MAIMOTOMONTANTUL US009752129B2DIN TUDI MINI (12 ) United States Patent ( 10 ) Patent No. : US 9 ,752 , 129 B2 Bell , III et al. (45 ) Date of Patent: * Sep . 5 , 2017 (54 ) HOST CELL MODIFICATION WITH 8 ,021 , 662 B2 9 / 2011 Szalay et al . 8 , 159 , 224 B2 4 / 2012 Fontius ARTIFICIAL ENDOSYMBIONTS 8 , 361, 437 B2 1 / 2013 Sharma et al . 8 ,406 ,498 B23 / 2013 Ortyn et al . @(71 ) Applicant: BELL BIOSYSTEMS, INC ., Palo 8 , 828 ,681 B2 * 9 /2014 Bell , III ...... A61K 49 / 1896 Alto , CA (US ) 424 / 9. 34 8 , 859 , 281 B2 * 10 / 2014 Bell , III ...... A61K 49/ 1896 @(72 ) Inventors: Caleb B . Bell, III, San Mateo , CA 435/ 252 . 1 (US ) ; Alexey Bazarov , Fremont, CA 8 ,956 ,873 B2 2/ 2015 Bell , III et al. 9 ,023 ,612 12 B2B2 "* 55 / 20152015 Bell , III ...... A61K 49 /0097 (US ) ; Abdul Wakeel, Fremont, CA 435 / 29 (US ) ; Joyce Barrozo , San Bruno , CA 9 ,085 ,764 B2 ** 7 / 2015 Bell , III C12N 5 /00 (US ) 9 ,137 ,975 B2 ** 9 /2015 Bell, III ...... A61K 49/ 1896 9 , 315 , 780 B2 ** 4 / 2016 Bell , III ...... C12N 5 / 16 @( 73 ) Assignee : Bell Biosystems, Inc. , Palo Alto , CA 9 , 370 ,566 B2 ** 6 / 2016 Bell , III C12N 15 / 03 (US ) 9 , 446 , 154 B2 ** 9 / 2016 Bell , III ...... A61K 49 /0097 9 ,458 ,432 B2 ** 10 /2016 Bell , III . . .. C12N 5 / 00 @( * ) Notice : Subject to any disclaimer, the term of this 9 ,481 , 869 B2 ** 11 / 2016 Bell , III ...... C12N 5 / 16 patent is extended or adjusted under 35 9 ,528 ,980 B2 * 12 /2016 Bell , III ...... A61K 49 / 1896 2002 /0012698 A1 1 /2002 Bauerlein et al . U . S . C . 154 ( b ) by 0 days. 2004 /0234455 A1 11/ 2004 Szalay This patent is subject to a terminal dis 2005 / 0069491 Al 3 / 2005 Szalay et al . claimer . 2007 / 0202572 AL 8 /2007 Szalay et al. 2007 /0258886 A1 11 /2007 Ahrens et al. 2007 /0275924 Al 11/ 2007 Khan (21 ) Appl . No. : 15 /331 , 847 2009 /0181101 A1 7/ 2009 Rademacher et al . ( 22 ) Filed : Oct. 22 , 2016 2009 /0311194 Al 12 /2009 Hu et al . ( Continued ) (65 ) Prior Publication Data FOREIGN PATENT DOCUMENTS US 2017 /0037378 A1 Feb . 9 , 2017 EP 1807517 B1 10 /2011 Related U .S . Application Data WO WO 2010 / 109187 9 / 2010 (63 ) Continuation of application No. 14/ 476 , 584 , filed on ( Continued ) Sep . 3 , 2014 , now Pat . No . 9 ,481 , 869 . OTHER PUBLICATIONS (60 ) Provisional application No .61 / 873 ,308 , filed on Sep . 3 , 2013 . Bazylinski et al , Magnetosome formation in prokaryotes , 2004 , Nature Reviews vol. 2 , pp . 217 - 230 . (51 ) Int. Ci. Bell , Tracking cardiac engraftment and viability ofMiPSC by MRI, C12P 21/ 02 ( 2006 .01 ) 2014 , Abstract for Grant Application , Project No. 1R43EB019239 C12N 15 /87 ( 2006 . 01 ) 01. C12N 5 / 16 ( 2006 .01 ) Brewer, et al. , Relaxometry of Bacterially Derived Organelles : A CO7K 14 / 195 ( 2006 . 01 ) Novel Class ofMRI Contrast Agent for Cell Labeling and Tracking , C12N 5 / 00 ( 2006 .01 ) May 2014 , Joint Ann Mtg ISMRM - ESMRMB , Italy . CI2N 15 /02 ( 2006 .01 ) (Continued ) C12N 15 /09 ( 2006 .01 ) (52 ) U . S . CI. CPC ...... C12N 5 / 16 (2013 . 01 ) ; CO7K 14 / 195 Primary Examiner — Nancy J Leith (2013 .01 ); C12N 15 / 87 ( 2013 .01 ); COZK ( 74 ) Attorney , Agent , or Firm — HelixIP 2319/ 034 (2013 .01 ); C12N 2510/ 00 (2013 .01 ) ( 58 ) Field of Classification Search None ( 57 ) ABSTRACT See application file for complete search history . The present invention is directed generally to host cells with References Cited artificial endosymbionts , wherein the artificial endosymbi (56 ) ont and the host cell communicate with each other to alter a U . S . PATENT DOCUMENTS phenotype of the host cell. In some embodiments , the communication comprises the secretion of a polypeptide 4 , 385 , 119 A 5 / 1983 Blakemore 4 ,677 , 067 A 6 / 1987 Schwartz et al. from the artificial endosymbiont into the host cell . The 5 , 843 ,643 A 12 / 1998 Ratner secreted polypeptide can be a selectable marker , a reporter 6 , 004 ,815 A 12 / 1999 Portnoy et al. protein , a transcription factor, a signal pathway protein , a 6 , 287 , 556 B1 9 / 2001 Portnoy et al. 6 , 436, 694 B1 8 / 2002 Tally et al. receptor, a growth factor, a cytokine , an effector molecule or 6 , 599 , 502 B27 / 2003 Portnoy et al . other factors that can produce a phenotype in the host cell . 7 , 390 ,646 B2 6 /2008 Andino -Pavlovsky et al . 7 , 470 ,427 B2 12 / 2008 Cocking 7 ,754 ,221 B2 7 / 2010 Szalay et al . 20 Claims, 35 Drawing Sheets US 9, 752 , 129 B2 Page 2

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Ordway et al, Animal models of mycobacteria infection , Current Stewart , F . J. et al ., Lateral symbionts acquisition in a maternally Protocols in Immunology (2011 ) Chapter 19 , p . 19 .5 . 1. transmitted chemosynthetic clam endosymbiosis , Mol. Biol. Evol. , 2008 , 25 (4 ): 673 -687 . * cited by examiner U . S . Patent Sep . 5 , 2017 Sheet 1 of 35 US 9 ,752 , 129 B2

WP009869454.1 WP008619475.1 WP009868028.1 WP009868027.1 WP009868025.1 WP017102899.1 001092641.1YP WP006387942.1 002395108.1YP 017031543.1WP WP017109466.1 YP005048901.) WP009866462.1 WP018907567.1 017029526.1WP WP007606711.1 YP006446912.1| WP008955916.1 YP_421364.1 YP420631.1 YP_420638.1 WP_009868207.1 YP_420640.1 YP421357.1 YP421356.1 YP421362.1 CAM76579.1 YP485257.1 008223511.1_WP

97% 96% 86% 90% 84% 84% 86% 82% 42% Ident |100% |74% |76%

-163|42%9e 40%le-143 le-12843% %125|385e- 70-12345% 3e-11837% %40-11839 1e-11641% 56%3e-162 2e-15744% %13248|le- %130|373e- %324432e- 5e-12444% le-11960% 6-31744% 40-11646% Evalue 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. |le-164

100% Query cover 46% 30% 51% 63% 46% 59% 36% %50 39% 43% 51% 35% 63%1060 250643% 355052% 327445% 220748%320852% 267057% 249440% 319249% 37%1554 121243% 198344% 200049% 202429% 2103.41% 196741%

Total score |7176 13522 6654 2249 3488 2589 2302 3709 3861 1291 3408 1725 1648

Max score |2959 2061 1976 |1672 917 646 643 626 624 574 579 534 562 507 446 436 454 431 452 448 402 413 417 424 428 402

FIG.1A

TypeVsecretorypathway,adhesinAidA(MagnetospirillummagneticumAMB-1]7176 TypeVsecretorypathway,adhesinAidApartial[Magnetospirillumsp.SO-1] RTXtoxinsandrelatedCa2+-bindingprotein[MagnetospirillummagneticumAMB1385 cellsurfaceglycoproteinprecursor(MagnetospirillummagneticumAMB-1] cellsurfaceglycoproteinprecursor(MagnetospirillummagneticumAMB-1] Autotransporteradhesin[MagnetospirillumgryphiswaldenseMSR-1] Description TypeVsecretorypathway(MagnetospirillummagneticumAMB-1] TypeVsecretorypathway,partial(VibrionalesbacteriumSWAT-3] outermembraneadhesinlikeproteiin,partial[Pseudogulbenkianiaferrooxidans] COG2931:RTXtoxinsandrelatedCa2+-bindingproteins[Magnetospirillum flagellin-likeprotein[MagnetospirillummagneticumAMB1] COG2931:RTXtoxinsandrelatedCa2+-bindingproteins[Magnetospirillum hypotheticalproteinRPB_1638[RhodopseudomonaspalustrisHaA2] outermembraneadhesin-likeprotein(ShewanellaloihicaPV41 hypotheticalproteinVS110512(VibriosplendidusLGP32] VCBSrepeat-containingprotein[DesulfomoniletiedjeiDSM6799] magneticumAMB-1)Magnetospirillunexoprotein[|large COG5295:Autotransporteradhesin[Magnetospirillummagnetotacticum hypotheticalprotein,partial[Magnetospirillummagnetotacticum) COG5651:PPE-repeatproteinsMagnetospirillummagnetotacticum] hypotheticalprotein,partial[Variovoraxparadoxus] VCBSrepcat-containingprotein(Bradyrhizobiumsp.WSM471 hypotheticalprotein,partialMagnetospirillummagnetotacticum hypotheticalprotein,partial[Vibriotasmaniensis] |hypotheticalprotein[Achromobacterxylosoxidans] hypotheticalprotein,partialVibriobreoganii] hypotheticalprotein(Vibriotasmaniensis] Largecxoprotein[VibriofurnissiiNCTC11218] hypotheticalprotein,partial(Vihriobreoganii] magnetotacticum)

11 magnetotacticum U . S . Patent Sep . 5 , 2017 Sheet 2 of 35 US 9 ,752 , 129 B2

AccessionNo. 004747418.1|WP WP017081492.1 WP008224673.1 YP007065062.1 YP008030908.1 WP008341635.1 WP008340241.1 YP002313811.1 YP293166.] WP017082048.1 WP004731498.1 WP_006366861.1 WP006218690.1 WP009845528.1 WP017099027.1 006223938.1WP_ WP020195639.1 YP001770167.1 WP007828145.1 WP019350838.1 WP017086433.1 YP006019007.1 YP004120666.1 WP019350792.1 |YP421361.1 017101375.)|WP YP286399.1 |WP007606142.1 |WP006228189,1 YP470132.1 YP0029477971 WP_0062

36% 38% 40% 34% 39% 36% 35% 37% Ident |35% |34%

2e-11537% 2e-11497% 4e-11444% 6e-11441% 2e-11339% %ul|39le- 6e-11134% 7e-10734% 5e-10436% 4e-10334% 1e-10234% 2e-10241% 101|40%10- 2e-10039% Se-9938% 3e-98|37% |6c-11134% 6e-11038% 9e-10541% |2e-10040% 1e-93 Evalue 8e-102 2e-97 5e-96 2e-95 Se-95 |2e-94 3e-94 6e-94 2e-93

Querycover 28% 45% 48% 51% 49% 47% 47% 41% 64% 31% 56% 44% 44% 52% 39% 58% 41% 42% 46% 59% 61% 52% 65% 49% 47% 49% 42% 32% 62% 51%

Totalscore 2628 2838 1423 1307 920 2338 1294 1325 2165 3809 1017 |1173 1638 2732 2923 915 |1833 2355 1692 2741 1125 |2665 |1017 1606 1802 |1517 |1655 950 |1347 1396 2564

Maxscore 419 377 401 417 415 394 404 404 404 396 389 383 383 382 380 379 378 374 370 367 362 |365 364 352 343 345 353 348 347 352 335

FIG.1B

type1secretiontargetdomain-containngprotein,partial(Rhizobiumsp.PDO1076] hypotheticalproteinamb1998[MagnetospirillummagneticumAMB-1] TISSsecretedagglutinin(RTX[AchromobacterxylosoxidansNH44784-1996) outermembraneadhesin-likeproteinDesulfovibrioaespoeensisAspo2] Description TypeVsecretorypathway,partial(VibrionalesbacteriumSWAT-3) hemolysin-typecalciumbindingrepeatfamilyprotein3,partial(Achromobacter outermembraneadhesin-likeprotein[Methylobacteriumsp.446] outermembraneadhesin-likeprotein[Calothrixsp.PCC75071 outermembraneadhesin-likeprotein(Sulfurimonasgotlandica] RTXtoxinhemolysin-typeprotein[RhizobiumetliCFN42] hypotheticalprotein,partial(Vibriosplendidus] hypotheticalprotein,partial(Vibriotasmaniensis) hypotheticalprotein,partial(Sulfurimonasgotlandica] outermembraneadhesinlikeproteiinVariovoraxparadoxusS1107 VCBSrepeatprotein[ShewanellapiezotoleransWP3] JMP1341Ralstoniaeutrophahypothetical_C5971[proteinReut hypotheticalprotein,partial(Achromobacterpiechaudii] hypotheticalprotein,partial(Vibriotasmaniensis) VCBSrepeat-containingprotein(Variovoraxsp.CF3137 hypotheticalprotein,partial(Vibriosplendidus) outermembraneadhesin-likeprotein(ShewanellabalticaBA1751 hypotheticalprotein(Vibriosplendidus) iron-regulatedproteinFrpC[Vibriotubiashii] hypotheticalprotein,partial(Vibriosplendidus Largeexoprotein,partial(Achromobacterpiechaudii] hypotheticalprotein,partial[Vibriosplendidus VCBSDechloromonasaromaticaRCBI VCBSrepeatprotein(Vibriosplendidus VCBS(Chlorobiumferrooxidans] owensiilVibrioproteinhypothetical(| toxin(Vibriosp.MED2221 picchaudii] U . S . Patent Sep . 5 , 2017 Sheet 3 of 35 US 9 ,752 ,129 B2

WP006366860.1| WP020477147.1 WP017243041.1 YP007551444.1 YP008104634.1 YP002008092.1 YP002827036.1 WP003580127.1 WP004744158.1 WP020334262.1 WP008216195.1 WP017054796.1 WP016361080.1 WP006228258.1 006228197.1WP 009867477.1WP_ WP017092000.1 YP004680854.1 017017027,1WP 009301782.1wp 009301784.1WP WP017015203.1 WP_010452358.1 IdentAccessionNo. YP728854.1 NP899981.1 NP719678.1 NP770354.1 YP_857965.1 004848254.1|YP

37% 34% 32% 33% 37% 32% 35% 36% 36% 33% 33% 38% 34% 33% 34% 38% 43% %39 %46 %35 39% 33% 34%

1c-8037% 60-7941% 4e-8436% 4e-8236% 7934%le- %7932|2e- 2e-93 9e-90 Evalue 4e-92 2e-90 2e-90 2e-90 6e-90 |92-90 3e-89 2e-88 5e-88 10-86 2e-86 2e-86 5c-86 |12-85 2e-85 85|3e- 7e-85 79|8e- 2e-78| 3e-78 8e-78

Query 46% 65% 65% 57% 66% 60% 53% 37% 62% 63% 56% 69% 57% 63% 59% 44% 32% 40% 56% 52% 48% 46% 42% 48% 57% 43% 48% 65% cover |24%

Total score 1674 1549 4678 965 2061 1092 1043 928 1657 3285 2701 2870 2872 3931 2906 |1303 1303 1488 1792 719 1732 1530 1484 1839 1839 1945 1265 |1760 3682

Max score 337 342 342 341 341 336 333 333 338 335333 329 325 328 327 323 307 |316 312 320 313 308 304 303 303 298 296 300 300

FIG.1C

hypotheticalproteinAHA_3491(Aeromonashydrophilasubsp.ATCC Description hypotheticalproteinPCA1042970[PseudomonasresinovoransNBRC1065537 metalloprotease,hemolysin-typecalciumbindingregion(Cupriavidustaiwanensis secretedVCBSdomainprotein(ShewanellaoneidensisMR-1] RTXtoxincxportedproteinRtxA[CupriavidusnccatorN-l] outermembraneadhesinlikeprotein[Pseudogulbenkianiasp.NH83] hypotheticalproteinAZKH_2157[Azoarcussp.KH132C] RTXtoxinhemolysin-typeprotein(SinorhizobiumfrediiNGR234] hypotheticalprotein(VibrionalesbacteriumSWAT-3] COG2931:RTXtoxinsandrelatedCa2+-bindingproteins[Magnetospirillum |hypotheticalproteinbil3714[BradyrhizobiumdiazoefficiensUSDA1101 hypotheticalprotein[Desulfovibriosp.6_146AFAA] 46AFAA)Desulfovibriosp.61hypothetical(protein RTXtoxinexportedprotein[RalstoniaeutrophaH16] hypotheticalprotein,partial(Vibriobreoganii] structuraltoxinChromobacteriumviolaceumATCC124721 |hypotheticalprotein,partial[Vibriogenomosp.F6] hypotheticalprotein,partial[Enterovibriocalviensis) hypotheticalprotein,partial[Enterovibriocalviensis] hypotheticalprotein(Vibriosplendidus_ hypotheticalprotein[Vibrionatriegens) Largeexoprotein,partial(Achromobacterpiechaudii piechaudiiAchromobacter]exoproteinpartial(,|Large iron-regulatedproteinFrpC[Vibriorotiferianus) RTX(oxinRhizobiumleguminosarum] |hypotheticalprotein[Vibriotubiashii] VCBSprotein(BilophilaWadsworthia |VCBS(Chlorobiumferrooxidans] magnetotacticum) splendidus)Vibriotoxinpartial(,| LMG194241 79661 U . S . Patent Sep . 5 , 2017 Sheet 4 of 35 US 9 ,752 , 129 B2

AccessionNo. WP016959134.1 WP006072371.1 WP009301783.1 008684592.1WP |YP004379207.1 WP004400669.1 .1002156318YP WP017361689.1 WP018926333.1 YP204889.1 WP_017003860.1

33% %34 32% 34% 33% 32% 34% 32% Ident |33% |33% 33%|

Evalue le-77| 5e-77 60-77 80-77 2e-76 le-75 le-75 3c-75 62-74 3e-73 le-72 Query 180673% 209043% 55%2422 168346% 180464% 182248% 250557% 126236% cover|score 58% |2320 303970% |188756% Total |1806

Max score 299 297 296 296 290 293 292 290 286 |285 |281

FIG.1D

outermembraneadhesinlikeprotein[PseudomonasmendocinaNK-01) RTXrepeat-containingcalciumbindingcytotoxinRtxAl[VibriofischeriES114] Description hypotheticalprotein,partial(Desulfovibriosp.6146AFAA] largeexoprotein(Desulfovibriosp.31syn3] hypotheticalprotein,partial(Pseudomonasumsongensis] iron-regulatedproteinFrpC,partial[Vibrionigripulchritudo] iron-regulatedproteinFrpC[VibriofischeriMJ1l] hypotheticalprotein,partial[Enterovibrionorvegicus hypotheticalprotein,partial[Enterovibrionorvegicus |lrypotheticalprotein(Pseudomonasmendocinal VCBSrepeatprotein(Vibrioshilonii] U . S . Patent Sep . 5 , 2017 Sheet 5 of 35 US 9 ,752 , 129 B2

AccessionNo. WP009869454.1 WP008619475.1 WP009868028.1 WP009866462.1 YP003452293.1 WP006072371,1 017819225.1WP WP017013208.1 WP017242742.1 WP017029493.1 WP017031546.1 WP017081491.1 WP017077555.] WP010245949.1 WP003596515.1 WP005442621,1 WP017081492.1 WP009704723.1 WP020195639.1 005375591.1WP 420640.1YP_ YP421364.1 WP_009868027.1 CAM76579.1 YP420631,1 YP420638.1 YP421362.1 YP_005040816.1 YP269146.1 017821990.1WP|

100% 78% 90% 59% 79% 84% %6666% 71% 31% 30% 28% 43% 31% 27% 42% 42% 42% 42% 42% 32% 30% 27% 40% 28% 40% Ident |37% |47% |27%

70-180 2e-156 3e-88 7650- 5e-76 4e-67 3e-49 2e-45 4e-41 3e-39 20-37 3e-37 Je-37 5e-37 3e-36 4e-36 4e-36 4e-36 Evalue 0. 0. |3e-179 |1c-55 |8e-40 |2e-37 |2e-37 |2e-36 |3e-36 |0. |70-45

Query 100% 99% 55% 49% 27% 50% 64% 27% 27% 24% 96% 85% 68% 52% 76% 44%44% 50% 48% 36% 31% 33% 58% 55% 69% 74% 13% 59% cover |36% |76% |78%

Total score 746 1231 1538 214 700 3583 3813 2215 846 920 2139 1517 689 1935676 829 1110 1381 384 581 715 518 836 455 878 |1451 438 1270 1253 1428

Max score 3583 2085 1390 598 559 539 331 291 291 244 224 191 189 177 172 169 170 165 164 165 164 164 163 161 161 160 160 160 |160

FIG.2A

|TypeVsecretorypathway,adhesinAidA[MagnetospirilluinmagneticumAMB-1] TypeVsecretorypathway,adhesinAidApartial(Magnetospirillumsp.SO-11 Autotransporteradhesin(MagnetospirillumgryphiswaldenseMSR-1) polymer-formingcytoskeletalfamilyprotein(Vibriosp.HENC03] Description RTXtoxinsandrelatedCa2+-bindingprotein[MagnetospirillummagneticumAMB largecxoprotein(MagnetospirillummagneticumAMB-1] TypeVsecretorypathway(MagnetospirillummagneticumAMB-1] |COG5295Autotransporter:adhesin[Magnetospirillummagnetotacticum) hypotheticalprotein,partial[Magnetospirillunmagnetotacticum] COG2931:RTXtoxinsandrelatedCa2+-bindingproteins[Magnetospirillum flagellin-likeprotein(MagnetospirillummagneticumAMB1] putativeCabindingRTXtoxin(Azospirillumlipoferum4B] hemolysin-typecalciumbindingregion(Azospirillumsp.B5101 VBCSrepeat-containingprotein(Colwelliapsychrerythraea34H] polymer-formingcytoskeletalfamilyprotein(Vibrioharveyi] outermembraneadhesin-likeprotein,partial(Acetivibriocellulolyticus polymer-formingcytoskeletalfamilyprotein[Vibrioharveyi polymer-formingcytoskeletalfamilyprotein[Vibrioalginolyticus) COG5651:PPE-repeatproteinsMagnetospirillum[magnetotacticum] hypotheticalprotein,partial(Enterovibriocalviensis] hypotheticalprotein,partial[Vibrioalginolyticus) hypotheticalprotein,partial(Vibriobreoganii] hypotheticalprotein,partial(Vibriobreoganii] hypotheticalprotein,partial(Vibriosplendidus] |outermembraneadhesinlikeproteiin[Methylobacteriunextorquens hypotheticalprotein,partial(Vibriosplendidus] VCBSrepeatprotein[Vibrioshilonii] breoganjilVibrioproteinpartial(hypothetical,| |hypotheticalprotein,partial[Vibriosplendidus] magnetotacticum] owensiiVibrio]proteinhypothetical(| U . S . Patent Sep . 5 , 2017 Sheet 6 of 35 US 9 ,752 , 129 B2

AccessionNo. YP004290562.1 WP007092538.1 YP008104634.1 YP006294136.1 YP006254384.1 YP001981885.1 WP017079130.1 008291998.1WP WP003886432.1 WP017188870.1 WP017086433.1 YP001280066.1 YP002395438.1 WP009023297.1 WP017082341.1 WP017092649.1 WP019350981.1 WP004730529.1 YP003125000.1 WP017003861.1 WP009672533.1 WP017361689.1 WP008294433.1 YP004379207.1 WP017081150.1 YP002008092.1 WP017068684.1 WP017072194.1 WP017064674.1 WP006366861.1

38% 27% 30% 36% 32% 38% 27% 38% 32% 27% 38% 27% 31% 27% 27% 40% 27% 37% 28% 30% 28% 27% 27% 26% 26% 26% 28% Ident |38% |27% |27%

Evalue le-35 40-35 5c-35 8e-35 le-34 3e-34 Se-34 1e-33 50-336e-33 2e-32 2e-31 2e-31 5e-31 |8e-31 le-30 28-30 2e-30 2e-30 30- 40-30 70-30 72-30 9e-30 3e-28 4e-28 70-28 8e-28 8e-28 94-28

Query cover 29% 181% 54% 18% 50% 28% 73% 16% 53% 74% 28% 35% 76% 48% 75% 63% 70% 70% 24% 45% 29% 51% 61% 51% 77%67% 67% 77% 63% 64% 45%

Total score 415 464 797 156 534 521 1410 152 368 |1418 858 536 1505 858 1418 1555 1128 1131 436 323 311 620 823 543 1525292 292 1232 1556 1468 792

Max score 158| 157 156 155 154 154 150 |150 148 |145 |145 144 143143 142 142 142 142 140 140 140 140 139 134134 | 133 133 133 |133

FIG.2B

hypotheticalproteinPCA1042970PseudomonasresinovoransNBRC1065531157 typeIsecretiontargetGGXGXDXXXrepeatproteindomain[Methylophaga152 putativeoutermembraneadhesin-likeprotein[Psychrobactersp.PRwf1] Description hypotheticalproteinMetbo_1350[Methanobacteriumsp.AL-21] polymer-formingcytoskeletalfamilyprotein,partial(Vibriosplendidus] polymer-formingcytoskeletalfamilyprotein,partial[Vibriosplendidus] outermembraneadhesin-likeprotein(ChitinophagapinensisDSM2588] outermembraneadhesinlikeprotein(PseudomonasmendocinaNK-011 |metalloprotease,hemolysin-typecalciumbindingregionCupriavidus[taiwanensis134 polymer-formingcytoskeletalfamilyprotein,partial(Vibriocrassostreae] hemolysin-typecalciumbindingregion,partial[Thalassospiraxiamenensis) RTXtoxins-relatedCa2+hindingprotein[Methylophagasp.JAM7] polymer-formningcytoskeletalfamilyprotein(Vibrioharveyi] polymer-formingcytoskeletalfamilyprotein[Vibriosplendidus] polymer-formingcytoskeletalfamilyprotein(Vibriosplendidus] splendidus]cytoskeletal,partial(Vibriofamilyprotein-forming|polymer |polymer-formingcytoskeletalfamilyprotein,partial(Vibriocrassostreae] canadensis34037SolitaleaDSMcontainingprotein(repeat-|VCBS putativehemolysinCellvibriojaponicusUeda107] outermembraneadhcsin-likeprotein[Mycobacteriumphlei] proteobacteriumNOR5-37gammahypothetical(protein| splendidus!cytoskeletalprotein[Vibriofamilypolymer-forming| splendidus)proteinVibriolrypothetical,partial[| LGP321splendidusVibriohypotheticalVS110856[protein| hypotheticalprotein,partial(Enterovibrionorvegicus |hypotheticalprotein[Paenibacillussp.HGH00391 hypotheticalprotein[Pseudomonasmendocina hypotheticalprotein(Congregibacterlitoralis] hypotheticalprotein,partial[Vibriocrassostreae) |VCBSChlorobiumferrooxidans thiooxydans LMG19424 U . S . Patent Sep . 5 , 2017 Sheet 7 of 35 US 9 ,752 , 129 B2

AccessionNo. WP017100400.1 0015221911.YP WP007642508.1 001770167.1YP 007551444.1YP WP004731498.1 YP003881756.1 WP017094008.1 YP005025033.1 YP007065062.1 YP004120666.1 WP006103065.1 WP009301783.1 WP020334262.1 WP005301102.1 009696193.1WP YP002947797.1 WP017094769.1 WP006218690.1 WP009622276.1 WP018907567.1 |YP005497395.1 |WP017519457,1 009817742.1WP_ 003124906.YP YP_857965.1 NP719678.1 |WP017102899.1 WP_006223938.1 NP899981.1

27% 40% 37% %28 %42 39% 41% 27% 37% 36% 42% 34% 28% 36% 37% 32% %33 %38 33% %26 46% 36% 36% 36% 40% 28% 40% 37% 26% Ident |35%

Evalue 9e-28 3e-27| 275e- 7e-27 8e-27 -le26 2e-26 2e-26 4e-26 5e-26 5e-26 8e-26 2e-25 22-25 2e-25 20-25 22-25 3e-25 4e-25 2e-24 4e-24 6e-24 le-23 3e-23 30-2330-23 le-22 le-2210-22 24-22

Query 77% 35% 66% 44%44% 38% 21% 30% 72% 31% 28% 27% 51% 44% 39% 44% 41% 36% 68% 35% 60% 29% 50% 38% 23% 56% cover |71% |32% |76% |19%

Total score |1497 436 408 446 596 1088 438 1389 934 835 372 757 029 651 692 501 282 336 1053 798 705 755 605 390 489 |1089 866 785 968

Max score 133 131 130 130 130 129 129 129 127 127 127 126 125 125 125 125 125 124 124 122 121 120 119 118 117 118 |116 115 115 115

FIG,2C

cadherinprodomainlikefamilyprotein,partial(Vibriosp.HENC-01] outermembraneadhesin-likeprotein[Methylobacteriunisp.446] RTXtoxin(Vibriosp.EJY3]>refWP01423512111EJY31 outermembraneadhesin-likeprotein[DesulfovibrioaespoeensisAspo2] Description polymer-formingcytoskeletalfamilyprotein,partialVibriotasmaniensis] polymer-formingcytoskeletalfamilyprotein,partial(Vibriosplendidus) hypotheticalprotein,partial(Desulfovibriosp.6_146AFAA) outermembraneadhesin-likeprotein(ChitinophagapinensisDSM2588] hypotheticalproteinAHA_3491(Aeromonashydrophilasubsp.ATCC secretedVCBSdomainprotein(ShewanellaoneidensisMR-1) hemolysin-typecalciumbindingrcpcatfamilyprotein3,partial(Achromobacter outermembraneadhesinlikeproteiin(Cellvibriosp.BR) hypotheticalproteinAZKH2157(Azoarcussp.KH32C) hemagglutinin/hemolysin-likeprotein(Dickeyadadantii3937] Conservedrepeatprotein[BacillusmegateriumWSH-002] outermembraneadhesin-likeprotein(Calothrixsp.PCC75071 hypotheticalprotein,partial[PseudomonasnitroreducensTX1] MBIC110171Acaryochlorismarinahypothetical60108[proteinAMI VariovoraxparadoxusS1101membranelikeproteiin(adhesinouter hypotheticalprotein,partial(Achromobacterpiechaudii] ljypotheticalprotein,partial(Variovoraxparadoxus] violaceum124721ChromobacteriumATCCstructuraltoxin( VCBSrepeatprotein[Vibriosplendidus] FG-GAPrepeatdomainprotein(Coleofasciculuschthonoplastes hypotheticalprotein(Vibrionatricgens] putativeRTXtoxin(Photobacteriumdamselae hypotheticalprotein,partial(Vibriosplendidus |hypotheticalprotein,partial(Vibriotasmaniensis) VCBS(Roseovariussp.2171 |VCBS(Pelagibacabermudensis] piechaudii] 7966) U . S . Patent Sep . 5 , 2017 Sheet 8 of 35 US 9 ,752 , 129 B2

AccessionNo. WP008164882.1 WP008891487.1 WP017082048.1 YP002946474.1 YP003505219.1 YP006446912,1

55% 39% 37% 38% 40% Ident |26%

Evalue 20-22 |6e-22 2e-21 4e-21 4e-21 4e-21

37%406 48445% 11531735% 112121243% 111113236% MaxTotalQuery score|cover 24461%|114

FIG.2D

Description Thalassospiraprofundimaris)calcium-bindingregion,partial(hemolysintype outermembraneadhesin-likeprotein(VariovoraxparadoxusS1101 outermembraneadhesin-likeprotein(DenitrovibrioacetiphilusDSM128091 VCBSrepeat-containingprotein(DesulfomoniletiedjeiDSM6799] hemolysin(Achromobacterarsenitoxydans] hypotheticalprotein[Vibriosplendidus) U . S . Patent Sep . 5 , 2017 Sheet 9 of 35 US 9, 752 , 129 B2

AccessionNo. WP009869271.1 WP008615895.1 WP_009869323.1 CAM75368.1 YP001830991.1 WP008888391.1 WP009467822.1 CAM76811.1 YP001867978.1 YP_003051754.1 YP005082876.1 WP017749095.1 YP001864081.1 WP008029683.1 WP017744127.1 WP017990332.1 YP_001976973.1 YP004975543.1 WP019646374,1 YP_423419.1 YP422662.} WP0105870001 YP914368.1 |YP001789672.1

100% 54% 91% 79% 28% 29% 30% 30% 30% 35% Ident 65% 50% 32% 30% 30% 32% 36% 29% 31% 31% |31% 31% 5e-2931% 2e-2831% le-116 6e-91 5e-86 36-37 le-34 3e-31 3e-31 90-31 2e-27 30-26 268e- 1e-25 le-25 2e-25 le-24 18-24 2e-24 2e-24 0. 0. 6e-25 QueryE cover|value 100%0.

