Molec. gen. Genet. 152, 2 0 5 - 2 1 0 (1977) © by Springer-Verlag 1977
A Specialized Transducing 2 Phage Carrying the Escherichia coli Genes for Phenylalanyl-tRNA Synthetase H. Hennecke 1, M. Springer 2, and A. B6ck 1 i Lehrstuhl ftir Mikrobiologie der Universitfit Regensburg, D-8400 Regensburg, Federal Republic of G e r m a n y 2 Institut de Biologic Physico-Chimique, 13, rue Pierre et Marie Curie, F-75005 Paris, France
Summary. A 2 phage has been isolated which specif-
ically transduces the Escherichia coli pheS and pheT genes coding for the c~ and/3 subunits of the phenylalanyl-tRNA synthetase (PRS). This phage transduces with high frequency (i) several temperaturesensitive PRS mutants to thermoresistance and (ii) a p-fluorophenylalanine resistant PRS mutant to sensitivity against this amino-acid analog. The in vitro PRS activities of such lysogens suggest that the c~ and /3 subunits coded by the transducing phage complement the mutant host PRS-subunits in vivo by means of formation of hybrid enzymes. The transducing Z phages were also used to infect UV light irradiated cells. The SDS-gel electrophoretic analysis of the proteins synthesized in such cells revealed that the phage codes at least for four different E. coli proteins. Two proteins with molecular weights of 94,000 and 38,000 daltons cross-reacted with an anti PRS serum and were thus identified as the /3 and c~ subunits of PRS, respectively. A third protein with a molecular weight of 22,000 daltons is identical with the ribosomal initiation factor IF3 (Springer et al., 1977b). The other protein ( M r 7 8 , 0 0 0 ) is still unidentified.
Introduction
The pheS and pheT genes coding for the ~ and /3 subunits of the tetrameric phenylalanyl-tRNA synthetase (PRS) map close together at 38min on the Escherichia coli chromosome (B6ck and Neidhardt, 1967; Russell and Pittard, 1971; Comer and B6ck, 1976). It is, however, unknown (i) whether these two genes lie on one transcriptional unit, and if so, in what direction they are transcribed, and (ii) how the transcription of the genes and the translation into the individual PRS polypeptides are regulated. One approach to answer these questions is the isolation of a specialized transducing 2 phage carrying the PRS
genes. A phage strain of this type can then be used as a starting strain for the selection and analysis of deletion mutants, and its D N A can be prepared and used as template for the study of in vitro transcription and translation. In this paper it is demonstrated that )~ phages carrying the PRS genes can be selected by their ability to complement in vivo several defined temperature-sensitive PRS mutants.
Materials and Methods Organisms and Media Table 1 shows the bacterial and phage strains used in this study together with their genotype and their source or derivation. The bacteriophage 2ci857 s RI;t ° sRI2 ° sRI). ° (int xis exofl7 cIII) A(Kourilsky, P., Perricaudet, M. and Tiollais, P., manuscript in preparation) which has only two sites cleavable by restriction endonuclease EcoRI in the non-essential region of its genome was used as a starting vector phage for the in vitro construction of ,tiE. coli D N A hybrids (Cameron et al., 1975). This phage originated from Zplac ci857 sRI2 ° sRI,~ ° sRI2 ° ( R a m b a c h and Tiollais, 1974) and has the deletion between the two sites sBamIZ 3 and sBamI2~ (Perricaudet and Tiollais, 1975). After transfection plaque forming hybrid phage lines were obtained. A total of nine such lysates have been prepared and kindly donated to us by Dr. Max Gottesman. Media and growth conditions for bacteria and 2 phages have been described in recent publications (Comer and B6ck, 1976; Hennecke, 1976; Springer et al., 1977a; Shimada et al., 1972; Schrenk and Weisberg, 1975).
Preparation of UVL-induced Lysates and Infection of Recipient Strains Lysogenic strains of AB1361 were grown at 40 ° C in LB medium (Miller, 1972) to a density of 2-3 x l0 s cells/ml. After centrifugation cells were resuspended in half the original volume of 0.1 M M gSO4. 5-ml portions of this suspension were irradiated with UV light (UVL) in open glass Petri dishes for 25 sec with 3200 erg/sec/cm 2. These cells were then transferred into an aluminium foil-covered Erlenmeyer flask containing 0.5 ml of ten-fold concentrated LB medium and aerated vigorously for 2.5 h at 4 0 ° C which results in spontaneous lysis of the cells. Cell debris was spun down and the supernatant (lysate) was stored over several drops of chloroform.
