Absence of ribonuclease in Alcaligenes faecalis and a possible mechanism of RNA degradation in this bacterium

337

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 95553

ABSENCE OF R I B O N U C L E A S E IN A L C A L I G E N E S F A E C A L I S AND A P O S S I B L E MECHANISM OF RNA D E G R A D A T I O N IN T H I S BACTERIUM S H U N J[ N A T O R I , TADAO H O R I U C H I " AND D E N ' I C H I MIZUNO

Faculty o/ Pharmaceutical Sciences, University o[ Tokyo, Hongo, Tokyo (Japan) (Received May 24th, 1966)

SUMMARY

I. Alcaligenes/aecalis RN- 4 was shown to have no detectable intra- or extracellular ribonuclease I (EC 2.7.7.I6) activity. In this strain, ribosomal and messenger RNA's were degraded by polynucleotide phosphorylase (EC 2.7.7.8) and potassiumactivated phosphodiesterase (EC 3.I-4.I) in vitro as demonstrated by the detection of nucleoside diphosphates and 5'-nucleotides as degradation products. 2. Mitomycin C induced the degradation of the ribosomal RNA of A. /aecalis RN- 4 in the same way as in Escherichia coli which is known to have a ribonuclease I. 3. In view of these results, the possibility that the ribonuclease I in Escherichia coli does not actually participate in the degradation of ribosomal RNA is discussed.

INTRODUCTION

We previously reported a marked degradation of ribosomal RNA in Escherichia coli, when cells were in a phosphate 1- or magnesium2-deficient state. Mitomycin C and other drugs were also shown to induce the degradation of rRNA (refs. 3, 4). The enzymes which participate in this degradation of rRNA are not yet known, although ribonuclease I (EC 2.7.7.16) has been shown to be involved 5 in phosphate deficiency. WADE AND ROBINSON6 could detect no ribonuclease I in Pseudomonas [luorescens. GRONLUND AND CAMPBELL7 reported that Pseudornonas aeruginosa has no ribonuclease I activity, although degradation of rRNA was observed during endogenous respiration. He proposed that in this strain polynucleotide phosphorylase (EC 2.7.7.8 ) is responsible for the degradation. The present work shows that a strain of Alcaligenes/aecalis contains negligible ribonuclease I activity and that mitomycin C induces the degradation of rRNA in this strain. Hence, a study was made on the enzymes which degrade rRNA and m R N A in this strain. Abbreviations: NMP, N D P and N T P ; nucleoside mono-, di-, and triphosphates, repectively; r R N A , ribosomal RNA; m R N A , messenger RNA. * P r e s e n t address: F a u l t y of P h a r m a c e u t i c a l Sciences, University of K y u s h u , F u k u o k a (Japan).

Biochim. Biophys. Acta, 134 (x9(~7) 337-346

338

s. NATORI, T. HORIUCHI, D. MIZUNO

Recently, m u t a n t s of E. coli lacking ribonuclease I have been isolated s,9 a n d v a r i o u s p a p e r s using these m u t a n t s were publishedS, TM.

MATERIALS AND .~fETHODS

Bacteria and media E. coli, W 25 (adenine- or guanine-requiring) was a gift from Dr. J. TOMIZAWA, N a t i o n a l I n s t i t u t e of H e a l t h of J a p a n . A./aecalis RN- 4 was o b t a i n e d in our labora t o r y d u r i n g the isolation of a ribonuclease I-less m u t a n t a n d identified as A./aecalis b y Dr. R. NAKAYA, N a t i o n a l I n s t i t u t e of H e a l t h of J a p a n . A./aecalis was given b y Dr. S. TAI
Spheroplast /ormation S p h e r o p l a s t s were p r e p a r e d following the description of NEu .aND HEPPEL 11.

