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Microbiological Research166 ( 2 0 1 1 ) 3 6 9 3 7 9

Available online at www.sciencedirect.com

www.elsevier.de/micres

Role of-factor (orf21) in clavulanic acidproduction inStreptomyces clavuligerusNRRL3585

Hum Nath Jnawali, Kwangkyoung Liou, Jae Kyung Sohng

Institute of Biomolecule Reconstruction (iBR), Department of Pharmaceutical Engineering, SunMoon University,

# 100, Asansi, Chungnam 336-708, Republic of Korea

R e c e i v e d 2 J u n e 2 0 1 0 ; r e c e i v e d i n r e v i s e d f o r m 1 5 J u l y 2 0 1 0 ; a c c e p t e d 1 7 J u l y 2 0 1 0

KEYWORDS

C l a v u l a n i c a c i d ;Replicatio n;S i g m a f a c t or ;Streptomyces

clavuligerus

N RRL 3585

SummaryA p u t a t i v e s i g m a f a c t o r g e n e , o r f 2 1 , w a s d i s r u p t e d o r o v e r e x p r e s s e d i n t h e w i l d - t y p ec l a v ul a n i c a c i d ( C A ) p r o du c e rStreptomyces clavuligerus N R R L 35 8 5 a n d c h a r ac t e r -i ze d. A n o rf 21 m ut an t (Streptomyces clavuligerus H N1 4 ) o f S. clavuligerus waso b t a in e d b y i n s e rt i o n a l i n a c t i v a t io n v i a d o u b le - c r os s o v er. A l t h ou g h t h e r e w a s l i t -t l e r e d u c t i o n o f s p o r u l a t i o n i n t h e m u t a n t , t h e g r o w t h p a t t e r n w a s s i m i l a r b e t w e e nm u ta n t a n d w i ld - ty p e. T h e p r od u ct i on w a s r e du c ed b y 1 0 1 5 % i n S. clavuligerusH N 14 c o mp a re d t o t h at i n w i ld - ty p e. O v er e xp r es s io n o f o r f2 1 i n w i ld - ty p e c e ll sc a us e d h y pe r pr o du c ti o n o f s p or e s o n s o li d m e di u m a n d i n cr e as e d c l av u l an i c a c idp r od u ct i on b y 1 . 43 - fo l d. T h e o v er e xp r es s io n o f o r f2 1 i n w i ld - ty p e S. clavuligeruss t im u la t ed t h e e x pr e ss i on o f t h e e a rl y c l av u la n ic a c id g e ne s , c e as 2 a n d c a s2 , a n dt h e r e g u l a t o r y g e n e , c c a R , a s d e m o n s t r a t e d b y R T - P C R . T h e e l e v a t i o n o f t h e c e a s 2 ,c a s 2 a n d c c a R t r a n s c r i p t s w a s c o n s i s t e n t w i t h t h e e n h a n c e d p r o d u c t i o n o f c l a v u l a n i cacid. 2 0 1 0 E l s e v i er G m b H . A l l r i g h ts r e s e rv e d .

Introduction

B a c t er i a a r e w i d es p r ea d o r g an i s ms c a p ab l e o f c o l -o n iz i ng i n d i ff e re nt e nv i ro n me n ts a n d c a n s u rv i ve

C orresponding author.T e l . : + 8 2 4 1 5 3 0 2 2 4 6 ; f a x : + 8 2 4 1 5 4 4 2 9 1 9 .

E-mail address: [email protected] ( J . K . S o h n g) .

u nd e r v a ri o us t y pe s o f s t re s se s . T h is c a pa c it y i s c o n-f e rr e d i n p a rt b y a l te rn a ti v e s i gm a f a ct o rs , w h ic hp e r m i t t h e r a p i d a d a p t a t i o n o f b a c t e r i a t o e n v i r o n -m e nt a l c h an g es b y s wi t ch i ng t h ei r g e ne t ic p r og r am si n r e s p o n s e t o i n t e r n a l o r e x t e r n a l s i g n a l s . A l t e r n a -t i v e s i g m a f a c t o r s o f t h e e x t r a c y t o p l a s m i c f u n c t i o n( E C F ) s u b f a m il y w e re i n i ti a l l y d e s c r ib e d a s a d i v e r-g e n t g r o u p o f t h e 70 f a m i ly i n v ol v e d i n r e s po n s est o c h a n g e s i n t h e e x t r a c y t o p l a s m i c c o m p a r t m e n t o f

0 9 4 4 -5 0 1 3 /$ s e e f r o n t m a t t er 2 0 1 0 E l s e v i er G m b H. A l l r i g h t s r e s e r v e d.doi:10.1016/j.micres.2010.07.005
http://localhost/var/www/apps/conversion/tmp/scratch_10/dx.doi.org/10.1016/j.micres.2010.07.005http://localhost/var/www/apps/conversion/tmp/scratch_10/dx.doi.org/10.1016/j.micres.2010.07.005http://localhost/var/www/apps/conversion/tmp/scratch_10/dx.doi.org/10.1016/j.micres.2010.07.005mailto:[email protected]://localhost/var/www/apps/conversion/tmp/scratch_10/dx.doi.org/10.1016/j.micres.2010.07.005http://localhost/var/www/apps/conversion/tmp/scratch_10/dx.doi.org/10.1016/j.micres.2010.07.005mailto:[email protected]://localhost/var/www/apps/conversion/tmp/scratch_10/dx.doi.org/10.1016/j.micres.2010.07.005

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370 H.N. Jnawali e t al.

Figure 1. T h e c l a v u la n i c a c i d b i o sy n t h e ti c p a t h w a y i nS. clavuligerus. S o l i d l i n e s r e p r e s e n t k n o w n s t e p s a n d b r o k e nl i n e s i n d i c a t e u n k n o w n p o r t i o n s o f t h e p a t h w a y .

t h e c e l l (L on e tt o e t a l . 1 9 94). B a ct e r ia l R N A p o l y -me rase containing the primary factor, 70 , d i r e c t sa nd i ni ti at es t ra ns cr ip ti on o f a l ar ge n um be r o f g e n es c o m mo n l y e x p re s s ed i n e x p o ne n t ia l g r o wt hp ha se (B o ru k ho v a a n d S ev e ri n ov 2 0 02 ; Yo u ng e t

