Tech,. Bull. Fac. Agr.. Kagawa Univ., Vol.. 37, No, 1,17-20, 1985
C E L L U L A R P R O T E I N S A R E L I T T L E DEGRADED DURING A U T O L Y S I S
O F
ESCHERICHIA COLI AND STREPTOMYCES GRISEOLUS
Masayuki S A T O , Yoshihiro O ~ A D A and Kazumi
TAOKA
Escherichia coli
2
Streptomiyces griseolus
0
;5
-
1)9
:/
I+I:,
Cells of Escherichza colz and Streptomyces grzseolus autolysed by the incubation in 0.1 M sodium phosphate buffer (pH 7 0) a t 2 7 "C It was tested by the electrophoresis methods whether the cellular proteins were modified during the autolysis The results were shown that the cellular p r o t e i n s of t h e s e organisms' were little degraded during the autolysis. lntracellular proteinase activity of
E.
coli was remarkably decreased during the autolys~sEscherzchza colz 2 Streptomyces grzseolw
0k;BRbikO
1MI) >@i%Q%j%@ (pH 7.0) @, 27°C Dt
24
+
~ 4t - t d 2 , - ;5- b I) 2 2 L f : o+a,$-
i. II . / X + l : Z h G O I B l $ 9 / / f 7 A f l + # $ ; ( h a $ y i h ' % , &XC?&@J?&.T%%$LI':. +0142%, r h ~ 0 @ 1 $ 9 ~ ~ f 7 ! R r ; s : c b ~ ~ r % - @ $ $ h . . d ~ ~ z ~ : f l k ) h ' ~ f : . $ 7 ; ~ .colia)El$& proteinase r&l%id: iS - ]I 2 2 + I - $ $ L
L
(Ef;
L f:,Introduction
In general, it is known that the bacterial autolysis results from the cell wall lysis by autolysins.il1 B u t it have been rarely marked whether the cellular macromolecules a r e degraded during the autolysis of bacteria L ~ t t l e is reported concerning autolysis of actinomycetes e x c e p t t h a t th: e e a u t o l y s i n s of Mycobacterium smegmatis were characterized by Kilburn e t a1 ( 2 ) In this study we investigated whether the cellular proteins of Escherzchza coli and Streptomyces grzseolus were degraded or not during their autolp sis
Materials and Methods
Organisms and cultivatioa Escherichia coli I F 0 3044 and Streptomyces griseolus I F 0 3415 were used in this study These organisms were grown in the culture medium containing 1 0 g of glucose, 1 0 g of polypeptone, 1 0 g of meat extract and 5 g of NaCl per liter of deionized water (pH 7..0) on a rotary shaker a t 27 "C ..
Autolysis Cells in the exponential growth phase were harvested by centrifugation and washed twice with cold 0 . 1 M (for E . . c o l i ) or 0.05 M (for S.. griseolus) sodium phosphate buffer (pH 7..0). T h e washed cells were resuspended in these buffer and incubated on a rotary shaker a t 27 "C for E. coli or on a monod shaker a t 30 'C for S. griseolus. These procedures were performed under pure culture conditions..
