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Analysis of Potential Essential Genes Using the Escherichia Coli K-12 Keio Mutant Collection

Permanent Link: http://ncf.sobek.ufl.edu/NCFE004069/00001

Material Information

Title: Analysis of Potential Essential Genes Using the Escherichia Coli K-12 Keio Mutant Collection
Physical Description: Book
Language: English
Creator: Dague, Darryl
Publisher: New College of Florida
Place of Publication: Sarasota, Fla.
Creation Date: 2009
Publication Date: 2009

Subjects

Subjects / Keywords: E. Coli
Essential Genes
Keto Collection
Transduction
Genre: bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The identification of essential Escherichia coli genes furthers our understanding of fundamental cellular processes. It is of practical importance because it reveals a set of targets that facilitate the discovery of antibiotic compounds. Unfortunately, the current literature leaves us with conflicting results as to the essentiality of many E. coli gene products. Dr. K. Rudd's laboratory set out to resolve these discrepancies and identify the complete essential gene set. The present paper reports the results of my contribution to this project. As a starting point, we examined the Keio collection, a resource of 3985 single-gene knockouts. We used P1 bacteriophage transduction to transfer these knockout mutations from the mutagenized background to our wild-type strain. The transduction frequency is inversely proportional to the missing gene's essentiality. For the majority of Keio mutants, we verified the non-essentiality of the disrupted gene. In cases where transductions failed, the mutants were investigated further. We circumvented factors that mask the 'true' phenotype of an essential gene deletion (e.g. tandem duplications, polar effects, and suppressor mutations). In addition, we redesigned a number of mutations whose construction was faulty in the Keio collection. The project was successful insofar as it verifies that the phenotype of each Keio strain is the result of its respective gene deletion, irrespective of the chromosomal background. As will be shown, P1 transduction is particularly applicable to this end.
Statement of Responsibility: by Darryl Dague
Thesis: Thesis (B.A.) -- New College of Florida, 2009
Electronic Access: RESTRICTED TO NCF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE
Bibliography: Includes bibliographical references.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Local: Faculty Sponsor: Walstrom, Katherine

Record Information

Source Institution: New College of Florida
Holding Location: New College of Florida
Rights Management: Applicable rights reserved.
Classification: local - S.T. 2009 D1
System ID: NCFE004069:00001

Permanent Link: http://ncf.sobek.ufl.edu/NCFE004069/00001

Material Information

Title: Analysis of Potential Essential Genes Using the Escherichia Coli K-12 Keio Mutant Collection
Physical Description: Book
Language: English
Creator: Dague, Darryl
Publisher: New College of Florida
Place of Publication: Sarasota, Fla.
Creation Date: 2009
Publication Date: 2009

Subjects

Subjects / Keywords: E. Coli
Essential Genes
Keto Collection
Transduction
Genre: bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The identification of essential Escherichia coli genes furthers our understanding of fundamental cellular processes. It is of practical importance because it reveals a set of targets that facilitate the discovery of antibiotic compounds. Unfortunately, the current literature leaves us with conflicting results as to the essentiality of many E. coli gene products. Dr. K. Rudd's laboratory set out to resolve these discrepancies and identify the complete essential gene set. The present paper reports the results of my contribution to this project. As a starting point, we examined the Keio collection, a resource of 3985 single-gene knockouts. We used P1 bacteriophage transduction to transfer these knockout mutations from the mutagenized background to our wild-type strain. The transduction frequency is inversely proportional to the missing gene's essentiality. For the majority of Keio mutants, we verified the non-essentiality of the disrupted gene. In cases where transductions failed, the mutants were investigated further. We circumvented factors that mask the 'true' phenotype of an essential gene deletion (e.g. tandem duplications, polar effects, and suppressor mutations). In addition, we redesigned a number of mutations whose construction was faulty in the Keio collection. The project was successful insofar as it verifies that the phenotype of each Keio strain is the result of its respective gene deletion, irrespective of the chromosomal background. As will be shown, P1 transduction is particularly applicable to this end.
Statement of Responsibility: by Darryl Dague
Thesis: Thesis (B.A.) -- New College of Florida, 2009
Electronic Access: RESTRICTED TO NCF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE
Bibliography: Includes bibliographical references.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Local: Faculty Sponsor: Walstrom, Katherine

Record Information

Source Institution: New College of Florida
Holding Location: New College of Florida
Rights Management: Applicable rights reserved.
Classification: local - S.T. 2009 D1
System ID: NCFE004069:00001


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A N A L Y S IS O F P O T E N T IA L E S S E N T IA L G E N E S U S I N G T H E E S CH E R I CH I A CO L I K 12 K E IO M U T A N T CO L L E CT IO N B Y D A R R Y L D A G U E A T he s i s S ubm i t t e d t o t he D i vi s i on o f N a t ur a l S c i e nc e s N e w C ol l e ge of F l o r i da i n pa r t i a l f ul f i l l m e nt of t he r e qui r e m e nt s f o r t he de gr e e B a c he l or of A r t s U nde r t he s pons or s hi p of D r K a t he r i ne W a l s t r om S a r a s ot a F l or i da M a r c h 2009

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i i A CK N O W L E D G E M E N T S T hi s r e s e a r c h w a s pe r f or m e d unde r t he s upe r vi s i on of D r K e nne t h R udd P I a nd D r Y us uf K a ya P os t D oc t or a l F e l l ow D e pa r t m e nt of B i oc he m i s t r y a nd M ol e c ul a r B i ol og y U ni ve r s i t y of M i a m i S c hool of M e di c i ne M i a m i F L

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i i i T A B L E O F CO N T E N T S A c k n ow l e d ge m e n t s i i A b s t r ac t vi I n t r od u c t i on O ve r vi e w 1 T he K e i o C ol l e c t i on 2 D i s c r e pa nc i e s i n t he L i t e r a t ur e 6 E s s e nt i a l F unc t i ons a nd M e t a bol i c E nz ym e s 9 A S t r a t e gy t o V a l i da t e t he K e i o C ol l e c t i on 14 P 1 P ha ge a nd G e ne r a l i z e d T r a ns duc t i on 15 M ut a t i ons M a s k e d by T a nde m D upl i c a t i ons a nd S uppr e s s or M ut a t i ons 19 D i f f e r e nc e s B e t w e e n t he B a c kgr ounds o f t he D ono r a nd R e c i pi e nt 24 B a c t e r i opha ge M e di a t e d R e c om bi na t i on 26

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i v M at e r i al s an d M e t h od s S t r a i ns 29 P r e pa r a t i on of P 1 L ys a t e s 29 P 1 T i t e r D e t e r m i na t i on 29 T r a ns duc t i ons 31 C onf i r m i ng T a nde m D upl i c a t i ons 33 P r e pa r a t i on of C om pe t e nt C e l l s 34 C he m i c a l T r a ns f or m a t i on 34 C ons t r uc t i on of R e d R e c om bi na nt 35 P r e pa r a t i on of E l e c t r oc om pe t e nt C e l l s 35 P C R P r i m e r D e s i gn f or M ut a ge ne s i s 36 E l e c t r opor a t i on 37

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v R e s u l t s an d D i s c u s s i on P ar t 1 P 1 O u t c r os s R e s t r i c t e d C r os s e s 39 T a nde m D upl i c a t i ons 34 D e t e r m i ni ng t he c a us e of t he t r a ppe d m ut a t i ons 41 S u p p r e s s or M u t a t i on s 46 P ar t 2 C om p l e m e n t a t i on S t u d i e s C i r c um ve nt i ng P 1 P ha ge R e s i s t a nc e 47 P ol a r E f f e c t s 48 P ar t 3 B ac t e r i op h age M e d i at e d R e c om b i n a t i on L i nke d B a c kgr ound M ut a t i ons 52 N e w D e l e t i on C ons t r uc t s 55 C on c l u s i on s 57 R e f e r e n c e s 58

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v i A N A L Y S IS O F P O T E N T IA L E S S E N T IA L G E N E S U S I N G T H E E S CH E R I CH I A CO L I K 12 K E IO M U T A N T CO L L E CT IO N D a r r yl D a gue N e w C ol l e ge of F l o r i da 2009 A B S T R A C T T he i de nt i f i c a t i on of e s s e nt i a l E s c he r i c hi a c ol i ge ne s f ur t he r s our unde r s t a ndi ng of f unda m e nt a l c e l l ul a r pr oc e s s e s I t i s of p r a c t i c a l i m por t a nc e be c a us e i t r e ve a l s a s e t of t a r ge t s t ha t f a c i l i t a t e t he di s c ove r y of a nt i bi ot i c c om pounds U nf or t una t e l y t he c ur r e n t l i t e r a t ur e l e a ve s us w i t h c on f l i c t i ng r e s ul t s a s t o t h e e s s e nt i a l i t y of m a ny E c ol i ge ne pr oduc t s D r K R udd s l a bor a t o r y s e t out t o r e s ol ve t he s e di s c r e pa nc i e s a nd i de nt i f y t he c om pl e t e e s s e nt i a l ge ne s e t T he pr e s e nt pa pe r r e p or t s t he r e s ul t s of m y c ont r i but i on t o t hi s pr oj e c t A s a s t a r t i ng poi nt w e e xa m i ne d t he K e i o c ol l e c t i on, a r e s our c e of 3985 s i ngl e ge ne knoc kout s W e us e d P 1 ba c t e r i opha ge t r a ns duc t i on t o t r a ns f e r t he s e knoc kout m ut a t i ons f r om t he m ut a ge ni z e d ba c kgr ound t o ou r w i l d t ype s t r a i n T he t r a ns duc t i on f r e que nc y i s i nve r s e l y pr opo r t i ona l t o t he m i s s i ng ge ne s e s s e nt i a l i t y. F or t he m a j or i t y of K e i o m ut a nt s w e ve r i f i e d t he non e s s e nt i a l i t y of t he di s r upt e d ge ne I n c a s e s w he r e t r a ns duc t i ons f a i l e d, t he m ut a nt s w e r e i nve s t i ga t e d f ur t he r W e c i r c um ve nt e d f a c t or s t ha t m a s k t he t r ue phe not ype of a n e s s e nt i a l ge ne de l e t i on ( e g t a nde m dupl i c a t i ons pol a r e f f e c t s a nd s uppr e s s or m ut a t i ons ) I n a ddi t i on, w e r e de s i gne d a num be r of m ut a t i ons w hos e c ons t r uc t i on w a s f a ul t y i n t he K e i o c ol l e c t i on T he pr oj e c t w a s s uc c e s s f ul i ns of a r a s i t ve r i f i e s t ha t t he phe not ype o f e a c h K e i o s t r a i n i s t he r e s ul t of i t s r e s pe c t i ve ge ne de l e t i on, i r r e s pe c t i ve of t he c hr om os om a l ba c kgr o und. A s w i l l be s how n, P 1 t r a ns duc t i on i s pa r t i c ul a r l y a ppl i c a bl e t o t hi s e nd D r K a t he r i ne W a l s t r om N a t ur a l S c i e nc e s

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1 I n tr od u c t i on F or a l ong t i m e t he ba c t e r i um w a s c ons i de r e d a ba g of e nz ym e s T hi s op i ni on be ga n t o c ha nge i n 1944 w he n O T A ve r y de m on s t r a t e d t ha t a t r a ns f or m i ng pr i nc i pl e c oul d t r a ns f e r ge ne s be t w e e n ba c t e r i a ( L i n e t al 1 984) T hi s t r a ns f or m i ng pr i nc i pl e w a s s oon know n a s D N A ( L i n e t al 1984) I t s i de nt i f i c a t i on i n ba c t e r i a l a i d t he g r oundw or k f or t he e l uc i da t i on of i t s s t r uc t ur e by W a t s on a nd C r i c k ( W a t s on a nd C r i c k, 1953) A r ound t hi s t i m e L e de r be r g a nd c ow or ke r s di s c ove r e d ba c t e r i a l c onj uga t i on, w hi c h s how e d t ha t t he ge ne s of ba c t e r i a a s w e l l a s of hi ghe r or ga ni s m s a r e l i nke d i n a c hr om os om e ( L e de r be r g e t al 1952) A s a r e s ul t of t he s e a nd ot he r s t udi e s r e s e a r c he r s be ga n t o r e c ogni z e t he c ons e r va t i on of c or e m e t a bol i c pa t h w a ys a nd he r e di t a r y m e c ha ni s m s i n al l or ga ni s m s ( L i n e t al 1984 ) C ons e que nt l y, t he ba c t e r i um s pe c i f i c a l l y E s c he r i c hi a c ol i be c a m e a m ode l s ys t e m f or s t udyi ng ba s i c bi ol ogi c a l pr oc e s s e s M a ny hope d t h i s know l e dge c oul d a dva nc e our unde r s t a ndi ng o f hu m a n di s e a s e s I n t he f ol l ow i ng de c a de s t hi s hope w a s r e a l i z e d t h r ough t he a c c um ul a t i on of da t a t ha t f or m s t he ba s i s of ou r bi ol ogi c a l s c i e nc e s Y e t t he E c ol i m ode l r e m a i ns i nc om pl e t e M a ny of i t s ge ne s a r e unc ha r a c t e r i z e d a nd w e kno w not hi ng a bout t he f unc t i on or r e gul a t i on of a f e w hundr e d ot he r ge ne s T he r e a l s o r e m a i ns t he m o r e f unda m e nt a l que s t i on c onc e r ni ng w hi c h ge ne s ar e r e qui r e d f or s ur v i v al A n a ns w e r t o t hi s que s t i on r e ve a l s a t t he f i ne s t l e ve l ba s i c phys i ol ogi c a l i nf o r m a t i on a bout c e l l ul a r m e t a bol i s m unde r s pe c i f i c gr ow t h c ondi t i ons A t a m o r e a bs t r a c t l e ve l i t a l l ow s f o r a c om pa r a t i ve a na l ys i s of t he c or e ba c t e r i a l r e pe r t oi r e a nd t he i r hi e r a r c hi c a l or ga ni z a t i on ( G e r de s e t al 2003) T hi s know l e dge c oul d hope f ul l y be a ppl i e d t o t he s t udy o f hi ghe r or ga ni s m s T he

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2 i de nt i f i c a t i on of e s s e nt i a l ge ne s i n E c ol i i s of p r a c t i c a l c onc e r n i ns of a r a s i t f a c i l i t a t e s t he di s c ove r y of ne w a nt i bi ot i c c om pounds G i ve n t he i nc r e a s i ngl y f r e que nt r e po r t s of dr ug r e s i s t a nt ba c t e r i a s uc h e f f or t s be c om e ne c e s s a r y. O ne m e t hod t o c l a s s i f y a ge ne a s e s s e nt i a l i nvol ve s t a r ge t i ng i t f or de l e t i on t o s e e i f t he c e l l di e s I f a de l e t i on m ut a nt gr ow s on gl uc o s e i t s knoc kout ge ne i s non e s s e nt i a l no m a t t e r how s i c kl y i t m i ght be c om e a s a r e s ul t I n 2004, B a ba e t al a ppl i e d t hi s m e t hod t o a l l E c ol i ge ne s us i ng t a r ge t e d m ut a ge ne s i s T h e i r e f f o r t s r e s ul t e d i n a gr oup of E c ol i s t r a i ns w i t h s i ngl e ge ne de l e t i ons c a l l e d t he K e i o c ol l e c t i on T he a ut hor s a l s o r e por t e d a l i s t of c a ndi da t e e s s e nt i a l ge ne s w hos e di s r upt i on c a us e d c e l l de a t h ( B a ba e t al 2006) U nf or t una t e l y, a f t e r c om pa r i ng t he s e r e s ul t s t o t he publ i s he d l i t e r a t ur e a nd e xa m i ni ng t he c ons t r uc t i on of s om e o f t he m ut a t i ons w e f ound a num be r of pr obl e m s w i t h t he s e s t r a i ns T h e K e i o C ol l e c t i on T he K e i o c ol l e c t i on i s a l i b r a r y o f 3985 s i ngl e ge ne knoc kout m ut a nt s i n E s c he r i c hi a c ol i K 12 ( B a ba e t al 2006 ) T he c on s t r uc t i on of e a c h K e i o s t r a i n s t a r t e d w i t h t he de s i gn of a de l e t i on f r a gm e nt t ha t w oul d r e pl a c e t he r e a di ng f r a m e of a s pe c i f i c ge ne t a r ge t w i t h a ge ne e nc odi ng a nt i bi ot i c r e s i s t a nc e T he e nds o f t he D N A f r a gm e nt w e r e m a de hom ol ogous t o t he ge ne t a r ge t ups t r e a m a nd dow ns t r e a m of t he i n i t i a t i on a nd t e r m i na t i on c odons r e s pe c t i ve l y ( B a ba e t al 200 6 ) B e t w e e n t he ho m ol ogi e s t he D N A f r a gm e nt c ons i s t s of a ka na m yc i n c a s s e t t e w hi c h pr ovi de s r e s i s t a nc e t o t he a nt i bi ot i c ka na m yc i n. U s i ng e l e c t r opor a t i on t he de l e t i on f r a gm e nt w a s dr i ve n i nt o a ba c t e r i um e xpr e s s i ng hi gh l e ve l s of a r e c om bi na s e e nz ym e T hr ough hom ol ogous r e c om bi na t i on, t he

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3 ka na m yc i n c a s s e t t e r e pl a c e d t he r e a di ng f r a m e o f t he ge ne t a r ge t i na c t i va t i ng t he t a r ge t e d ge ne I n p r a c t i c e t h i s pr oc e dur e i s h i ghl y e f f i c i e nt yi e l di ng t hous a nds of r e c om bi na nt s on a ve r a ge w hi c h c a n be s e l e c t e d us i ng ka na m yc i n r e s i s t a nc e I n E c ol i s t r a i n B W 25113, B a ba e t al t a r ge t e d 4288 a nnot a t e d ge ne s a nd obt a i ne d 3985 nul l i ns e r t i on/ de l e t i on m ut a nt s T he va l ue of t he r e s ul t i ng gr oup o f m ut a n t s i s t w o f ol d: ( 1) i t f a c i l i t a t e s r e s e a r c h a i m e d a t E c ol i s r e m a i n i ng ge ne f unc t i ona l que s t i ons a nd ( 2) i t p r e di c t s w hi c h ge ne s a r e e s s e nt i a l f or gr ow t h on gl uc os e E a c h of t he s e f a c t or s m a ke s t he K e i o c ol l e c t i on a t r e m e ndous r e s our c e f or de ve l opi ng our unde r s t a n di ng of t he E c ol i m ode l T he f ol l ow i ng t w o s t udi e s gi ve a n i de a o f how t he K e i o c ol l e c t i on ha s be e n us e d: 1. F unc t i onal c har ac t e r i z at i ons I n 2006 X i e e t al r e por t e d t he us e of a n E c ol i m ut a nt a s a hos t f or t he bi oc a t a l yt i c c onve r s i on o f m ona c o l i n J a c i d t o s i m va s t a t i n a c i d. I n t hi s r e a c t i on, t he t hi oe s t e r di m e t hyl but y r yl S m e t h yl m e r c a pt opr opr i ona t e ( D M B S M M P ) t r a ns f e r s i t s a c yl gr oup t o m ona c ol i n J f or m i ng s i m va s t a t i n a c i d ( Z i e e t a l 2007) S i m va s t a t i n i s a c hol e s t e r ol l ow e r i ng c om p ound a nd one of t he be s t s e l l i ng dr ugs i n t he w o r l d ( Z i e e t al 2007) C ur r e nt l y s i m va s t a t i n i s m a de ( s e m i s ynt he t i c a l l y) f r om t he na t ur a l p r oduc t l ova s t a t i n, t hr ough a m u l t i s t e p c he m i c a l s ynt he s i s ( Z i e e t al 2007 ) T he a ut hor s not i c e d t h a t a n unknow n E c ol i e nz ym e hydr ol yz e s t he m e t hyl e s t e r bond i n D M B S M M P f o r m i ng a l e s s e f f i c i e nt a c yl donor ( S e e F i gur e 1) T he c om pe t i ng pa t hw a y r e duc e d t he yi e l d of t he s i m va s t a t i n pr oduc t I n a n e f f or t t o i de nt i f y t he unknow n e nz y m e Z i e e t al s c r e e ne d a g r oup o f K e i o m ut a nt s w i t h ge ne de l e t i ons t ha t i na c t i va t e t h e i r a bi l i t y t o c a t a l yz e t hi s t ype of r e a c t i on ( i e ge ne s e nc odi ng t he c a r boxyl e s t e r a s e s ) T he y f ound t ha t a bi oH m u t a nt i s c om pl e t e l y de f i c i e nt i n t he c om pe t i ng r e a c t i on ( Z i e e t al 2007 ) F o r t he w hol e c e l l

