- Email: [email protected]
H.
Bioelearoc~emistry a n d B i o ~ . r g e t i c s 7 ( I 9 8 o ) 6 5 9 - 6 7 0 J . ElectroanaL C ~ m . 116 ( I 9 8 o ) 6 5 9 - 6 7 0 Elsevier Sequoia S.A.. Lausanne
-- P r i n t e d
in Italy
365 - The Temperature Dependence of the Production of Penicillinase in Bacillus eere~cs 569/H. * by R. W. KUENNEN, Department
h i . J . SLMONE a n d G. P . KREISH.MAN o f C h e m i s t r y , U n i v e r s i t y o f CirLcinnati, C i n c i n n a t i , O h i o 45221, US.~.
~lanuscript received ~ovember
~_4th 1979
Summary
The activity and amount of both the exo form and the cell-bound f o r m s o f p e n i c i l l i n a s e (penicillin a m i d o - [ 3 - 1 a c t a m a s e , E C 3.5.2.6) p r o d u c e d by the constitutive strain Bacillus cereus 569/H have been measured as a f u n c t i o n o f t h e t e m p e r a t u r e _ T h e a c t i v i t y o f b o t h f o r m s is r e l a t i v e l y c o n s t a n t f r o m 33 t o 42 °C- A b o v e t h i s t e m p e r a t u r e , h o w e v e r , a r a p i d d e c r e a s e in t h e a c t i v i t y is o b s e r v e d a n d v i r t u a l l y n o a c t i v i t y c a n b e d e t e c t e d in cells g r o w n a t 4 6 °C. A t all t e m p e r a t u r e s , t h e e x o - p e n i c i l l i n a s e is e x c r e t e d i n t o t h e m e d i u m o n l y in t h e a c t i v e f o r m . C o n c u r r e n t ~sith t h e d e c r e a s e in t h e a m o u n t o f e x o - p e n i c i l l i n a s e s e c r e t e d is a p a r a l l e l d e c r e a s e in a c t i v i t y o f t h e c e l l - b o u n d penicillinase, w h i c h i m p l i e s t h a t the exo-penicillinase can only be formed from active cell-bound penicillinase. A l t h o u g h t h e a c t i v i t y o f t h e c e l l - b o u n d p e n i c i l l i n a s e d e c r e a s e s a b o v e 42 °C, t h e r e is a m a r k e d i n c r e a s e o f a p r o t e i n in t h e m e m b r a n e of the same apparent electrophoretic mobility as purified venicillinase. I t is p o s t u l a t e d t h a t t h e p r o d u c t i o n o f p e n i c i l l i n a s e o c c u r s - a b o v e 42 °C b u t t h a t t h e loss o f a c t i , d t y is t h e r e s u l t o f d e n a t u r a t i o n o f t h e p r o t e i n i n a n a l t e r e d m e m b r a n e . T h e loss o f a c t i v i t y c o r r e l a t e s w e l l w i t h a d r a m a t i c c h a n g e i n t h e f a t t y a c i d c o m p o s i t i o n o f t h e m e m b r a n e a b o v e 42 °C-
Introduction P e n i c i l l i n a s e (penicillin a m i d o - ~ b - l a c t a m a s e , E C 3 . 5 . 2 _ 6 ) is s e c r e t e d into the growth medium by Gram-positive bacteria of the genus Bacillus. I n B . c e r e u s , i t h a s b e e n s h o w n t o d a t e t h a t t h e p r o t e i n e x i s t s in a t l e a s t t w o f o r m s , ~ - p e n i c i l l i n a s e ( ~ - p e n ) w h ichis f o u n d f r e e in t h e c u l t u r e m e d i u m * Presented at the 5th International 3--8 S e p t e m b e r 1979, W e i m a r ( D . D . R . )
o3o':'--4598/8o/o659-o67o
~
Symposium
E l s e v i e r S e q u o i a S.A_
orL B i o e l e c t r o c h e m i s t r y ,
66o
I4:uennen, S i m o n e a n d K r e i s h m a n
or can be loosely bound to the membrane surface, and y-penicillinase (y-pen} w h i c h is t i g h t l y b o u n d t o t h e m e m b r a n e a n d c a n n o t b e n e u t r a l i z e d b y a n t i s e r u m p r e p a r e d a g a i n s t m-pen [1-3]. I t h a s b e e n s h o w n t h a t y - p e n is a c o n f o r m e r o f m-pen, t h e t w o f o r m s b e i n g r e a d i l y i n t e r c o n v e r t a b l e [4]- T h e y - p e n m a y e x i s t i n v a r i o u s p o l y m e r i c f o r m s i n t h e m e m b r a n e [5]- I n o t h e r species o f B a c i l l u s , n a m e l y B . l i c h e n i f o r m i s , a ~ - p e n i c i l l i n a s e (~-pen) h a s b e e n f o u n d a n d s h o w n t o b e a l i p o p r o t e i n . g - P e n is p r o d u c e d b y a specific c l e a v a g e o f a p o r t i o n o f [~-pen b y a p r o t e a s e n e a r t h e a m i n o t e r m i n u s w h e r e t h e l i p i d is a t t a c h e d [6]. N o s u c h l i p o p r o t e i n h a s y e t b e e n f o u n d i n B_ c e r e u s b u t i t s e x i s t e n c e h a s b e e n i n f e r r e d f r o m s t r u c t u r a l h o m o l o g i e s 6f m - p e n i c i l l i n a s e s f r o m v a r i o u s B a c i l l u s species. BERNSTEIN et aL h a v e m e a s u r e d t h e t e m p e r a t u r e d e p e n d e n c e o f m-pen a c t i v i t y as a f u n c t i o n of t h e t e m p e r a t u r e for t h e n o r m a l inducible a n d c o n s t i t u t i v e s t r a i n s o f B . c e r e u s [7]- I n b o t h o r g a n i s m s , t h e r e w a s a n i n c r e a s e i n a c t i v i t y With i n c r e a s i n g t e m p e r a t u r e u p t o 4 z oC, w h e r e a m a x i m u m a m o u n t of m-pen was produced. Above this t e m p e r a t u r e , a drastic decline in activity was observed. The authors speculate that t h e d i f f e r e n c e i n t h e r a t e o f i n c r e a s e f o r e a c h strain, is c o n s i s t e n t ~s~th a c o n t r o l o f p r o t e i n s y n t h e s i s a t t w o levels, n a m e l y a t t h e t r a n s c r i p t i o n a l a n d a t t h e t r a n s l a t i o n a l levels, e a c h b e i n g e f f e e t e d b y t e m p e r a t u r e . I n t h e inducible strain, t h e larger incremental increase in penicillinase a c t i v i t y w i t h t e m p e r a t u r e can be a t t r i b u t e d to t h e r m a l derepression b y inactivation of t h e repressor protein i t s d L F o r the constitutive strain, t h e smaller incremental change was attributed to the temperature sensitivity of the t r a n s l a t i o n a l process. T h e a u t h o r s d o n o t s p e c u l a t e as t o t h e c a u s e s f o r t h e d r a m a t i c d e c r e a s e i n p e n i c i l l i n a s e a c f i ~ S t y a b o v e 4z °C. T h e p u r p o s e of this study was to address this question. Materia~ and methods
Organisms,
inoculg and media
T h e c o n s t i t u t i v e s t r a i n 5 6 9 / H o f B . c e r e u s w a s a k i n d g i f t o f Dr. R . A. DAY. B a c t e r i a l p r e c u l t u r e s w e r e g r o w n f r o m a s p o r e i n n o c u l u m i n S. p e p t o n e b r o t h [8]. P o l y c a r b o n a t e e r l e n m e y e r f l a s k s (SOO c m n) c o n t a i n i n g 5o c m a o f c a s e i n h y d r o l y s a t e g r o w t h m e d i u m [9] w e r e i n o c u l a t e d •~5th x % (vol/vol) o f t h e p r e c u l t u r e a n d i n c u b a t e d i n a GCA-P r e c i s i o n Scientific M o d e l 50 s h a k e r b a t h c o n t r o l l e d t o --_o 5 oC a n d i i o osc_/min_ B a c t e r i a l g r o w t h w a s d e t e r m i n e d b y o p t i c a l d e n s i t y w h i c h w a s m o n i t o r e d a t 650 n m w i t h a BAUSH a n d LO.XZB M o d e l 340 s p e c t r o m e t e r . B a c t e r i a l cells w e r e h a r v e s t e d i n l a t e log p h a s e a t a n o p t i c a l d e n s i t y o f a p p r o x i m a t e l y 1. 3 . Enzyme
assays
Penicillinase was assayed iodometrically essentially by a modified m e t h o d o f PERR~--r [10] f o r t h e i o d i n e i n s e n s i t i v e f o r m o f t h e e n z y m e
Penicillina~e Transport= in
B.
