Biosynthesis of bacitracin and protein

533

SHORT COMMUNICATIONS TABLE IONIC

STRENGTH

OF

DNA

V22B V22B P238

DNA

SOLUTIONS

IN

I

SALT WATER

BEFORE

Transition

AND

n

native -+ denatured at 60 ° n a t i v e --> r e n a t u r e d a t 22 ° n a t i v e -+ w h o l l y d e n a t u r e d *

AFTER

HEAT

DENATURATION

n"

t,

2 . 2 . lO 21

2. 5" 1021

1021

~2.

2.2'

9

1 0 21

~I

5" lO2°

~,i

I O 21

1 . 2 . lO 21

• l O 2o

5 . 5 ' lO2°

n" h, 5

• B y a l e n g t h y h e a t i n g a t 9 7 ° f o l l o w e d b y a q u i c k c o o l i n g a t o °.

f=lMHz K

CpP-•

/

,

tg~5 3

/

10C

gc DNA V 22 B

2

8C

I

io

~0

1.5

Temperature

F i g . 3. D i e l e c t r i c b e h a v i o u r

of DNA

solution in 8 M urea.

solutions in pure formamide and in 8 M urea. No melting characteristics were observed. These results are in agreement with other worksZ, 3, which indicated that, in these solvents, DNA is always in a denatured state.

Laboratoire d'Electronique et de Physique du Solide de l'Universitd de Lyon, Lyon (France)

GuY ]~ESNARD DANE VASILESCU

1 ]V[. HANSS, R . VIOVY AND C. SADRON, Compt. Rend., 2 5 6 (1963) 4 5 1 o . 2 p . O . P . T s ' o , G . K . HELMKAMP AND C. SANDERS, Biochim. Biophys. Acta, 55 ( 1 9 6 2 ) 5 8 4 . 3 G . K . HELMKAMP AND 1). O. P . TS'O, Biochim. Biophys. Acta, 74 ( 1 9 6 2 ) 1 7 2 4 .

Received May I5th, 1964 Biochim. Biophys. Acta, 91 ( 1 9 6 4 ) 5 3 1 - 5 3 3 SC 93030

Biosynthesis of bacitracin and protein The use of puromycin and chloramphenicol, antibiotics which specifically inhibit protein synthesis1, ~, has resulted in the demonstration that the process of assembling amino acids into the polypeptides tyrocidine and gramicidine-S involves a mechanism Biochim. Biophys. Acta, 91 ( 1 9 6 4 ) 5 3 3 - 5 3 6

534

SHORT COMMUNICATIONS

other than that used for protein synthesis3, 4. We have carried out similar studies on the synthesis of the antibiotic bacitracin. Results have been obtained which show that the synthesis of protein and bacitracin can be dissociated and provide evidence that the synthesis of bacitracin also involves a mechanism other than that used in protein synthesis. The organism used was a bacitracin producing strain of Bacillus licheni/ormis (ATCC lO716) which was grown in a synthetic medium. The medium, modified from that described by H E N D L I N 5, contained in I 1, I2.O g L-glutamic acid, 2.o g citric acid, o.I g MgSO4, o.oi g MnSO4, o.5 g K2I-[PO4, and 0.025 g FeC1a, and was adjusted to pI-[ 7.o with NaOH. The culture of B. licheni/ormis in synthetic medium was grown for 16 h at 36o with agitation by a reciprocating shaker at 12o cycles:"min. Formation of bacitracin was studied either by transferring a io-ml sample of the culture into 5o-ml flasks and continuing the shaking at 36°, or by shaking io ml of an incubation mixture to which were added cells which were harvested and twice washed with 0.85 o/ /0 saline. The growth of bacterial cultures was determined by measuring the absorbancv at 62o m# using cylindrical cells 15 mm in diameter. Samples were diluted so that the absorbancy readings were in the proportional range. The determination of bacitracin was made by a microbiological assayL In experiments where protein synthesis was being measured, the incorporation of [14Clleucine into protein was determined in the manner described by MACH et al."L DL-I2J~Clleucine, 0.5 mC" mmole was obtained from Tracerlab, and D-phenylalanine was a product of California Corporation for Biochemical Research. 500-