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4 bi oc onve r s i on of s i m va s t a t i n f r om m ona c ol i n J a c i d a nd D M B S M M P t he y r e por t a 94 pe r c e nt r e c ove r y us i ng t he bi oH m ut a nt T he a ut hor s a r gue t ha t t he i r b i oc a t a l yt i c pr oc e s s i s l e s s e xpe n s i ve a nd ha s a hi ghe r yi e l d t ha n t he c om m onl y us e d c he m i c a l s ynt he s i s F i g u r e 1 W h o l e c e l l b i o c o n v e r s i o n o f M o n a c o l i n J a c i d a n d d i m e t h y l b u t y r y l S m e t h y l m e r c a p t o p r o p i o n a t e ( D M B S M M P ) t o f r e e s i m v a s t a t i n a c i d B i o H c a r b o x y l e s t e r a s e c a t a l y z e s t h e u n d e s i r a b l e h y d r o l o y s i s o f D M B S M M P t o t h e l e s s e f f i c i e n t a c y l d o n o r d i m e t h y l b u t y r y l S m e r c a p t o p r o p i o n i c a c i d ( Z i e e t a l 2 0 0 7 ) L o v D i s a n a c y l t r a n s f e r a s e c a t a l y z i n g t h e l a s t s t e p i n t h e b i o s y n t h e s i s o f l o v a s t a t i n 2. H i gh t hr oughput A nal y s e s A not he r s t udy m e a s ur e d t he gr ow t h of a l l 3985 K e i o m ut a nt s i n hum a n s e r um ( S e e F i gur e 2 ) I t s r e s ul t s i ndi c a t e t ha t de nov o nuc l e ot i de s ynt he s i s r e pr e s e nt s t he m os t c r i t i c a l m e t a bol i c f un c t i on f or ba c t e r i a l g r ow t h i n bl ood ( S a m a nt e t al 2008) T he c or r e s pondi ng e nz ym e s a r e pl a us i bl e t a r ge t s f or nove l c om pounds t ha t w oul d s l ow t he pr ol i f e r a t i on of ba c t e r i a l pa t hoge ns i n t he bl oods t r e a m

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5 F i g u r e 2 D y e b a s e d s c r e e n i n g f o r m u t a n t s w i t h a g r o w t h d e f e c t i n h u m a n s e r u m ( S a m a n t e t a l 2 0 0 8 ) M u t a n t s t h a t g r e w i n t h e s e r u m g e n e r a t e d a d e e p b l u e c o l o r ; l a c k o f a b l u e c o l o r a s s e e n w i t h g e n e s i n v o l v e d i n p y r i m i d i n e a n d p u r i n e s y n t h e s i s i n d i c a t e d t h e i n a b i l i t y o f a m u t a n t t o g r o w ( S a m a n t e t a l 2 0 0 8 ) H a vi ng c ons i de r e d s om e of t he a ppl i c a t i ons of t he K e i o c ol l e c t i on, w e c a n a r t i c ul a t e w ha t t he s e s t r a i ns c ont r i but e t o t he a na l ys i s of ge ne e s s e nt i a l i t y. E s s e nt i a l ge ne s a r e une xpe c t e dl y di f f i c ul t t o s t udy ge ne t i c a l l y be c a us e t he c e l l di e s i f t he y a r e d i s r upt e d. T o a de gr e e t he f a i l ur e of a s i ngl e ge ne knoc kout t o gr ow i ndi c a t e s t he e s s e nt i a l i t y of t he di s r upt e d ge ne ( K a ya pe r s ona l c om m uni c a t i on) O n t he ot he r ha nd, t he gr ow t h of a knoc kout s t r a i n i ndi c a t e s t he di s pe ns a bi l i t y of t he di s r upt e d ge ne C ons i de r i ng t hi s B a ba

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6 e t al pur por t e d t ha t t he 3985 ge ne s i na c t i va t e d w e r e non e s s e nt i a l A l t e r na t i ve l y t he 303 ge ne s w hos e di s r upt i on w a s l e t ha l t o t he ba c t e r i u m w e r e c ons i de r e d e s s e nt i a l R e s ul t s f r om t he s e e xpe r i m e nt s how e ve r a r e s ubj e c t t o bot h f a l s e pos i t i ve s a nd ne ga t i ve s : e s s e nt i a l ge ne s c a n be f a l s e l y r e por t e d a s non e s s e nt i a l a nd vi s a ve r s a I nde e d, ot he r l a bor a t or i e s ha ve be e n a bl e t o c ons t r uc t vi a bl e knoc kout s t r a i ns i n a f e w o f t he s e c a ndi da t e e s s e nt i a l ge ne s by a ddi ng pa r t i c ul a r nu t r i e nt s t o t he m e di um ( K a ya pe r s ona l c om m uni c a t i on) I n ot he r c a s e s t he gr ow t h of a K e i o de l e t i on m ut a nt c onf l i c t s w i t h e xpe c t a t i ons ba s e d on a n e s s e nt i a l f unc t i on of t he i na c t i va t e d ge ne T he pur pos e of t hi s t he s i s pr oj e c t w a s t o i nve s t i ga t e t he s e di s c r e pa nc i e s D i s c r e p an c i e s i n t h e L i t e r at u r e A n e s s e nt i a l ge ne c a n be pos i t i ve l y i de nt i f i e d by t a ki ng t he r i gor ous a ppr oa c h o f de l e t i ng t he ge ne on t he c hr om os om e w hi l e ha vi ng a f unc t i ona l c opy on a pl a s m i d w i t h a t i ght l y r e gul a t e d p r om ot e r W e know t he ge ne i s e s s e nt i a l i f e l i m i na t i on of ge ne e xpr e s s i on on t he pl a s m i d l e a ds t o c e l l de a t h or gr ow t h c e s s a t i on. T hough t hi s m e t hod w oul d not be p r a c t i c a l f o r ge nom e w i de a s s e s s m e n t va r i ous l a bor a t or i e s ha ve de t e r m i ne d t h e e s s e nt i a l i t y of a num be r of ge ne s t hi s w a y. T he s e r e s ul t s c a n be c om pi l e d, but a n i nt e r pr e t a t i on ba s e d on va r i ous s our c e s c a n be pr o bl e m a t i c gi ve n e xpe r i m e nt a l di f f e r e nc e s i n s t r a i ns a nd gr ow t h c ondi t i ons W e r e f e r t o t he P E C ( P r of i l i ng of t he E c ol i C h r om os om e ) da t a ba s e a t ht t p: / / w w w s hi ge n. ni g a c j p/ e c ol i / pe c / i nde x. j s p ) w hi c h ha s ga t he r e d e s s e nt i a l i t y da t a f r om r e s e a r c h r e por t s a n d de l e t i on m ut a t i on s t udi e s ( Y a m a r a ki e t al 2008)

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7 T o da t e t he r e ha ve be e n a bout t w o e xpe r i m e nt a l g e nom e w i de ge ne e s s e nt i a l i t y s t udi e s R e pr e s e nt i ng t he f i r s t o f t he s e G e r de s e t a l ( 2003 ) e m pl oye d hi gh t h r oughput gl oba l t r a ns pos on i ns e r t i on m ut a ge ne s i s a l s o kno w n a s ge ne t i c f oot pr i nt i ng. I n t hi s e xpe r i m e nt a t r a ns pos on r a ndom l y i ns e r t s i nt o t he ge nom e a nd t he pos i t i on of e a c h i ns e r t i on i n l i ve c e l l s c a n be de t e r m i ne d I f a ge ne i s not e s s e nt i a l i t s houl d ha ve m or e t r a ns pos on i ns e r t i ons i n a c ol l e c t i on o f l i ve c e l l s t h a n doe s a n e s s e nt i a l ge ne H e r e i n, t hi s s t udy pr e di c t e d 620 e s s e nt i a l ge ne s ba s e d on t he f a i l ur e t o a c c um ul a t e i ns e r t i on m ut a t i ons ( G e r de s e t al 2003 ) T hi s s t r a t e gy m e a s ur e s c e l l popul a t i ons ( S e e F i gu r e 3) T he r e f or e t he c l a s s i f i c a t i ons of t hi s s t udy m i ght be i nc or r e c t f or m ut a t i ons t ha t c a us e s l ow gr ow t h be c a us e t he s m a l l c ol oni e s c oul d be m i s s e d a nd r e s ul t i n t he i nc or r e c t c onc l us i on t ha t t he ge ne i s e s s e nt i a l T hi s m i ght e xpl a i n t he r e l a t i ve l y hi gh num be r o f ge ne s t he y c l a s s i f i e d a s e s s e nt i a l ( B a ba e t al 2006) T hi s i l l us t r a t e s how d i f f e r e nc e s i n e s s e nt i a l i t y pr e di c t i ons c a n be a t t r i but e d t o t he us e o f di f f e r e nt m ut a ge ne s i s s t r a t e gi e s or t he m e t hod us e d t o di s c r i m i na t e be t w e e n e s s e nt i a l a nd non e s s e nt i a l ge ne s ( K a ya pe r s ona l c om m uni c a t i on)

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8 F i g u r e 3 D i s t r i b u t i o n o f t r a n s p o s o n i n s e r t i o n d e n s i t i e s d e n s i t i e s o f e s s e n t i a l g e n e s a n d t h e i r r e s p e c t i v e e v o l u t i o n a r y r e t e n t i o n i n d e x a l o n g t h e E c o l i c h r o m o s o m e ( G e r d e s e t a l 2 0 0 3 ) E s s e n t i a l g e n e s w e r e p r e d i c t e d u s i n g t r a n s p o s o n i n s e r t i o n d e n s i t i e s c a l c u l a t e d a s t h e n u m b e r o f t r a n s p o s i t i o n e v e n t s p e r 1 0 0 k b s l i d i n g w i n d o w A n a l g o r i t h m w a s u s e d t o c o m p u t e a n e v o l u t i o n a r y r e t e n t i o n i n d e x f r o m e a c h g e n e s t r a n s p o s o n i n s e r t i o n d e n s i t y ( G e r d e s e t a l 2 0 0 3 ) N o t e : o r i C a n d d i f d e n o t e t h e o r i g i n o f c h r o m o s o m a l r e p l i c a t i o n a n d t h e r e p l i c a t i o n t e r m i n a t i o n a r e a r e s p e c t i v e l y T he K e i o c ol l e c t i on r e pr e s e nt s t he s e c ond ge nom e w i de e s s e nt i a l i t y s t udy. I t w a s t he f i r s t t o pr ovi de a n e va l ua t i on o f ge ne e s s e nt i a l i t y i n a s i ngl e s t r a i n us i ng c ons i s t e nt e xpe r i m e nt a l c ondi t i ons ( K i m a nd C opl e y, 2007) T he P E C da t a ba s e a nd B a ba e t al a gr e e i n t he i r c l a s s i f i c a t i on of 2 59 e s s e nt i a l ge ne s T he r e a r e 24 ge ne s t ha t a r e c l a s s i f i e d a s none s s e nt i a l i n t he P E C da t a ba s e but c ons i de r e d e s s e nt i a l by B a ba e t al O n t he ot he r

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9 ha nd, a l a r ge num be r of ge ne s c l a s s i f i e d a s non e s s e nt i a l by B a ba e t al ha ve e vi de nc e f or t he i r be i ng e i t h e r e s s e nt i a l or c ondi t i ona l l y e s s e nt i a l ( de f i ne d be l ow ) W e be ga n t he e xpe r i m e nt a l po r t i on o f t he pr oj e c t w i t h a ge ne s e t of c a ndi da t e e s s e nt i a l ge ne s A s w e pr ogr e s s e d, t hi s l i s t w a s m o di f i e d on D r R udd s da t a ba s e e c oge ne or g, w hi c h p r ovi de s c ont i nuous upda t e s o n E c ol i ge ne a nnot a t i on a nd ge ne a nd pr ot e i n f unc t i ona l c ha r a c t e r i z a t i ons A t t he s t a r t o f t he pr oj e c t he r e duc e d t he s e t of 303 c a ndi da t e e s s e nt i a l ge ne s t o 290. ( F o r ge ne by ge n e di s c us s i on of t he 13 ge ne s r e m ove d f r om t he e s s e nt i a l ge ne s e t r e f e r t o E s s e nt i a l G e n e s unde r t he E c oT op i c bul l e t i n a t ht t p: / / e c oge ne or g) E s s e n t i al F u n c t i on s an d M e t ab ol i c E n z ym e s A l t hough t he f oc us o f t hi s p r oj e c t i s on e s s e nt i a l ge ne s w e c a n a l s o c ons i de r t he e s s e nt i a l i t y of ge ne f unc t i ons I t i s pos s i bl e t ha t a s i ngl e e s s e nt i a l f unc t i on c a n be e nc ode d by t w o or m or e ge ne s w hi c h c a n be e i t he r hom o l o gous or of i nde pe nde nt or i g i ns ( R udd, pe r s ona l c om m uni c a t i on) G e ne s e que nc e s c a n be c l a s s i f i e d w i t hi n c l us t e r s of or t hol ogous gr oups or C O G s ( S e e F i gu r e 4) M os t e s s e nt i a l ge ne s be l ong t o t he C O G s i nvol ve d i n c e l l di vi s i on, l i pi d m e t a bol i s m t r a ns l a t i on, t r a ns c r i pt i o n, a nd c e l l e nve l ope bi oge ne s i s O f t he f our o r t hol ogous gr oup c a t e gor i e s m os t e s s e nt i a l g e ne s a r e a s s oc i a t e d w i t h m e t a bol i s m ( B a ba e t al 2006 )

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1 0 F i g u r e 4 C O G c l a s s i f i c a t i o n o f E c o l i K 1 2 g e n e s ( B a b a e t a l 2 0 0 6 ) H e r e w e of f e r a br i e f a c c ount of f a c t or s t ha t de t e r m i ne t he e s s e nt i a l i t y of 227 ge ne s i n t he E c ol i c e nt r a l m e t a bol i c pa t hw a ys A l t hough m os t of t he s e ge ne s a r e ne e de d f or g r o w t h on gl uc os e a bout 80 a r e non e s s e nt i a l ( B a ba e t al 2006) T he i r non e s s e nt i a l i t y c a n be a t t r i but e d t o a l t e r na t i ve m e t a bol i c pa t hw a ys or t he p r e s e nc e of i s oz ym e s a l t e r na t i ve e nz ym e s br oa d s pe c i f i c i t y e nz ym e s or m ul t i f unc t i ona l e nz ym e s t ha t c a n c om p e ns a t e f or t he di s r upt e d ge ne p r oduc t ( K i m a nd C opl e y, 2007 ) I n s om e c a s e s i nt e r c onne c t i ng m e t a bol i c pa t hw a ys a l l ow E c ol i t o bypa s s a de f e c t i ve m e t a bol i c s t e p. T ha t i s s t e ps i n a n i ndi vi dua l pa t hw a y m a y n ot be e s s e nt i a l i f t he r e i s a n i nt e r c onne c t i ng pa t hw a y f or s ynt he s i z i ng c e r t a i n m ol e c ul e s T hi s i s m o r e l i ke l y t o oc c ur f or c a t a bol i c pa t hw a ys w hi c h a r e hi gh l y c onne c t e d, t ha n b i os ynt he t i c pa t hw a ys w hi c h

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1 1 t e nd t o be l i ne a r ( K i m a nd C opl e y, 2007) F i g u r e 5 O v e r v i e w o f t h e c e n t r a l m e t a b o l i c p a t h w a y s o f E c o l i ( K i m a n d C o p l e y 2 0 0 7 ) B a s e d o n m e t a b o l i c p a t h w a y s a l o n e a n e n z y m e f u n c t i o n m a y a p p e a r r e d u n d a n t o r n o n e s s e n t i a l ; h o w e v e r t h e e n z y m e m i g h t b e u n i q u e i n i t s a b i l i t y t o p r o d u c e a p r e c u r s o r f o r a b i o s y n t h e t i c p a t h w a y A n i m por t a nt pur pos e of c e nt r a l m e t a bol i c pa t hw a ys i s t o pr oduc e m e t a bol i c i nt e r m e di a t e s I n a ddi t i on t o pr oduc i ng e ne r gy, c a t a bol i c pa t hw a ys s uppl y ke y pr e c ur s or s f or bi os ynt he t i c pa t hw a ys ( K i m a nd C opl e y, 2007) E xa m pl e s of t hi s c a n be s e e n i n F i gur e 5 f or pa t hw a ys t ha t pr oduc e s uc h p r e c ur s or s a s di hydr oxya c e t one phos pha t e ( D H A P ) e r yt hr os e 4 phos pha t e ( E 4P ) r i bos e 5 ph os pha t e ( R 5P ) a c e t yl C oA ( A c C oA ) oxa l oa c e t a t e ( O A A ) ke t ogl ut a r a t e ( K G ) a nd s uc c i nyl C oA ( S C oA ) E nz ym e s r e qui r e d t o m a ke t he s e m ol e c ul e s m a y be e s s e nt i a l

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1 2 E ve n i f i nt e r c onne c t i ng pa t hw a ys a r e a va i l a bl e t o bypa s s a m i s s i ng e nz ym e t he a c c um ul a t i on of a s ubs t r a t e f o r a m i s s i ng e nz ym e m a y c a us e l e t ha l t ox i c i t y ( K a ya pe r s ona l c om m uni c a t i on) C ons i de r t he t hr e e pa t h w a ys E c ol i c a n us e t o de g r a de gl uc os e t o gl yc e r a l de hyde 3 phos pha t e ( G A P ) gl yc ol yt i c pe nt os e phos pha t e a nd E nt ne r D oudor of f pa t hw a ys ( K i m a nd C opl e y 2007) G i v e n t he ba c t e r i um s f l e xi bl e m e t a bol i s m w e m i ght e xpe c t t ha t t he e nz ym e s of t he i ni t i a l pa r t of g l yc ol ys i s w oul d be non e s s e nt i a l f or g r ow t h on gl uc os e ( S e e F i gu r e 5) H ow e ve r du e t o t he t ox i c e f f e c t of m e t a bol i t e s t ha t a c c um ul a t e w i t hout a pa r t i c ul a r e nz ym e p hos phogl uc os e i s om e r a s e ( P gi ) i s t he onl y non e s s e nt i a l e nz ym e of t he s e ( K i m a nd C opl e y, 2007) T he t oxi c i t y c a us e d by t he l os s of t he ot he r e nz ym e s i s of t e n due t o e f f e c t s on ge ne e xpr e s s i on c a us e d by a l t e r a t i ons i n l e ve l s of m e t a bol i t e s be f or e a nd a f t e r t he bl oc k i n t he pa t h w a y ( K i m a nd C opl e y, 2007 ) T o he l p i l l us t r a t e t he t oxi c i t y of m e t a bol i t e s w e b r i e f l y di s c us s t w o e s s e nt i a l ge ne s : f baA ( f r uc t os e bi s phos pha t e a l dol a s e ) a nd t pi A ( t r i os e phos pha t e i s om e r a s e ) 1. F r uc t os e bi s phos pha t e a l dol a s e ( F ba A ) c a t a l ys e s t he a l dol c l e a v a ge of f r uc t os e 1, 6 bi s phos pha t e ( F D P ) E c ol i ha s a n i s oz ym e of F ba A c a l l e d F ba B w hi c h i s e xpr e s s e d onl y unde r gl uc one oge ni c c ondi t i ons A s a r e s ul t t he i s oz ym e i s not pr e s e nt dur i ng gr ow t h on gl uc os e ( S c a m uf f a a nd C a pr i ol i 1980) W he n f ba A i s de l e t e d t he F D P c onc e nt r a t i on i nc r e a s e s T oxi c i t y oc c ur s w he n F D P a l l os t e r i c a l l y i nhi bi t s 6 phos phogl uc ona t e de hydr oge ne a s e a n e nz ym e i n t he pe nt os e phos pha t e pa t hw a y. H e nc e a bl oc k i n t he gl yc ol yt i c pa t hw a y a t f ba A a l s o i m pa i r s t he f l ux t hr ough t he a l t e r na t i v e pe nt os e phos pha t e pa t hw a y ( K i m a nd C opl e y, 2007 ) T hi s i l l us t r a t e s how r e dunda nt e nz ym e a c t i vi t y ( i e F ba B ) m i ght not m a ke up f or l os t f unc t i ona l a c t i vi t y. F ur t he r i t s how s t he c onne c t i vi t y be t w e e n e nz ym e s i n