Preparation o/ the crude extract, cell sap and ribosomes i g r a m of cells was s u s p e n d e d in 25 ml of Tris-HC1 buffer, d i s r u p t e d b y sonic oscillation a n d centrifuged for 30 rain at 20 ooo ×g. The s u p e r n a t a n t was used as t h e crude e x t r a c t . I t was centrifuged for 9 ° rain at Io5 ooo × g to give a p r e c i p i t a t e (ribosomes) a n d a s u p e r n a t a n t (cell sap).

Preparation o~ ribonuclease and assay method Ribonuclease I was e x t r a c t e d from E. coli a n d A./aeealis following the m e t h o d of SPA.FIRAND HOLLINGWORTH 12. The e n z y m e a c t i v i t y was d e t e r m i n e d b y the a m o u n t of acid-soluble digestion p r o d u c t s formed from y e a s t R N A , as e s t i m a t e d b y t h e increase in a b s o r b a n c e at 260 m/~. I ml of i n c u b a t i o n m i x t u r e c o n t a i n e d 2.5 m g of R N A , ioo/~moles Tris-HC1 (pH 8.I) a n d e n z y m e solution. The a s s a y m i x t u r e was i n c u b a t e d at 37 ° for 4 ° rain a n d t h e n o.25 ml of 25 o/,o perchloric acid (containing 0.75 o,/ ,,o of u r a n y l a c e t a t e ) was added. One e n z y m e unit was defined as t h a t a m o u n t of e n z y m e which caused an increase of I in the a b s o r b a n c e at 260 mff u n d e r the a b o v e a s s a y conditions.

Separation o~ N M P , N D P and N I ' P and other methods NMP, N D P a n d N T P were s e p a r a t e d b y tile m e t h o d of COliN AXD CAterER 13. P r e p a r a t i o n of pulse-labelled R N A a n d f r a c t i o n a t i o n b y column c h r o m a t o g r a p h y on D o w e x I a n d p a p e r c h r o m a t o g r a p h y were carried out essentially as described previo u s l y 14. Biochim. Biopl~ys. _4cta, 134 (1967) 337 346

R N A DEGRADATION IN A. /aecalis

339

RESULTS

Ribonuclease activity o/ A. /aecalis RN- 4 A./aecalis R N - 4 was grown on n u t r i e n t a g a r c o n t a i n i n g 0.5 O//oR N A . Colonies were w a s h e d w i t h w a t e r a n d t h e n 25 O//operchlorie acid was s p r a y e d on the surface of t h e plates. I t became t u r b i d when R N A was unchanged, while b a c t e r i a l colonies p r o d u c i n g ribonuclease I such as E. coli W 25 gave clear spots. The crude e x t r a c t of A. /aecalis R N - 4 was shown to have no d e t e c t a b l e ribonuclease I a c t i v i t y . T r e a t m e n t for a c t i v a t i o n a n d purification of possibly l a t e n t ribonuclease I in A./aecalis R N - 4 was carried out according to the SPAHR AND HOLLINGWORTH12 m e t h o d . The crude e x t r a c t was t r e a t e d with 4 M u r e a a n d o.oi M NaC1, d i a l y z e d a g a i n s t w a t e r a n d a d j u s t e d to p H 4.5 with a c e t a t e buffer. A p r e c i p i t a t e which f o r m e d was r e m o v e d b y c e n t r i f u g a t i o n a n d the clear s u p e r n a t a n t was fract i o n a t e d w i t h a m m o n i u m sulfate at p H 4.5. The m a t e r i a l p r e c i p i t a t i n g b e t w e e n 0. 4 to 0.8 O/,o s a t u r a t i o n was dissolved in w a t e r a n d dialyzed. N e i t h e r this solution nor a n y o t h e r fractions h a d a n y d e t e c t a b l e ribonuclease I a c t i v i t y . NEU A N D HEPPEL 11 r e p o r t e d a m a r k e d release of ribonuclease I into the m e d i u m when E. coli was t r e a t e d w i t h E D T A a n d l y s o z y m e (EC 3.2.1.17). W h e n A. /aecalis R N - 4 was t r e a t e d w i t h E D T A a n d l y s o z y m e , no ribonuclease I a c t i v i t y was released into the m e d i u m . Therefore, it was concluded t h a t A./aecalis R N - 4 h a d no d e t e c t a b l e intra-, or e x t r a c e l l u l a r ribonuclease I (Table I). A n o t h e r stock s t r a i n of A. /aecalis given b y Dr. S. T A K A H A S H I , w a s also e x a m i n e d a n d the same r e s u l t s were o b t a i n e d as w i t h A. /aecalis R N - 4. TABLE

1

COMPARISON

OF

RIBONUCLGASE

ACTIVITIES

IN

E . coli

AND

.zl.