56% 63% 58% 63% |15% 51% 67% 61% 67% 65% 32% 50% 58% 54% 64% 69% 65% 63% 50% 63% 65% 66% 20%

Total score 2585 910 751 377 569 602 381 1219 224 1160 135 271 371 203 367 641 368 810 568 680 309 801 121

Max score 601 377 163 155 143 144 135 134 130 127 125 125 4 124 122 121 122 121

FIG.3A

142HEMAGGLUTININ/HEMOLYSIN-RELATEDPROTEIN[Magnetospirillum rhizobiocin/RTXtoxinandhemolysin-typecalciumbindingprotein[Rhizobiumetli|122 Description RTXtoxinsandrelatedCa2+-bindingprotein(MagnetospirillummagneticumAMB2585 RTXtoxinsandrelatedCa2+-bindingprotein[MagnetospirillummagneticumAMB331 ATCC9039]Beijerinckiasubsp.indicaproproteinconvertaseP( putativerhizobiocin/RTXtoxinandhemolysin-typecalciumbindingprotein hemolysin-typecalciumbindingprotein(Pseudovibriosp.FOBEGI) 910COG2931:RTXtoxinsandrclatedCa2+-bindingproteins[Magnetospirillum COG4254:Uncharacterizedproteinconservedinbacteria(Magnetospirillum RTXtoxinsandrelatedCa2+-bindingproteins[Magnetospirillumgryphiswaldense310 hemolysin-typecalciumbindingregion[Roseibiumsp.TrichSKD4] hemolysin-typecalciumbindingregion[NostocpunctiformePCC73102] hemolysin-typecalciumbindingregion[NostocpunctiformePCC73102] Hemolysin-typecalciumbindingregion[Rhodobactersp.SW2] hemolysin-typecalciumbindingprotein(ParacoccusdenitrificansPD1222] calciumbindinghemolysinprotein(Thalassospiraprofundimaris] hemolysin-typecalciumbindingregion[Schlesneriapaludicola] Na-Caexchanger/integrinbeta4[LeptothrixcholodniiSP6]. |lrypotheticalproteinAZOLI240606[Azospirillumlipoferum4B] RTXtoxin(Magnetospirillumsp.SO-1] [MethylovorusglucosetrophusSIP3-4] hypotheticalprotein(Novispirillumitersonii] gryphiswaldenseMSR-1] hypotheticalproteinScytonemahofinanni hypotheticalprotein(Rhizobiumleguminosarum magnetotacticum] |hypotheticalprotein(Scytonemahofmannil CIAT652] magnetotacticum MSR-1) 11| U . S . Patent Sep . 5 , 2017 Sheet 10 of 35 US 9, 752 ,129 B2

AccessionNo. YP004180362.1 YP002826836.1 007088891.1WP WP018236938.1 YP007052011.1 WP018236607.1 WP008030394.1 WP008548480.1 010180036.1WP WP003545660.1 WP020582427.1 WP008047127.1 WP006714325.1 WP003532128.1 YP007551607.1 YP005189185.1 WP008110911.1 YP003050467,1 WP009385092.1 WP008614612.1 YP005189512.1 WP019867288.1 WP017677435.1 YP_003450680.1 002281223.1YP_ WP019864974, 017023581.1WP| 003168877.1YP_| WP_005594452.1

29% 30% 29% 32% 30% 32% 29% 30% 33% 29% 26% 29% 28% 29% 29% 30% 30% 30% 28% 28% 28% 29% 31% 43% 30% 31% 28% 30% 28%

3e-24 3e-24 4e-24 5e-24 82-24 le-23 20-23 22-23 2e-23 22-23 3e-23 3e-23 4e-23 4e-23 5e-23 6e-23 84-23 8e-23 8-23 le-22 2e-22 3e-22 4e-22 82-22 le-21 2e-21 2c-21 2e-21 QueryE valuecover| 63%3e-24

56% 63% 61% 64% 55% 62% 50% 48% 63% 61% 63% 62% 61% 42% 49% 56% 48% 63% 63% 71% 52% 49% 78% 64% 61% 64% 27351% 50863%

Total score 298 700 409 445 |823 343 272 322 398 318 950 426 |1493 115 346 399 499 160 852 158 456 672 471 653

Max score 120 120 120 120 120|120 118 117 117 117 117 118 117 117 [16 115 116 115 115 115 |115 115 114 114 114 112 |112 112 |111 111

FIG.3B

Description rhizobiocin/RTXtoxinandhemolysin-typecalciumbindingprotein[Azospirilluin hemolysin-typecalciumbindingprotein(IsosphaerapallidaATCC43644] hemolysin-typecalciumbindingprotein(Rhizobiumleguminosarumbv.trifolii type1secretionproteinwithC-terminaltargetdomain[Pseudomonassp.GM747 |hemolysin-typecalciumnbindingproteinMethylovorusglucosetrophusSIP341 hypotheticalproteinNGR_c23240[SinorhizobiumfrediiNGR234] hemolysin-typecalciumbindingprotein,partial[Thalassospiraxiamenensis] hemolysin-typecalciumbindingproteinNostocsp.PCC71071 Hemolysin-typecalciumbindingregion(Rhodobactersp.SW21 Hemolysin-typecalciumbindingregion(Glaciecolasp.HTCC2999] hypotheticalproteinAZKH_2325[Azoarcussp.KH32C) Collagenalpha-2(1)chainAlphatypeIcollagen[SinorhizobiumfrediiHH1031 hypotheticalprotein(Magnetospirillumsp.SO-1] |hypotheticalproteinSFHH10302192[SinorhizobiumfrediiHH103] Hemolysin-typecalciumnbindingdomaincontainingprotein(Candidatus AccumulibacterphosphatiscladellAstr.UW-11 Ca2+-bindingprotein,RTXtoxin(Herbaspirillumsp.YR522] hypotheticalprotein(Rhizobiumsp.BR816] hypotheticalprotein(Rhizobiumsp.BR8161 type1secrctionprotein[Pscudovibriosp.JE062) Ca2-+bindingprotein,RTXtoxinpartial[Rhizobiunileguminosarum] hypotheticalprotein,partial[Endozoicomonaselysicola hypotheticalprotein[Methylovulummiyakonense) hypotheticalprotein[Pseudomonasalcaliphila] typeIsecretionprotein(Vibrioichthyoenteri] type1secretionprotein(Vibriosp.N418] hypotheticalprotein[Methylovulummiyakonensel typeIsecretionprotein(Vibrioscophthalmi] melilotilSinorhizobiumhemolysin[D| logeilAliivibrioproteinhypothetical[| B5101sp. WSM23041 U . S . Patent Sep . 5 , 2017 Sheet 11 of 35 US 9 ,752 , 129 B2

AccessionNo. WP006231502.1 WP006274635.1 YP001944163.1 WP006274634.1 YP007511038.1 YP004120097.1 WP018240167.1 WP017265529.1 WP008622436,1 YP005723848.1 YP008104687,1 WP005052115.1 WP017717249.1 WP004210915.1 WP007091705.1 YP007515571.1 WP017274029.1 WP018043892.1 WP007675223.1 YP005716684.1 WP017263229.1 WP018679436.1 WP006833885.1 WP_010060462.1 YP002310757,1 WP006102484.1 NP436619.1 YP004556664. YP_468266.1 |WP003545662.1

Ident 28% 29% 29% 29% 28% 27% 29% 29% 29% 28% 29% 31% 29% 29% 28% 30% 29 29% 29% 29% 27% 29% 30%30% 30% 28% 28% 27% 29% 28%

E 30-21 48-21 215e- 6e-21 7e-21 7-21 8e-21 le-20 le-20 le-20 le-20 le-20 14-20 le-20 le-20 2e-20 2e-20 4e-20 4e-20 Se-20 5e-20 5e-20 6e-20 7e-20 82-20 98-20 le-19 4e-19 value 48-21 |3e-20

68% 66% 50% 70% 50% 51% 61% 50% 63% %65 66% 47% 61% 52% 51% %54 %54 75% 59% 52% 64% 48% TotalQuery scorecover 62%417 77857% 67%863 76%1221 84%371 50%445 51664% 50%444 417 678 639 300 319 442 212 382 485 303 597 472 383 246 303 106 358 254 465 648 316 744744 437

110) Max score 110110 110 110 110 109 108 108 108 108 108 108 108 108 108 107 108 108 |107 |106 |106 105 106 106 106 106 106 105 105 103

FIG.3C

calciumbindinghemolysinprotein,putativeErythrobacter[sp.SD-21] rhizobiocin/RTXtoxinandhemolysin-typecalciumbindingprotein(Rhizobiumetli putativecalciumbindinghemolysinprotein(BradyrhizobiumoligotrophicumS58] Description hemolysin-typecalciunbindingprotein(ChlorobiumlimicolaDSM245] hypotheticalprotein,partialMagnetospirillumsp.SO-1) probabableiype1secretiontargetrepeatprotein(SinorhizobiummelilotiSMIL hypotheticalproteinPCA10_43500[PscudomonasresinovoransNBRC1065531 hemolysin-typecalciumbindingprotein(SinorhizobiummelilotiBL225C] rhizobiocin/RTXtoxinandhemolysin-typecalciumbindingprotein,partial BradyrhizobiumoligotrophicumS581bindingregion[hemolysin-typecalcium Fecrprotein[DesulfovibrioaespoeensisAspo-2] hemolysin-adenlyatecyclase(Sinorhizobiummeliloti1021] hemolysin-typecalciumbindingregion,partial[Thalassospiraxiamenensis putativecalcium-bindingprotein,partial[Rhizobiumleguminosarum] RTXtoxinsandrelatedCa2+-bindingprotein[alphaproteobacteriumBAL1991 Hemolysin-typecalciumbindingregion(ShewanellapiezotoleransWP31 typeIsecretiontargetGGXGXDXXXrepeatproteindomain SinorhizobiummelilotiAK837bindingprotein(hemolysin-typecalcium| hypotheticalprotein,partial(Acinetobacterbeijerinckii] |hypotheticalprotein(Oscillatoriasp.PCC10802] 88A]proteinMethylobacteriumsp.hypothetical[| tjernbergiaelAcinetobacierhypotheticalpartial(protein, typeIsecretionprotein[Photobacteriumprofundum) rhizobiocinRZCAAsticcacaulisbiprosthecium) hemolysin(Rhizobiumsp.BR816) |hypotheticalprotein(Sphingobiumyanoikuyae] rhizobiocinRZCA(Asticcacaulisbiprosthecium hemolysinD(Sinorhizobiummeliloti] melilotipartialSinorhizobiumhemolysin(D,| hemolysinDSinorhizobiummeliloti [Rhizobiumetli) CFN42} U . S . Patent Sep . 5 , 2017 Sheet 12 of 35 US 9 ,752 , 129 B2

AccessionNo. WP018095958.1 YP766400.1 YP003158921.1 YP002826449.1 WP006519062,1 WP010480511.1 WP008758430,1 WP017804098.1 YP005189186.1

Ident 4e-1927% 295e-%19 197e-|28% 27%7e-19 70-1928% 29%9e-19 2e-1830% 2e-1827% 2e-1827% E value TotalQuery scorecover 56% 63% 61% 63% 63% 62% 61% 48% 63% 381 536 250 452 555511 540 519 244 Max score 103 103 103 102 101

FIG.3D

Description hemolysin-typecalciumbindingprotein[DesulfomicrobiumbaculatumDSM4028] |hemolysin-typecalciumbindingrepeatprotein(Leptolyngbyasp.PCC7375] hemolysin-typecalciumbindingregion[RhodobacteraceaebacteriumKLHIl] rhizobiocin(Rhizobiumleguminosarumbv.viciae38411 Collagenalpha-1(XXVII)chain[SinorhizobiumfrediiHH103] calcium-bindingprotein[Acaryochlorissp.CCMEE54101 |hypotheticalprotein,partial(Nodulariaspumigena] hemolysinD[Sinorhizobiummeliloti] hemolysin[SinorhizobiumfrediiNGR234] (Coleofasciculuschthonoplastes U . S . Patent Sep . 5 , 2017 Sheet 13 of 35 US 9, 752 ,129 B2

AccessionNo. YP006446912.1 WP006218690.1 WP008684592.1 WP016361080.1 YP003558595.1 YP001770167.1 WP_004731498.1 WP010452358.1 ABM63821.1 YP002156318.1 005048901.1YP WP004747418.1 WP007942972.1 WP006070242.1 0080309081.YP WP020334262.1 WP020621311.1 WP017082048.1 WP018058402,1 YP_420631.1 YP420638.1 YP421364.1 YP286399.1 NP899981.1 857965.1YP_ NP719678.1 YP204889.1 YP485257.1 WP0054196581 YP421356.1 YP561402.1

Ident 100% 94% 97% 46% 45% 34% 35% 36% 39% 34% 34% 40% 39% 34% 43% 46% 33% 99% 35% 33% 32% 32% 32% 34% 36% |38% |37% |35% |34% |44% |36%

E value 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0.

|77% TotalQuery |coverscore 10086|100% 3163795% 1231190% 14277|86% 767490% 839787% 92%4982 67%4571 502080% 781189% 95%6732 466392% 243465% 661089% 1149905693% |87%45121144 111115837|88% 497984%1110 1127487%1087 1060393787% 1011429787% 998239585% 992972290% 965695086% 9327 |1238359485% 12011509090% |1185664690% |1158667094% |1016250859% 543289%1005|

Max score 10086 |5598 2958 2012 1758 1706 1519 1445 1434 1360 1303 1280 1248| 1240 1172

FIG.4A

TypeVsecretorypathway,adhesinAidA[MagnetospirillummagneticumAMB-1) cellsurfaceglycoproteinprecursorMagnetospirillummagneticumAMB-1] TISSsecretedagglutinin(RTX[AchromobacterxylosoxidansNI144784-1996) Description TypeVsecretorypathway[MagnetospirillummagneticumAMB-1] outermembraneadhesin-likeprotein[Methylobacteriumsp.4461 hypotheticalproteinAHA_3491[Aeromonashydrophilasubsp.ATCC secretedVCBSdomainprotein(ShewanellaoneidensisMR-1] RTXrepeat-containingcalciumbindingcytotoxinRtxA1[VibriofischeriES1141 hypotheticalprotein,partial[Pseudomonassp.35MFCvill] hypotheticalprotein,partial(OxalobacteraceaebacteriumJG10001004-K231 largeexoprotein[MagnetospirillummagneticumAMB-1] VCBSrepeat-containingprotein(DesulfomoniletiedjeiDSM6799] structuraltoxin(ChromobacteriumviolaceumATCC12472) VCBSrepeat-containingprotein(Pseudomonassp.GM21] hypotheticalproteinSden0384[ShewanelladenitrificansOS217] hypotheticalprotein,partial(Achromobacterpicchaudii] |largeexoprotein(Desulfovibriosp.3_1syn3] HaA21Rhodopseudomonaspalustrislrypothetical1638[proteinRPB| iron-regulatedproteinFroC[Vibriorotiferianus] iron-regulatedproteinFrpC[VibriofischeriMJ11] hypotheticalprotein[Vibrionatriegens] hypotheticalprotein[Vibriosplendidus) VCBSprotein[Bilophilawadsworthia) RTXtoxin(ShewanellaviolaceaDSS12] VCBSDechloromonasaromaticaRCB) VCBSrepeatprotein(Vibriosplendidus] MadA[Mesorhizobiumsp.R88B] 112181furnissiiNCTCexoprotein(Vibrio|Large iron-regulatedproteinFrpC[Vibriotubiashii] iron-regulatedproteinFrpc[Vibrioshilonii] RTXtoxin[Aliivibriofischeril 79661 U . S . Patent Sep . 5 , 2017 Sheet 14 of 35 US 9 ,752 , 129 B2

AccessionNo. WP017021259.1 WP017011038.1 WP009301783.1 WP017109466.1 WP018926333.1 WP006366861.1 WP017099027.1 WP020195639.1 WP009845528.1 WP017017027.1 WP017003860.1 WP017102899.1 WP009869454.1 WP007606142.1 WP004749162.1 WP017243041.) WP017015203.1 WP004744158.1 WP017029498.1 YP002947797.1 YP374597.1 WP017019291.1 |WP004400669.1 YP001092641.) YP002313811.1 YP002395108.1 YP421357.1 YP421362.1 |WP017053340.1 YP008104634.1 |WP009868025.1 |YP005025033.1

Ident 38% 32% 32% 32% 34% 34% 35% 35% 33% 39% 40% 36% 35% 31% 34% 91% 32% 33% 35% 36% 52% 33% 29% 40% 32% 29% 57% 33% 32% 31% 31% |83%

E

0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0, 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0, value |0. |0.

Query cover 79% 92% 82% 86% 85% 85% 89% 87% 84% 88% 88% 91% 87% 69% 83% 87% 78% 83% 88% 88% 70% 85% 62% 90% 84% 84% 90% 85% 85% 86% 87% 89% Total score 5136 6776 3584 4569 5168 77506 7019 5072 2556 6644 5554 7524 7602 5374 4627 16303 4790 2841 4934 5353 3950 24960 3809 1746 5887 6239 5398 9349 2259 5392 4513 8014

Max score 888 895 905 885 861 865 838 838 808 796 789 790 748 768 754 665 737 707 707 694 709 635 689 682 690 631| 668 667 667 653

FIG.4B

cellsurfaceglycoproteinprecursor[MagnctospirillummagneticumAMB-1] type1secretiontargetdomain-containngprotein,partial[Rhizobiumsp.PDO10761673 Description hypotheticalproteinPlut_0676[ChlorobiumluteolumDSM273] hypotheticalprotein,partial[Desulfovibriosp.6_146AFAA) outermembraneadhesin-likeprotein(ShewanellaloihicaPV41 |hypotheticalproteinPCA1042970PseudomonasresinovoransNBRC1065531703 outermembraneadhesinlikeproteiin(VariovoraxparadoxusS110) hypotheticalproteinVS_110512[VibriosplendidusLGP32] COG5295:Autotransporteradhesin(Magnetospirillummagnetotacticum] hypotheticalprotein,partial(Vibriogenoniosp.F6] hypotheticalprotein,partial(Aliivibriologei] calviensisEnterovibrio]hypothetical,partial[protein hypotheticalprotein,partial[Pseudomonasumsongensis) hypotheticalprotein,partial(Vibriotasmaniensis] hypotheticalprotein,partial[Enterovibriocalviensis) tasmaniensis]proteinpartialVibriohypothetical,( magneticumAMB-11Magnetospirillumflagellinlikeprotein[| hypotheticalprotein,partial[Vibriobreoganii] hypotheticalprotein,partial[Magnetospirillummagnetotacticum] hypotheticalprotein,partial[Enterovibriocalviensis] fischeriAliivibrio)proteinhypothetical,partial(| |iron-regulatedproteinFrpC,partial(Vibrionigripulchritudo) hypotheticalprotein(Vibriotasmaniensis VCBSrepeatprotein(ShewanellapiezotoleransWP31 hypotheticalprotein[Vibrioowensii] hypotheticalprotein,partial[Enterovibrionorvegicus tubiashii}partial(VibrioregulatedproteinFrpC,|iron- hypotheticalprotein(Vibriotubiashii] VCBS(Chlorobiumferrooxidans] |toxin(Vibriosp.MED222] EJY31Vibriosp.RTXtoxin( Vibriobreoganülproteinhypothetical(| U . S . Patent Sep . 5 , 2017 Sheet 15 of 35 US 9 ,752 , 129 B2

AccessionNo. WP006072371.1 WP017092000.1 WP017081492.1 WP008216195.1 WP008341635.1 WP018907567.1 WP009301782.1 WP005594382.1 WP006228189,1 WP017031543.1 WP009868027.1 WP007606711.1 WP008224673.1 WP006228197.1 WP006387942.1 |WP008340241.1 NP800463.1 YP004680854,1 |YP001758872.1 YP007065062.1 YP269146.1 YP004120666.1 |WP019825879.1 YP006019007.1 YP007551444.1 YP001092444.1 |YP001758871.1 WP(20398403.1 YP293166.1 |YP0035585941 |YP470132.1

44% 42% 43% 45% 37% 38% 45%

58%3e-18039 2e-150 2e-147 1e-146 9e-145 le-144 4e-143 3e-141 2e-17128%903630 87%23865e-157|32 |value 485887%0.29 682290%0.36 79%0.354064 500288%3e-17931 5e-17933%113043 474076%le-17841 529089%7e-17734 88%le-172314587 82202e-16941%83% 412788%le-16832 89%2954382e-166 290569%3e-16434 478183%20-16129 299286%1e-15839 le-15745187%4718 800383%2e-15444 73%40-15329789 214869%4e-15331 75%|le-150312178 254550%2e-15049 TotalQueryEIdent 36%-1715281le90| sover 81% 87% 61% 85% 87% 90% 87% 2948 14756 7321 2060 4610 5109 5154 6194 Max score 643 624 632 630 631 632 625 609 613592 609 563 586 592 585 575 544 531 559 520 550 548 540 531 530 500 524 494 503 508 474

FIG.4C

Description RTXtoxinexportedproteinRtxA[CupriavidusnecatorN-1] outermembraneadhesin-likeprotein(ShewanellawoodyiATCC519081 outermembraneadhesin-likeproteinCalothrixsp.PCC7507] hypotheticalprotein[VibrionalesbacteriumSWAT-3] DesulfovibrioaespoeensisAspo-21membranelikeprotein(adhesin|outer hypotheticalproteinAZKH_2157(Azoarcussp.KH32C) outermembraneadhesin-likeprotein(ShewanellaloihicaPV41 outermembraneadhesin-likeprotein(ShewanellawoodyiATCC51908] COG2931:RTXtoxinsandrelatedCa2+-bindingproteins[Magnetospirillum TypeVsecretorypathway,partial[VibrionalesbacteriumSWAT-31 biofiln-associatedsurfaceproteinVibrioparahaemolyticusRIMD22106331 outermembraneadhesin-likeprotein(Sulfurimonasgotlandica] VBCSrepeat-containingprotein(Colwelliapsychrerythraea34H] 46AFAALDesulfovibriosp.61hypothetical(protein VBCSrepeat-containingprotein[Vibrioscophthalmi] ShewanellaBA1757balticamembrane-likeprotein(adhesinouter VCBSrepeat-containingproteinBradyrhizobiumsp.WSM4717 JMP1341RalstoniaeutrophalvpotheticalReutC5971{protein hypotheticalprotein,partial[Sulfurimonasgotlandica] hypotheticalprotein,partial(Vibriosplendidus) paradoxusVariovorax)hypothetical,partial(protein| Largeexoprotein,partial(Achromobacterpiechaudii] hypotheticalprotein,partial[Vibriobreoganii] hypotheticalprotein[Kordiimonasgwangyangensis) RhizobiumCFN421etlihemolysin-typeprotein[toxin|RTX Vibriosplendidus]proteinhypothetical( RTXtoxin(ShowanellaviolaceaDSS12) piechaudiiAchromobacterexoproteinpartial(,Large hypotheticalprotein(Achromobacterxylosoxidans VCBSrepcatprotein[Vibrioshilonii toxin,partialVibriosplendidus] magnetotacticum U . S . Patent Sep . 5 , 2017 Sheet 16 of 35 US 9 ,752 , 129 B2

AccessionNo. WP017029526.1 WP_006366860.1 WP017077555.1 WP006962234.1 WP008223511.1 770354.1NP

Ident 38% |40% 3e-140|28% 1e-13938% 4e-13729% le-13651% 8e-13338% E value8e-141 1338e-

82% 85% 85% 88% 41%41% TotalQuery scorecover53% 2074 2630 5025 3596 7873 2856 Max score 498 506 482 496 458 482

FIG.4D

Description VCBS[Chlorobiumferrooxidans]>gb:EAT58497.1 TypeVsecretorypathway,partial(VibrionalesbacteriumSWAT-3] hypotheticalproteinbll3714[BradyrhizobiumdiazoefficiensUSDA110] hypotheticalprotein,partial(Vibriosplendidus) hypotheticalprotein,partial(Vibriobreoganii] ferrooxidansDSM13031] hypotheticalprotein(Vibriocoralliilyticus U . S . Patent Sep . 5 , 2017 Sheet 17 of 35 US 9 ,752 , 129 B2

AccessionNo. WP006218690.1 WP004731498.1 WP008684592.1 WP016361080.1 WP018058402.1 WP007942972.1 WP020334262.1 YP420638.1 YP420631.1 YP421364.1 YP006446912,1 421356.1YP YP001770167.1 NP899981.1 |YP005048901.1 NP719678.1 |WP004747418.1 YP003558595.1 YP008030908.1 YP485257.1 YP561402.1 YP002156318.1 |WP005419658.1 WP010452358.1 |WP017082048.1 ABM65821.1 YP002947797.1 YP_286399.1 |YP_857965.1 |YP204889.1

100% 100% ldent 94% 96% 45% 43% 39% 38% 38% 35% 34% 40% 47% 34% 35% 35% 35% 44% 41% 36% 33% 32% 36% 33% 33% 34% 34% 39% 38% 32%

0. .0 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0.) 0. 0. 0. 0. 0. 0. E value

100% Query 92% 87% 83% 89% 84% 87% 80% 88% 81% 82% 85% 87% 87% 85% 80% 85% 88% 82% 82% 84% 86% 89% 83% 88% 82% 86% 80% 84% cover |89% Total score 6895 31712 9344 6831 3568 4700 11419 3611 3871 4321 5537 3229 4348 9652 7756 3731 5469 3734 23693119 1986 6052 24472447 2915 53402883 5373 2463 4564 2536

Max score 6895 5612 1810 1673 1451 1398 |1347 1293 1199 |1168 1137 1118 1111 1092 1069 1041 1013 991 976 981 974 978 978 966 885 896

FIG.5A

hypotheticalproteinAHA_3491[Aeromonashydrophilasubsp.ATCC1164 TypeVsecretorypathway,adhesinAidA[MagnetospisillummagneticumAMB-1]2065 0112889251:VCBSaromaticaref]WP.DechloromonasRCB1>VCBS cellsurfaceglycoproteinprecursor(MagnetospirillummagneticumAMB-1] 1016TISSsecretedagglutinin(RTXAchromobacterxylosoxidansNH44784-1996) RTXrepeat-containingcalciumbindingcytotoxinRtxAl[VibriofischeriES114)1004 hypotheticalprotein,partial(OxalobacteraceaebacteriumJGI0001004-K23] Description TypeVsecretorypathway(MagnetospirillummagneticumAMB-1] largeexoprotein(MagnetospirilluminagneticumAMB-1] Methylobacteriumsp.4-461membranelikeprotein(adhesinouter secretedVCBSdomainprotein[ShewanellaoneidensisMR-1] VCBSrepeat-containingprotein[DesulfomoniletiedjeiDSM6799] VCBSprotein[Bilophilawadsworthia]>ybEPC05682.11 hypotheticalproteinSden0384(ShewanelladenitrificansOS217] |VCBSrepeal-containingprotein(Pseudomonassp.GM21] outermembraneadhesinlikeproteiin(VariovoraxparadoxusS1101 hypotheticalprotein,partial(Achromobacterpiechaudii] structuraltoxin(ChromobacteriumviolaceumATCC124721 largeexoprotein[Desulfovibriosp.3_1syn3] palustrisHaA21Rhodopseudomonashypothetical1638(proteinRPB| VCBSrepeatprotein(Vibriosplendidus] |Largeexoprotein(VibriofurnissiiNCTC11218] tubiashii]protein[VibrioregulatedFrpCiron- iron-regulatedproteinFrpC[VibriofischeriMJ11] iron-regulatedproteinFrpC[Vibriorotiferianus] hypotheticalprotein(Vibriosplendidus] RTXtoxin(ShewanellaviolaceaDSS121 MadA(Mesorhizobiumsp.R88B] |hypotheticalprotein[Vibrionatriegens [Dechloromonasaromatical RTXtoxinAliivibriofischeri? 7966] U . S . Patent Sep . 5 , 2017 Sheet 18 of 35 US 9, 752 ,129 B2

AccessionNo. WP006070242.1 WP017019291.1 WP004400669.1 WP017011038.1 WP017021259.1 WP009301783.1 0206213111.WP WP009869454.1 WP017109466.1 WP018926333.1 YP001092641.1 WP006366861.1 WP009868027.1 YP002395108.1 WP017099027.1 WP017102899.1 WP020195639.1 WP009845528.1 WP017003860.1 YP008104634.1 YP004680854.1 WP007606142.1 WP017053340.1 WP017015203.1 WP009868025.1 YP374597.1 002313811.1YP YP421357.1 WP0170294981 YP421362,1 WP_017243041.1 |

31% 32% 34% 31% 35% 50% 35% 33% 34% 40% 38% %55 33% 35% 36% 31% 94% 35% 31% 36% 29% %83 40% 29% Ident |34% |32% |36% 33 |3276

E

0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. value 0. |0. 0. 0. 0.