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1 3 di f f e r e nt pa t hw a ys w hi c h, i n t hi s c a s e r e s ul t s i n t he une xpe c t e d bl oc k i n bot h pa t hw a ys f or gl uc os e c a t a bol i s m 2. T r i os e phos pha t e i s om e r a s e ( T pi A ) c a t a l yz e s t he i nt e r c onve r s i on be t w e e n gl yc e r a l de hyde 3 phos pha t e a nd di hydr oxya c e t one phos pha t e ( D H A P ) T he t oxi c e f f e c t s of di s r upt i ng t hi s e nz ym e a r e c a us e d by t he m e t hyl gl yoxa l f or m e d w he n D H A P s pont a ne ous l y de c om pos e s ( S e e F i gur e 5; F e r gus on e t al 1998 ) A m ut a nt i n t pi A w i l l ge ne r a l l y not g r ow on gl uc os e ; how e ve r s pont a ne ous a da pt i ve m ut a t i ons c a n r e s ul t i n t he e xpr e s s i on of t he m e t hyl gl yoxa l pa t hw a y ge ne s T hi s l e a ds t o t he c onve r s i on of t hi s t oxi c m e t a bol i t e t o D l a c t a t e ( F e r gus on e t al 1998) T hi s e xa m pl e i l l us t r a t e s t he a c t i vi t y o f s uppr e s s o r m ut at i ons ( or s e c ond s i t e m ut at i ons ) w hi c h c a n a l l ow knoc kout m ut a nt s i n e s s e nt i a l ge ne s t o s ur vi v e S e c ond s i t e m ut a t i ons w e r e e nc ount e r e d f r e que nt l y t hr oughout t he c our s e of our e xpe r i m e nt s S uc h obs e r va t i ons f or c e us t o m odi f y our de f i ni t i on of a n e s s e nt i a l ge ne W e c a l l ge ne s w hos e di s r upt i on c an be s uppr e s s e d c ondi t i onal l y e s s e nt i al F i g u r e 5 S y n t h e s i s a n d D e t o x i f i c a t i o n o f M e t h y g l y o x a l ( F e r g u s o n e t a l 1 9 9 8 ) D i h y d r o x y a c e t o n e p h o s p h a t e ( D H A P ) i s c o n v e r t e d t o m e t h y g l y o x a l ( M G ) b y t h e a c t i o n o f m e t h y l g l y o x a l s y n t h a s e G l u t a t h i o n e ( G S H ) s p o n t a n e o u s l y r e a c t s w i t h m e t h y l g l y o x a l t o f o r m h e m i t h i o a l a c e t a l ( H T A ) T h i s b e c o m e s a s u b s t r a t e

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1 4 f o r g l y o x a l a s e I p r o d u c i n g S l a c t o y l g l u t a t h i o n e ( S L G ) w h i c h i s a s u b s t r a t e f o r g l y o x a l a s e I I ; g l y o x a l a s e I I t h e n p r o d u c e s D l a c t a t e ( D L a c ) a n d f r e e g l u t a t h i o n e ( F e r g u s o n e t a l 1 9 9 8 ) A l t e r na t i ve l y, t he ba c t e r i um c a n s ur vi ve w i t hout a n e nz ym e e s s e nt i a l f or gr ow t h on gl uc os e i f i t i s pr ov i de d a n a l t e r na t i ve m e di a or c ondi t i on. W e e nc ount e r e d a f e w e xa m pl e s of a uxot r ophy ( S e e R e s ul t s ) f i r s t i n t he l i t e r a t ur e a nd l a t e r ve r i f i e d e xpe r i m e nt a l l y: di a m i nopi m e l i c a c i d a nd m i ni m a l m e di a s us t a i n a # gapA ( e nc ode s G A P de hyr oge na s e ) m ut a nt ( F r a e nke l e t al 1996) ; t hy m i di ne s us t a i ns a m ut a nt i n f ol E ( e nc ode s G T P c yc l ohydr ol a s e I ) ( K l a us e t al 200 5) ; a nd t e m pe r a t u r e s be l ow 20 C s us t a i n a n # r poH ( e nc ode s R N A pol ym e r a s e s i gm a H ) m u t a nt ( Z hou e t al 1988) T he s e r e s ul t s a r e di s c us s e d i n de t a i l l a t e r A S t r at e gy t o V al i d at e t h e K e i o C ol l e c t i on W e ha ve di s c us s e d t he i m por t a nc e of i de nt i f yi ng e s s e nt i a l ge ne s t he c om pl i c a t i ons i nhe r e nt t o a ge ne e s s e nt i a l i t y s t udy, a nd f a c t or s t h a t c ont r i but e t o t he e s s e nt i a l i t y of a ge ne or f unc t i ona l r ol e I t r e m a i ns t o be s how n, how e ve r t ha t a n e xpe r i m e nt a l va l i da t i on of t he K e i o c ol l e c t i on c a n pr ovi de us w i t h t he ve r i t a bl e i de nt i f i c a t i on of e s s e nt i a l ge ne s w e s e t out t o f i nd. A n e xpe r i m e nt a l va l i da t i on of a m ut a nt m us t ( 1) e xa m i ne t he de s i gn of t he de l e t i on c ons t r uc t us e d i n m ut a ge ne s i s ; ( 2) pr ove t ha t t he m ut a nt phe not ype i s t he r e s ul t o f i t s r e s pe c t i ve m ut a t i on; a nd ( 3) ve r i f y t ha t t he m ut a nt phe not ype i s not t he r e s ul t of t he c hr om os om a l ba c kgr ound ( e g. s pont a ne ous s uppr e s s or m ut a t i ons or i nhe r i t e d m ut a t i ons ) O nl y i n t h i s w a y c a n a s e r i e s of t e s t s on a K e i o m ut a nt i ndubi t a bl y c l a s s i f y t he e s s e nt i a l i t y of t he di s r upt e d ge ne T o t hi s e nd w e t r a ns f e r r e d e a c h

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1 5 m ut a t i on f r o m t he m ut a ge ni z e d ( K e i o) ba c kgr ound s t r a i n ( E c ol i K 12 B W 25113) t o our w i l d t ype s t r a i n ( E c ol i K 12 M G 1655_s e q_ r ph + 1 ) T he m ut a t i ons w e r e t r a ns f e r r e d us i ng a ba c t e r i a l vi r us c a l l e d P 1 ba c t e r i opha ge P 1 P hage and G e ne r al i z e d T r ans duc t i on T he a c t i on of vi r us e s i n a ba c t e r i a l c ul t u r e w a s f i r s t de s c r i be d i n 1915 ( T w or t 1915) I n 1917 F e l i x d H e r e l l e c oi ne d t he t e r m ba c t e r i ophage ( ba c t e r i a e a t e r ) f or t he vi r us he i s ol a t e d f r om dys e nt e r y B ac i l l us ( d H e r e l l e 1917) C ur r e nt l y, i t i s m os t c om m on t o us e t he na m e phage A pha g e i s a pa r t i c l e c ons i s t i ng of i t s ge nom e s ur r ounde d by pr ot e i n s ubuni t s t ha t f or m a pr ot e c t i ve s he l l or c ap s i d ( L i n e t al 1984) L i ke a l l v i r us e s i t i s a s ubc e l l ul a r pa r a s i t e t ha t p r ovi de s i t s hos t c e l l w i t h t he ge ne t i c i n f or m a t i on r e qui r e d f or i t s r e pl i c a t i on. T he r e a r e t w o t ype s of pha ge s l y t i c a nd t e m pe r at e T he i nf e c t i on o f a ba c t e r i um by a l yt i c pha ge a l w a ys l e a ds t o c e l l de a t h. A t e m p e r a t e pha ge how e ve r c a n be c om e e s t a bl i s he d nonvi r ul e nt l y i n t he c hr om os om e of i t s hos t t hr ough a pr oc e s s c a l l e d l ys oge ni z a t i on ( L i n e t al 1984 ) T hi s i s c a l l e d t he l y s oge ni c r e s pons e w he r e i n t he pha ge c hr om os om e e nt e r s a pr ophage s t a t e ( L i n e t al 19 84) I n t hi s c a s e c om p l e x r e pr e s s i on s ys t e m s ge ne t i c a l l y s i l e nc e t he pr opha ge a l l ow i ng t he hos t c e l l t o r e pl i c a t e T he s e r e pr e s s i on s ys t e m s a r e i nf l ue nc e d by s pe c i f i c e nvi r onm e nt a l or c e l l ul a r c ondi t i ons t ha t c a n r e a c t i va t e t he pr opha ge a nd c a us e l yt i c de ve l opm e nt A s a s i de not e s om e pr o pha ge s c onf e r t oxi ge ni c i t y t o ha r m l e s s ba c t e r i a gi vi ng us bot ul i s m c hol e r a di pht he r i a a nd t oxi c s hoc k s ynd r om e ( Y a r m ol i ns ky, 2004) I n t he

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1 6 pr e s e nt s t udy, w e us e d t he t e m pe r a t e pha ge s P 1 a n d ( f o r ge ne r a l i z e d t r a ns duc t i ons a nd R e d m e di a t e d m ut a ge ne s i s r e s pe c t i ve l y) P 1 pha ge ha s pl a ye d a p r om i ne nt r ol e i n t he de ve l o pm e nt o f m o l e c ul a r bi ol ogy I t t a ke s c ont r ol of i t s hos t a t 42 C but r e m a i ns l ys oge ni z e d ( l a t e nt ) a t l ow e r t e m pe r a t ur e s T hi s i s a n i m po r t a nt e xpe r i m e nt a l c ont r ol poi nt T he l yt i c c yc l e i s r e pr e s e nt e d i n F i gu r e 7 ( L i n e t al 1984 ) L a t e i n i n f e c t i on, D N A i s pa c ke d i nt o c a ps i d s t r uc t ur e s by one of t w o ge ne r a l pr oc e s s e s I n s i t e t o s i t e pa c ka gi ng, pha ge e nc ode d e ndonuc l e a s e s c l e a ve a t s pe c i f i c ba s e s e que nc e s on t he D N A ( L i n e t al 19 84) pha ge us e s t hi s m e t hod. T he s e c ond t ype w hi c h i s us e d by P 1 pha ge i s de s c r i b e d a s he a df ul pa c ka gi ng. I n t hi s pr oc e s s t he pha ge pa r t i c l e i s f i l l e d t o c a pa c i t y w i t h D N A a nd t he D N A i s c ut nons pe c i f i c a l l y ( L i n e t al 1984) T he a bi l i t y t o us e P 1 pha ge f o r ge ne t i c m a ni pul a t i on i s a n i m m e di a t e r e s ul t of i t s he a df ul pa c ka gi ng m e c ha ni s m B e c a us e t he pa c ka gi ng i s nons pe c i f i c t he pha ge ha s t he pot e nt i a l t o e nc l os e a ny D N A t ha t i s pr e s e nt i n t he ba c t e r i um l a t e i n t he i nf e c t i on i nc l udi ng a ny r e m a i ni ng hos t D N A ( L i n e t al 198 4) M os t t e m pe r a t e pha ge s ha ve e xt e ns i ve nuc l e a s e a c t i vi t y. A s a r e s ul t t he hos t D N A i s de gr a de d by t he t i m e t he ba c t e r i um l ys e s ( L i n e t al 1984) H ow e ve r P 1 ph a ge e xpr e s s e s f a r f e w e r nuc l e a s e s a l l ow i ng hos t D N A t o be pa c ka ge d. A f t e r a P 1 l yt i c i nf e c t i on, a m i xt u r e of D N A pa c ke d pha ge pa r t i c l e s i s r e l e a s e d a f t e r t he ba c t e r i um l ys e s T he s e pa r t i c l e s c ons i s t of bot h nor m a l vi r us e s a nd vi r a l pa r t i c l e s c a r r yi ng he a df ul f r a gm e nt s of t he hos t c hr om os om e ( S e e F i gur e 8) T he s e ps e udo v i r us e s e f f e c t i ve l y be c om e ve hi c l e s f or t r a ns f e r r i ng a ny r e m a i ni ng ba c t e r i a l ge nom i c D N A t o ot he r hos t s T he hos t D N A i s i nj e c t e d i nt o a s e ns i t i ve ba c t e r i um a nd i nc or po r a t e d i nt o t he r e c i pi e nt s c hr om os om e t hr ough hom ol ogous

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1 7 r e c om bi na t i on ( L i n e t al 1984 ) T he ph a ge m e di a t e d t r a ns f e r o f hos t D N A be t w e e n ba c t e r i a i s c a l l e d ge ne r al i z e d t r ans duc t i on ( S e e F i gur e 8) A uni f or m pe r c e nt a ge of t he s e ps e udo vi r us e s w i l l ha ve a r bi t r a r i l y pa c ka ge d ge nom i c D N A c ont a i ni ng t he K e i o m ut a t i ons M or e ove r a uni f or m pe r c e nt a ge o f r e c i pi e nt s w i l l i nc or po r a t e t hi s D N A f r a gm e nt i n t o t he i r c h r om os om e I n t hi s s c e na r i o, r e c i pi e nt s c a n be s e l e c t e d f or on t he ba s i s of ka na m yc i n r e s i s t a nc e e nc ode d by t he i r a nt i bi ot i c c a s s e t t e t ha t r e pl a c e d t he de l e t e d ge ne ( s e e a bove di s c us s i on of K e i o m ut a t i on de s i gn) A s r e l a t e s t o t he pr e s e nt r e po r t a t r ans duc t ant i s a ba c t e r i um t ha t ha s be e n i nf e c t e d w i t h a t r a ns duc i ng pa r t i c l e c ont a i ni ng a K e i o m ut a t i on f r om t he donor s t r a i n D N A T hi s t r a ns duc t a nt ha s t he s a m e m ut a t i on a nd ka na m yc i n r e s i s t a nc e a s t h e donor K e i o s t r a i n, e xc e pt i t i s i n our w i l d t ype c hr om os om a l ba c kgr ound. T he a s s um pt i on t ha t s t r i c t l y non e s s e nt i a l ge ne s c a n be di s r upt e d w i t h e qua l e a s e i s c onf i r m e d be c a us e m ut a t i ons i n s t r i c t l y non e s s e nt i a l ge ne s yi e l d a n e qua l num be r o f ka na m yc i n r e s i s t a nt t r a ns duc t a nt s ( K a ya pe r s ona l c om m uni c a t i on)

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1 8 F i g u r e 7 G e n e r a l i z e d t r a n s d u c t i o n ( D a l e 1 9 8 9 ) F i g u r e 8 P 1 p h a g e t r a n s d u c t i o n ( L i n e t a l 1 9 8 4 ) H e a d f u l D N A p a c k a g i n g m a k e s t h e p h a g e a v e h i c l e f o r t r a n s f e r r i n g b a c t e r i a l D N A f r o m i t s h o s t t o a n o t h e r b a c t e r i u m N o t e t h e r e l a t i v e l y m i n u t e p o p u l a t i o n o f p h a g e t h a t b e c o m e t r a n s d u c i n g p h a g e h e n c e t h e i n e f f i c i e n c y o f g e n e r a l i z e d t r a n s d u c t i o n

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1 9 S om e t e r m i nol ogy w i l l be us e f ul i n t he p r e s e nt di s c us s i on: w e de f i ne a n out c r os s t o be t he pha ge m e di a t e d t r a ns f e r o f D N A f r om a donor s t r a i n t o a r e c i pi e nt of a di f f e r e nt ge nom i c ba c kgr ound. A bac k c r os s i s t he pha ge m e di a t e d t r a ns f e r o f D N A f r om a donor s t r a i n t o a r e c i pi e nt s t r a i n o f t he s am e c hr om os om a l ba c kgr ound. A f t e r a ny m ut a ge ne s i s pr oc e dur e i t i s c o m m on t o ba c kc r os s t he ne w m ut a t i ons f r om t he m ut a ge ni z e d s t r a i n t o a w i l d t ype s t r a i n ( K a ya pe r s ona l c om m un i c a t i on) B ot h of t he s e pr oc e dur e s c l e a r t he c hr om os om a l ba c kgr ound of unw a nt e d s e c onda r y m ut a t i ons a nd e ns ur e s t ha t t he phe not ype of t he m ut a nt i s t he r e s ul t o f i t s r e s pe c t i ve de l e t i on. T hi s w a s not done f or t he K e i o c ol l e c t i on s o w e pe r f or m e d i t pos t hoc B y c om pl e t i ng t he 3985 t r a ns duc t i ons w e t r a ns f e r r e d t he 3985 K e i o m ut a t i ons i nt o a ne w ba c kgr ound, e f f e c t i ve l y c r e a t i ng a ne w c ol l e c t i on, w hi c h w e c a l l t he K R E c ol l e c t i on M u t at i on s M as k e d b y T an d e m D u p l i c at i o n s an d S u p p r e s s or M u t at i o n s T o c l a r i f y t he r e a s on t o out c r os s e a c h of t he 3985 K e i o m ut a t i ons w a s t w o f ol d F i r s t a f t e r a n out c r os s w e c oul d obs e r ve w he t he r t he m ut a nt phe not ype w a s t he r e s ul t of onl y i t s l oc a l i ns e r t i on/ de l e t i on m ut a t i on A c ha nge i n m ut a nt phe not ype a f t e r a n out c r os s w oul d i ndi c a t e t ha t ot he r i n f l ue nc e s a s i de f r om t h e s i ngl e ge ne de l e t i on, w e r e a t ha nd. I n t hi s s e ns e a n out c r os s i s ol a t e s t he K e i o m ut a t i ons f r om t he i r ba c kgr ound a m bi gui t i e s ( e g. i ns e r t i on s e que nc e e l e m e nt s c a pa bl e of i nde pe nde nt t r a ns pos i t i on, de l e t i ons s e que nc e / a nnot a t i on a m bi gui t i e s unknow n m ut a t i ons i nduc e d by m ut a ge ne s i s ) A s t r a ns duc t i on i s c l e a n a nd uni f o r m a nd doe s not i nt r oduc e r a ndom or e xt r a ne ous m ut a t i ons i t i s a pa r t i c ul a r l y us e f ul t e c hni que f or c he c ki ng t h e de l e t i on m ut a nt s

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2 0 T he s e c ond i m pe t us t o out c r os s e a c h of t he K e i o m ut a t i ons w a s t o i de nt i f y pr obl e m a t i c c a s e s of i ne f f i c i e nt t r a ns duc t i on c a l l e d r e s t r i c t e d c r o s s e s B e c a us e non e s s e nt i a l ge ne s a r e di s pe ns a bl e w e s houl d be a bl e t o di s r upt t he m w i t h e qua l e a s e A s w e know m ut a nt s w i t hi n t he K e i o c ol l e c t i on a r e pu r p or t e d t o ha ve knoc kout m ut a t i ons i n t he c odi ng r e gi on o f non e s s e nt i a l ge ne s ( B a ba e t al 2006) H e r e i n w e e xpe c t t r a ns duc t i ons of t he K e i o m ut a nt s t o y i e l d a n un r e s t r i c t e d a nd e q ua l num be r o f t r a ns duc a nt s I f t h i s doe s not oc c ur t he n ot he r f a c t or s a l l ow t he hos t K e i o s t r a i n t o g r ow no r m a l l y by m a s ki ng t he phe not ype of t he m ut a t i on. I n m os t c a s e s t he s e m a s ki ng f a c t or s c a nnot be out c r os s e d. T he t w o m os t i m por t a nt of t he s e f a c t or s w e r e t a nde m d upl i c a t i ons a nd s uppr e s s or m ut a t i ons w hi c h w e w i l l e xa m i ne i n t u r n. A s w i l l be s how n, e a c h of t he s e f a c t or s i s a s s oc i a t e d w i t h a c e r t a i n de gr e e of t r a ns duc t i on r e s t r i c t i on t ha t c a n b e r e pr e s e nt e d by a l ow t r a ns duc t i on f r e que nc y. T he e xi s t e nc e of a s i ngl e ge ne knoc kout w a s e vi de nc e e nough f or t he a ut hor s o f t he K e i o a r t i c l e t o c l a s s i f y a di s r upt e d ge ne a s non e s s e nt i a l ( B a ba e t al 2006) O f t e n how e ve r a n e s s e nt i a l ge ne c a n be dupl i c a t e d t hr ou gh hom ol ogous r e c om bi na t i on dur i ng D N A s ynt he s i s ( S e e F i gur e 9) S uc h pa r t i a l di pl oi d s m a s k t he phe not ype of a n e s s e nt i a l ge ne de l e t i on: c e l l de a t h. T ha t a K e i o s t r a i n ha s a t a nde m dupl i c a t i on i ndi c a t e s t ha t t he f ol l ow i ng e ve nt s oc c ur r e d: ( 1) a n e s s e nt i a l ge ne w a s t a r ge t e d f or di s r upt i on; ( 2) a popul a t i on of ba c t e r i a i n t he t a r ge t e d c ul t u r e ( a bou t 1 pe r c e nt ) ha d t w o c opi e s of t he e s s e nt i a l ge ne t a r ge t ; ( 3) onl y t hos e c e l l s w i t h t he dupl i c a t e d ge ne s ur vi ve d a f t e r m ut a ge ne s i s ; a nd ( 4) t he dup l i c a t i on w e nt unnot i c e d by B a ba e t al a nd t he t a r ge t e d ge ne w a s c l a s s i f i e d a s non e s s e nt i a l T hi s l ogi c a s s um e s t ha t ge ne dupl i c a t i ons w i l l be r e m ove d

