1 * agains t a n cestor s tr ain Sg GmmB4 s howing 99.99 % 299 si mil ar ity, with the single lar ge d el et i on expanded to show details of t he g enes delet ed. The 300 top f i gu re s ho w s the alignment of th e ent i r e c h romosome o f each strain, s howing the high 301 level of s imilari t y between the seque ncies, with t he l ar ge del etion ci r cle d . T h e b ottom 302 diagram show s t he c od i n g genes (or ange) an d ps eudogene s (yellow) flanking and inc lud e d 303 in the del et i o n. G enome po s itio n s (bp) a r e rela tive to t he S gGmmB4 r efer ence genome. 304 Figures were produ c ed u sing ea syfig. 305 306 307 308 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 27, 2025. ; https://doi.org/10.1101/2025.06.26.659281doi: bioRxiv preprint Table 1 – G ene s within the 17,209 b p d el et ed region of Sg GmmC1* ac c o rd ing to th e SgGmmB4 r eference (Goodhead et al , 2020). Gene names 309 and pro ducts wer e ann otated using P ROKKA; con served ort hol ogous gr oup s wer e as sig n ed us in g the STRI N G dat abas e; gene onto l o g y a nd 310 con s er ved regions w er e assigned u s in g the Int erpr o database. K e y: C D S – coding D N A s equen ce; p seudo – ps eu d ogene; C O G – c lu ster ed 311 ort hologous group; G O – gene ont ology. Yellow – pseudogene s ; blue – CD S . 312 313 314 CDS/ pseudo COG GO terms Conserved regions Code Description Biological Molecular Cellular ba eS_ 1 S i g n a l tr a ns d u c t io n hi s t id in e - pr o te i n k in a se BaeS ps e u d o COG 0 6 42 S i gn al t ransducti o n histi d i ne k i n a s e - - - - ba eS_ 2 S i g n a l tr a ns d u c t io n hi s t id in e - pr o te i n k in a se BaeS ps e u d o COG 0 6 42 S i gn al t ransducti o n histi d i ne k i n a s e GO :000 7 1 65 s ig n a l t r a n s du c t i o n GO: 000 0 1 55 p hosp ho r e lay se n s or k ina s e acti vity GO: 001 6 0 21 i n t e g r a l c o m p on e nt of me m br an e IPR0 0 3 661 S ign a l t ra n sd uc t i on h i s t i d ine ki na s e , d i m e r i s a t i on / ph o s ph oa c c ep t o r do m ai n IPR0 0 3 660 H AM P dom ain (s e n so r a nd c hem o t ax i s) ba eS_ 3 S i g n a l tr a ns d u c t io n hi s t id in e - pr o te i n k in a se BaeS ps e u d o COG 0 6 42 S i gn al t ransducti o n histi d i ne k i n a s e - - - IPR0 0 5 467 H istid in e ki na se d o ma i n S y s t em ati c G en e ID : 0 3996 Hy po th eti ca l prot ei n ps e u d o COG 0 8 26 C ol l ag en a s e-li ke pr ot ea s e, Prt C f a mi ly - - - - y hbU _2 P u tativ e pr ot e as e YhbU p r e cu rs o r ps e u d o COG 0 8 26 C ol l ag en a s e-li ke pr ot ea s e, Prt C f a mi ly - - - IPR0 0 1 539 Pe p t i d a se U 32 S y s t em ati c G en e ID : 0 3998 Pe p t i da se f a m il y U 3 2 ps e u d o COG 0 8 26 C ol l ag en a s e-li ke pr ot ea s e, Prt C f a mi ly - - - IPR0 0 1 539 Pe p t i d a se U 32 thi M Hyd rox y et hy l thi azol e ki n a s e CDS COG 2 1 45 Hyd roxy e t hylth iaz ol e k ina se, s u g ar k i n a s e fa mi ly GO :000 9 2 28 th iam in e biosynth e t i c p roc e ss GO: 000 4 4 17 hy dr ox y eth y l thia zol e k i n a se ac t iv it y - IPR0 0 0 417 H y dr ox y eth y l thi azol e k i n as e es aR _2 T r an sc r ip t i on a l a c t iva t o r p ro te i n E sa R CDS COG 2 1 97 DNA -bindi ng r esp o n s e r eg ulato r, N a rL /F ix J f am i ly , co n t a in s REC a nd H T H do m ai ns GO :000 6 3 55 r e g ul ati on of trans cri p t i on , DN A- tem p l a t ed GO: 000 3 6 77 DNA b indi ng - IPR0 0 0 792 T rans cri p ti on r e gul a t or L u x R, C- te r mi n al .