;Preparation of cell free extract. The incubated cells were harvested by centrifugation and disrupted by ultrasonic radiation in the fresh 0.05 M tris-HC1 buffer (pH 7.5) for 3 min a t 0 C . The cell free extract
18 Tech. Bull. Fac.. Agr. Kagawa Univ., Vol.. 37, No. 1, 1985
suspensions were centrifuged at about 10,000 X g for 1 0 mln The supernatant solutlon was used for the enzyme assay and the electrophoresls
Assay meihods P r o t e ~ n a s e a c t ~ v ~ t y was assayed by the methods described In the prevlous paper131 except for uslng the denaturated hemoglob~n solutlon adjusted at pH 7 0 w~thout T r ~ t o n X - 1 0 0 a s t h e
substrate Intr acellular and extr acellular nltr ogen compounds ipr otetns, peptldes and amlno a c ~ d s ) were determined by Lomry-Fohn methods4 w ~ t h bovlne serum album~n a s standard
SDS-polyacr ylamzde gel electrophores zs SDS-polyacrylamlde gel electrophoresls was carried out according to I aemm115 wlth a p o l y a c r ~ l a m ~ d e 112 5 % slab gel contalnlng 0 1 % sodlum dodecll sulfate These gels were stalned wlth 0 025 % Coomassle brllllant blue and destalned by dlffus~on In methanol-acetlc ac~d-water 150 75 875)
Results
1 4utolysls of E cola and S gr zseolus
As shown in F i g 1, about 50 % of initial absorbance in E colz cell suspension was decreased by
Incubation time (h) Incubation time (h)
Fig 1 Autolysis of Escherzchza colz I F 0 3044 Flg 2 Autolysis of Streptomyces grzseolus I F 0 in 0 1 M sodium phosphate buffer (pH 7 0) 3415 in 0 0 5 M sodium phosphate buffer
0,
Absorbance of cell suspension at 600 nm; A, (pH 7 0 )absorbance of extracellular solution a t 280 nm; @,
0,
Absorbance a t 600 nm; @, extracellular proteinase activity in cell f r e e extract nitrogen compoundautolysis after the incubation for 48 h And the intracellular proteinase activity was remarkably decreased during the incubation About 8 0 % decrease in turbidity of S griseolus cell suspension was observed after a 48 h of incubation a s shown in Fig 2 And about 8 4 % of the intracellular nitrogen compounds was excreted outside cells during the autolysis
M.
SATO, Y. OKADA and K. TAOKA : Cellular proteins during bacterial autolysis2. Cellular proteins during autolysis.
Figures 3 and 4 shonr the patterns of electrophoretic migration of cellular proteins in SDS-polyacrylamide
Fig. 3. SDS-polyacrylamide gel electrophoresis Fig. 4. SDS-polyacrylamide gel electrophoresis of intracellular proteins of Escherichia of cellular proteins of Streptomyces
coli. griseolus.
Amount of sample proteins is 40 big for No. 1, 2 A is initial cellular proteins. B and C a r e and 3, 1 2 0 pccg for No. 4, 5 and 6. Incubation time intracellular and extracellular proteins, respective- is zero 11 for No. 1 and 4. 24 h for No. 2 and 5, 72 ly, a f t e r incubation of cell f o r 48 h.
M
i s the h f o r No. 3 and 6. No. 7 is standrt~xl proteins (a, standards described under Fig. 3.phosphorylase; b, bovine serum albumin; c, ovalbumin; d, carbonic anhydrase; e, trypsin inhibitor; f, a-lactalbumin) purchased from Pharmacia F i n e Chemicals.
gels before and a f t e r the incubations of cell suspensions in the sodium phosphate buffer ( pH 7.0 ). The patterns of cellular proteins of E. coli and S. griseolus {vere rarely changed during the autolysis except that only a little of protein bands faded. In addition, ire estimated the amount of nitrogen compounds that filtered through the membrane filter (DAIAFLO Ubl 1 0 ) or \ r e r e soluble in 2.5 % trichroloacetic acid. The ratio of these low molecular nitrogen compounds to the totals changed negligibly \\.it11 the autolysis.
Discussion
In studies on bacterial autolysis, little have been reported concerning whether cellular proteins and nucleic acids a r e degraded o r not. Rlany investigations i r e r e focused on cell wall lysis during autolysis of E.colik6) S t r e ~ t o c o c c u s faecalisf7.8) and Bacillus sublillis(9) etc. The results in this study suggest that most of cellular proteins of E. c o l . and S. griseolus a r e little degraded during their autolysis. T h e s e intact proteins a r e probably releasecl from cells by cell mall lysis. In the autolysis of Saccharomyces cerevisiae and Aspergillus niger, cellular proteins seemed to be degraded and proteinase activity #increased a s reported in the previous paper.is.10) T h e behavior of cellular proteins during autolysis in procaryotic cells appears to he different from that in eucaryotic cells.
20
Tech. Bull.. Fac.. Agr. Kagawa Univ., Vol. 37, No. 1, 1985Acknowledgment
W e thank to Professor ShGjir6 Iwahara of Kagawa University for his valuable discussions and cncoura- gement
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