cereus
66x
a n d b y t h e m e t h o d of SARGENT [II] f o r b o t h i o d i n e s e n s i t i v e a n d i n s e n s i t i v e f o r m s of t h e e n z y m e _ U n i t a c t i v i t y of p e n i c i i l l n a s e is e q u a l t o t h e mount o f penicillina.se w h i c h h y d r o l y z e s I m i c r o m o l e o f b e n z y l p e n i cillin in 6o m i n u t e s a t 3o oC a n d p H 7.o [I2].
Puril~cation o f the ~t-penicillinase i ~ the culture m e d i u m by gel chromatography The ~-pen was isolated from the culture medium using a Tygon c o l u m n (65 c m x o c m ) pack~.a w i t h S e p h a d e x G - 7 5 - 1 o o - T h e c o l u m n w a s e i u t e d w i t h O.lO M s o d i u m l~_~..~sphate b u f f e r , p H 7.00, a t a flow r a t e o f o-5 c m 3 / m i n -
Preparatio~ o f protolblasts for the determination of cell-bound penicillinase P r o t o p l a s t s w e r e p r e p a r e d f r o m B . cereus 5 6 9 / H b y t h e r a p i d p r o c e d u r e o f LA~IPE,~ et al. [I3]- T h i s t e c h n i q u e i n v o l v e s t h e u s u a l p r o t o p l a s t f o r m a t i o n b y t r e a t m e n t w i t h l y s o z y m e in a h y p ~ r t o n i e s o l u t i o n . F u r t h e r s y n t h e s i s of p e n i e i l l i n a s e w a s a l s o p r e v e n t e d b y t h e p r e s e n c e o f e h l o r a m p h e n i c o l in t h e p r o t o p l a s t i n g s o l u t i o n . A p r o c e d u r e a s d e s c r i b e d b y LAMPEN et al. [14] f o r g r o w i n g p r o t o p l a s t s w a s u s e d .
Preparation o f membrane proteins .for electrophoresis Cell m e m b r a n e s w e r e p r e p a r e d b y t h e r e l a t i v e l y r a p i d p r o c e d u r e o f KO,~I,~GS et al. [15], w h e r e t h e ceils a r e t r e a t e d w i t h l y s o z y m e in a h y p o t o r A c m e d i u m w h i c h r e s u l t s in a n i m m e d i a t e lysis o f t h e cells a n d f o r m a t i o n o f m e m b r a n e vesicles. T h e s e m e m b r a n e vesicles a r e c o l l e c t e d b y c e n t r i f u g a t i o n f o r 30 rain. a t 16,ooo x g a t 5 °C- S D S p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s w a s p e r f o r m e d a s d e s c r i b e d b y NEVILLE [16], w i t h m o d i f i c a t i o n s o f RXLFORS et al. [17]- T h e S D S g e l s y s t e m utilizes a s u l f a t e b o r a t e d i s c o n t i n u o u s b u f f e r s y s t e m ( r u n n i n g p H 9-5) w h i c h s t a c k s a n d u n s t a c k s protein SDS complexes over a range of 2,3oo to 32o,ooo daltons, providing high resolution fractionation. Reference proteins used for molecular weight determination were b o v i n e s e r u m a l b u m i n (68,ooo), o v a l b u m i n (43,ooo), d e o x y r i b o n u c i e a s e 1 (3x,4oo), c a r b o n i c a n h y d r a s e (29,0o0) a n d l y s o z y m e (14,3oo)A GILFORD d e n s i t o m e t e r e q u i p p e d w i t h a g e l s c a n n e r w a s u s e d f o r s c a n n i n g gels a t v a r i o u s g r o w t h t e m p e r a t u r e s a n d q u a l i t a t i v e t r a n s m i s s i o n of various proteins was recorded.
Gas chromatography-mass spectrometry Fatty acid methyl esters were prepared following the procedure d e s c r i b e d "by MACGEE et al. [18]. T h e g a s c h r o m a t o g r a p h w a s a VARL~,~ A e r o g r a p h M o d e l 17.4o d u a l f l a m e u n i t w i t h 3.8 m × 3-2 m m i n s i d e d i a m e t e r s t a i n l e s s s t e e l c o l u m n p a c k e d : w i t h 3 ~o silicone O V - z 7 o n 8o t o zoo G a s C h r o m Q (Ar-TECH ASSOCIATES). T h e c a r r i e r g a s w a s h e l i u m a t 20 e m a / m i n . T h e i n j e c t i o n p o r t a n d d e t e c t o r w e r e h e l d a t 29o a n d .3oo-°C r e s p e c t i v e l y .
66z
Kuennen,
Simone and
Kreishman
A 8 - 9 m m s (EL1)s a m p l e w a s i n j e c t e d i n t o a s t a x t i n g c o l u m n t e m p e r a t u r e o f x45 °C w h i c h w a s p r o g r a m m e d to increase at a rate of 8 oC/min, to a final t e m p e r a t u r e o f 3oo oC h e l d , i s o t h e r m a l l y for 3 vain.
T h e gas c h r o m a t o g r a p h w a s i n t e r f a c e d w i t h a HITACHI type RMU-- 7 m a s s s p e c t r o m e t e r o p e r a t e d a t 70 e V w i t h a V G DATASYSTEM 2000 c o m p u t e r . P e a k a r e a s r e p r e s e n t i n g a m o u n t s o f iso-~4 a n d norma~-C~a w e r e e s t i m a t e d u s i n g b e s t t r i a n g u l a r fit.
Results ~
disen-~b~_~'on
T h e t e m p e r a t u r e d e p e n d e n c e o f m-pen a c t i v i t y i n c u l t u r e m e d i u m of t h e c o n s t i t u t i v e s t r a i n o f B . c e r e u s i s s h o w n in Fig. x. T h e a c t i v i t y o f = - p e n in t h e c u l t u r e m e d i u m of b a c t e r i a g r o w n t o t h e s a m e o p t i c a l d e n s i t y i n t h e l a t e log p h a s e o f g r o w t h r e m a i n s v i r t u a l l y u n c h a n g e d ,~atiI 4z oC, w h e n a b o v e t h i s t e m p e r a t u r e a d r a s t i c d e c r e a s e is o b s e r v e d . T o r u l e o u t t h e p o s s i b i l i t y t h a t t h e d e c r e a s e is d u e t o penicillinase d e n a turation, the conformational properties, as assumed b y U V spectroscopy, a n d t h e a c t i v i t y o f i s o l a t e d peniciUinase w e r e s t u d i e d as a f u n c t i o n o f
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Fig. z. T h e t e m p e r a t u r e d e p e n d e n c e of ce-pen a c t l v i t y i n t h e c u l t u r e m e d i u m .