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 27, 2025. ; https://doi.org/10.1101/2025.06.26.659281doi: bioRxiv preprint CDS/ pseudo COG GO terms Conserved regions Code Description Biological Molecular Cellular S y s t em ati c G en e ID : 0 4001 P u tativ e a c etyl tra n sf era s e CDS COG 0 4 54 N - a ce ty l t r an s fe ra se , GN AT s u pe rf a mil y (in clu des histo ne ac etylt ran sfer as e H P A 2 ) - GO: 000 80 8 0 N - a c et y l t r an s f e r a s e a c t i v i t y - IPR0 0 0 182 G NA T d om a i n S y s t em ati c G en e ID : 0 4002 Hy po th eti ca l prot ei n C D S C O G 04 2 5 Tu s A - r el at e d s u l f u r t r a ns f er a s e - - - IPR0 0 1 455 T us A -li k e dom ain S y s t em ati c G en e ID : 0 4003 Hy po th eti ca l prot ei n CDS COG 2 3 91 U n c h ar a c te rize d mem b r a ne p ro te i n Yed E / Y e eE, c o nta ins t wo sulfu r tr a ns p o rt do m ai ns - - - IPR0 0 7 272 Su lphur t ran sp o rt d omai n S y s t em ati c G en e ID : 0 4004 P u tativ e i n n e r m e m b rane p r ote in CDS COG 2 3 91 U n c h ar a c te rize d mem b r a ne p ro te i n Yed E / Y e eE, c o nta ins t wo sulfu r tr a ns p o rt do m ai ns - - - IPR0 0 7 272 Su lphur t ran sp o rt d omai n yeeE _1 L ys R su b str at e bi nding d o ma i n p r ot ein ps e u d o COG 0 5 83 DNA -bindi ng tr ans c ripti on a l r e gulato r, Ly s R fa mil y - - - IPR0 0 5 119 Ly s R , substrat e-bi nd i ng yeeE _2 L ys R su b str at e bi nding d o ma i n p r ot ein ps e u d o COG 0 5 83 DNA -bindi ng tr ans c ripti on a l r e gulato r, Ly s R fa mil y - - - G3D SA: 3.40. 1 9 0.1 0 Per ipla sm ic b i ndin g p rot ei n-li ke I I dt ha dh D-thr e o -3-hy d ro xy as par ta t e de h yd rata s e ps e u d o COG 3 6 16 D - s er i ne de am i na s e , p y r i d ox a l ph os p h a te - de p e n d e n t - - - IPR0 4 2 208 D -s eri ne deh y drat a s e-l ik e d o main S y s t em ati c G en e ID : 0 4008 Hy po th eti ca l prot ei n ps e u d o COG 1 2 88 U n c h ar a c te rize d mem b r a ne p ro te i n Y f cC , ion tr an s port er su p erf a mi ly - - GO: 001 6 0 21 i n t e g r a l c o m p on e nt of me m br an e IPR0 1 838 5 C 4 - d i carboxyl a t e anae ro b i c c a rri er- lik e IPR0 1 838 7 Un ch a rac te r ise d p ro tei n Yc g A / Y f c C S y s t em ati c G en e ID : 0 4009 Hy po th eti ca l prot ei n CDS COG 1 2 88 U n c h ar a c te rize d mem b r a ne p ro te i n Y f cC , ion tr an s port er su p erf a mi ly - - GO: 001 6 0 21 i n t e g r a l c o m p on e nt of me m br an e IPR0 1 838 5 C 4 - d i carboxyl a t e anae ro b i c c a rri er- lik e IPR0 1 838 7 Un ch a rac te r ise d p ro tei n Yc g A / Y f c C yx e P _2 P ut ativ e hydr ola s e Yx e P CDS COG 1 4 73 Metal-d e pen den t - GO: 001 6 7 87 h y d r o la s e - IPR0 0 2 933 Pe p t i d a se M2 0 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 27, 2025. ; https://doi.org/10.1101/2025.06.26.659281doi: bioRxiv preprint CDS/ pseudo COG GO terms Conserved regions Code Description Biological Molecular Cellular a mi das e/ a mi noacyl as e/ carb oxy p e p ti das e activity IPR0 1 7 439 A min o hydr olas e Sys t e ma t ic Ge ne I D: _ 04 01 1 H y po t h et i c al p r ot ei n CDS - - - - - - ya b J_ 2 Enami n e /i min e dea minas e ps e u d o COG 0 2 51 E na mine dea m i nas e Ri dA, hous e cl e a ni n g o f r eacti ve en am i ne i n t er m ed i at es, Y j gF/Y E R057 c/U K 1 1 4 fam il y - - - IPR0 0 6 175 Yj gF/ YE R05 7c/U K11 4 famil y S