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2 1 w he n t he c e l l di vi de s i f t he y a r e unne e de d, a s i s o f t e n t he c a s e f or s t r i c t l y non e s s e nt i a l ge ne s F i g u r e 9 T h i s u p p e r h a l f o f t h i s f i g u r e d i s p l a y s h o w r e c o m b i n a t i o n b e t w e e n h o m o l o g o u s s e q u e n c e s l e a d s t o t h e d u p l i c a t i o n o f t h e g e n e s b e t w e e n t h e s e s e q u e n c e s D u r i n g D N A r e p l i c a t i o n u n e q u a l c r o s s i n g o v e r b e t w e e n t h e h o m o l o g o u s s e q u e n c e s c a n y i e l d t a n d e m d u p l i c a t i o n s I n t h i s c a s e t h e p o s t r e p l i c a t e d c h r o m o s o m e w i l l c o n t a i n t w o c o p i e s o f t h e e s s e n t i a l g e n e D u r i n g m u t a g e n e s i s o r t r a n s d u c t i o n o n e o f t h e g e n e c o p i e s c a n b e r e p l a c e d b y a k a n a m y c i n c a s s e t t e b u t t h e b a c t e r i a w i l l s u r v i v e b e c a u s e i t h a d a p r e e x i s t i n g t a n d e m d u p l i c a t i o n F or a K e i o m ut a nt w i t h a t a nde m dupl i c a t i on, i t s k a na m yc i n c a s s e t t e c a nnot be c o t r a ns duc e d w i t h t he e s s e nt i a l ge ne T hi s i s s i m p l y be c a us e t he l e ngt h of t he c hr om os om e be t w e e n t he w i l d t ype e s s e nt i a l ge ne a nd t he ka na m yc i n c a s s e t t e i s t oo l ong f or i t t o be l i ke l y pa c ka ge d i nt o a P 1 pha ge C ons e que nt l y, t he t r a ns duc t i ons w i l l not yi e l d a ny t r a ns duc t a nt s be c a us e t he ba c t e r i um di e s i n on e of t w o w a ys : ( 1) i t ob t a i ns t he G e n e d u p l i c a t i o n f o l l o w e d b y

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2 2 ka na m yc i n r e s i s t a nc e ge ne but l os e s t he e s s e nt i a l ge ne or ( 2 ) i t r e t a i ns t he e s s e nt i a l ge ne but doe s not ha ve r e s i s t a nc e t o t he s e l e c t i ve m e di a c ont a i ni ng ka na yc i n. T he e xc e pt i on t o t hi s s c e na r i o i s w he n t he r e c i pi e nt c e l l a l r e a dy ha s t w o c opi e s of t he t a r ge t e d e s s e nt i a l ge ne T hi s oc c ur s i n a bout one pe r c e nt of t he r e c i pi e nt c e l l s ( K a ya pe r s ona l c om m uni c a t i on) T he r e f or e t he t r a ns duc t i on i s l i m i t e d not on l y by t he a l r e a dy l ow e f f i c i e nc y of t r a ns duc t i on, but a l s o by t he s m a l l popul a t i on o f r e c i pi e nt ba c t e r i a w i t h a pr e e xi s t i ng dupl i c a t e c opy of t he t a r ge t e d e s s e nt i a l ge ne T he e ve nt s out l i ne d i n t he pr e vi ous pa r a gr a ph a ppl y t o t he l a t e r of t he s e l i m i t a t i ons : on l y 1 i n 100 ba c t e r i a ha ve a t a nde m dupl i c a t i on i n t he ge ne t a r ge t G i ve n t h i s w e c a n d e t e r m i ne t he num be r of t r a ns duc t a nt s w e e xpe c t t o obt a i n ( f or t he t r a ns du c t i on of a n e s s e nt i a l ge ne de l e t i on) i f w e know t he e xpe c t e d t r a ns duc t a nt yi e l d f or a non e s s e nt i a l ge ne de l e t i on. E m pi r i c a l l y f or ou r w i l d t ype s t r a i n, P 1 t r a ns duc t i on of a ka na m yc i n c a s s e t t e i nt o a s t r i c t l y non e s s e nt i a l ge ne c ons i s t e nt l y yi e l ds 200 t o 400 knoc kout m ut a nt s ( K a ya pe r s ona l c om m uni c a t i on) ( I n t he s e c ons i de r a t i ons w e a s s um e a s t a r t i ng c ul t ur e o f 10 9 c e l l s ) I f w e m ul t i p l y t hi s unr e s t r i c t e d o r uppe r l i m i t r a nge by t he pe r c e nt a ge ( ~ 1% ) o f c e l l s w i t h a pr e e xi s t i ng dupl i c a t i on i n t he e s s e nt i a l ge ne t a r ge t a r a nge of 2 t o 4 t r a ns duc t a nt s i s obt a i ne d ( S e e F i gur e 10) H e nc e f or K e i o m ut a t i ons t ha t t r a ns duc e t o yi e l d 2 t o 4 ka na m yc i n r e s i s t a nt r e c i pi e nt s w e de s i gne d de l e t i on p r i m e r s t o t e s t f or t he pr e s e nc e of a dupl i c a t e w i l d t ype c opy of t he t a r ge t e d ge ne H ow e ve r w e c a nnot a s s um e a uni f o r m c onc e nt r a t i on of t r a ns duc i ng pha ge pa r t i c l e s a nd s o t he t r a ns duc t a nt c ount m us t be r e l a t e d t o t he t i t e r o f t he P 1 l ys a t e by c a l c ul a t i ng a nor m a l i z e d t r a ns duc t i on f r e que nc y f or e a c h m u t a t i on. T hi s pr oc e dur e w i l l be de s c r i be d s hor t l y.

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2 3 F i g u r e 1 0 A n a l y s i s b y c o u n t i n g k a n a m y c i n r e s i s t a n t t r a n s d u c t a n t s T h e a c t u a l c o m p a r a t i v e a n a l y s i s w a s n o r m a l i z e d u s i n g t r a n s d u c t i o n f r e q u e n c i e s ; h o w e v e r f o r t h e e a s e o f d i s c u s s i o n w e a s s u m e h e r e t h e s a m e p h a g e c o n c e n t r a t i o n ( p f u / m L ) a n d r e c i p i e n t s t a r t i n g c u l t u r e ( 1 0 9 c e l l s ) P 1 t r a ns duc t i on i s a be t t e r m e t hod t o a na l yz e e s s e nt i a l ge ne s be c a us e i t yi e l ds c ol ony num be r s t ha t a r e e a s y t o c ount I n c ont r a s t m or e e f f i c i e nt m ut a ge ne s i s m e t hods c a n pr oduc e 10 5 r e c om bi na n t s w i t h t he s a m e s t a r t i ng c ul t ur e A bout 1 pe r c e nt o r 1000 of t he s e c e l l s pos s e s s a t a nde m dupl i c a t i on. W he n B a ba e t al t a r ge t e d a ge ne f o r de l e t i on, t he y ba s e d t he i r e s s e nt i a l i t y pr e di c t i on on t he 'ye s or no' qua l i t y of gr ow t h or de a t h on a pl a t e c ont a i n i ng ka na m yc i n. I n t h i s c a s e i t i s e a s y t o ove r l ook t he di f f e r e nc e be t w e e n 1000 a nd 10, 000 c ol oni e s on a P e t r i di s h be c a us e t he r e a r e t oo m a ny t o c ount O n t he ot he r ha nd, a s a c ons e que nc e of t he i ne f f i c i e nc y of t r a ns duc t i on, w hi c h r e s ul t s f r om t he a r bi t r a r y pa c ka gi ng o f D N A by P 1 pha ge w e c a n e a s i l y di s c e r n t he gr ow t h o f 2 c ol oni e s f r om 200 c ol oni e s f or e xa m pl e

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2 4 T he ot he r f a c t or t ha t m a s ke d t he phe not ype of m a n y K e i o m ut a t i ons w a s t he pr e s e nc e of s uppr e s s or or s e c ond s i t e m ut a t i ons T he y a r e de f i ne d a s s e c onda r y m ut a t i ons t ha t c or r e c t t he e f f e c t o f t he pr i m a r y m ut a t i ons a nd c a n be i nt r age ni c a nd i n t r ac odoni c i n t he s a m e ge ne a nd c odon, r e s pe c t i ve l y, a s t he o r i gi na l m ut a t i on, or e x t r age ni c out s i de t he or i gi na l ge ne ( L i n e t al 1984) C ons i de r a m u t a t i o n t ha t a l t e r s t he c ha r ge of a n e nc ode d ge ne m a ki ng i t nonf unc t i ona l ; he r e a c onve nt i ona l e xa m pl e of a n i nt r ac odoni c s uppr e s s or m ut a t i on w oul d be a n a m i no a c i d s ubs t i t ut i on ( e g a r gi ni ne t o gl yc i ne ) a t a pos i t i on ne a r t he m u t a t i on i n t he t e r t i a r y s t r uc t u r e of a p r ot e i n t ha t c om pe ns a t e s f or t he pr i m a r y m ut a t i on ( e g. by r e s t or i ng t he p r ot e i n s n a t i ve c ha r ge ) ( L i n e t al 1984 ) E a r l i e r w e c om m e nt e d on a t ype of e x t r age ni c s uppr e s s or m ut a t i on f or a p r i m a r y m ut a t i on i n t pi A w hi c h e l i m i na t e d t he t oxi c e f f e c t s t ha t r e s ul t e d f r om l os i ng T pi A a c t i vi t y W he n a m ut a nt r e qui r e s a s uppr e s s or m ut a t i on t o s ur vi ve t he di s r upt e d ge ne i s c l a s s i f i e d a s c ondi t i ona l l y e s s e nt i a l I t i s di f f i c ul t t o t r a ns f e r bo t h t he K e i o m ut a t i on a nd t he s e c ond s i t e m ut a t i on s i m pl y be c a us e t he P 1 pha ge pa c ka ge s a l i m i t e d a m ount o f D N A C ons e que nt l y, ve r y f e w t r a ns duc t a nt s a r e pr oduc e d. T he num be r of t r a ns duc t a nt s va r i e s w i t h t he di s t a nc e be t w e e n t he s e c ond s i t e m ut a t i on a nd t he ka na m yc i n c a s s e t t e of t he K e i o m ut a t i on on t he c hr om os om e D i f f e r e n c e s b e t w e e n t h e B ac k gr ou n d s of t h e D o n or an d R e c i p i e n t T he di f f e r e nc e s be t w e e n E c ol i B W 25113 a nd M G 1655_s e q_ r ph + 1 a r e i m por t a nt B ot h s t r a i ns a r e K 12 de r i va t i ve s ; t he y di f f e r how e ve r a t s e ve r a l ge ne t i c l oc i B W 25113

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2 5 ha s # l ac Z # r haB A D # ar aB A D r ph 1 a nd # hs dR m ut a t i ons ; M G 1655_s e q_ r ph + 1 c a r r i e s a n i ns e r t i on e l e m e nt I S 1H a t t he f l hD C l oc us c a us i ng hype r m ot i l i t y I n t r a ns duc i ng t he m ut a t i ons of t he K e i o c ol l e c t i on i t w a s ne c e s s a r y t o e ns ur e t ha t t he s e ba c kg r ound m ut a t i ons f r om t he K e i o c ol l e c t i on w e r e not i nt r oduc e d t o t he ne w K R E c ol l e c t i on. I n ot he r w or ds w e ne e de d t o ke e p t he e nt i r e ne w m ut a nt c ol l e c t i on i s oge ni c e xc e pt f or t he pa r t i c ul a r ka na m yc i n i ns e r t i on/ de l e t i on. I s oge ne t i c i t y i s f unda m e nt a l t o a ny c om pa r i s on be t w e e n knoc kout m ut a nt s T hi s i s i m por t a nt t o ke e p i n m i nd be c a us e P 1 pha ge c a n pa c ka ge a bout 100 ki l oba s e s of c hr o m o s om e ( K a ya pe r s ona l c om m uni c a t i on) C ons e que nt l y, i t c a n c ot r ans duc e unw a nt e d l ac r h a ar a or hs d m ut a t i ons w i t hi n r a nge of t he K e i o m ut a t i ons F or K e i o m u t a t i ons ne a r t he # l ac Z # r haB A D a nd # ar aB A D m ut a t i ons w e e xa m i ne d w he t he r t he s e ba c kgr ound m ut a t i ons w e r e c ot r a ns duc e d w i t h t he i nt e nde d K e i o m ut a t i on ( i e w he t he r t he y w e r e bot h pa c ke d i nt o one pha ge ) L i nke d o r c ot r a ns duc e d m ut a t i ons w e r e obs e r ve d i n t he r e c i pi e nt us i ng nut r i t i ona l c ol or t e s t s : # l ac Z # r haB A D a nd # ar aB A D f or m w hi t e c ol oni e s on L B pl us X g a l + I P T G M a c C onke y + R ha m nos e a nd M a c C onke y + A r a bi nos e m e di a r e s pe c t i ve l y. T o t e s t f o r r ph 1 a nd # hs dR m ut a t i on s a nd t he pr e s e nc e of t he I S 1H e l e m e nt w e pe r f or m e d c ol ony P C R on t he r e c i pi e nt s t r a i ns t o a m pl i f y a nd c onf i r m t he w i l d t ype s e que nc e a t e a c h of t he s e l oc i N e a r l y 30 of t he de l e t i ons w e r e s o c l os e t o t he ba c kgr ound m ut a t i ons i n t he dono r ( K e i o) s t r a i n t h a t t he y w e r e a l w a ys t r a ns f e r r e d t og e t he r a nd w e c oul d not us e t r a ns duc t i on. S i nc e a gr oup of m ut a nt s c a n be ve r i t a bl y c om pa r e d onl y w he n t he y a r e i s oge ni c e x c e pt f or t he gi ve n ge ne de l e t i on i t w a s ne c e s s a r y t o us e a di f f e r e nt s t r a t e gy t o a dd t h i s gr oup of m ut a t i ons t o our K R E c ol l e c t i on O ur s t r a t e gy w a s t o a m pl i f y t he de l e t i on f r a gm e nt of t he

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2 6 m ut a t i ons i n t he o r i gi na l K e i o s t r a i ns T he r e s ul t i n g D N A f r a gm e nt s w e r e us e d t o de l e t e e a c h of t he 30 pr obl e m a t i c ge ne s i n our M G 1655 w i l d t ype s t r a i n us i ng t he s a m e r e c om bi na t i on m ut a ge ne s i s m e t hod t ha t w a s us e d t o pr oduc e t he K e i o c ol l e c t i on. T he r e s ul t i ng m ut a t i ons w e r e t he n ba c kc r os s e d us i ng P 1 t r a ns duc t i on t o M G 1655 t o r e m ove a ny e xt r a ne ous m ut a t i ons B ac t e r i op h age m e d i at e d R e c om b i n at i on A n ove r vi e w of t he E c ol i hom ol ogous r e c om bi na t i on s ys t e m w i l l be o f us e gi ve n i t s i m por t a nc e t o t he ge ne t i c m a ni pul a t i ons us e d i n t hi s s t udy. H om o l ogous r e c om bi na t i on i s a f unda m e nt a l r e ge ne r a t i ve pr oc e s s t ha t o c c ur s i n e ve r y l i vi ng c e l l I n m os t o r ga ni s m s i t oc c ur s i nf r e que nt l y, a s i t r e qui r e s a c om pl e x s e t of r e a c t i ons a nd a n e xt e ns i ve D N A s e que nc e hom ol ogy. I n E c ol i r e c om bi na t i on w i t h l i ne a r D N A i s l i m i t e d by t he a c t i vi t y of t he ba c t e r i a l R e c B C D nuc l e a s e ( E l l i s e t al 200 1) T he goa l of pha ge m e di a t e d r e c om bi na t i on s ys t e m s ha s be e n t o i nc r e a s e t he f r e que nc y of r e c om bi na t i on ( E l l i s e t al 2001) W e us e d t he ba c t e r i opha ge R e d s ys t e m ( D a t e s e nko a nd W a nne r 2000) a s w a s us e d t o c ons t r uc t t he o r i gi na l K e i o m ut a t i ons T he R e d m e di a t e d r e c om bi na t i on r e qui r e s t he t hr e e ge ne p r oduc t s e nc ode d on t he pr opha ge : G a m E xo, a nd B e t a ( S e e F i gur e 11 ; D a t e s e nko a nd W a nne r 2000) E a c h of t he s e r e c om bi na t i on f unc t i ons i nc r e a s e s t he e f f i c i e nc y of ho m ol ogous r e c om bi na t i on w i t h l i ne a r doubl e s t r a nde d D N A ( S e e T a bl e 1 )

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2 7 F i g u r e 1 1 R e c o m b i n e e r i n g H o m o l o g o u s r e c o m b i n a t i o n i s t h e r e s u l t o f t h e a s s o c i a t i o n a n d e x c h a n g e o f t w o r e g i o n s o f n e a r l y i d e n t i c a l g e n e s e q u e n c e ( D a t s e n k o a n d W a n n e r 2 0 0 0 )

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2 8 P h a g e P r o t e i n A c t i v i t y G a m I n h i b i t s t h e n u c l e a s e a c t i v i t y o f R e c B C D E x o ( d s D N A d e p e n d e n t e x o n u c l e a s e ) D i g e s t s t h e l i n e a r D N A i n t h e 5 t o 3 d i r e c t i o n l e a v i n g 3 o v e r h a n g s t h a t a c t a s s u b s t r a t e s f o r r e c o m b i n a t i o n B e t a ( s s D N A b i n d i n g p r o t e i n ) P r o m o t e s h o m o l g o u s r e c o m b i n a t i o n b e t w e e n t h e c h r o m o s o m e a n d t h e s y n t h e t i c c o m p l e m e n t a r y s s D N A d o n o r T a b l e 1 A c t i o n o f t h e P h a g e p r o t e i n s a s t h e y c o n t r i b u t e t o p r o v i d i n g h o m o l o g o u s r e c o m b i n a t i o n w i t h l i n e a r D N A ( E l l i s e t a l 2 0 0 1 ) N o t e : d s D N A a n d s s D N A r e p r e s e n t d o u b l e a n d s i n g l e s t r a n d e d D N A r e s p e c t i v e l y I t w a s ne c e s s a r y t o ke e p t r a c k o f t he pr obl e m s t ha t m i ght l e a d t o e r r one ous e s s e nt i a l i t y c l a s s i f i c a t i on. F l ow c har t 1 i l l us t r a t e s a f e w of t he s our c e s of e r r o r w e s us pe c t e d m i ght unde r m i ne t he r e s ul t s obt a i ne d f r o m us e of t he K e i o c ol l e c t i on. F l o w c h a r t 1 O v e r v i e w o f t h e s c h e m e u s e d b y B a b a e t a l t o o b t a i n g e n e e s s e n t i a l i t y d a t a P o s s i b l e s o u r c e s o f e r r o r a r e g i v e n i n b o x e s b o r d e r e d i n r e d F r o m t h e t o p d o w n : t h e B W 2 5 1 1 3 s t r a i n h a s n u m e r o u s t r a n s f e r a b l e b a c k g r o u n d m u t a t i o n s s o m e o f t h e s e a r e u n c h a r a c t e r i z e d ; t h e d e l e t i o n c o n s t r u c t w h i c h w i l l r e c o m b i n e w i t h t h e b a c t e r i a l c h r o m o s o m e o n e i t h e r s i d e o f t h e t a r g e t e d g e n e c a n i n f l u e n c e e x p r e s s i o n o f d o w n s t r e a m g e n e s o r d e l e t e t h e m a l t o g e t h e r i f i t i s d e s i g n e d i n c o r r e c t l y ; a n d a s d e s c r i b e d i n a p r e v i o u s s e c t i o n t h e m u t a g e n e s i s s t r a t e g y c a n p r o d u c e m i s l e a d i n g r e s u l t s