[aecalis

I g ( w e t w e i g h t ) o f c e l l s w a s c o n v e r t e d t o s p b e r o p l a s t s f o l l o w i n g N E U AND H E P P E L ' S m e t h o d n and ribonuclease 1 activities were compared. Alternatively, i g of cells was disrupted by sonic oscillation and the ribonuclease I was extracted as described in the text and the activities of the two strains were compared.

Spheroplast medium Crude extract

E. coli W-2 5 (units per g cell)

A. [aecalis R N - 4 (units per g cell)

355 --

o o

-414

o 1. 4

(NH4)~SO 4 [ractionalion 0-0. 4 saturation 0. 4 o . 8 s a t u r a t i o n

Stability o/ribosomes R i b o s o m e s were p r e p a r e d from cells in t h e s t a t i o n a r y phase, w a s h e d once w i t h saline, r e s u s p e n d e d in Tris-HC1 buffer (o.I M Tris, o . o o i M MgC12, p H 7-4), a n d dial y z e d a g a i n s t the same buffer at 4 ° for IO h. The r i b o s o m a l suspension t h u s o b t a i n e d was i n c u b a t e d at 37 ° with 2 mM E D T A in order to t e s t the s t a b i l i t y of t h e ribosomes. The increase in the cold perchloric acid-soluble a n d u l t r a v i o l e t - a b s o r b i n g m a t e r i a l released d u r i n g t h e i n c u b a t i o n p e r i o d was m e a s u r e d ( F i g . l ) . R i b o s o m e s of E. coli were v e r y u n s t a b l e a n d were a l m o s t Biochim. Biophys. Acta, I 3 4 ( I 9 6 7 ) 3 3 7 - 3 4 6

34 °

S. NATORI, T. HORIUCHI, D. MIZUNO

completely broken down d u r i n g a 3o-min i n c u b a t i o n period, while less t h a n IO 0o of those of A . /aecalis RN-4 were destroyed on i n c u b a t i o n for 6o min. This result shows t h a t the ribosomes of A . / a e c a l i s RN- 4 are very stable even in the presence of E D T A .

10C

~

E. coli

9C

100 9O

8C '-,~

80 5 •.~ 70

7C

~6C

o ~ 60

5c

.

50

4c

g S 40

~_ @ 3c

c

a_ 2C

~ 30 $ A. foecGis

1C

0,__,----¢---r~70

10 20 Incubation

30

n

, 40

50

. o0

time (min)

20

~o ~ ~ j . ~ 0

4

5

6

pH

7

/

/

8

9

i. S t a b i l i t y of ribosomes o f .4. ]aecalis R N - 4. R i b o s o m e suspensions f r o m .4./aecc*lis and 1,2. coli were i n c u b a t e d w i t h 2 m M E D T A in T r i s H C ! (o.1 M Tris, o. 3 n l M MgC12, p H 8.1) a t 37 °. Fig.

I nil of incubation mixture contained 0. 3 mg of ribosomes as rRNA. © .... C), F.. coli \V 25; • - - - • , A. /aecalis RN 4Fig. 2. The autodegradation of ribosomes in crude extracts of E. coli W-25(O) and .4. /aecalis RN- 4 ( • ) . Crude extracts were incubated at 37° in the presence of to nlM EDTA. Samples were taken after ~o rain (E. coli) and 90 rain (d. [aecalis).