y s t em ati c G en e ID : 0 4013 Hy po th eti ca l prot ei n ps e u d o - - - - - - S y s t em ati c G en e ID : 0 4014 S u lf i te e xp o r te r T au E /S a f E p se ud o C O G 0 73 0 U n c h ar a c te rize d mem b r a ne p ro te i n Y f c A - - GO: 001 6 0 21 i n t e g r a l c o m p on e nt of me m br an e IPR0 0 2 781 T ransm e mbran e p r ot ein Ta u E-li k e S y s t em ati c G en e ID : 0 4015 S u lf i te e xp o r te r T au E /S a f E C D S C O G 0 73 0 U n c h ar a c te rize d mem b r a ne p ro te i n Y f c A - - GO: 001 6 0 21 i n t e g r a l c o m p on e nt of me m br an e IPR0 0 2 781 T ransm e mbran e p r ot ein Ta u E-li k e S y s t em ati c G en e ID : 0 4016 Hy po th eti ca l prot ei n CDS - - - - - - S y s t em ati c G en e ID : 0 4017 PT S s y s tem , l ac to se /ce llo b i o s e s pec if ic I IB s ub u ni t CDS COG 3 4 14 P h o sp ho t ran sfer a s e s sy st em, g ala ctito l- s p ec i f ic I I B c om p o ne n t GO :000 9 4 01 p h os p hoe no lp yr u v a t e - dep end e nt s ugar ph o sp h otransf era s e syst em GO: 000 89 8 2 p r ot ein-N ( PI )- ph o sp h o hi st idi ne-su gar p ho s ph ot r a n s f e r a s e activity - IPR0 0 3 501 Ph o sp h otr an sf er as e syst em, EII B c om p o ne n t , t yp e 2/ 3 ga tC Gal a ctitol per m e as e I I C co m pon en t ps e u d o COG 3 7 75 P h o sp ho t ran sfer a s e s sy st em, g ala ctito l- s p ec i f ic I I C c o mp o ne n t - - - IPR0 0 4 703 P h os ph o t ra n s fe ra se s y s te m , s u g a r - sp ec i f ic pe rme ase c o m p o ne n t IPR0 1 3 853 G a l a cti t o l p er me as e II C c o mpon e nt S y s t em ati c G en e ID : 0 4019 Hy po th eti ca l prot ei n .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 27, 2025. ; https://doi.org/10.1101/2025.06.26.659281doi: bioRxiv preprint CDS/ pseudo COG GO terms Conserved regions Code Description Biological Molecular Cellular CDS - - - - - - S y s t em ati c G en e ID : 0 4020 P T S syst e m gal actit ol-sp e cifi c tra n s po r t er s u bunit II A CDS COG 1 7 62 Ph os p h ot r a n s f e r as e s y s t e m m anni tol/fr u cto s e-s p eci fic I IA domai n (Nt r-t y p e) - - - IPR0 0 2 178 PT S EI IA type-2 domai n S y s t em ati c G en e ID : 0 4021 Hy po th eti ca l prot ei n CDS N OG 33 1 0 3 n on su p e rvis e d orth o l og ou s gr oup - - - - fa b G _4 3 -o xo a cyl-[ acyl- car ri er- p rot e in] r e ductas e FabG ps e u d o COG 1 0 28 NAD ( P )-dep end e nt d eh y drog e n as e , sh ort- ch a in al c ohol de hy d ro gen a s e f a m i ly - GO: 001 6 4 91 ox i dor e d u ctas e acti vity - IPR0 0 2 347 Sh o rt- c hai n d e h yd rog e na s e /r edu cta s e SD R S y s t em ati c G en e ID : 0 4023 T ra n sposas e IS 116/IS 1 1 0/I S9 0 2 f a m i ly p ro t ei n p s eu do C O G 35 4 7 Tr a n s po s a s e GO :000 6 3 13 t ransp o si ti on , DN A-m edi at e d GO: 000 3 6 77 DNA b indi ng GO: 000 4 803 tr a n s po s ase activity - IPR0 0 3 346 T ransp o sa s e, I S116/I S110 /IS 9 0 2 S y s t em ati c G en e ID : 0 4024 T ra n sposas e p s eu do C O G 35 4 7 Tr a n s po s a s e GO :000 6 3 13 t ransp o si ti on , DN A-m edi at e d GO: 000 3 6 77 DNA b indi ng GO: 000 4 803 tr a n s po s ase activity - IPR0 0 3 346 T ransp o sa s e, I S116/I S110 /IS 9 0 2 S y s t em ati c G en e ID : 0 4025 H yp o t he t ic al p r o te in ps e u d o COG 3 5 47 T ra ns p os ase - - - - 315 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 27, 2025. ; https://doi.org/10.1101/2025.06.26.659281doi: bioRxiv preprint Table 2 - Snippy output w it h al l insertions , delet ions and single-nucleotide polymor phis ms in Sodalis glossinidius s t rain SgG mmC1*, in 316 comparison with compar a t or strain SgGmmB4. Gene names and p rodu cts were annotat ed us ing PRO KKA; con se rved ort ho logous group s were 317 assigned u sing t he STRI NG databa se; g en e on tology and c o n s er v ed r egions were as s igned u sing the Int erpro databas e. Ke y: *** - Tier 1 318 pseudogeni s at ion (mor e t han two-thi rds of the gene as a n notated in SgGmmB4 is r emoved); ** - Tier 2 pseudogenisation (disrupt ion o f a 319 con s er ved region by tru nc at ion); * - T ier 3 pseudogenisa t ion (disrupt i o n of a c on served r egi o n by a mis s en s e mu t a t ion). CD S – C od in g DN A 320 sequence. P seudo – pseudogene. In s – insert ion. Del – dele tion. Co m – c o mplex polymor phism. SN P – si n gl e nu cleotide po l ymo rphism. A A – 321 amino acid. B – biol ogic al pr oc es s. M – molec u l ar process . C – cell u l ar proces s . Yellow – ps eud o ge n es ; blue – CD S ; o range – potent i al n ovel 322 pseudogene s, shaded b y tier. 323 324 B 4 p os i t ion Ty p e CD S/ p s eu do GO Te rm s COG s SN P eff ec t Disru pte d c on ser v e d re gi on s ⎯ Chromosome ⎯ * ** S y s te mati c G e n e I D: 0 0 037 h y po t h et i c al p r ot ei n 26 ,5 7 7 ins CDS - - - - Fra m e s hif t truncat e s at A A 59/ 1 9 9 - * ** h srA _1 pu tati ve transp o rt p rot e in H sr A 35 1 ,610 ins CDS GO:00 5 5 085 B: tr an s m e mb r an e tr a n sp o r t C O G 0477 MFS fam i ly p e rm eas e Fra m e s hif t r em o v es hs rA_ 1 on an t i s e n s e st rand, r e p l ac e s wi th t wo over l a p pin g h y po t h et i c al s o n s en s e stran d ( no c o ns erv ed r egi ons on ne w gen es) IP R 0 1170 1 ; P F 0 7690 (m ajor fa cil it ator supe rfamil y ) IP R 0 2084 6 ; PS 5 0850 ( majo r fac il itato r supe rfamil y p r ofi l e) IP R 0 3625 9 ; SS F 1 03 47 3 (M F S gen e ra l s u b s tr a te tr a ns p or te r) PT H R 23 50 1 : S F 180 (m u lt id r u g r e sis t a nce pro t ei n B h omol og ) G 3 D S A : 1. 20 . 1 7 20 . 1 0 (m u l tid ru g r esi stanc e pro t ei n D ) GO:00 2 2 857 M: tr an s m em b r an e t ra ns port er a cti vi ty Sys t e ma t ic Ge ne I D: 00 81 0 Tai l fib e r p r ot ein g p37 C t e r mina l 58 8 ,917 ins ps e u d o - - C O G 4675 Mi cr o c ysti n -d epen dent pro t e in (functi on unkno wn) Fra m e s hif t truncat e s at A A 17 1 /2 60 IP R 0 2224 6 ; PF12 6 0 4 (b act eri o pha g e tail f ib re pr o te i n g p 37 C te rm i na l f a m ily ) nn r_ 3 NA D( P )H- h ydr a t e r epai r en zyme N n r 61 9 ,593 ins ps e u d o GO:00 5 2 855 M: AD P -d epe nde n t N AD ( P) H-hy dr at e de h yd ratas e a ctivi ty C O G 0062 Ca r bohydr a t e t r a ns port a nd me ta bo l i sm ; N A D H X ep i me ra se activity Fra m e s hif t r em o v es fi rst 53/ 1 1 9 AA s an d a l t ers AA s 54-73 IP R 0 0063 1 ; PF01 2 5 6 ( ca rb o hy d rat e k inas e famil y ) PS 5 13 83 ( Yj eF C-te rm ina l domain ) IP R 0 2905 6 (r i bo k in a se - l ik e s u pe r fa m il y) C O G 0063 Ca r bohydr a t e t r a ns port a nd me ta bo l i sm pr o P _2/ proP _1 Pr oli ne /beta in e tr anspo rt e r 86 9 ,745 ins CDS GO:00 5 5 085 M: AD P -d epe nde n t C O G 0477 Ca r bohydr a t e t r a ns port a nd R e m ov es pr o p _2 st op Al l cons erv ed r e g i ons i n ta ct .