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T a b l e z. T h e r m o s t a b i l i t y temperature.
of exopen/cillinase as a function of time at a given
Temperature oC
Initial activity unit/cm s
Activity at 6 h unit/cm s
38
6630 unit/cm s
626x u n i t / c m s
48
6 8 9 7 u n i t / c m ~t
42
6585 unit/cmS
44
6885 u n i t / c m 3
~ ' e t loss unit/cm s
Loss ~/o
369 urt[cm a
5-6
6585 u n i t / c m s
3xz un/cm ~
4-5
6323 u n l t / c m 3
262 u n / c m ~
4-0
6zoo unitlcm~
635 u n / c m ~
9-2
46
62o 7 unit/cm3
5927 unit/cma
280 u n / c m a
4-5
48
5935 unitJcmX
5666 unit/cm a
268 u n / c m 3
4-5
664
"
Kuennen. Simone and Kreishman
t e m v e r a t u r e . N o d i s c e r n a b l e c h a n g e in t h e a b s o r b a n c e a t z S o n m c o u l d b e d e t e c t e d i n NaC1--H±O u n t i l ~ 5 5 oC w h e r e t h e p r o t e i n p r e s u m a b l y a g gregates. In addition to the lack of a detectable c0nformational change i n t h e p r o t e i n , t h e a c t i v i t y of t h e i s o l a t e d p r o t e i n r e m a i n e d , c o n s t a n t u n t i l ~ 5 2 oC w h e r e t o s s o f a c t i v i t y w a s o b s e r v e d (Fig. 2). T h e p r o t e i n can also be incubated at elevated temperatures for prolonged periods of t i m e w i t h m i n i m a l loss o f a c t i v i t y ( T a b l e I). = - P e n i t s e l f is t h e r e f o r e extremely thermostable as compared to other proteins and the marked d e c r e a s e in a c t i v i t y a t 42 °C c a n n o t b e a t t r i b u t e d t o p r o t e i n d e n a t u r a t i o n at elevated temperatures.
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S i n c e o n l y t h e p r o t e i n a c t i v i t y is b e i n g m e a s u r e d , t h e p o s s i b i l i t y t h a t t h e =--pen is b e i n g r e l e a s e d i n t o t h e m e d i u m i n a n i n a c t i v e f o r m m u s t b e r u l e d o u t . T h e m o n o m e r i c g - p e n c a n .he. p u r i f i e d f r o m o t h e r p r o t e i n s in t h e m e d i u m b y u t i l i z i n g g e l c h r o m a t o g r a p h y (Fig. 3)- A t a l l t e m p e r a t u r e s , t h e specific a c t i v i t y o f t h e m o n o m e r i c g - p e n p e a k w a s a p p r o x i m a t e l y 3 x I o s u n i t s / r a g of p r o t e i n w h i c h i s t h e specific a c t i v i t y o f t h e p u r i f i e d e n z y m e Ix9]- S i n c e b o t h t h e a b s o r b a n c e a n d a c t i v i t y o f t h a t p e a k d e c r e a s e d c o n c u r r e n t l y , a b o v e 4 z °C, ~ - p e n is s e c r e t e d i n t o t h e m e d i u m ha *Jae a c t i v e f o r m o n l y b u t a t s u p p r e s s e d l e v e l s .
Penlei!!inase Transport in B.c.ev~us
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F i g . 4-
The temperature dependence of the total ceil-bound penicillinase actisity measured after
protoplasting.
The temperature dependence of the activity of the total cell-bound p e n i c i l l i n a s e (i.e. y - p e n p l u s 0 - p e n ) m e a s u r e d a f t e r p r o t o p l a s t i n g (Fig. 4) a n d r e m a i n i n g c e l l - b o u n d p e n i c i l l i n a s e meast~red a f t e r h o m o g e n / z a t i o n (Fig. 5) p a r a l l e l s t h a t f o r ~ - p e n w i t h a r a p i d d e c r e a s e in a c t i v i t y a b o v e 42 °C- S i n c e t h e r a t i o s o f a c t i v i t y o f t h e ~ - p e n t o t h e c e l l - b o u n d penicill i n a s e a r e t h e s a m e a b o v e 4 2 oC a s b e l o w , w e i n f e r t h a t t h e p r o t e a s e h a s n o t d e n a t u r e d b u t t h a t t h e d e c r e a s e in s e c r e t i o n in a c t i v e ~ - p e n i n t o t h e m e d i u m is t h e r e s u l t o f a d e c r e a s e i n t h e s u b s t r a t e f o r t h e p r o t e a s e , n a m e l y t h e active form of ~-pen. It is interesting to note that after homogenization o f cells g r o w n a t 46 °C, i e s i d u a l a c t i v i t y c a n b e d e t e c t e d i m p l y i n g t h a t t h e o r g a n i s m is c a p a b l e o f p r o d u c i n g a c t i v e p e n i c i l l i n a s e i n s o m e f o c m a t 4 6 oC, a l b e i t a t a d e p r e s s e d l e v e l . T o d e t e r m i n e if a n i n a c t i v e f o r m o f p e n i c i U i n a s e is a c c u m u l a t i n g i n t h e m e m b r a n e a b o v e 42 oC, t h e p r o t e i n s f o u n d in t h e m e m b r a n e o f c e i l s g r o w n a t 35 °C a n d 4 6 oC h a v e b e e n a n a l y z e d b y S.D.S_ g e l e l e c t r o p h o r e s i s ( F i g , 6). M o s t , i f n o t a l l o f t h e p r o t e i n s , t h a t a r e p r e s e n t a t 35 °C a r e p r e s e n t a t 46 °C. B r e a k d o w n o f t h e t r a n s l a t i o n a l p r o c e s s a t
666
Kuennen,=Simone
and Kreishman
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150
._> 100 °
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50
I 33
! 35
I 37
39
41
43
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! 47
Fig. 5Remaining cell-bound penicillinase activity measured after homogenization as a function of temperature.
4 6 oC a s t h e c a u s e f o r d e c r e a s e in p e n i e i l l i n a s e p r o d u c t i o n is u n l i k e l y . The relative amounts of each type of protein do, however, vary with a predominant increase in the lower molecular weight proteins and a d e c r e a s e i n t h e h i g h e r m o l e c u l a r w e i g h t p r o t e i n s a t 4 6 °C. T h i s m a y b e d u e t o e i t h e r t h e r m a l sensitix~ity o f t h e t r a n s c r i p t i o n a l o r t r a n s l a t i o n a l p r o c e s s e s f o r t h e l a r g e r p r o t e i n s o r reflect b r e a k d o w n of o l i g o m e r i c p r o t e i n s i n t o t h e i r s u b t m i t s . T h e gels o f t h e m e m b r a n e p r o t e i n s f r o m t h e cells g r o w n a t t h e t w o t e m p e r a t u r e s w e r e o p t i c a l l y s c a n n e d a n d t h e s e r e s u l t s a r e s h o w n i n F i g . 7- A p r o t e i n w h i c h h a s a n a p p a r e n t m o l e c u l a r weight of 33.ooo and the same electrophoretic mobility as partially p u r i f i e d [~-pen f r o m B . c e r e u s m a r k e d l y i n c r e a s e s . W e t e n t a t i v e l y a s s i g n t h i s p e a k t o b e {~-pen. W h e t h e r : t h e d e c r e a s e i n t h e p e a k a t 6 6 , o 0 o m . w . (i.e_ p o s s i b l y t h e d i m e r ) r e p r e s e n t s a s h i f t i n t h e monomer-oligomer e q u i l i b r i u m is n o t clear. S i n c e v i r t u a l l y n o a c t i v i t y can: b e d e t e c t e d i n c e l l s g r o w n a t 4 6 oC; a n d t h e r e is a n i n c r e a s e i n t h e 33,000 m . w , p r o t e i n
Penicillinase T r a n s p o r t i n B . cereus
667
i n t h e m e m b r a n e , w e p o s t u l a t e t h a t peniciPlinase is still b e i n g p r o d u c e d a t 46 oC b u t t r a n s p o r t across t h e m e m b r a n e a n d s u b s e q u e n t c l e a v a g e b y t h e p r o t e a s e d o e s n o t occur. SVhether b l o c k a g e o f t r a n s p o r t is t h e r e s u l t of (0 a conforrnational c h a n g e in t h e [~-pen, (ii) t h e b u i l d u p of t h e p r o t e i n on t h e inside of t h e m e m b r a n e b e c a u s e it d o e s n o t t r a v e r s e t h e m e m b r a n e , a shift in t h e monomer--oligomer e q u i l i b r i u m effecting p r o t e a s e a c t i v i t y , (ii~) o r f o r o t h e r r e a s o n s h a s y e t t o b e d e t e r m i n e d .
