Molec. gen. Genet. 180, 357 360 (1980) © by Springer-Verlag 1980
Derepression of Alkaline Phosphatase in Hfr of Escherichia coli During Conjugation P.N. Bhavsar and O. Siddiqi Molecular Biology Unit, Tata Institute of F u n d a m e n t a l Research, Bombay 400 005, India
Summary. In a mating mixture of Hfr and F- bacteria the gene for alkaline phosphatase undergoes a transient derepression at the time of transfer. It is shown that this escape from repression occurs in the donor cells and is probably connected with the synchronous duplication of the transferred genome.
Introduction
The alkaline phosphatase of Esc.herichia coli is repressed by inorganic phosphate (Horiuchi et al. 1959; Torriani 1960). The enzyme is a dimer composed of two identical subunits (Rothman and Byrne 1963). A single structural gene phoA specifies the polypeptide (Garen 1960), while at least four cistrons phoR, phoB, phoS, and phoT located in two distinct regions of the chromosome R1 and R2 regulate repression (Echols et al. 1961; Garen and Otsuji 1964; Aono and Otsuji 1968; Yagil et al. 1970; Willsky et al. 1973; Bracha and Yagil 1973). The exact role played by these genes in the synthesis of phosphatase is not yet fully understood (Schlesinger and Olsen 1968; Wilkins 1972; Morris et al. 1974; B rickman and Beckwith 1975). While examining the expression of the structural gene for phosphatase after its transfer by conjugation to a constitutive recipient, Joshi and Siddiqi (1968) found that phosphatase activity in the mating mixture increases in an unusual manner. A small rise in phosphatase activity at the time of transfer of phoA gene is followed by a prolonged quiescence of about 30 min after which extensive enzyme synthesis takes place. Joshi and Siddiqi ascribed the early burst to transient expression of the transferred phoA gene and the delayed synthesis to phoA + Offprint requests to. P.N. Bhavsar
recombinants. Subsequently, when similar experiments were carried out with repressible recipients in phosphate-rich broth, it was found that, although the delayed extensive expression ofphoA + gene was abolished, the transient early synthesis persisted. Since both donors and recipients in these matings were repressible, we were led to investigate the possibility that the transient synthesis of phosphatase is, in fact, due to a temporary derepression of the donors engaged in mating. In this paper we present evidence to show that this is indeed so.
Materials and Methods Strains. The genotypes of the E. coli strains used are listed in
Table 1. Media. L-broth was used for suspension cultures and mating. The
Tris minimal m e d i u m contained 0.2% glucose as a sole source of carbon and 1.25 x 10-3 M K H z P O ~. For the selection of phoA + recombinants, glucose in the Tris m e d i u m was replaced with 1.1 x 10-2 M Na-/~-glycerophosphate (Joshi and Siddiqi 1968). Suspension Buffer. For all suspensions and for making sucrose
gradients 0.01 M Tris (hydroxymethyl) amino methane-HC1 buffer pH 8.0 containing 0.001 M MgSO4 was used. Conjugation. Overnight cultures of donors and recipients were diluted and regrown at 37 ° C in L-broth. In a 500-ml flask 3 ml of
Table 1. Escheria coIi strains used Strain
Genotype
K10 K10 met FK10W1 CR34 FU56 NS65 P678-54
HfrC str ~ HfrC met str r F FF F F-
thrleustr r thr leu thy s t / thr l e u s t / p h o A R2 lacprostr~ thr leu lac gal x y l mal mini-cell former
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358
P.N. Bhavsar and O. Siddiqi: Derepression of Alkaline Phosphatase in E. coli
4 x 10S/ml Hfr cells were mixed with 7 ml of 8 x 108/ml F - . Five minutes later 5 ml of the mixture was added to 95 ml of prewarmed L-broth in a one-litre flask and swirled gently in a gyrotory water bath. To prevent multiplication of donors 100 pg/ml streptomycin was added at 30 min. Samples for assaying enzyme and recombinants were removed at suitable intervals. A mixture of 1 ml mating mixture and 3 ml chilled saline was sheared in a Waring blender. The cells were sedimented, washed and suspended in 1 ml of the suspension buffer and plated for phoA + recombinants.
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Isolation of Mini Cells. Mini cells were isolated from the strain P678-54 by differential centrifugation from broth-grown cultures. A litre of exponentially growing culture was centrifuged at 1,000 x g for 10 rain to remove large cells. The supernatant was removed and spun at 10,000xg for 15min. The cells were resuspended in 20 ml of 0.01 M Tris-HC1 buffer containing 0.001 M MgSO4 and centrifugation at 1,000 x g was repeated. The supernatant containing small cells was then spun in a sucrose gradient. Of the suspension 1 ml was layered over 15 ml of a linear 5%-20% gradient of sucrose in buffer and centrifuged in a fixed angle rotor at 1,000 x g for 35 min. The mini cells formed a broad band in the middle of the tube and were removed with a sterile Pasteur pipette. The collected cells were suspended in L-broth and were used immediately for mating. The proportion of contaminating normal cells was about 1%.