H. Hennecke et al. : A 2 Phage Transducing the PRS Genes
206 Table 1. E. coli and phage ,~ strains used Strain
Genotype"
Reference
AB1360
F-, thi-1, argE3, his-4, proA2, aroD6, lacY1, galK2, mtl-1, xyl-5, tsx-29, supE44? 2
Taylor and Thoman (1964)
AB1365
F , thi-1, argE3, his-4, proA2, lacY1, galK2, mtl-1, xyl-5, tsx-29, supE44, ,~-
Springer et al. (1977a)
JPlll6
HfrH, pheT354 b, thi , galE-PL5, rel-1, 2
Russell and Pittard (1971)
AB1361
F - , pheSl8 c, all other markers like AB1365
Springer et al. (1977a)
HH1
F - , pheS5 ~, all other markers like AB1365
H. Hennecke (1976)
AB1601
isogenic with HH1, but independently isolated
Springer et al. (1977a)
AB1701
F , pheT354 b, all other markers like AB1365
Springer et al. (1977a)
MC106
F - , pheS12 d, pheT354 b, pps-4, all other markers like AB1365
Comer and B6ck (1976)
RW262
supF +, tonA, 2
Schreuk and Weisberg (1975)
159
rpsL, su , gal-, UV s, 2-
P. Ptashne (1967)
159 (2 ind )
rpsL, su-, gal , UV ~, 2 ind
Springer et al. (1977b) M. Gottesman
wt
Y199
ci857, xis6, $7, (b515, b519, nin5) A
Schrenk and Weisberg (1975) via M. Gottesman
2 vir
vl, v2, v3
Jacob and Wollmann (1954) via M. Yarmolinsky
W30
b2 c
Gottesman and Yarmolinsky (1968)
2 ind-
cI + ind-
Jacob and Campbell (1959) via A. Goze
2 p2
ci857, sRI2 ° sRI2 ° sRI2 °, (int xis exo/? 7 clII) A E. coli D N A between the two EcoRI sites sRI24 and sRIlac
M. Gottesman; Kourilsky, Perricaudet and Tiollais (manuscript in preparation); this work
a Gene symbols were adopted from Bachmann et al. (1976) b Temperature-sensitive PRS (altered/~ subunit) c Temperature-sensitive PRS (altered c~ subunit) '~ p-Fluorophenylalanine-resistant PRS (altered c~ subunit)
For infection the recipient strains were grown in TB medium containing 0.2% maltose (Schrenk and Weisberg, 1975), centrifuged and resuspended in 0.4 volumes of 10 mM MgSO4. About 2 x 108 cells were infected with 4 x 10 s specialized transducing 2 phages together with 2 x 109 wild-type 2 phages as helper. The mixture was incubated for 20 min at 30 ° C. Selection for transductants is described in Results.
Preparation of Crude Cell-free Extracts Crude extracts were prepared as described by Comer and B6ck (1976) or as given by Springer et al. (1977a) who made use of the DEAE elution step of Muench and Berg (1966). The protein contents of the extracts were determined by the Folin phenol procedure (Lowry et al., 1951) with bovine serum albumin as the standard.
Purification of Enzyme, Preparation of Enzyme Subunits and o f tRNA Phenylalanyl-tRNA synthetase was purified to near homogeneity (about 350-400 fold) from Escherichia coli as described previously (Kosakowski and B6ck, 1970). The c~and/~ subunits of this enzyme were separated as described by Hennecke and B6ck (1975). Crude
t R N A from E. coli was isolated by phenol extraction (Ehrenstein and Lipmann, 1961) and isopropanol precipitation (Zubay, 1962).
Enzyme Assays The activity of phenylalanyl-tRNA synthetase was determined by aminoacylation of t R N A with L-[14C]phenylalanine as described by Kosakowski and B6ck (1970) or by Muench and Berg (1966). The p-fluorophenylalanine resistant (or sensitive) character of the enzyme was measured using p-fluoro-D,L@4C]phenylalanine as substrate (Hennecke and B6ck, 1975).
In vitro Complementation The in vitro complementation of PRS-activity was performed as worked out by Comer and B6ck (1976). Prior to the actual reconstitution step the mutant crude extracts were incubated for 5 min at 40 ° C.
Infection of UVL-irradiated Cells and Analysis of Proteins Synthesized The methods of Murialdo and Siminovitch (1971) were adapted for this purpose as described by Springer et al. (1977b). Labelling
H. Hennecke et al. : A 2 Phage Transducing the PRS Genes was performed with 2.5 gCi of [14C]-L-leucine per ml (specific radioactivity 280 ~tCi/gmole). The labelled cells were broken by sonication and the extracts were analyzed on 10% sodium dodecylsulfate (SDS) polyacrylamide slab gels (Studier, 1973; Laemmli, 1970). After staining and destaining, the gels were processed according to Bonner and Laskey (1974) and Laskey and Mills (1975) and finally exposed for at least 36 h to Kodak RP Royal X Omat films. Immunoprecipitation with anti PRS Immunoglobulins The preparation of antibodies specific for the phenylalanyl-tRNA synthetase was performed as described recently (Hennecke et al., 1977). The antibody preparation used for this experiment was the 7-immunoglobulin fraction obtained after three consecutive ammonium sulfate precipitations, at 33% saturation, of the crude antiserum (Campbell etaI., 1970). After infection of UVL-irradiated cells with 2 the isotopically labelled ribosome-free crude extract was mixed with anti PRS immunoglobulins. The antibody.PRS complex formed was precipitated with an anti rabbit immunoglobulin antiserum. The precipitate was pelleted, washed and dissolved in SDS containing buffer. The anti PRS cross reacting material was then analysed on SDS polyacrylamide gels. The complete procedure is described in detail by Springer et al. (i977b).
207 Table 2. In vitro complementation of the PRS-activity from tem-
perature-sensitive E. coli strains Crude extracts a and purified subunits AB1361 AB1601 AB1701
Specific PRS-activity b 333 592 651
AB1601 +AB1701 AB1361 +AB1601 AB1361 +AB1701
2655 424 2051
AB1601+ c~ AB1601+ ]~ AB1701+ e AB1701+/~ AB1361+ c~ AB136I+ p
3005 579 700 3241 2434 361
a The crude extracts used were prepared as described by Springer et al. (1977a) and by Muench and Berg (1966). b Expressed as cpm/5 ~tg crude extract protein determined in the aminoacylation assay of Muench and Berg (1966).