Degradation o/ ribosomal R N A in the presence o/ cell supernatant, E D T A , KCI, N a ~ H P O 4 and K H 2 P O 4 A l t h o u g h the ribosomal fraction of A. /aecalis RN-4 had no ribonuclease I activity, this enzyme might have been present in some other fractions of the cell. To test this possibility, crude extracts were prepared from cells in the logarithmic phase of growth a n d the percentage degradation of R N A in the presence of IO mM E D T A was d e t e r m i n e d at various p H values (Fig. 2). The ribosomes of E. coli were b r o k e n down m a r k e d l y at pH 7 in the presence of IO mM E D T A , while those of A. /aecalis RN- 4 were not, b u t the percentage d e g r a d a t i o n of the latter increased g r a d u a l l y with increase in p H value. To test which enzymes p a r t i c i p a t e d in the degradation of the ribosomes of A . / a e c a l i s RN-4, various substances were added to the i n c u b a t i o n m i x t u r e a n d their effects on the d e g r a d a t i o n of the ribosomes were examined. Cells were labelled with 32p for three generations a n d t h e n resuspended a n d i n c u b a t e d for I h in a m e d i u m not c o n t a i n i n g isotope. A ribosomal suspension was prepared from these cells. The influences of E D T A , KC1, Na2HPO 4 and KH2PO 4 on the d e g r a d a t i o n of A. /aecalis RN- 4 ribosomes was followed b y i n c u b a t i o n of ribosome suspensions in Tris HC1 (pH 8.i) c o n t a i n i n g these substances a n d b y measurem e n t of the cold perchloric acid-precipitable r a d i o a c t i v i t y (Fig. 3). Biochi~. l~ioph~,s..,Icla, 134 (~9~7) 337 346

RNA

DEGRADATION IN

A. /aecalis

341

100 b

100- o 90-

90-

KH2PO4

80-

80-

70-

/ /

70-

Kcl

60-

g

i

50

50-

40-

40-

30-

30,

n~ 20.

20.

1

0

.

0,

o

10

~ ,

,

1

2

0

~

Incubation time (h)

Incubation time (h)

Fig. 3. D e g r a d a t i o n of ribosomes on addition of Na=HPO 4, IKH2PO4, E D T A and NC1. Labelled ribosome suspensions p r e p a r e d from A. [aecalis RN- 4 were incubated in the presence of Na,HPO4, C)-(2); KH2PO~, (1 (1; E D T A , ® ®; KC1, & - • and w i t h o u t a n y addition O-O. F o r (b) b u t n o t (a) 2.8 mg protein of the lO 5 ooo X g s u p e r n a t a n t were added t o i ml of incubation mixture. C o m p o n e n t s were added at concentrations of 50 raM, except for E D T A which was added at a c o n c e n t r a t i o n of 2 mM. After incubation, cold perchloric acidprecipitable radioactivity was counted.

When KC1, Na2HP Q and KH=POa were added to the incubation mixture, a marked degradation of ribosomes was observed. The degradation of the ribosomes resulting from addition of Na2HP Q was greatly increased when cell sap was added. These results suggest that the enzyme responsible for degradation of ribosomes in the presence of orthophosphate is polynucleotide phosphorylase and that in the presence of potassium it is potassium-activated phosphodiesterase (EC 3-1.4. I), as has been shown in E. coli15,16 and Lactobacillus caseW. To confirm the participation of the above enzymes, the degradation products were analyzed by column chromatography on Dowex-i (HCOO-) (Fig. 4a). The majority of the radioactivity in the degradation products was found in the NMP and N D P fractions when ribosomes were incubated with Na2HP Q and cell sap. In this case, the ratio of NMP to N D P increased with time (Table II).