a l . 2 0 02). O v er t h e p a st s e ve ra l d e ca d es , a l te r na -tive f ac to rs i n t he 70 s u pe rf a mi l y h a ve b e ens ho wn t o d ir ec t t he r es pe ct iv e p oo l o f R NA p ol y-m e r a s e m o l e c u l e s t o t r a n s c r i b e s u i t e s o f g e n e s t h a ta l l o w a d a p ta t i on t o c h a ng i n g e n v ir o n me n t al c o n di -t i o n s (L o ne t to e t a l . 1 9 94 ; G r ub e r a n d G r os s 2 0 03 ;P a g e t a n d H e l m a n n 2 0 0 3). T h e r a t e o f t r a n s c r i p t i o ni n it i at i on f r om v a ri o us p r om o te r s h a s b e en k n ow nt o b e r eg ul at ed b y a n um be r o f D NA -b in di ng p ro -t e in s , w hi c h e i th er b i nd t o s p ec i fic s eq u en ce s o nD N A ( a c t i v a t o r s o r r e p r e s s o r s ) ( B r o w n i n g a n d B u s b y2 0 0 4 ; G r a i n g e r a n d B u s b y 2 0 0 8 ; B a l l e z a e t a l . 2 0 0 9 )o r r a th e r b i nd i n a n o ns p ec ifi c m a nn er ( n uc l eo i d-associate d prote ins) (M c L e o d a n d J o h n s o n , 2 0 0 1) orb y m ec h an is m s t h at d o n o t i nv o lv e d i re c t i nt e ra c -t i on o f t r an s cr i pt i on f a ct o rs w i th D N A (H a ug e n e ta l . 2 0 08). T h e b i n d i n g o f t h e c o g n a t e R s e A t o S i g Eh a s b e en d e mo n st r at e d (D on a e t a l. 2 00 8). Rse A,t h e S i gE s p ec i fic a n ti - si g ma f a ct o r i s i na c ti v at e dby phosphory lation-de pe nde nt C lpC 1P2 prote oly sis(B a r i k e t a l . 2 0 1 0 ).

Streptomyces clavuligerus N R RL 3 58 5 i s a G r am -p o si t iv e fi l am e nt o us b a ct e ri um t h at i s w e ll k n ow nf o r i t s a b i l i t y t o p r o d u c e a n a r r a y o f - l a c t a m c o m -

p o un d s, i nc l ud i ng c ep h am y ci n C , c l av u la n ic a ci d( C A ) a n d o t h e r s t r u c t u r a l l y r e l a t e d c l a v a m s . C A h a sp o o r a n t i ba c t er i a l a c t i vi t y, b u t i t b i n d s i r r ev e r si b l yt o t he s er in e h yd ro xy l g ro up a t t he a ct iv e c en te rof - l a c ta m a se , p r o d uc i n g a s t a b le a c y l at e d i n t er -

m ed ia te a nd i na ct iv at in g t he e nz ym e (L i ra s a n dRodrguez-Garcia 2000). T h e b i o s yn t h et i c p a t h wa yl e ad i ng f r om t h e p r im a ry m et a bo l it e s l-arginineand gly ce ralde hy de -3-phosphate to clavulanic acid,as shown in Fig. 1 (Khal eeli et al. 1999;Va le nt in e e t a l. 1 99 3), i s s ti ll u nd er i nt en si vei nv es ti ga ti on . T he fi rs t s te p i n t he b io sy nt he ti cp a t h wa y g i v es r i s e t o N 2 - (2 - c a rb o x ye t h yl ) - a rg i n in ea n d i s c a t al y z e d b y c a r b ox y e th y l a rg i n in e s y n th a s e( Ce as ) (K ha le el i e t a l. 1 99 9; P r ez -Re do nd o e ta l. 1 99 9). - L ac t am s y nt h et a se ( B ls ) c a ta l yz e st h e s e c on d s t e p c o n ve r t in g c a r bo x y et h y l ar g i ni n et o d e o x yg u a ni d i no - p r oc l a v am i n ic a c i d, w h i ch n o wc o nt a in s t h e -l ac ta m r ing (B ac hm an n e t a l.1998). D e o x yg u a ni d i no - p r oc l a v am i n ic a c i d i s t h e nh y d ro x y l at e d t o g i v e g u a ni d i ne - p r oc l a v am i n ic a c i db y c la va mi na te s yn th as e ( Ca s) (B al dw in e t a l.1991; Marsh et al. 1992). S ub se qu en tl y, p ro -c l a v am i n ic a c i d a m i d in o h y d ro l a s e ( Pa h ) c a t al y z est h e r e a ct i o n f r o m g u a ni d i ne - p r oc l a v am i n ic a c i d t op ro cl av am in ic a ci d, w he re t he g ua ni di no g ro upf ro m t he a rg in in e- de ri ve d e nd o f t he m ol ec ul ei s r em ov ed (E ls on e t a l. 1 99 3). C l av a mi n at e s y n-t ha se ( Ca s) t he n c at al yz es t he f or ma ti on o f t he

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R o l e o f - f a c t o r (orf21) i n c l a v u l a n i c a c i d p r o d u c t i o n i n Streptomyces clavuligerus NRRL3585 371

fi r st b i cy c li c i n te rm e di a te t h ro u gh o x id a ti v e r i ngc lo su re o f p ro cl av am in ic a ci d t o g iv e d ih yd ro -c l av a mi ni c a c id f o ll o we d b y d e sa t ur a ti o n t o f o rmc l a v a m i n i c a c i d ( B a l d w i n e t a l . 1 9 9 1 ; S a l o w e e t a l .1990). C l a v a ld e h yd e , t h e l a s t k n o wn i n t er m e di a t eo f t h e p a t h w a y , u l t i m a t e l y g e t s c o n v e r t e d t o c l a v u -l a ni c a c id b y c l av u la ni c a c id d e hy d ro g en a se ( C ad )

(N i ch o ls o n e t a l . 1 9 94). Howe ver, the me chanismb y w hi c h c l av a mi ni c a c id i s c o nv e rt e d t o c l av a ld e -h y d e i s s t i l l n o t c l e a r . S o , w e e x a m i n e d t h e r e g i o n so f t h eS. clavuligerus c h r o m o s o m e i n t h e v i c i n i t y o f t h e C A c l u s t e r t o i d e n t i f y a d d i t i o n a l g e n e s t h a t a r ei n v ol v e d i n t h e b i o s y n th e s is a n d r e g u l a t io n o f C A .

I n t h e p r e s e n t s t u d y , w e e x a m i n e d t h e e f f e c t s o f t h e s i g m a f a c t or g e ne (orf21) b y d i sr u pt i ng orf21,w h i c h w a s f o u n d d o w n s t r e a m o f t h e c l a v u l a n i c a c i d( CA ) g en e c lu st er s i n S. clavuligerus NRRL 3585.We a l so e x am i ne d t h e e f fe c ts o f o v er ex p re ss i onof orf21 o n C A p r o d uc t i on . A l t h ou g h t h e f u n ct i o n s

o f t he m aj or it y o f t he s ig ma f ac to r g en es r em ai nu n kn ow n , t h e e x is t en c e o f a s i gm a f a ct o r g e n e i nS. clavuligerus N R RL 3 58 5 i m pl i es t h at i t m a y h a vea n i m p o rt a n t p h y s i o l og i c al r o l e. T h i s w o r k d e m on -s t ra t es t h at t h e o v er ex p re ss i on o f orf21 encodingR NA P s ig ma f ac to r s li gh tl y e nh an ce s C A p ro du c-tion.