Query 87% 86% 84% 89% 82% 83% 87% 81% 80% 75% 85% 81% 89% 60% 82% 84% 85% 80% 82% 83% 81% 83% 90% 83% 79% 73% 81% 77% 80% 85% cover %80| Total score 2962 2558 49034903 3923 2391 7361 2870 2549 4468 2758 3074 3547 5024 5217 3446 3064 5366 4232 2886 10927 2929 4623 877 2660 3466 15101 4527 6195 6495 8403 3666 Max score 893 879 862 847 860 835 855 847 835 800 810 783 784 783 746 762 744 734 754 669735 710 728710 717 636 672 681 671 634 686

FIG.5B

Description cellsurfaceglycoproteinprecursor(MagnetospirillummagneticumAMB-1] hypotheticalproteinPCA1042970[PscudomonasresinovoransNBRC106553 |type1secretiontargetdomain-containngprotein,partial[Rhizobiumsp.PDO10761 hypotheticalprotein,partial[Desulfovibriosp.6146AFAAL COG5295:Autotransporteradhesin[Magnetospirillummagnetotacticum) hypotheticalproteinPlut0676[ChlorobiumluteolumDSM2731 outermembraneadhesin-likeprotein(ShewanellaloihicaPV47 COG2931:RTXtoxinsandrelatedCa2+-bindingproteins(Magnetospirillum RTXtoxinexportedproteinRtxA[CupriavidusnecatorN-1] flagellin-likeprotein[MagnetospirillummagneticumAMB1] iron-regulatedproteinFrpC,partial(Vibrionigripulchritudo] hypotheticalprotein,partial[Enterovibriocalviensis] 35MFCvilj]Pseudomonassp.hypotheticalpartial(protein,| hypotheticalprotein,partial[Pseudomonasumsongensis] LGP321splendidus>hypotheticalVS_110512[Vibrioprotein| hypotheticalprotein,partial[Enterovibrionorvegicus] |hypotheticalprotein,partial(Vibriogenomosp.F6] fischeriAliivibrio]proteinpartialhypothetical,(| hypotheticalprotein,partial(Aliivibriologci] VCBSrepeatprotein(ShewanellapiezotoleransWP3] hypotheticalprotein,partial[Vibriotasmaniensis] hypotheticalprotein,partial(Vibriotasmaniensis] |hypotheticalprotein,partial[Enterovibriocalviensis] hypotheticalprotein,partial[Magnetospirillummagnetotacticum hypotheticalprotein,partial[Vibriobreoganii] iron-regulatedproteinFrpc(Vibrioshilonii} tasmaniensis)proteinVibriohypothetical| VCBSChlorobiumferrooxidans] hypotheticalprotein(Vibrioowensii] hypotheticalprotein(Vibriobreoganii] magnetotacticum toxin(Vibriosp.MED222] U . S . Patent Sep . 5 , 2017 Sheet 19 of 35 US 9, 752 ,129 B2

AccessionNo. WP004749162.1 WP004744158.1 WP017017027.1 YP001758872.1 YP007065062.1 WP017092000.1 WP006072371.1 WP008216195.1 017081492,1WP YP004120666.1 WP009301782,1 WP008341635.1 WP005594382.1 WP019825879.1 YP006019007.1 WP006228189.1 YP003558594.1 YP007551444.1 YP001092444.1 WP008224673.1 0203984031.WP WP_006228197.1 |YP005025033.1 NP800463.1 |WP008340241.1 |YP269146.1 |WP018907567.1 |WP017031543.1 001758871.)YP| |WP007606711.1 470132.1YP YP293166,1

30% Ident 32% 33% |32% 2e-17935% 3e-17731% 31%1742e- 2e-17128% 7e-166|33% 39%1652e- 9e-16329% le-16029% 7e-16039% Se-15644% 1e-15531% 6e-15130% 60-15030% 1c-14944% le-14945% le-14844% le-14545% 6e-14138% 0.33% |3e-18042% |3e-17528% |12-17440% |le-17334% 8e-16940% 6e-16734% |2e-15745% |le-15033% 38%|2e-144 E value 0. 0. 0. 0.

Query 79% 84% 83% 78% 62% 84% 83% 83% 84% 52% 80% 80% 82% 77% 86% 78% 53% 80% %85 80% 81% 76% 81% 90% 89% 72% 85% 77% 79% 68% 85% cover |77%

Total score 3859 3458 3112 1195 1694 4317 2237 4290 3907 2983 3055 2563 |2638 3541 2175 1697 |1212 3121 3250 1960 |4054 6044 2108 2704 |2180 674 1903 4894 1823 3024 |2327 1866

Max score 668 674 661 639 648 628 626 623 620 601 617 598 607 560 592 576 572 578 571 546 530 523 555 540 537 537 527 505 529 495 |500 501

FIG.5C

Description outermembraneadhesin-likeprotein[DesulfovibrioaespoeensisAspo21 outermembraneadhesin-likeprotein(ShewanellawoodyiATCC51908] outermembraneadhesin-likeprotein(Calothrixsp.PCC7507] biofilm-associatedsurfaceprotein(VibrioparahaemolyticusRIMD2210633] hypotheticalproteinAZKH2157(Azoarcussp.KH32C) outermembraneadhesin-likeprotein[ShewanellaloihicaPV41 |TypeVsecrctorypathway,partial[VibrionalesbacteriumSWAT-3] outermenibraneadhesin-likeprotein(ShewanellawoodyiATCC519081 hypotheticalprotein[VibrionalesbacteriumSWAT-3] VBCSrepeat-containingprotein(Colwelliapsychrerythraca34H] hypotheticalprotein(Desulfovibriosp.6_146AFAA outermembraneadhesin-likeprotein(ShewanellabalticaBA175] |VCBSrepcat-containingprotein[Bradyrhizobiumsp.WSM471] RTXtoxinhemolysin-typeprotein(RhizobiumetliCFN421 hypotheticalproteinReutC5971(RalstoniaeutrophaJMP134) tubiashii]Vibriopartial(FrpCprotein,ironregulated- calviensisEnterovibrio)hypothetical,partial[protein hypotheticalprotein,partial[Sulfurimonasgotlandica] hypotheticalprotein,partial(Vibriosplendidus) hypotheticalprotein,partial[Variovoraxparadoxus] outermembraneadhesin-likeprotein(Sulfurimonasgotlandical VBCSrepeat-containingprotein(Vibrioscophthalmi] hypotheticalprotein(Vibriotubiashii] hypotheticalprotein(Vibriosplendidus] Largeexoprotein,partial(Achromobacterpiechaudii breoganjilVibrioproteinpartial(hypothetical, hypotheticalprotein(Kordiimonasgwangyangensis) Largeexoprotein,partial(Achromobacterpiechaudii] RTXtoxin(Vibriosp.EJY3] toxin,partial[Vibriosplendidus] |VCBSrepeatprotein[Vibrioshilonii] DSS121violaceaShewanellatoxin(RTX U . S . Patent Sep . 5 , 2017 Sheet 20 of 35 US 9 ,752 , 129 B2

AccessionNo. WP006387942.1 WP008223511.1 WP017101375.1 WP006366860.1 WP017029526.1 WP017077555.1 WP006962234.1

|

EIdent value 6e-14144% 1e-13951% 4e-13940% 4e-13939%6e-138|38% le-13528% 2e-13529%

345183% 457283% 537479% 215871% TotalQuery scorecover |287983% 1870 3784 Max score 472 466 475 491 476 490 489

FIG.5D

Description TypeVsecretorypathway,partial[VibrionalesbacteriumSWAT-3] hypotheticalprotein[Achromobacterxylosoxidans] hypotheticalprotein,partial(Vibriotasmaniensis] hypotheticalprotein,partial(Vibriobreoganii] hypotheticalprotein,partial(Vibriosplendidus] hypotheticalprotein(Vibriocoralliilyticus] VCBS(Chlorobiumferrooxidans U . S . Patent Sep . 5 , 2017 Sheet 21 of 35 US 9 , 752 ,129 B2

AccessionNo. WP_009869271.1 YP001830991.1 WP019646374.1 WP009758714.1 WP008614612,1 WP008888391.1 WP017719322.1 WP007254292.1 WP003615611,1 YP004556664.1 WP009564177.1 YP007076420.1 YP007076007,1 YP005723848.1 WP010180036.1 422662.1YP WP_008615895.1 YP_423419,1 WP_009869323.1 CAM74103.1 CAM76248.1 CAM75368.1 YP_004866759.1 |YP_007642128.1 NP436619.1

57% 63% 80% 38% 66% 34% 47% 53% 36% 32% 43% 31% 31% 32% 30% 31% 29% 30% 29% 31% 29% 32% Ident 100%| |31% |31%

E 3e-126 8e-117 le-100 7e-59 5e-45 27le- 20-26 9e-26 4e-25 3e-22 46-21 5e-21 9e-21 2c-19 5e-19 le-18 3e-18 7e-18 8e-18 le-17 le-17 le-17 le-17 2e-17 value |0.

100% 16% 19% 36% 30% 34% 32% 34% Query 55% 32% 55% 9% 29% 11% 21% 42% 59% 31% 37% 54% 34% 31% 33% 40% cover |37%

Total 407 332 170 124 123 152 99.0 368 190 543 1034 score 2448 |754 408 2 402 224 504 415 1342 291 278Házi 434 541 316 501 Max score 2448 437 332 170 131 124 123 114 110 110 109 103102 102 99.0 99.4 99.8 98.6 97.4 98.6

FIG.6A

regulatoryPdomainofsubtilisin-likeproproteinconvertases[Nostocsp.PCC7524]990 |98.2probababletype1secretiontargetrepeatprotein[SinorhizobiummelilotiSMill RTXtoxinsandrelatedCa2+-bindingprotein[MagnetospirillummagneticumAMB407 proteinconservedinbacteria[MagnetospirillumgrynhiswaldenseMSR-1] Description RTXtoxinsandrelatedCa2+-bindingprotein[MagnetospirillummagneticumAMB RTXtoxin[Magnetospirillumsp.SO-]>gb|EME706921 COG2931:RTXtoxinsandrelatedCa2+-bindingproteins(Magnetospirillum232 proteinconservedinbacteria[MagnetospirillumgryphiswaldenseMSR-11 proproteinconvertaseP(Beijerinckiaindicasubsp.ATCC90391 hemolysin-typecalciumbindingrepeatfamilyprotein[Micavibrioaeruginosavorus104 calciumbindinghemolysinprotein,putative[Oceanicolagranulosus) COG4254:Uncharacterizedproteinconservedinbacteria[Magnetospirillum RTXtoxinsandrelatedCa2+-bindingproteins[Magnetospirillumgryphiswaldense124 hypotheticalprotein(Magnetospirillumsp.SO-11 calciumbindinghemolysinprotein[Thalassospiraprofundimaris] Hemolysin-typecalciumbindingregion(MicavibrioaeruginosavorusEPB] Hemolysin-typecalciumbindingregion(Methylosinustrichosporium] hemolysin-typecalciumbindingprotein[SinorhizobiummelilotiAK83] putativecalcium-bindingprotein[Nostocsp.PCC7524] Hemolysin-typecalciumbindingregion(Glaciecolasp.HTCC2999] hypotheticalprotein[Rhodobactersp.AKP1] hypotheticalproteinNovispirillumitersonii] rhizobiocinRZCA(Ahrensiasp.R2A130] hypotheticalprotein(Oscillatoriasp.PCC108021 hemolysin-adenlyatecyclase(Sinorhizobiummeliloti10211 [Magnetospirillumsp.SO-1} magnetotacticum] magnetotacticum) MSR-11 ARL-131 1 11 U . S . Patent Sep . 5 , 2017 Sheet 22 of 35 US 9, 752 ,129 B2

AccessionNo. WP017718625.1 YP002520640.1 WP019867288.1 WP018095958.1 YP001312798.1 YP001045054.1 WP002724414.1 WP017140389.1 WP008758430.1 YP003886691.1 WP017265529.1 YP002518066.1 YP007195216.1 WP017274029.1 WP019906466,1 YP003450312.1 WP018207839,1 WP007088891.1 YP002495897.1 YP005716684.1 YP004975543.1 YP551701.1 YP_006370736.1 WP009490921.1 914368.1YP| |WP020582427.1 |WP003532128.1 |WP009467822,1 4214111.NP EQB15964.1 |WP018009580.1

31% 32% 28% 29% 34% 29% 29% 30% 28% 29% 30% 31% 31% 31% 28%31% 29% 32% 30% 28% 36% 30% 32% 28% 29% Ident |37% |31% |31% |28% |34% |31%

E 2e-17 3e-17 40-17 5e-17 9e-17 2e-16 2e-16 3c-16 5e-16 7e-16 9e-16 3e-15 3e-15 3e-15 5-3e 4c-15 64-15 82-15 8e-15 1e-14 2e-14 3e-14 4e-14 4e-}4 5c-14 6e-14 60-14 le-13 le-13 2e-13 value |9e-16

Query 29% 34% 30% 35% 32% 30% 30% 30% 32% 34% 30% 45% 34% 37% 32% 31% 31% 35% 30% 32% 31% 29% 32% 37% 29% 33% 30% 32% 34% cover %37|

94.4 89.4 86.3 Total score 246 591 143 1326 407 151 152 408 |549 244 |1162 596 320 257 417 658 333 894 377 822 210 |326 812 531 160 421 316

97.8 97.4 97.1 96.7 96.3 94.7 94.7 94.4 94.0 92.8 92.8 92.0 91.3 90.9 90.1 90.1 89.7 89.4 88.6 89.4 87.8 88.2 86.7 87.0 87.4 86.7 86.3 84.7 85.5 85.1 Max score |90.5

FIG.6B

Description Hemolysin-typecalciumbindingregion,RTX(RhodobactersphaeroidesKD131] Hemolysin-typecalciumbindingrcpcat(2copies)[SinorhizobiummelilotiGR4] hypotheticalproteinRLDS_09615[SphingobiumlactosutensD$20] outermembraneadhesin-likeprotein[RhodobactersphaeroidesATCC17029] hemolysin-typecalciumbindingregion(RhodobacteraceaebacteriumKLHI) hemolysin-typecalciumbindingregion[Roseibiumsp.TrichSKD4] Azospirillumsp.B510]hemolysinprotein[bindingcalcium hemolysin-typecalciumbindingprotein,partial[Thalassospiraxiamenensis) hemolysin-typecalciumbindingregion[Polaromonassp.JS666] rhizobiocin/RTXtoxinandhemolysin-typecalciumbindingprotein[Tistrella hemolysin-typecalciumbindingprotein[ParacoccusdenitrificansPD1222] Hemolysin-typecalciumbindingprotein(SinorhizobiummedicaeWSM4191 proproteinconvertaseP(Cyanothecesp.PCC7822] proproteinconvertaseP[MethylobacteriumnodulansORS20601 hemolysin-typecalciunbindingprotein[SinorhizobiummelilotiBL225C] hypotheticalproteinAZOLI_p40606[Azospirillumlipoferum4B1 hypotheticalprotein[Methylovulummiyakonense] hypotheticalprotein,partial(Endozoicomonaselysicola] iron-regulatedproteinFrpCaulobactercrescentusNA1000] hypotheticalprotein[Methylobacteriumsp.77] hypotheticalprotein(Oscillatoriasp.PCC10802] Ca2+-bindingprotein,RTXtoxin[Microvirgasp.WSM3557) hemolysinD[Sinorhizobiummeliloti] hemolysinD[Sinorhizobiummeliloti] hemolysinD(Sinorhizobiummeliloti] calcium-bindingprotein(CaulobactercrescentusCB151 hemolysinD,partial(Sinorhizobiummeliloti] hemolysinD[Sinorhizobiummedicae] hemolysinD(Sinorhizobiuinmedicac] adhesin(Rhodobactersphacroides) adhesin[Rhodobactersphaeroides) mobilisKA081020-065) U . S . Patent Sep . 5 , 2017 Sheet 23 of 35 US 9 , 752 ,129 B2

AccessionNo. WP004210915.1 WP008622436.1 WP019903186.1 WP006519062.1 WP004207719.1 WP017263229.1 YP005030312,1 YP002992972.1 WP018716239.1 YP002310757,1 WP008029683.1 WP005332196.1 EPX86685.1 AAR38409,1 EKD61936.1 WP_019867241.1 WP_020655477.1 WP_019867066.1 EPX78170,1 YP004680854.1 WP020480612.1 YP006370739.1 |WP008030394.1 |YP003693926.1 WP017717249.1 |YP681508.1 |YP007643803.1 |YP_355041.1 |YP006370737.1

|

Ident 28% 29% 29% 29% 30% 28% 33% 31% 31%37% 32% 33% 30% 30% 30% 29% 28% 32% 37% 37% 27% 34% 30% 29% 33% %31 %29 30% 31%

E 40-13 le-12 le-12 1e-122e-12 le-11 le-11 le-11 3e-11 5e11- 6e-11 6e-11 value 3e-13 4e-13 4e-13 7e-13 70-13 Se-12 58-12 |7e-12 8e-12 12-11 2e-11 2e-11 4e-11 4c-11 4e-11 5e-11 115e-

36% 33% 30% 31% 37% 29% 31% 29% 15% 29% 36% 31% |42% |30% 33% 30% |33% |34% 32232% 37%191 35%277 15034% 506|34% 33%406 30%239 25033% TotalQuery score|cover 83,2|28% 84.031% 81.615%

78.6 420 204 |120 172 314 175 142 132 126 139 143 261 |307 238 251 260

Max score 84,0 84.0 83.2 82.4 82.8 82,4 81,6 81.6 79.3 79.0 79.0 78.6 78.2 78.6 77.4 77.8 77.4 77.0 77.4 76.6 77.0 76.6 76.3

FIG.6C

typeIsecretiontargetrepeat-containingprotein(RoseobacterdenitrificansOch114183.2 84.0hemolysin-typecalciumbindingregion[DesulfovibriosalexigensDSM2638] .580typeIsecretiontargetGGXGXDXXXrepeatprotein(unculturedmarinebacterium hemolysin-typecalciumbindingrepeatfamilyprotein,partial(unculturedbacterium]79.3 RTXtoxins/relatedCa2+-bindingprotein[RubellimicrobiumthermophilumDSM79.7 hemolysin-typecalciumbindingregion,RTXpartial[Rhodobactersphaeroides78.6 hemolysin-typecalciumbindingrepeatprotein(Leptolyngbyasp.PCC7375) Description Hemolysin-typecalciumbindingregion[ShewanellapiezotoleransWP3] hypotheticalprotein,partial[Magnetospirillumsp.SO-1] calciumbindinghemolysinprotein(TistrellamobilisKA081020-0651 RTXtoxinexportedproteinRtxa(CupriavidusnecatorN-1] hemolysin-typecalciumbindingprotein(StarkeyanovellaDSM506) proteinofunknownfunction(AzospirillumbrasilenseSp245] Hemolysin-typecalciumbindingregion[Rhodobactersp.SW21 hemolysin-typecalciumbindingregion[Aeromonasmedia] hypotheticalprotein(Methylobacteriumsp.771 Alkalinephosphatase(ThalassobacterarenaeDSM195931 hypothcticalprotein(Oscillatoriasp.PCC10802] proproteinconvertaseP[TistrellamobilisKA081020-0651 RhodobacterSW21sp.Hemolysinbindingregion(-typecalcium| hypotheticalprotein(Sphingobiumyanoikuyael hypotheticalprotein(Brachymonaschironomi AlkalinephosphataseMicavibrioaeruginosavorusEPB1 hypotheticalprotein(Methylovulummiyakonense) hypotheticalprotein(Massilianiasiensis] hypotheticalprotein(Methylovulummiyakonense] hypotheticalproteinSphingobiumyanoikuyae) |hypotheticalprotein(Oscillatoriasp.PCC10802] hemolysinD[Sinorhizobiummeliloti

166841 582] 2.411 U . S . Patent Sep . 5 , 2017 Sheet 24 of 35 US 9 ,752 , 129 B2

AccessionNo. YP004110428.1 YP006370049.1 WP017023581.1 YP004295065.1 YP007512736.1 YP002548098.1 EPY02797.1 YP003158921.1 WP017677435.1 WP009385092.1 WP020583217.1 WP017721018.1 YP003158384.1 WP019903589.1 YP005605535.1

EIdent value 6e-1129% 8e-1130% 8e-1132% 8e-1128% le-10|28% 1e-1029% 29%le-10 10-30% 30%2e-10 30%2e-10 2e-1030% 4e-1028% le-0927% le-0928% 2e-0932%

|28% |37% 12032% 302 |33% 34%306 36333% 26128% 29%249 35%283 36%734 29237% |TotalQuery scorecover 32%|230 |34339% 71.229% |185 242

75.9 76.3 75.5 76.375.9 75.9 75.9 .575 75.1 75.1 973. 72.8 72.4 71.2 Max score |77.0

FIG.6D

Description calciumbindinghemolysinprotein[TistrellamobilisKA081020-065] calciumbindinghemolysinprotein,putative(BradyrhizobiumoligotrophicumS58] hemolysin-typccalciumbindingprotcin(DesulfomicrobiumbaculatumDSM4028] hemolysin-typecalciumbindingprotein(DesulfomicrobiumbaculatumDSM4028] 5'-nucleotidasedomaincontainingprotein[RhodopseudomonaspalustrisDX11 hemolysin-typecalciumbindingregion(Nitrosomonassp.AL212] Hemolysin-typecalciumbindingprotein(AgrobacteriumvitisS4] hemolysin-adenylatecyclase(PhaeospirillumfulvumMGUK5) hypotheticalproteinBJ6T_06520(BradyrhizobiumjaponicumUSDA6 hypotheticalprotein,partial[Endozoicomonaselysicola) hypotheticalprotein(Oscillatoriasp.PCC10802] hypotheticalprotein(Methylobacteriumsp.771 hypotheticalprotein(Aliivibriologei] hypotheticalprotein(Pseudomonasalcaliphila] typeIsecretionprotein[Vibriosp.N418] U . S . Patent Sep . 5 , 2017 Sheet 25 of 35 US 9, 752 ,129 B2

AccessionNo. WP009043135,1 YP422785.1 WP008614612.1 WP_009870351.1 EPY02541.1 CAM76811,1 WP006216951,1 YP006254384.1 WP010602925.1 YP001522191.1 YP001981885.1 YP007656731.1 Yp374310.1 WP018896697.1 YP003881756.1 WP019646374,1 WP006274635,1 YP003125000.1 CAX84233.1 YP003890797.1 WP018230537.1 WP008281368.1 WP007560973.1 WP007329466.1 YP007614514.1 WP008064234.1 YP003453433.1 YP001867978.1 WP007605159.1 0018161581YP|

100% 64% 58% 41% 30% 32% 34% 29% 34% 31% 30% 30% 32% 31% 28% 40% 29% 30% 29% 28% 27% 28% 30% 39% 29% 27% 35%29% Ident |31%

E le-75 3e-26 2e-14 3e-14 8e-14 70-13 le-12 le-0 le-10 9e-10 4e-08 5e-08 7e-08 7e-08 4e-06 le-05 3e-05 6e-04 value 0. 0. 0. 0.005 0.007 0.012 0.099 0.13 0.36 3.2

74% 33% 34% 28% 34% 36% 28% 40% 35% 28% 24% 37% 25% 29% 35% 48% |34% 27% |11% |31% |33% |14% |37% |23% |12% TotalQuery coverscore 1925100% 1211100% 98%1045 |9% 82.8 66.6 50.4 50.1 49,3 46.2 45.8 44.3 41.6 41,2 40.0 272 249 880 806 1252 505 |321 134 1046 450 122 458 107 117 103 140

88.2 87.0 85.9 82.8 82.0 75.5 72.8 67.0 66.6 59.3 53.5 49.3 46.2 44.3 40.0 67.0 60.8 57.8 1 45.8 416 41.2 Max Score 1830 |1082 688 272 66.2 50.

FIG.7

outermembraneautotransporterbarreldonainprotein[Methylobacteriumsp.GXF41504 outermembraneadhesin-likeproteinPseudomonas(denitrificansATCC75.5138671 hypotheticalproteinamb3422(MagnetospirillummagneticumAMB-1] 126HEMAGGLUTININ/HEMOLYSIN-RELATEDPROTEIN[Magnetospirillum outermembraneautotransporter[BurkholderiaambifariaMC40-6] Description hypotheticalprotein[Magnetospirillumsp.SO-1]> hypotheticalproteinK67805578[PhaeospirillumfulvumMGUK5] outermembraneadhesinlikeprotein,partial(Achromobacterpiechaudii] hypotheticalproteinAM1E0108[AcaryochlorismarinaMBIC11017) hypotheticalproteinPlut0379(ChlorobiumluteolumDSM273] hemagglutininhemolysin-likeprotein[Dickeyadadantii3937] ouiermeinbrancadhesin-likeprotein(ChitinophagapincnsisDSM2588] outermembranetransportbarrel(Sphingomonassp.MM-11 hypotheticalproteinAZL101290(Azospirillumsp.B510] hemolysin-typecalciumbindingregion[NostocpunctiformePCC73102] COG2931:RTXtoxinsandrelatedCa2+-bindingproteins[Magnetospirillum VCBSrepeat-containingprotein(SolitaleacanadensisDSM34031 hypotheticalprotein(Rhizobiumsp.2MFC0131] outermembraneadhesin-likeprotein(Cyanothecesp.PCC78221 hypotheticalprotein[Bradyrhizobiumsp.WSM471] hypotheticalprotein,partial(Pedobacteragri) Uedal071japonicusCellvibrioputativehemolysin[ hypotheticalprotein(Novispirillumitersonii] hypotheticalprotein[Methyloversatilisuniversalis] Largeexoprotein[Roseovariussp.TM1035] hypotheticalprotein[Methyloversatilisuniversalis] gryphiswaldenseMSR-1] rhizobiocinRzcAAsticcacaulisbiprosthecium uncharacterizedprotein(unculturedbacterium cadherin(Rhodopirellulabaltica] transporter(Pseudomonaschlororaphis] magnetotacticum) U . S . Patent Sep . 5 , 2017 Sheet 26 of 35 US 9 , 752 ,129 B2

AccessionNo. WP009539978.1 WP003293555.1 WP019813773.1 WP002931922.1 WP002944457.1 WP018882170.1 WP018867039,1 WP018937733.1 WP018987943,1 YP003158388.1 WP019563530.1 YP007553126.1 WP018877499.1 YP_422838.1 WP_009870208.1 WP_008619118.1 EPY00949.1 WP_019643622.1 YP_005416034.1 CAM77380.1 WP_010180038.1 WP_008248572.1 WP_019592978.1

100% Ident 95% 91% 79% 56% 54% 55% %70 39% 39% 35%

40-14342% 6e-108|36% 4e-10737% 5e-10737% 8e-10735% 3e-10635% 4e-10635% 6e-10635% 9e-106|37% 10-10535% le-105|35% le-10538% E 1809c- le-146 7e-146 2e-105 value 0. 0. 0. 0. 0. 0.