TABLE II CHANGE

IN

THE

RATIO

OF

N M P TO N D P

DURING

INCUBATION

Labelled ribosomes were incubated with Na2HPO 4 and cell sap. After 9o min and I5 o min incubation, acid soluble degradation p r o d u c t s were analyzed b y D o w e x i c o l u m n c h r o m a t o g r a p h y . The ratio of N M P to N D P was calculated from the radioactivities of the fractions. Incubation time

UMP/UDP CMP/CDI' GMP/GDP

9 ° min

I5o min

1.5 2. 5 I.O

4-3 8.0 2.6 t?iochim. Biophys. Acta, 134 (1967) 337-34(>

342

S. NATORI, T. HORIUCHI, D. MIZUNO

1.1 1.0 0.9 -0.8 o 0.7 0.6 05

CMP

122 42O 18 16 ×

a

IMp

D-

Q.4i ~0.3 ~2 o.~

10 c GDP 8 &-. ,.~

ADP

cDP G~P u~P ~

:~i 6

0

< o 1.C O.C 0.~ o o.;

2'o

,

,

,

,

,

40 60 80 100 120 160 Tube number (12ml fraction)

2O 18 16 0

CMP I'

0

180

AMP /tUMP

×

12

o.e

'

8 o.~ g o.~ 0.G C

CDP

~

A

I

GfMP

10 r-

6

o ll

C0

20

o 40 60 80 100 120 140 160 Tube number (12ml fraction)

5

8

Fig. 4. C h r o m a t o g r a p h i c separation of acid-soluble degradation product. Labelled ribosomes were incubated in the presence of 5 ° mM Na=HPO~ and cell sap (a) or NC1 (b) at 37 ° . After 9o rain incubation, the 5 ~{) cold perchloric acid-soluble fractions were analyzed b y column c h r o m a t o g r a p h y on D o w e x i and c o m p a r e d w i t h a u t h e n t i c samples. • • , absorbance at e6o mff; C ) - - - - © , c o u n t s / m i n per ml.

(a)

C:)

(b) 2~ 3'

o

C)

cycl i c AMP

C

CD C

I 5CAMI

2"or, 3t AMP

2 ~,3' cyclic AMP

O

2'or 3 ~ AMP

(2

ISampl

C

1500 1200

E

E

900 -~ 600 o

5~-AMp

Sample 500 -400

c

- 3 0 0 - ~E. 200

300 U

Fig. 5. Identification of the position of o r t h o p h o s p h a t e in AMP. The AMP fraction was collected by a d s o r p t i o n on Norit SX-3o , followed b y elution with 5 ° o/o a m n l o n i u m ethanol (pH II.5) and evaporation. P a p e r c h r o m a t o g r a p h y was carried o u t w i t h the buffer s y s t e m described b y PLESNER 'z°. (a) is the AMP fraction from Fig. 4 a, (b) is from Fig. 4 b.

Biochim. Biophys..4eta, 134 (1907) 337 346

RNA DEGRADATIONIN A. /aecalis

343

It can be concluded that in this case RNA was degraded to N D P by polynucleotide phosphorylase and then further converted to NMP. On the other hand, the only degradation product was NMP on incubation in the presence of KC1, as shown in Fig. 4b. The AMP fraction was concentrated and analyzed by paper chromatography to examine the position of the orthophosphate (Fig. 5). The only product was 5'AMP and no traces of 2'3'-AMP or 2'3'-cyclic AMP were found in either case. These results show that orthophosphate stimulates the activity of polynucleotide phosphorylase while KC1 stimulates that of potassium-activated phosphodiesterase. The percentage degradation was greatest when KH2PO 4 was added. Therefore in this case both the above enzymes participate in the degradation of ribosomes. When EDTA was added there was a slight increase in the degradation of the ribosomes, presumably because the ribosomes became susceptible to polynucleotide phosphorylase or phosphodiesterase in the presence of EDTA. Adenylate kinase (EC 2.7.4.3) and cytidylate kinase (EC 2.7.4.3 type) activities were detected in the crude extract of A. /aecalis RN-4, and presumably these enzymes participate partly in the conversion of NDP to NMP (Table III). TABLE