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2 9 M at e r i a l s an d M e th od s S t r ai n s M G 1655_s e q_ r ph + 1 T he m ut a t i o n # r ph w a s r e pa i r e d i n t hi s s t r a i n t o l i m i t t he c hr om os om a l ba c kgr ound s i nf l ue nc e i n t hi s s t udy I n t he K e i o c ol l e c t i on s t r a i n B W 25113, t he f r a m e s hi f t m ut a t i on i n r ph s e ve r e l y r e duc e s R N a s e P H a c t i vi t y, l e a di ng t o a n i nc om pl e t e m a t ur a t i on of t R N A ne e de d f or gr o w t h. T he ot he r m ut a t i on i n r ph i s a s i ngl e ba s e pa i r de l e t i on c a us i ng a pol a r e f f e c t on t he dow ns t r e a m py r E ge ne w hi c h e nc ode s or ot a t e phos phor i bos yl t r a ns f e r a s e T hi s r e s ul t s i n pa r t i a l u r a c i l s t a r va t i on w he n gr ow n on m i ni m a l m e di um ( J e ns e n, 1993) T oge t h e r t he s e f a c t or s r e duc e t he gr ow t h of a # r ph m ut a nt by a bout 15 pe r c e nt ( J e ns e n, 1993) P r e p ar at i on o f P 1 L ys at e s I n t hi s s t e p, w e i nduc e d t he l y t i c c y c l e of P 1 phage by he at i ng a c ul t ur e of e ac h K e i o m ut ant w i t h a di l ut i on of t h e v i r us H os t D N A be c om e s nons pe c i f i c al l y pac k age d i nt o P 1 ph age par t i c l e s A n ove r ni ght c ul t u r e of t he K e i o donor s t r a i n w a s gr ow n i n 2 m L L ur i a B e r t a ni br ot h ( L B ) F r om t hi s 150 $ L w a s a dde d t o a t ube c ont a i ni ng L B 5 m M C a C l 2 a nd 1x10 6 p f u/ m L P 1 ba c t e r i opha ge a nd i nc uba t e d f or 5 6 hour s a t 37C T he t ube w a s ne xt de c a nt e d i nt o a 2 m L c e nt r i f uge t ube w i t h 100 150 $ L c hl or o f or m t he n c e nt r i f uge d a nd s t or e d a t 4C P 1 T i t e r D e t e r m i n at i on ( S e e F i gur e 12 ) I n t he i n i t i al out c r os s of t he 3985 K e i o m ut at i ons t he P 1 t i t e r w as not de t e r m i ne d. I n a s e c ond r ound of t r an s duc t i ons w e t i t e r e d e ac h of t he P 1 l y s at e s I n t hi s t i t e r s t e p, no k anam y c i n i s pr e s e nt s o t he t ot al num be r of phage par t i c l e s c an be de t e r m i ne d. A n ove r ni ght w i l d t ype c ul t ur e w a s gr ow n i n 2 m L

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3 0 L B T he P 1 l ys a t e w a s di l ut e d i n T C M buf f e r ( 10 m M f i na l c onc e nt r a t i ons of T r i s H C l C a C l 2 a nd M gC l 2 ) by a f a c t or of 10 6 a nd 10 4 I n E ppe ndor f t ube s 100 $ L of t he ove r ni ght c ul t ur e w a s m i xe d w i t h 100 $ L o f t he P 1 pha ge di l ut i on A f t e r a f i ve m i nu t e i nc uba t i on a t 37C t he pha ge ba c t e r i a m i xt ur e w a s a dde d t o t ube s c ont a i ni ng 2 5 m L L B t op a ga r ( 0 6% a ga r ; 5 m M C a C l 2 ) vor t e xe d br i e f l y a nd pou r e d ove r L B pl a t e s O n t he f ol l ow i ng da y, t he num be r of pl a que f or m i ng uni t s m ul t i pl i e d by t he di l u t i on f a c t or a nd di v i de d by t he pl a t e d vol um e ( m L ) ga ve t he pha ge c onc e nt r a t i on i n pf u ( pl a que f or m i ng uni t s ) pe r m L

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3 1 F i g u r e 1 2 P 1 t i t e r d e t e r m i n a t i o n ( L i n e t a l 1 9 8 4 ) A d i l u t i o n o f t h e p h a g e l y s a t e s u s p e n s i o n i s m i x e d w i t h a s u s p e n s i o n o f c e l l s a n d g i v e n t i m e t o a d s o r b ( i n f e c t ) T h e m i x t u r e i s t h e n d i l u t e d w i t h a m o l t e n s o l u t i o n o f a g a r a n d i m m e d i a t e l y p o u r e d o v e r t h e s u r f a c e o f a h a r d e n e d n u t r i e n t a g a r T h e p l a t e s a r e i n c u b a t e d f o r s e v e r a l h o u r s A f t e r w a r d s t h e n u m b e r o f p l a q u e s i s c o u n t e d t o d e t e r m i n e t h e p h a g e c o n c e n t r a t i o n T r an d s u c t i on I n t hi s s t e p, t he P 1 phage i nf e c t s t h e r e c i pi e nt s t r ai n. T r ans f e r of hos t D N A c ont ai ni ng t he i nt e nde d K e i o m ut at i ons i s s e l e c t e d f or on t he bas i s of k anam y c i n r e s i s t an c e I n t hi s c as e t he m e t hod s e l e c t s f or P 1 p hage c ont ai ni ng k now n D N A w i t h a k anam y c i n r e s i s t an c e ge ne T hi s D N A r e c om bi ne s w i t h t he M G 1655 c hr om os om e t o m ak e

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3 2 a bac t e r i um t hat gr ow s i nt o a c ol ony T he i ni t i al o ut c r os s of 3985 K e i o m ut at i ons w as f ac i l i t at e d by hi gh t hr oughput m e t hods i nc l udi ng t he us e of 24 w e l l C O ST A R di s he s ( Se e F i gur e 13) U nr e s t r i c t e d m ut at i ons t r a ns duc e d t o y i e l d ov e r 100 agar e m be dde d c ol oni e s M ut at i ons w hos e t r ans duc t i on w as r e s t r i c t e d w e r e obv i ous y i e l di ng f e w e r t han 20 c ol oni e s I n t he s e c ond r ound of t r ans duc t i ons hav i ng t i t e r e d e ac h of l y s at e s w e c al c ul at e d a t r ans duc t i on f r e que nc y f or e a c h K e i o m ut at i on. T h i s al l ow e d us t o c om par e t he t r ans duc t i on f r e que nc i e s of t he m ut at i on i n e ac h K e i o s t r ai n. T he r e c i pi e nt s t r a i n M G 1655_s e q_ r ph + 1 w a s gr ow n ove r ni ght i n L B di l ut e d i nt o a 1: 20 s ol ut i on a nd t he n gr ow n f or 6 7 hour s A 1: 10 di l ut i on of t he P 1 l ys a t e i n T C M buf f e r w a s pr e pa r e d. A m i xt ur e of 100 $ L of t he pha ge di l ut i on a nd 100 $ L f r e s h c e l l c ul t ur e w a s i nc uba t e d f or 20 m i nut e s a t 37C N e xt 1 m L o f L B pl us 10 m M s odi um c i t r a t e w a s a dde d a nd m i xe d f or 1 hour a t 37C A f t e r out gr ow t h t he s a m pl e w a s c e nt r i f uge d a t 4000X g f or 5 m i nu t e s F i na l l y, t he s upe r na t a nt w a s r e m ove d a nd t he c e l l s w e r e r e s us pe nde d i n 100 $ L pl a t i ng buf f e r a nd s pr e a d ont o one w e l l of a C O S T A R d i s h c ont a i ni ng t he s e l e c t i ve a nt i bi ot i c ka na m yc i n.

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3 3 F i g u r e 1 3 T h e u s e o f 2 4 w e l l C O S T A R d i s h e s f a c i l i t a t e d t h e i n i t i a l o u t c r o s s o f t h e 3 9 8 5 K e i o m u t a t i o n s B a c t e r i a l c o l o n i e s a r e v i s i b l e i n t h e e n l a r g e d w e l l C on f i r m i n g T an d e m D u p l i c at i on s I f a t r ans duc t i on y i e l de d v e r y f e w k anam y c i n r e s i s t ant c ol oni e s w e t e s t e d bot h t he K e i o hos t s t r ai n and t he r e c i pi e nt s t r ai ns f or dupl i c at e c opi e s of t he t ar ge t ge ne T he pr e s e nc e o f a t ande m dup l i c at i on i ndi c at e s t hat t he r e s pe c t i v e ge ne i s not di s pe ns abl e I t i s e i t he r e s s e nt i al or r e qui r e s s om e s pe c i al c ondi t i on f or gr ow t h. M os t pr i m e r s de s i gne d t o c o nf i r m t a nde m dupl i c a t i ons w e r e 20 nt i n l e ngt h; t he y w e r e or de r e d t hr ough t he D N A c or e l a b a t t he D e pa r t m e nt of B i oc he m i s t r y of t he U ni ve r s i t y of M i a m i A pa i r o f p r i m e r s ( f or w a r d a nd r e ve r s e ) w a s de s i gne d c om pl e m e nt a r y t o e i t he r s i de of t he ge ne t a r ge t s o pe n r e a di ng f r a m e E a c h pr i m e r pa i r w a s us e d t o r un a po l ym e r a s e c ha i n r e a c t i on. F or e a c h s a m pl e one c ol ony w a s s us pe nde d i n 20 $ L o f di s t i l l e d w a t e r O f t hi s t e m pl at e m i xt u r e 1. 5 $ L w a s t a ke n a nd l oa de d i nt o a P C R t ube c ont a i ni ng t he f or w a r d a nd r e ve r s e pr i m e r s ( 1. 25 $ L e a c h f r om a 0 1 M s t oc k s ol ut i on) a nd 6 $ L di s t i l l e d w a t e r N e xt 10 $ L of 2x P C R M a s t e r M i x, or de r e d f r o m S i gm a A l dr i c h, w a s a dde d t o e a c h P C R t ube m i xe d, a nd pl a c e d i n a t he r m oc yc l e r T he

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3 4 t he r m oc yc l e r w a s s e t t o a l i d t e m pe r a t u r e of 104C ; t he c yc l e of t he t e m pe r a t ur e c ha nge s w a s : 94C f or 5 m i nut e s ; 94C f or 45 s e c onds ; 61C f or 45 s e c onds ; 72C f or 5 m i nut e s T hi s s e que nc e w a s r e pe a t e d 29 t i m e s T he t he r m oc yc l e r he l d t he s a m pl e s a t 4C a f t e r t he pr ogr a m w a s c om pl e t e T h e r e a c t i on pr oduc t s w e r e ha r ve s t e d a nd r un t hr ough a 1% s ol ut i on of a ga r os e i n T B E c ont a i ni ng 3 $ L of 1 M e t hi di um b r om i de T o e a c h ge l 100 vol t s w e r e a ppl i e d f o r a bout 1 hour P r e p ar at i on o f C om p e t e n t C e l l s T hi s s t e p pr e pa r e s c om pe t e nt c e l l s f or t r ans f e c t i ng w i t h an A SK A c l one or pS I M 6 p l as m i d A n ove r ni ght c ul t ur e o f t he w i l d t ype M G 1655_s e q_ r ph + 1 w a s gr ow n ove r ni ght I t w a s t he n di l ut e d 1: 100 i n L B g r ow n t o a n opt i c a l de ns i t y, a t 600 nm of 0. 600 a nd put on i c e f or 30 m i nu t e s N e xt 5 m L o f t he c ul t ur e w a s pl a c e d i nt o a c e nt r i f uge t ube a nd s pun f or 5 m i nut e s a t 5000X g a nd 4C T he c e l l s w e r e t he n r e s us pe nde d i n 5 m L o f 0 1 M C a C l 2 a nd i nc uba t e d on i c e f or 30 m i nut e s T he c e nt r i f uga t i on w a s r e pe a t e d t o y i e l d a f i na l vol um e of 0 5 m L 0 1 M C a C l 2 ( T he c e l l s a t t hi s poi nt w e r e c om pe t e nt ) C h e m i c al T r an s f or m at i on ( S e e F i gur e 14 ) T o 0. 1 m L of t he C a C l 2 c om pe t e nt c e l l s 2. 5 $ L of pl a s m i d D N A w e r e t r a ns f e r r e d T he m i xt u r e w a s ke pt on i c e f or 30 m i nut e s he a t s hoc ke d a t 42C f or 2 m i nut e s a nd i c e d onc e m o r e f or 2 m i nu t e s N e xt 1 m L o f L B m e di a w a s a dde d a nd t he c e l l s w e r e i nc uba t e d f or 1 hour a t 37C F i na l l y, 200 $ L of t he c ul t ur e w a s a dde d t o L B pl us s e l e c t i ve a nt i bi ot i c p l a t e s N ot e : t he A S K A c l one s c a r r y c hl or a m phe ni c ol r e s i s t a nc e ; t he pS I M 6 r e c om bi n a t i on pl a s m i d c onf e r s r e s i s t a nc e t o a m pi c i l l i n.

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3 5 F i g u r e 1 4 B a s i c p r o c e d u r e f o r p l a s m i d t r a n s f o r m a t i o n i n E c o l i ( D a l e 1 9 8 9 ) C on s t r u c t i on of R e d R e c om b i n a n t T o pr e pa r e i nduc e d a nd c om pe t e nt c e l l s f or e l e c t r opor a t i on, M G 1655_s e q_ r ph + 1 w a s t r a ns f or m e d w i t h pS I M 6, a he a t i nduc i bl e R e c om bi na s e pl a s m i d ( us i ng m e t hods de s c r i be d i n t he pr e vi ous t w o s e c t i ons ) T r a ns f or m a nt s w e r e s e l e c t e d us i ng r e s i s t a nc e t o t h e a nt i bi ot i c a m pi c i l l i n P r e p ar at i on o f E l e c t r oc om p e t e n t C e l l s T he goal of t hi s pr oc e dur e i s ( a) t o i nduc e R e c om bi nas e and ( b) t o r e m ov e s al t s f r om t he m e d i um T hi s s t e p i s ne c e s s ar y t o e ns ur e a hi gh e f f i c i e nc y t r ans f or m at i on. T o t hi s e nd, pS I M 6 c om pl e m e nt e d c e l l s w e r e gr ow n ove r ni ght i n 5 m L S O B ( 2% B a c t ot r ypt one ; 0. 5 % Y e a s t e xt r a c t ; 10 m M N a C l ; 2 5 m M K C l ; 10 m M M gC l 2 ; 10 m M M gS O 4 ; 20 m M gl uc os e ) a t 30C N e xt t he c ul t ur e w a s di l ut e d 1: 40 i n 20 m L S O B pl us 50 $ g/ m L a m pi c i l l i n a nd gr ow n t o a n opt i c a l de ns i t y ( O D ) a t 600 n m of 0 600. T o i nduc e t he e xpr e s s i on of R e c om bi na s e t he c e l l s w e r e

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3 6 he a t e d t o 42C f o r 15 m i nut e s ; f r om t hi s a vol um e of 8 m L w a s a dde d i n e a c h o f t w o c e nt r i f uge t ube s a nd put on i c e f or 30 m i nu t e s T he c e l l s w e r e w a s he d t hr e e t i m e s w i t h 10% gl yc e r ol t o r e m ove s a l t A f t e r t he t hi r d c yc l e of c e nt r i f uga t i on t he c e l l s w e r e s u s pe nde d i n 0. 3 m L of 10% gl yc e r ol t o r e a c h a f i na l c onc e nt r a t i on of 150 O D / m L I nduc e d a nd c om pe t e nt c e l l s w e r e ke pt on i c e unt i l e l e c t r opor a t i on. P C R P r i m e r D e s i gn f or M u t age n e s i s W e c oul d not t r ans duc e a f e w of t he m ut at i ons w i t hout c ar r y i ng a s e c ond s i t e m ut at i on ( s e e s e c t i on t i t l e d D i f f e r e nc e s be t w e e n t he B ac k gr ound of t he D onor and R e c i pi e nt ) W e r e di d t he m ut age ne s i s i n our w i l d t y pe s t r ai n us i ng t he s am e i ns e r t i on s e que nc e of t he K e i o m ut ant s W e s i m pl y am pl i f i e d t he k anam y c i n c as s e t t e of t he pr obl e m at i c K e i o m ut at i ons us i ng c ol ony P C R T he f or w ar d and r e v e r s e de l e t i on pr i m e r s w e r e de s i gne d w i t h at l e as t 50 bp hom ol ogi e s on e i t he r e nd of t he k anam y c i n c as s e t t e T o m a ke ne w ge ne de l e t i ons bot h N t e r m i na l a nd C t e r m i na l de l e t i on p r i m e r s w e r e de s i gne d w i t h a t l e a s t 30 nt e xt e ns i ons a w a y f r o m t he i ni t i a t i on a nd t e r m i na t i on c odons r e s pe c t i ve l y, of t he t a r ge t ge ne A t l e a s t 50 bp ho m ol ogi e s on e i t he r e nd of t he t a r ge t e d ge ne a r e r e qui r e d t o i nduc e r e c om bi na t i on ( D a t s e nko a nd W a nne r 2000) T o ge ne r a t e P C R f r a gm e nt s c ont a i ni ng t he ka na m c yi n c a s s e t t e t he 20 nt s e que nc e 5 A T T C C G G G G A T C C G T C G A C C 3 a nd t he 21 nt s e que nc e 5 T G T A G G C T G G A G C T G C T T C G 3 w e r e a dde d t o t he 30 nt N t e r m i na l a nd C t e r m i na l de l e t i on pr i m e r s r e s pe c t i ve l y. P C R f r a gm e nt s w e r e a m pl i f i e d us i ng c ol ony P C R

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3 7 E l e c t r op or at i on F r om t he s ol ut i on o f e l e c t r oc om pe t e nt M G 1655_s e q_ r ph + 1 + pS I M 6 c e l l s 40 $ L w a s m i xe d w i t h 10 100 ng o f D N A ( ~ 1 $ L ) f r om t he P C R p r oduc t a nd pl a c e d i n a dr y C e l l P or a t o r c uve t t e T he c e l l s w e r e s hoc ke d w i t h 1. 75 ki l ovol t s a nd i nc uba t e d i n 1 m L S O C f or 1 hou r a t 37C T he n 3 00 $ L w a s s pr e a d ont o L B pl us t he a nt i bi ot i c t o s e l e c t f o r k an R t r a ns f or m a nt s

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3 8 R e s u l ts an d D i s c u s s i on P ar t 1: P 1 O u t c r os s I n t he i n i t i a l t r a ns duc t i on of t he 3985 K e i o m ut a t i o ns ( or i gi na l l y c l a s s i f i e d a s non e s s e nt i a l ) s t r i c t l y non e s s e nt i a l ge ne s yi e l de d ove r 100 w i l d t ype s i z e c ol oni e s or t r a ns duc t a nt s I n t he s e c a s e s t he r e w a s no di s c e r na bl e di f f e r e nc e be t w e e n t he m ut a nt phe not ype of t he K e i o hos t a nd t he w i l d t ype r e c i p i e nt F o r 2 364 K e i o m ut a t i ons w e c onc l ude t he i r r e s pe c t i ve ge ne s t o be non e s s e nt i a l T r a ns duc t i ons of t he ot he r 1621 K e i o m ut a t i ons ha d t o be f ur t he r i nve s t i ga t e d ( S e e T a bl e 2) : 1. A gr oup o f 1536 K e i o m ut a t i ons t r a ns duc e d t o yi e l d ove r 100 t r a ns duc t a nt s but gr e w s l o w l y i n t he r e c i pi e nt ba c kgr ound T he s e m ut a nt s w e r e not i nve s t i ga t e d f ur t he r 2. A gr oup o f 8 K e i o m ut a t i ons c oul d not be t r a ns duc e d f r om t he K e i o s t r a i n be c a us e t he di s r upt e d ge ne w a s ne c e s s a r y f or t he l ys oge ni z a t i on of t he P 1 pr opha ge W e us e d pl a s m i d c om pl e m e nt a t i on t o r e s ol ve t hi s l i m i t a t i on ( S e e s e c t i on t i t l e d C om pl e m e nt a t i on S t udi e s ) 3. A gr oup o f 77 K e i o m ut a t i ons w a s m os t p r obl e m a t i c T r a ns duc t i on of t he i r r e s pe c t i ve m ut a t i ons w a s r e s t r i c t e d, yi e l di ng f e w e r t ha n 100 ka na m yc i n r e s i s t a nt knoc kout s T he s e a r e di s c us s e d be l ow