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 27, 2025. ; https://doi.org/10.1101/2025.06.26.659281doi: bioRxiv preprint B 4 p os i t ion Ty p e CD S/ p s eu do GO Te rm s COG s SN P eff ec t Disru pte d c on ser v e d re gi on s N AD ( P) H-hy dr at e de h yd ratas e a ctivi ty m e t a b olis m, m a j o r fa cil i tator supe rfamil y c od o n , merg i ng p r op_ 2 a n d pro p_1 GO:00 2 2 857 M: tr an s m em b r an e t ra ns port er a cti vi ty GO:00 1 6 021 C: int eg ral comp o n en t o f me m b ra ne ** p g l_2 Po l ygala ctu ronas e 1 ,04 8 , 5 09 i n s C D S G O : 0 00 59 7 5 B : c a r b oh y dr a te me t a b o l i c pro c ess C O G 2706 Ca r bohydr a t e t r a ns port a nd m e t a b olis m, 6- ph o sp h ogl uco n ol act on a s e Fra m e s hif t truncat e s at A A 17 1 /1 82 IP R 0 0074 3 ; PF00 2 9 5 ( gly c osi de h yd rol a s e f a m i ly 2 8 ) IP R 0 1105 0 ; S S F 51 1 2 6 (pe c t in ly a se - like supe rfamil y ) GO:00 0 4 650 M: poly gal act u r onas e acti v i ty * S y st em ati c Gen e ID : 0 160 3 B a c te r io ph age l ys i s p ro te in 1 ,21 5 , 3 24 c o m C D S - - N O G 2 69 7 2 D NA - p ac k ag ing p r o te i n gp 3 M is se nse a t A A 23 , R t o K IP R 0 3206 6 ; PF16 6 7 7 (D NA-pa ck a g in g pro t ei n gp 3 ) 1,2 15 , 3 8 7 SN P CDS - - NOG 2 6 972 DN A-pa c ka gi n g prot ein g p3 Synony m ous - * n ag C _ 2 N -a cetyl gl uc osami ne r ep r e s so r 1 ,42 4 , 4 93 S NP C D S G O : 0 00 63 5 5 B : re g u l a ti o n o f t ra ns c ripti on, D NA- te mp l a ted C O G 1940 Ca r bohydr a t e t r a ns port a nd me ta bo l i sm , R O K f a m ily Mi ss en s e a t AA 2 6 3/ 419, C to F IP R 0 0060 0 ; PF00 4 80 (ROK f a m ily ) IP R 0 4312 9 ; S S F 53 0 6 7 (ac t i n - l ike ATP a se do m ain ) GO:00 0 3 700 M: D N A-bin d i ng t ra ns c ripti on fa ctor a c t ivity * pt sG _ 1 PTS syst e m glu cos e-sp eci fi c EII CBA c o m po n e n t 1,4 27 , 6 7 6 SN P CDS GO:00 0 9 401 B: phosph oenolpy ruv at e- de pen den t s ugar phosphotra n sf e ra s e syst em C O G 1263 c ar b oh y dr a te t ra n sp o r t a n d m e t a b olis m, PTS s yst em M i s s e n s e at A A 2 8/ 6 7 8, M t o I IP R 0 1097 4 ; TIG R 0 1 998 ( N- ac e tyl g l ucos am i ne-sp eci fi c PTS tr anspo rt er s u bu ni t I I B C ) IP R 0 1301 3 ; P S 5 1103 (phos p ho enol py r uvat e - dep end e nt s ugar phosphotr an sf e ras e syst em ( PTS) ) IP R 0 0335 2 ; PF02 3 7 8 ( ph os p h o t r an s fe ra se syst em , EI I C) PT H R 30 00 9 ( cytoch rom e c-t y p e sy n t hesi s pro t ei n a n d P T S tr ans m e m br ane co m p onent ) GO:00 0 8 982 M: pr o t ei n-N ( PI) - phosphohi stidi n e-suga r phosphotra n sf e ra s e acti v i ty C O G 1264 ca rb o h yd rat e tr an s port and m e t a b olis m, PTS s yst em GO:00 1 5 572 M: N-ac etyl glu c o sam in e t r an s m em b r a n e t ra ns port er a cti vi ty C O G 2190 ca rb o h yd rat e tr an s port and m e t a b olis m, PTS s yst em GO:00 1 9 866 C: org anel l e in ner me m b ra ne GO:00 1 6 021 C: int eg ral comp o n en t o f me m b ra ne GO:00 1 6 020 C: m emb rane * s uc B Co m p one nt of 2-ox ogluta rat e dehy d ro ge na s e 1,4 52 , 0 5 3 SN P CDS GO:00 0 6 099 B: tr ic ar bo xy li c aci d cycl e C O G 0508 Ene r gy pr o d u ct i o n and c o nve rsi o n, dehydro g enas e Mi ss en s e a t AA 2 7 4/ 396, P to S IP R 0 0625 5 ; TIG R 0 1 347 ( di hy d r ol i p oy l l y s i n e- r esi due s u cc inylt ransf eras e ) G O : 0 01 67 4 6 M : ac y ltr a n s f er ase a c t iv i t y .