Materials and methods

Bacterial strains, culture conditions andDNA manipulation

T he b ac te ri al s tr ai ns a nd p la sm id s u se d i n t hi ss tu dy a re l is te d i n Ta b l e 1. Escherichia coli wereg r o wn i n L u r ia B er t a n i ( L B ) b r o th a n d m a i nt a i ne don LB agar medium at 37 C . D N A m a ni p ul a ti o nw as c ar ri ed o ut i n E. coli XL 1-Blue (Stratage ne ).P l as m id s w e re p r op a ga t ed i n E. coli E T 12 5 67 t oo b t a in u n m et h y la t e d D N A f o r t r a ns f o rm a t i on i n t oS. clavuligerus NRRL 3585. S. clavuligerus w a s c ul -t u r e d o n d i f f e r e n t m e d i a f o r d i f f e r e n t p u r p o s e s . F o rp r ot o pl a st t r an s fo r ma t io n , T S B ( 2 5 m l , 1 . 7% t r yp -t o ne , 0 . 3% s o yt o ne , 0 . 25 % d e xt r os e , 0 . 5% s o di um

c hl or id e, a nd 0 .2 5% p ot as si um p ho sp ha te ) s up -p l em en te d w it h 1 % ( w/ v) m al to se w as i ni ti al l yi n oc ul a te d w i th s e ed c u lt u re o f S. clavuligerus at28 C f or 2 4 h . T he g ro wi ng s ee d c ul tu re w as t he nt ra ns fe rr ed t o R 2Y E m ed iu m ( 50 m l, 5 % s uc ro se ,0 . 0 2 % p o t a s s i um s u l f at e , 1 % m a g ne s i um c h l or i d e,1 % g lu co se , 0 .5 % y ea st e xt ra ct , a nd 0 .0 1% D if coc as am ino a cid ) at 25 0 r pm an d 2 8C for 36 h .Streptomyces t r an s f or m a nt s w e r e s u p p le m e nt e dw i t h a p r a m y c i n ( 2 5 g / m l ) o r n e o m y c i n ( 2 0 g/ml).F o r C A p r od u ct i on , t h e m e di um w as c o mp o se d o f 2 . 0% g l yc e ro l , s o yb e an fl ou r, 1 5 g ; s o lu b le s t ar c h,

4 .7 g ; K H2 PO4 , 0. 1 g ; a nd F eSO47H2 O, 0 .2 g . T hem ed iu m w as a dj us te d t o p H 6 .8 w it h N aO H. A llb ac te ri al s to ck s w er e s to re d i n 3 0% g ly ce ro l a t70 C . T he p GE M- T E as y v ect or w as t he ro u-t i ne c l on i ng v e ct o rs u s ed f o r D N A m a ni p ul a ti o n.p K C1 1 39 (K ie s er e t a l . 2 0 00) w as u se d f or t he d is -r u pt i on o f orf21. T he p IB R25 v ec to r (S t ha p it e t

a l. 2 00 4) w as u se d f or t he r ep li ca ti on o f orf21i n to w i ld - ty p e. T h e p l as m id s w e re i s ol a te d f r omE. coli u si ng t he a lk al in e l ys is m et ho d a nd p ur i-fi e d u s in g Q i ag e n i o n- e xc ha n ge c o lu m ns ( H il d en ,G e rm a ny ) . S t an d ar d m et h od s w er e u s ed f o r D NAc l o ni n g , p l a s mi d i s o l at i o n a n d r e s tr i c ti o n e n z ym ed i g e s t i o n (K i e s e r e t a l . 2 0 0 0 ; S a m b r o o k a n d R u s s e l l2001).

Disruption oforf21

The orf21 w as d is ru pt ed u si ng a h om ol og ou sr ec om bi na ti on a pp ro ac h w it h p KC 11 39 . F or t hei n ac t iv a ti o n o f orf21 in S. clavuligerus, theu ps tr ea m f ra gm en t, o rf 21 -U ( 15 67 b p) , a nd t hed ow ns tr ea m f ra gm en t, o rf 21 -D ( 14 91 b p) , w er ea mp l ifi ed b y P CR . T he p ri me r p ai rs a re s ho wn i nTa b le 2. T he a mp li fie d D NA f ra gm en t o f o rf 21 -U w as d ig es te d w it h Hind II I a nd XbaI a nd c lo ne di n to t h e c o rr es p on d in g s i te s o f p K C1 1 39 , r es u lt -i ng in p UC 21. T he P CR f ra gm ent or f21 -D wasd i ge s te d w i th XbaI and BamH I a nd l ig at ed i nt ot he s am e s it es o f p UC 21 t o g iv e p UD 21 . p UD 21

w a s d i ge s te d w i th XbaI a nd l ig at ed w it h t he f ra g-m e nt o f t h e n eo m yc i n- r es i st a nc e g e ne , r es u lt i ngi n p HN 14 . I n t hi s fi na l c on st ru ct , p HN 14 , orf21w a s r ep l ac e d b y neor i n -f r am e . p H N1 4 w as fi n al l yt r an s fo rm e d i n to t h e E. coli E T 1 2 5 6 7 d e m et h y l a-t i on h o st a n d t h en t r an s fo r me d i n to w i ld - ty p e S.clavuligerus N R R L3 5 85 f o r d e le t io n b y r e pl i ca t iv eplasmid-me diate d homologous re combination. Thec o n di t i o ns f o r p r o t op l a s t f o r m at i o n , r e g en e ra t i o n,a n d D NA t r an s fo rm a ti o n w er e s l ig h tl y m o di fi ed f r omt he m et ho ds o f H op wo od (K ie s er e t a l . 2 0 00 ; G a rc i a-Dominguez et a l. 19 87). A f te r t h ei r f o rm a ti o n,

t he p ro to pl as ts w er e t re at ed w it h 0 .1 m M a ur in -t r ic a rb o xy l ic a c id ( ATA ) ( S ig m a) f o r 1 0 m i n b e fo r eb ei ng m ix ed w it h t he p la sm id s. T he n, 2 00 l of4 0 % ( w/ v ) p o ly et h yl e ne g l yc o l 1 0 00 ( P EG , M er c k-S h u ch a r dt ) s o l u ti o n w a s p r o m pt l y a d d e d, f o l l o we db y b ri ef c en tr if ug at io n t o r em ov e t he P EG a ndthe n re suspe nsion in P-buffe r. The transforme d pro-t op l as ts w er e t he n p la te d o n R 2Y E r eg en er at io np la te s a nd i nc ub at ed a t 2 8 C . A ft er 2 4 h , p la te sw er e o v er l ai d w i th 0 . 3% a g ar s o lu t io n c o nt a in i ng20 g /m l o f n eo my ci n t o o bt ai n p HN 14 t ra ns fo r-mants.

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Table 1. S t r a in s a n d p l a s m id s u s e d i n t h i s s t u d y.