5.0-

2.5-

400-

2.0-

~300-

>

1.5-

~.. 2001.0-

5 ~) I 0 0

.5-

o

o

,

I0

,

20

MINUTES Fig. 1

,

30

i

40

0

o

5

I0

i5

20

MINUTES

Fig. 2

Fig. i. I n h i b i t i o n of p r o t e i n s y n t h e s i s b y p u r o m y c i n , c h l o r a m p h e n i c o l a n d tetracycline. T h e i n c u b a t i o n m i x t u r e s , IO ml in volume, p H 7-5, c o n t a i n e d cells f r o m io ml of c u l t u r e a n d t h e following c o m p o n e n t s (#moles): Tris, 5oo; glucose, IOO; MgSO 4, 5o; cysteine, 50; DL-[2-'4CI leucine, io; a n d 12.5 of each of t h e L-isomers of isoleucine, histidine, tysine, a s p a r t i c acid, asp a r a g i n e , g l u t a m i c acid, o r n i t h i n e a n d p h e n y l a l a n i n e . T h e s y m b o l s refer to: O - C ) , control; [7-[S], p u r o m y c i n , i o o / t g / m l ; ~ - ~ , c h l o r a m p h e n i c o l , 5oftg/ml; a n d O - Q , tetracycline, 5 ° #g/inl. T h e a d d i t i o n of t h e El~C]leucine (zero t i m e ) was preceded b y a i o - m i n i n c u b a t i o n period. Fig. 2. B a c i t r a c i n s y n t h e s i s in t h e presence of p u r o m y c i n , c h l o r a m p h e n i c o l a n d tetracycline. T h e e x p e r i m e n t a l c o n d i t i o n s were identical to t h o s e g i v e n for Fig. i.

Biochim. Biophys. ~4cl~t, 91 (1964) 533 ~ 536

SHORT COMMUNICATIONS

535

The three antibiotics, chloramphenicol, puromycin, and tetracycline, inhibit protein synthesis without appreciably affecting the formation of bacitracin. In Fig. I is shown the effect of these antibiotics on the incorporation of [14C]leucine into protein. At the levels of antibiotics used, there is practically a complete inhibition of leucine incorporation. On the other hand, the same concentrations of antibiotics are essentially without effect on the formation of bacitracin as is shown in Fig. 2. The possibility that the appearance of antibiotic activity during the incubation period was the result of a conversion of a po]ypeptide precursor into the active bacitracin is unlikely. It has been previously shown that the formation of bacitracin involves the incorporation of the constituent amino acids into the polypeptide8, 9. The formation of bacitracin by protoplasts of B. licheni/orrnis can be markedly inhibited by D-phenylalaninet When this amino acid is added to growing cultures of B. licheniformis which are producing bacitracin, complete inhibition of antibiotic 3.0-

-8.0

-6.0

>_ 2.ou z m n~

-4.0 tn

__

o

1.0-

D

<1

-2.0

o-

<1

-o

2'0

4'0

6'0

8'0

,60

,ao

,40

,60

M INUTES

Fig. 3. Effect of D-phenylalanine on growth of B. licheni/ormis and bacitracin formation. The incubation mixtures contained io ml of i6-h culture medium to which was added IOO/*moles of D-phenylalanine ( 0 - 0 and A - A ) . Growth, ( 3 - © and 0 - 0 ; increase in bacitracin,/X-A and A - A.