F i g . 6. S D S g e l e l e c t r o p h o r e s i g o f m e m b r a n e p r o t e i n s f r o m celia g r o w n a t 35 ~C a n d 4 6 oC. Arroxv i n d i c a t e s t h e e l e c t r o p h o r e t i c rnobilitx- o f p a r t i a l l y p u r i f i e d a - p e n .
T h e p r e m i s e t h a t b l o c k a g e o f t r a n s p o r t is r e l a t e d t o e v e n t s t h a t o c c u r in t h e m e m b r a n e is s u p p o r t e d b y p r e l i m i n a r y r e s u l t s p e r t a i n i n g t o t h e c o m p o s i t i o n of t h e f a t t y a c i d g r o u p s o f t h e lipids as a f u n c t i o n o f g r o w t h t e m p e r a t u r e a n d s o l v e n t conditions. As c a n b e seen in Fig. 8, t h e r a t i o of t h e n-C14 f a t t y a c i d t o t h e iso-Cl4 f a t t y a c i d is t e m p e r a t u r e dependent. %Vith increasing t e m p e r a t u r e , t h e r e l a t i v e a m o u n t of t h e higher melting-~-Cq4 c o m p o n e n t increases. T h i s p r e s u m a b l y o c c u r s t o m a i n t a i n p r o p e r m e m b r a n e f l u i d i t y ~Sth increasing t e m p e r a t u r e . The l a r g e s t i n c r e a s e o c c u r s a b o v e 42 °C w h i c h is c o i n c i d e n t w i t h t h e d e c r e a s e in penicillinase acti~aity. W e p o s t u l a t e t h a t t h e m a r k e d l y c h a n g e d c o m p o s i t i o n of t h e m e m b r a n e c o u l d effect t h e t r a n s p o r t process_
Kuennen.
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]Big. 7O p t i c a l se'~n~ o f t h e S D S g e l s s h o w n i n F i g . 6. ( h ) a n d ( - - - ) a r e f o r c e l l s g r o l v n a t 35 °C a n d 4 6 °C, r e s p e c t i v e l y - ,8~,-ro,w i n d i c a t e s t h e e l e c t r o p h o r e t i c m o b i l i t y o f p a r t i a l l y p u r i f i e d
[~-pen. Not only does the composition of the membrane .vary with temperature but apparently with the solvent structure as evidenced by the m a r k e d l y d i f f e r e n t n-C14/iso-C~4 r a t i o a t 38 °C f o r ceils g r o w n i n H ± O or DzO. These solvents have different properties, boiling point, melting p o i n t , h y d r o g e n b o n d s t r e n g t h : d i e l e c t r i c c o n s t a n t , etc., a n d t h e d i f f e r e n c e s in the composition of the membrane at the same temperature may reflect these differences . . . . . . . In summary, these preliminary studies of the l temperature dep e n d e n c e o f t h e p r o d u c t i o n o f p e n i c i l l i n a s e b y B . cereus 5 6 9 / H - c l e a r l y i n d i c a t e t h a t t h e r e is a d r a m a t i c d e c r e a s e i n t h e a c t i v i t y o f a l l f o r m s o f t h e e n z y m e s , n a m e l y , ~ - p e n , [3-pen a n d y - p e n , a b o v e 4 2 0 C . T h i s decrease in activity/cannot, :however. be attributed to heat denaturation o f t h e ce-pen i n - t h e c u l t u r e m e d i u m o r t o t h e b r e a k d o w n o f t h e t r a n s l a t i o n a l p r o c e s s e s o f t h e cell, T h e r e s u l t s d o , :howev_er, i m p l y , : b u t a r e
Penicillinase
Transport
in B_ cereus
669
in no means conclusive, that the decrease in activity is the result of blockage of transport across the cell membrane of the penicillinase, iMany drastic changes in the properties of biological systems in addition to those noted in this paper in H,O-NaCl solutions occur at 42 0C f2o-233 which -is coincident with the previously noted higher order phase change in bulk water structure [zo]. Whether subtle changes in the structure of the surrounding medium can illicit such drama& and detrimental changes in a living system will require further verification_
2Sr
2.0 -
t co 36
38
40
42
44
46
Fig_ S_ The temperature dependence of the ratio of ?A& xzxtiofor cells grcm-n in membrane- (0) Indicates
to iso-CzI fatty acids found in the
D=O
at
38 ‘C_
Aeknowledgemenis The anthors wish to thank R_ A. DAY for helpful discussions during the course of this work. T_ HECKLER for supplving us with a sample of partially purified (-75 Oh) $-penicillinase, A. ~ORWEL for assistance in running the GC-MS system. and E_ $5. KANESHIRO for use of her scanning R.W_K_ was the recipient of a Biomedical Research spectrophotometer. This work was supported in part by a _-mt Student _Assistantship. from the University Research Council of the University of Cincmnati (G.P.K.).
670
]Euennen, Simone and
Kreishman
]tege~enees Ix] [2] [3] [4]
[~] [6] [7] [8J [9]
[IO] [XX] Ix2] [x3] [x4] [xS]
[I6] [xT] [I8] [xgJ [2o] [2x] [22] [23]
~ I . R . POLLOCK, J . Gen_ _~ficrobiol. 15 ( I 9 5 6 ) I 5 4 R . SHEI,XIN, J . G e n . ~ [ i c r o b i o L 21 ( I 9 5 9 ) x24 ~/-* GSANYI, AgliB ~]/liGrobioL A c a d . Sai. H u n g . 17 (x97o) 9 r }I_B_ RUDZIK a n d J . IMSA~NDE. J_ Biol_ Chem_ 9-45 ( I 9 7 o) 3 5 5 6 M . G . SARGENT a n d J.O_ L A ~ P E N , Proc_ N a t l . Acad_ Sci.o U S A 65 (x97 o) 9 6 2 J . IMSANDE, 2VlicyobioL R~0. 4 2 (x978) 6 7 a n d r e f e r e n c e s t h e r e i n A_ B ~ R N E r E I ~ , K_~V. ~XCKERSON a n d R . A . DAY, A r c h . B i o 6 h e m . B i o p h y s . 119 (x967) 5 o M . R . POLLOCK a ~ d C . J . PERRET, B r i t . J_ tExp. P a t h o l . 3 2 (x95x) 3 8 7 ~ L K o ~ u T , b L l ~ POLLOCK a n d E _ J . TRXDG~*T, B i o c h e m . J_ 62 ( I 9 5 6 ) 39~ C.J. PERRL~T", N a f u r ~ (London) 174 (x954) I O I 2 M . G . SARG~.~T, J_ Bacteriol_ 95 ( I 9 6 8 ) x 4 9 3 M . R . POLLOCK, B r i t . J_ E x p _ P a t h o l . 39 (x957) 6 o 9 ~ I . G . SARGENT, B . K - GtlOSH a n d J . O . L_~,~P]~, J . Bacte~riol. 9 6 (x968) x 3 2 9 B . K . GHOSH, M . G . SARGENT a n d J . O . LAMPENo J . B a c t e r i o L 96 (~968) x 3 z 4 ~ V . ~ . KONINGS° fl-. BISSCHOP, M . VEENHUIS a n d C . A . V E R . ~ U L ~ N , jr. Bact6~riol_ 116 ( I 9 7 3 ) x 4 5 6 D . M . ,~EVILLE. J r . , J . B i o l . C h e m . 2 4 6 (x97x) 6 3 2 8 L . RXLFORS, _A- V~'IESL~'~DER a n d S. STAHL~ J . B a c t e r i o l . 135 ( I 9 7 8 ) IO43 J . M A c G E E , C. GLOZZeECK~R a n d C. v~VATANAKUNAKORN, C a n . J . B l i c r o biol. :~3 ( I 9 7 7 ) x478 M_ ~[ADALA~X ancl R._A_ DAY. B i o c k i m . B i o p h y s . A c t a 2 3 6 (x97 x) x9x G . P . KREISH~L~-NN, D . A . F o s s , K . I N o u ~ a n d L . LEXSER° B i o c h e m i s t r y 15 (x976) 5 4 3 x C.~V_ =A~D~RSON, H . B - H-~JLSM.L. ~V.R_ HEXNEMAN a n d G . P . KREXSHMAN. B i o c h e m . Bioplzys_ Res_ C o m m u n _ 76 ( I 9 7 7 ) 3 3 9 G . P . KREISHMAN, C _ ~ r_ .~'IkNDERSON. C . - - H . S v , H . B . HALSALL a n d ~V.R_ HEINEM_~,N, B i o e l e c t r o c h e m . Bio6-aerg. 5 ( I 9 7 8 ) I 9 6 G_P. KREISHMAS, C . - - H . S u , C.~V_ A~IDERSON, H . B . I-L~LSALL a n d ~ V . R . H£[NEMAN, A d v . C h e m . , 188 ( I 9 8 0 ) 169
-- P r i n t e d
in Italy
365 - The Temperature Dependence of the Production of Penicillinase in Bacillus eere~cs 569/H. * by R. W. KUENNEN, Department
h i . J . SLMONE a n d G. P . KREISH.MAN o f C h e m i s t r y , U n i v e r s i t y o f CirLcinnati, C i n c i n n a t i , O h i o 45221, US.~.