Results and Discussion
Characterization of the AB1361 PRS Lesion The thermosensitive E. coli mutant AB 1361 was previously described to exhibit two defects in vitro, one in the initiation factor IF3 activity and the other in the phenylalanyl-tRNA synthetase activity (Springer et al., 1977a). As the temperature-sensitive phenotype is characterized by a low reversion frequency strain AB1361 was considered to be a suitable recipient for the selection of those )~ phages out of a LFT-lysate which are able to specifically transduce AB1361 to thermoresistance. In order to further characterize the PRS lesion of ABI361 we first attempted to find out whether the ~ or the /3 subunit is affected by the mutation. For this purpose the AB1361 PRS-activity in crude extracts was complemented in vitro either with crude extracts of other well defined PRS-mutants (Comer and B6ck, 1976) or with purified ~ and [3 subunits of wild-type phenylalanyl-tRNA synthetase (Hennecke and B6ck, 1975; Comer and B6ck, 1976). The results of these experiments are listed in Table 2. The experiments show that PRS activity could only be generated from crude extracts of strain AB 1361 when purified wild type ~ subunit was added for reconstitution or when a crude extract of AB1701 (a/3 mutant) was used. Thus, the PRS of strain AB1361 is inferred to posses an altered ~ subunit.
Preparation of HFT-lysates A total of nine different hybrid phage lines (LFT lysates) obtained by transfection with 2/E. coli D N A
hybrids prepared in vitro were used to infect strain AB1361 at a m.o.i, of 2 together with wild-type helper phages at a m.o.i, of 10. The selection was made for growth on LB plates at 42 ° C. With one of the lysates a considerable number of thermoresistant transductant colonies appeared. These transductants were then purified and tested for )ovir sensitivity, 2clear (W30) resistance and maltose utilization. A total of 15 transductants were grown up and irradiated with UV light. The resulting UV induced lysates showed 2 phage titers between l09 and 101° pfu/ml on RW262. These lysates were able to transduce AB1361 to thermoresistance with high frequency ( H F T lysates). They were named HFTL-1, H F T L 2... HFTL-15. The H F T lysates are mixtures of wildtype and transducing phages which produce turbid and clear plaques on RW262 at 42 ° C, respectively. Several of the clear plaques were further purified as described (Springer et al., 1977b) and the resulting lines were then named 2pl, 2p2, etc.
In vivo Effect of the Transducing Phages on Other Genetically Defined PRS-mutants After infection of AB1361 the transducing 2 phages are apparently able to complement in vivo the mutation in the c~gene of this strain. To investigate whether the phages can also complement other well defined PRS mutations the following strains were chosen as recipient for the infection with HFTL-2, HFTL-7, HFTL-8 and HFTL-13 : (i) HH1, a temperature-sensitive c~ mutant different from AB1361, (ii) J P l l l 6 ,
208
H. Hennecke et al. : A 2 Phage Transducing the PRS Genes
a temperature-sensitive fi mutant and (iii) MC106 (Comer and B6ck, 1976) a strain which contains the fi mutation of J P l l l 6 in addition to a mutation in the ~ gene rendering the mutant resistant to p-fluorophenylalanine (Hennecke and B6ck, 1975). Selection was for growth on LB plates at 40 ° C. With all three strains and with all the lysates used thermoresistant transductants arose with high frequency. In addition, the thermoresistant transductants of MC 106 were sensitive to p-fluorophenylalanine. The pps- marker (inability to utilize pyruvate as sole carbon source) of MC106, which is located counterclockwise in the near neighborhood of the PRSgenes (Brice and Kornberg, 1968), was also tested for being complemented by the transducing phages. However, none of the thermoresistant transductants of MC106 were able to grow on pyruvate as sole carbon source.
PRS-activity of the Thermoresistant Tranductants Next we determined the PRS activities in crude extracts of the thermoresistant transductants described above in order to see whether the phenotypic reversion of temperature-sensitivity is correlated with a regenerated PRS activity. The results obtained are summarized in Table 3. The PRS assays were carried
Table 3. PRS activity in crude extracts" of thermoresistant transductants Receptor strains
Transducing phage
AB1360 AB1361 HH1 JP1116 MC106
Specific PRS-activity b using the substrate 1~C Phenylalanine
14C p-Fluorophenylalanine
at 28 ° C
at 37 ° C
at 28 ° C
at 37 ° C
2105 550 70 t015 918
2903 0 0 249 237
1063 n.d. n.d. 421 0
1596 n.d. n.d. 107 0
AB1361
HFTL-2 HFTL-7 HFTL-8 HFTL-13
2984 2627 4238 3457
3244 2985 4436 4843
n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d.