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3 9 S l o w G r o w t h P 1 P h a g e R e s i s t a n t R e s t r i c t e d C r o s s 1 5 3 6 8 7 7 T a b l e 2 S u m m a r y o f P 1 t r a n s d u c t i o n s a f t e r i n i t i a l o u t c r o s s o f K e i o m u t a t i o n s i n t o M G 1 6 5 5 s e q r p h + 1 S l o w g r o w t h p h e n o t y p e w a s b a s e d o n s m a l l c o l o n y s i z e c o m p a r e d t o t h e s i z e o f c o l o n i e s w i t h n o n e s s e n t i a l g e n e d e l e t i o n s ; P 1 p h a g e r e s i s t a n c e w a s b a s e d o n P 1 t i t e r a n d a P 1 s e n s i t i v i t y t e s t ; a r e s t r i c t e d c r o s s w a s d e t e r m i n e d b a s e d o n t h e n u m b e r o f t r a n s d u c t a n t s R e s t r i c t e d C r os s e s R e s t r i c t e d c r os s e s a r e i ne f f i c i e n t t r a ns duc t i ons T h e y a r e i ndi c a t i ve o f f a c t or s t ha t m a s k t he phe not ype of a l e t ha l K e i o m ut a nt of w hi c h t he m os t i m po r t a nt a r e t a nde m dupl i c a t i ons a nd s uppr e s s or m ut a t i ons ( s e e I nt r odu c t i on) I n t he c a s e of e s s e nt i a l ge ne s a s m a l l num be r of t r a ns duc t i on s a r e obt a i ne d f r om t he s m a l l num be r o f c e l l s w i t h t a nde m dupl i c a t i ons i n t he ge ne t a r ge t F or s uppr e s s or m ut a t i ons t he t r a ns duc t i ons a r e l i m i t e d by t he i m pr oba bi l i t y of t he P 1 pha ge pa c ka gi ng bot h t he ( ne c e s s a r y) s e c ond s i t e m ut a t i on a nd t he ka na m yc i n c a s s e t t e of t he K e i o m ut a nt F l ow c har t 2 gi ve s a n ove r vi e w of c a s e s of r e s t r i c t e d c r os s e s w e obs e r ve d dur i ng t he P 1 out c r os s N e w l ys a t e s w e r e pr e pa r e d a nd t i t e r e d f or e a c h of t he s e 77 pr obl e m a t i c m ut a t i ons D ur i ng a r e pe a t e d r ound o f t r a ns duc t i ons w e t a l l i e d t he num be r of t r a ns duc t a nt s pe r pl a que f or m i ng uni t

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4 0 F l o w c h a r t 2 O v e r v i e w o f t h e o b s e r v e d c a s e s o f r e s t r i c t e d c r o s s e s T h e n u m b e r o f t r a n s d u c t a n t s o b t a i n e d i s s h o w n C a s e s t h a t r e q u i r e d a n a l t e r n a t i v e t o P 1 t r a n s d u c t i o n ( t o b e d e s c r i b e d ) a r e s h a d e d i n g r e e n T an d e m d u p l i c at i on s D r R udd no t i c e d t ha t a w e l l know n e s s e nt i a l ge ne r poD ( t he hous e ke e pi ng s i gm a s ubuni t of R N A pol ym e r a s e ) w a s de l e t e d i n a K e i o s t r a i n t ha t gr e w L a t e r w e f ound t ha t t he K e i o de l e t i on s t r a i n ha d a t r a ppe d d upl i c a t i on. T ha t i s t he ka na m yc i n c a s s e t t e r e pl a c e d one of t w o r poD c opi e s T he du pl i c a t e c opy w oul d not ha ve be e n m a i nt a i ne d i f r poD w a s non e s s e nt i a l T o e xa m i ne w he t he r t he s e t a nde m dupl i c a t i ons w e r e c om m on, w e t e s t e d e ve r y K e i o s t r a i n w hos e out c r os s w a s s e ve r e l y r e s t r i c t e d. W e de s i gne d a pa i r of P C R de t e c t i on pr i m e r s f or 39 m ut a t i ons w hos e t r a ns duc t i on f r e que nc y w a s l e s s t ha n 10 t r a ns duc t a nt s pe r

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4 1 10 7 pf u P C R t e s t i ng r e ve a l e d 20 c a s e s w he r e a kn oc kout m ut a nt w a s s uppor t e d by a dupl i c a t e c opy of t he di s r upt e d ge ne T he dupl i c a t i ons c oul d be c onf i r m e d i n bo t h t he K e i o hos t s t r a i n a nd r e c i pi e nt w i l d t ype s t r a i n ( S e e T a bl e 3 ) F i gur e 15 s how s a n e xa m pl e of e a c h of t hr e e obs e r ve d c a s e s of t a nde m dup l i c a t i on w e e nc ount e r e d. ( F or a r e por t o f t he r e m a i ni ng 19 o f t he 39 m ut a t i ons ( i e t hos e w i t ho ut a t a nde m dupl i c a t i on) w e t e s t e d, s e e t he ne xt s e c t i on. ) F i g u r e 1 5 T h r e e d i f f e r e n t t y p e s o f o b s e r v e d t a n d e m d u p l i c a t i o n s i n K e i o c o l l e c t i o n : ( a ) h e m E i s a n e s s e n t i a l g e n e ; ( b ) d e l e t i o n o f r p s O r e s u l t s i n a s e v e r e g r o w t h d e f e c t ; ( c ) c o d e l e t i o n o f a n e s s e n t i a l g e n e n e x t t o b t u B T h e a r r o w s p o i n t t o w a r d s t h e w i l d t y p e a l l e l e T h e m u t a n t s w i t h t a n d e m d u p l i c a t i o n s d i s p l a y b o t h a w i l d t y p e a l l e l e a n d t h e k a n a m y c i n c a s s e t t e N o t e : t h e P C R p r o d u c t s f o r t h e k a n a m y c i n c a s s e t t e s a r e a l l t h e s a m e l e n g t h ; a s p i c t u r e d t h e m a r k e r s i z e s i n e a c h g e l a r e n o t a l i g n e d D e t e r m i n i n g t h e c au s e of t h e t r ap p e d d u p l i c a t i o n s T o r e i t e r a t e t he ge ne s de l e t e d a s pa r t of t he K e i o c ol l e c t i on a r e s uppos e dl y non e s s e nt i a l T hi s a s s e s s m e nt i s ba s e d on t he a bi l i t y o f t he s i ngl e ge ne knoc kout t o gr ow

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4 2 w he n a ka na m yc i n c a s s e t t e i na c t i va t e s t he gi ve n ge ne t a r ge t B y t r a ns duc i ng t he m ut a t i ons w e w e r e a bl e t o de t e c t t he f ol l ow i ng pr obl e m s : 1. E s s e n t i al ge n e s f al s e l y c l as s i f i e d as n on e s s e n t i al W e e xa m i ne d t he de s i gn of t he de l e t i on c ons t r uc t f o r e a c h s t r a i n be f or e a t t e m pt i ng t o c l a s s i f y t he m T he c ons t r uc t i on i s s ound i f t he ka na m yc i n c a s s e t t e r e pl a c e s t he r e a di ng f r a m e of t he ge ne t a r ge t be t w e e n i t s i ni t i a t i on a nd t e r m i na t i on c odons ( B a ba e t al 2006 ) I n t he s e c a s e s t he pr e s e nc e of a ge ne dupl i c a t i on i ndi c a t e s t ha t t he ge ne t a r ge t i s r e qui r e d f o r gr ow t h ( S e e T a bl e 3 ) 2. M u t at i on s t h at h ave a p ol ar e f f e c t on n e ar b y ge n e s O f t he 20 s t r a i ns w i t h c onf i r m e d dupl i c a t i ons f our ha d de l e t i ons i n w ha t w e s us pe c t e d t o be a non e s s e nt i a l ge ne ( S e e T a bl e 3) I n t he s e c a s e s t he pr e s e nc e of a dupl i c a t i on i n t he ge ne t a r ge t w a s puz z l i ng. T he de l e t i on c ons t r uc t w a s c or r e c t f or t he s e m ut a t i ons but i t bo r de r e d a n e s s e nt i a l ge ne T he r e f o r e w e c ons i de r e d t he pos s i bi l i t y t ha t t he non e s s e nt i a l ge ne m ut a t i on c oul d i nhi bi t or ha ve a pol a r e f f e c t on t he a dj a c e nt e s s e nt i a l ge ne T o t e s t t hi s pos s i bi l i t y, w e pe r f or m e d a s e t of c om pl e m e nt a t i on s t udi e s w hi c h a r e di s c us s e d i n t he ne xt s e c t i on. 3. C on d i t i on al l y e s s e n t i al ge n e s f al s e l y c l as s i f i e d a s n on e s s e n t i al W e c ont i nuous l y r e f e r e nc e d t he publ i s he d l i t e r a t ur e f o r i nf o r m a t i on c onc e r ni ng t he f unc t i ona l r ol e s of t he s e ge ne s a nd w e t e s t e d w a ys w e c oul d pos s i bl y s uppor t gr ow t h of c e l l s w i t h m ut a t i ons i n t he s e ge ne s A f t e r a s e r i e s of t r i a l a nd e r r or gr ow t h s t udi e s w e de t e r m i ne d t ha t t he di s r upt i on of f ou r of t he s e ge ne s r e s ul t s i n a uxot r ophy. K e i o m ut a nt s i n t he s e ge ne s r e qui r e s pe c i a l s uppl e m e nt a t i on ( S e e

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4 3 T a bl e 3) I ns of a r w e c l a s s i f y t he s e ge ne s a s c ondi t i ona l l y e s s e nt i a l : f ol E f ol B a nd f ol P W i t hout t he s uppl e m e nt a l nut r i e nt t he r e s pe c t i ve ge ne i s e s s e nt i a l a nd knoc kout m ut a nt s r e qui r e t he dupl i c a t e w i l d t ype c opy. W he n w e r e pe a t e d t he t r a ns duc t i on w i t h t he r e qui r e d s uppl e m e nt w e obs e r ve d a n unr e s t r i c t e d c r os s i n e a c h c a s e T he s e w e r e c a us e s of nut r i t i ona l a uxot r ophy. W e a l s o e nc ount e r e d a t e m pe r a t ur e s e ns i t i ve m ut a nt i n r poH w hi c h c a nn ot gr ow a t t e m pe r a t ur e s a bove 20C T he K e i o m ut a nt i n t hi s ge ne w a s s us t a i ne d a t 37C by a dup l i c a t e c opy. T he r poH ge ne pr oduc t i s a 32 k i l oda l t on s i gm a f a c t or t ha t c onf e r s upon t he c or e R N A pol ym e r a s e t he a bi l i t y t o i ni t i a t e t r a ns c r i pt i o n f r om he a t s hoc k pr om ot e r s ( G r os s m a n e t al 1985 ) B e c a us e s om e he a t s hoc k ge ne s ( e g. dnaK a nd gr oE ) e nc ode pr ot e i ns t ha t a r e e s s e nt i a l f or gr ow t h a m ut a nt i n r poH gr ow s onl y be l ow t he he a t s hoc k t e m pe r a t ur e ( G r os s m a n e t al 1985 ) H e nc e r poH i s c ondi t i ona l l y e s s e nt i a l 4. P os t m u t age n e s i s s e l e c t i on of r e c om b i n an t s A n e xc e pt i ona l c a s e w a s obs e r ve d f or t he # r ps O m ut a nt T he hos t K e i o s t r a i n ha d a t a r ge t ge ne dupl i c a t i on, but t he m ut a t i on t r a ns duc e d t o yi e l d a w i l d t ype r e c i pi e nt w i t h onl y t he de l e t i on a l l e l e W hi l e t he hos t K e i o s t r a i n gr e w no r m a l l y w i t h t he ge ne du pl i c a t i on, t he w i l d t ype r e c i pi e nt gr e w s l ow l y be c a us e i t l a c ke d t he 30S r i b os om a l s ubuni t e nc ode d by r ps O T hi s i l l us t r a t e s t ha t dupl i c a t i ons c a n be t r a pp e d w he n a ne a r l y e s s e nt i a l ge ne i s t a r ge t e d w i t h m ut a ge ne s i s E xpe r i m e nt a l l y l a r ge ka na m yc i n r e s i s t a nt c ol oni e s w i t h a dupl i c a t e d t a r ge t ge ne w i l l a ppe a r s e ve r a l hour s pos t m ut a ge ne s i s T he phe not ype of t he s e m ut a nt s i s f a l s e T r ue knoc kout s w i t hout t he ge ne dupl i c a t i on, a r e l i ke l y t o s how up m a ny hour s l a t e r a s t i ny or dus t s i z e c ol oni e s

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4 4 ( K a ya pe r s ona l c om m uni c a t i on) I t i s c om m on t o r e s ol ve t he s e c onc e r ns by t r a ns duc i ng t he t a r ge t e d de l e t i ons out of t he m ut a g e ni z e d ba c kgr ound. 5. M i s t ak e n d ou b l e ge n e d e l e t i on s c au s e d b y f a u l t y d e l e t i on c on s t r u c t A ge ne dupl i c a t i on w a s f ound i n t he bt u B de l e t i on s t r a i n. B e c a us e bt uB i s a w e l l know n non e s s e nt i a l ge ne w e l ooke d c l os e r a nd f ound t ha t t he de l e t i on a l l e l e r e m ove s t he s t a r t c odon o f t he a dj a c e nt e s s e nt i a l ge ne m ur I ( S e e F i gur e 16) A l s o, w e obs e r ve d a w i l d t ype c opy of t he non e s s e nt i a l ge ne hol D i n t he # hol D m ut a nt I n t hi s c a s e t he de l e t i on f r a gm e nt r e m ove d t he p r om ot e r of t he ne a r by e s s e nt i a l ge ne r i m I D r R udd us e d t he de l e t i on pr i m e r s e que nc e s pr ovi de d by B a ba e t al t o c he c k f or a l l s uc h a c c i de nt a l doubl e ge ne knoc kou t s i n t he K e i o c ol l e c t i on. B a s e d on t he i r p r i m e r s e que nc e s a nd ge ne a nnot a t i ons a bout 200 of t he s i ngl e ge ne knoc kout s a r e a c t ua l l y doubl e m ut a nt s ( R udd, pe r s ona l c om m uni c a t i on) T hi s e xc l ude s K e i o s t r a i ns w hos e de l e t i ons r e m ove d t he r i bos om e bi ndi ng s i t e but l e f t t he ope n r e a di ng f r a m e of t he ne i ghbo r i ng ge ne i nt a c t W e r e m a de t he m ut a t i ons i f t he m i s t a ke nl y i na c t i va t e d ge ne w a s pr e di c t e d t o be e s s e nt i a l M os t of t he s e f a ul t y doubl e ge ne de l e t i ons how e ve r di s r upt t w o non e s s e nt i a l ge ne s s o w e ha ve ye t t o r e m a ke t he m F i g u r e 1 6 G e n e m a p d i s p l a y i n g h o w a D N A f r a g m e n t d e s i g n e d t o r e p l a c e a n o n e s s e n t i a l g e n e ( b t u B ) m i g h t f a u l t i l y i n h i b i t a n e s s e n t i a l g e n e ( m u r I ) ( h t t p : / / e c o g e n e o r g ) A s a s i d e n o t e c o m p o u n d s t h a t i n h i b i t

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4 5 M u r I h a v e r e c e n t l y b e c o m e t h e o b j e c t o f a n t i b i o t i c r e s e a r c h I n a d d i t i o n t o c a t a l y z i n g a r e a c t i o n i n c e l l w a l l s y n t h e s i s M u r I h a s i n s o m e s t r a i n s e v o l v e d t h e a b i l i t y t o s t o p D N A g y r a s e w h i c h i n t u r n b l o c k s D N A r e p l i c a t i o n a n d c e l l d i v i s i o n ( S e n g u p t a e t a l 2 0 0 8 ) T h e c u r r e n t p a n e l o f a n t i b i o t i c s t h a t i n h i b i t D N A g y r a s e b i n d w h i l e t h e p r o t e i n i s a t t a c h e d t o D N A W h e n M u r I i s p r e s e n t t h e g y r a s e i s t a k e n a w a y f r o m t h e s u b s t r a t e D N A a n d t h e s e a n t i b i o t i c s d o n o t w o r k T h e e m e r g e n c e o f m u l t i f u n c t i o n a l c o m p e n s a t o r y e n z y m e s i n a p h e n o m e n o n c a l l e d m o o n l i g h t i n g c o n t r i b u t e s t o t h e e m e r g e n c e o f a n t i b i o t i c r e s i s t a n c e i n b a c t e r i a ( S e n g u p t a e t a l 2 0 0 8 ) T a b l e 3 T a n d e m d u p l i c a t i o n s i n t h e K e i o c o l l e c t i o n d o n o r a n d M G 1 6 5 5 s e q r p h + 1 r e c i p i e n t G e n e E n c o d e d P r o d u c t E s s e n t i a l G e n e s I n c o r r e c t l y c l a s s i f i e d a s n o n e s s e n t i a l i n t h e o r i g i n a l K e i o c o l l e c t i o n p a r C T o p o i s o m e r a s e I V s u b u n i t A l p t B p r o b a b l e A B C t r a n s p o r t e r h e m E U r o p o r p h y r i n o g e n d e c a r b o x y l a s e r p s T 3 0 S r i b o s o m a l p r o t e i n S 2 0 a l a S A l a n i n e t R N A l i g a s e p r f B P e p t i d e c h a i n r e l e a s e f a c t o r 2 r p s U 3 0 S r i b o s o m a l s u b u n i t p r o t e i n S 2 1 d n a G P r i m a s e f o r D N A r e p l i c a t i o n g l y S G l y c i n e t R N A l i g a s e b e t a s u b u n i t g r o L C h a p e r o n i n C p n 6 0 g l m M P h o s p h o g l u c o s a m i n e m u t a s e r h o T r a n s c r i p t i o n t e r m i n a t i o n f a c t o r R h o r p o D h o u s e k e e p i n g s i g m a s u b u n i t o f R N A p o l y m e r a s e M u t a t i o n s n e x t t o e s s e n t i a l g e n e s ( S e e P o l a r E f f e c t s ) c o a A P a n t o t h e n a t e k i n a s e c o a E D e p h o s p h o C o A k i n a s e p r i B P r i m o s o m a l p r o t e i n n i l e S I s o l e u c i n e t R N A l i g a s e M u t a t i o n s t h a t p r o d u c e a u x o t r o p h y o r t e m p e r a t u r e s e n s i t i v i t y C o n d i t i o n / S u p p l e m e n t r p s O 3 0 S r i b o s o m a l s u b u n i t p r o t e i n S 1 5 f o l E G T P c y c l o h y d r o l a s e I t h y m i d i n e r p o H R N A p o l y m e r a s e s i g m a 3 2 t e m p e r a t u r e s b e l o w 2 0 C f o l B D i h y d r o n e o p t e r i n a l d o l a s e f o l a t e f o l P D i h y d r o p t e r o a t e s y n t h a s e f o l a t e F a u l t y d o u b l e d e l e t i o n c o n s t r u c t s h o l D D N A p o l y m e r a s e p s i s u b u n i t ( d o u b l e w i t h r i m I ) b t u B B 1 2 u p t a k e ( d o u b l e m u t a n t w i t h m u r I )

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4 6 S u p p r e s s or m u t at i on s O f t he 39 s t r a i ns t ha t ha d a ve r y l ow t r a ns duc t i on r a t e a nd t ha t w e r e t e s t e d f or t a nde m dupl i c a t i ons 19 w e r e s how n t o ha ve onl y t he ka na m yc i n c a s s e t t e w i t h no w i l d t ype dupl i c a t e T he y pos s i bl y ha ve a s uppr e s s or or s e c ond s i t e m ut a t i on t ha t a l l ow s a f e w of t he de l e t i on m ut a nt s t o gr ow I f t he s e c ond s i t e m ut a t i on i s unl i nke d ( m or e t ha n 80 kb a pa r t ) i t c a nnot be out c r os s e d be c a us e t he t w o m u t a t i ons a r e t oo f a r a pa r t t o be pa c ka ge d i n t he s a m e P 1 pha ge pa r t i c l e T hi s w a s obs e r ve d f or t w o o f t he s t r a i ns w hi c h m us t ha ve de ve l ope d t he s uppr e s s or m ut a t i on af t e r t he t r a ns duc t i on. O ut c r os s e s of t he o t he r 17 s t r a i ns yi e l de d onl y one o r a f e w t r a ns duc t a nt s T hi s w a s t he r e s ul t of e i t he r ( a ) l i nke d/ c ot r a nduc e d m ut a t i ons w i t hi n 80 kb o r ( b) s pont a ne ous s uppr e s s or m ut a t i ons T he s e ge ne s a r e c ondi t i ona l l y e s s e nt i a l T o c l a r i f y a ge ne t ha t i s non e s s e nt i a l but t ha t m a ke s t he c e l l s gr ow s l ow l y, w i l l not r e s t r i c t t he t r a ns duc t i on r a t e I n t he s e c a s e s how e ve r w e obs e r ve d a r e s t r i c t e d t r a ns duc t i on, l i m i t e d by t he l ow num be r of pha ge pa r t i c l e s w i t h l i nke d s uppr e s s or m ut a t i ons o r by f o r m a t i on of s pont a ne ous s uppr e s s or m ut a t i ons S pont a ne ous s uppr e s s or m ut a t i ons a r e a ppa r e nt w he n t he s i z e s of t he ka na m yc i n r e s i s t a nt c ol oni e s c ha nge ove r a s e r i e s of ge ne r a t i ons