~lanuscript received ~ovember
~_4th 1979
Summary
The activity and amount of both the exo form and the cell-bound f o r m s o f p e n i c i l l i n a s e (penicillin a m i d o - [ 3 - 1 a c t a m a s e , E C 3.5.2.6) p r o d u c e d by the constitutive strain Bacillus cereus 569/H have been measured as a f u n c t i o n o f t h e t e m p e r a t u r e _ T h e a c t i v i t y o f b o t h f o r m s is r e l a t i v e l y c o n s t a n t f r o m 33 t o 42 °C- A b o v e t h i s t e m p e r a t u r e , h o w e v e r , a r a p i d d e c r e a s e in t h e a c t i v i t y is o b s e r v e d a n d v i r t u a l l y n o a c t i v i t y c a n b e d e t e c t e d in cells g r o w n a t 4 6 °C. A t all t e m p e r a t u r e s , t h e e x o - p e n i c i l l i n a s e is e x c r e t e d i n t o t h e m e d i u m o n l y in t h e a c t i v e f o r m . C o n c u r r e n t ~sith t h e d e c r e a s e in t h e a m o u n t o f e x o - p e n i c i l l i n a s e s e c r e t e d is a p a r a l l e l d e c r e a s e in a c t i v i t y o f t h e c e l l - b o u n d penicillinase, w h i c h i m p l i e s t h a t the exo-penicillinase can only be formed from active cell-bound penicillinase. A l t h o u g h t h e a c t i v i t y o f t h e c e l l - b o u n d p e n i c i l l i n a s e d e c r e a s e s a b o v e 42 °C, t h e r e is a m a r k e d i n c r e a s e o f a p r o t e i n in t h e m e m b r a n e of the same apparent electrophoretic mobility as purified venicillinase. I t is p o s t u l a t e d t h a t t h e p r o d u c t i o n o f p e n i c i l l i n a s e o c c u r s - a b o v e 42 °C b u t t h a t t h e loss o f a c t i , d t y is t h e r e s u l t o f d e n a t u r a t i o n o f t h e p r o t e i n i n a n a l t e r e d m e m b r a n e . T h e loss o f a c t i v i t y c o r r e l a t e s w e l l w i t h a d r a m a t i c c h a n g e i n t h e f a t t y a c i d c o m p o s i t i o n o f t h e m e m b r a n e a b o v e 42 °C-
Introduction P e n i c i l l i n a s e (penicillin a m i d o - ~ b - l a c t a m a s e , E C 3 . 5 . 2 _ 6 ) is s e c r e t e d into the growth medium by Gram-positive bacteria of the genus Bacillus. I n B . c e r e u s , i t h a s b e e n s h o w n t o d a t e t h a t t h e p r o t e i n e x i s t s in a t l e a s t t w o f o r m s , ~ - p e n i c i l l i n a s e ( ~ - p e n ) w h ichis f o u n d f r e e in t h e c u l t u r e m e d i u m * Presented at the 5th International 3--8 S e p t e m b e r 1979, W e i m a r ( D . D . R . )
o3o':'--4598/8o/o659-o67o
~
Symposium
E l s e v i e r S e q u o i a S.A_
orL B i o e l e c t r o c h e m i s t r y ,
66o
I4:uennen, S i m o n e a n d K r e i s h m a n
or can be loosely bound to the membrane surface, and y-penicillinase (y-pen} w h i c h is t i g h t l y b o u n d t o t h e m e m b r a n e a n d c a n n o t b e n e u t r a l i z e d b y a n t i s e r u m p r e p a r e d a g a i n s t m-pen [1-3]. I t h a s b e e n s h o w n t h a t y - p e n is a c o n f o r m e r o f m-pen, t h e t w o f o r m s b e i n g r e a d i l y i n t e r c o n v e r t a b l e [4]- T h e y - p e n m a y e x i s t i n v a r i o u s p o l y m e r i c f o r m s i n t h e m e m b r a n e [5]- I n o t h e r species o f B a c i l l u s , n a m e l y B . l i c h e n i f o r m i s , a ~ - p e n i c i l l i n a s e (~-pen) h a s b e e n f o u n d a n d s h o w n t o b e a l i p o p r o t e i n . g - P e n is p r o d u c e d b y a specific c l e a v a g e o f a p o r t i o n o f [~-pen b y a p r o t e a s e n e a r t h e a m i n o t e r m i n u s w h e r e t h e l i p i d is a t t a c h e d [6]. N o s u c h l i p o p r o t e i n h a s y e t b e e n f o u n d i n B_ c e r e u s b u t i t s e x i s t e n c e h a s b e e n i n f e r r e d f r o m s t r u c t u r a l h o m o l o g i e s 6f m - p e n i c i l l i n a s e s f r o m v a r i o u s B a c i l l u s species. BERNSTEIN et aL h a v e m e a s u r e d t h e t e m p e r a t u r e d e p e n d e n c e o f m-pen a c t i v i t y as a f u n c t i o n of t h e t e m p e r a t u r e for t h e n o r m a l inducible a n d c o n s t i t u t i v e s t r a i n s o f B . c e r e u s [7]- I n b o t h o r g a n i s m s , t h e r e w a s a n i n c r e a s e i n a c t i v i t y With i n c r e a s i n g t e m p e r a t u r e u p t o 4 z oC, w h e r e a m a x i m u m a m o u n t of m-pen was produced. Above this t e m p e r a t u r e , a drastic decline in activity was observed. The authors speculate that t h e d i f f e r e n c e i n t h e r a t e o f i n c r e a s e f o r e a c h strain, is c o n s i s t e n t ~s~th a c o n t r o l o f p r o t e i n s y n t h e s i s a t t w o levels, n a m e l y a t t h e t r a n s c r i p t i o n a l a n d a t t h e t r a n s l a t i o n a l levels, e a c h b e i n g e f f e e t e d b y t e m p e r a t u r e . I n t h e inducible strain, t h e larger incremental increase in penicillinase a c t i v i t y w i t h t e m p e r a t u r e can be a t t r i b u t e d to t h e r m a l derepression b y inactivation of t h e repressor protein i t s d L F o r the constitutive strain, t h e smaller incremental change was attributed to the temperature sensitivity of the t r a n s l a t i o n a l process. T h e a u t h o r s d o n o t s p e c u l a t e as t o t h e c a u s e s f o r t h e d r a m a t i c d e c r e a s e i n p e n i c i l l i n a s e a c f i ~ S t y a b o v e 4z °C. T h e p u r p o s e of this study was to address this question. Materia~ and methods
Organisms,
inoculg and media
T h e c o n s t i t u t i v e s t r a i n 5 6 9 / H o f B . c e r e u s w a s a k i n d g i f t o f Dr. R . A. DAY. B a c t e r i a l p r e c u l t u r e s w e r e g r o w n f r o m a s p o r e i n n o c u l u m i n S. p e p t o n e b r o t h [8]. P o l y c a r b o n a t e e r l e n m e y e r f l a s k s (SOO c m n) c o n t a i n i n g 5o c m a o f c a s e i n h y d r o l y s a t e g r o w t h m e d i u m [9] w e r e i n o c u l a t e d •~5th x % (vol/vol) o f t h e p r e c u l t u r e a n d i n c u b a t e d i n a GCA-P r e c i s i o n Scientific M o d e l 50 s h a k e r b a t h c o n t r o l l e d t o --_o 5 oC a n d i i o osc_/min_ B a c t e r i a l g r o w t h w a s d e t e r m i n e d b y o p t i c a l d e n s i t y w h i c h w a s m o n i t o r e d a t 650 n m w i t h a BAUSH a n d LO.XZB M o d e l 340 s p e c t r o m e t e r . B a c t e r i a l cells w e r e h a r v e s t e d i n l a t e log p h a s e a t a n o p t i c a l d e n s i t y o f a p p r o x i m a t e l y 1. 3 . Enzyme
assays
Penicillinase was assayed iodometrically essentially by a modified m e t h o d o f PERR~--r [10] f o r t h e i o d i n e i n s e n s i t i v e f o r m o f t h e e n z y m e
Penicillina~e Transport= in
B.