Strain or plasmid Descri pti on So urce o r refer ence

Bacter ial str ainsStreptomyces clavuligerus W i l d -t y p e , c e p h am y c i n, c l a v u la n i c a c i d a n d 5 S

clavam pr o ducerNRRL3585

S. clavuligerus HN14 or f21insertional disr uptio n mu tant Thi s study

S. clavuligerus/pICA2 o r f 2 1 o v e r ex p r e ss e d i n t oS. clavuligerus wi ld Thi s studyE. coli

XL 1-Blue General clo ning host Str atagene PBL co mpanyE T12567 General clo ning host Str atagene Lajolla CA, USA

P l a s mi d v e c t or sp G E M - T e a s y v e c t o r E. coli g e n e r a l c l o n i n g v e c t or, A m pr Pr o mega, USApHN14 Disr uptio n construct car rying or f21 di sr upted

w i t h n e o m yc i n - re s i s ta n c e g e n eT h i s s t u d y

pKC1139 Streptomyces E. coli bifunctio nal vecto r,apr amycin r esistance

Str atagene

pIBR25 Streptomyces expr essio n vecto r, co ntainst h i o st r e p to n r e s i st a n c e c a s se t t e a n dermE*pr o mo to r

S t h a p i t e t a l . ( 2 0 0 4 )

pICA2 pIBR25 with or f21 gen e insert Thi s study

Generation of mutant and Southern blothybridization

A ft er t he fi ft h g en er at io n o f s el ec ti on a t 3 7 C ,4 70 c ol on ie s w er e p ic ke d a t r an do m, a nd e ac hcol ony was plated on a separate R2YE pl atec o nt a in in g n eo m yc i n ( 2 0g /m l) a nd a pr am yc in(25 g /m l) . O ne o f t he m d is pl ay ed t he d ou bl e-

c r o ss o v er p h e no t y p e o f n e om y c in - r es i s ta n c e a n d

a p ra m yc i n s e ns i ti v it y. T h e d i sr u pt e d orf21 wasc o nfi r me d b y S o ut h er n b l ot h yb r id i za t io n a n al y -s is v ia s ta nd ar d p ro to co ls (Ki es er e t a l. 2 00 0).H y b r i d i z a t i o n w a s p e r f o r m e d u n d e r h i g h s t r i n g e n c yc o n di t i o ns ( 0 . 5 SS C, 6 8C ) a s d es cr ib ed e ls e-w he re ( Ro ch e) , u si ng a p ro be l ab el ed w it h t heD i g o x i g e n i n H i g h P r i m e D N A L a b e l i n g a n d D e t e c t i o nS t ar t er K it I I ( B oe hr i ng e r) f o r S ou t he rn b l ot a na l -

y s is o n H y bo nd - N n yl o n m e mb r an es ( A me r sh a m) .

Table 2. O l i g o nu c l e ot i d e p r i m e rs u s e d i n t h i s s t u d y.

Oli gonucleo ti de Sequence (5-3) Pur po se

o r f21-UFo r f21-UR

TCAAGCTTCGCGTATCCGCTGCCGATCAATTCTAGATTCCCTTCAGCCTCGCCGCC

A m p l i fic a t i on o f orf2 1 u p s t r e a mr e g i o n f o r d i s r u p t i o n

o r f21-DFo r f21-DR

ATTCTAGACGTACGGCAGCCGCACCACGAATAGGATCCTCACCGTCGTCCTGCTGACG

A m p l i fic a t i on o f orf2 1 d o w n s t r e a mr e g i o n f o r d i s r u p t i o n

o r f21-OF

o r f21-OR

ATGGATCCTGAAGGGAAACGGCCGTG

ACTGCAGGGCGGGGACGGGGACGGCCTA

A m p l i fic a t i on o f orf2 1 f o r

o ver expr essio n

ccaR-RT-FccaR-RT-R

CTCGCCCTGTGGAAGGGGCCCGCCCGTTTCTGCAGCGTTGGTTCAGGGA

A m p l i fic a t i on o f ccaR fo r RT-PCR

claR-RT-FclaR-RT-R

CGTGTCACCAGGATCTCCGAGCGCGGACGCATATCTTCGGCCAT

A m p l i fic a t i on o f claR fo r RT-PCR

cas2RT-Fcas2RT-R

GTGCCCTGCTGCGTGGACGTGGCCATCTCGAGTCAGCGGCGCGGCGAGAA

A m p l i fic a t i on o f cas2 fo r RT-PCR

ceas2RT-Fceas2RT-R

CCAGCCGACCTTCCTCATCGCGGGGAAGCTTTCAGATGCTCAGGGCGCCG

A m p l i fic a t i on o f ceas2 fo r RT-PCR

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A c hr om og en ic m et ho d w as u se d t o d et ec t t hep r ob e s o n t h e b l o t . T h e m u t a nt w a s d e s i gn a te d a sS. clavuligerus HN14.

Construction of recombinant plasmids andtransformation

P l as m id p I CA 2 w a s c o ns t ru ct e d a c co r di n g t o s t an -d a r d p r o ce d u re s (S t ha p it e t a l . 2 0 04 ; M a dd u ri a n dHutchinson 1995). T h e s e t s o f o l i g o n u c l e o t i d e s u s e df o r a m pl i fic a ti o n a r e s h ow n i n Ta b l e 2. T h e p u ri -fi ed P CR p ro du ct s w er e fi rs t i ns er te d i nt o t hepGEM-T-Easy ve ctor (Prome ga) and the norf21 frag-m en ts w er e i ns er te d i nt o p IB R2 5 v ia BamHI/PstIr e st r ic t io n s i te s t o y i el d p I CA 2 . p I C A2 a nd p I BR 2 5w e re fi n al l y t r an s fo r me d i nt o t h e E. coli ET12567d e me t hy l at i on h o st a n d t h en a g ai n t r an sf o rm e di n t o w i l d - t y p eS. clavuligerus. T h e s e t r a n s f o r m a n t s

w e re d e si g na t ed a s S. clavuligerus/ p IC A 2 a nd S.clavuligerus/ p I B R2 5 , r e s pe c t iv e l y. Tr a n sf o r m an t sw e r e o b t a i n e d b y s e l e c t i o n w i t h 5 g/ml thiostre p-ton.

RNA extractions and RT-PCR analysis

E a ch a l iq u ot ( 5 m l ) o f t h e c u l tu r es g r ow n f o r a b ou t6 0 h was sus pend ed i n R NA Pro tectTM Bacte riaR ea g en t ( Q ia g en , D u ss e ld o rf , G e rm a ny ) f o r 5 m i ni n o rd er t o i so la te t ot al R NA . T he R Ne as y m in i k it( Q ia g en ) w a s u s ed f o r R N A i s ol a ti o n a c co r di n g t o

t h e m a n uf a c t ur e rs i n s t ru c t io n . C o n ta m i na n t D N Ai n t h e s a mp l e w a s e l im i na t ed b y u s in g R N as e -f r eeD N as e ( Q ia g en ) a n d v e ri fi ed b y P C R a n al y si s u s in gR N A a s t h e t e mp l at e . T h e t o ta l R N A c o nc e nt r at i ona n d p u ri t y w e re d e te rm i ne d f r om t h e 2 6 0/ 2 80 n mr a ti o . R T- P CR w a s p e rf o rm e d w i th a Q u an t iTe c hS YB R G re en RT- PC R Ki t ( Qi ag en ). F or t ra ns cr ip -t i on a l a n al y si s o f C A b i os y nt h et i c g e ne s , p r im e rsfor ceaS2, cas2, ccaR, a n d claR w er e u s ed . A l l t h ep r i m e r s u s e d i n t h e R T - P C R e x p e r i m e n t a r e g i v e n i nTa b l e 2. E q ua l a mo un ts o f R NA f ro m e ac h s am pl ew e re u se d f o r R T- P CR a n al y si s . P C R r ea c ti o n w a s