Enzyme Assay. In 5 ml of 0.01 M Tris-HC1 buffer containing 0.001 M MgSO4 and 100 gg/ml of chloramphenicol, 5 ml aliquots of mating mixture were chilled. The cells were harvested and were resuspended in 1 ml of the same buffer. A drop of toluene was added and the sample shaken vigorously. Of the toluenised cell suspension 0.5 ml was added to 1.5 ml of 4 mg/ml p-nitrophenyl phosphate in 1 M Tis-HC1 buffer pH 8 and incubated in a water bath at 32° C. After sufficient colour had developed, the reaction was stopped with 2 ml of chilled 1 M KzHPO¢ and optical density at 410 nm was measured in a 1-cm cell. The phosphatase activity is expressed as rise in OD 410 h 1/ml of the suspension. This has been corrected for colour in control blanks and absorption at 550 nm by toluenised cells.
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Fig. 1. Increase in alkaline phosphatase activity in a mating mixture of Hfr K10 phoA + and F - phoA- R~ (FU56). • Phosphatase activity per 5 mI mating mixture; o phoA + recombinants per ml of the mating mixture
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T h e t w o - s t a g e i n c r e a s e in p h o s p h a t a s e a c t i v i t y in a mating mixture of phoA + repressible HfrC and an R2 phoA F - , d e s c r i b e d b y J o s h i a n d S i d d i q i ( 1 9 6 8 ) is s h o w n in Fig. 1. A t a b o u t 8 m i n , t h e t i m e w h e n t h e phoA ÷ g e n e o f H f r C is t r a n s f e r r e d , a s m a l l rise in p h o s p h a t a s e a c t i v i t y o c c u r s . T h e i n i t i a l b u r s t o f s y n t h e s i s is, h o w e v e r , s h o r t - l i v e d a n d e x t e n s i v e e n z y m e s y n t h e s i s is n o t r e s u m e d u n t i l a f t e r 30 m i n o r so. S i m i l a r e x p e r i m e n t s w i t h t w o d i f f e r e n t r e p r e s s i b l e r e c i p i e n t s a r e p r e s e n t e d i n F i g . 2. W h e n t h e r e c i p i e n t is r e p r e s s i b l e , t h e s e c o n d p h a s e o f s y n t h e s i s is a b o l ished but the early burst remains unaffected. This m u s t m e a n , t h a t e i t h e r t h e i n c o m i n g d o n o r g e n e is not accessible to repression or the limited initial synt h e s i s o c c u r s in t h e H f r i t s e l f w h e r e t h e p h o A + g e n e s o m e h o w e s c a p e s r e p r e s s i o n a t t h e t i m e o f its t r a n s f e r .
Effect o f Streptomycin. S t r e p t o m y c i n i n h i b i t s p r o t e i n synthesis but does not interfere with the progress of
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Fig. 2. Phosphatase synthesis during conjugation between repressible Hfr and repressible F - strains. A) HfrC (K10) x F-K10W1. B) HfrC (K10) x F CR34. × Phosphatase activity per 5 mI mating mixture. • Phosphatase activity in 5 ml culture of Hfr alone. • Phosphatase activity per 5 ml recipient culture alone
P.N. Bhavsar and O. Siddiqi: Derepression of.Alkaline Phosphatase in E. coli
ongoing chromosome transfer. We therefore examined phosphatase synthesis during conjugal transfer in HfrC x F - matings in which the donor and the recipients were alternately sensitive to streptomycin. The results are presented in Fig. 3. With the Hfr sensitive, the drug abolished the early burst of enzyme synthesis without interfering with chromosome transfer. But when the F - alone was sensitive, streptomycin had no effect on the initial rise in phosphatase activity. The same results are obtained regardless of whether the recipient is repressed, phosphatase positive or phosphatase negative. The site of enzyme synthesis must therefore be the Hfr and not the F - .
359
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Mating With Mini Cells. Mini cells are anucleate cells produced by certain mutants of E. coli defective in cell division (Cohen et al. 1967). These cells accept DNA during conjugation but, being devoid of RNA polymerase, cannot support protein synthesis (Cohen et al. 1968). Figure 4 shows that in HfrC × mini cell matings phosphatase synthesis at 8 rain persists.
Fig, 4. Increase in enzyme activity during mating between mini cells and phoA + repressible donor in phosphate rich broth, x phosphatase activity 5 ml conjugation mixture in cross ; • phosphatase activity in donor alone
Transfer of Suppressible phoA Mu,tations to Su + Recipient. In several experiments suppressible nonsense mutants of the phoA gene (Garen and Siddiqi 1962)
were transferred to a phosphatase negative F- containing a suppressor. No early synthesis of enzyme at the time of transfer was observed.