HH1 JPlll6 MC106
HFTL-8 HFTL-8 HFTL-8
3133 4745 5076
3576 5122 5466
n.d. n.d. 1555
n.d. n.d. 1699
" Crude extracts were prepared as described by C o m e r and B6ck (1976) b Expressed as cpm/5 gg crude extract protein determined in the aminoacylation assay of Kosakowski and B6ck (1970) n.d. = n o t determined
~--~20-
~15o
.~ 10i
5-
ncl_
2'2
2'g
33
37
42
Incubation temperature (°C) Fig. 1. Temperature-activity relationship of the PRS-activity in crude extracts of E. coli strains. ([]) AB1361, (©) AB1360, (e) thermoresistant transductant of AB1361 obtained with HFTL-8
out both at 28 ° C and 37 ° C in order to differentiate between thermostabile and thermolabile PRS species. In contrast to the PRS activities of the four recipient strains the specific activities of the enzymes of the thermoresistant transductants are greatly increased and, in addition, comparatively thermostabile. The transducing phage is able to complement the defective enzymes of both ct and fi temperature-sensitive mutants. In addition, the p-fluorophenylalanine-resistant trait of strain MC106 is abolished in the thermoresistant transductants of strain MC106 and their PRS is able to use p-fluorophenylalanine as a substrate. However, the specific attachment activity of p-fluorophenylalanine to t R N A is about 30% of the value obtained with phenylalanine as substrate, whereas the PRS of strains with wild-type e genes (AB1360, J P l l l 6 ) show a value of 50% (Table 3; Hennecke and B6ck, 1975). This result and the finding that thermoresistant transductants exhibit about twice the specific PRS-activity of control strain AB1360 (Table 3) favour the conclusion that the transductants have regained their active PRS via the formation of hybrid enzymes containing both wild-type and mutant enzyme subunits. Supporting evidence comes from the experiment of Figure 1 which shows the temperature-activity relationship of the PRS-activity of a thermoresistant transductant derived from AB1361 and those of its parental strains. Here the PRS of the transductant is only partially thermostabile, a behaviour which was recently observed in the case of hybrid phenylalanyl-tRNA synthetases present in merodiploid strains (Hennecke, 1976) and which points to the presence of the temperature-sensitive c~ subunit in the hybrid enzyme. The fact that about double the amount of PRSactivity is generated in the transductants as compared
H. Hennecke et al. : A 2 Phage Transducing the PRS Genes
209
with a wild-type PRS strain, indicates that the transductants are merodiploid for the PRS genes (Piepersberg etal., 1975; Hennecke, 1976; Springer etal., 1977a). However, there is no genetic evidence at the moment which could decide between an integration of the transducing phage DNA into the PRS region or into the normal attachment site (att2) of the host genome. IF3 activity is also reverted in the thermoresistant transductants of AB1361; the detailed results are reported by Springer et al. (1977b). Analysis of Proteins Synthesized in UVL-irradiated Cells The results presented above have indicated that the transducing 2 phages might carry the structural genes for the c~ and fl subunits of phenylalanyl-tRNA synthetase. Final proof for this inference was brought about by the SDS gel electrophoretic analysis of radioactively labelled proteins synthesized in UVL-irradiated cells (Murialdo and Siminovitch, 1971) upon infection with a purified 2p2 lysate. The infection stimulates [14C]-L-leucine incorporation into proteins several fold over the low background of the host. Two different E. coli strains were used as hosts: (i) a UV sensitive strain 159, and (ii) strain 159 lysogenized with a 2ind- bacteriophage. Since 2p2 contains E. coli DNA in the b2 region, we used Y199, a phage carrying several deletions in the b2 region, as control. After labelling, the extracts of infected and non-infected strains were analyzed by SDS slab gel electrophoresis and by subsequent autoradiography (Fig. 2). Lane 3 of Figure 2 demonstrates the residual background protein synthesis of the extract of the non-infected strain 159 2ind . Likewise, when control phage Y199 infects strain 159 2ind- only background protein synthesis is visible (Fig. 2 lane 5) indicating that the repressor of 2ind- is still active after the UVL-irradiation. Infection of strain 159 by )@2, on the other hand, results in the synthesis of several proteins, among them 2 coded proteins as well as proteins coded by the E. coli DNA which is integrated in the specialized transducing phage (Fig. 2, lane 6). When 2 p2 infects strain 159 2ind , four bands appear (Fig. 2, lane 4) which are not repressible by the cI gene product of 2ind . The expression of these proteins is therefore assumed to be under the control of E. coli promoters and genes. The molecular weights of these four proteins from top to bottom are 94,000, 78,000, 38,000 and 22,000. The molecular weight values of the first and the third proteins are identical with those of the fl and c~subunits of PRS, respectively (Hanke et al., 1974; Fayat et al., 1974). Furthermore, the positions of the first and third proteins coincide
Fig. 2. Scintillation autograph (fluorograph) of the SDS gel electrophoretic analysis of radioactively labelled crude extracts from E. coli strains infected with 2 strains. Samples 1, 2, 11, and 12 contain the non-labelled marker proteins bovine serum albumin (Mr 69,000), alcohol dehydrogenase (M r 35,000), chymotrypsinogen A (Mr 25,000), and cytochrome c (M s 12,500), which have been used for molecular weight determination (not shown). Samples 7 and 8 contain non-labelled purified c~ and /3 subunits of PRS, respectively. Their position on the Coomassie blue-stained gel (not shown) is given by the arrows. Samples 3 6 contain the following crude extracts: (3) non-infected strain 1592ind(3480 cpm on the gel), (4) 1592ind- infected with 2p2 (3200 cpm), (5) 159)0ind- infected with Y199 (3200 cpm), (6) strain 159 infected with 2p2 (5800 cpm). Sample 9 contains the anti-PRS cross-reacting material from the high speed supernatant (S100) of the extract of strain 159 infected with Y199 (521 cpm), and sample 10 shows the anti-PRS cross-reacting material from the SI00 of the extract of 159 infected with ,~p2
exactly with the positions of purified non-labelled fi and c~ subunits, respectively, that have been applied on lanes 8 and 7 (Fig. 2) and localized by staining the gel with Coomassie blue (not shown). Finally, the electrophoretic analysis of the immunoprecipitated anti-PRS cross reacting material in the crude extracts of the UVL-irradiated strain 159 infected with 2p2 clearly revealed two protein bands coinciding with the position of the fi and c~ subunits of PRS (Fig. 2, lane 10). These proteins are not precipitated by PRS specific antibodies from crude extracts of strain 159 infected with phage Y199 (Fig. 2, lane 9). For the appearance of the low molecular weight bands in samples 9 and 10 of Figure 2 we have no other explanation at the moment except that they could represent unspecifically precipitated proteins or degradation products.