c a rr ie d o u t a s f o ll o ws : fi r st s t ra n d c DN A s y nt h es i sa t 5 0 C f o r 3 0 m i n; i ni t ia l d e na t ur a ti o n a t 9 5 C f o r1 5 m in; a nd 45 cyc les o f 1 m in at 94 C , 1 min at63 C a n d e l o n g a t i o n a t 7 2 C f o r 2 m i n. T h e R T- P CRp r o d u c t s w e r e e l e c t r o p h o r e s e d o n 1 . 0 % a g a r o s e g e la n d v i s u a l i z e d b y e t h i d i u m b r o m i d e s t a i n i n g . N e g a -t i v e c o n tr o l s i n c l ud e d Ta q D N A p o l y me r a se w i t ho u tr e ve rs e t r an s cr i pt s t o c o nfi r m t h at t h e a m pl i fie dp r od u ct s w e re n o t d e ri v ed f r om c h ro m os a l D N A,w h ic h c o ul d c o nt a mi n at e t h e R N A p r ep a ra t io n s.T he 1 6S r RN A g en e f ro m S. venezuelae w a s u s eda s a p o s it i v e i n t e r n al c o n tr o l .

Analysis of CA production

A 2 -m l s am pl e w as t ak en f ro m e ac h c ul tu re o nc ee v er y 1 2 h d u ri ng t h e f e rm e nt a ti o n p r o c es s ( 4 8 h ) .T h e s u p e r n a t a n t w a s u s e d f o r a s s a y s o f C A ( A o k i e ta l . 1 9 7 6). C A w a s a n a l y z e d b y r e a c t i o n w i t h i m i d a -z o l e . T h e fi l t e re d f e r me n t at i o n s u p e r n at a n t ( 9 0 m l )

w as r ea ct ed w it h 3 0 m l o f 3 M i mi da zo le s ol ut io n ( pH6 .8 ) a t 3 7 C f o r 4 0 m i n. T h e i m i d az o le d e ri v at i vew a s d i l u t e d 1 5 - f o l d w i t h d d H 2 O , a n d i t s a b s o r b a n c ew as m ea su re d a t 3 12 n m (L i a n d To w ns e nd 2 0 06).C A w a s a n a ly z e d b y h i g h- p e r fo r m an c e l i q u i d c h r o-m at og ra ph y ( HP LC ) u si ng a C 18 r ev er se d- ph as ec o l um n ( 4 . 6 m m 2 50 m m, 5 0m KANTO Re age nts,J ap an ) a t 3 12 n m. T he m ob il e p ha se c on si st ed o f 0 . 1 M K H2 PO4 ( p H 3 . 3 a d j u s t e d w i t h H3 PO4 ) a n d 6 %m e t ha n o l ( fl o w r a t e 1 m l / m in ) . T h u s , t h e m y c el i u md r y w e i g h t o f S. clavuligerus/pIC A2,S. clavuligerusH N 14 a n d w i ld - ty p e w e re d e te rm i ne d a t d i ff e re nt

i n c u b a t i o n t i m e s i n R 2 Y E m e d i a . M y c e l i a w e r e h a r -v es te d b y c en tr if ug at io n a t 6 00 0 r pm f or 1 7 m in .T he m yc el ia w er e w as he d w it h d dH2 O an d t hend r i e d a t 8 0 C t o i n v a r i a b l e w e i g h t .

Nucleotide sequence accession numbers

T he n uc le ot id e s eq ue nc e o f orf21 h as b eend e p o si t e d i n G e n Ba n k u n d er t h e a c c es s i on n u m be rEU594508.

Results

Sequence analysis oforf21

I t h as b ee n p re vi ou sl y r ep or te d t ha t a s er ie s o f p ar ti al s tr uc tu ra l g en es a re i nv ol ve d i n t he C Ab i o s yn t h et i c p a t h wa y (J en se n e t a l. 2 00 4). Whilet he se g en es a cc ou nt f or m an y o f t he e xp ec te da c t i v i t i e s , i t i s n o t c l e a r w h e t h e r a l l o f t h e g e n e t i ci nf or ma ti on n ee de d t o f or m C A i s p re se nt i n t hi sr e g i o n . B e c a u s e a d d i t i o n a l g e n e s m i g h t b e r e q u i r e df or t he p ro du ct io n o f C A, w e i nv es ti ga te d t heD NA s eq ue nc e o f t he r eg io n b ey on d t hi s p re vi -

o u sl y r e po r te d a r ea . C o mp l et e s e qu en c e a n al y si so f p C LVA 9 r e v ea l e d a 4 3 , 88 9 - b p s e g m e n t t h a t c o n -s is te d o f n in e n ew p ut at iv e O RF s d ow ns tr ea m o f t h e g e n e s f o r t h e C A b i o s y n t h e t i c p a t h w a y ( Jnawalie t a l. 2 00 8). The orf21 g en e l i es d o wn s tr ea m o f t he C A g en e c lu st er. T he orf21 c od i ng r eg i on i s6 06 b p i n l en gt h a nd e nc od es 2 01 a a, w it h a p re -d i ct e d m o le cu l ar m a ss o f 2 2 .7 5 k D a. T h e d e du c edprote in se que nce ofOrf21 s h o w s p u t a t i v e R N A p o l y -m e r as e s i g ma f a c t or. Orf21 s h ow ed 4 7 % i d en t it yw i th t h e p u ta t iv e R N A p o ly m er a se s i gm a f a ct o r o f Streptomyces coelicolor A 3 (2 ) ( N P 6 2 71 7 8) a nd 4 7 %

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Figure 2. ( A ) G en e r a ti o n of orf21- d e l e te d m u t a nt (S. clavuligerus H N 1 4) v i a d o u b l e- c r os s o v er i n t e g ra t i o n o f p H N 1 4a n d ( B ) S o u t h e r n a n a l y s i s o f g e n o m i c D N A f r o m w i l d - t y p e a n d orf21- d e l e te d m u t a n ts a f t e rKpnI d i g e s ti o n . L a n e M - 3 5 ,orf21 m u t a n t s ; l a n e M , m o l e c u l a r s i z e m a r k e r a n d l a n e W , w i l d - t y p e .

i d en t it y w it h t h e p u ta t iv e R N A p o ly m er a se s i gm af a c t o r o f Streptomyces avermitilis ( N P 8 2 6 30 0 ) .