100% Query cover 93% 98% 93% 91% 94% 92% 59% 91% 95% 93% 90% 92% 92% 92% 92% 92% 92% 92% 94% 92% 92% 92%

Total score 1220 1090 |1076 896 |611 592 581 525 449 448 439351 | 350 350 345 343 345 346 346 346 345 345 343

Max score 1220 1090 1076 896 611 592 581 525 449 448 439 351 350 |350 345 343 345 346 346 346 345 345 343

FIG.8A

toxinsecretionABCtransporterpermeaseandATP-bindingprotein[Azoarcussp. Description COG2274:ABC-typebacteriocin/lantibioticexporters,containanNterminal ABCtransporterATP-binding/permeaseprotein(Thauerasp.281 ABCtransporterATP-binding/permeaseprotein[Thauerasp.27] ABC-typeprotease/lipasetransportsystem,ATPaseandpermeasecomponents ABC-typeprotease/lipasetransportsystem,ATPaseandpermeasecomponent ABC-typeprotease/lipasetransportsystem,ATPaseandpermeasecomponent ABC-typeprotease/lipasetransportsystem,ATPaseandpermeasecomponents proteincontainingABCtransporterdomain[Magnetospirillumgryphiswaldense ABC-lypeprotease/lipasetransportsystem,ATPaseandpermeasecomponents ABC-typeprotease/lipasetransportsystem,ATPaseandpermeasecomponents typeIsecretionsystemATPasc(DesulfomicrobiumbaculatumDSM4028] MagnetospirillummagneticumAMB-1] ABC-typeprotease/lipasetransporter(Caenispirillumsalinarum] ABC-typeprotcasc/lipasetransportsystem(Pseudomonasstutzeri] ABCtransporterrelatedprotein(OsedaxsymbiontRsl] double-glycinepeptidasedomain[Magnetospirillummagnetotacticum protein(Magnetospirillumsp.SO-1] hypotheticalprotein(Novispirillumitersonii] [RhodospirillumphotometricumDSM122] |hypotheticalproteinThioalkalivibrio(sp,ALgr5] |hypotheticalprotein[Thioalkalivibriosp.ALE27] ABCtransporter[Thioalkalivibriosp.ALE25] ABCtransporter[Thioalkalivibriosp.AKL3] ABCtransporter[Thioalkalivibriosp.ALM92] ABCtransporter[Thioalkalivibriosp.ALE28] [PhaeospirillumfulvumMGU-K5] toluclasticus)Azoarcustransporter[ABC Glaciecolasp.HTCC29991 [Limnobactersp.MED1051 MSR-1 KH32C) U . S . Patent Sep . 5 , 2017 Sheet 27 of 35 US 9 ,752 , 129 B2

AccessionNo. WP020331854.1 WP019594102.1 WP018951739.1 WP020286188.1 WP019628234.1 WP018861453.1 WP018873589.1 WP007022486.1 WP009100771.1 WP018868670.1 WP019626728.1 WP018939823.1 WP019024347.1 WP003069985.1 WP018863861.1 WP003075146.1 WP019590480.1 WP009724196.1 019568356).WP YP_006292562.1 YP003303859.1 006295629.1YP| 0196241851.WP WP004318584.1 WP019592005.1 WP008220329.1 WP017926137.1 YP_005431238.1 004157431.1YP WP018139367.1

Ident 32% 35% 35% 35% 34% 35% 36% 36% 35% 35% 34% 35% 36% 35% 34% 35% 35% 35% 34% 34% 35% 35% 33% 35% 34% 35% 35% 35% 36% 35%

E value 3e-105 5e-105 5e-105 5e-105 6e-105 6e-105 7e-105 9e-105 3e-104 3e-104 4e-104 4e-104 8e-104 le-103 2e-103 2e-103 7e-103 9e-103 9e-103 le-102 2e-102 2c-102 3e-102 5e-102 5e-102 5e-102 -1027e 70-102 -1029e 1e-101

Query cover 91% 92% 92% 92% 93% 92% 92% 92% 91% 93% 91% 92% 92% 92% 93% 91% 92% 92% 92% 93% 92% 92% 91% 92% 93% 91% 92%90% 92% 92%

Total score 344 344 |344 344 343 343 343 343 342 342 342 342 340 341 340 340 338 |338 338 338 337 338 337| |336 337 336 335335 332 335 Max score 344 344 344 344 343 343 343 343 342 342 342 342 340 341 340 340 338 338 338 338 337 338 337 336 337 335335 335 332 335

FIG.8B

ABCtransporter,transmembraneregion:PeptidaseC39bacteriocin Description typeIsecretionsystemATPase(SulfurospirillumdeleyianumDSM6946] ABC-typebacteriocin/lantibioticexporter(Vibriofluvialis] ALMg13-21Thioalkalivibriosp.transporter[ABC symbiont]OsedaxR$2transporterrelatedprotein[|ABC ABCtransporter[Thioalkalivibriosp.HL-Eb18] ABCtransporterATP-bindingproteinMarinobacterhydrocarbonoclasticusATCC ABCtransporter[Thioalkalivibriosp.ALM2T] |ABCtransporter[Thioalkalivibriosp.AKLIO] ABCtransporter[Thioalkalivibriosp.ALJ16] processing[Mcthylophagasp.JAM7]> ABCtransporter[Thioalkalivibriosp.ALJIO] ABCtransporter[Thioalkalivibriosp.ALIT] |ABCtransporter(Thioalkalivibriosp.AKL11] ABCtransporter[Thioalkalivibriosp.ARh5] |ABCtransporter[Thioalkalivibriosp.ALI20] |ABCtransporter[Thioalkalivibriosp.ALE20] ABCtransporter[Thioalkalivibriosp.ALE10] ABCtransporter(VariovoraxparadoxusEPS] |ABCtransporter[Thioalkalivibriosp.ALJ11] ABCtransporter[Thioalkalivibriosp.AKL121 ABCtransporter(Thioalkalivibriosp.AL331 ABCtransporter(Neptuniibactercaesariensis] ATPase(Halomonassp.GFAJ-11 |ABCtransporter[Methylophagasp.JAMI] ABCtransporter(Campylobacterrectus]testosteroni]Comamonastransporter[ ABC |ABCtransporter(Thioalkalivibriosp.ARh31 nanhaiensis)RheinheimerapeptidaseC39| |ABCtransporter[Comamonastestosteroni] |ATPase[Halomonassp.TD01]

49840] U . S . Patent Sep . 5 , 2017 Sheet 28 of 35 US 9, 752 ,129 B2

AccessionNo. WP016915006.1 WP009670226.1 WP019878733.1 YP003460560.1 WP019592545.1 YP003279062.1 WP018233111.1 WP016954151.1 WP018993876.1 YP005552492.1 WP010553238.1 WP007583161.1 WP018136203.1 WP009650523.1 WP003053581.1 WP009294535.1 YP001466532.1 YP437190.1 YP001408654.1 WP019043539.1 WP007838295.1 WP002961326.1 |WP002804835.1 WP005913568.1 |WP007145632.1 WP008164875.1 WP016902305.1 WP007972241.1 WP006449299.1 WP_004332497.1 YP_002946334,1

35% 35% 135%

2e-10134% 4c-10034% 6e-10034% 9e-10033% |35%90le-101 QueryEIdent 92%2e-10135 2e-10135%92 90%4e-10136 6e-10135%92 90%8e-10134 91%le-10034 92%4e-100|33 92%5e-10033 34%93Se-100 91%6e-10034 91%7e-10034 92%le-9933 93%34le-99 992e-37%90 34%912e-99 91%24-9933 2e-9995%35 3e-9995%|35 89%3e-9934 91%4e-99|33 42-99 994e- 95%4e-9934 91%70-9936 8e-99 valuecover| 92%2e-10135%

93% 91% 92% 91% 95% 95% 91%

Total score 335 335 335 335 335 333 333 333 332 331 331 331 330 330 332 330 331 330 331 326 330 329 329 328 329 328 328 328 328 328 325 Max score 335 335 335 335 335 333 333 333 332 331 331 331 330 330 332 330 331 330 331 326 330 329 329 328 329 328 328 328 328 328 325

FIG.8C

Description toxinsecretionATP-bindingprotein(ComamonastestosteroniCNB2] hypotheticalproteinCCC13826_0891(Campylobacterconcisus13826] toxinsecretionATP-bindingprotein(Campylobactercurvus525.92] ABCtransporterpermease/ATP-bindingprotein[Xanthomonasfragariae] toxinsecretionABCtransporterpermeaseandATP-bindingprotein[Thauera typeisecretionsystemATPase[Thioalkalivibriosp.K90mix] |ABCtransporterATP-bindingcomponentArcobacter[sp.L] chcjuensisKCTC23961transporterHahellabacteriocinlantibiotic(/ABC |ABCtransporterpermease/ATP-bindingproteinXanthomonas(axonopodis ABCtransporter(Campylobactersp.FOBRC14] |ABCtransporter(Campylobactersp.10_150] ABCtransporter(Thioalkalivibriosp.ALRh] |ABCtransporter[Thioalkalivibriothiocyanodenitrificans] hypotheticalprotein(Catenovulumagarivorans] ABCtransporter[Thioalkalivibriosp.ALgrl] ABCtransporter(Pseudoalteromonassp.BSi204297 ABCtransporter[Comamonastestosteroni] |ABCtransporter[gammaproteobacteriumIMCC1989) hypotheticalprotein[Succinispiramobilis] aminisulfidivorans]MethylophagapeptidaseC39( ABCtransporter[Achromobacterarsenitoxydans] arcticaPseudoalteromonas]transporter(|ABC ABCtransporter(Campylobactercurvus |ABCtransporterComamonas[testosteroni] CF3337Variovoraxsp.transporter[|ABC ABCtransporter[Pseudoalteromonashaloplanktis peptidaseC39Xanthomonasarboricola peptidaseC39[Xanthomonasfuscans] peptidaseC39[Xanthomonasgardneril ABCtransporter[VariovoraxparadoxusS1101 ATPase(Halomonasstevensii linaloolentis U . S . Patent Sep . 5 , 2017 Sheet 29 of 35 US 9 ,752 , 129 B2

AccessionNo. WP003802991.1 WP005374556.1 WP010089615.1 WP017634537.1 WP005383569.1 YP001791124.1 WP008074445.1 WP010628832.1 YP003799068.1 WP010325122.1 WP018719059.1 WP008297682.1 YP_004852936.1 |WP018906841.1 YP_158002,1 WP_005973599.1

34% 34% 34% 35% 34% 35% 34% 36% 33% 35% 34% 37% 35% 33% Ident |34% |35%

E value 8e-99 9e-99 98-99 1e-98 1e-98 2e-98 2e-98 2e-98 2e-98 2e-98 3e-98 3e-98 3e-98 3e-98 3e-98 3e-98

Query cover 91% 92% 91% 95% 92% 92% 91% 91% 92% 91% 91% 91% 90% 92% 90% 92%

Total score 327 327 328 327 327 327 327 323 326 327 326 326 325 327 327 322 Max score 327 327 327 327 327 323 326 327 326 326 325 327 327 322

FIG.8D

Description |ABCtransporterpermease/ATP-bindingprotein[Xanthomonasaxonopodispv. ABCtransporterATP-bindingpermease(AromatoleumaroniaticumEINI] typeIsecretionsystemATPase(LeptothrixcholodniSP-6] typeIsecretionsystemAtPaseHlyB(CandidatusNitrospiradefluvii] type1secretionsystemATPase,partial[CandidatusNitrosopurilussalarial ABCtransporter[Halomonassp.KM-1] ABCtransporter[Alcaligenesfaecalis ABCtransporter(Vibrioalginolyticus] ABCtransporter[Burkholderiaubonensis] ABCtransporter(Vibriosp.712il] ABCtransporter(Vibrioalginolyticus) ABCtransporter(Variovoraxparadoxus] ABCtransporter(Vibriosinaloensis) |ABCtransporter[Marinobacteriumstanieri] peptidaseC39[Arhodomonasaquaeolei] ABCtransporter[Pectobacteriumwasabiae] citrumeloF11 U . S . Patent Sep . 5 , 2017 Sheet 30 of 35 US 9, 752 ,129 B2

AccessionNo. CAM74531.1 WP009869454.1 WP008622436.1 WP_009869271.1 WP(09868027.1 YP420631.1 |WP008615895.1 CAM77170.1 WP008614612.1 09868207.1WP( 008621158.1WP WP008622382.) WP008617421.1 009865035.1|WP WP_009864918.1 CAM76579,1 |YP_420640.1 |YP422785.1 |YP421364.1 423419.1YP_ YP_420638.1 YP_422662.1

|

40% 45% 39% 36% 44% 44% 29% 30% 36% 52% 33% 35% 33% 32% 47% Ident 46% 37% 42% 47% /40% 45% |38%

E le-05 4e-05 7e-05 12-04 le-04 le-04 6e-04 6e-04 value 0.001 0.003 0.008 0.014 0.019 0.019 0.026 0420. 0.045 0.049 0.051 054|0. 0.064 0.12

Query 19% 15% 19% 32% 46% 13% 15% 42% 23% 16% 11% 10% 13% 22% 14% cover |13% |15% |11% |19% %6 9% |16%

78.6 36.6 70.5 35.4 63.) 35.4 34.3 33.9 Total score 105 105 132 174 192 183 288 |115 107 |196 404 125 324

Max score 46.6 44.7 43.9 43.9 43.5 41.6 40.8 37.7 37.0 36.6 36.2 35.4 35.4 34.3

FIG.9A

|TypeVsecretorypathway,adhesinAidA[MagnetospirillummagneticumAMB-1]41.6 RTXtoxinsandrelatedCa2+-bindingprotein[MagnetospirillummagneticumAMB45.1 RTXtoxinsandrelatedCa2+-bindingprotein(MagnetospirillummagneticumAMB39.7 Hemolysin-typecalçiumbindingregion[MagnetospirillumgryphiswaldenseMSR37.0 RTXtoxinsandrelatedCa2+-bindingprotein[MagnetospirillummagneticunAMB35.4 hemolysin-typecalciumbindingrepeatcontainingprotein,partial[Magnetospirilluin35.4 Autotransporteradhesin(MagnetospirillumgryphiswaldenseMSR-1] hypotheticalproteinamb3422MagnetospirillummagneticumAMB-1] VBCSrepeat-containingprotein,partial(Magnetospirillumsp.SO1] outermembraneadhesin-likeprotein,partial(Magnetospirillumsp.SO1] COG2931:RTXtoxinsandrelatedCa2+-bindingproteins[Magnetospirillum34.3 COG2931:RTXtoxinsandrelatedCa2+-bindingproteins(Magnetospirillum33.9 Autotransporteradhesin(MagnetospirillumgryphiswaldenseMSR-1] Description |hypotheticalprotein,partial[Magnetospirillumsp.S0-11> TypeVsecretorypathway[MagnetospirillummagncticumAMB-1] COG5295:Autotransporteradhesin(Magnetospirillummagnetotacticum) COG2931:RTXtoxinsandrelatedCa2+-bindingproteinsMagnetospirillum( COG2931:RTXtoxinsandrelatedCa2+-bindingproteins(Magnetospirillum magneticumAMB-11Magnetospirillumexoprotein(large SO-1]Magnetospirillumsp.lrypotheticalprotein(| COG2931:RTXtoxinsandrelatedCa2+-bindingproteins(Magnetospirillum RTXtoxin(Magnetospirillumsp.S0-11 magnetotacticum)

sp.S0-1] magnetotacticum magnetotacticum 11 11 magnetotacticum 11 magnetotacticum U . S . Patent Sep . 5 , 2017 Sheet 31 of 35 US 9 ,752 , 129 B2

009865351,1IWP AccessionNo. WP009865796.1 WP008621034.1 WP008622434.1 WP_009865727.1 WP_009870351.1 WP_008619475.1 WP_008622842.1 YP_422579.1 WP_009865379.1 WP_009863686.1 WP_009865975.1 WP_009865835.1 WP_009868232.1 WP_009862535.1 CAM75368.1 008615635.1WP_ WP_009861891.1 CAM74983.1 CAM76862.1

32% 35% 32% 49% 47% 26% 31% 39% 31% 28% 34% 31% 35% 40% 37% 31%37% Ident |47% 45%

E 0.61 value 0.16 0.22 0.36 0.62 0.66 0.70 0.81 0.83 0.90 1.0 1.3 2. 6.16.3

14% 18% 16% 11% 13% 19% Query 13% 11% 7% 7% 22% 24% 18% 21% 8% 7% cover %13| 8% %6

Totalscore 97.4 158 33.1 90.5 31.6 31.2 60.1 60.4 150 57.8 30.8 59.7 30.8 85.9 28.5 57.4 28.1 28.528,1

33.1 31.2 31.2 30.4 29.3 28.1 Max score |32.0

FIG.9B

HollidayjunctionDNAhelicaseRuvB[MagnetospirillummagneticumAMB-1]|31.6 hypotheticalproteinMGR1109(MagnetospirillumgryphiswaldenseMSR-1)28.5 COG4953:Membranecarboxypeptidase/penicillin-bindingproteinPbpC,partial30.8 COG2931:RTXtoxinsandrelatedCa2+-bindingproteins,partialMagnetospirillum(|29.3 28.1periplasmiccontainC-terminaltypically,serineproteasesCOG0265:Trypsinlike COG2931:RTXtoxinsandrelatedCa2+-bindingproteins,partial[Magnetospirillum33.1 COG2931:RTXtoxinsandrelatedCa2+-bindingproteins,partial[Magnetospirillum31.6 COG2931:RTXtoxinsandrelatedCa2+-bindingproteins,partial[Magnetospirillum31.2 31.2COG2931:RTXtoxinsandrelatedCa2+-bindingproteins(Magnetospirillum COG2931:RTXtoxinsandrelatedCa2+-bindingproteins,partial[Magnetospirillum30.4 calciumbindinghemolysinprotein,partial[Magnetospirillumsp.SO-1] COG2931:RTXtoxinsandrelatedCa2+-bindingproteins,partial[Magnetospirillum28.5 Description 33.9COG2931:RTXtoxinsandrelatedCa2+-bindingproteins[Magnetospirillum TypeVsecretorypathway,adhesinAidapartialMagnetospirillumsp.SO-11 HollidayjunctionDNAhelicaseRuvB(Magnetospirillumsp.SO-11 RTXtoxinsandrelatedCa2+-bindingproteins[Magnetospirillumgryphiswaldense30.8 hypotheticalprotein,partial(Magnetospirillumsp.SO-1] COG2931:RTXtoxinsandrelatedCa2+-bindingproteins[Magnetospirillum hypotheticalprotein,partial(Magnetospirillumsp.SO-1] lipoproteinreleasingsystemATP-bindingprotein[Magnetospirillum gryphiswaldenseMSR-1] magnetotacticuni) magnetotacticum) magnetotacticum) magnetotacticum) magnetotacticum) magnetotacticum] [Magnetospirillummagnetotacticum magnetotacticum) magnetotacticum) MSR-11 magnetotacticum U . S . Patent Sep . 5 , 2017 Sheet 32 of 35 US 9, 752 , 129 B2

AccessionNo. |WP_009870006.1 WP_009862419.1 WP_008618626.1

Ident 42% 50% 28%

E

6.9

Query cover 77% % 14% 5

28.1 26. 28.1 Total score 2

Max score 28.1 26.2 28.1

FIG.IC

Description asparaginesynthase[Magnetospirillumsp.S0-1]>gb|EME6933511 COG1680:Beta-lactamaseclassCandotherpenicillinbindingproteins COG2931:RTXtoxinsandrelatedCa2+-bindingproteins(Magnetospirillum PDZdomain,partial[Magnetospirillummagnetotacticum) synthase(Magnetospirillumsp.SO-11 [Magnetospirillummagnetotacticum] magnetotacticum) U . S . Patent Sep . 5 , 2017 Sheet 33 of 35 US 9, 752 ,129 B2

Green Fluorescence in Supernatant

recensioni successive convivencia

...... :

Ex485/20,Em528

ACHE AMO- 1 Nee Cho WWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWW FIG . 10 Supernatantvs. Bacterial cells in Suspension 1400

1200 ......

1000

Ex$85/20,Ers:52828 * Susematent Bacan 90ansion 400 200

...... AC16 AMBINes Ctrl FIG . 11 U . S . Patent Sep . 5 , 2017 Sheet 34 of 35 US 9, 752 ,129 B2