210
The E. coli DNA-coded protein with the molecular weight of 22,000 daltons was identified as the protein synthesis initiation factor IF3 (Springer et al., 1977b). The protein with the molecular weight of 78,000 daltons has not yet been identified. It will be interesting to see whether it is also a component of the protein synthesizing system. Acknowledgements. We are greatly indebted to Dr. Max Gottesman
for his generous gift of the LFT lysates which have made possible this study. We also wish to thank Monique Graffe and Heidi Franz for their expert assistance. This work has been supported by a grant from the Deutsche Forschungsgemeinschaft and by an EMBO short-term fellowship.
References Bachmann, B.J., Low, K.B., Taylor, A.L.: Recalibrated linkage map of Escherichia coli K-12. Bact. Rev. 40, 116-167 (1976) B6ck, A., Neidhardt, F.C.: Genetic mapping of phenylalanylsRNA synthetase in Escherichia eoli. Science (Wash.) 157, 7 8 3 9 (1967) Bonner, W.M., Laskey, R.A. : A film detection method for tritiumlabelled proteins and nucleic acids in polyacrylamide gels. Europ. J. Biochem. 46, 83-88 (1974) Brice, C.B., Kornberg, H.L.: Genetic control of isocitrate lyase activity in Escheriehia coli. J. Bact. 96, 2t85-2186 (1968) Cameron, J.R., Panasenko, S.M., Lehman, I.R., Davis, R.W.: In vitro construction of bacteriophage 2 carrying segments of the Eseheriehia coli chromosome: Selection of hybrids containing the gene for DNA ligase. Proc. nat. Acad. Sci. (Wash.) 72, 3416-3420 (1975) Campbell, D.H., Garvey, J.S., Cremer, N.E., Sussdorf, D.H.: Methods in immunology, 2nd ed. NewYork: W.A. Benjamin Inc. 1970 Comer, M.M., B6ck, A.: Genes for the e and fl subunits of the phenylalanyl-transfer ribonucleic acid synthetase of Eseherichia coli. J. Bact. 127, 923-933 (1976) Ehrenstein, G. von, Lipmann, F.: Experiments on hemoglobin biosynthesis. Proc. nat. Acad. Sci. (Wash.) 47, 941 950 (1961) Fayat, G., Blanquet, S., Dessen, P., Batelier, G., Waller, J.-P.: The molecular weight and subunit composition of phenylalanyltRNA synthetase from Escherichia coli K12. Biochimie 56, 3541 (1974) Gottesman, M.E., Yarmolinsky, M.B.: Integration-negative mutantsofbacteriophage lambda. J. molec. Biol. 31,487 505 (1968) Hanke, T., Bartmann, P., Hennecke, H., Kosakowski, H.M., Jaenicke, R., Holler, E., B6ck, A. : L-phenylalanyl-tRNA synthetase of Escherichia eoli K10. A reinvestigation of molecular weight and subunit structure. Europ. J. Biochem. 43, 601-607 (1974) Hennecke, H. : Use of mutant enzymes to demonstrate the presence of two active sites on phenytalanyl-tRNA synthetase from E. coli. FEBS Lett. 72, 182=186 (1976) Hennecke H., B6ck, A. : Altered c~subunits in phenylalanyl-tRNA syiithetases from p-fluorophenylalanine-resistant strains of Escherichia eoli. Europ. J. Biochem. 55, 431 437 (1975) Hennecke, H., Walther, I., Franz, H.: Immunochemical studies on phenylalanyl-tRNA synthetase from Escherichia eoli. Hoppe-Seylers Z. physiol. Chem. 358, 197 208 (1977) Jacob, F., Campbell, A.: Sur le syst6me de r6pression assurant l'immunit6 chez les bact6ries lysog6nes. C. R. Acad. Sci. (Paris) 248, 3219-3221 (1959)
H. Hennecke et al. : A ,t Phage Transducing the PRS Genes Jacob, F., Wollmann, W.L.: Etude g6n+tique d'un bact6riophage temper+ d'Escherichia coli. I. Le systbme g6n6tique du bact6riophage 2. Ann. Inst. Pasteur 87, 653~690 (1954) Kosakowski, H.M., B6ck, A.: The subunit structure of phenylalanyl-tRNA synthetase of Escheriehia eoli. Europ. J. Biochem. 12, 67-73 (1970) Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.) 227, 680-685 (1970) Laskey, R.A., Mills, A.D.: Quantitative film detection of aH and 14C in polyacrylamide gels by fluorography. Europ. J. Biochem. 56, 335-341 (1975) Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. : Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275 (1951) Miller, J.H. : Experiments in molecular genetics. New York: Cold Spring Harbor Laboratory 1972 Muench, K.H., Berg, P. : Preparation of aminoacyl-ribonucleic acid synthetases from Escheriehia eoli. In: Procedures in nucleic acid research, Vol. 1, pp. 375-383 (Cantoni, G.L. and Davis, D.R., eds.). New York: Harper and Row, Publishers 1966 Murialdo, H., Siminovitch, L.: The morphogenesis of bacteriophage lambda. III. Identification of genes specifying morphogenetic proteins. In: The bacteriophage lambda, pp. 711-723 (Hershey, A.D., ed.). New York: Cold Spring Harbor Laboratory 1971 