cereus
66x
a n d b y t h e m e t h o d of SARGENT [II] f o r b o t h i o d i n e s e n s i t i v e a n d i n s e n s i t i v e f o r m s of t h e e n z y m e _ U n i t a c t i v i t y of p e n i c i i l l n a s e is e q u a l t o t h e mount o f penicillina.se w h i c h h y d r o l y z e s I m i c r o m o l e o f b e n z y l p e n i cillin in 6o m i n u t e s a t 3o oC a n d p H 7.o [I2].
Puril~cation o f the ~t-penicillinase i ~ the culture m e d i u m by gel chromatography The ~-pen was isolated from the culture medium using a Tygon c o l u m n (65 c m x o c m ) pack~.a w i t h S e p h a d e x G - 7 5 - 1 o o - T h e c o l u m n w a s e i u t e d w i t h O.lO M s o d i u m l~_~..~sphate b u f f e r , p H 7.00, a t a flow r a t e o f o-5 c m 3 / m i n -
Preparatio~ o f protolblasts for the determination of cell-bound penicillinase P r o t o p l a s t s w e r e p r e p a r e d f r o m B . cereus 5 6 9 / H b y t h e r a p i d p r o c e d u r e o f LA~IPE,~ et al. [I3]- T h i s t e c h n i q u e i n v o l v e s t h e u s u a l p r o t o p l a s t f o r m a t i o n b y t r e a t m e n t w i t h l y s o z y m e in a h y p ~ r t o n i e s o l u t i o n . F u r t h e r s y n t h e s i s of p e n i e i l l i n a s e w a s a l s o p r e v e n t e d b y t h e p r e s e n c e o f e h l o r a m p h e n i c o l in t h e p r o t o p l a s t i n g s o l u t i o n . A p r o c e d u r e a s d e s c r i b e d b y LAMPEN et al. [14] f o r g r o w i n g p r o t o p l a s t s w a s u s e d .
Preparation o f membrane proteins .for electrophoresis Cell m e m b r a n e s w e r e p r e p a r e d b y t h e r e l a t i v e l y r a p i d p r o c e d u r e o f KO,~I,~GS et al. [15], w h e r e t h e ceils a r e t r e a t e d w i t h l y s o z y m e in a h y p o t o r A c m e d i u m w h i c h r e s u l t s in a n i m m e d i a t e lysis o f t h e cells a n d f o r m a t i o n o f m e m b r a n e vesicles. T h e s e m e m b r a n e vesicles a r e c o l l e c t e d b y c e n t r i f u g a t i o n f o r 30 rain. a t 16,ooo x g a t 5 °C- S D S p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s w a s p e r f o r m e d a s d e s c r i b e d b y NEVILLE [16], w i t h m o d i f i c a t i o n s o f RXLFORS et al. [17]- T h e S D S g e l s y s t e m utilizes a s u l f a t e b o r a t e d i s c o n t i n u o u s b u f f e r s y s t e m ( r u n n i n g p H 9-5) w h i c h s t a c k s a n d u n s t a c k s protein SDS complexes over a range of 2,3oo to 32o,ooo daltons, providing high resolution fractionation. Reference proteins used for molecular weight determination were b o v i n e s e r u m a l b u m i n (68,ooo), o v a l b u m i n (43,ooo), d e o x y r i b o n u c i e a s e 1 (3x,4oo), c a r b o n i c a n h y d r a s e (29,0o0) a n d l y s o z y m e (14,3oo)A GILFORD d e n s i t o m e t e r e q u i p p e d w i t h a g e l s c a n n e r w a s u s e d f o r s c a n n i n g gels a t v a r i o u s g r o w t h t e m p e r a t u r e s a n d q u a l i t a t i v e t r a n s m i s s i o n of various proteins was recorded.
Gas chromatography-mass spectrometry Fatty acid methyl esters were prepared following the procedure d e s c r i b e d "by MACGEE et al. [18]. T h e g a s c h r o m a t o g r a p h w a s a VARL~,~ A e r o g r a p h M o d e l 17.4o d u a l f l a m e u n i t w i t h 3.8 m × 3-2 m m i n s i d e d i a m e t e r s t a i n l e s s s t e e l c o l u m n p a c k e d : w i t h 3 ~o silicone O V - z 7 o n 8o t o zoo G a s C h r o m Q (Ar-TECH ASSOCIATES). T h e c a r r i e r g a s w a s h e l i u m a t 20 e m a / m i n . T h e i n j e c t i o n p o r t a n d d e t e c t o r w e r e h e l d a t 29o a n d .3oo-°C r e s p e c t i v e l y .
66z
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A 8 - 9 m m s (EL1)s a m p l e w a s i n j e c t e d i n t o a s t a x t i n g c o l u m n t e m p e r a t u r e o f x45 °C w h i c h w a s p r o g r a m m e d to increase at a rate of 8 oC/min, to a final t e m p e r a t u r e o f 3oo oC h e l d , i s o t h e r m a l l y for 3 vain.
T h e gas c h r o m a t o g r a p h w a s i n t e r f a c e d w i t h a HITACHI type RMU-- 7 m a s s s p e c t r o m e t e r o p e r a t e d a t 70 e V w i t h a V G DATASYSTEM 2000 c o m p u t e r . P e a k a r e a s r e p r e s e n t i n g a m o u n t s o f iso-~4 a n d norma~-C~a w e r e e s t i m a t e d u s i n g b e s t t r i a n g u l a r fit.
Results ~
disen-~b~_~'on
T h e t e m p e r a t u r e d e p e n d e n c e o f m-pen a c t i v i t y i n c u l t u r e m e d i u m of t h e c o n s t i t u t i v e s t r a i n o f B . c e r e u s i s s h o w n in Fig. x. T h e a c t i v i t y o f = - p e n in t h e c u l t u r e m e d i u m of b a c t e r i a g r o w n t o t h e s a m e o p t i c a l d e n s i t y i n t h e l a t e log p h a s e o f g r o w t h r e m a i n s v i r t u a l l y u n c h a n g e d ,~atiI 4z oC, w h e n a b o v e t h i s t e m p e r a t u r e a d r a s t i c d e c r e a s e is o b s e r v e d . T o r u l e o u t t h e p o s s i b i l i t y t h a t t h e d e c r e a s e is d u e t o penicillinase d e n a turation, the conformational properties, as assumed b y U V spectroscopy, a n d t h e a c t i v i t y o f i s o l a t e d peniciUinase w e r e s t u d i e d as a f u n c t i o n o f
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T a b l e z. T h e r m o s t a b i l i t y temperature.
of exopen/cillinase as a function of time at a given
Temperature oC
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t e m v e r a t u r e . N o d i s c e r n a b l e c h a n g e in t h e a b s o r b a n c e a t z S o n m c o u l d b e d e t e c t e d i n NaC1--H±O u n t i l ~ 5 5 oC w h e r e t h e p r o t e i n p r e s u m a b l y a g gregates. In addition to the lack of a detectable c0nformational change i n t h e p r o t e i n , t h e a c t i v i t y of t h e i s o l a t e d p r o t e i n r e m a i n e d , c o n s t a n t u n t i l ~ 5 2 oC w h e r e t o s s o f a c t i v i t y w a s o b s e r v e d (Fig. 2). T h e p r o t e i n can also be incubated at elevated temperatures for prolonged periods of t i m e w i t h m i n i m a l loss o f a c t i v i t y ( T a b l e I). = - P e n i t s e l f is t h e r e f o r e extremely thermostable as compared to other proteins and the marked d e c r e a s e in a c t i v i t y a t 42 °C c a n n o t b e a t t r i b u t e d t o p r o t e i n d e n a t u r a t i o n at elevated temperatures.