FIG . 12

FIG . 13 U . S . Patent Sep . 5 , 2017 Sheet 35 of 35 US 9 ,752 , 129 B2

FIG , 14 US 9 ,752 , 129 B2 HOST CELL MODIFICATION WITH cells containing longer lasting changes, including heritable ARTIFICIAL ENDOSYMBIONTS changes, typically requires the use of homologous recom bination , which despite the advances in technology , continue CROSS REFERENCE TO RELATED to have low efficiency rates. In addition , introduction of APPLICATIONS 5 multiple , different heterologous nucleic acids is complicated by the higher complexity of eukaryotic cells (e . g ., presence This application is a continuation of U .S . Ser . No . 14 /476 , of organelles) , greater degree of unpredictability in 584 filed Sep . 3 , 2014 , which claims benefit of U . S . Provi- responses to multiple heterologous factors , and the techno sional application Ser . No. 61 / 873 , 308 , filed Sep . 3 , 2013, all logical disadvantages of selecting eukaryotic cells having of which are incorporated by reference in their entirety for 10 multiple genetic /phenotypic changes . Thus , it is desirable to all purposes . find alternative methods of engineering eukaryotic cells for introducing new functionalities into the cells , where the REFERENCE TO SEQUENCE LISTING , TABLE methods can be applied independently of or in combination OR COMPUTER PROGRAM with recombinant DNA technology for modifying eukary 15 otic cells. The official copy of the Sequence Listing is submitted concurrently with the specification as an ASCII formatted SUMMARY text file via EFS -Web , with a file name of “ BELL .018 _ ST25 . txt" , a creation date of Sep . 3 , 2014 , and The present invention is related to host cells that have a size of 962 kilobytes . The Sequence Listing filed via 20 been altered or reprogrammed by communication and /or EFS -Web is part of the specification and is hereby incorpo transfer of chemical information with artificial endosymbi rated in its entirety by reference herein . onts . In some embodiments , the artificial endosymbionts of the invention secrete to and /or transport from the host cell TECHNICAL FIELD polypeptides, nucleic acids , lipids, carbohydrates , amino 25 acids , or other factors. In some embodiments , the artificial The present invention relates generally to host cells with endosymbiont secretes a protein into the host cell . In some artificial endosymbionts . The artificial endosymbionts of the enembodiments , the secreted protein is a heterologous protein host cell communicates with the host cell and changes to the artificial endosymbiont . In some embodiments , the aspects of the host cell. secreted protein from the artificial endosymbiont causes a 30 phenotype in the host cell . In some embodiments , the BACKGROUND artificial endosymbiont of the invention secretes a nucleic acid into the host cell. In some embodiments , the nucleic The advent of recombinant DNA technology created the acids a recombinant nucleic acid . In some embodiments , the ability to alter the genetic makeup of organisms, eliminating nucleic acid secreted from the artificial endosymbiont causes the natural barriers that prevented transmission of genetic 35 a phenotype in the host cell . material between unrelated organisms. The ease of growing The invention also relates to methods for changing a host and manipulating bacteria and the numerous techniques cell by the introduction of an artificial endosymbiont that available for introducing heterologous genes makes bacteria secretes to and / or transports from the host cell polypeptides , model organisms for genetic manipulation using recombi- nucleic acids, lipids, carbohydrates, amino acids , or other nant DNA technology . Gram - negative and Gram - positive 40 factors . In some embodiments, the method introduces a bacteria have been subjected to successful manipulation protein into the host cell from the artificial endosymbiont. In with recombinant DNA technology . Because of the well some embodiments , the method introduces a nucleic acid developed technology for transforming bacterial cells , the into the host cell from the artificial endosymbiont . In some relative ease of genetically manipulating different bacteria , embodiments , the method produces a phenotype in the host short reproduction times, and the comparatively small 45 cell resulting from the polypeptide and /or nucleic acid genomes , bacteria have been used as vehicles for synthetic introduced from the artificial endosymbiont. biology applications, which aims to create novel artificial In some embodiments , the host cells and methods of the biological pathways , organisms or devices, or the redesign invention are used to make medically and industrially of existing natural biological systems. Moreover, bacteria important recombinant peptides/ proteins that will be useful exist with a wide range of functionalities, such as unique 50 for therapeutic , biopharmaceutical, agricultural, and indus metabolic pathways ( e . g ., photo - and chemoautotrophism ) , trial applications . In some embodiments , the artificial endo magnetotactic properties (Blakemore , R ., “ Magnetotactic symbionts and methods of the invention are used to intro bacteria ,” Science 24 : 377 - 379 , 1975 ) , and extremophiles duce into host cells phenotypes that require the introduction ( e. g ., thermophiles) , which allows creation of recombinant of multiple factors and /or multiple genes. bacteria with the properties present in the bacterial host cell. 55 In some embodiments , the host cell is a mammalian cell . Recombinant DNA techniques have also been used to In some embodiments , the host cell is a human , mouse , rat , manipulate the genetic makeup of eukaryotic cells , tran - canine , primate , or rodent cell. In some embodiments , the siently or as a heritable property . For example , homologous host cell is a fibroblast cell, epithelial cell, keratinocyte , and targeted recombination allows the generation of recom - hepatocyte , adipocyte or endothelial cell. In some embodi binant animals containing heterologous genes . Despite the 60 ments , the host cell is a stem cell, or pluripotent ES cell , or advances in technology, manipulation of eukaryotic cells by pluripotent iPS cell , a multipotentmesenchymal stem cell or recombinant DNA techniques has greater challenges as multipotent hematopoietic stem cell. In some embodiments , compared to manipulation of bacteria . Some eukaryotic cells the host cell is a progenitor cell , such as for example , a targeted for recombinant DNA manipulation , e . g ., mamma - neural progenitor cell, an angioblast, an osteoblast , a chon lian cells, are slower to grow than bacterial cells, making 65 droblast , a pancreatic progenitor cell , or an epidermal pro selection of eukaryotic cells containing a heterologous genitor cell . In some embodiments , the host cell is a solid nucleic acid more time consuming . Creation of eukaryotic tumor cell or a hematopoietic cancer cell . In some embodi US 9 ,752 , 129 B2 ments , the host cell is from a carcinoma, sarcoma, leukemia , containing GFP fused to the C - terminal transport sequence lymphoma, or glioma. In some embodiments , the host cell is of NCBI Accession No. YP _ 422785 ) as compared to that of obtained from a prostate cancer, breast cancer, lung cancer, the M . magneticum AMB - 1 negative control (denoted as colorectal cancer, pancreatic cancer, melanoma, glioblas - AMB - 1 Neg Ctrl ) ( see Example 4 ) . toma, liver cancer , or the NCI 60 panel of cancer cell lines. 5 FIG . 11 shows levels of green fluorescent protein fluo rescence in supernatants of cultures of AC16 cells as com BRIEF DESCRIPTION OF THE FIGURES pared to fluorescence levels of suspensions of AC16 cells . Also shown are fluorescence levels of supernatant of M . FIGS. 1A - 1D depict BLAST results for a search with the magneticum AMB - 1 Neg Ctrl cells as compared to M . amino acid sequence for Type V secretory pathway adhesin 10 magneticum AMB- 1 Neg Ctrl cells in suspension . AidA (SEQ ID NO : 1 ) from Magnetospirillum magneticum FIG . 12 shows fluorescence microscopy of AMB - 1 AMB - 1 . Exemplary sequences of homologous regions labeled MDA -MB231 cells (40x images ) . MDA -MB231 retrieved in the search , which included the search query cells were labeled with AMB - 1 , where the AMB - 1 contained Magnetospirillum magneticum AMB - 1 adhesin AidA , are ( A ) only GFP plasmid or ( B ) GFP - fused to C - terminal 200 presented as SEQ ID NOS: 1 - 103 . 15 amino acids of YP _ 422785 (i . e. , AC16 ) , and dually stained FIGS. 2A -2D depict BLAST results for a search with the for GFP and AMB - 1 one day post - labeling with bacteria and amino acid sequence for protein accession no . YP _ 420640 , examined by fluorescence microscopy (see Example 4 for annotated as RTX toxin from Magnetospirillum magneticum details ). Both panels are merged images of staining of GFP AMB - 1 (SEQ ID NO : 104 ). Exemplary sequences of ( red ), AMB - 1 (green ), and DAPI (blue ). homologous regions retrieved in the search , which included 20 FIG . 13 shows fluorescence microscopy of AMB - 1 the search query Magnetospirillum magneticum AMB - 1 labeled MDA -MB231 cells ( single channel and composite RTX toxin YP _ 420640 , are presented as SEQ ID NOS : 104 - images ) (40x images ) . MDA -MB231 cells were labeled 122 . with AMB - 1 containing GFP plasmid only , and dually FIGS . 3A -3D depict BLAST results for a search with the stained one day post - labeling and examined by fluorescence amino acid sequence for protein accession no . YP _ 423419 , 25 microscopy . Top left panel ( A ) represents AMB - 1 staining annotated as RTX toxin from Magnetospirillum magneticum (green channel) , ( B ) top right panel GFP staining ( red AMB - 1 (SEQ ID NO : 123 ) . Exemplary sequences contain - channel ) , ( C ) bottom left panel DAPI staining (blue chan ing homologous regions retrieved in the search , which n el ) , and (D ) merged images of AMB - 1 (green channel ), included the search query Magnetospirillum magneticum GFP ( red channel) , and DAPI (blue channel) staining . AMB - 1 RTX toxin YP _ 423419 , are presented as SEQ ID 30 FIG . 14 shows fluorescence microscopy of AMB - 1 NOS: 123 - 129 . labeled MDA -MB231 cells ( single channel and composite FIGS . 4A -4D depict BLAST results for a search with the images ) (40x images ) . MDA -MB231 cells were labeled amino acid sequence for large exoprotein from Magneto with AMB - 1 containing GFP- fused to C - terminal 200 amino spirillum magneticum AMB - 1 (SEQ ID NO : 130 ) . acids of YP _ 422785 (AC16 ), and dually stained one day FIGS . 5A -5D depict BLAST results for a search with the 35 post- labeling and examined by fluorescence microscopy. amino acid sequence for Type V secretory pathway protein Top left panel ( A ) represents AMB - 1 staining ( green chan (accession no . YP _ 420638 ) ( SEQ ID NO : 131 ) from Mag - nel) ; top right panel ( B ) represents GFP staining (red chan netospirillum magneticum AMB - 1 . nel) ; bottom left panel (C ) represents DAPI staining ( blue FIGS. 6A -6D depict BLAST results for a search with the channel ) ; and bottom right panel ( D ) represents merged amino acid sequence for protein accession no . YP _ 422662 40 images of AMB- 1 (green channel ), GFP ( red channel) , and (SEQ ID NO : 132 ) , annotated as RTX toxin from Magneto - DAPI (blue channel ) staining . spirillum magneticum AMB - 1 . Exemplary sequences of homologous regions retrieved in the search , which included DETAILED DESCRIPTION the search query Magnetospirillum magneticum AMB - 1 RTX toxin YP _ 422662, are presented as SEQ ID NOS : 132 - 45 The present invention is directed to host cells that contain 140 . an artificial endosymbiont, wherein the artificial endosym FIG . 7 depicts BLAST results for a search with the 949 biont secretes into and / or transports from the host cell a C -terminal amino acids of YP 422785 (SEQ ID NO : 141) polypeptide , nucleic acid , or other factor. In some embodi from Magnetospirillum magneticum AMB - 1 . Exemplary ments , the factor, e . g . , a protein or nucleic acid , is secreted sequences of homologous regions retrieved in the search , 50 into the host cell and produces a phenotype in the host cell, which included YP 422785 , are presented as SEQ ID NOS : particularly a phenotype not present prior to the introduction 141 - 253 . of the artificial endosymbiont into the host cell. In some FIGS. 8A -8D depict BLAST results for a search with the embodiments , the artificial endosymbiont secretes into and / amino acid sequence for ABC - type protease/ lipase transport or transports from the host cell two or more factors ( i. e . , system , ATPase and permease components (SEQ ID 55 multiple factors ) . In some embodiments , secretion into the NO : 254 ) from Magnetospirillum magneticum AMB - 1 . host cell can use the endogenous secretion machinery and Exemplary sequences of homologous regions retrieved in secretion signals ( e . g . , sequences ) of the artificial endosym the search , which included the search query Magnetospiril - biont, or the artificial endosymbiont engineered to contain lum magneticum AMB - 1 ATPase and permease , are pre - the secretion systems for transport of the factor into the host sented as SEQ ID NOS :254 - 268. 60 cell. FIGS . 9A - 9C depict BLAST results for a search with the Before various embodiments of the present invention are amino acid sequence for thermostable lipase TliA ( SEQ ID further described , it is to be understood that this disclosure NO : 269) from Pseudomonas flourescens . Exemplary is not limited to the particular embodiments described , as sequences of homologous regions retrieved in the search are such may, of course , vary . It is also to be understood that the presented as SEQ ID NOS :270 - 286 . 65 terminology used herein is for the purposes of describing FIG . 10 shows levels of green fluorescence in extracel- particular embodiments only , and is not intended to be lular supernatants of AC16 cells ( M . magneticum AMB - 1 limiting . US 9 ,752 , 129 B2 It is also to be noted that as used in the present disclosure ence to a host cell , such as an artificial endosymbiont, refers and in the appended claims, the singular terms “ a ” , “ an " , and to a nucleic acid or polypeptide not naturally present in the “ the ” include plural referents unless context clearly indicates given host cell . For example , a nucleic acid molecule otherwise . Similarly , the word “ or ” is intended to include containing a non -host nucleic acid encoding a polypeptide " and ” (and vice versa ) unless the context clearly indicates 5 operably linked to a host nucleic acid comprising a promoter otherwise . In addition , the use of “ or” means “ and /or ” unless is considered to be a heterologous nucleic acid molecule . stated otherwise . Conversely , a heterologous nucleic acid molecule can com In addition , the words “ comprise , " " comprises, " " com - prise an endogenous structural gene operably linked with a prising, ” “ include, " " includes ,” and “ including ” are inter non -host (exogenous ) promoter . Similarly, a peptide or changeable and not intended to be limiting . Where descrip - 10 polypeptide encoded by a non - host nucleic acid molecule , or tions of various embodiments use the term “ comprising, " an endogenous polypeptide fused to a non -host polypeptide those skilled in the art would understand that in some is a heterologous peptide or polypeptide. specific instances, an embodiment can be alternatively Unless specific definitions are provided , all other techni described using language “ consisting essentially of” or cal and scientific terms used herein have the same meaning “ consisting of. " 15 as commonly understood by one of ordinary skill in the art . Numerical limitations given with respect to concentra - Definitions of common terms in molecular biology may be tions or levels of a substance are intended to be approximate . found in , for example , Benjamin Lewin , Genes V , Oxford Thus, where a concentration is indicated to be at least ( for University Press ( 1994 ); The Encyclopedia of Molecular example ) 10 ug , it is intended that the concentration be B iology , John Kendrew , ed . , Blackwell Science Ltd . ( 1994 ) ; understood to be at least approximately or about 10 ug . 20 and Molecular Biology and Biotechnology : a Comprehen The section headings used herein are for organizational sive Desk Reference , Robert A . Meyers , ed ., VCH Publish purposes only and not to be construed as limiting the subject e rs , Inc. ( 1995 ) . matter described . Artificial Endosymbionts Definitions In one aspect , the artificial endosymbionts of the present As used herein , the term “ artificial endosymbiont” refers 25 invention include single -celled organisms, e . g. , bacteria , that to a single -celled organism that is or has been introduced are capable of surviving in a eukaryotic cell , wherein the into a host cell through human intervention . The single - artificial endosymbiont secretes into and / or transports from celled organism secretes to and /or transports from the host the host cell a polypeptide (s ), nucleic acid ( s ), or other cell polypeptide ( s ), nucleic acid ( s ) , lipid ( s ), carbohydrate ( s ), factor( s ). In some embodiments, the polypeptide and /or amino acid ( s ) , or other factor ( s ) . And this communication 30 nucleic acid are recombinant or heterologous to the artificial between the single cell organism and the host cell results in endosymbiont. In some embodiments , the artificial endos a phenotype for the host cell and /or artificial endosymbiont. ymbiont introduces a phenotype into the host cell through As used herein , the term “ cellular life cycle ” refers to the secretion and / or transport between the host cell and the series of events involving the growth , replication , and divi artificial endosymbiont. In some embodiments , this pheno sion of a eukaryotic cell . It is generally divided into five 35 type introduced into the host cell by the artificial endosym stages , known as Go, in which the cell is quiescent, G , and biont is maintained in daughter cells of the host cell . In some G2, in which the cell increases in size , S , in which the cell embodiments , the host cell maintains the functionality for at duplicates its DNA , and M , in which the cell undergoes least 1 day, 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11, 12 , 13 , 14 , 15 , 16 , mitosis and divides . 17 , 18 , 19 or 20 days . In some embodiments, the artificial As used herein , the term “ daughter cell” refers to cells that 40 endosymbiont can stably maintain the phenotype in host are formed by the division of a cell . daughter cells through at least 3 cell divisions, or at least 4 As used herein , the term " essential molecule” refers to a cell divisions, or at least 5 cell divisions , or at least 6 , 7 , 8 , molecule needed by a cell for growth or survival. 9 , 10 , 11, 12 , 13 , 14 , 15, 16 , 17 , 18 , 19 , 20 or more cell As used herein , the term “ genetically modified ” refers to divisions. In some embodiments , the artificial endosymbiont altering the DNA of a cell so that a desired property or 45 can stably maintain phenotype in the host daughter cells characteristic of the cell is changed . through 3 - 5 divisions, or 5 - 10 divisions, or 10 - 15 divisions , As used herein , the term “ magnetosome” refers to par ticles of magnetite ( i. e ., Fe3O4) or greigite (FezS4 ) enclosed In some embodiments , the artificial endosymbionts are by a sheath or membrane, either as individual particles or in Proteobacteria , particularly a - Proteobacteria . In the current chains of particles . 50 taxonomic scheme based on 16S rRNA , C - proteobacteria As used herein , the term “ magnetotactic bacteria ” or are recognized as a Class within the phylum Proteobacteria , “ MTB ” refers to bacteria which are able to orient in a and are subdivided into 7 main subgroups or orders (Cau magnetic field . lobacterales , Rhizobiales , Rhodobacterales, Rhodospirilla As used herein , the term " mammal” refers to warm - les, Rickettsiales, Sphingomonadales and Parvularculales ) blooded vertebrate animals all of which are characterized by 55 (Gupta , R . S . , " Phylogenomics and signature proteins for the hair on the skin and , in the female , milk producing mam - alpha Proteobacteria and its main groups ,” BMC Microbiol. mary glands. 7 :106 , 2007 ; incorporated herein by reference ). As used herein , the term “ phenotype ” refers to the set of A large number of a -proteobacterial genomes that cover observable characteristics of an individual resulting from the all of the main groups within a - proteobacteria have been interaction of its genotype with the environment. 60 sequenced , providing information that can be used to iden As used herein , the term “ secrete ” refers to the passing of tify unique sets of genes or proteins that are distinctive molecules or signals from one side of a membrane to the characteristics of various higher taxonomic groups ( e. g ., other side. families, orders, etc . ) within a - proteobacteria . (Gupta , As used herein , the term " heterologous” when used in supra ) . Analyses of these sequenced genomes show that reference to a nucleic acid or polypeptide refers to a nucleic 65 many species of a -proteobacteria contain transport systems acid or polypeptide not normally present in nature . Accord - useful in the present invention . FIGS . 1 - 9 show BLAST ingly , a heterologous nucleic acid or polypeptide in refer - homology searches performed at NCBIprotein blast website US 9 , 752 , 129 B2 ( blast. ncbi . nlm .nih . gov /Blast . cgi ) , and these searches show The genomes of a number of artificial endosymbionts that many a -proteobacteria species have protein coding have been or are being sequenced : M . frigidum , M . burtonii, sequences homologous to proteins of Type 1 and 5 secretion C . symbiosum , C . psychrerythraea , P . haloplanktis , Haloru systems. Thus, in some embodiments , the artificial endos- brum lacusprofundi , Vibrio salmonicida, Photobacterium ymbionts comprise a - proteobacteria . 5 profundum , S . violacea , S . frigidimarina , Psychrobacter sp . In some embodiments , an exemplary a -proteobacteria is 273 - 4 , S . benthica , Psychromonas sp . CNPT3, Moritella sp ., magnetotactic bacteria (MTB ) . MTB are a diverse group of Desulfotalea Psychrophila , Exiguobacterium 255 - 15 , Fla bacteria that belong to different subgroups of the Proteo vobacterium psychrophilum , Psychroflexus torquis , Polari bacteria and Nitrospirae phylum , and are mostly represented bacter filamentous , P . irgensii , Renibacterium salmoni within the a - proteobacteria . MTB have a Gram -negative 10 narum , Leifsonia -related PHSC20 -cl , Acidithiobacillus cell wall structure ( e . g ., inner membrane , periplasm , and ferrooxidans, Thermoplasma acidophilum , Picrophilus tor outer membrane ) . They inhabit water bodies or sediments ridus , Sulfolobus tokodaii , and Ferroplasma acidarmanus. with vertical chemical concentration gradient, predomi- Secretion Systems of Artificial Endosymbionts nantly at the oxic - anoxic interface , and thus in some In the present invention , secretion of protein (s ), nucleic instances categorized as microaerophiles, anaerobic , facul- 15 acid (s ) , other molecule (s ) and other factor( s ) from the arti tative aerobic or some combination of the three . All MTB ficial endosymbiont, particularly a - proteobacteria and more are chemoorganoheterotrophic although somemarine strains particularly magnetotactic bacteria , can make use of the can also grow chemolithoautotrophically (Bazylinski et al ., endogenous secretion systems of the artificial endosymbi “ Chemolithoautotrophy in the marine , magnetotactic bacte - ont . In some embodiments , the artificial endosymbiont can rial strains MV - 1 and MV- 2 , ” Arch Microbiol. 182 ( 5 ): 373 - 20 be engineered with heterologous secretion systems, or por 87 , 2004 ; Williams et al. , “ Evidence for autotrophy via the tions thereof, for directed secretion of these target mol reverse tricarboxylic acid cycle in the marine magnetotactic ecules. In some embodiments, these transport systems of the coccus strain MC - 1 , ” Appl Environ Microbiol . 72 ( 2 ): 1322 - invention are used to transport medically and industrially 9 , 2006 ). All MTB contain magnetosomes, which are intra - important genes , recombinant peptides /proteins , and /or cellular structures comprising magnetic iron crystals envel- 25 other factors that will be useful for therapeutic , biopharma oped by a phospholipid bilayer membrane (Gorby et al. , ceutical, agricultural, and industrial applications . However, “ Characterization of the bacterial magnetosome mem - although various secretion systems are known in the art and brane , " J Bacteriol. 170 ( 2 ) : 834 -41 , 1988 ) . In some embodi- described in the present disclosure , it is to be understood that ments , the transport pathways of MTB deliver endogenous practice of the present invention does not require knowledge and exogenous proteins and / or nucleic acids to the host cell . 30 or understanding of a specific secretion system . Generally , A large number of MTB species are known to those of the signals or sequences that cause secretion of polypeptides ordinary skill in the art since their initial discovery or other factors are known for many single celled organisms; (Blakemore , R ., “ Magnetotactic bacteria , ” Science 24 : 377 - they can also be found on known secreted proteins or nucleic 79 , 1975 ; Faivre , D . and Schüler , D . , “Magnetotactic bac - acids, or identified based on similarities to sequences found teria and magnetosomes ," Chem Rev. 108 : 4875 - 4898 , 2008 ; 35 on secreted proteins or nucleic acids. These secretion signals publications incorporated herein by reference ) . MTB have can then be used , particularly as fusions, to direct a target been identified in different subgroups of the Proteobacteria molecule for secretion in the artificial endosymbiont into the and the Nitrospira phylum , with most of the phylotypes host cell without prior knowledge of the specific transport grouping in a - Proteobacteria . Currently , culturable MTB system that directs secretion using the particular secretion strains assigned as a -Proteobacteria by 16S rRNA sequence 40 signal. similarity include the strain originally isolated by Blakemore Various secretion systems can be used in the artificial in 1975 , Magnetospirillum magnetotactium (formerly endosymbionts of the disclosure , e . g . , a magnetotactic bac Aquasprillium magnetotactium ) , M . gryphiswaldense , M . teria . For example , the a - proteobacteria have transport path magneticum strain AMB - 1 , M . polymorphum , Magneto ways that include ABC transporter- based pathways (includ spirillum sp . MSM - 4 and MSM - 6 , Magnetococcus marinus, 45 ing the type I secretion system , TiSS ) , type II secretion marine vibrio strains MV - 1 and MV- 2 , a marine spirillum systems ( T2SS ), type III secretion systems ( T3SS ) , type IV strain MMS- 1 and Magnetococcus sp . strain MC - 1 , as well secretion systems ( T4SS ) , type V secretion systems ( T5SS ) , as others . In some embodiments , all such MTB can be used type VI secretion systems ( T6SS ) , type VII secretion sys in the present invention . tems ( T7SS ) , and other transport systems that are known in Other bacteria useful as artificial endosymbionts include , 50 the art . In some embodiments , these transport systems of for example , Anabaena , Nostoc , Diazotroph , Cyanobacteria , A -proteobacteria are used to transport proteins , nucleic Trichodesmium , Beijerinckia , Clostridium , Green sulfur acids, and other factors from the artificial endosymbiont into bacteria , Azotobacteraceae, Rhizobia , Frankia , flavobacte the host cell . In Gram -negative bacteria , secreted proteins ria , Methanosarcinales, aerobic halophilic Archaea of the are exported across the inner and outer membrane in a single order Halobacteriales, the fermentative anaerobes of the 55 step via the TISS , T3SS , T4SS , and TOSS pathways . Pro order Halanaerobiales (low G + C brand of the Firmicutes ) , teins are exported into the periplasmic space across inner the red aerobic Salinibacter ( Bacteroidetes branch ) , Marino - membrane via Sec or two - arginine ( Tat ) pathways . Proteins bacter, Halomonas, Dermacoccus, Kocuria , Micromono - are transported across the outer membrane from the peri spora , Streptomyces , Williamsia , Tskamurella , Alteromonas, plasmic space by T2SS , T5SS or less commonly by T1SS or Colwellia , Glaciecola , Pseudoalteromonas, Shewanella , 60 T4SS . Polaribacter, Pseudomonas, Psychrobacter, Athrobacter, TISS consists of three proteins ; an inner membrane Frigoribacterium , Subtercola , Microbacterium , Rhodoccu , protein with a cytoplasmic ATPase domain operating as an Bacillus, Bacteroides, Propionibacterium , Fusobacterium , ATP - binding cassette (ABC ) transporter (Escherichia coli Klebsiella , lecithinase - positive Clostridia , Veillonella , Fuso - HlyB ) , a periplasmic adaptor (also known as membrane bacteria , Chromatiaceae , Chlorobiceae, Rhodospirillaceae , 65 fusion protein , MFP ; E . coli HlyD ) , and an outer membrane thiobacilli , nitrosomonas , nitrobacter , methanogens , aceto - channel protein of the ToiC family ( E . coli TolC ) (reviewed gens, sulfate reducers , and lactic acid bacteria . in Delepelaire , P . , “ Type I secretion in gram -negative bac US 9 , 752 , 129 B2 10 teria ,” Biochem Biophy Acta 1694 :149 -61 , 2004 ). These in the extracellular medium , which in turn generates the proteins form a pore through the periplasm via which an biologically active form of the toxin . The T1SS substrates unfolded protein may be translocated . T1SS protein sub contain a translocation signal at the C - terminus ( last 27 to strates typically contain carboxy - terminal, glycine - and 218 - amino acid fragment of HlyA , minimal secretion signal aspartate - rich repeats known as repeat- in - toxin (RTX ) (Lin - 5 is located within the last ~ 60 C - terminal amino acids and is hartova et al. , " RTX proteins : a highly diverse family both necessary and sufficient to direct secretion ) (Kenny , B . secreted by a common mechanism , " FEMS Microbiol Rev. et al. , “ Analysis of the haemolysin transport process through 34 : 1076 - 1112, 2010 ; incorporated herein by reference ) and the secretion from E . coli of PCM , CAT or beta - galactosi are often located close to ABC and MFP genes . Due to its dase fused to the C - terminal signal domain , ” MolMicrobiol . simplicity , TiSS has been used to transport heterologous 10 5 : 2557 -68 , 1991 ; incorporated herein by reference in its proteins . Several studies have shown the utility of TiSS to entirety ) . Proteins targeted to the translocator carry an transport exogenous proteins to the extracellular medium uncleaved , poorly conserved secretion signal at the extreme ( see , e . g ., Delepelaire , P . , supra ; Reed , B and Chen , R ., C -terminus ( absolutely required for secretion ) that binds to “ Biotechnological applications of bacterial protein secre - the nucleotide binding domain of ABC -ATPase ( E . coli tion : from therapeutics to biofuel production ,” Res Micro - 15 HlyB ) in a reaction reversible by ATP that mimic initial biol. 164 :675 -682 , 2013 ; all of which are incorporated movement of HlyA into the translocation channel and rapid herein by reference in their entirety ) . Low et al. found that transport of HlyA to the extracellular medium (Holland B . et cyclodextrin glucanotransferase secretion could be al. , “ Type 1 protein secretion in bacteria , the ABC - trans improved by overexpression of the E . coli a -haemolysin porter dependent pathway (Review ), ” Mol Membr Biol . transporter (Low , K . O . et al. , “ An effective extracellular 20 22 : 29 - 39 , 2005 ; incorporated herein by reference in its protein secretion by an ABC transporter system in Escheri entirety ) . chia coli : statistical modeling and optimization of cyclodex - TISS from Pseudomonas fluorescens is also known and trin glucanotransferase secretory production ,” J Ind Micro - has been used to secrete recombinant proteins. Park et al. biol Biotechnol. 38 : 1587 - 97 , 2011; incorporated herein by found a 105 amino acid polypeptide as the minimal region reference in its entirety ) . Su and coworkers also used E . coli 25 for recognition and transport by the lipase ABC transporter . a - haemolysin secretion systems to secrete protein . By over - A fusion of a target protein to this minimal region allowed expressing HlyBD , two strain -specific components of TiSS , secretion of recombinant protein ( Park , Y . et al . , “ Identifi they showed that the recombinant T1SS secreted a high level cation of the minimal region in lipase ABC transporter ( 1 . 5 g per liter ) of protein from an E . coli (Su , L . et al. , recognition domain of Pseudomonas fluorescens for secre “ Extracellular overexpression of recombinant Thermobifida 30 tion and fluorescence of green fluorescent protein ,” Microb . fusca cutinase by alpha -hemolysin secretion system in E . Cell. Fact. 11 : 60 , 2012; incorporated herein by reference in coli BL21 (DE3 ) ," Microb Cell Fact. 11 : 8 , 2012 ; incorpo - its entirety ) . The versatility of T1SS for protein secretion is rated herein by reference in its entirety ) . The E . coli hemoly - seen in its wide array of transport substrates, which vary sin transporter is also known to secrete other heterologous from small proteins like the hemophore Has A ( 19 kDa ) to TISS substrates expressed in E . coli, including exotoxins 35 huge surface layer proteins up to 900 kDa in size (Linhar Cya of Bordatella . pertussis (Sebo , P ., and Ladant, D ., tova et al ., “RTX proteins: a highly diverse family secreted “ Repeat sequences in the B . pertussis adenylate cyclase by a common mechanism ,” FEMS Microbiol Rev . 34 : 1076 toxin can be recognized as alternative carboxy - proximal 1112 , 2010 ; Satchell , K . J ., “ Structure and function of secretion signals by the E . coli alpha - haemolysin transloca - MARTX toxins and other large repetitive RTX proteins, " tor, ” Mol Microbiol. 9 : 999 - 1009 , 1993 ) , LtkA of Aggre - 40 Annu Rev Microbiol. 65 : 71- 90 , 2011 ; all of which are gatibacter actinomycetemcomitans (Lally , E . T . et al . , incorporated herein by reference in their entirety ) . Other “ Analysis of the Actinobacillus actinomycetemcomitans leu - proteins secreted by TiSS include, for example adenylate kotoxin gene , Delineation of unique features and compari - cyclases , lipases and proteases. son to homologous toxins, ” J Biol Chem . 264 : 15451 -456 , Another T1SS system that can be used in the invention is 1989 ) , PaxA of Pasteurella aerogenes (Kuhnert , P . et al. , 45 RaxABC from Xanthomonas oryzae pv. oryzae . Phyloge “ Characterization of PaxA and its operon : a cohemolytic netic analysis identifies RaxB as an ABC transporter ( da RTXtoxin determined from pathogenic Pasteurella aero - Silva , F . G . et al ., “ Bacterial genes involved in type I genes, ” Infect. Immun . 68 :6 - 12 , 2000 ) , and FrpA of Neis - secretion and sulfation are required to elicit the rice Xa21 seria meningitidis ( Thompson , S . A . and Sparling, P . E ., mediated innate immune response, ” Mol Plant Microbe “ The RTX cytotoxin -related Frp , protein of Neisseria min - 50 Interact. 17 :593 -601 , 2004 ; incorporated herein by refer ingitidis is secreted extracellularly by meningococci and by e nce in its entirety ) , equivalent to HlyB from E . coli. The HlyBD + Escherichia coli. ” Infect Immun . 61 :2906 - 11 , 1993 ; RaxABC transport system is used to secrete AvrXa21 mol all of which are incorporated herein by reference in their ecules ( small sulfated polypeptides ) , metalloproteases , entirety ) . The assembly of the TiSS complex (best exem - adhesion factors and glycanases (Delepelaire , P . , “ Type I plified by E . coli hemolysin (Hly ) secretion system ) is 55 secretion in gram - negative bacteria ,” Biochim Biophys Acta nucleotide - independent, and the translocation of Hly ( the 1694 : 149 -161 , 2004 ; Reddy , J. D . et al. , “ To1C is required TISS substrate ) requires ATP hydrolysis catalyzed by HlyB for pathogenicity of Xylella fastidioa in Vitis vinifera grape ( Thanabalu , T . et al. , “ Substrate - induced assembly of a vines, ” Mol Plant Microbe Interact. 20 : 403 -410 , 2007 ; all of contiguous channel for the protein export from E . coli : which are incorporated herein by reference in their entirety ) . reversible bridging of an inner -membrane translocase to an 60 Autotransporter systems, a subset of T5SS , provide poten outer membrane exit pore , " EMBO J . 17 :6487 - 96 , 1998 ; tially the simplest mechanism for extracellular secretion of incorporated by reference in its entirety ) . HlyA is member of recombinant proteins. An autotransporter consists of an the RTX (repeats in toxin ) protein family and contains N - terminal Sec -dependent signal sequence , a passenger glycine - rich peptide repeats in the C -terminal domain , domain and a C -terminal beta -motif . The translocation is a which have the consensus sequence GGXGXD ( X repre - 65 two -step process. The target protein is transported into the sents any amino acid ) (SEQ ID NO : 287 ) and are important periplasm using Sec - dependent transport whereupon the for the binding of Ca2 + ions . This triggers folding of Hly beta- motif forms a transmembrane pore through which the US 9 , 752 , 129 B2 12 passenger domain is secreted out of the periplasm (Dautin , - continued N . and Barnstein , H . D . , “ Protein secretion in gram -negative bacteria via the autotransporter pathway, ” Annu Rev Micro LSGNINVODKGTVTLGGEGELSPDLTLONOMLYSLFNGYRNIWSGSLNA biol. 61: 89 - 112 , 2007 ; Thanassi, D . G . , et al. , " Protein PDATVSMTDTQWSMNGNSTAGNMKLNRTIVGFNGGTSPFTTLITDNLDA secretion in the absence of ATP : the autotransporter , two - 5 partner secretion and chaperone /usher pathways of gram VQSAFVMRTDLNKADKLVINKSATGHDNSIWVNFLKKPSNKDTLDIPLV negative bacteria (Review ), ” Mol Membr Biol. 22 :63 - 72 , 2005; all of which are incorporated herein by reference in SAPEATADNLFRASTRVVGFSDVTPILSVRKEDGKKEWVLDGYQVARND their entirety ) . The beta -motif can be cleaved , allowing GQGKAAATFMHISYNNFITEVNNLNKRMGDLRDINGEAGTWVRLLNGSG translocation of the target protein out of the cell (Leyton , D . 10 L . et al. , “ From self sufficiency to dependence : mechanism SADGGFTDHYTLLOMGADRKHELGSMDLFTGVMATYTDTDASADLYSGK and factors important for autotransporter biogenesis , ” Nat TKSWGGGFYASGLFRSGAYFDVIAKYIHNENKYDLNFAGAGKONFRSHS Rev Microbiol. 10 : 213 -25 , 2012 ; incorporated herein by reference in its entirety ) . LYAGAEVGYRYHLTDTTFVEPQAELVWGRLQGQTFNWNDSGMDVSMRRN For transport in the T5 SS system , the target protein is 15 SVNPLVGRTGWSGKTFSGKDWSLTARAGLHYEFDLTDSADVHLKDAAG fused with the N - terminal signal sequence and the C - termi nal signal, beta - domain that mediate translocation of a EHOINGRKDSRMLYGVGLNARFGDNTRLGLEVERSAFGKYNTDDAINAN recombinant protein through the inner and outer membranes , respectively . This chimera gene has the N - terminal signal IRYSF sequence fused in frame to the N -terminal end of the target 20 Many E . coli autotransporters can also be used in the gene , and a second , in frame fusion to DNA encoding the present invention . Ko et al. used the YfaL autotransporter beta -domain sequence at the C -terminal end of the target (NCBI accession no . P45508 ) to secrete proteins ranging gene . In some embodiments , the passenger domain may be from 25 . 3 to 143 kDa from E . coli (Ko . H . J ., et al. . replaced in the fusion protein . For example , Jong et al . “ Functional cell surface display and controlled secretion of defined passenger domains of E . coli autotransporter hemo- 25 diverse agarolytic enzymes by Escherichia coli with a novel globin -binding protease (Hbp ) that could be replaced in a ligation - independent cloning vector based on the autotrans fusion protein to facilitate secretion , along with an intact porter YfaL ,” Appl Environ Microbiol. 78 :3051 -58 , 2012 ; beta - domain (Jong , W . S . et al . , “ A structurally informed incorporated herein by reference in its entirety ). The YfaL autotransporter platform for efficient heterologous protein 2 autotransporter comprises the following sequence : secretion and display, ” Microb Cell Fact. 11 :85 , 2012 ; incor- 304 porated herein by reference in its entirety ) . As such , the Hbp (NCBI Accession no . 088093, 1377 amino acids) passenger ( SEQ ID NO : 289 ) domains that can be replaced by heterologous proteins MRIIFLRKEYLSLLPSMIASLFSANGVAAVTDSCQGYDVKASCQASROS include the following: ( 1 ) 53 - 308 ; ( 2 ) 533 -608 ; ( 3 ) 657 -697 ; ( 4 ) 735 -766 ; (5 ) 898 -922 amino acids : 35 LSGITQDWSIADGQWLVFSDMTNNASGGAVFLOQGAEFSLLPENETGMT LFANNTVTGEYNNGGAIFAKENSTLNLTDVIFSGNVAGGYGGAIYSSGI ( SEQ ID NO : 288 ) NDTGAVDLRVTNAMFRNNIANDGKGGAIYTINNDVYLSDVIFDNNQAYT MNRIYSLRYSAVARGFIAVSEFARKCVHKSVRRLCFPVLLLIPVLFSAG STSYSDGDGGAIDVTDNNSDSKHPSGYTIVNNTAFTNNTAEGYGGAIYT SLAGTVNNELGYQLFRDFAENKGMFRPGATNIA ' YNKQGEFVGTLDKAA 40 NSVTAPYLIDISVDDSYSQNGGVLVDENNSAAGYGDGPSSAAGGFMYLG MPDFSAVDSEIGVATLINPQYIASVKHNGGYINVSFGDGENRYNIVDRN LSEVTFDIADGKTLVIGNTENDGAVDSIAGTGLITKTGSGDLVLNADNN NAPSLDFHAPRLDKLVTEVAPTAVTAQGAVAGAYLDKERYPVFYRLGSG DFTGEMQI ENGEVTLGRSNSLMNVGDTHCQDDPQDCYGLTIGSIDQYQN TQYIKDSNGQLTKMGGAYSWLTGGTVGSLSSYONGEMISTSSGLVFDYK 45 QAELNVGSTOQTFVHALTGFQNGTLNIDAGGNVTVNQGSFAGIIEGAGO LNGAMPIYGEAGDSGSPLFAFDTVONKWVLVGVLTAGNGAGGRGNNWAV LTIAQNGSYVLAGAQSMALTGDIVVDDGAVLSLEGDAADLTALQDDPQS IPLDFIGQKFNEDNDAPVTFRTSEGGALEWSFNSSTGAGALTOGTTTYA IVLNGGVLDLSDFSTWQSGTSYNDGLEVSGSSGTVIGSQDVVDLAGGDN MHGQQGNDLNAGKNLIFQGQNGQINLKDSVSQGAGSLTFRDNYTVTTSN 50 LHIGGDGKDGVYVVVDASDGOVSLANNNSYLGTTQIASGTLMVSDNSQL GSTWTGAGIVVDNGVSVNWOVNGVKGDNLHKIGEGTLTVOGTGINEGGL GDTHYNRQVIFTDKQQESVMEITSDVDTRSDAAGHGRDI EMRADGEVAV KVGDGKVLNQQADNKGQVQAFSSVNIASGRPTVVLTDERQVNPDTVSW DAGVDTQWGALMADSSGQHQDEGSTLIKTGAGTLELTASGTTQSAVRVE GYRGGTLDVNGNSLTFHOLKAADYGAVLANNVDKRATITLDYALRADKV 55 EGTLKGDVADILPYASSLWVGDGATFVTGADQDIOSIDAISSGTIDISD ALNGWSESGKGTAGNLYKYNNPYTNTTDYFILKOSTYGYFPTDOSSNAT GTVLRLTGODTSVALNASLFNGDGILVNATDGVILTGELNINLETDSLT WEFVGHSQGDAQKLVADRFNTAGYLFHGOLKGNLNVDNRLPEGVTGALV YLSNVTVNGNLTNTSGAVSLONGVAGDTLTVNGDYTGGGTLLLDSELNG