Perricaudet, M., Tiollais, P. : Defective bacteriophage chromosome, potential vector for DNA fragments obtained after cleavage by Bacillus amyloliquefaciens endonuclease (BAM I). FEBS Lett. 56, 7-11 (1975) Piepersberg, A., Hennecke, H., Engelhard, M., Nass, G., B6ck, A.: Cross-reactivity of phenylalanyl-transfer ribonucleic acid ligases from different microorganisms. J. Bact. 124, 1482-1488 (1975) Patshne, M. : Isolation of the 2 phage repressor. Proc. nat. Acad. Sci. (Wash.) 57, 306~13 (1967) Rambach, A., Tiollais, P. : Bacteriophage 2 having EcoRI endonuclease sites only in the nonessential region of the genome. Proc. nat. Acad. Sci. (Wash.) 71, 3927 3930 (1974) Russell, R.R.B., Pittard, A.J.: Mutants of Escherichia coli unable to make protein at 42 ° C. J. Bact. 108, 790 798 (1971) Schrenk, W.J., Weisberg, R.A. : A simple method for making new transducing lines of coliphage 2. Molec. gen. Genet. 137, 101-107 (1975) Shimada, K., Weisberg, R., Gottesman, M.: Prophage lambda at unusual chromosomal locations. I. Location of the secondary attachment sites and the properties of the lysogens. J. molec. Biol. 63, 483-503 (1972) Springer, M., Graffe, M., Grunberg-Manago, M. : Characterization of an E. coli mutant with a thermolabile initiation factor IF3 activity. Molec. gen. Genet. 151, 17 26 (1977a) Springer, M., Graffe, M., Hennecke, H.: Specialized transducing phage for initiation factor IF3 gene in E. coli. Proc. nat. Acad. Sci. (Wash.), in press (1977b) Studier, F.W.: Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J. molec. Biol. 79, 2/37 248 (1973) Taylor, A.L., Thoman, M.S.: The genetic map of Escherichia coli K-12. Genetics 50, 659-677 (1964) Zubay, G.J. : The isolation and fractionation of soluble ribonucleic acid. J. molec. Biol. 4, 347-356 (1962)
Communicated by H.G. Wittmann
Received January 20 / February 14, 1977
A Specialized Transducing 2 Phage Carrying the Escherichia coli Genes for Phenylalanyl-tRNA Synthetase H. Hennecke 1, M. Springer 2, and A. B6ck 1 i Lehrstuhl ftir Mikrobiologie der Universitfit Regensburg, D-8400 Regensburg, Federal Republic of G e r m a n y 2 Institut de Biologic Physico-Chimique, 13, rue Pierre et Marie Curie, F-75005 Paris, France
Summary. A 2 phage has been isolated which specif-
ically transduces the Escherichia coli pheS and pheT genes coding for the c~ and/3 subunits of the phenylalanyl-tRNA synthetase (PRS). This phage transduces with high frequency (i) several temperaturesensitive PRS mutants to thermoresistance and (ii) a p-fluorophenylalanine resistant PRS mutant to sensitivity against this amino-acid analog. The in vitro PRS activities of such lysogens suggest that the c~ and /3 subunits coded by the transducing phage complement the mutant host PRS-subunits in vivo by means of formation of hybrid enzymes. The transducing Z phages were also used to infect UV light irradiated cells. The SDS-gel electrophoretic analysis of the proteins synthesized in such cells revealed that the phage codes at least for four different E. coli proteins. Two proteins with molecular weights of 94,000 and 38,000 daltons cross-reacted with an anti PRS serum and were thus identified as the /3 and c~ subunits of PRS, respectively. A third protein with a molecular weight of 22,000 daltons is identical with the ribosomal initiation factor IF3 (Springer et al., 1977b). The other protein ( M r 7 8 , 0 0 0 ) is still unidentified.
Introduction
The pheS and pheT genes coding for the ~ and /3 subunits of the tetrameric phenylalanyl-tRNA synthetase (PRS) map close together at 38min on the Escherichia coli chromosome (B6ck and Neidhardt, 1967; Russell and Pittard, 1971; Comer and B6ck, 1976). It is, however, unknown (i) whether these two genes lie on one transcriptional unit, and if so, in what direction they are transcribed, and (ii) how the transcription of the genes and the translation into the individual PRS polypeptides are regulated. One approach to answer these questions is the isolation of a specialized transducing 2 phage carrying the PRS
genes. A phage strain of this type can then be used as a starting strain for the selection and analysis of deletion mutants, and its D N A can be prepared and used as template for the study of in vitro transcription and translation. In this paper it is demonstrated that )~ phages carrying the PRS genes can be selected by their ability to complement in vivo several defined temperature-sensitive PRS mutants.