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 27, 2025. ; https://doi.org/10.1101/2025.06.26.659281doi: bioRxiv preprint B 4 p os i t ion Ty p e CD S/ p s eu do GO Te rm s COG s SN P eff ec t Disru pte d c on ser v e d re gi on s GO:00 0 4 149 M: d i h yd rol ipoyl lys in e - re s i d ue su c ci n y l tr a ns f er a se acti v i ty IP R 0 0107 8; PF00 1 9 8 ( 2-ox o ac id dehydro g enas es acyl t ra n sfe ras e ( ca t a l y t ic do m ain ) GO:00 4 5 252 C: o x o g l utarat e de h yd rog e n as e c ompl ex ** yd c V I nne r m e mbr a n e t ransp ort er pr o t ein Yd c V 1 ,53 5 , 7 73 i n s C D S G O : 0 05 50 8 5 B : t ra n smem b ra ne tr a n sp o r t C O G 1177 Ino r gani c i o n tr ans p o rt an d m e t a b olis m; b i n di ng-p ro t e in- dep end e nt t r a ns port sy st e ms i n n e r m e mb ra ne com p o nent M is se nse a t A A 26 4 (V t o G) and tr u n cat ed at AA 266/ 2 8 1 IP R 0 3590 6 ( M e tI -li ke sup e r fami ly ) PT H R 43 84 8 ( Pu t r es cin e tra n sp ort s yst em p e r mea se p ro te i n) GO:00 1 6 020 C: m emb rane S y s t em ati c G en e ID : 0 2100 hy p otheti cal p r ot ein 1,5 66 , 2 6 0 ins CDS - - C O G 5464 Trans po s as e, fun cti on u nkno wn Tw o hypothet i cal g e n e s me rge d Al l cons erv ed r e g i ons i n ta ct * w c aJ U D P- g l uc os e:und eca p r enyl-phosp h at e glu c o s e-1- p ho sp hat e tra n sf era s e 1,6 38 , 8 6 4 SN P CDS - - C O G 2148 Cel l wal l / m emb ra ne / e nv el op e biog en esi s , su gar tr an sf e ras e Mi ss en s e a t AA 3 7 4/ 464, G to A IP R 0 1747 3 ; TIG R 0 3 023 ( u nde ca p r e ny l - ph o sp hat e g lu cos e phos p hot ransfe ra s e ) IP R 0 1747 5 ; TIG R 0 3 025 ( exopoly sa ccha rid e b i o s y n t he s is p o ly p ren y l gly cosyl phosph o tra nsf e r a s e ) IP R 0 0336 2 ; PF02 3 9 7 (b act eri a l s ugar transf eras e ) PT H R 30 57 6 ( c o lo n i c b i o s y n t h e s i s U D P - g l uco se l i p id ca rr ie r tra ns fe r a se ) za p C_ 2 Ce ll di visi o n p ro t e i n Z ap C 1,7 01 , 4 6 2 ins ps e u d o - - NOG 0 1 298 Cel l divi si on Fra m e s hif t r em o v ed ge n e IP R 0 0980 9 ; PF07 1 2 6 (Ce ll - d iv is io n p r o tei n ZapC ) * ** m a n C 1 M an n o s e-1-p h o sp h at e gu a ny l ylt ransf eras e 1 1,8 67 , 8 9 8 ins CDS GO:00 0 9 058 B: bi o s yntheti c p ro c e ss C O G 0662 Ca r bohydr a t e t r a ns port a nd me ta bo l i sm Fra m e s hif t truncat e s at A A 1 2 9/ 4 7 1 an d m ov e s A A s 13 9 -47 1 onto n ext fram e w it h c lo se s t p r o mo te r > 5 0b p upstr eam f r o m s e cond h a l f IP R 0 0637 5 ; TIG R 0 1 479 ( m a nn ose - 1 - ph o sp hat e g u an y l yltr an sf eras e /mann o s e-6- ph o sp hat e is om eras e ) IP R 0 0583 5 ; PF00 4 8 3 (n ucl e oti dyl transf eras e ) IP R 0 2904 4 ( n u cl eoti d e - d i phosph o -su gar transf eras e s) GO:00 0 0 271 B: poly sa ccha rid e biosy n th eti c pro c ess C O G 0836 Cel l wal l / m emb ra ne / e nv el op e biog en esi s GO:00 0 5 976 B: poly sa ccha rid e m etaboli c p roc es s GO:00 1 6 779 M: nuc l eoti dy l tr an sf er as e acti v i ty S y s t em ati c G en e ID : 0 2481 hy p otheti cal p r ot ein 1,8 96 , 4 2 1 ins CDS - - - - Fra m e s hif t at A A 2 20 /246, a l te r s re ma i ni ng AA s a nd e x ten d s to 25 0 - S y s t em ati c G en e ID : 0 2937 hy p otheti cal p r ot ein 2,2 05 , 2 0 6 ins ps e u d o - - - - Fra m e s hif t alt er s f rom AA 4 2 / 46 , e x t e n d s t o 49 Al l cons erv ed r e g i ons i n ta ct sh l A H e m ol ys i n pr e c u r s or .