MDGAADISGTFTQENGRLTLQGHPVIHAYNTOSVADKLAASGDHSVLTO GI DDSVSDQLVMNGNTAGNITVVVNSITGIGEPTSTGIKVVDFAADPTQFQ PTSFSQEDWENRSFTFDRLSLKNTDFGLGRNATLNTTIQADNSSVTLGD NNAQFSLAGSGYVNMGAYDYTLVEDNNDWYLRSQEVTPPSPPDPDPTPD SRVFIDKNDGQGTAFTLEEGTSVATKDADKSVFNGTVNLDNOSVLNIND PDPTPDPDPTPDPEPTPAYQPVLNAKVGGYLNNLRAANQAFMMERRDHA IFNGGIQANNSTVNISSDSAVLGNSTLTSTALNLNKGANALASQSFVSD 65 GGDGQTLNLRVIGGDYHYTAAGQLAQHEDTSTVQLSGDLFSGRWGTDGE GPVNISDATLSLNSRPDEVSHTLLPVYDYAGSWNLKGDDARLNVGPYSM US 9 , 752 , 129 B2 13 14 of which are hereby incorporated by reference in their WMLGIVGGYSDNQGDSRSNMTGTRADNONHGYAVGLTSSWFQHGNQKOG- continued entirety ) and can be divided into three groups (Fronzes , R . et al. , supra ; incorporated herein by reference in its entirety ) . AWLDSWLQYAWFSNDVSEQEDGTDHYHSSGIIASLEAGYQWLPGRGVVI Group 1 T4SSs mediate the conjugative transfer of plasmid EPQAQVIYQGVQQDDFTAANRARVSQSQGDDIQTRLGLHSEWRTAVHVI 5 DNA or transposons into a wide range of bacterial species . For example , E . coli and Agrobacterium tumefaciens can PTLDLNYYHDPHSTEIEEDGSTISDDAVKORGEIKVGVTGNISORVSLR deliver DNA substrates into fungal, plant of human cells GSVAWQKGSDDFAQTAGFLSMTVKW (Grohmann , E . et al. , " Conjugative Plasmid Transfer in Gram - positive Bacteria , ” Microbiol Mol Biol Rev 67 ( 2 ) : In some embodiments , a protease (e . g. , a tobacco etch 10 277 -301 , 2003; Lawley, T. D . et al ., “ F Factor Conjugation virus protease ) is used to cleave the C - terminus of the fusion Is a True Type Iv Secretion System , ” FEMS Microbiol Lett . proteins to remove the beta - domain and autotransporter , 224 ( 1 ) : 1 - 15 , 2003 ; Trieu -Cuot , P . et al. , “ In Vivo Transfer of resulting in secretion from the cell . Sevastsyanovich et al. Genetic Information between Gram - positive and Gram utilized the E . coli serine protease Pet to cleave fusion Negative Bacteria , ” EMBO J. 4 ( 13A ) :3583 - 7 , 1985 ; all of proteins and provide for complete secretion of a range of 15 which are hereby incorporated by reference in their entirety ) . proteins varying in sizes and structures , including multi - T4SSs in group 2 , such as those found in Helicobacter pylori component proteins (Sevastsyanovich , Y . R . et al . , “ A gen - and Neisseria gonorrhea , mediate the uptake and release of eralized module for the selective extracellular accumulation DNA into the extracellular environment (Smeets , L . C . and of recombinant proteins ," Microb Cell Fact. 11 :69 , 2012 ; J . G . Kusters, “ Natural Transformation in Helicobacter incorporated herein by reference in its entirety ) . Pet is one 20 Pylori: DNA Transport in an Unexpected Way ,” Trends of the serine protease autotransporters of the Enterobacte - Microbiol. 10 ( 4 ) 159 -62 , 2002 (discussion 162) ; Hamilton , riaceae (SPATEs ) that releases passenger domain from the H . L . and J . P . Dillard , “ Natural Transformation of Neisseria beta - domain . Gonorrhoeae : From DNA Donation to Homologous Recom An application of an autotransporter for consolidated bination , ” Mol Microbiol . 59 ( 2 ) : 376 -85 , 2006 ; all of which bioprocessing uses an E . coli autotransporter Antigen 43 25 are hereby incorporated by reference in their entirety ) . ( Ag43 ) engineered to secrete a target protein . This auto Group 3 T4SSs deliver effector molecules into eukaryotic transporter system is unique in that the passenger domain cells during infection . H . pylori, Brucella suis and Legion Ag43alpha is self- cleaved , yet the secreted domain is non ella pneumophila are examples of bacteria that use their covalently attached to the beta - domain , forming an integral T4SSs to inject virulence proteins into mammalian host cells outer -membrane protein . In order to secrete the recombinant 30 (Backert , S . and Meyer, T . F ., “ Type IV Secretion Systems protein , the segment of Ag43alpha containing the cleavage and Their Effectors in Bacterial Pathogenesis ,” Curr Opin mechanism was fused to a target sequence . When engi- Microbiol. 9 ( 2 ) : 207 - 17 , 2006 ; Corbel, M . J . , “ Brucellosis : neered into an E . coli strain , the self- cleaving autotrans - An Overview ,” Emerg Infect Dis 3 ( 2 ) : 213 - 21, 1997 ; Ninio , porter secreted target protein out of the cell (Wargacki , A . J . S . and C . R . Roy, “ Effector Proteins Translocated by Legio et al ., “ An engineered microbial platform for direct biofuel 35 nella Pneumophila : Strength in Numbers , ” Trends Micro production from brown macroalgae ," Science 335 :308 - 13 , biol. 15 (8 ): 372 -80 , 2007; all of which are hereby incorpo 2012 ; incorporated herein by reference in its entirety ) . rated by reference in their entirety ) . A . tumefaciens uses its Avery large number of proteins are secreted via the T5SS, group 3 T4SS to deliver oncogenic DNA and proteins into more even than the T2SS (Jacob -Dubuisson , F . et al ., plant cells (Fronzes , R . et al ., “ The Structural Biology of “ Protein secretion through autotransporter and two -partner 40 Type IV Secretion Systems, ” Nat Rev Microbiol. 7 ( 10 ) : 703 pathway, ” Biochem Biophy Acta 1694 :235 - 57 , 2004 ; Dautin , 14 , 2009 ; incorporated herein by reference in its entirety ) . N . and Bernstein , H . D . , “ Protein secretion in the gram Genes encoding components of the T4SS are usually negative bacteria via autotransporter pathway , ” Ann Rev arranged in a single or several operons . H . pylori is an Microbiol . 61: 89 - 112 , 2007 ) . Most of the T5SS secreted example of a bacterium that encodes multiple T4SSs. H . proteins characterized to date are virulence factors . Proteins 45 pylori has an effector protein delivery system encoded by the secreted via the T5SS include adhesins such as AIDA - I and cag pathogenicity island and a DNA release and uptake Ag43 of E . coli , Hia of Haemophilus influenzae , YadA of system encoded by the comB gene cluster ( Backert , S . and Yersinia enteroliticola , and Pm of Bordetella pertussis ; T . F . Meyer, “ Type IV Secretion Systems and Their Effectors toxins such as VacA of Helicobacter pylori ; proteases such in Bacterial Pathogenesis, ” Curr Opin Microbiol . 9 ( 2 ) 207 as IgA proteases of Neisseria gonorrheae and Neisseria 50 17 , 2006 ; Smeets , L . C . and Kusters , J. G ., “ Natural Trans meningitides, SepA of Shigella flexneri and PrtS of Serratia formation in Helicobacter Pylori: DNA Transport in an marcescens ; and S - layer proteins such as rOmpB of Rick - Unexpected Way, ” Trends Microbiol. 10 ( 4 ) : 159 -62 , 2002 ; ettsia sp . and Hsr of Helicobacter pylori . T5bSS ( TPS ) all publications incorporated herein by reference in their secreted proteins include adhesins such as Hec A /HecB of entirety ). the plant pathogen Dickeya dadantii ( Erwinia chrysan - 55 Depending on the structural components that compose a themii ) and cytolysins such as Sh1A / Sh1B of Serratia T4SS , the systems can be broadly classified as either type marcescens, HpmA/ HpmB of Proteus mirabilis and EthA ) IVA or type IVB systems (Voth , D . E . et al . , “ Bacterial Type EthB of Edwardsiella tarda . IV Secretion Systems: Versatile Virulence Machines, ” The type IV secretion system ( T4SS ) is a versatile , Future Microbiol. 7 ( 2 ): 241 -57 , 2011 ; incorporated herein by multi - component secretion system used by both gram -nega - 60 reference in its entirety ). A . tumefaciens T4SS and those that tive and gram - positive bacteria to secrete proteins , DNA , resemble it fall into the type IVA secretion systems. The and protein -DNA complexes into a wide range of targeted prototypic T -DNA or VirB secretion system of A . tumefa eukaryotic and bacterial cells ( Fronzes, R . et al. , “ The ciens is the most well characterized T4SS . The A . tumefa Structural Biology of Type IV Secretion Systems, ” Nat Rev ciens T4SS transporter complex and others similar to it Microbiol 7 ( 10 ) :703 -14 , 2009 ; Backert, S . and T . F . Meyer. 65 typically consist of 11 VirB proteins ( encoded by the virB1 “ Type IV Secretion Systems and Their Effectors in Bacterial virB11 genes ) and the coupling protein VirD4 , an NTPase Pathogenesis ,” Curr Opin Microbiol 9 ( 2 ): 207 - 17 , 2006 ; all ( Tegtmeyer , N ., S . Wessler and S . Backert , “ Role of the US 9 , 752 , 129 B2 15 16 Cag -Pathogenicity Island Encoded Type Iv Secretion Sys reference in its entirety ). These substrates have been anno tem in Helicobacter Pylori Pathogenesis , " FEBS J . 278 ( 8 ) : tated as large exoprotein , type 5 secretory pathway, repeats 1190 - 202 , 2011 ; incorporated herein by reference in its in - toxin (RTX ) toxins and related Ca² + binding protein , entirety ) . Agrobacterial VirB proteins are grouped into three adhesion AidA , and hypothetical protein amb3422 with categories : core components , pilus -associated components 5 putative functions such as aminomethyltransferase , adhe and energetic components . T4SSs that fall under the type IV sion , and cadherin , consistent with type 1 secretion sub B classification were demonstrated in Legionella pneumo strates in other bacteria . Exemplary sequences are presented phila to consist of twenty -two structural proteins and 5 in the Sequence Listing. Moreover, genes responsible for chaperone proteins (Vogel , J . P . et al. , “ Conjugative Transfer constituting the TISS complex (such as YP _ 422838 , by the Virulence System of Legionella Pneumophila , ” Sci- 10 YP _ 421739, homolog of E . coli HlyB ; E . coli HlyD mem ence 279 (5352 ) : 873 - 6 , 1998 ; Segal, G . , M . et al. , “ Host Cell brane fusion protein homologs in MTB gi: 83311477 , Killing and Bacterial Conjugation Require Overlapping Sets 83312258 , 83312575 , 83313156 with locus tag amb2378 ; of Genes within a 22 -Kb Region of the Legionella Pneu amb3159 ; amb3476 ; amb4057 ; E . coli TolC outer mem mophila Genome, ” Proc Natl Acad Sci . USA 95 ( 4 ) : 1669 - 74 , brane protein homologs in MTB gi : 83311344 ; 83312160 ; 1998 ; all publications incorporated herein by reference in 15 83312256 with locus tag amb2245 : amb3061: amb3157 ) are their entirety ) . present in MTB genome. Type IVA secretion systems and Type IVB secretion FIGS . 1 - 9 show BLAST alignments of nine MTB proteins systems recognize different but overlapping translocation that are either secreted or are part of the cells TiSS secretion signals. A recent study showed that two Brucella effectors machinery with the sequenced genes of other a -proteobac can be translocated by L . pneumophila demonstrating that a 20 teriate and other organisms. Each of the sequences, as type IVB secretion system can recognize translocation sig described by the accession numbers , and the exemplary nals from type IVA secretion system effectors (de Jong, M . sequences ( e . g . , SEQ ID NOS : 1 - 286 ) can be used in the F . et al ., “ Identification of Vcea and Vcec , Two Members of present invention . the Vjbr Regulon That Are Translocated into Macrophagesnages In some embodiments , the protein for secretion can be by the Brucella Type Iv Secretion System ,” Mol Microbiol1. 25 fullfused to C - terminal 200 amino acids of YP _ 420640 (RTX 70 ( 6 ) : 1378 - 96 , 2008 ; incorporated herein by reference in its toxins and related Ca² + binding protein ) , YP _ 423419 (RTX entirety ) . toxins and related Ca2 + binding protein ) , YP _ 422785 The following are examples of T4SS translocation sig (amb3422 ), or other MTB TISS substrates. Alternatively , nals. The A . tumefaciens translocation signal resides in a the proteins in MTB - YP _ 420502 , YP _ 420631 . 1 , hydrophilic C -terminal region with a consensus R - X ( 7 ) - R - 30 YP _ 4206384 .1 , YP _ 420640 . 1, YP _ 421364. 1 , YP _ 422662 . 1, X -R - X -R - X -X (n )motif (SEQ ID NO :290 ) (Vergunst , A . C . YP _ 422785 . 1 , and YP _ 423419. 1 may be used for the fusion et al. , " Positive Charge Is an Important Feature of the by taking their C - terminus ( e . g . , the 200 C - terminal amino C - Terminal Transport Signal of the Virb /D4 - Translocated acids which contains the secretion signal ) to target the Proteins of Agrobacterium , " Proc Natl Acad Sci. USA recombinant fusion protein to the secretion system . An 102 ( 3 ) : 832 - 7 , 2005 ; incorporated herein by reference in its 35 exemplary C - terminal 200 amino acid sequence containing entirety ) . Bartonella has a BID domain and a short posi a secretion signal is that of YP _ 422785 , which has the tively charged tail sequence that together form a bipartite following sequence : C - terminal translocation signal ( Schulein , R . et al ., “ A Bipartite SignalMediates the Transfer of Type IV Secretion Substrates of Bartonella Henselae into Human Cells , ” Proc 40 ( SEQ ID NO : 291 ) Natl Acad Sci . USA 102 ( 3 ): 856 -61 , 2005 ; incorporated DSGTGWIWDPTLNGTSLSVTAHAATTVNGNSTYNGGATADYVIGLGONE herein by reference in its entirety ) . In Helicobacter pylori, there is evidence that both the N - and C -terminal ends of the MVHLGGGADVYAGHGSYNTIGVSDMNFHFLDGGAGGGNTLQWENLNNQA CagA protein have translocation signals . Hohlfeld et al. FDLTGLWNNAVONFDVLDLSHANNNNAVLDLAHLQSMLNGTNAVTGTGN observed that residues 6 - 26 of CagA are important for 45 translocation (Hohlfeld , S . et al ., “ A C - Terminal Transloca - ALWIGGNSVTLAGSGWQANGTAELTVNGQHDSYTQYSNGDTHVYVENH tion Signal Is Necessary , but Not Sufficient for Type IV Secretion of the Helicobacter Pylori Caga Protein , " Mol THVG Microbiol. 59 (5 ) : 1624 - 37 , 2006 ; incorporated herein by As illustrated by the foregoing , bacteria use diverse reference in its entirety ) . Hohlfeld et al. also show that CagA 50 machinery to secrete proteins as a means for interacting with translocation depends on the presence of its 20 C - terminal their environment, which in case of endosymbionts, includes amino acids (Hohlfeld , S . et al ., supra ) . the environment of the host cell. In some embodiments , the Type 1 , 4 , and 5 secretion system genes have also been present invention modifies the morphology or the physiol identified in the MTB Magnetospirillum sp ., strain AMB - 1 ogy of the host cell (as well as organism ) via protein (s ), genome by sequence alignments . AMB - 1 contains 83 genes 55 nucleic acid ( s ) , and /or other factor( s ) secreted from the that are involved in cell motility and secretion (Matsunaga , artificial endosymbiont through a secretion system . T . et al ., " Complete genome sequence of the facultative Modified Artificial Endosymbionts anaerobic magnetotactic bacterium Magnetospirillum sp . In some embodiments , the artificial endosymbionts , such AMB - 1 , " DNA Res. 12 : 157 - 166 , 2005 ; incorporated herein as a -proteobacteria , are modified to achieve a higher level of by reference in its entirety ) . There are at least seven genes 60 recombinant protein secretion . Methods for genetically present in MTB that encode for RTX proteins . Several modifying artificial endosymbionts can use methods well putative TiSS substrates ( YP _ 420631. 1 , YP _ 420638 . 1 , known in the art for recombinantly modifying single celled YP _ 420640 . 1 , YP _ 421364 . 1 , YP _ 422662 . 1 , YP _ 422785 . 1 , organisms. Typically , the artificial endosymbiont is geneti and YP _ 423419 . 1 ) have been identified in MTB ( M . mag - cally modified to improve secretion of target molecules from neticum AMB - 1 ) (Linhartova et al ., “ RTX proteins: a highly 65 the artificial endosymbiont into the host cell . Alternatively, diverse family secreted by a common mechanism , ” FEMS the artificial endosymbiont is engineered to increase trans Microbiol. Rev. 34 : 1076 - 1112 , 2010 ; incorporated herein by port of a target molecule from the host into the artificial US 9 , 752 , 129 B2 17 18 endosymbiont . Modifications may also involve increasing engineered into the artificial endosymbiont. The target pro production of proteins or RNA through changing promoter tein is fused N -terminally to a 104 residue, minimal region or ribosome binding sequences, or deleting or silencing of comprising: certain genes in the artificial endosymbiont. In some embodiments , the flagellar proteins of an artifi- 5 cial endosymbiont are modified so that the flagellar proteins ( SEQ ID NO : 293 ) are no longer expressed . Flagellar proteins have high homol GSDGNDLIQGGKGADFIEGGKGNDTIRDNSGHNTFLFSGHFGQDRIIGY ogy to bacterial secretion systems suggesting a common QPTDRLVFQGADGSTDLRDHAKAVGADTVLSFGADSVTLVGVGLGGLWS evolutionary ancestor. In some embodiments , the flagellar proteins of an artificial endosymbiont are modified to create 10 EGVLIS . a secretion system . In some embodiments , the single- celled This fusion of target protein and minimal region allows organism is modified so that it can no longer synthesize an secretion of recombinant protein . It is interesting to note that essential molecule that is preferably provided by the eukary - there are several proteins exhibiting substantial sequence otic host cell. In some embodiments , the single - celled organ - homology to P . fluorescens lipase TliA such as YP _ 420640 , ism is genetically modified so that its cell cycle is coordi YP _ 422785 , YP _ 421364 , YP _ 420631 , and others that are nated with the cell cycle of the eukaryotic host cell so that present in the MTB genome (see , e. g. , FIG . 9 and sequences copy number of the single - celled organism can be main in Sequence Listing ) . Most of this homology is located in the tained at a sufficient level to impart the phenotype to C -terminal domain of the proteins , which contains the daughter cells . 20 transport signal . In some embodiments , the MTB lipase Molecular biology tools have been developed for genetic 2 ABC transport system is used to transport recombinant manipulationsm of MTB most extensively in AMB - 1 and M . proteins by fusing target protein to a C - terminal signal of gryphiswaldense strain MSR - 1 ( reviewed in Jogler, C . and Schtiler, D . in Magnetoreception and Magnetosomes in MTB lipase ABC transport system substrates (also see FIG . Bacteria , New York , Springer, p 134 - 138 , 2007 ; incorpo 9 , accession numbers and corresponding sequences in the rated herein by reference ). The genomes of two other 25 Sequence Listing for sequence homology ). In some embodi Magnetospirillum strains and Magnetococcus sp . strain ments , the C - terminal signal sequence comprises : MC - 1 have also been recently sequenced . In some embodi ments, genes from these strains or other MTB strains, ( SEQ ID NO : 294 ) presently culturable or unculturable , sequenced or unse GAIGGAGAIPGITLVGNAGNDDIIGTNGNDLLLGGKGGATYRFSGGGCG quenced , know or unknown , can be used in the present invention . SGGGWSIVOSDINDVISAGAGDDVIYGDARLVNGNIQITGSGNDVLDGG Several engineering approaches are used to modify arti SGNDQIHGGAGNDTIIGGTGDDVMFGDQGNDTFLFDFGFGHDVVDGGRG ficial endosymbionts , including : ( 1 ) engineering into the artificial endosymbiont dedicated secretion systems that SNWTDTLDLTHDNOISSVNIEGVSGWAVSVDAOGHHVAQATNGAHDANG naturally exist in other bacteria ; ( 2 ) engineering cell enve - » lope mutations into the artificial endosymbiont so that it TIVTNHDGSODTIEFHNVEKVVW . alters the outer membrane or peptidoglycan layer perme InIn somesom embodiments , the RaxA , RaxB and RaxC genes ability ( e . g . , Shin , H . and Chen , R . R . , “ Extracellular recom - of Xanthomonas oryzae pv . oryzae are recombinantly binant protein production from and Escherichia coli lpp expressed in the artificial endosymbiont. The target protein deletion mutant, ” Biotechnol Bioeng . 101 : 1288 - 96 , 2008 ; 40 is fused to RaxST, which is the recognition sequence for the incorporated herein by reference in its entirety ); and ( 3 ) RaxABC secretory system . In some embodiments , the co - expression of a lysis - promoting protein that removes the RaxST recognition sequence comprises : outer membrane (Ni , Y . and Chen , R ., “ Extracellular recom binant protein production from and Escherichia coli, ” Bio technol Lett. 31: 1661 -70 , 2009 ; incorporated herein by ( SEQ ID NO : 295 ) reference in its entirety ) . MLMGMSEEHPSNVQIDDAQRVRLLRAVFDAYYQNRQELGTVFDTNRAWC In some embodiments , the E . coli a -haemolysin trans porter genes HlyB and HlyD are recombinantly expressed in SRLTGLARLFPRSRMICCVRDVGWIVDSFERLAQSQPLRLSALFGYDPE the artificial endosymbiont. Target proteins are then engi DSVSMHADLLTAPRGWGYALDGLRQAFYGDHADRLLLLRYDTLAQRPA neered by fusing them with T1SS substrate secretion signal, 3 which is located in C -terminal of HlyA to target them to the QAMEQVYAFLQLPAFAHDYAGVQAEAERFDAALQMPGLHRVRRGVHYVP a -haemolysin transporter system . The Hly A secretion signal RRSVLPPALFDOLQELAFWESAPSHGALLV . comprises : In some embodiments , the target protein is fused at the 55 N -terminal end to a Sec - dependent signal sequence , and the ( SEO ID NO : 292 ) C -terminal end of the target protein is fused to a B -motif . GNSLAKNVLSGGKGNDKLYGSEGADLLDGGEGNDLLKGGYGNDIYRYLS Translocation of such target protein fusions is a two -step process . The target protein is transported into the periplasm GYGHHIIDDDGGKDDKLSLADIDFRDVAFRREGNDLIMYKAEGNVLSIG using Sec -dependent transport, whereupon the B -motif HKNGITFRNWFEKESGDISNHQIEQIFDKDGRVITPDSLKKALEYOOSN 60 forms a transmembrane pore in the outer -membrane through which the target protein is secreted from the periplasmic NKASYVYGNDALAYGSODNLNPLINEISKIISAAGNFDVKEERAAASLL space . The B -motif is cleaved , allowing translocation of the target protein . QLSGNASDFSYGRNSITLTASA . In some embodiments , the target protein is fused to an In some embodiments, the TiSS from Pseudomonas 65 autotransporter ( target protein replaced the passenger fluorescens is used to transport recombinant proteins. The domain region ( 29 to 685 amino acids ), such as the YfaL T1SS genes such as TliA for the lipase ABC transporter are autotransporter (Ko , H . J . et al. , “ Functional cell surface US 9 , 752 , 129 B2 19 20 display and controlled secretion of diverse agarolytic - continued enzymes by Escherichia coli with a novel ligation - indepen dent cloning vector based on the autotransporter YfaL ,” App NTTLNGGEQWMHEGAIATGTVINDKGWOWKPGTVATDTVNTGAEGGP Environ Microbiol. 78 : 3051 - 3058 , 2012 ; incorporated DAENGDTGOFVRGDAVRTTINKNGROIVRAEGTANTTVVYAGGDOTVHG herein by reference in its entirety ) . The YfaL autotransporter 5 sequence comprises : HALDTTLNGGYQYVHNGGTASDTVVNSDGWQIVKNGGVAGNITVNQKGR LQVDAGGTATNVILKQGGALVISTAATVTGINRLGAFSVVEGKADNVVL ( SEQ ID NO : 296 ) MRIIFLRKEYLSLLPSMIASLFSANGVAAVTDSCQGYDVKASCQASROS ENGGRLDVLIGHTATNTRVDDGGTLDVRNGGTATTVSMGNGGVLLADSG LSGI TQDWSIADGQWLVFSDMTNNASGGAVFLOQGAEFSLLPENETGMT AAVSGTRSDGKAFSIGGGQADALMLEKGSSFTLNAGDTATDTTVNGGLF LPANNTVTGEYNNGGAIFAKENSTLNLTDVIFSGNVAGGYGGAIYSSGI TARGGTLAGTTTLNNGAILTLSGKTVNNDTLTIREGDALLQGGSLTGNG NDTGAVDLRVTNAMFRNNIANDGKGGAIYTINNDVYLSDVIFDNNQAYT SVEKSGSGTLTVSNTTLTOKAVNLNEGTLTLNDSTVTTDVIAQRGTALK STSYSDGDGGAIDVTDNNSDSKHPSGYTIVNNTAFTNNTAEGYGGAIYT LTGSTVLNGAIDPINVTLASGATWNIPDNATVQSWDDLSHAGQIHFTS NSVTAPYLIDISVDDSYSQNGGVLVDENNSAAGYGDGPSSAAGGFMYLG TRTGKFVPATLKVKNLNGONGTISLRVRPDMAQNNADRLVIDGGRATGK LSEVTFDIADGKTLVIGNTENDGAVDSIAGTGLITKTGSGDLVLNADNN TILNLVNAGNSASGLATSGKGIQVVEAINGATTEEGAFVQGNRLQAGAF DFTGEMQIENGEVTLGRSNSLMNVGDTHCQDDPODCYGLTIGSIDOYON 20 NYSLNRDSDESWYLRSENAYRAEVPLYASMLTQAMDYDRIVAGSRSHQT QAELNVGSTOQTFVHALTGFONGTLNIDAGGNVTVNOGSFAGIIEGAGQ GVNGENNSVRLSIQGGHLGHDNNGGIARGATPESSGSYGFVRLEGDLMR LTIAQNGSYVLAGAQSMALTGDIWDDGAVLSLEGDAADLTALQDDPOS TEVAGMSVTAGVYGAAGHSSVDVKDDDGSRAGTVRDDAGSLGGYLNLVH IVLNGGVLDLSDFSTWOSGTSYNDGLEVSGSSGTVIGSQDVVDLAGGDN 25 ISSGLWADIVAQGTRHSMKAS SDNNDFRARGWGWLGSLETGLPFSITDN LHIGGDGKDGVYVVVDASDGOVSLANNNSYLGTTQIASGTLMVSDNSQL LMLEPQLQYTWQGLSLDDGKDNAGYVKFGHGSAQHVRAGFRLGSHNDMT GDTHYNROVIFTDKQQESVMEITSDVDTRSDAAGHGRDIEMRADGEVAV FGEGTSSRAPLRDSAKHSVSELPVNWWVOPSVIRTFSSRGDMRVGTSTA DAGVDTQWGALMADSSGQHQDEGSTLIKTGAGTLELTASGTTOSAVRVE 30 GSGMTFSPSQNGTSLDLQAGLEARVRENITLGVQAGYAHSVSGSSAEGY EGTLKGDVADILPYASSLWVGDGATFVTGADQDIOSIDAISSGTIDISD NGQATLNVTF . In some embodiments , the translocase of the outer mito GTVLRLTGODTSVALNASLFNGDGILVNATDGVILTGELNINLETDSLT chondrialmembrane ( TOM complex ) is engineered into the YLSNVTVNGNLTNTSGAVSLONGVAGDTLTVNGDYTGGGTLLLDSELNG 35 artificial endosymbiont. The TOM complex includes the receptors Tom20 , Tom22 , Tom70 and the channel- forming DDSVSDQLVMNGNTAGNITVVVNSITGIGEPTSTGIKVVDFAADPTQFQ protein Tom 40 , and several other small subunits ( reviewed NNAQFSLAGSGYVNMGAYDYTLVEDNNDWYLRSQEVTPPSPPDPDPTPD in Hoogenraad , N . J . et al. , Biochem Biophy Acta 1592 : 97 - 105 , 2002 ; Neupert W . and Herrmann , J . M . , Annu Rev PDPTPDPDPTPDPEPTPAYQPVLNAKVGGYLNNLRAANQAFMMERRDHA 40 Biochem . 76 :723 -749 , 2007; and Chacinska , A . et al. , Cell GGDGQTLNLRVIGGDYHYTAAGQLAQHEDTSTVQLSGDLFSGRWGTDGE 138 :628 - 44 , 2009 ; all publications incorporated herein by reference ) . Tom20 recognizes the substrate and transfers to WMLGIVGGYSDNQGDSRSNMTGTRADNQNHGYAVGLTSSWFQHGNQKQG centrally located Tom22 , where the substrate is inserted into AWLDSWLQYAWFSNDVSEQEDGTDHYHSSGIIASLEAGYQWLPGRGVVI 45 the Tom40 channel . Upon substrate import , TOM complex forms a complex with the translocase of the inner membrane EPQAQVIYQGVQODDFTAANRARVSQSOGDDIQTRLGLHSEWRTAVHVI ( TIM complex ) ( Chacinska , A . et al. , EMBO J 22 :5370 -81 , PTLDLNYYHDPHSTEIEEDGSTISDDAVKORGEIKVGVTGNISORVSLR 2003 ; incorporated herein by reference ) . The TIM complex consists of four integral membrane proteins , Tim23 , Tim17 , GSVAWQKGSDDFAQTAGFLSMTVKW . 50 Tim50 , and Tim21 . Tim23 forms the protein - conducting In some embodiments , a protease is included to cleave the channel of the translocase and is tightly associated with B -motif from the target protein , such as for example , a Tomil, whereas, limou acts as regulator for the lim23 tobacco etch virus protease , E . coli serine protease Pet, and channel and Tim21 transiently interacts with the TOM serine protease autotransporters of the Enterobacteriaceae complex via Tom22 (Milisav , I . et al . , “Modular structure of (SPATES ), that releases passenger domain from the B -do - 55 the Tim23 preprotein translocase of mitochondria ,” J Biol main , without requiring exogenous protease . In some Chem . 276 : 25856 - 25861, 2001; incorporated herein by ref embodiments , an E . coli autotransporter Antigen 43 ( Ag43) erence ). is used with the target protein . The Antigen 43 autotrans Nucleic Acids porter sequence comprises : In another aspect , the present invention relates to the 60 nucleic acids that encode, at least in part, the individual peptides, polypeptides and proteins secreted in the methods ( SEO ID NO : 297 ) of the present invention . In some embodiments, the nucleic MKRHLNTCYRLVWNHMTGAFVVASELARARGKRGGVAVALSLAAVTSLP acids may be natural, synthetic or a combination thereof. In some embodiments , the nucleic acids of the invention also VLAADIVVHPGETVNGGTLANHDNQIVFGTTNGMTISTGLEYGPDNEAN 65 include the nucleic acids that are secreted from the artificial TGGQWVQDGGTANKTTVTSGGLQRVNPGGSVSDTVISAGGGOSLQGRAV endosymbiont into the host cell. The nucleic acids of the invention may be RNA , mRNA, DNA or cDNA . US 9 , 752 , 129 B2 21 22 In some embodiments , the nucleic acids of the invention the complement of the polynucleotides including a nucleo also include expression vectors , such as plasmids, or viral tide sequence that has at least about 80 % , more typically at vectors, or linear vectors , or vectors that integrate into least about 90 % , and even more typically at least about 95 % , chromosomal DNA . Expression vectors can contain a sequence identity to a polynucleotide encoding a polypep nucleic acid sequence that enables the vector to replicate in 5 tide recited above . The polynucleotide can be DNA ( ge one or more selected host cells . Such sequences are well nomic , cDNA , amplified , or synthetic ) or RNA . Methods known for a variety of cells . The origin of replication from and algorithms for obtaining such polynucleotides are well the plasmid PBR322 is suitable for most Gram - negative known to those of skill in the art and can include , for bacteria . In eukaryotic host cells , e . g . , mammalian cells , the example , methods for determining hybridization conditions expression vector can be integrated into the host cell chro - 10 which can routinely isolate polynucleotides of the desired mosome and then replicate with the host chromosome sequence identities . Similarly , vectors can be integrated into the chromosome of Nucleic acids which encode protein analogs or variants in prokaryotic cells . accordance with this invention ( i . e . , wherein one or more Expression vectors also generally contain a selection amino acids are designed to differ from the wild type gene, also termed a selectable marker . Selectable markers 15 polypeptide ) may be produced using site directed mutagen are well - known in the art for prokaryotic and eukaryotic e sis or PCR amplification in which the primer ( s ) have the cells , including host cells of the invention . Generally, the desired point mutations . For a detailed description of suit selection gene encodes a protein necessary for the survival able mutagenesis techniques, see Sambrook et al. , Molecular or growth of transformed host cells grown in a selective Cloning : A Laboratory Manual, Cold Spring Harbor Labo culture medium . Host cells not transformed with the vector 20 ratory Press , Cold Spring Harbor, N . Y . ( 1989 ) and / or Cur containing the selection gene will not survive in the culture rent Protocols in Molecular Biology , Ausubel et al. , eds , medium . Typical selection genes encode proteins that ( a ) Green Publishers Inc . and Wiley and Sons , N . Y ( 1994 ) . confer resistance to antibiotics or other toxins, e. g ., ampi Chemical synthesis using methods well known in the art, cillin , neomycin , methotrexate , or tetracycline, ( b ) comple - such as that described by Engels et al. , Angew Chem Intl Ed . ment auxotrophic deficiencies , or ( c ) supply critical nutri- 25 28 :716 - 34 , 1989 , may also be used to prepare such nucleic ents not available from complex media , e . g . , the gene acids. encoding D - alanine racemase for Bacilli . In some embodi In some embodiments , the nucleic acids encode a variant ments , an exemplary selection scheme utilizes a drug to ( i . e . , recombinant variant) of the polypeptides of the present arrest growth of a host cell. Those cells that are successfully invention . As used herein " variant” or “ recombinant variant" transformed with a heterologous gene produce a protein 30 refers to any polypeptide differing from naturally occurring conferring drug resistance and thus survive the selection polypeptides by amino acid insertions , deletions, and sub regimen . Other selectable markers for use in bacterial or stitutions, created using recombinant DNA techniques . eukaryotic ( including mammalian ) systems are well - known Guidance in determining which amino acid residues may be in the art. In some embodiments , the selectable marker is the replaced , added , or deleted without abolishing activities of target protein or encoded by the nucleic acid secreted by the 35 interest, such as enzymatic or binding activities , may be artificial endosymbiont into the host cell. found by comparing the sequence of the particular polypep In some embodiments , the expression vector for produc - tide with that of homologous peptides and minimizing the ing a heterologous polypeptide also contains a promoter, number of amino acid sequence changes made in regions of which can be a constitutive or an inducible promoter, that is high homology . recognized by the host RNA polymerase and is operably 40 In some embodiments , amino acid “ substitutions" for linked to the nucleic acid encoding the target protein . creating variants are preferably the result of replacing one Inducible or constitutive promoters ( or control regions ) with amino acid with another amino acid having similar structural suitable enhancers , introns, and other regulatory sequences and /or chemical properties, i. e . , conservative amino acid are well -known in the art. replacements Amino acid substitutions may be made on the In some embodiments , it may be desirable to modify the 45 basis of similarity in polarity , charge , solubility , hydropho polypeptides of the present invention . One of skill will bicity , hydrophilicity , and / or the amphipathic nature of the recognize many ways of generating alterations in a given residues involved . For example , nonpolar (hydrophobic ) nucleic acid construct to generate variant polypeptides Such amino acids include alanine , leucine , isoleucine , valine , well -known methods include site - directed mutagenesis , proline , phenylalanine , tryptophan , and methionine ; polar PCR amplification using degenerate oligonucleotides , expo - 50 neutral amino acids include glycine , serine , threonine , cys sure of cells containing the nucleic acid to mutagenic agents teine , tyrosine , asparagine , and glutamine ; positively or radiation , chemical synthesis of a desired oligonucleotide charged ( basic ) amino acids include arginine, lysine, and ( e . g . , in conjunction with ligation and/ or cloning to generate histidine ; and negatively charged ( acidic ) amino acids large nucleic acids ) and other well -known techniques ( see , include aspartic acid and glutamic acid . e . g ., Gillam and Smith , Gene 8 :81 - 97 , 1979 ; Roberts et al ., 55 In some embodiments , amino acid “ insertions” or “ dele Nature 328 : 731 -734 , 1987 ) . tions” for the variants are typically in the range of about 1 In some embodiments , the recombinant nucleic acids to 5 amino acids. The variation allowed may be experimen encoding the polypeptides of the invention are modified to tally determined by systematically making insertions , dele provide preferred codons which enhance translation of the tions, or substitutions of amino acids in a polypeptide nucleic acid in a selected organism . 60 molecule using recombinant DNA techniques and assaying The polynucleotides of the invention also include poly - the resulting recombinant variants for activity . nucleotides including nucleotide sequences that are substan - Alternatively , in some embodiments, where alteration of tially equivalent to the polynucleotides of the invention . function is desired , insertions, deletions or non -conservative Polynucleotides according to the invention can have at least alterations can be engineered in the nucleic acid to produce about 80 % , more typically at least about 90 % , and even 65 altered polypeptides or chimeric polypeptides, e .g ., variant more typically at least about 95 % , sequence identity to a polypeptides. Such alterations can , for example , alter one or polynucleotide of the invention . The invention also provides more of the biological functions or biochemical character US 9 ,752 , 129 B2 23 24 istics of the polypeptides of the invention . For example , such Hanna , J . et al. , “ Treatment of sickle cell anemia mouse alterations may change polypeptide characteristics such as model with iPS cells generated from autologous skin , " ligand -binding affinities or degradation /turnover rate . Fur Science 318 : 1920 -23 , 2007 ; Holtzman , D . M . et al ., ther, such alterations can be selected so as to generate “ Expression of human apolipoprotein E reduces amyloid - ß polypeptides that are better suited for expression , scale up , 5 deposition in a mouse model of Alzheimer ' s disease . ” J Clin and the like in the host cells chosen for expression . Invest. 103 (6 ): R15 - R21, 1999 ; Warren , R . S . et al. , “ Regu Alternatively , recombinant nucleic acids encoding these lation by vascular endothelial growth factor of human colon same or similar polypeptides may be synthesized or selected by making use of the “ redundancy ” in the genetic code . cancer tumorigenesis in a mouse model of experimental Various codon substitutions, such as the silent changes 10 liver metastasis , ” J Clin Invest. 95 : 1789 - 1797 , 1995 ; each which produce various restriction sites , may be introduced publication incorporated herein by reference ) Animal cells to optimize cloning into a plasmid or viral vector or expres include , for example, fibroblasts , epithelial cells ( e . g . , renal, sion in a particular prokaryotic or eukaryotic system . Muta mammary, prostate , lung ), keratinocytes , hepatocytes , tions in the polynucleotide sequence may be reflected in the adopicytes , endothelial cells , hematopoietic cells . In some polypeptide or domains of other peptides added to the 15 emembodiments , the animal cells are adult cells ( e . g . , termi polypeptide to modify the properties of any part of the nally differentiated , dividing or non -dividing ) or embryonic polypeptide , to change characteristics such as ligand -bind cells ( e .g ., blastocyst cells , etc .) . ing affinities, or degradation / turnover rate . In some embodiments , the host cell is a cancer cell , In some embodiments , nucleic acid or polynucleotides including human cancer cells . Many cancer cell lines that are encoding the novel nucleic acids are changed via site - 20 well known to those of ordinary skill in the art can be used directed mutagenesis . This method uses oligonucleotide as a host cell , including common epithelial tumor cell lines sequences that encode the polynucleotide sequence of the such as Coco - 2 , MDA -MB231 and MCF7 , and non - epithe desired amino acid variant, as well as a sufficient adjacent lial tumor cell lines, such as HT- 1080 and HL60 , the nucleotide on both sides of the changed amino acid to form NCI60 - cell line panel ( see, e . g . , Shoemaker, R . , " The NC160 a stable duplex on either side of the site of being changed . 25 human tumor cell line anticancer drug screen , " Nature In general, the techniques of site -directed mutagenesis are Reviews Cancer 6 :813 - 23 , 2006 ; incorporated herein by well known to those of skill in the art and this technique is reference ). Additionally , those of ordinary skill in the art are exemplified by publications such as, Edelman et al ., DNA familiar with obtaining cancer cells from primary tumors . 2 : 183 , 1983 . A versatile and efficient method for producing Cancer cells also include , for example , solid tumor cell site - specific changes in a polynucleotide sequence is 30 types , hematopoietic cancer cells , carcinomas , sarcomas , described in Zoller and Smith , Nucleic Acids Res. 10 :6487 - leukemias , lymphomas , gliomas, as well as specific tissue 500 , 1982 . related cancers such as prostate cancer, breast cancer, lung In some embodiments , PCR may also be used to create cancer, colorectal cancer, pancreatic cancer, melanoma , amino acid sequence variants of the novel nucleic acids . glioblastoma, liver cancer, and the NCI 60 panel of cancer When small amounts of template DNA are used as starting 35 cell lines . material , primer ( s ) that differs slightly in sequence from the In some embodiments , the host cells are stem cells . corresponding region in the template DNA can generate the Variety of stem cells types are known in the art and can be desired amino acid variant. PCR amplification results in a used as host cells , including for example , embryonic stem population of product DNA fragments that differ from the cells , inducible pluripotent stem cells , hematopoietic stem polynucleotide template encoding the target at the position 40 cells , neural stem cells , epidermal neural crest stem cells , specified by the primer. The product DNA fragments replace mammary stem cells , intestinal stem cells , mesenchymal the corresponding region in the plasmid and this gives the stem cells , olfactory adult stem cells , testicular cells , and desired amino acid variant. progenitor cells ( e . g ., neural, angioblast , osteoblast, chond A further technique for generating amino acid variants is roblast , pancreatic, epidermal, etc. ) . the cassette mutagenesis technique described in Wells et al ., 45 In some embodiments , the host cell is a cell found in the Gene 34 : 315 , 1985 ; and other mutagenesis techniques well circulatory system of a mammal, including humans . Exem known in the art, such as, for example , the mutagenesis plary circulatory system cells include, among others , red techniques described in Sambrook et al ., supra , and Current blood cells , platelets , plasma cells , T - cells , natural killer Protocols in Molecular Biology , referenced herein . cells , or the like , and precursor cells of the same. As a group , Host Cells 50 these cells are defined to be circulating host cells of the In another aspect , the present invention provides a invention . The present invention may be used with any of eukaryotic host cell containing an artificial endosymbiont, these circulating cells . In some embodiments , the host cell is wherein the artificial endosymbiont imparts a phenotype to a T -cell . In some embodiments , the host cell is a B - cell. In the host cell by secreting proteins , nucleic acids , and/ or some embodiments , the host cell is a neutrophil . In some other factors from the artificial endosymbiont into the host 55 embodiments , the host cell is a megakaryocyte . cell. In some embodiments , the artificial endosymbiont is In some embodiments , the host cell is a plant cell . In some heritable. embodiments , the host cells are cells of monocotyledonous In the present invention , various eukaryotic cells can be or dicotyledonous plants including, but not limited to , used as the host cell . In some embodiments , the host cells of maize , wheat, barley , rye , oat, rice , soybean , peanut, pea , the invention are animal cells. In some embodiments , the 60 lentil and alfalfa , cotton , rapeseed , canola , pepper, sun host cells are mammalian cells , such as that of mouse , rat, flower, potato , tobacco , tomato , eggplant, eucalyptus , a tree , rabbit, hamster, porcine, bovine , feline , or canine . In some an ornamental plant, a perennial grass , or a forage crop . In embodiments , the mammalian cells are cells of primates , some embodiments , the host cells are algal, including but including but not limited to , monkeys , chimpanzees , goril - not limited to algae of the genera Chlorella , Chlamydomo las, and humans. In some embodiments , the mammalians 65 nas, Scenedesmus, Isochrysis, Dunaliella , Tetraselmis , Nan cells are mouse cells , as mice routinely function as a model nochloropsis , or Prototheca . In some embodiments , the host for other mammals , most particularly for humans (see , e. g ., cells are fungi cells , including but not limited to , fungi of the US 9 , 752 , 129 B2 25 26 genera Saccharomyces , Klyuveromyces, Candida , Pichia , baculovirus, protein factors , genetic knock - ins, etc . ) ( see , Debaromyces , Hansenula , Yarrowia , Zygosaccharomyces , e . g . , Salminen , M . et al. , “ Improvement in nuclear entry and or Schizosaccharomyces. transgene expression of baculoviruses by disintegration of In some embodiments , at least one gene from the host cell microtubules in human hepatocytes, ” J Virol. 79 ( 5 ) :2720 - 8 , is genetically altered . In some embodiments ,mutual nutri - 5 2005 ; Modalsli, K . R . et al. , “Microinjection of HEP - 2 cells tional dependence ( biotrophy ) may be established between with coxsackie B1 virus RNA enhances invasiveness of the artificial endosymbiont and the host cell by genetic Shigella flexneri only after prestimulation with UV - inacti modification of the host cell , using the appropriate molecu - vated virus, ” APMIS 101 :602 -6 , 1993 ; Hayward , R . D . and lar biology techniques specific to the target host cell type Koronakis , V . , “ Direct nucleation and bundling of actin by known to those of ordinary skill in the art, to create host cell 10 the SipC protein of invasive Salmonella , ” EMBO J . dependence on the artificial endosymbiont for some essen - 18 :4926 -34 , 1999 ; Yoshida , S . et al. , “ Shigella deliver an tial macromolecule thus establishing the environmental effector protein to trigger host microtubule destabilization , pressures for biotrophy . In some embodiments , nutritional which promotes Racl activity and efficient bacterial inter dependence for an artificial endosymbiont on the host cell nalization , ” EMBO J . 21 : 2923 - 35 , 2002 ; Bigildeev et al. , can be established by genetically altering the host cell to 15 Exp Hematol. 