Disruption oforf21

606 b p of the orf21 g en e f ro m S. clavuligerusN R R L 3 5 8 5 w e r e i n s e r t i o n a l l y i n a c t i v a t e d b y h o m o l -o go us r ec om bi na ti on b et we en p HN 14 a nd t heS. clavuligerus ch ro mo so me , a nd t he d ou bl e-c ro ss ov er m ut an t w as s el ec te d a s d es cr ib ed i n

M a te r ia l s a n d m e th o ds . T h e g e ne t ic o r ga n iz a -t io n a nd t he r es tr ic ti on e nd on uc le as e m ap a res h ow n i n F ig . 2A. p HN 14 w as i nt ro du ce d i nt o S.clavuligerus b y P E G - me d i at e d p r o t op l a s t t r a ns f o r -m a t i on . A f t er p l a s mi d c u r in g , o n e p u t a t i v e m u t a n t(S. clavuligerus H N 1 4) t h a t w a s a p r am y c in - s en s i ti v ea n d n e o my c i n- r e si s t an t w a s s e l ec t e d. I n s er t i on a linactivation oforf21 v i a d o u b l e - c r o s s o v e r w a s v e r i -fi e d b y S o ut h er n b l ot h y br i di z at i on u s in g t h e D N Af ra gm en t f ro m t he u ps tr ea m r eg io n o f t he orf21a s t h e p r ob e . R es t ri c ti o n w i th KpnI r es ul te d i n ah y br i di z at i on b a nd a t 6 . 72 3 k b w he n p r ob i ng w il d -t y pe D NA , w he r ea s a 6 . 93 7 -k b s i gn a l w a s o b ta i ne d

f r om t h e m u t an t s ( F ig . 2B) . T h e g e n e t i c e x c h a ng ew a s f u rt h er c o nfi r me d b y P C R a m pl i fic a ti o n o f t h en eo my ci n m ar ke r g en e f ro m t he g en om ic D NA o f t h e a p r am y c in - s en s i ti v e c o l on i e s ( d a t a n o t s h o wn ) .

Replication oforf21intoS. clavuligerus

p IC A2 (orf21) w a s c o ns t ru c te d i n p I BR 2 5, w hi c h c on -t a in s a s t ro n germE* p r om o te r. A s s h ow n i n F i g . 3,t h os e t r an s fo r ma n ts h a rb o ri n g m ul t ip l e c o pi e s o f orf21 s h ow e d g r e at e r i m p r o v e me n t s i n C A p r o d uc -

t i o n . W e f o u n d t h a t C A p r o d u c t i o n w a s i n c r e a s e d b y1 . 4 3 -f o l d i n S. clavuligerus/ p IC A 2 c o mp a re d w i tht h at o f S. clavuligerus/ p I B R2 5 . A l s o , g r o w t h r a t eso f o v er e xp r es s in g s t ra i ns w er e h i gh er t h an t h at o f c o n t r o l (F i g . 4). T h e s e r e s u l t s w e r e r e p r o d u c i b l e i nse parate e xpe rime nts.

Comparison of morphology

T he g ro wt h a nd m or ph ol og y o f S. clavuligerus

H N1 4 a nd S. clavuligerus/ pI CA 2 w er e t es te d i ns po ru la ti on I SP 2 a nd I SP 4 m ed ia . T he m ut an tf o rm e d a e ri a l m y ce l ia b u t p r od u ce d f e we r s p or es

Figure 3. C o mp a ri s on o f C A p r od u ct s . M e as u re m en to f CA p rod uc ts a t d if fe re nt g ro wt h s tag es ( ex po-n en ti al p ha se , 4 8 h ; s ta ti on ar y p ha se , 6 0 h ; p ha se o f d ec li ne , 7 2 h ). S ym bo ls : , p ro du ct a mo un t f ro mS.clavuligerus/pIBR25; , fr om S. clavuligerus/pICA2and , f rom S. clavuligerus HN 14.

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Figure 4. M y ce l i um d r y w e i gh t o f t h e orf21 g e n e d i s-r u p t e d m u t a n t , r e p l i c a te d s t r a in s a n d t h e c o n t r o l s t r a i na t d i f f e r e n t i n c u b a t i on t i m e s i n C A p r o d u c t i on m e d i a.

t ha n w il d- ty pe o n b ot h m ed ia (F ig . 5). Howe ve r,S. clavuligerus/ p I C A2 s p o ru l a t ed p r e ma t u r el y a n da b u nd a n t ly c o m p ar e d w i t h b o t h t h e w i l d -t y p e a n dp l as m id c o nt r ol s t ra i ns (F ig . 5). C el l d en si ty w asm ea su re d a s d ry w ei gh t. A s s ho wn i n F ig . 4, theb i o m as s e s o f S. clavuligerus H N 14 a nd t h e w i ld - ty p es t r a i n w e r e q u i t e s i m i l a r i n t h e m e d i a t e s t e d . S t r a i nS. clavuligerus/ p I C A 2, w h ic h o v e re x p re s s es orf21f r om a m u lt i co p y p l as m id , s h ow ed h ig h er b i om a sst h a n t h e c o n t r o l (F i g . 4). T h e s e r e s u l t s w e r e r e p r o -d u c ib l e i n s e p ar a t e e x p er i m en t s .

Transcriptional analysis of CA biosyntheticgenes

S. clavuligerus H N 14 , t h eorf21 de fe ctive mutant,s ho we d r ed uc ed l ev el s o f C A p ro du ct io n, w hi le

Figure 6. RT-PCR analysis o fcas2,ceas2,claR andccaRw i th e x pr e ss i on o f 1 6 S r R NA a s i n te r na l r e fe r en c e. R T-P C R o f ( I ) cas2, ( II )ceas2, ( I II ) claR, a nd ( IV ) ccaR fr o m(A) S. clavuligerus H N 14 , ( B ) S. clavuligerus w i l d - ty p e a n d(C) S. clavuligerus/ p I C A 2 . R N A i s o l a t e d f r o m e a c h s t r a i nw a s s u b j e c t e d t o R T - P C R o f 1 6 S r R N A a s w e l l . T h e r e l a t i v ei n te n si t i es o f t h e t r an s cr i pt i on a l b a nd s w e re m e as u re du si ng a Ph os ph oI ma ge r a nd w er e c al cu la te d w it h t heImagequant pr o gr am.

o v e re x p re s s io n y i e ld e d h i g he r p r o d uc t i o n i n w i l d -t y pe . I n o r de r t o d e te rm i ne t h e e f f e ct o f orf21 onC A b i o s y n t h e t i c g e n e s , R T - P C R a n a l y s i s w a s c a r r i e d

o u t. A m on g g e ne s i nv o lv e d i n C A b i os y nt h es i s, ceaS2and cas2 e n co d i n g b i o s yn t h et i c e n z ym e s s p e ci fi cf or t he e ar ly s ta ge s o f C A p ro du ct io n (Ale xande ra nd J en se n 1 99 8; Pa ra dk ar e t a l. 1 99 8), weresubje cte d to transcriptional analy sis. Transcriptionl e v e l s o f ccaR andclaR w e r e a l s o d e t e r m i n e d , s i n c e

Figure 5. M o r p ho l o g i ca l p h e n o t yp e s o f ( I ) S. clavuligerus/ p I C A2 , ( I I )S. clavuligerus w i l d - t yp e , ( I I I )S. clavuligerusH N 1 4 g r o w i n g i n ( A ) I S P 2 a n d ( B ) I S P 4 a g a r p l a t e s .