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S i n c e o n l y t h e p r o t e i n a c t i v i t y is b e i n g m e a s u r e d , t h e p o s s i b i l i t y t h a t t h e =--pen is b e i n g r e l e a s e d i n t o t h e m e d i u m i n a n i n a c t i v e f o r m m u s t b e r u l e d o u t . T h e m o n o m e r i c g - p e n c a n .he. p u r i f i e d f r o m o t h e r p r o t e i n s in t h e m e d i u m b y u t i l i z i n g g e l c h r o m a t o g r a p h y (Fig. 3)- A t a l l t e m p e r a t u r e s , t h e specific a c t i v i t y o f t h e m o n o m e r i c g - p e n p e a k w a s a p p r o x i m a t e l y 3 x I o s u n i t s / r a g of p r o t e i n w h i c h i s t h e specific a c t i v i t y o f t h e p u r i f i e d e n z y m e Ix9]- S i n c e b o t h t h e a b s o r b a n c e a n d a c t i v i t y o f t h a t p e a k d e c r e a s e d c o n c u r r e n t l y , a b o v e 4 z °C, ~ - p e n is s e c r e t e d i n t o t h e m e d i u m ha *Jae a c t i v e f o r m o n l y b u t a t s u p p r e s s e d l e v e l s .
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The temperature dependence of the total ceil-bound penicillinase actisity measured after
protoplasting.
The temperature dependence of the activity of the total cell-bound p e n i c i l l i n a s e (i.e. y - p e n p l u s 0 - p e n ) m e a s u r e d a f t e r p r o t o p l a s t i n g (Fig. 4) a n d r e m a i n i n g c e l l - b o u n d p e n i c i l l i n a s e meast~red a f t e r h o m o g e n / z a t i o n (Fig. 5) p a r a l l e l s t h a t f o r ~ - p e n w i t h a r a p i d d e c r e a s e in a c t i v i t y a b o v e 42 °C- S i n c e t h e r a t i o s o f a c t i v i t y o f t h e ~ - p e n t o t h e c e l l - b o u n d penicill i n a s e a r e t h e s a m e a b o v e 4 2 oC a s b e l o w , w e i n f e r t h a t t h e p r o t e a s e h a s n o t d e n a t u r e d b u t t h a t t h e d e c r e a s e in s e c r e t i o n in a c t i v e ~ - p e n i n t o t h e m e d i u m is t h e r e s u l t o f a d e c r e a s e i n t h e s u b s t r a t e f o r t h e p r o t e a s e , n a m e l y t h e active form of ~-pen. It is interesting to note that after homogenization o f cells g r o w n a t 46 °C, i e s i d u a l a c t i v i t y c a n b e d e t e c t e d i m p l y i n g t h a t t h e o r g a n i s m is c a p a b l e o f p r o d u c i n g a c t i v e p e n i c i l l i n a s e i n s o m e f o c m a t 4 6 oC, a l b e i t a t a d e p r e s s e d l e v e l . T o d e t e r m i n e if a n i n a c t i v e f o r m o f p e n i c i U i n a s e is a c c u m u l a t i n g i n t h e m e m b r a n e a b o v e 42 oC, t h e p r o t e i n s f o u n d in t h e m e m b r a n e o f c e i l s g r o w n a t 35 °C a n d 4 6 oC h a v e b e e n a n a l y z e d b y S.D.S_ g e l e l e c t r o p h o r e s i s ( F i g , 6). M o s t , i f n o t a l l o f t h e p r o t e i n s , t h a t a r e p r e s e n t a t 35 °C a r e p r e s e n t a t 46 °C. B r e a k d o w n o f t h e t r a n s l a t i o n a l p r o c e s s a t
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Fig. 5Remaining cell-bound penicillinase activity measured after homogenization as a function of temperature.
4 6 oC a s t h e c a u s e f o r d e c r e a s e in p e n i e i l l i n a s e p r o d u c t i o n is u n l i k e l y . The relative amounts of each type of protein do, however, vary with a predominant increase in the lower molecular weight proteins and a d e c r e a s e i n t h e h i g h e r m o l e c u l a r w e i g h t p r o t e i n s a t 4 6 °C. T h i s m a y b e d u e t o e i t h e r t h e r m a l sensitix~ity o f t h e t r a n s c r i p t i o n a l o r t r a n s l a t i o n a l p r o c e s s e s f o r t h e l a r g e r p r o t e i n s o r reflect b r e a k d o w n of o l i g o m e r i c p r o t e i n s i n t o t h e i r s u b t m i t s . T h e gels o f t h e m e m b r a n e p r o t e i n s f r o m t h e cells g r o w n a t t h e t w o t e m p e r a t u r e s w e r e o p t i c a l l y s c a n n e d a n d t h e s e r e s u l t s a r e s h o w n i n F i g . 7- A p r o t e i n w h i c h h a s a n a p p a r e n t m o l e c u l a r weight of 33.ooo and the same electrophoretic mobility as partially p u r i f i e d [~-pen f r o m B . c e r e u s m a r k e d l y i n c r e a s e s . W e t e n t a t i v e l y a s s i g n t h i s p e a k t o b e {~-pen. W h e t h e r : t h e d e c r e a s e i n t h e p e a k a t 6 6 , o 0 o m . w . (i.e_ p o s s i b l y t h e d i m e r ) r e p r e s e n t s a s h i f t i n t h e monomer-oligomer e q u i l i b r i u m is n o t clear. S i n c e v i r t u a l l y n o a c t i v i t y can: b e d e t e c t e d i n c e l l s g r o w n a t 4 6 oC; a n d t h e r e is a n i n c r e a s e i n t h e 33,000 m . w , p r o t e i n
Penicillinase T r a n s p o r t i n B . cereus
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i n t h e m e m b r a n e , w e p o s t u l a t e t h a t peniciPlinase is still b e i n g p r o d u c e d a t 46 oC b u t t r a n s p o r t across t h e m e m b r a n e a n d s u b s e q u e n t c l e a v a g e b y t h e p r o t e a s e d o e s n o t occur. SVhether b l o c k a g e o f t r a n s p o r t is t h e r e s u l t of (0 a conforrnational c h a n g e in t h e [~-pen, (ii) t h e b u i l d u p of t h e p r o t e i n on t h e inside of t h e m e m b r a n e b e c a u s e it d o e s n o t t r a v e r s e t h e m e m b r a n e , a shift in t h e monomer--oligomer e q u i l i b r i u m effecting p r o t e a s e a c t i v i t y , (ii~) o r f o r o t h e r r e a s o n s h a s y e t t o b e d e t e r m i n e d .
F i g . 6. S D S g e l e l e c t r o p h o r e s i g o f m e m b r a n e p r o t e i n s f r o m celia g r o w n a t 35 ~C a n d 4 6 oC. Arroxv i n d i c a t e s t h e e l e c t r o p h o r e t i c rnobilitx- o f p a r t i a l l y p u r i f i e d a - p e n .