formation occurs without any marked effect on the growth of the organism. These results are shown in Fig. 3. The increase in the growth of the organism during the incubation period can be taken as an indication that the D-phenylalanine has little effect on protein synthesis. At the present time the site of D-phenylalanine inhibition on bacitracin synthesis is not known. However, since the inhibition is overcome by L-phenylalanine 9, it is reasonable to assume that bacitracin synthesis involves the utilization of L-phenylalanine and that the D-isomer interferes with this process. If this assumption is correct, then the inhibition of bacitracin synthesis by D-phenylalanine and its failure to inhibit protein synthesis (utilization of L-phenylalanine) would provide additional evidence that the pathways of protein and bacitracin synthesis are different. At the present time the details of polypeptide antibiotic synthesis are still to be Biochim. Biophys. Acta, 91 (1964) 533-536

536

BIOCHIMICA ET BIOPHYSICA ACTA

elucidated. However, it would appear likely that the assembling of amino acids is a step-wise process involving the participation of a series of enzymes.

Department o/Biological Chemistry, School o/Medicine, University o/ Cali/ornia, Los Angeles, Cali/. (U.S.A.)

NEAL CORNELL JOHN E. SNOKE

B. YARMOLINSKY AND G. L. DE LA HABA, Biochim. Biophys. Acla, 49 (1961) 451. RENDI AND S. OCHOA, J. Biol. Chem., 237 (1962) 3711. MACH, E. ]~EICH AND E. L. TATUM, Proc. Natl. Acad. Sci. U.S., 5 ° (1963) 175. S. EIKHOM, J. JONSEN, S. LALAND AND T. REFSVIK, Biochim. Biophys. Aeta, 76 (1963) 465 . HENDLIN, Arch. Biochem., 24 (1949) 435. W. BERNLOHR ANn G. J~. NOVELLI, Arch. Biochem. Biophys., 87 (196o) 232. D. DARKER, H. t3. BROWN, A. H. FREE, B. BIRO AND J. T. GOORLEY, J..4~1. Pharm. Assoc. Sci. Ed., 37 (1948 ) 1568 J. E. SNOKE, J. Bacteriol., 80 (196o) 5521 2 3 4

M. R. B. T. D. I~. 7 G.

9 j . E. SNOKE, J. Bacteriol., 81 (I96I) 986.

Received July 29th, 1964 Biochim. Biophys. Acta, 91 (1964) 533-536

Preliminory Notes PN 91035

The effect of heporin on the titer of the infectious nucleic ocid from the lactic dehydrogenase agent Recently it was shown that incubation of infectious nucleic acid 1,2 from the lactic dehydrogenase agent with ribonuclease (EC 2.7.7.16) or normal plasma resulted in complete loss of infectivity. In the course of these experiments it was observed that extraction of crystalline ribonuclease with phenol led to the elimination of the ribonuclease activity from the aqueous phase, whereas extraction of normal mouse plasma with phenol resulted in only partial loss of its nucleic acid inactivating capacity. Since the nucleic acid inactivating capacity of plasma was thought to be due to ribonuclease, the failure of phenol to completely eliminate this activity suggested that a factor in addition to ribonuclease might be present 2. The present study was undertaken to investigate the nature of this factor. The term infectious nucleic acid as used throughout this paper refers to an infectious moiety which is completely inactivated by IO/~g/ml of ribonuclease at room temperature for 15 min. Infectious nucleic acid was prepared from the lactic dehydrogenase agent by extraction with ether 2 and was assayed 1,3 by intracerebral injection into 3-6-week-old CAF-I male mice. Phenol extraction of the various reagents was carried out as described previously 2. Blood was collected either in 0.5 mg/ml of heparin (Hynson, Westcott, and Dunning) or in 1.5 mg/ml of E D T A (Fischer). Serum and plasma were diluted I in 3 in 0.02 M sodium phosphate buffer (pH 7.0) prior to phenol treatment. The nucleic acid inactivating capacity of each reagent before and after treatment with phenol was evaluated by incubation with infectious nucleic acid. As seen in Table I, heparinized plasma completely inactivated the infectious nucleic acid prepared from the lactic dehydrogenase agent. Since previous exBiochim. Biophys. Acla, 91 (1964) 536-538