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t he se g en es e nc od e r eg ul at or s o f C A b io sy nt he -sis (Pa ra dk ar e t a l. 1 99 8; P r ez -Re do nd o e t a l.1998). T h e w i l d -t y p e, S. clavuligerus H N 14 a n dS.clavuligerus/ p IC A 2 s t ra i ns w e re h ar v es t ed a t t h ep ea k o f C A p ro du ct io n i n s ta ti on ar y p ha se a ndu se d t o p re pa re t ot al R NA f or RT- PC R a na ly si s.W h en t r an s cr i pt i on l e ve l s o f b i os y nt h et i c g e ne s

i n v o lv e d i n a n t ib i o t ic p r o d uc t i o n w e r e e v a l ua t e d ,t h e m u ta n t d i sp l ay ed c o mp a ra t iv e ly l o we r l e ve l sand S. clavuligerus/ p IC A 2 s t ra i ns s h ow ed h i gh ert r a n s c r i p t i o n l e v e l s c o m p a r e d t o w i l d - t y p e ( F i g . 6).T h e r eg u la t or y g e ne , claR, w as s im il ar ly t ra n-s cr ib ed i n a ll s tr ai ns . O n t he o th er h an d, ccaRt r a n s c r i p t i o n l e v e l s i n t h eorf21 m u t a n t s t r a i n w e r el ow er t ha n t ho se i n t he w il d -t yp e s tr ai n, w hi leS. clavuligerus/ p IC A 2 h a d h i gh er ccaR transcrip-t i on l e ve l s b a s e d o n R T- P CR (F i g . 6). The se RT-PC Rr e su l ts s u gg e st t h at O R F2 1 w a s n o t i n vo l ve d i n t h et r an s cr i pt i on o f l a te b i os y nt h et i c e n zy me s f o r C A

p ro du ct io n, b ut m ay a ff ec t t he t ra ns cr ip ti on o f t h e e a rl y b i os y nt h et i c e nz y me s a n d ccaR, a reg-u la to r o f t he e ar ly b io sy nt he ti c e nz ym es f or C Aproduction.

Production, isolation, and analysis of CA

T h e e f f e c t o f orf21 o n C A p r o d uc t i on w a s a s s e s s edb y u si n g w i ld - ty p e, S. clavuligerus H N1 4 a nd S.clavuligerus/ pI CA 2 a s b at ch c ul tu re s i n C A p ro -d uc ti on m ed ia . A ft er 4 8- h i nc ub at io n, s am pl esw ere w it hd rawn ev ery 12 h i n o rd er t o a ss es s

t he yi el d o f s eco nd ary me ta bo lit es b y H PLCa na ly si s. H PL C a na ly si s o f c ul tu re s up er na ta nt sfrom orf21 d i sr up ta nt g ro wn i n s oy m ed iu m a nds amp led at 4 8, 60 , 7 2 and 8 4 h a ft er in oc ul a-t io n r ev ea le d s li gh tl y d ec re as ed p ro du ct io n o f c l av u la n ic a c id . H ow e ve r, i n t h e orf21-re plicate dwild-ty pe strain,S. clavuligerus/ p I C A 2 , C A p r o d u c -t io n w as i nc re as ed b y 1 .4 3- fo ld c om pa re d t o t hec o nt r ol (F ig . 3). C A p r od u ct i on i n cr e as e d i n it i al l yf ro m 4 8 h b ec om es m or e a nd m or e p ro no un ce da s t im e p er io d i nc re as es , u lt im at el y r ea ch ed t ot he h ig he st C A p ro du ct io n a t 6 0 h . O n r ea ch in g t ot h e h i g he s t s a t u ra t e d l e v e l , i t d e c l in e s c o n t i n ua l l y

s h ow in g a s l ig h t d e c re a se o f C A p r od u ct i on a t 7 2 h(F i g . 3).

Discussion

G e ne s i n vo l ve d i n t h e b i os y nt h es i s o f C A m e ta b o-l it es i n S. clavuligerus h av e b ee n l oc at ed i n t heC A g en e c lu st er, b ut s om e s te p i n t he p ro po se db i o s yn t h et i c p a t h wa y h a v e n o c o r re s p on d i ng g e n esi n t hi s c lu st er f or C A p ro du ct io n a re k no wn . I n

v i ew o f t h e p o ss i bi l it y t h at a d di t io n al g e ne s m i gh tb e r eq ui re d f or t he p ro du ct io n o f C A, w e i nv es -t ig at ed t he D NA s eq ue nc e o f t he r eg io n b ey on dt h is p r ev i ou s ly r e po r te d a r ea a nd f o un d n i ne n ewp u t a t i v e O R F s d o w n s t r e a m o f t h e g e n e s f o r t h e C Ab i os y nt h et i c p a th wa y a n d w e a n al y ze d orf22 andorf23 a n d f o u n d t h e i r i n v o l v e m e n t i n C A b i o s y n t h e -

s is (J na wa li e t a l. 2 00 8). H e re , w e d i d d i sr u pt i ona s w el l a s o v er ex p re s si o n o f orf21 ( s i g ma f a c to r )g e ne a n d a n al y ze d t o a s se ss i t s i nv o lv e me nt i n C Ab i o s y n t h e s i s . T h e p r e d i c t e d a m i n o a c i d s e q u e n c e o f O r f 2 1 i s s i m i l a r t o p r o t e i n s p o s s e s s i n g s i g m a f a c t o r sdomains.

T h e m e ch a ni s ms u nd e rl y in g r eg u la t io n o f t h ee x tr a cy t op l as m ic f u nc t io n s i gm a f a ct o rs r em a inl a r g e l y u n k n o w n i nStreptomyces. W h i l e a n t i - s i g m af a c t o r s h a v e b e e n i d e n t i fi e d f o r S i g H , S i g E , S i g K a n dS i gL , t h ei r r e gu l at i on i n t h e c o n t ex t o f s i gm a f a c-t o r f u n ct i o n a w a it s e l u ci d a t io n . T h e s t r es s - in d u ce d

e x t ra c y to p l a sm i c f u n ct i o n s i g m a f a c to r S i g E s h o wsi n c re a s ed e x p re s s io n u n d er h e a t, s u r fa c e a n d o x i d a-t iv e s tr es s, a nd S ig E i s k no wn t o b e r eg ul at ed b yS i gH u nd e r o x id a ti v e s t re ss (R am an e t a l. 2 00 1).D e l et i o n o f sigE r e su l ts i n r e du ce d l e th a li t y w i thp e rs i st e nc e o f t h e b a ct e ri u m i n m i ce (A nd o e t a l.2003).

In B. subtilis, a u t op h o sp h o ry l a t io n o f t h e a d a p -t o r M cs B i n hi b it s t h e h e a t s ho c k r ep r es s or C t sR b yt a r g e t i n g i t f o r d e g r a d a t i o n b y C l p C P (K i r s t e i n e t a l .2007). C l p CP c o n tr o l s s p o r ul a t i on i nB. subtilis byd e g r ad i n g t h e a n t i- s i g ma f a c t or S p o II A B w h ic h r e g -ulate s F (Pa n e t a l. 2 00 1). Ki m e t a l . ( 2 0 0 9) have

r ec en tl y d em on st ra te d t he r ol e o f t he C lp P1 /P 2prote ase s ofS. coelicolor i n i n d u c i n g r a p i d t u r n o v e rof R

, a n i so form o f R f or mi ng p ar t o f a n eg -a ti ve f ee db ac k r eg ul at or y l oo p. I n S. coelicolora m on g d i ff e re nt s i gm a f a ct o rs , H i s i nv ol v ed i ns p o r ul a t i on a n d s a l t -s t r es s r e s po n s e ( S e v ci k o v a e ta l. 2 00 1; Vi ol li er e t a l. 2 00 3b), and B p la ys ak e y r o l e i n o s m ot i c a n d o x i d at i v e r e s p o ns e s , w h i c ha r e r eq u ir ed f o r b o th a e ri a l h yp h al f o rm a ti o n a n da n t ib i o t ic b i o s yn t h es i s (C h o e t a l . 2 0 0 1 ; L e e e t a l .2 0 05 ; V io l li e r e t a l . 2 0 03 a). F and WhiG a r e t h es p o r u l a t i o n - s p e c i fi c s i g m a f a c t o r s ( H o m e r o v a e t a l .