Materials and Methods Organisms and Media Table 1 shows the bacterial and phage strains used in this study together with their genotype and their source or derivation. The bacteriophage 2ci857 s RI;t ° sRI2 ° sRI). ° (int xis exofl7 cIII) A(Kourilsky, P., Perricaudet, M. and Tiollais, P., manuscript in preparation) which has only two sites cleavable by restriction endonuclease EcoRI in the non-essential region of its genome was used as a starting vector phage for the in vitro construction of ,tiE. coli D N A hybrids (Cameron et al., 1975). This phage originated from Zplac ci857 sRI2 ° sRI,~ ° sRI2 ° ( R a m b a c h and Tiollais, 1974) and has the deletion between the two sites sBamIZ 3 and sBamI2~ (Perricaudet and Tiollais, 1975). After transfection plaque forming hybrid phage lines were obtained. A total of nine such lysates have been prepared and kindly donated to us by Dr. Max Gottesman. Media and growth conditions for bacteria and 2 phages have been described in recent publications (Comer and B6ck, 1976; Hennecke, 1976; Springer et al., 1977a; Shimada et al., 1972; Schrenk and Weisberg, 1975).
Preparation of UVL-induced Lysates and Infection of Recipient Strains Lysogenic strains of AB1361 were grown at 40 ° C in LB medium (Miller, 1972) to a density of 2-3 x l0 s cells/ml. After centrifugation cells were resuspended in half the original volume of 0.1 M M gSO4. 5-ml portions of this suspension were irradiated with UV light (UVL) in open glass Petri dishes for 25 sec with 3200 erg/sec/cm 2. These cells were then transferred into an aluminium foil-covered Erlenmeyer flask containing 0.5 ml of ten-fold concentrated LB medium and aerated vigorously for 2.5 h at 4 0 ° C which results in spontaneous lysis of the cells. Cell debris was spun down and the supernatant (lysate) was stored over several drops of chloroform.
H. Hennecke et al. : A 2 Phage Transducing the PRS Genes
206 Table 1. E. coli and phage ,~ strains used Strain
Genotype"
Reference
AB1360
F-, thi-1, argE3, his-4, proA2, aroD6, lacY1, galK2, mtl-1, xyl-5, tsx-29, supE44? 2
Taylor and Thoman (1964)
AB1365
F , thi-1, argE3, his-4, proA2, lacY1, galK2, mtl-1, xyl-5, tsx-29, supE44, ,~-
Springer et al. (1977a)
JPlll6
HfrH, pheT354 b, thi , galE-PL5, rel-1, 2
Russell and Pittard (1971)
AB1361
F - , pheSl8 c, all other markers like AB1365
Springer et al. (1977a)
HH1
F - , pheS5 ~, all other markers like AB1365
H. Hennecke (1976)
AB1601
isogenic with HH1, but independently isolated
Springer et al. (1977a)
AB1701
F , pheT354 b, all other markers like AB1365
Springer et al. (1977a)
MC106
F - , pheS12 d, pheT354 b, pps-4, all other markers like AB1365
Comer and B6ck (1976)
RW262
supF +, tonA, 2
Schreuk and Weisberg (1975)
159
rpsL, su , gal-, UV s, 2-
P. Ptashne (1967)
159 (2 ind )
rpsL, su-, gal , UV ~, 2 ind
Springer et al. (1977b) M. Gottesman
wt
Y199
ci857, xis6, $7, (b515, b519, nin5) A
Schrenk and Weisberg (1975) via M. Gottesman
2 vir
vl, v2, v3
Jacob and Wollmann (1954) via M. Yarmolinsky
W30
b2 c
Gottesman and Yarmolinsky (1968)
2 ind-
cI + ind-
Jacob and Campbell (1959) via A. Goze
2 p2
ci857, sRI2 ° sRI2 ° sRI2 °, (int xis exo/? 7 clII) A E. coli D N A between the two EcoRI sites sRI24 and sRIlac
M. Gottesman; Kourilsky, Perricaudet and Tiollais (manuscript in preparation); this work
a Gene symbols were adopted from Bachmann et al. (1976) b Temperature-sensitive PRS (altered/~ subunit) c Temperature-sensitive PRS (altered c~ subunit) '~ p-Fluorophenylalanine-resistant PRS (altered c~ subunit)
For infection the recipient strains were grown in TB medium containing 0.2% maltose (Schrenk and Weisberg, 1975), centrifuged and resuspended in 0.4 volumes of 10 mM MgSO4. About 2 x 108 cells were infected with 4 x 10 s specialized transducing 2 phages together with 2 x 109 wild-type 2 phages as helper. The mixture was incubated for 20 min at 30 ° C. Selection for transductants is described in Results.
Preparation of Crude Cell-free Extracts Crude extracts were prepared as described by Comer and B6ck (1976) or as given by Springer et al. (1977a) who made use of the DEAE elution step of Muench and Berg (1966). The protein contents of the extracts were determined by the Folin phenol procedure (Lowry et al., 1951) with bovine serum albumin as the standard.
Purification of Enzyme, Preparation of Enzyme Subunits and o f tRNA Phenylalanyl-tRNA synthetase was purified to near homogeneity (about 350-400 fold) from Escherichia coli as described previously (Kosakowski and B6ck, 1970). The c~and/~ subunits of this enzyme were separated as described by Hennecke and B6ck (1975). Crude
t R N A from E. coli was isolated by phenol extraction (Ehrenstein and Lipmann, 1961) and isopropanol precipitation (Zubay, 1962).
Enzyme Assays The activity of phenylalanyl-tRNA synthetase was determined by aminoacylation of t R N A with L-[14C]phenylalanine as described by Kosakowski and B6ck (1970) or by Muench and Berg (1966). The p-fluorophenylalanine resistant (or sensitive) character of the enzyme was measured using p-fluoro-D,L@4C]phenylalanine as substrate (Hennecke and B6ck, 1975).