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 27, 2025. ; https://doi.org/10.1101/2025.06.26.659281doi: bioRxiv preprint B 4 p os i t ion Ty p e CD S/ p s eu do GO Te rm s COG s SN P eff ec t Disru pte d c on ser v e d re gi on s S y s t em ati c G en e ID : 0 3153 hy p otheti cal p r ot ein 2,3 60 , 3 2 6 ins CDS - - C O G 3210 Intra c e l lula r tr affi ckin g , s e cr e ti o n an d v e si cula r tr ans p o rt Fra m e s hif t at A A 2 33 /248 o f shlA alt e rs l ast 1 5 AA s and add s PR O K K A _ 0 315 3 unchang e d onto end Al l cons erv ed r e g i ons i n ta ct CDS - - - - Al l cons erv ed r e g i ons i n ta ct S y s t em ati c G en e ID : 0 3155 hy p otheti cal p r ot ein S y s t em ati c G en e ID : 0 3156 hy p otheti cal p r ot ein 2,3 61 , 5 1 5 ins CDS - - - - Fra m e s hif t m er ge s P RO KKA _0 3 1 55 and P RO KKA _0 3 1 56 with 17 ext ra A As b et w e en th em Al l cons erv ed r e g i ons i n ta ct CDS - - - - Al l cons erv ed r e g i ons i n ta ct * p h e S P heny l alani ne--tR NA li gas e al pha subu ni t 2,3 67 , 3 0 9 SN P CDS GO:00 4 3 039 B: t RN A am ino a c ylati o n C O G 0016 Transl ati on, ri bo s om a l st ru ctur e a n d biog en esi s M i s s e n s e at A A 5 9/ 3 2 7, V t o G IP R 0 2291 1 ; MF_ 00281 ( P hen y l a l anin e — tR NA li g as e alp h a s u b u nit ph eS) IP R 0 0452 9 ; x ( Phe n yl a l an in e— tR NA l iga se alph a su bu nit ) IP R 0 04188 ; PF02 9 1 2 ( Am in oa cy l tR N A syn t heta s e cla ss II , N -t er minal d o ma in) IP R 0 1097 8; S S F 46 5 8 9 ( tRN A -b in d i ng a rm ) G 3 D S A : 3. 30 . 9 3 0. 1 0 (Bi r a b if u nc t io n a l pro t ei n; doma in 2 ) GO:00 0 6 432 B: ph en y lala nyl-t RNA amin oacylat i o n GO:00 0 5 524 M: AT P bin d i ng GO:00 0 0 049 M: t RN A b i ndin g G O : 0 00 48 1 2 M : am in o ac y l- tR N A lig a se acti v i ty G O : 0 00 01 6 6 M : n u cle o t ide b i n d in g GO:00 0 4 826 M: ph en yl al anin e-t R N A li ga se a c t ivi t y GO:00 0 5 737 C: c y topl as m sl yA _2 Tr an s c ri p ti o na l r e g u l ator Sl yA 2 ,40 7 , 9 04 i n s C D S G O : 0 00 63 5 5 B : re g u l a ti o n o f t ra ns c ripti on, D NA- te mp l a ted C O G 1846 Trans cr ipti on r e gul a t or I n s e rtion 50bp i n fr ont of ge n e, u na l te red A A se que n c e but move s n ear est start codo n f r o m 50 b p a w ay t o 9 3b p aw ay Al l cons erv ed r e g i ons i n ta ct GO:00 0 3 700 M: D N A-bin d i ng t ra ns c ripti on fa ctor a c t ivity * f nr Fu m a r a te a n d n it ra t e re d uc ti on re g ul a t o ry p r o te in 2 ,46 7 , 5 83 S NP C D S G O : 0 00 63 5 5 B : re g u l a ti o n o f t ra ns c ripti on, D NA- te mp l a ted C O G 0664 Si gn al t ransdu cti on me chanis ms, t r a n scr ip t i o n al re g ul a t o r, c rp f n r famil y Mi ss en s e a t AA 5 2 /2 51 , P to T IP R 0 0059 5 ; cd00 0 3 8 ( effe ctor domai n of th e CAP fa mil y o f tran scri p t ion facto rs ), P F 000 27 (cy cl i c n u cl e otid e - b i ndi ng d o mai n), P S 50 04 2 ( c A M P /c GMP b i n d ing mo t i f p ro f ile ) IP R 0 1471 0 (R m l C - li ke je ll y r o ll f o l d) IP R 0 1849 0 (c yc lic n uc le o t i d e - b i nd in g - li ke) PT H R 24 56 7 ( C R P f a m i ly t r a n s c r ip t i o n a l reg u la to r y p ro t e in ) GO:00 0 3 700 M: D N A-bin d i ng t ra ns c ripti on fa ctor a c t ivity GO:00 0 3 677 M: D N A bi ndin g qu iA_ 1 Qui n at e/ shi kim at e deh y dr o genas e ( q ui- ) 2,5 62 , 2 8 2 ins ps e u d o - - C O G 4993 Ca r bohydr a t e t r a ns port a nd m e t a b olis m, de hydro g enase Fra m e s hif t ad d s 2 2 AA s to start o f g e n e Al l cons erv ed r e g i ons i n ta ct S y s t em ati c G en e ID : 0 3881 hy p otheti cal p r ot ein 2,8 73 , 8 8 5 ins ps e u d o - - NOG 2 9 7256 Non sup ervi s ed o rth ol o g ous gr o ups Fra m e s hif t at 23 2 / 844 - .