2 0 0 0 ; T a n e t a l . 1 9 9 8 ).I n t hi s s tu dy, i n o rd er t o b et te r u nd er st an dt he r ol es o f t he s ig ma f ac to r (orf21) i n c l av u la n ica c i d b i o sy n t he s i s, w e c o n st r u ct e d S. clavuligerusm u t a nt w i t h d i s r up t e dorf21 a n d c h a ra c t er i z ed i t sf u nc t io n . T h e m u ta n t w a s c o ns t ru ct e d b y d i sr up t -ing orf21 s e q ue nc e s b y i n se rt i on o f t h e n e om y ci ng e ne , a n d t h en r e pl a ci n g t h e w i ld - ty p e c hr o mo -somal orf21 w i th t h e d i sr up t ed c o py, in vivo, byh o m ol o g o us r e c om b i na t i o n. T h e m u t at i o n r e s ul t e di n a 1 0 1 5 % d e c r e a s e i n c l a v u l a n i c a c i d p r o d u c t i o ni n C A p r o d uc t i on m e d i um .

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We a l so s t ud i ed t h e e f f e ct o f orf21 ove re xpre s-s io n i n S. clavuligerus. Tr a n sf o r m an t s c o n ta i n in gt h e a m pl i fie d orf21 g en e i n t he m ul ti co py p la s-m i d p I C A 2 s h o w e d a 1 . 4 3 - f o l d i n c r e a s e i n c l a v u l a n i ca c i d p r o d uc t i on d u r in g f e r me n t at i o n. O v e re x p r es -s io n o f orf21 p la ye d a n i mp or ta nt p ar t i n t hi ss t ud y b e ca u se t h e e f fe c ts o f orf21 o n C A p r od u c-

t i on h a ve n o t b e en i nv e st i ga t ed y e t. T h e e f fe c tso f v ar io us e nv ir on me nt al f ac to rs o n t he s ig maf a c t or - m ut a n t (S. clavuligerus H N1 4 ) s t ra i ns a n do v er ex p re ss i ng s t ra i ns w er e a l so e x am i ne d u n de rv a ri o us g r ow t h c o nd i ti o ns . A l th o ug h g r ow t h a n dm o rp h ol o gi c al a n al y se s r e ve a le d n o d i ff e re nc e sb et we en t he k no ck ou t m ut an t a nd w il d- ty pe , as li gh t i nc re as e i n C A p ro du ct io n a nd p re ma tu res p o r ul a t i on w a s o b s er v e d i n S. clavuligerus/pIC A2o v er ex p re ss i ng s t ra i ns . We s ug g es t t h at o v er e x-p ress io n o f th e g ene m ay b e p ro mo ted b y t hes t r o ng p r o m ot e r ermE* , w hi ch a ct iv at es t he C A

p ro du ct io n c as ca de . T he g ro wt h r at e m ay h av ec o nt r ib u te d s l ig h tl y t o t h e i n cr e as e d C A p r od u c-t i o n b e c au s e t h e r e p l ic a t ed s t r ai n s s h o we d s l i g ht l yh ig he r g ro wt h r at e t ha n t he c on tr ol (F ig. 4). As i gm a f a ct o r ( f a ct o r ) i s a p r o k ar y o ti c t r a ns c r ip -t i on i ni t ia t io n f a ct o r t h at e na b le s s p ec i fic b i nd i ng o f R N A p o ly m er a se t o g e ne p r om o te rs . D i ff e re nt s i gm af a c t o r s a r e a c t i v a t e d i n r e s p o n s e t o d i f f e r e n t e n v i -r o n me n t al c o n di t i on s . B a c t er i a l R N A p o l y me r a se sc o n ta i n in g t h e p r i m ar y f a c to r d i r ec t a n d i n i ti a t et ra ns cr ip ti on o f a l ar ge n um be r o f g en es c om -m o nl y e x pr es s ed i n t h e e x po ne n ti a l g r ow t h p h as e(B o r u k h o v a a n d S e v e r i n o v 2 0 0 2 ; Y o u n g e t a l . 2 0 0 2 ).

A s e s ta b li s he d b y R T- P CR a n al y se s , i t i s c o nc e iv -a b le t h at Orf21 i n du c ed C A p r od u ct i on b y a c ti v at i ngo t he r g e ne s i n vo l ve d i n C A b i os y nt h es i s. S t im u la -t io n o f c la vu la ni c a ci d p ro du ct io n s ug ge st s t ha te x p r e s s i o n o f t h e e a r l y g e n e s i n t h e C A b i o s y n t h e t i cp a t h w a y i s a c t i v a t e d b yorf21, a s d e m o n s t r a t e d b yR T- P C R a n a l ys e s . F u r th e r mo r e , t h e a b u nd a n t s p o ref o r m a t i o n a n d h i g h e r C A p r o d u c t i o n w a s c o n s i s t e n tw i t h t h e e l e v a t e d l e v e l o f t r a n s c r i p t i o n o f ccaR ando t h e r g e n e s i n t h e e a r l y s t a g e s o f b i o s y n t h e s i s . O u rfi n d i ng s s u g g es t t h a tOrf21 m a y b i nd t o p r om o te r si n t h e C A g e n e c l u s t e r t o a c t i v a t e t h e C A p r o d u c t i o n

c a s ca d e . O v e r ex p r es s i on o f R N A p o l y me r a se s i g m af a c t o r s i n a b i o s y n t h e t i c g e n e c l u s t e r m a y e n h a n c ea n t i bi o t ic p r o d uc t i o n. A l t h ou g h g e n e d i s r up t i o n o f orf21 s ho we d o nl y 1 0 15 % l es s C A p ro du ct io n i nS. clavuligerus N R RL 3 58 5 , w e w e re i nt e re st e d t ok n ow t h at w o ul d h ap p en w he n t h e s a m e g e ne w a so v er ex p re ss e d i n t h e w il d -t y pe s t ra i n. A s a r e su l t,w e f ou nd t ha t C A p ro du ct io n w as i nc re as ed b y1 .4 3- fo ld o ve r t he n or ma l l ev el . H en ce , w e c on -c l u d ed t h a tOrf21 s o m e h o w i n d u c e d C A p r o d u c t i o nb y a c t i v a t i n g o t h e r g e n e s i n v o l v e d i n C A b i o s y n t h e -sis.

Acknowledgments

T h is w o rk w a s s up p or t ed b y A g en d a p r og r am ( N o.2 0 0 90 5 F P 01 4 ) , R u ra l D e v el o p m en t A d m i ni s t ra t i o n,R e p u b l i c o f K o r e a .

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