T h e p r e m i s e t h a t b l o c k a g e o f t r a n s p o r t is r e l a t e d t o e v e n t s t h a t o c c u r in t h e m e m b r a n e is s u p p o r t e d b y p r e l i m i n a r y r e s u l t s p e r t a i n i n g t o t h e c o m p o s i t i o n of t h e f a t t y a c i d g r o u p s o f t h e lipids as a f u n c t i o n o f g r o w t h t e m p e r a t u r e a n d s o l v e n t conditions. As c a n b e seen in Fig. 8, t h e r a t i o of t h e n-C14 f a t t y a c i d t o t h e iso-Cl4 f a t t y a c i d is t e m p e r a t u r e dependent. %Vith increasing t e m p e r a t u r e , t h e r e l a t i v e a m o u n t of t h e higher melting-~-Cq4 c o m p o n e n t increases. T h i s p r e s u m a b l y o c c u r s t o m a i n t a i n p r o p e r m e m b r a n e f l u i d i t y ~Sth increasing t e m p e r a t u r e . The l a r g e s t i n c r e a s e o c c u r s a b o v e 42 °C w h i c h is c o i n c i d e n t w i t h t h e d e c r e a s e in penicillinase acti~aity. W e p o s t u l a t e t h a t t h e m a r k e d l y c h a n g e d c o m p o s i t i o n of t h e m e m b r a n e c o u l d effect t h e t r a n s p o r t process_
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Molecular
]Big. 7O p t i c a l se'~n~ o f t h e S D S g e l s s h o w n i n F i g . 6. ( h ) a n d ( - - - ) a r e f o r c e l l s g r o l v n a t 35 °C a n d 4 6 °C, r e s p e c t i v e l y - ,8~,-ro,w i n d i c a t e s t h e e l e c t r o p h o r e t i c m o b i l i t y o f p a r t i a l l y p u r i f i e d
[~-pen. Not only does the composition of the membrane .vary with temperature but apparently with the solvent structure as evidenced by the m a r k e d l y d i f f e r e n t n-C14/iso-C~4 r a t i o a t 38 °C f o r ceils g r o w n i n H ± O or DzO. These solvents have different properties, boiling point, melting p o i n t , h y d r o g e n b o n d s t r e n g t h : d i e l e c t r i c c o n s t a n t , etc., a n d t h e d i f f e r e n c e s in the composition of the membrane at the same temperature may reflect these differences . . . . . . . In summary, these preliminary studies of the l temperature dep e n d e n c e o f t h e p r o d u c t i o n o f p e n i c i l l i n a s e b y B . cereus 5 6 9 / H - c l e a r l y i n d i c a t e t h a t t h e r e is a d r a m a t i c d e c r e a s e i n t h e a c t i v i t y o f a l l f o r m s o f t h e e n z y m e s , n a m e l y , ~ - p e n , [3-pen a n d y - p e n , a b o v e 4 2 0 C . T h i s decrease in activity/cannot, :however. be attributed to heat denaturation o f t h e ce-pen i n - t h e c u l t u r e m e d i u m o r t o t h e b r e a k d o w n o f t h e t r a n s l a t i o n a l p r o c e s s e s o f t h e cell, T h e r e s u l t s d o , :howev_er, i m p l y , : b u t a r e
Penicillinase
Transport
in B_ cereus
669
in no means conclusive, that the decrease in activity is the result of blockage of transport across the cell membrane of the penicillinase, iMany drastic changes in the properties of biological systems in addition to those noted in this paper in H,O-NaCl solutions occur at 42 0C f2o-233 which -is coincident with the previously noted higher order phase change in bulk water structure [zo]. Whether subtle changes in the structure of the surrounding medium can illicit such drama& and detrimental changes in a living system will require further verification_
2Sr
2.0 -
t co 36
38
40
42
44
46
Fig_ S_ The temperature dependence of the ratio of ?A& xzxtiofor cells grcm-n in membrane- (0) Indicates
to iso-CzI fatty acids found in the
D=O
at
38 ‘C_
Aeknowledgemenis The anthors wish to thank R_ A. DAY for helpful discussions during the course of this work. T_ HECKLER for supplving us with a sample of partially purified (-75 Oh) $-penicillinase, A. ~ORWEL for assistance in running the GC-MS system. and E_ $5. KANESHIRO for use of her scanning R.W_K_ was the recipient of a Biomedical Research spectrophotometer. This work was supported in part by a _-mt Student _Assistantship. from the University Research Council of the University of Cincmnati (G.P.K.).
670
]Euennen, Simone and
Kreishman
]tege~enees Ix] [2] [3] [4]
[~] [6] [7] [8J [9]
[IO] [XX] Ix2] [x3] [x4] [xS]
[I6] [xT] [I8] [xgJ [2o] [2x] [22] [23]
~ I . R . POLLOCK, J . Gen_ _~ficrobiol. 15 ( I 9 5 6 ) I 5 4 R . SHEI,XIN, J . G e n . ~ [ i c r o b i o L 21 ( I 9 5 9 ) x24 ~/-* GSANYI, AgliB ~]/liGrobioL A c a d . Sai. H u n g . 17 (x97o) 9 r }I_B_ RUDZIK a n d J . IMSA~NDE. J_ Biol_ Chem_ 9-45 ( I 9 7 o) 3 5 5 6 M . G . SARGENT a n d J.O_ L A ~ P E N , Proc_ N a t l . Acad_ Sci.o U S A 65 (x97 o) 9 6 2 J . IMSANDE, 2VlicyobioL R~0. 4 2 (x978) 6 7 a n d r e f e r e n c e s t h e r e i n A_ B ~ R N E r E I ~ , K_~V. ~XCKERSON a n d R . A . DAY, A r c h . B i o 6 h e m . B i o p h y s . 119 (x967) 5 o M . R . POLLOCK a ~ d C . J . PERRET, B r i t . J_ tExp. P a t h o l . 3 2 (x95x) 3 8 7 ~ L K o ~ u T , b L l ~ POLLOCK a n d E _ J . TRXDG~*T, B i o c h e m . J_ 62 ( I 9 5 6 ) 39~ C.J. PERRL~T", N a f u r ~ (London) 174 (x954) I O I 2 M . G . SARG~.~T, J_ Bacteriol_ 95 ( I 9 6 8 ) x 4 9 3 M . R . POLLOCK, B r i t . J_ E x p _ P a t h o l . 39 (x957) 6 o 9 ~ I . G . SARGENT, B . K - GtlOSH a n d J . O . L_~,~P]~, J . Bacte~riol. 9 6 (x968) x 3 2 9 B . K . GHOSH, M . G . SARGENT a n d J . O . LAMPENo J . B a c t e r i o L 96 (~968) x 3 z 4 ~ V . ~ . KONINGS° fl-. BISSCHOP, M . VEENHUIS a n d C . A . V E R . ~ U L ~ N , jr. Bact6~riol_ 116 ( I 9 7 3 ) x 4 5 6 D . M . ,~EVILLE. J r . , J . B i o l . C h e m . 2 4 6 (x97x) 6 3 2 8 L . RXLFORS, _A- V~'IESL~'~DER a n d S. STAHL~ J . B a c t e r i o l . 135 ( I 9 7 8 ) IO43 J . M A c G E E , C. GLOZZeECK~R a n d C. v~VATANAKUNAKORN, C a n . J . B l i c r o biol. :~3 ( I 9 7 7 ) x478 M_ ~[ADALA~X ancl R._A_ DAY. B i o c k i m . B i o p h y s . A c t a 2 3 6 (x97 x) x9x G . P . KREISH~L~-NN, D . A . F o s s , K . I N o u ~ a n d L . LEXSER° B i o c h e m i s t r y 15 (x976) 5 4 3 x C.~V_ =A~D~RSON, H . B - H-~JLSM.L. ~V.R_ HEXNEMAN a n d G . P . KREXSHMAN. B i o c h e m . Bioplzys_ Res_ C o m m u n _ 76 ( I 9 7 7 ) 3 3 9 G . P . KREISHMAN, C _ ~ r_ .~'IkNDERSON. C . - - H . S v , H . B . HALSALL a n d ~V.R_ HEINEM_~,N, B i o e l e c t r o c h e m . Bio6-aerg. 5 ( I 9 7 8 ) I 9 6 G_P. KREISHMAS, C . - - H . S u , C.~V_ A~IDERSON, H . B . I-L~LSALL a n d ~ V . R . H£[NEMAN, A d v . C h e m . , 188 ( I 9 8 0 ) 169