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 27, 2025. ; https://doi.org/10.1101/2025.06.26.659281doi: bioRxiv preprint B 4 p os i t ion Ty p e CD S/ p s eu do GO Te rm s COG s SN P eff ec t Disru pte d c on ser v e d re gi on s br ea k i ng i n t o t w o prot ei ns ch a A _2 S od i u m/ pr o ton an t iport e r Cha A ch a A _3 3,2 07 , 2 6 2 ins ps e u d o - - C O G 0387 Ino r gani c i o n tr ans p o rt an d m e t a b olis m, ca l ci um p ro t on Fra m e s hif t m er ge s chaA _ 2 and ch aA _ 3 wi th an ext ra 7 AA s b e twee n Al l cons erv ed r e g i ons i n ta ct ps e u d o - - Al l cons erv ed r e g i ons i n ta ct S y s t em ati c G en e ID : 0 4953 N M T1 / THI 5 lik e pr ot e i n 3,6 32 , 8 6 3 ins CDS - - - - Fra m e s hif t truncat e s at A A 26 9 /2 91 Al l cons erv ed r e g i ons i n ta ct * S y st em ati c Gen e ID : 0 520 4 h y po t h e t ic a l p r o te i n 3 ,83 0 , 3 47 S NP C D S G O : 0 00 59 7 5 B : c a r b oh y dr a te me t a b o l i c pro c ess C O G 0726 Ca r bohydr a t e t r a ns port a nd m e t a b olis m, 4-ami no-4-deo xy - alph a- L-ara b i nopyran o syl un d eca pr enyl pho s pha t e biosynth e ti c p roc e ss Mi ss en s e a t AA 2 5 2/ 324, A to E G 3 D S A : 3. 20 . 2 0 . 37 0 ( gl y c osi d e hy dr ola s e /d e a c et ylas e ) cd 1 0 935 (puta t i v e ca tal y t i c domain of li p o po l ysa c ch a ri de bio syn th e sis pr ot e i n Wa l W a n d it s ba c te r ia l ho m ol og s) G O : 0 00 38 2 4 M : c a ta ly ti c a c ti v it y yji R _ 3 putativ e H TH- ty pe t ran s cri ptional r e gulat or Yji R 3,9 62 , 5 8 4 ins ps e u d o GO:00 0 3 824 M: ca t a l yt ic act ivi ty C O G 1167 Trans cr ipti ona l r e gu l a to r, gn t R famil y Fra m e s hif t r em o v es fi rst 46/ 1 0 3 AA s IP R 0 1542 2 ; G 3D SA: 3 . 9 0. 1150. 10 (as p a rtat e amin o tra nsf er a s e, d o main 1) IP R 0 1542 4 ; S S F 53 3 83 ( P L P -d epe nde n t transf eras e s) PT H R 42 79 0 : S F 7 (t r a ns c ripti onal r e gulato r- re l a te d ) * * S yst e m a t i c G en e ID: 05385 h y po t he t ic al p r o te i n 3,9 67 , 5 4 7 ins CDS GO:00 3 2 506 B: cy to kin eti c p ro ce ss C O G 3266 Fu n ctio n unkn o w n, D a mX-r el at ed pro t ei n Fra m e s hif t r em o v es fi rst 38/ 2 8 8 AA s IP R 0 3289 9 ; MF_ 0202 1 ( C e l l div i si o n prot ei n Dam X ) GO:00 4 2 834 M: pe ptid ogl ycan bindin g GO:00 3 0 428 C: c el l s e ptum ⎯ pSG4/3 ⎯ S y s t em ati c G en e ID : 0 5911 hy p otheti cal p r ot ein 636 ins CDS - - - - Fra m e s hif t at A A 5 8/ 64 , e x ten d s to 17 3 - * * S yst e m a t i c G en e ID: 0591 2 h y po t he t ic al p r o te i n 3 ,68 0 i n s C D S G O : 0 00 82 3 7 M : me ta llo p e p t id a se acti v i ty C O G 3291 P K D r epe at Fra m e s hif t truncat e s at A A 56 0 /5 73 IP R 0 2407 9 ; G3D SA : 3 . 4 0.3 90.10 (c oll ag e n as e , c atalyti c dom a i n) S SF55 4 86 (Me t a llo p ro te a se s ("z i nc i n s") , catal ytic do ma i n ) PF13 6 88 ( M etal lo-p eptida s e fami ly M1 2) S y s t em ati c G en e ID : 0 5921 hy p otheti cal p r ot ein 10 ,1 4 0 del CDS - - - - Fra m e s hif t truncat e s at A A 92/ 1 0 6 - 1 0 ,2 69 S NP Mi ss en s e a t AA 5 0 , T to P .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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