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Discussion
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Fig, 3. Effect of streptomycin on phosphatase synthesis during conjugation: A a and A2, Hfr K10str~ x FU56, donor sensitive to streptomycin; B 1 and B 2 Hfr K 1 0 s t / x N S 6 5 recipient sensitive to streptomycin. × phospatase activity per 5 ml mating mixture; • Hfr alone, • F - alone; o number of phoA + recombinants per ml of the mating mixture
Our experiments indicate that limited phosphatase synthesis at the time of conjugal transfer of the phoA gene occurs in the donor cells and not in the recipients as originally assumed (Joshi and Siddiqi 1968). Mating triggers the initiation of a fresh round of DNA replication in the Hfr (Sarathy and Siddiqi 1973). As the majority of donors initiate transfer within a few minutes of being mixed, gene replication during conjugation must exhibit a high degree of synchrony. The possibility suggests itself that the short burst of phosphatase synthesis in repressed donor cells is caused by synchronised replication of the phoA gene during transfer. Kuempel et al. (1965) have examined the effect of synchronous duplication on the synthesis of a number of enzymes in E. coli. They found that the uninduced level of alkaline phosphatase (autogenous level) increases in bursts, once during each duplication cycle. Because transfer replication leaves the number of gene copies in the Hfr unchanged, an altered generepressor ratio (Epstein 1967) is not a likely explanation of the transient release from repression. Reference Aono H, Otsuji N (1968) Genetic mapping of regulator gene phoS for alkaline phosphatase in Escherichia curl. J Bacteriol 95:118~1183
360
P.N. Bhavsar and O. Siddiqi: Derepression of Alkaline Phosphatase in E. coli
Bracha M, Yagil E (1973) A new type of alkaline phosphatasenegative mutants in E. coli K12. Mol Gen Genet 122:53-60 Brickman E, Beckwith J (1975) Analysis of the regulation of E. coli alkaline phosphatase synthesis using deletions and d?80 transducing phages. J Mol Biol 96: 307-316 Cohen A, Allison DP, Adler HI, Curtiss III R (1967) Genetic transfer of minicells of Escherichia coli K12. Genetics 56: 550551 Cohen A, Fisher WD, Curtiss III R, Adler HI (1968a) DNA isolated from Escherichia coli minicells mated with F ÷ cells. Proc Natl Acad Sci USA 61:61-68 Cohen A, Fisher WD, Curtiss III R, Adler HI (1968b) The properties of DNA transferred to minicells during conjugation. Cold Spring Harbor Syrup Quant Biol 33:635 641 Echols H, Garen A, Garen S, Torriani A (1961) Genetic control of repression of alkaline phosphatase in E. coli. J Mol Biol 3:425-438 Epstein W (1967) Transposition of the lac region of Escherichia coli IV: Escape from repression in bacteriophage-carried lac genes. J Mol Biol 30:52%543 Garen A (1960) Genetic control of the specificity of the bacterial enzyme alkaline phosphatase. Syrup Soc Gen Microbiol 11:239 247 Garen A, Otsuji N (1964) Isolation of a protein specified by a regulator gene. J Mol Biol 8:841 852 Garen A, Siddiqi O (1962) Suppression of mutations in the alkaline phosphatase structural cistron of E. coli. Proc Natl Acad Sci USA 48 : 1121-1127 Horiuchi T, Horiuchi S, Mizuno D (1959) A possible negative feedback phenomenon controlling formation of alkaline phosphomonoesterase in Escherichia coll. Natur 183:1529-1530 Joshi GP, Siddiqi O (1968) Enzyme synthesis following conjugation and recombination in Escherichia coll. J Mol Biol 32:201 210
Kuempel PL, Masters M, Pardee AB (1965) Bursts of enzyme synthesis in the bacterial duplication cycle. Biochem Biophys Res Commun 18:858-867 Morris H, Schlesinger MJ, Bracha M, Yagil E (1974) Pleiotropic effects of mutations involved in the regulation of Escherichia coli K12 alkaline phosphatase. J Bacteriol 119: 583-592 Rothman F, Byrne R (1963) Fingerprint analysis of alkaline phosphatase of E. coIi K12. J Mol Biol 6:330-340 Sarathy PV, Siddiqi O (1973a) DNA synthesis during bacterial conjugation. I. Effect of mating on DNA replication in Escherichia coli Hfr. J Mol Biol 78:427-441 Schlesinger M, Olsen R (1968) Expression and localization of Escherichia coli alkaline phosphatase synthesized in Salmonella typhimurium cytoplasm. J Bacteriol 96:1601-1605 Torriani A (1960) Influence of inorganic phosphate on the formation of phosphatases by Escherichia coli. Biochim Biophys Acta 38:460-470 Wilkins AS (1972) Physiological factors in the regulation of alkaline phosphatase synthesis in E. coli. J Bacteriol 110:616-623 Willsky GR, Bennett RL, Malamy MH (1973) Inorganic phosphate transport in Escherichia coli. Involvement of two genes which play a role in alkaline phosphatase regulation. J Bacteriol 113 : 529-539 Yagil E, Bracha M, Silberstein N (1970) Further genetic mapping of the phoA-phoR region for alkaline phosphatase synthesis in Escherichia coli K12, Mol Gen Genet 109:18 26.
Communicated
b y L.S. L e r m a n
Received July 16, 1980