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4 7 P ar t 2: C om p l e m e n t at i on S t u d i e s C i r c u m ve n t i n g P 1 P h age R e s i s t an c e A f e w of t h e de l e t i on s t r a i ns of t he K e i o c ol l e c t i on w e r e f ound t o be r e s i s t a nt t o P 1 ba c t e r i opha ge I n s om e c a s e s t he r e s i s t a nc e ha d pr e vi ous l y be e n s how n. F or e xa m pl e t ol Q e nc ode s a pr ot e i n i nvo l ve d i n out e r m e m b r a ne s t a bi l i t y a nd i s r e qui r e d f o r t r a ns l oc a t i on of pha ge D N A f r om t he e xt r a c e l l ul a r c a ps i d t hr ough t he out e r a nd i nne r m e m br a ne s i nt o t he c yt opl a s m ( L a z z a r oni e t al 1999) A m ut a nt i n s s p ( s t r i nge nt s t a r va t i on pr ot e i n) s how s a de f e c t i n t he P 1 l yt i c c yc l e a s i t s hi f t s f r om e a r l y t o l a t e ge ne e xpr e s s i on ( W i l l i a m s e t al 2001 ) B e c a us e t he s e m ut a nt s w e r e r e s i s t a nt t o P 1 ba c t e r i opha ge w e c oul d not m a ke P 1 l ys a t e s t o t r a ns duc e t he i r r e s pe c t i ve m ut a t i ons T he r e f or e w e ne e de d t o p r ovi de t he K e i o B W 25113 s t r a i n w i t h a w i l d t ype a l l e l e of t he m i s s i ng g e ne T hi s w a s done us i ng c he m i c a l t r a ns f or m a t i on t o c om pl e m e nt t he c e l l s w i t h t he a ppr op r i a t e A S K A c l one pl a s m i d e nc odi ng t he de l e t e d ge ne T r a ns f o r m a nt s w e r e s e l e c t e d us i ng c hl or a m phe ni c ol r e s i s t a nc e e nc ode d on t he A S K A pl a s m i d. U s i ng t he s e c e l l s w e pr od uc e d a nd t i t e r e d ne w P 1 l ys a t e s T he l ys a t e s w e obt a i ne d w e r e us e d, a s be f or e t o i nf e c t our w i l d t ype s t r a i n I n s ubs e que nt t r a ns duc t i ons t he K e i o m ut a t i ons t r a ns duc e d t o yi e l d ove r 100 t r a ns duc t a nt s ( a s de s c r i be d i n t he m e t hods s e c t i on) w i t h t he e xc e pt i on of t w o s t r a i ns w e f a i l e d t o c om pl e m e nt ( S e e T a bl e 4) B e s i de s va l i da t i ng t he pr e di c t i on t ha t t he s e ge ne s a r e non e s s e nt i a l w e m a de t he K e i o m ut a t i ons a va i l a bl e i n our w i l d t ype r e c i pi e nt M ut a nt s f r om

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4 8 t he K R E c ol l e c t i on ha ve a l r e a dy be e n s e nt t o t he C ol i G e ne t i c S t oc k C e nt e r ( C G S C ) a t Y a l e U ni ve r s i t y. K e i o M u t a n t P 1 T i t e r ( W i t h o u t c o m p l e m e n t ) p f u / m L P 1 T i t e r ( C o m p l e m e n t e d ) p f u / m L T o t a l N u m b e r o f C o l o n i e s # c o l o n y / 1 0 7 p f u ( W i t h o u t c o m p l e m e n t ) # c o l o n y / 1 0 7 p f u ( C o m p l e m e n t e d ) d n a K 1 x 1 0 5 1 x 1 0 8 3 0 3 0 0 l p c A 0 1 3 x 1 0 8 2 0 1 5 4 t o l Q 0 6 x 1 0 7 1 4 2 3 3 r n t 1 6 x 1 0 7 3 x 1 0 5 1 6 5 3 3 3 3 u b i G 3 x 1 0 6 9 x 1 0 7 2 4 3 3 3 2 6 7 u b i X 0 1 x 1 0 7 2 0 2 0 0 0 r e c A 0 F a i l e d t o C o m p l i m e n t u b i H 1 x 1 0 6 F a i l e d t o C o m p l i m e n t 1 0 0 0 r f a E 0 3 x 1 0 7 1 0 3 3 3 s s p A 0 2 x 1 0 7 1 5 7 5 0 r f a D 0 2 1 x 1 0 8 1 9 9 0 r f a C 0 1 1 x 1 0 8 2 4 2 1 8 r f a I 0 1 4 x 1 0 8 1 2 8 6 p r i A 4 x 1 0 6 3 x 1 0 5 1 1 0 3 6 6 6 6 h s d M 2 x 1 0 7 1 6 x 1 0 6 1 8 1 0 0 1 8 0 0 0 d n a T 2 x 1 0 6 1 3 x 1 0 8 3 7 1 0 0 0 2 8 5 T a b l e 4 R e s u l t s o f c o m p l e m e n t i n g P 1 r e s i s t a n t s t r a i n s a n d v e r y r e s t r i c t e d o u t c r o s s e s T o c l a r i f y t h e p u r p o s e o f c o m p l e m e n t i n g t h e s t r a i n s w a s o n l y t o o b t a i n a P 1 l y s a t e S u b s e q u e n t t r a n s d u c t i o n s w e r e p e r f o r m e d u s i n g o u r w i l d t y p e s t r a i n P ol ar E f f e c t s I n s t r a i ns w i t h t a nde m dupl i c a t i ons w e l ooke d a t n e i ghbor i ng ge ne s t o c he c k f o r pol a r e f f e c t s t he de l e t i on c ons t r uc t m i gh t ha ve on t he m T h i s be c a m e i m por t a nt w he n c ons i de r i ng t he c a ndi da t e e s s e nt i a l ge ne s t ha t B a ba e t al c oul d not di s r upt W e de s c r i be he r e a n i nve s t i ga t i on of t r a ns duc t a nt s w i t h dup l i c a t i ons i n non e s s e nt i a l ge ne s W he n t a r ge t i ng a ge ne i n a m ul t i ge ni c ope r on t he pr om o t e r of t he ka na m yc i n c a s s e t t e c a n s om e t i m e s i nt r oduc e pol a r e f f e c t s t ha t i nf l ue nc e ne a r by ge ne s T he s e a r e p r i m a r i l y a

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4 9 c ons e que nc e of t he c l us t e r i ng of ba c t e r i a l ge ne s i n ope r ons t ha t a r e t r a ns c r i be d j oi nt l y t o f or m a pol yc i s t r oni c m R N A t r a ns c r i pt ( L i n e t al 1984) T ha t i s r i bos om e s bi nd t o t he R N A a nd pr ogr e s s dow n t he w hol e of a n ope r on w i t hout pa us e I n t ot a l w e f ound s i x de l e t i on s t r a i ns t ha t s ha r e t he c onf l i c t o f a pa r t i a l di pl o i dy i n a non e s s e nt i a l ge ne I n t he s e c a s e s t he a dj a c e nt e s s e nt i a l ge ne t ha t w a s di s r upt e d i s a l s o dupl i c a t e d. T o i nve s t i ga t e t he c ont r i but i on o f pol a r e f f e c t s w e c om p l e m e nt e d M G 1655_s e q_ r ph + 1 s t r a i ns w i t h pl a s m i ds e xpr e s s i ng t he non e s s e nt i a l a nd e s s e nt i a l ge ne s i n que s t i on ( S e e T a bl e 5) A s de s c r i be d e a r l i e r t hi s w a s done us i ng c he m i c a l t r a ns f or m a t i on w i t h t he a ppr op r i a t e A S K A c l one p l a s m i d. N e xt f or e a c h pa i r o f c om pl e m e nt e d s t r a i ns ( or g r oup o f t h r e e i n t he c a s e of c oaA ) w e di s r upt e d t he non e s s e nt i a l ge ne by P 1 t r a ns duc t i on. I n t hi s w a y, w e obs e r ve d t he e f f e c t of t he t r a ns duc e d m ut a t i on i n s t r a i ns t ha t c ons t i t ue nt l y e xpr e s s e a c h of t he a dj a c e nt ge ne s I f t he non e s s e nt i a l ge ne c a n onl y be de l e t e d i n a s t r a i n c om p l e m e nt e d by a n a dj a c e nt ge ne w e s uppos e t ha t t hi s ne a r by ge ne i s i nhi bi t e d by a pol a r e f f e c t c a us e d by t he t r a ns duc e d m ut a t i on ( S e e F i gur e 16 ) T he f i r s t e xa m pl e w a s t he pr i B de l e t i on K e i o s t r a i n, w hi c h w a s f ound t o ha ve a t a nde m dupl i c a t i on, bu t t he l i t e r a t ur e ( a nd i t s pr e s e nc e i n t he K e i o c ol l e c t i on) s uppos e t he ge ne t o be non e s s e nt i a l W e s a w t ha t pr i B i s u ps t r e a m of t he e s s e nt i a l ge ne r ps R by 4 ba s e pa i r s I n t h i s c a s e a ddi t i ona l s t udy w a s r e qui r e d be c a us e t he pr e s e nc e of a t a nde m dupl i c a t i on doe s not a l l ow a f i r m c onc l us i on a bout t he e s s e nt i a l i t y of t he pr i B ge ne A s e c ond c a s e w a s t he K e i o de l e t i on s t r a i n f o r c oa A a w e l l know n non e s s e nt i a l ge ne B e c a us e c oaA i s non e s s e nt i a l w e w e r e s ur pr i s e d t o s e e t ha t a de l e t i on m ut a nt r e qui r e d t he w i l d t ype a l l e l e I nt e r e s t i ngl y, D r Y K a ya s a w t ha t c oaA r uns i n t he

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5 0 oppos i t e di r e c t i on of t w o a dj a c e nt e s s e nt i a l ge ne s bi r A a nd m ur B ( S e e F i gu r e 17) M ur B c a t a l yz e s a r e a c t i on i n pe pt i dogl yc a n s ynt he s i s a nd i s e s s e nt i a l ( Z oe i by e t al 2002 ) A bi r A m ut a nt c a nnot t a ke up bi o t i n a nd w i l l di e w i t h out hi gh c onc e nt r a t i ons o f bi ot i n ( C a m pbe l l e t al 1972) T he r e pl a c e m e nt of c oaA w i t h t he ka na m yc i n c a s s e t t e w a s hypot he s i z e d t o dow n r e gul a t e t he e xpr e s s i on of t h e s e t w o e s s e nt i a l ge ne s t hr ough a nt i s e ns e i nhi bi t i on. T ha t i s t he s t r ong p r om ot e r o f t he a nt i bi o t i c r e s i s t a nc e c a s s e t t e m i ght i nduc e t r a ns c r i pt i on pa s t t he de l e t i on c ons t r uc t ; i n t hi s c a s e t he r e s ul t i ng a nt i s e ns e m R N A i na c t i va t e s t he e s s e nt i a l ge ne by bi ndi ng t o t he c od i ng m R N A of a n e s s e nt i a l ge ne T hi s t ype of a nt i s e ns e i nhi bi t i on i nvol vi ng a ka na m yc i n c a s s e t t e i ns e r t i on ha s ye t t o be r e por t e d. B e f o r e de s c r i bi ng ou r r e s ul t s w e r e f e r t h e r e a de r t o F i gur e 16, w hi c h c l a r i f i e s s ubs e que nt di s c u s s i on. G e n e D e l e t e d b y P 1 T r a n s d u c t i o n G e n e C o m p l e m e n t e d b y T r a n s f o r m a t i o n N u m b e r o f T r a n s d u c t a n t s m u r B ( e s s e n t i a l ) ~ 2 0 0 b i r A ( e s s e n t i a l ) 0 c o a A c o a A ( n o n e s s e n t i a l ) 0 p r i B ( n o n e s s e n t i a l ) 1 p r i B r p s R ( e s s e n t i a l ) 4 8 ( t i n y ) i l e S 3 i l e S l s p A 8 c o a E 2 4 5 c o a E y a c F 0 T a b l e 5 P o l a r e f f e c t s : P 1 t r a n s d u c t i o n i n t o c o m p l e m e n t e d s t r a i n s

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5 1 F i g u r e 1 7 R e s u l t s t h a t p r o v i d e t h e f i r s t e x a m p l e o f a n t i s e n s e i n h i b i t i o n d u e t o k a n i n s e r t i o n F i g u r e 1 8 G e n e m a p f r o m D r K R u d d s d a t a b a s e s h o w i n g t h e p o s i t i o n i n g a n d d i r e c t i o n o f c o a A w i t h r e s p e c t t o t w o e s s e n t i a l g e n e s b i r A a n d m u r B ( h t t p : / / e c o g e n e o r g ) T he m os t s t r i ki ng of t he r e s ul t s i n T a bl e 5 i nvol ve s c oaA a ge ne w hi c h c oul d onl y be de l e t e d i n a s t r a i n c om pl e m e nt e d w i t h t he ne a r b y e s s e nt i a l ge ne m ur B T he ka na m yc i n c a s s e t t e c oul d not r e pl a c e c oaA w i t hout a t a nde m dupl i c a t i on, de s pi t e t he f a c t t ha t c oaA c ode s f or a non e s s e nt i a l ki na s e ( V a l l e r i a nd R oc k, 1987) T hi s s ugge s t s t ha t t he r e i s

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5 2 s om e t hi ng i n t he c oa A de l e t i on c ons t r uc t t ha t i s t o xi c t o t he c e l l W e p r opos e t ha t t he pr om ot e r o f t he k an c a s s e t t e i nduc e s t r a ns c r i p t i on be yond i t s i ns e r t i on s i t e t ow a r ds t he t w o e s s e nt i a l ge ne s T hi s m a y i nhi bi t t he t r a ns l a t i on of bot h m ur B a nd bi r A A s di s pl a ye d i n F i gur e 16, w e w e r e a bl e t o di s r upt c oaA i n t he m ur B c om pl e m e nt e d s t r a i n, bu t not i n t he bi r A c om pl e m e nt e d s t r a i n P e r ha ps t he e xpr e s s i on of t he ka na m yc i n c a s s e t t e f r om c oaA i s s uf f i c i e nt t o r e duc e t he e ndoge nous bi r A e xpr e s s e d a nd bi r A e xpr e s s e d f r om t he pl a s m i d. O ne e xpl a na t i on i s t ha t m ur B a s a r e p r e s s or pr ot e i n, i s r e qui r e d a t l ow c onc e nt r a t i ons w hi l e B i r A i s r e qui r e d a t hi ghe r c o nc e nt r a t i ons T he a bi l i t y t o c om pl e m e nt m ur B on t he pl a s m i d m i ght a l s o be c a us e d by t he g r e a t e r di s t a nc e be t w e e n m ur B a nd c oaA t ha n be t w e e n bi r A a nd c oa A ( S e e F i gur e 17) I t i s i nt e r e s t i ng t ha t bot h t he s e ge ne pr oduc t s ha ve be e n r e s e a r c he d a s t a r ge t s f or a nt i b i ot i c i nhi b i t or s T r a ns duc t i ons pe r f or m e d w i t h t he ot he r ge ne pa i r s i n T a bl e 5 i ndi c a t e pol a r e f f e c t s w hi c h a r e pr oba bl y t he c ons e que nc e of p e r t ur be d t r a ns c r i pt i on H ow e ve r t he s e t r i a l s w oul d ne e d t o be r e pe a t e d t o m a ke a m or e de f i ni t i ve c l a s s i f i c a t i on. P ar t 3: B ac t e r i op h age m e d i at e d R e c om b i n at i on L i n k e d B ac k gr ou n d M u t at i on s A s di s c us s e d i n t he i nt r oduc t i on t he di f f e r e nc e s be t w e e n E c ol i B W 25113 a nd M G 1655_s e q_ r ph + 1 a r e i m por t a nt W hi l e t r a ns f e r r i ng m ut a t i ons w i t h P 1 ba c t e r i opha ge i t w a s i m por t a nt t o ke e p i n m i nd t he ba c kgr ound m ut a t i ons of t he P 1 donor T he K e i o c ol l e c t i on s t r a i n, B W 25113, ha s ba c kgr ound m ut a t i ons a t # l ac Z # r haB A D # ar aB A D

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5 3 r ph 1 a nd # hs dR I t a l s o i s m i s s i ng a n i ns e r t i on e l e m e nt ( I S 1H ) a t t he f l hD C l oc us ( S e e F i gur e 16) T o e ns u r e t ha t t he s e ba c kgr ound m ut a t i ons a r e not i nt r oduc e d f r om B W 25113 i nt o t he K R E c ol l e c t i on, w e s e t a s i de a gr oup of 37 K e i o m ut a nt s w hos e ka na m yc i n i ns e r t i on/ de l e t i on w a s w i t hi n ~ 80 kb of one t he B W 25113 ba c kgr ound m ut a t i ons F or t he s e m ut a nt s P 1 t r a ns duc t i on a l w a ys c a r r i e s bot h t he ka na m yc i n c a s s e t t e a nd one of t he B W 25113 ba c kgr ound m ut a t i ons F or ot he r m ut a t i ons t ha t w e r e onl y s om e t i m e s l i nke d t o B W 25113 ba c kgr ound m ut a t i ons w e us e d t he nut r i t i ona l c ol or t e s t s or P C R de s c r i be d i n t he i nt r oduc t i on t o i s ol a t e t r a ns duc t a nt s w i t h j us t t he de s i r e d m ut a t i on. F or t he K R E c ol l e c t i on t o be a r e l i a bl e r e s our c e i t s m ut a nt s m u s t be i s oge ni c w i t h t he e xc e pt i on of t he i r r e s pe c t i ve ge ne de l e t i ons T hi s w a y, w e know t he phe not ype o f a m u t a nt i s c a us e d by t he i na c t i va t i on of i t s t a r ge t e d ge ne a nd not by a c ha nge i n t he c hr om os om a l ba c kgr ound. F i g u r e 1 9 A s t h i s f i g u r e d i s p l a y s t h e r e c i p i e n t M G 1 6 5 5 s e q r p h + 1 s t r a i n h a s t h e i n s e r t i o n s e q u e n c e I S 1 H a t t h e f l h C D l o c u s b u t t h e h o s t K e i o s t r a i n d o e s n o t T o e n s u r e t h a t t r a n s d u c t i o n f r o m t h e K e i o m u t a n t s d o e s n o t r e m o v e o u r w i l d t y p e r e c i p i e n t s I S 1 H e l e m e n t w e o p t e d t o r e c o n s t r u c t s o m e o f t h e K e i o m u t a t i o n s i n o u r w i l d t y p e w i t h o u t u s i n g t r a n s d u c t i o n W e d i d t h i s f o r a l l t h e g e n e s i n t h e p i c t u r e d c h r o m o s o m a l f r a m e ( S e e T a b l e 6 ; h t t p : / / e c o g e n e o r g )