111

ADENYLATE-KINASE AND CYTIDYLATE-KINASE ACTIVITIES IN A CRUDE EXTRACT OF A./aecalis R N - 4 i m l of i n c u b a t i o n m i x t u r e ( o . i M T r i s - H C l , i m M MgCI~, p H 8.1) c o n t a i n i n g 0. 5 # m o l e s of A D P o r C D I " a n d c r u d e e x t r a c t (0.9 m g p r o t e i n ) w a s i n c u b a t e d a t 37 ° f o r 45 m i n . T h e a c i d s o l u b l e f r a c t i o n w a s a n a l y z e d f o l l o w i n g t h e m e t h o d o f COHN A N D C A R T E R 13, a n d t h e r a t i o s o f t h e f r a c t i o n s as determined by distribution percentage were calculated from their absorbances at 260 m#. o

time

(%)

45-rain incubation

(%)

AMP ADP ATP

I 1.9 84"3 3.8

38.3 44.4 17. 3

CMP CDP CTP

1 I.O 89.o o

35.8 43.8 20. 4

Degradation o~ the mRNA of A. /aecalis RN-4 in vitro Cells were grown in Medium B. At the logarithmic phase of growth I14C]uracil or [a4C]adenine was added for 4 ° sec to label the mRNA, and the culture was rapidly chilled in an ice bath. The cells were collected by centrifugation, washed once with saline, suspended ill Tris-HC1 (o.I M Tris, o.oi M MgC12, pH 7.4), and disrupted by sonic oscillation. The ribosome pellet was obtained as described previously and resuspended in Tris-HC1 (o.I M Tris, 0.005 M MgCI~, pH 7.4). It was dialyzed against the same buffer at 4 ° for 12 h. This 14C-labelled ribosome suspension represents 14C-labelled mRNA. To obtain uniformly labelled ribosomes, a ribosome suspension was prepared from cells labelled with [14C]uracil or E14C]adenine for 4 h and then resuspended and incubated for I h in a medium not containing isotope. These two kinds of ribosome suspension were incubated in Tris-HC1 (o.I M Tris, 0.005 M MgC12, pH 7.4) at 37 ° for 60 min. Na2HPO 4 and KC1 were added to the t3iochim. Biophys. Acta, 134 (1967) 3 3 7 3 4 6

344

S. NATORI, T. HORIUCHI, D. MIZUNO

TABLE

1V

DEGRADATION

OF

m R N A AND r R N A I N L ¢. cell A N D A . /aecalis RN- 4 in vitro

The degradation of pulse labelled R N A in vitro in E. coli W 25 and A./aecalis RN- 4 was examined. Pulse-labelled ribosomes were incubated under conditions where m R N A was degraded selectively. I ml of i n c u b a t i o n m i x t u r e (o.1 M Tris HC1, 5 mM MgC12, p H 7-4) contained 0. 3 mg r i b o s o m e as ribosomal R N A and 5 ° m M of each c o m p o n e n t , and cell sap was added to a concentration of 0.9 mg protein per ml.

Componenl added

N o addition

Na2HPO 4 KC1 Cell sap Cell s a p - - Na2HPO ~ Cell sap + KC1

E. coli

.4. /aecalis R N - 4

mRNA rRNA (degradation °o)

mRNA rRA:A (degradation ~!'£})

48.2 71.0 0o. 2 57.0 72.8 06.2

28. 5 03.4 4 o. 0 35.2 58.0 43.0

o 13-4 13. I o 18.6 14.(}

4.9 14.3 14..5 o 16.8 2

incubation mixture at concentrations of 50 mM. The results are shown in Table IV. More than 6o o~ of the m R N A was degraded under these conditions while the r R N A was not degraded so much. The same result was obtained with both E. coli and A. /aecalis. In these experiments in vitro, the participation of ribonuclease I in the degradation of m R N A is negligible.