39 : 187 , 2011 ; Finlay, B . B . and Falkow , S ., eliminate the ability of the host cell to synthesize various “ Common themes in microbial pathogenicity revisited . metabolites, cofactors, vitamins, nucleotides, or other essen - Microbiol Mol Biol Rev. 61: 136 -69 , 1997 ; each publication tial molecules . In such embodiments , the essential molecule incorporated herein by reference in its entirety ) . may be provided by the artificial endosymbiont. For In some embodiments , the related process of macropino example , an exemplary host cell gene for modification is the 20 cytosis or " cell drinking, " a method numerous bacteria and gene encoding the enzymeserine hydroxymethyltransferase , viruses employ for intracellular entry, can be used to intro which converts serine into glycine at the terminus of the duce the artificial endosymbiont into the host cell (Zhang , 3 -phosphoglycerate biosynthetic pathway for amino acid In : Molecular Imaging and Contrast Agent Database (MI production . Inactivation of this gene in the host cell would CAD ) [database online ] (2004 ); Bethesda (Md . ) : National create a host cell dependent on or favoring an artificial 25 Library of Medicine (US ) , NCBI 2004 - 2011 ; each publica endosymbiont that produces glycine . tion incorporated herein by reference ) . Various protocols can Methods of Introducing Artificial Endosymbionts into be employed to induce cells to take up bacteria . Several Host Cells agents , such as nucleic acids, proteins , drugs and organelles The single - celled organisms of the invention can be have been encapsulated in liposomes and delivered to cells introduced into host cells by a number of methods known to 30 (Ben -Haim , N . et al ., “ Cell -specific integration of artificial those of skill in the art including, but not limited to , organelles based on functionalized polymer vesicles, ” Nano microinjection , natural phagocytosis , induced phagocytosis , Lett. 8 (5 ): 1368 - 73 , 2008 ; Lian , W . et al ., “ Intracellular macropinocytosis , other cellular uptake processes , liposome delivery can be achieved by bombarding cells or tissues with fusion , erythrocyte ghost fusion , electroporation , receptor accelerated molecules or bacteria without the need for mediated methods, and the like (see , e . g. , Microinjection 35 carrier particles, ” Exp Cell Res. 313 (1 ) :53 -64 , 2007 ; Heng , and Organelle Transplantation Techniques , Celis et al ., eds . , B . C . and Cao , T , “ Immunoliposome- mediated delivery of Academic Press, New York ( 1986 ), and references cited neomycin phosphotransferase for the lineage - specific selec therein ; incorporated herein by reference in its entirety ) . tion of differentiated / committed stem cell progenies : Poten In some embodiments , a single -celled organism is intro tial advantages over transfection with marker genes, fluo duced into the host cell by microinjection into the cytoplasm 40 rescence - activated and magnetic affinity cell - sorting, ” Med . of the host cell . A variety of microinjection techniques are Hypotheses 65 ( 2 ) : 334 - 6 , 2005 ; Potrykus , Ciba Found Symp . known to those skilled in the art . Microinjection is an Vol. 1 54 : 198 , 1990 ; each publication incorporated herein by efficient transfer technique (essentially 100 % ) and has no reference ). This method is inexpensive, relatively simple cell type restrictions (Id . ; Xi, Z . and Dobson , S ., " Charac and scalable . Additionally , liposome uptake can be enhanced terization of Wolbachia transfection efficiency by using 45 by manipulation of incubation conditions, variation of lipo microinjection of embryonic cytoplasm and embryo homo - some charge , receptor mediation , and magnetic enhance genate, ” Appl Environ Microbiol. 71 ( 6 ): 3199 - 204 , 2005 ; ment ( see , e . g . , Pan et al. , Int J Pharm . 358 : 263, 2008 ; Goetz , M . et al ., “ Microinjection and growth of bacteria in Sarbolouki, M . N . and Toliat , T. , “ Storage stability of the cytosol of mammalian host cells , ” Proc Natl Acad Sci . stabilized MLV and REV liposomes containing sodium USA 98 : 12221- 6 , 2001 ; each publication incorporated 50 methotrexate (aqueous & lyophilized ) , ” J Pharm Sci herein by reference ) . Techno. 52( 10 ) : 23 -27 , 1998 ; Elorza , B . et al. , “ Comparison Naturally phagocytotic cells have been show to take up of particle size and encapsulation parameters of three lipo bacteria , including MTB (Burdette , D . L . et al ., “ Vibrio somal preparations .” J Microencapsul. 10 ( 2 ): 237 - 48 , 1993 ; VopQ induces P13 -kinase independent autophagy and Mykhaylyk , O . et al. , “ Liposomal Magnetofection , ” Meth antagonizes phagocytosis , ” Mol Microbiol. 73 :639 , 2009 ; 55 ods Mol Bio . 605 :487 -525 , 2010 ; each publication incorpo Wiedemann , A . et al ., “ Yersinia enterocolitica invasin trig rated herein by reference ) . gers phagocytosis via B1 integrins, CDC42Hs and WASp in Erythrocyte -mediated transfer is similar to liposome macrophages , " Cell Microbiol. 3 :693 , 2001 ; Hackam , D . J . fusion and has been shown to have high efficiency and et al. , “ Rho is required for the initiation of calcium signaling efficacy across all cell types tested (Microinjection and and phagocytosis by Fcy receptors in macrophages, " J Exp 60 Organelle Transplantation Techniques ; Celis et al. Eds . ; Med . 186 ( 6 ) :955 -66 , 1997 ; Matsunaga , T . et al. , “ Phagocy - Academic Press : New York ( 1986 ) , incorporated herein by tosis of bacterial magnetite by leucocytes . Appl Microbiol reference ) . Typically erythrocytes are loaded by osmotic Biotech. 31( 4 ): 401 -5 , 1989; all publications incorporated shock methods or electroporation methods ( Schoen , P . et al ., herein by reference ). " Gene transfer mediated by fusion protein hemagglutinin In some embodiments , non - phagocytotic cell types can be 65 reconstituted in cationic lipid vesicles , ” Gene Therapy induced to endocytose bacteria by co - culturing with various 6 : 823 - 32 , 1999 ; Li, L . H . et al. , “ Electrofusion between factors : media , chemical factors, and biologic factors ( e . g ., heterogeneous - sized mammalian cells in a pellet : potential US 9 , 752 , 129 B2 27 28 applications in drug delivery and hybridoma formation , factors can alter gene transcription or translation, post trans Biophysical J. 71: 479 -86 , 1996 ; Carruthers , A . and Mel - lational modifications , host cell differentiation , remodeling, chior, D . L ., “ A rapid method of reconstituting human proliferation , sensitivity or response to external and / or inter erythrocyte sugar transport proteins, ” Biochemistry nal stimuli , metabolic , anabolic or other biochemical pro 23 : 2712 - 18 , 1984 ; each publication incorporated herein by 5 cesses . In some embodiments , the artificial endosymbiont reference ) . Alternatively, erythrocytes may be loaded indi can control host cells through expression , availability , and rectly by loading hematopoietic progenitors with single delivery of certain transcription factors , growth factors , celled organisms and inducing them to differentiate and cytokines, signaling pathway molecules , or other recombi expand into erythrocytes containing single - celled organ - nant proteins. The artificial endosymbiont may also intro isms. 10 duce a desirable phenotype to the host cell through the In some embodiments , electroporation , a commonly used , proteins, nucleic acids or other factors that are secreted into inexpensive method to deliver factors to cells , can be used the host cell from the artificial endosymbiont. to introduce artificial endosymbionts into host cells In some embodiments , the proteins , nucleic acids, or other ( Potrykus , I. , “ Gene transfer methods for plants and cell factors secreted from an artificial endosymbiont into the host cultures, " Ciba Found Symp 154 : 198 - 208 ; discussion 208 - 15 cell can be used to affect cell viability , proliferation , differ 112 , 1990 ; Wolbank , S . et al. , “ Labeling of human adipose - entiation , de - differentiation , growth , angiogenesis , neuro derived stem cells for non - invasive in vivo cell tracking, " genesis , osteogenesis, and detoxification . In some embodi Cell Tissue Bank 8 : 163- 177 , 2007 ; each publication incor ments , the artificial endosymbiont can be used to label the porated herein by reference ) . cell , treat a pathology or deficiency, induce cell death , In some embodiments , a host cell that naturally endocy - 20 induce wound healing ,modify cell signaling, modify gene toses bacteria ( e . g . , Chinese hamster ovary (CHO ) is used . expression , neutralize intracellular proteins or nucleic acids , In some embodiments , the modified single - celled bacteria or provide metabolic pathways for creating biofuels or new are added to the CHO culture directly . CHO cells are energy sources. In some embodiments , the artificial endos cultured by standard procedures , such as in Ham ' s F - 12 ymbiont is used to modify cell function by providing , among media with 10 % fetal calf serum media , prior to infection 25 others , nutrients , growth factors , proteins, minerals , nucleic with the MTB . Post infection , the media is augmented with acids, therapeutic agents , small molecules , ions , chemok additional iron (40 to 80 uM ) as either ferric malate or FeCiz . ines , polysaccharides, lipids, metals , cofactors , and hor Numerous other cell types internalize bacteria by endocy - mones. In some embodiments , the artificial endosymbiont tosis or more specifically phagocytosis ; endosymbionts or may also be used to manufacture with the host cell biore parasites have their own methods for cellular entry, and 30 mediation agents, enzymes, neurotransmitters , polypeptides , these natural processes can be exploited for internalization carbohydrates , pesticides , fertilizers , and other desirable of the artificial endosymbionts resulting in the generation of compounds . In some embodiments , an endosymbiont can so - called symbiosomes. In some embodiments, symbio - secrete proteins related to energy generation or exchange , somes from one cell can be transplanted to another cell type such as hydrogenase, nitrogenase , and laccase . (i . e . , one incapable of endocytosis of artificial endosymbi- 35 In some embodiments , the artificial endosymbiont can onts ) using microinjection , organelle transplantation , and secret an amino acid into the host cell, including, alanine , chimera techniques. These host cells are cultured in typical arginine, asparagine , aspartic acid , cysteine , glutamine , glu media and with the techniques for the specific cell type . tamic acid , glycine, histidine , isoleucine , leucine , lysine , In some embodiments , a single - celled organism is intro - methionine , phenylalanine , proline , serine , threonine, tryp duced to the host cell by a liposome mediated process . 40 tophan , tyrosine , or valine . Mitochondria and chloroplasts, which are larger than MTB , In some embodiments , the artificial endosymbiont can have been efficiently introduced into eukaryotic cells when secrete into a host cell a nucleic acid , RNA ,mRNA , hnRNA , encapsulated into liposomes (see , e . g . , Bonnett, H . T . , Planta SARNA , siRNA , microRNA , antisense RNA or DNA , or 131 : 229 , 1976 ; Giles, K . et al. , “ Liposome- mediated uptake DNA . Genetic material exchange between an artificial endo of chloroplasts by plant protoplasts , " In Vitro Cellular & 45 symbiont and a host cell can be used for gene therapy , to Developmental Biology - Plant 16 ( 7 ) :581 - 584 ; each publi - silence a gene , to transcribe a gene , to replace a gene , to cation incorporated herein by reference ). Numerous lipo - modify expression of a gene , to modify a gene , to introduce some fusion protocols and agents are available and can be a gene or nucleic acid fragment, to bind nucleic acids , or to used by the skilled artisan without undue experimentation interact with nucleic acids. ( see, e . g . , Ben -Haim , N . et al. , “ Cell - specific integration of 50 In some embodiments , the artificial endosymbiont can artificial organelles based on functionalized polymer introduce into the host cell signal pathway molecules , such vesicles ,” Nano Lett. 8 (5 ): 1368 -73 , 2008 ; Lian , W . et al. , as, for example , receptors , ligands (hormones , neurotrans “ Intracellular delivery can be achieved by bombarding cells m itters , cytokines , chemokines ) , ion channels , kinases , or tissues with accelerated molecules or bacteria without the phosphatases , DNA bindings proteins ( e . g ., transcription need for carrier particles, ” Experimental Cell Research 55 factors , repressors , enhancers ), and the like . An exemplary 313 ( 1 ) : 53 -64 , 2007 ; Heng , B . C . and Cao , T ., “ Immunoli set of proteins for reprogramming cells are the Yamanaka posome- mediated delivery of neomycin phosphotransferase factors , which include Oct4 , Oct3 , Sox2 , K1f4 , c -Myc , for the lineage - specific selection of differentiated / committed NANOG , and Lin28 . Expression of all or a subset of the stem cell progenies: Potential advantages over transfection Yamanaka factors can reprogram mature cells into iPS cells with marker genes , fluorescence -activated and magnetic 60 ( see , e . g ., Takahashi et al. , “ Induction of Pluripotent Stem affinity cell - sorting , ” Med . Hypotheses 65 ( 2 ) : 334 - 6 , 2005 ; Cells from Adult Human Fibroblasts by Defined Factors, " Potrykus, Ciba Found Symp , Vol. 1 54 : 198 , 1990 ; each Cell 131 ( 5 ) : 861- 72 , 2007 ) . publication incorporated herein by reference ) . In some embodiments , an artificial endosymbiont can be Use of Reprogrammed Host Cells used to sense intracellular moieties to quantify intracellular The artificial endosymbionts of the present invention 65 levels of such moiety , drive release of an agent, drive cell introduce into host cells peptides /proteins , nucleic acids, death , drive expression of a gene , label the cell , or sense cell and / or other factors . These proteins, nucleic acids, or other viability , differentiation , or cell cycle stage. US 9 ,752 , 129 B2 29 30 In some embodiments , an artificial endosymbiont can ( low G + C brand of the Firmicutes ) and the red aerobic secrete transcription factors used to reprogram a cell , for Salinibacter (Bacteroidetes branch ). example Oct4 . In some embodiments, an artificial endos In some embodiments , artificial endosymbionts can be ymbiont can secrete an enzyme to replace enzyme deficien used to impart molecular and physiological adaptations, cies , for example , in a lysosomal storage disease or ALDH2. 5 such as tolerability to extreme environments . For example , In some embodiments , an artificial endosymbiont can thermophilic and psychrophilic microbes have colonized secrete a moiety to label a cell , such as Calcein , superpara temperature and climate extreme environments . Thermo magnetic iron oxide , gadolinium containing reagents , fluo - philes have evolved their proteins , membranes and cellular rescent proteins , luminescent proteins, magnetic reporters , components to withstand temperatures , in the most extreme other reporter proteins (Gilad et al. , “ MRI Reporter Genes , " 10 cases , above 100° C . Artificial endosymbionts could extend J Nucl Med . 49 : 1905 - 08 , 2008 ; incorporated herein by the temperature range available to some crops . Artificial reference ) . In some embodiments, the artificial endosymbi- endosymbionts derived from psychrophiles or psychro ont can secrete a protein or other factor that provide a beacon trophs, may provide cold resistance to plants and fungi. for the host cell from a reporter, such as a fluorescent protein These microorganismshave a range ofmolecular and physi ( e . g ., GFP , RFP , YFP , CFP, etc . ), and / or luciferase . Other 15 ological adaptations : cold adapted enzymes , unique lipids reporters that can be secreted include , among others, for membranes that maintain fluidity at lower temperatures , enzymes , epitope tags , and heterologous proteins not nor cryoprotectants such as exopolysaccharides and cold shock mally expressed in the host cell . or antifreeze proteins. The secretion of cryoprotectants into Prokaryotes have evolved to survive in many ecological host cells would provide a cold tolerant phenotype . Genera niches and thus have a staggeringly large biodiversity . 20 of such bacteria include , among others , Marinobacter , Halo Conversely , eukaryotes have a much more limited biochem - monas , Dermacoccus, Kocuria , Micromonospora , Strepto istry but have the ability to create complex structures, tissues myces, Williamsia , Tskamurella , Clostridium , Alteromonas , and organisms. Thus , in some embodiments , the present Colwellia , Glaciecola , Pseudoalteromonas, Shewanella , invention is used to integrate the biodiversity of prokaryotes Polaribacter , Pseudomonas, Psychrobacter, Athrobacter, into the organismal complexity of eukaryotes . This capabil - 25 Frigoribacterium , Subtercola , Microbacterium , Rhodoccus ity relies on the ability of the artificial endosymbiont and and Bacillus. The genomes of a number of these have or are host cell to communicate or transfer various substances or being sequenced : M . frigidum , M . burtonii, C . symbiosum , signals from one to the other and vise versa . Specific C . psychrerythraea , P. haloplanktis , Halorubrum lacuspro problems encountered by host cells that are solved by fundi, Vibrio salmonicida , Photobacterium profundum , S . artificial endosymbionts can create a number of new host 30 violacea , S . frigidimarina , Psychrobacter sp . 273 - 4 , S . ben cells . For example , issues in plant agriculture today include , thica , Psychromonas sp . CNPT3 , Moritella sp ., Desulfo among others , use of fertilizers ; shrinking fresh water sup - talea psychrophila , Exiguobacterium 255 - 15 , Flavobacte plies ; temperature and climate fluctuations; insect, pest and rium psychrophilum , Psychroflexus torquis , Polaribacter fungal infections; toxic soil environments and diminishing filamentous, P . irgensii, Renibacterium salmoninarum and nutritional value . These are all issues that bacteria have 35 Leifsonia -related PHSC20 -cl . solved . In some embodiments , artificial endosymbionts are used An example of an application of the present invention is as remedial measures against pollution and other toxic providing a source of fixed nitrogen , which is the main components in the environment. For example , soil environ component of fertilizers, for the growth of plants and fungi. ments can become unsuitable for agriculture due to the There is a large and diverse class of microbes that fix 40 presence of toxic levels of heavy metals or other pollutants . atmospheric nitrogen using the enzyme nitrogenase . These Artificial endosymbionts that metabolize these or concen include , among others , Anabaena, Nostoc , Diazotroph , Cya - trate them and thus provide a resistant phenotype to the host nobacteria , e . g . the highly significant Trichodesmium , Bei - plant . Classes of microorganisms known as Acidophiles jerinckia , Clostridium , Green sulfur bacteria , Azotobacter have the ability to tolerate high concentrations of metals aceae, Rhizobia , and Frankia . Thus , in some embodiments , 45 ( e . g ., arsenic , mercury , copper , iron , etc . ) and metalloids . artificial endosymbionts derived from such organisms can be The genomes of Acidithiobacillus ferrooxidans , Thermo used to fix nitrogen from the air and transport a nitrogen plasma acidophilum , Picrophilus torridus, Sulfolobus toko containing compound compatible with the host cell' s daii and Ferroplasma acidarmanus have been sequenced , metabolism . and such organisms can be introduced into host cells to Another application of the present invention is to address 50 provide tolerance against such toxic environments . concerns regarding fresh water supplies, which could in In some embodiments, artificial endosymbionts can be principle be alleviated if crop plants could be irrigated or used to produce and deliver essential vitamins , for example , grown hydroponically with seawater. This would create a B12 , biotin , folic acid , and pantothenate to host cells. Such large osmotic stress on the plant cells . Surviving high salt nutrients are synthesized by several intestinal genera of environments is an ecological niche in which halophilic 55 bacteria , including Bacteroides , Eubacterium , Propionibac bacteria thrive, including members from flavobacteria , cya - terium , and Fusobacterium (Hooper , L . V . et al. , “ How nobacteria , proteobacteria , firmicutes , methanosarcinales host- microbial interactions shape the nutrient environment and halobacteriales. Generally two types of osmotic adap - of the mammalian intestine, ” Annu Rev Nutr. 22 : 283 - 307 , tation have been reported : accumulation of inorganic ions or 2002 ). Pseudomonas, Klebsiella , lecithinase -positive synthesis of organic osmotic solutes . The former “ high -salt 60 Clostridia , Veillonella and Fusobacteria produce consider in ” strategy provided by artificial endosymbionts could able amounts of Vitamin B12 like material ( Albert , M . J . et reduce the osmotic stress on plant cells , allowing the crop to al. , “ Vitamin B12 synthesis by human small intestinal bac survive seawater irrigation by concentrating salt ions into teria ,” Nature 283 :781 - 82 , 1980 ; incorporated herein by the artificial endosymbiont, thus lowering the host cell reference ) . cytoplasmic salt levels below lethal. This strategy is used by 65 In some embodiments , the present invention can be used the aerobic halophilic Archaea of the order Halobacteriales , in synthetic biology applications. Over the past decades , the fermentative anaerobes of the order Halanaerobiales advances in genetics, synthetic biology and microbiology US 9 ,752 , 129 B2 31 32 have created a large number of engineered microbes for sizes and diverse functions (their cognate substrates ). The various bioindustrial, biopharmaceutical, and other commer MTB genome encodes T1SS genes (Matsunga , T. et al. , cial applications . A limitation of these systems is that the “ Complete genome sequence of the facultative anaerobic engineered microbe needs to support the genetics and magnetotactic bacterium Magnetospirillum sp . AMB - 1 , " metabolism for both cell viability ( e . g . , housekeeping func - 5 DNA Res. 12 : 157 -66 . 2005: incorporated herein by reference tions ) and function of the engineered pathways . Artificial endosymbionts provide an alternative strategy because the in its entirety ). housekeeping functions are provided by the host, which Target protein , green fluorescent protein (GFP ) , or red allows more resources to be dedicated to the engineered fluorescent protein (RFP ) , is N - terminally fused to C - termi functionality in the artificial endosymbiont. In a medical nal 200 amino acids of YP _ 420640 (RTX toxins and related setting , drug eluting artificial endosymbionts could replace Ca2 + binding protein ), YP _ 423419 (RTX toxins and related cellular functions underlying numerous diseases; returning Ca2 + binding protein ) , YP _ 422785 (amb3422 ) , or other insulin production to pancreatic cells , restoring hormone or MTB TISS substrates . Alternatively , the proteins in MTB metabolic deficiencies, etc . In an industrial setting , artificial endosymbionts could increase the yields of numerous bio YP _ 420502 , YP _ 420631 . 1 , YP _ 420638 . 1 , YP _ 420640 . 1 , synthesized materials , biofuels , etc . YP _ 421364 . 1 , YP _ 422662 . 1 , YP _ 422785 . 1 , and In some embodiments , artificial endosymbionts that pro YP _ 423419 .1 can be used for the fusion by taking their vide carbon , energy ( like the endosymbiotically derived C -terminus ( e . g . , the 200 C - terminal amino acids which mitochondria , hydrogensomes , plastids , mitosomes and contains the secretion signal) to target the recombinant mitochondrion - derived organelles ) or other metabolites fusion protein to the secretion system . The DNA encoding could be useful commercially , for example, enabling animal 20 the fusion ofGFP or RFP with 200 C -terminal amino acids cells to derive carbon and / or ATP though the Calvin cycle of YP _ 420640 (RTX toxins and related Ca2+ binding pro and photosynthesis , thereby allowing use of in - lab grown tein ), YP _ 423419 (RTX toxins and related Ca2 + binding food stocks . Attempts to transfer chloroplast from plant cells protein ) , YP 422785 (amb3422 ) , or other MTB TISS sub to animal cells have been reported previously ( Bonnett, H . strates is cloned into pBBR -MSC (Kovach , M . E . et al, T ., “ On the mechanism of the uptake of Vaucheria ; chloro - 25 PBBR1MCS : a broad - host -range cloning vector, ” Biotech plasts by carrot protoplasts treated with polyethylene gly col. ” Planta 131 :229 - 33 , 1976 ; Giles , K . et al . , “ Liposome niques 16 : 800 - 2 , 1994 ) . Translocation of the fusion protein mediated uptake of chloroplasts by plant protoplasts ,” In out of the artificial endosymbiont is detected by fluores Vitro Cellular & Developmental Biology - Plant 16 : 581 - 4 , cence , or immunofluorescence and /or immunoblotting . 1980 ; incorporated herein by reference ) . Since the majority In another specific example , the hemolysin (Hly ) secre of the chloroplasts proteins are encoded in the nuclear 30 tion system of E . coli is one of the best studied type I genome it is not surprising that these have yet to be secretion systems ( TISS ) . Secretion of the hemolysin A successful . Indeed the definition of an endosymbiotically toxin (HlyA ) is catalyzed by a membrane protein complex derived organelle is transfer of genetic material to the host• ( Bakkes, P . J ., et al. , “ The rate of folding dictates substrate nuclear genome. secretion by the Escherichia coli hemolysin type 1 secretion In some embodiments, ATP production using an electron 35 system ,” J Biol Chem . 285 (52 ) :40573 - 80 , 2010 ) that con donor other than oxygen could be used to enable various sists of HlyB , an inner membrane protein that is an ATP host cells to inhabit new niches, potentially even extrater binding cassette transporter (Davidson , A . L . and J . Chen restrial. Microorganisms have many ways to produce ATP : " ATP -binding cassette transporters in bacteria , ” Annu Rev phototrophy, chemotrophy, photolithotrophy (examples : Biochem . 73 :241 -68 , 2004 ) , Tolc , the outer membrane pro cyanobacteria , Chromatiaceae , Chlorobiceae ) , photoorgano - 40 tein (Koronakis , V . , et al. “ Crystal structure of the bacterial trophy ( example Rhodospirillaceae ) , chemolithotrophy ( ex - membrane protein TolC central to multidrug efflux and amples : hydrogen -oxidizing bacteria , thiobacilli , nitrosomo protein export ,” Nature 405 (6789 ) : 914 - 9 , 2000 ) , and HlyD , nas, nitrobacter, methanogens , acetogens ) and the membrane fusion protein that is anchored to the inner chemoorganotrophy ( examples: pseudomonads, bacillus, membrane ( Johnson , J . M . and G . M . Church , “ Alignment sulfate reducers , clostridiana , lactic acid bacteria ), and artiniartifi - 1545. and structure predictionprediction of divergent protein familiesfamilies neriperi . cial endosymbionts containing such metabolic pathway or plasmic and outer membrane proteins of bacterial efflux artificial endosymbionts engineered to contain such path pumps ,” J Mol Biol. 287 ( 3 ) :695 - 715 , 1999 ) . Export of HlyA ways can be introduced into appropriate host cells . Recently bacteria that produce or conduct electricity have requires ATP hydrolysis by HlyB ( Thanabalu , T ., et al. , been described . Artificial endosymbionts derived from these “ Substrate - induced assembly of a contiguous channel for bacteria could enable complex tissues to be created that have 50 protein export from E . coli : reversible bridging of an inner the ability to interact directly with electronics. membrane translocase to an outer membrane exit pore ,” The invention will be better understood from the experi EMBO J . 17 ( 22 ) :6487 - 96 , 1998 ) . The last 218 C -terminal mental details which follow . However , one skilled in the art amino acids of HlyA have been shown to direct the secretion will readily appreciate that the specific methods and results of a large variety of polypeptides through the TiSS (Kenny , discussed are merely illustrative of the invention as 55 B . et al. , “ Analysis of the haemolysin transport process described , and therefore are not intended to be exhaustive or through the secretion from Escherichia coli of PCM , CAT or to limit the invention to the precise forms disclosed . beta - galactosidase fused to the Hly C - terminal signal domain ,” Mol Microbiol. 5 :2557 -68 , 1991 ; Mackman , N . et EXAMPLES al. , “ Release of a chimeric protein into the medium from Example 1 60 Escherichia coli using the C -terminal secretion signal of haemolysin ,” EMBO J . 6 , 2835 -41 , 1987 ; Holland , I. B ., et Translocation of Proteins into Host Cells Using a al. , “ The mechanism of secretion of hemolysin and other Type I Secretion System from an Artificial polypeptides from gram - negative bacteria ,” J Bioenerg Endosymbiont Biomembr. 22 (3 ) :473 -91 , 1990 ). 65 In order to translocate a protein , such as GFP or RFP, out The type I secretion systems (T1SS ) in Gram -negative of the bacterial cell into a target host cell via the TISS , the bacteria can be used to export a variety of proteins of various protein , GFP, for example (GenBank : ABG78037 . 1) will be US 9 , 752 , 129 B2 33 34 fused to the last 218 C - terminal amino acids of Hly into the host cell is monitored by observation of the hum (HlyAN - term 218 , UniProtKB /Swiss - Prot : P09983 . 1 , mingbird phenotype caused by CagA in the host cell. The bolded - underlined sequence ) : hummingbird phenotype is described in Segal et al. as " characterized by spreading and elongated growth of the 5 cell, the presence of lamellipodia ( thin actin sheets present ( SEO ID NO : 298 ) at the edge of the cell ), and filapodia ( finger - like protrusions MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFIC containing a tight bundle of actin filaments ( see Segal, E . D . TTGKLPVPWPTLVTILTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERT et al. , “ Altered States: Involvement of Phosphorylated Caga in the Induction of Host Cellular Growth Changes by IFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYN Helicobacter Pylori, " Proc Natl Acad Sci. USA 96 (25 ) : SHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYOONTPIGDGPVLL 14559 -64 , 1999 ) The hummingbird phenotype resembles the morphologi PDNHYLSTOSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGNSLAK cal changes induced by hepatocyte growth factor (HGF ) or NVLSGGKGNDKLYGSEGADLLDGGEGNDLLKGGYGNDI YRYLSGYGHHI (Sugiyama , T . , “ Development of gastric cancer associated IDDDGGKDDKLSLADIDERDVAFRREGNDLIMYKAEGNVLSIGHKNGIT with Helicobacter pylori infection , ” Cancer Chemother EKNWEEKESGDISNHOIEOIFDKDGRVITPDSLKKALE YOOSNNKASYV Pharmacol. 54 (Suppl 1 ): S12 -20 , 2004 ) . Hepatocyte growth factor, also known as scatter factor, evokes a unique mor YGNDALAYGSOGNLNPLINEISKIISAAGNFDVKEERAAASLLOLSGNA phogenic activity , e . g ., induces kidney, or mammary gland derived epithelial cells , to form branching ducts in three SDFSYGRNSITLTASA dimensional collagen gels (Ohmichi , H . et al. , " Hepatocyte For, example , the GFP - Hly AC - term 218 fusion will be growth factor (HGF ) acts as a mesenchyme- derived mor secreted out of the bacterial cell into the host cell via the phogenic factor during fetal lung development, ” Develop HlyB -HlyD - TO1C complex . The GFP -Hly AC - term 218 25 ment 125 : 1315 - 1324 , 1998 ) . fusion as well as the HlyB -HlyD - TolC complex will be Other proteins, including GFP, are fused to the C - terminal engineered into the pBBR1MCS - 2 plasmid and be residues of MobÀ ( see sequence below ) in order to be expressed under the control of the tac promoter. Transloca translocated from Helicobacter pylori with a T4SS into a tion of the GFP - HlyAC - term 218 fusion into target host cells MDM -MB231 host cell. In the case of GFP, translocation of will be monitored by fluorescence microscopy. 30 this protein into the MDM -MB231 host cell can be moni Translocation /Secretion assays ( to demonstrate that pro tored by looking for the presence of GFP fluorescence teins are secreted from MTB ) . Dilute ( 1 : 10 ) overnight outside of the artificial endosymbiont. cultures of MTB strains harboring the appropriate recombi GFP amino acid sequence (GenBank : ABG78037 .1 , nant plasmids into fresh MG supplemented with antibiotics sequence ) fused to MobA residues 684 - 709 (GenBank : ( for culture conditions, see Greene, S . E . et al. , “ Analysis of 35 AAA26445- . 1, bolded -underlined sequence ) is shown below : the CtrA pathway in Magnetospirillum reveals an ancestral role in motility in alpha proteobacteria ,” J Bact. 194 : 2973 ( SEQ ID NO : 299 ) 86 , 2012 ) . Cells carrying the defined plasmid combinations MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFIC are grown to an optical density of 400 nm (OD400 ) of 0 .2 before IPTG or arabinose is added to induce the expression 40 TIGKLPVPWPTLVTILTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERT of target protein fusions . Proteins in the supernatants are IFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYN precipitated with 10 - 20 % trichloroacetic acid for 30 min at 4° C . The precipitated proteins are collected by centrifuga SHNVYIMADKOKNGIKUNFKIRHNIEDGSVQLADHYQONTPIGDGPVLL tion and washed in 80 % acetone . Cell pellets are washed once in 20 mM Tris ( pH 8 . 0 ) - 1 mM EDTA . Cell pellets and 45 PDNHYLSTOSALSKDPNEKRDHMVLLEFVTAAGI TLGMDELYKLARAEL precipitated supernatants are resuspended in 1x sodium ARAPAPRORGMDRGGPDFSM . dodecyl sulfate -polyacrylamide gel electrophoresis (SDS PAGE ) loading buffer and amounts equivalent to 0 . 2 OD Alternatively , other proteins, including GFP , could be unit are analyzed by SDS -PAGE and immunoblotting . Pro fused to the C - terminal 50 amino acid residues of VirD5 in teins are stained with Coomassie Brilliant Blue and / or 50 order to be translocated from A . tumefaciens via a T4SS into probed by immunoblotting using specific antibodies . MTB a MDM -MB231 host cell . In the case ofGFP , translocation isolates that secrete GFP are introduced to the mammalian of this protein into the MDM -MB231 host cell can be cell line MDA -MB231 , using the magnet assisted entry monitored by looking for the presence of GFP fluorescence . method . Cells with artificial endosymbionts are obtained , GFP amino acid sequence (GenBank : ABG78037 . 1 , and translocated protein is detected by fluorescence . or 55 sequence ) fused to VirD5 residues 787 - 836 (NCBI Refer immunofluorescence or immunoblotting . ence Sequence : YP _001967549 . 1 , bolded -underlined sequence ) is shown below : Example 2 Translocation of a Protein into a Host Cell Using a 60 MS ( SEO ID NO : 300 ) Type IV Secretion System from an Artificial MVSKGEELFTGVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFIC Endosymbiont TIGKLPVPWPTLVTILTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERT CagA is secreted through a T4SS system by engineering IFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYN a CagA protein by replacing the last 20 amino acids with 2465 SHNVYIMADKOKNGIKVNFKIRHNIEDGSVOLADHYOONTPIGDGPVLL amino acids (residues 684 -709 ) from the C - terminal end of the RSF1010 MobA protein . Translocation of CagA -MOLA US 9 , 752 , 129 B2 35 36 GFP without a stop codon , referred to as GFPmut3 gene. The PDNHYLSTOSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKISALDR- continued polynucleotide encoding the fusion protein was operably linked to the lac promoter and the fusion protein expressed TARLISISPSKARSKAETEKAIDELDDRRVYDPRDRAQDKAFKR . using the extrachromosomal plasmid pBBR1MCS- 2 . This construct was sequence validated and conjugated into M . Example 3 magneticum AMB - 1 to create M . magneticum AMB - 1 strain AC16 . Translocation of a Nucleic Acid into a Host Cell Measuring green fluorescence of bacterial cells and extra Using a Type IV Secretion System from an cellular supernatant. M . magneticum AMB - 1 strain AC16 Artificial Endosymbiont 10 and non - fluorescent M . magneticum AMB - 1 were grown in standard media (see , e . g . , US patent publication US2013 / Caga The type 4 secretion system ( T4SS ) can be used to 0183758 ) . Green fluorescence of M . magneticum AMB - 1 transfer plasmid DNA from an artificial endosymbiont into strain AC16 , non -fluorescent M . magneticum AMB - 1, and a mammalian host cell . Sequence analysis reveals that their respective aliquots of extracellular supernatant were components of the T4SS system are present in MTB . 15 measured using a BioTek® Synergy H1 Microplate Reader Plasmid pBBR - MSC is engineered to include ( oriT + with excitation wavelength of 485 / 20 nm and emission trwABC ) for transfer of the plasmid by a T4SS system , and wavelength of 528 /20 nm . Both M . magneticum AMB - 1 the plasmid is engineered to contain an expression cassette strains assessed were adjusted to optical density at wave encoding the target protein under control of the HCMV IE1 length of 400 nm (OD400 ) of 0 . 195 to ensure all samples promoter -enhancer - first intron . The target protein is a select- 20 contained the same amount of bacterial cells per unit vol able marker such as DHFR or glutamate synthetase, or a ume. To assess the presence of green fluorescent protein reporter such as GFP , or a transcription factor such as cMyc . (GFP ) that has been transported out from M . magneticum A mammalian selectable marker can be used as the target AMB - 1 and into the extracellular space , 1 ml aliquots of gene. These include puromycin N - acetyl- transferase gene supernatant from each M . magneticum AMB - 1 strain with for puromycin resistance; blasticidin S deaminase for blas - 25 no cells present were obtained via centrifugation at 13 ,000 ticidin S resistance ; and aminoglycoside 3 -phosphotrans - rpm for 10 min . Without disturbing the bacterial pellet , 200 ferase for G418 resistance . If MDA -MB231 is used as a host ul of the supernatant were pipetted out and dispensed into cell line , 2 ug/ ml puromycin , 5 ug /ml blasticidin S , or 1 wells of a black 96 -well microplate . In parallel , 200 ul of M . mg/ml G418 is used for selection . magneticum AMB - 1 cells in suspension of each strain were Plasmid DNA harboring a DNA fragment of interest along 30 dispensed into wells of the microplate in order to assess total with an antibiotic resistance cassette (the choice of cassette green fluorescence from both intracellular and extracellular will vary depending on the conditions needed ) can be GFP . This experiment was done in biological and technical introduced into MTB via conjugation with a mating strain of triplicates . Measurements of GFP fluorescence in superna E . coli that is auxotrophic to diaminopimelic acid (DAP ) . tants of AC16 and AMB - 1 (negative control) are shown in Successful transfer of the plasmid DNA to MTB will result 35 FIG . 10 . Results show that there were significantly higher in growth of MTB on MG agar plates in the presence of levels of green fluorescence in the extracellular supernatant antibiotic . The E . coli mating strain auxotrophic to (DAP ) of AC16 as compared to that of the M . magneticum AMB -1 will ensure that any growth seen on the MG agar platest negative control (denoted as AMB - 1 Neg Ctrl) . Comparison antibiotic will be strictly MTB as no E . coli colonies should of fluorescence levels in supernatant of AC16 cells and cells survive on plates not containing DAP. MTB colonies har- 40 in suspension are shown in FIG . 11. Results show that there boring the plasmid DNA will be cultured in liquid MG was no significant difference between levels of green fluo growth medium to be used in subsequent experiments where rescence detected in the supernatant of AC16 cells and that MTB will be introduced into mammalian cells . MTB with detected with AC16 cells in suspension , which indicates that the plasmid are introduced into mammalian cells , such as the the majority ofGFP signal is from the extracellular medium . human breast cancer cell line MDA -MB231 . The plasmid 45 There is no significant difference between green fluores will then integrate into the host cell chromosome via non cence detected in the supernatant of the AMB - 1 Neg Ctrl and homologous recombination . Transfer of plasmid nucleic that detected with the same bacterial cells in suspension , acid from MTB , the artificial endosymbiont, to the host cell which suggests that this low fluorescence is due to back (MDA -MB231 ) is detected by selection for host cells that ground . grow in the presence of 2 ug /ml puromycin , 5 ug /ml 50 Immunohistochemisty to detect translocation of GFP. To blasticidin S , or 1 mg/ ml G418 . Selected cells are grown and examine the translocation of fusion protein (GFP fused to then suitable assays are performed to detect chromosomal C -terminal 200 amino acids of YP _ 422785 ) from magneto integration of the plasmid into the host cell genome, such as toactic bacteria Magnetospirillum magneticum AMB - 1 to for example , in situ hybridizations or Southerns. eukaryotic cells (human breast cancer cells MDA -MB231 ) , MDA -MB231 cells were grown in standard cell culture Example 4 medium ( e . g ., DMEM media supplemented with 10 % fetal calf serum ) and the cells labeled with M . magneticum Transport of Reporter Protein into a Eukaryotic AMB - 1 strain AC16 , for example by mixing AC16 cells Host Cell with MDA -MB231 cells and then using a NdFeB , Grade 60 N52 magnet to assist in introduction of the M . magneticum Reporter protein transport construct . A 600 base pair AMB - 1 cells into the MDA -MB231 cells . The transfer of the polynucleotide encoding the transport sequence denoted as fusion protein from magnetotactic bacteria into the extra 422785 ( from YP _ 422785 ) , which is the 200 amino acid cellular environment was monitored by immunofluores pod2785( SEQ ID( froNo NO: :291 291 ) ) C - terminal end of protein amb3422 cence microscopy. Briefly , MDA -MB231 cells were labeled ( accession no . YP _ 422785 ) , a putative Type 1 Secretion 65 with magnetotactic bacteria , and at various time points System ( T1SS ) protein native to Magnetospirillum magne - post - labeling the labeled MDA -MB231 cells were fixed in ticum AMB -1 , was fused to a polynucleotide encoding a 4 % paraformaldehyde, permeabilized and blocked with US 9 , 752 , 129 B2 37 38 0 . 1 % Triton X - 100 and 1 % bovine serum albumin . Eukary nel) The translocation of GFP protein ( in Panel B ) to the otic cells were stained with GFP and AMB - 1 antibodies for extracellular environment was not observed by double 1 h at room temperature. Slides were washed and incubated immunoflourescent staining of MDA -MB231 cells contain with Alexa Fluor 488 donkey anti - rabbit and Alexa Fluor ing AMB - 1 cells containing GFP only. 594 donkey anti -mouse IgG ( H + L ) secondary antibodies for 5 FIG . 14 shows fluorescence microscopy ofMDA -MB231 30 min . Slides were washed and mounted with Vectashield cells labeled with AC16 cells ( single channel and composite with DAPI, and fluorescence images were obtained using images ) (40x images ) . MDA -MB231 cells were labeled fluorescence microscope . with AC16 cells (AMB - 1 containing GFP - fused to C -termi FIG . 12 shows fluorescence microscopy images (40x ) of nal 200 amino acids of YP _ 422785 ) and dually stained one MDA -MB231 cells labeled with AMB - 1 containing ( A ) 10 day post -labeling and examined by fluorescence micros only GFP plasmid and ( B ) GFP - fused to C - terminal 200 copy : ( A ) AMB - 1 ( green channel) , ( B ) GFP ( red channel) , amino acids of YP _ 422785 , one day after labeling with the (C ) DAPI (blue channel ), and (D )merged images of AMB -1 M . magneticum AMB - 1 cells and dually stained with the ( green channel) , GFP ( red channel) , and DAPI (blue chan antibodies . Both panels are merged images of anti- GFP nel) The translocation of GFP - fusion protein ( in Panel B ) to (red ), anti - AMB - 1 ( green ) , and DAPI (blue ) . The translo - 16 the extracellular environment is revealed by double immu cation ofGFP -fusion protein ( in panel B ) to the extracellular noflourescent staining of AMB - 1 labeled MDA -MB231 environment is revealed by double immunoflourescent stain cells , demonstrating the ability of AMB - 1 to transfer pro ing of AMB - 1 labeled MDA -MB231 cells , demonstrating teins into the host cells using type 1 secretion system . the ability of AMB - 1 to transfer proteins into the host cells The foregoing descriptions of specific embodiments of the using type 1 secretion system . present invention have been presented for purposes of FIG . 13 . shows fluorescence microscopy of AMB -120 illustration and description . They are not intended to be labeled MDA -MB231 cells (single channel and composite exhaustive or to limit the invention to the precise forms images ) (40x images ) . MDA -MB231 cells were labeled disclosed , and many modifications and variations are pos with AMB - 1 containing GFP plasmid only (without the sible in light of the above teaching . secretion sequence ) , and the cells dually stained one day All patents , patent applications, publications, and refer post- labeling and examined by fluorescence microscopy : 25 ences cited herein are expressly incorporated by reference to ( A ) AMB -1 only (green channel ), (B ) GFP (red channel) , the same extent as if each individual publication or patent (C ) DAPI ( blue channel) , and ( D ) merged images of AMB - 1 application was specifically and individually indicated to be ( green channel) , GFP (red channel) , and DAPI (blue chan incorporated by reference .