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5 4 W e de c i de d t o r e m a ke t he 37 m ut a nt s i n our w i l d t ype s t r a i n M G 1655_s e q_ r ph + 1 us i ng r e c om bi na t i on ( Se e M at e r i al s and M e t hods ) U nf or t una t e l y I w a s a bl e t o r e m a ke onl y 12 of t he m ut a nt s be f or e m y t i m e w a s up. A f t e r t he r e t u r n o f D r Y K a ya w ho w a s on l e a ve t ow a r ds t he e nd of m y s t a y, t he r e m a i ni ng m ut a nt s w i l l l i ke l y be m a de O nc e t he m ut a nt s a r e c ons t r uc t e d, t he y c a n be ba c kc r os s e d t o i s ol a t e t he m ut a t i ons a ny e xt r a ne ous m ut a t i ons I s us pe c t t ha t t he s e t r a ns duc t i ons w i l l b e unr e s t r i c t e d a nd e a c h of t he t a r ge t e d ge ne s w i l l be non e s s e nt i a l s i nc e none of t he ge ne s a r e pr e di c t e d t o be e s s e nt i a l G e n e N e a r b y B a c k g r o u n d M u t a t i o n c h e R M i s s i n g I S 1 H a t f l h D C c h e W M i s s i n g I S 1 H a t f l h D C c h e A M i s s i n g I S 1 H a t f l h D C m o t B M i s s i n g I S 1 H a t f l h D C m o t A M i s s i n g I S 1 H a t f l h D C f l h C M i s s i n g I S 1 H a t f l h D C f l h D M i s s i n g I S 1 H a t f l h D C u s p C M i s s i n g I S 1 H a t f l h D C o t s A M i s s i n g I S 1 H a t f l h D C o t s B M i s s i n g I S 1 H a t f l h D C a r a H M i s s i n g I S 1 H a t f l h D C a r a F M i s s i n g I S 1 H a t f l h D C f t n A M i s s i n g I S 1 H a t f l h D C y e c H M i s s i n g I S 1 H a t f l h D C t y r P M i s s i n g I S 1 H a t f l h D C y e c A M i s s i n g I S 1 H a t f l h D C y e c C M i s s i n g I S 1 H a t f l h D C f l i Y M i s s i n g I S 1 H a t f l h D C f l i C M i s s i n g 1 S 1 H a t f l h D C f l i S M i s s i n g I S 1 H a t f l h D C y e d D M i s s i n g I S 1 H a t f l h D C y e d N M i s s i n g I S 1 H a t f l h D C f l i F M i s s i n g I S 1 H a t f l h D C f l i J M i s s i n g I S 1 H a t f l h D C y i b T r p h m i n u s k b l r p h m i n u s r f a B r p h m i n u s r f a P r p h m i n u s m u t M r p h m i n u s r p m G r p h m i n u s y i c R r p h m i n u s s l m A r p h m i n u s p y r E r p h m i n u s y i c C r p h m i n u s

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5 5 d i n D r p h m i n u s y i c G r p h m i n u s l i g B r p h m i n u s T a b l e 6 G e n e s i n a c t i v a t e d b y m u t a g e n e s i s t h a t c o u l d n o t b e d e l e t e d b y t r a n s d u c t i o n w i t h o u t a t r a n s f e r r i n g a n e a r b y B W 2 5 1 1 3 b a c k g r o u n d m u t a t i o n s T h e 1 2 s t r a i n s t h a t I c o m p l e t e d a r e b o l d i n t h e t a b l e ; t h e y w e r e s t r e a k e d t w i c e o n L B p l a t e s p l u s k a n a m y c i n a n d t h e n f r o z e n i n D M S O N e w D e l e t i on C on s t r u c t s T he 303 ge ne s t ha t B a ba e t al c oul d not m a ke w e r e pur por t e d t o be e s s e nt i a l D r K a ya r e vi e w e d t he l i t e r a t u r e c onc e r ni ng e a c h o f t h e s e ge ne s a nd f ound t ha t 33 of t he 303 w e r e e l s e w he r e s how n t o be non e s s e nt i a l T o r e a s s e s s t he s e a nd ot he r pr e di c t i ons w e de s i gne d de l e t i on f r a gm e nt s t o p r oduc e ne w m ut a t i ons i n t he M G 1655 s t r a i n ( S e e T a bl e 7) A f t e r s e gr e ga t i ng t he r e c om bi na nt s w e us e d P 1 ba c t e r i opha ge t o t r a ns duc e t he 33 m ut a t i ons i n t o B W 25113 a nd M G 1655_s e q_ r ph + 1 W e obt a i ne d nor m a l i z e d t r a ns duc t i on f r e que nc i e s a nd, i n 25 c a s e s s a w no r e s t r i c t e d c r os s e s F or 8 o t he r s t r a i ns w e f a i l e d t o obt a i n w or ki ng l ys t a t e s a nd, f or l a c k o f t i m e di d n ot ba c kc r os s s t he m ut a ge ni z e d s t r a i ns I n s u m w e c onf i r m e d t he non e s s e nt i a l i t y of t he s e ge ne s r e ga r dl e s s of ba c kgr ound. D e l e t i o n P 1 T i t e r / 1 0 9 ( p f u / m l ) T o t a l c o l o n i e s ( S u m ) M G 1 6 5 5 B W2 5 1 1 3 # c o l o n y / 1 0 7 p f u M G 1 6 5 5 B W2 5 1 1 3 b t u B 0 1 4 9 2 7 6 4 0 0 1 8 5 0 0 2 6 8 0 0 i n t Z y f f S 0 0 1 4 1 3 5 3 0 0 9 6 4 0 2 1 4 0 h o l D 0 2 9 1 1 5 2 0 0 5 1 6 8 r i m I 0 0 9 8 4 2 8 2 3 0 0 2 8 0 0 3 0 4 0 y d f B 0 0 4 8 1 8 3 0 0 3 7 5 6 2 0 y h h Q 0 0 1 6 1 1 2 3 1 2 8 7 6 3 0 7 9 0 0 y h b V 0 0 3 0 2 3 5 2 0 0 1 1 0 0 6 6 2 0 b c s B 0 0 5 0 9 6 0 1 3 1 1 1 0 0 2 5 0 0 y f i T 0 0 1 6 4 2 7 8 7 1 6 0 0 5 3 0 0 y r b B 0 0 6 4 4 5 2 3 0 0 8 0 0 4 6 0 0 y d i L 0 1 6 8 1 2 9 5 7 1 5 6 0 e n t D 0 0 5 2 4 2 4 2 5 4 0 0 4 0 0

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5 6 y q g T 0 0 9 6 2 0 2 0 0 2 0 0 2 0 0 0 y a g G 0 0 4 0 6 2 0 0 1 3 4 4 9 0 0 3 3 0 0 y i g P 0 0 0 0 3 4 8 6 0 1 6 0 0 0 0 2 0 0 0 0 0 u b i B 0 0 0 0 2 2 0 0 2 0 0 1 1 1 1 0 0 1 1 1 1 0 0 y m f K 0 0 0 0 2 3 0 0 5 1 6 6 0 0 2 7 0 0 y r a L 0 0 3 6 8 1 6 6 2 8 4 4 5 0 0 7 7 0 0 a l s K 0 0 2 7 2 3 0 0 2 0 0 1 2 0 0 8 0 0 c y d A 0 0 0 0 9 3 0 0 3 0 0 3 7 0 0 0 3 7 0 0 0 c y d C 0 0 0 0 3 2 3 2 5 2 8 5 9 0 0 1 9 2 0 0 w a a U 0 0 0 5 1 3 0 0 3 0 0 5 8 8 0 0 5 8 8 0 0 w z y E M u t a g e n e s i s F a i l e d m i n D 0 0 0 1 6 3 9 5 5 2 7 0 0 3 8 2 0 m i n E M u t a g e n e s i s F a i l e d T a b l e 7 N e w d e l e t i o n s n o t i n K e i o c o l l e c t i o n a n d t h e i r t r a n s f e r ( u s i n g P 1 t r a n s d u c t i o n ) i n t o K e i o a n d K R E c o l l e c t i o n b a c k g r o u n d s W e f ound t ha t s om e of t he c ons t r uc t e d m ut a nt s r e q ui r e d s pe c i a l m e di a a ddi t i o na l s uppl e m e nt s or ot he r uni que c ondi t i ons t o gr ow F or e xa m pl e gapA e nc ode s f or gl yc e r a l de hyde s 3 phos pha t e de hy dr oge na s e ( G A P D H ) w hi c h c onve r t s gl yc e r a l de hyde 3 phos pha t e ( G A P ) i n t o 1 3 bi s phos phogl yc e r a t e ( S e t a e t al 1997 ) W e obs e r ve d t ha t a gapA m ut a nt c oul d onl y gr ow on m i ni m a l m e di a s uppl e m e nt e d w i t h di a m i nopi m e l i c a c i d ( D A P ) T he b i os ynt he s i s of di a m i nopi m e l i c a c i d a n e s s e nt i a l c om pone nt of pe pt i dogl yc a n i n t he ba c t e r i a l c e l l w a l l i s l i m i t e d by t he pr e s e nc e of py r uva t e ( F r a e nke l e t al 1996) I n a gapA m u t a nt py r va t e i s l i m i t e d; m o r e ove r t he i n c r e a s e d c onc e nt r a t i on of G A P ( w i t hout G A P H ) l e a ds t o a h i gh e r r a t e of i s om e r i z a t i on t o di hydr oxya c t one phops pha t e ( D H A P ) A s de s c r i be d i n t he i nt r oduc t i on, D H A P s pont a ne ous l y de c om pos e s t o t he t oxi c i nt e r m e di a t e m e t hyl gl yoxa l ( K a ya pe r s ona l c om m uni c a t i on) W e w e r e onl y a bl e t o i s ol a t e a gapA m u t a nt on m i ni m a l m e d i a w hi c h m i ght be t he r e s ul t of t he l ow e r gl yc ol yt i c a c t i vi t y i n l i m i t i ng c ondi t i ons a nd c ons e que nt l y l o w e r c onc e nt r a t i ons of t he t oxi c i nt e r m e di a t e

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5 7 A l s o not e w or t hy, w e e xa m i ne d a n unknow n e s s e nt i a l ge ne y f f S w hi c h bor de r s t he pr opha ge r e m na nt E ut / C P Z 55 E c ol i K 12 s t r a i n M G 1655 ha s s e ve n s uc h pr opha ge e l e m e nt s ( ht t p: / / e c oge ne or g) W e f ound t ha t t he i n a c t i va t i on of t hi s ge ne a l one c a us e s c e l l de a t h, w hi l e t he de l e t i on of t he w hol e pr opha ge e l e m e nt ( 9 ge ne s t ot a l ) doe s not s e e m t o a f f e c t t he c e l l H e nc e one f unc t i on of t he y f f S ge ne m i ght be t o r e pr e s s t he E ut / C P Z 55 pr opha ge C on c l u s i o n s T hough P 1 t r a ns duc t i on i s a m uc h us e d l a bor a t or y t e c hni que t o da t e i t ha s not be e n a ppl i e d t o a s s e s s ge ne e s s e nt i a l i t y. I t i s uni qu e l y a pt t o s uc h c ha r a c t e r i z a t i ons i n i t s uni f or m i t y a nd i ne f f i c i e nc y. I n a ddi t i on ou r a na l y s i s of e s s e nt i a l i t y w a s m os t l y qua nt i t a t i ve i n c ont r a s t t o t he l e s s s t r i nge nt qua l i t a t i ve a s s e s s m e nt us e d i n t he or i gi na l m ut a ge ne s i s c a us e d by r e c om bi na t i on. I t s houl d a l s o be r e i t e r a t e d t ha t a n e f f i c i e nt l y m ut a ge ni z e d c e l l i s pr one t o s e c onda r y m ut a t i ons t ha t a t r a ns duc t a nt i s not A f t e r t he f e w r e m a i ni ng e x pe r i m e nt s a r e c om pl e t e d, D r s R udd a nd K a ya w i l l r e por t i n a ddi t i on t o t he e s s e nt i a l ge ne s e t ne w c l a s s e s of e s s e nt i a l i t y: ( 1) non e s s e nt i al ge ne s i nc l udi ng a l l t he unr e s t r i c t e d c r os s e s ; ( 2) e s s e nt i al ge ne s t hos e w hi c h c a nnot be i na c t i va t e d i n a ha pl oi d; ( 3) c ondi t i onal l y e s s e nt i al ge ne s t hos e w hos e i na c t i va t i on r e qui r e s s om e s e c ond s i t e m ut a t i ons T he r e vi e w e d a s s i gnm e nt s of ge ne e s s e nt i a l i t y a r e i nc or por a t e d i nt o D r R udd s w e bs i t e w hi c h c a n b e a c c e s s e d a t ht t p: / / e c oge ne or g I n t he i n i t i a l out c r os s w e ve r i f i e d m os t of t he r e s ul t s of t he K e i o c ol l e c t i on. G e ne s w hos e di s r upt i on w a s r e s t r i c t e d i n t hi s s t e p w e r e r e t e s t e d a nd c l a s s i f i e d a s e i t he r

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5 8 e s s e nt i al or c ondi t i onal l y e s s e nt i al F o r ge ne s w i t h a l ow t r a ns duc t i on f r e que nc y, w e pr oc e e de d t o c he c k f or a t a nde m dupl i c a t i on or s e c ond s i t e m ut a t i on. I f t hi s s t e p w a s i nc onc l us i ve w e c ons i de r e d t he pos s i bi l i t y of pol a r e f f e c t s or P 1 r e s i s t a nc e I f t he de l e t i on c ons t r uc t of a K e i o m ut a nt w a s a t f a ul t ( e g doubl e m ut a nt s ne a r by ba c kgr ound m ut a t i ons ) or i f a ge ne de l e t i on w a s not pr e s e nt ( d ue t o l a c k o f s e que nc e a nnot a t i on a t t he t i m e of t he B a ba e t al s t udy) w e m ut a ge ni z e d t he ge ne a nd s ubs e que nt l y t r a ns duc e d e a c h r e c om bi na nt t o obs e r ve t he e f f i c i e nc y o f ge ne t r a ns f e r T he s e e xpe r i m e nt s de m ons t r a t e a num be r of p r obl e m s t ha t c onf r ont a ge nom e w i de s t udy. T he y s how t he i m po r t a nc e of r e f e r e nc i ng t he l i t e r a t u r e t o c om pa r e r e s ul t s a nd c he c k f or pr ob l e m s R e f e r e n c e s B ab a e t al 2006. K e i o c ol l e c t i on of E c ol i de l e t i o n m ut a nt s M ol e c ul ar Sy s t e m s B i ol ogy 21 : 1 11. C am p b e l l A D e l C a m p i l l o C am p b e l l A an d C h an g R 1972. A m ut a nt of E s c he r i c hi a c ol i t ha t r e qui r e s hi gh c onc e nt r a t i ons o f bi ot i n P N A S 69 : 676 680. d H e r e l l e F 1917. S ur un m i c r obe i nvi s i bl e a nt a g oni s t i c de s bac i l l e s dy s e nt e r i que C R A c ad. Sc i P ar i s 165 : 373 375 D al e JW 1989 M ol e c ul ar G e ne t i c s of B ac t e r i a J ohn W i l e y & S ons L t d D at s e n k o K A an d Wan n e r B L 2000. O ne s t e p i na c t i va t i on of c hr o m os om a l ge ne s i n E s c he r i c hi a c ol i K 12 us i ng P C R p r oduc t s P N A S 97 : 6640 6645.

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5 9 E l l i s H M Y u D D i T i z i o T a n d C ou r t D L 2001 H i gh e f f i c i e nc y m ut a ge ne s i s r e pa i r a nd e ngi ne e r i ng of c hr om os om a l D N A us i ng s i ngl e s t r a nde d ol i gonu c l e ot i de s P N A S 98 : 6742 6746 F e r gu s on e t al 1998 M e t hyl gl yoxa l p r oduc t i on i n ba c t e r i a A r c h M i c r obi ol 170 : 209 219. F r ae n k e l e t al 1996 G l yc ol ys i s i n E s c he r i c hi a c ol i and Sal m one l l a 2 e d. W a s hi ngt on, D C : A SM P r e s s 189 198. G e r d e s e t al 2003 E xp e r i m e nt a l de t e r m i na t i on a n d s ys t e m l e ve l a na l ys i s of e s s e nt i a l ge ne s i n E s c he r i c hi a c ol i M G 1655. J B ac t e r i ol 18 5 : 5673 5684. G r os s m an e t al 1985. M ut a t i ons i n t he r poH G e n e of E s c he r i c hi a c ol i K 12 phe not ypi c a l l y s uppr e s s a t e m pe r a t ur e s e ns i t i ve m ut a nt J B ac t e r i ol 161 : 939 943. Je n s e n K F 1993. T he E c ol i K 12 w i l d t ype s W 3110 a nd M G 1655 ha ve a n r ph f r a m e s hi f t m ut a t i on t ha t l e a ds t o pyr i m i d i ne s t a r va t i on due t o l ow py r E e xpr e s s i on l e ve l s J B ac t e r i ol 175 : 3401 3407. K aya Y an d R u d d K 2008 N ot e s a n d pe r s ona l c om m uni c a t i on K i m J an d C o p l e y S D 2007 W hy m e t a bol i c e nz ym e s a r e e s s e nt i a l or none s s e nt i a l f or gr ow t h of E s c he r i c hi a c ol i K 12 on G l uc os e B i oc he m i s t r y 46 : 12501 12511. K l au s e t al 2005 H i ghe r pl a nt pl a s t i ds a nd c ya no ba c t e r i a ha ve f ol a t e c a r r i e r s r e l a t e d t o t hos e of t r ypa nos om a t i ds J B i ol C he m 46 : 38457 38463. L az z ar on i e t al 1999 T he T o l pr o t e i ns of E s c he r i c hi a c ol i a nd t he i r i nvol ve m e nt i n t he upt a ke of bi om ol e c ul e s a nd out e r m e m b r a ne s t a bi l i t y. F E M S M i c r obi ol L e t t 177 : 191 197.

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6 0 L e d e r b e r g e t al 1952 S e x c om pa t i bi l i t y i n E c ol i G e ne t i c s 37 : 720 730. L i n E C C G ol d s t e i n R an d S yvan e n M 1984 B ac t e r i a, P l as m i ds and P hage s H a r va r d U ni ve r s i t y P r e s s S am an t e t al 2008 N ut r i e nt s ne e de d f or ba c t e r i a l bl oods t r e a m gr ow t h. P L oS P a t h oge ns 4 : 1 10. S c am u f f a M D an d C ap r i ol i R M 1980 C om pa r i s on of t he m e c ha ni s m s of t w o d i s t i nc t a l dol a s e s f r om E c ol i gr ow n on gl uc one oge ni c s ubs t r a t e s B i oc he m B i ophy s A c t a 614 : 583 590. S e n gu p t a e t al 2008 M oonl i ght i ng f unc t i on o f gl ut a m a t e r a c e m a s e f r om M y c obac t e r i um t ube r c ul os i s : r a c e m i z a t i on a nd D N A gyr a s e i nhi bi t i on a r e t w o i nde pe nde nt a c t i vi t i e s of t he e nz ym e M i c r obi ol ogy 154 : 2796 2803 S e t a e t al 1997 C ha r a c t e r i z a t i on of E s c he r i c hi a c ol i s t r a i ns w i t h gap A a nd gapB ge ne s de l e t e d. J B ac t e r i ol 179 : 5218 5221 T w or t F W 1915 A n i nve s t i ga t i on on t he na t ur e of ul t r a m i c r os c opi c vi r us e s L anc e t 2 : 1241 1243. V al l e r i D S an d R oc k C O 1987 I s ol a t i on a nd c ha r a c t e r i z a t i on of t e m pe r a t ur e s e ns i t i ve pa nt ot he na t e ki na s e ( c oaA ) m u t a nt s of E s c he r i c hi a c ol i J B ac t e r i ol 169 : 5795 5800. Wat s on JD an d C r i c k F H 1953. M ol e c ul a r s t r uc t ur e of nuc l e i c a c i ds ; a s t r uc t ur e f or de oxyr i bos e nuc l e i c a c i d N at ur e 171 : 737 738

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6 1 Wi l l i am s M D F u c h s JA an d F l i c k i n ge r M C 20 00. N ul l m ut a t i on i n t he s t r i nge nt s t a r va t i on pr ot e i n o f E s c he r i c hi a c ol i di s r upt s l yt i c de ve l opm e nt of ba c t e r i opha ge P 1. G e ne 109 : 21 30 X i e e t al 2007. I m p r ovi ng s i m va s t a t i n bi oc onve r s i on. M e t abol i c E ngi ne e r i ng 9 : 379 386 Y am ar ak i e t al 2008 P r of i l i ng o f t he E c ol i C hr om os om e D a t a ba s e Y ar m ol i n s k y M B 2004 B a c t e r i opha ge P 1 i n r e t r os pe c t a nd i n pr os pe c t J B ac t e r i ol 186 : 7025 7028. Z h ou e t al 1988 I s ol a t i on a nd c ha r a c t e r i z a t i on o f E c ol i m ut a nt s t ha t l a c k t he he a t s hoc k s i gm a f a c t or s i gm a 32. J B ac t e r i ol 170 : 364 0 3649. Z oe i b y A E S an s c h agr i n F an d L e ve s q u e R C 2 002. S t r uc t ur e a nd f unc t i on o f M ur e nz ym e s : de ve l opm e nt of nove l i nh i bi t or s M ol e c u l ar M i c r obi ol ogy 47 : 1 12.


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