Degradation o/ RNA in vitro in the presence o~ mitomycin C Mitomycin C is known to induce the degradation of rRNA in E. celia, ~8. The m e c h a n i s m of this r R N A degradation is not yet known, but ribonuclease I should be 5000

3.7

iiio°

3.6

3000 I-/-

1.0

Q

3.9 4000 ~

.

oooT

3.8

.j

1.0 0.9 0.8 O.7 ~" 3.6 0

:).5 0.4

9.5 © 200(

3.4

0.3 02

0.3 fl 100(

0.2

0.1

0

1

2

3

Incubation time (h)

<

0.1 I

0

I

I

I

I

I

I 2 3 Incubation time (h)

Fig. 6. Changes in radioactivity of R N A and D N A after addition of m i t o m y c i n C. Cells labelled with 3zp were resuspended in cold medium. After 20 min incubation 5 / , g / m l of m i t o m y c i n C were added. Aliquots of 3 ml were taken at intervals and fractionated by the GCHMIDT-THANNHAUSER m e t h o d 19, and the radioactivity of each fraction w a s counted. (a) treated with mitomycin C at o time, (b) control. O O, RNA; ~) ®, DNA; (D - - ~ , acid-soluble; • • , g r o w t h (A650 m#)-

Biochim. B i o p h y s . . 4 c t a ,

134 (1967) 337 346

RNA DEGRADATIONIN A. /aecalis

345

essential for degradation of the ribosomes. Therefore, tests were made in order to determine whether or not mitomycin C induced the degradation of RNA in RN- 4 which has no ribonuclesae I activity. Cells were incubated in 6o ml of medium A .containing 5o #C of 32p for 4 h. Then the cells were washed once with buffered saline and resuspended in the same medium without isotope. After 2o min incubation mitomycin C was added at a concentration of 5 ~g per ml. Aliquots of 3 ml were pipetted out at intervals and fractionated by the SCHMIDT-THANNHAUSERmethod 19. Labelled cells were examined serially for 3 h in a medium containing mitomycin C and the radioactivities of the RNA and DNA fractions were found to decrease markedly (Fig. 6). This phenomenon was essentially the same as with E. coli treated with mitomycin C (ref. 3)-

DISCUSSION

A./aecalis RN-4 was shown to have no detectable ribonuclease I activity. This is similar to the result with P. aeruginosa. It was of interest to examine whether or not ribonuclease I is in general essential for the degradation. Therefore the enzymes participating in the degradation of rRNA in this strain were studied. The ribosomes of A./aecalis RN- 4 are very stable and are not degraded even in the presence of EDTA. However, when Na2HPO 4 and KC1 were added, a marked stimulation of degradation of ribosomes was observed. This is because, in place of ribonuclease I, polynucleotide phosphorylase and potassium-activated phosphodiesterase participate in the degradation of ribosomes in this strain. The ratio of NMP to N D P increased with time when ribosomes were incubated in the presence of Na2HPO ~ and cell sap. This result suggests that the following sequence operates in this case. RNA~NDP~NMP Adenylate kinase and cytidylate kinase activities were detected in A. /aecalis RN- 4 (Fig. 7), and so these enzymes presumably participate to some extent in the conversion of NDP to NMP. Thus, it is clear that in this strain mRNA and rRNA are broken down by enzymes other than ribonuelease I. When E. coli and A./aecalis were subjected to mitomycin C, the same extent of degradation of the rRNA was observed. Since one of them, namely A. /aecalis RN-4, lacks ribonuclease I, the question as to whether or not ribonuclease I is really responsible for the degradation of ribosomal RNA in E. coli is unsolved. However, evidence has been presented that ribonuclease I was involved in the degradation of rRNA in cells in the phosphate-deficient state 5. GRONLUND AND CAMPBELL 7 reported that the degradation of rRNA in P. aeruginosa was induced by polynucleotide phosphorylase, but in the case of A. /aecalis evidence was given that both polynucleotide phosphorylase and potassiumactivated phosphodiesterase were involved. It is probable that polynucleotide phosphorylase and potassium-activated phosphodiesterase are the enzymes which participate most actively in the degradation of RNA's in A./aecalis RN-4.

Biochim. Biophys..4c/a, 134 (1967) 337-346

346

S. NATORI, T. H O R I U C H I ,

D. MIZUNO

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