SEQUENCE LISTING The patent contains a lengthy “ Sequence Listing ” section . A copy of the “ Sequence Listing” is available in electronic form from the USPTO web site (http :/ / seqdata .uspto . gov /? pageRequest= docDetail & DocID = US09752129B2 ). An electronic copy of the “ Sequence Listing ” will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1. 19 (b )( 3 ).

We claim : mammalian cell comprising a magnetotactic bacterium , 1 . An isolated mammalian cell comprising a magnetotac expressing a protein in the magnetotactic bacterium , secret tic bacterium ,wherein the magnetotactic bacterium provides ing the protein from the magnetotactic bacterium to the a protein to the host cell and thereby provides a phenotype 4 mammalian cell whereby the protein produces a phenotype to the mammalian cell . 2 . The mammalian cell of claim 1 , wherein the mamma in the mammalian cell . lian cell is a stem cell . 12 . The method of claim 11 , wherein the protein is a 3 . The mammalian cell of claim 2 , wherein the stem cell selectable marker. is a neural stem cell . 13. The method of claim 11 , wherein the protein is a 4 . The mammalian cell of claim 2 , wherein the stem cell 50 reporter protein . is a mesenchymal stem cell . 14 . The method of claim 13, wherein the protein is a 5 . The mammalian cell of claim 2 , wherein the stem cell magnetosome protein . is a neural crest stem cell . 15 . The method of claim 11 , wherein the mammalian cell 6 . The mammalian cell of claim 1 , wherein the mamma is stem cell . lian cell is a neural progenitor cell . 55 16 . The method of claim 15 , wherein the stem cell is a 7 . The mammalian cell of claim 1 , wherein the mamma neural stem cell . lian cell is selected from the group consisting of a murine 17 . The method of claim 15 , wherein the stem cell is a cell, a rat cell , a rabbit cell , a hamster cell , a porcine cell, a mesenchymal stem cell. bovine cell, a feline cell , a canine cell and a human cell . 18 . The method of claim 17, wherein the stem cell is a 8 . The mammalian cell of claim 7 . wherein the mamma . 60 neural crest stem cell . lian cell is a human cell . 19 . The method of claim 11 , wherein the mammalian cell 9 . The mammalian cell of claim 7 , wherein the mamma is a neural progenitor cell. lian cell is a murine cell. 20 . The method of claim 11 , wherein the mammalian cell 10 . The mammalian cell of claim 1, wherein the protein is selected from group consisting of a murine cell, a rat cell , is a magnetosome protein . a rabbit cell , a hamster cell, a porcine cell , bovine , a feline 11 . A method for introducing a phenotype into a mam cell, a canine cell and a human cell . malian cell , comprising the steps of : culturing an isolated * * * * *