In vitro Complementation The in vitro complementation of PRS-activity was performed as worked out by Comer and B6ck (1976). Prior to the actual reconstitution step the mutant crude extracts were incubated for 5 min at 40 ° C.
Infection of UVL-irradiated Cells and Analysis of Proteins Synthesized The methods of Murialdo and Siminovitch (1971) were adapted for this purpose as described by Springer et al. (1977b). Labelling
H. Hennecke et al. : A 2 Phage Transducing the PRS Genes was performed with 2.5 gCi of [14C]-L-leucine per ml (specific radioactivity 280 ~tCi/gmole). The labelled cells were broken by sonication and the extracts were analyzed on 10% sodium dodecylsulfate (SDS) polyacrylamide slab gels (Studier, 1973; Laemmli, 1970). After staining and destaining, the gels were processed according to Bonner and Laskey (1974) and Laskey and Mills (1975) and finally exposed for at least 36 h to Kodak RP Royal X Omat films. Immunoprecipitation with anti PRS Immunoglobulins The preparation of antibodies specific for the phenylalanyl-tRNA synthetase was performed as described recently (Hennecke et al., 1977). The antibody preparation used for this experiment was the 7-immunoglobulin fraction obtained after three consecutive ammonium sulfate precipitations, at 33% saturation, of the crude antiserum (Campbell etaI., 1970). After infection of UVL-irradiated cells with 2 the isotopically labelled ribosome-free crude extract was mixed with anti PRS immunoglobulins. The antibody.PRS complex formed was precipitated with an anti rabbit immunoglobulin antiserum. The precipitate was pelleted, washed and dissolved in SDS containing buffer. The anti PRS cross reacting material was then analysed on SDS polyacrylamide gels. The complete procedure is described in detail by Springer et al. (i977b).
207 Table 2. In vitro complementation of the PRS-activity from tem-
perature-sensitive E. coli strains Crude extracts a and purified subunits AB1361 AB1601 AB1701
Specific PRS-activity b 333 592 651
AB1601 +AB1701 AB1361 +AB1601 AB1361 +AB1701
2655 424 2051
AB1601+ c~ AB1601+ ]~ AB1701+ e AB1701+/~ AB1361+ c~ AB136I+ p
3005 579 700 3241 2434 361
a The crude extracts used were prepared as described by Springer et al. (1977a) and by Muench and Berg (1966). b Expressed as cpm/5 ~tg crude extract protein determined in the aminoacylation assay of Muench and Berg (1966).
Results and Discussion
Characterization of the AB1361 PRS Lesion The thermosensitive E. coli mutant AB 1361 was previously described to exhibit two defects in vitro, one in the initiation factor IF3 activity and the other in the phenylalanyl-tRNA synthetase activity (Springer et al., 1977a). As the temperature-sensitive phenotype is characterized by a low reversion frequency strain AB1361 was considered to be a suitable recipient for the selection of those )~ phages out of a LFT-lysate which are able to specifically transduce AB1361 to thermoresistance. In order to further characterize the PRS lesion of ABI361 we first attempted to find out whether the ~ or the /3 subunit is affected by the mutation. For this purpose the AB1361 PRS-activity in crude extracts was complemented in vitro either with crude extracts of other well defined PRS-mutants (Comer and B6ck, 1976) or with purified ~ and [3 subunits of wild-type phenylalanyl-tRNA synthetase (Hennecke and B6ck, 1975; Comer and B6ck, 1976). The results of these experiments are listed in Table 2. The experiments show that PRS activity could only be generated from crude extracts of strain AB 1361 when purified wild type ~ subunit was added for reconstitution or when a crude extract of AB1701 (a/3 mutant) was used. Thus, the PRS of strain AB1361 is inferred to posses an altered ~ subunit.
Preparation of HFT-lysates A total of nine different hybrid phage lines (LFT lysates) obtained by transfection with 2/E. coli D N A
hybrids prepared in vitro were used to infect strain AB1361 at a m.o.i, of 2 together with wild-type helper phages at a m.o.i, of 10. The selection was made for growth on LB plates at 42 ° C. With one of the lysates a considerable number of thermoresistant transductant colonies appeared. These transductants were then purified and tested for )ovir sensitivity, 2clear (W30) resistance and maltose utilization. A total of 15 transductants were grown up and irradiated with UV light. The resulting UV induced lysates showed 2 phage titers between l09 and 101° pfu/ml on RW262. These lysates were able to transduce AB1361 to thermoresistance with high frequency ( H F T lysates). They were named HFTL-1, H F T L 2... HFTL-15. The H F T lysates are mixtures of wildtype and transducing phages which produce turbid and clear plaques on RW262 at 42 ° C, respectively. Several of the clear plaques were further purified as described (Springer et al., 1977b) and the resulting lines were then named 2pl, 2p2, etc.
In vivo Effect of the Transducing Phages on Other Genetically Defined PRS-mutants After infection of AB1361 the transducing 2 phages are apparently able to complement in vivo the mutation in the c~gene of this strain. To investigate whether the phages can also complement other well defined PRS mutations the following strains were chosen as recipient for the infection with HFTL-2, HFTL-7, HFTL-8 and HFTL-13 : (i) HH1, a temperature-sensitive c~ mutant different from AB1361, (ii) J P l l l 6 ,
208
H. Hennecke et al. : A 2 Phage Transducing the PRS Genes
a temperature-sensitive fi mutant and (iii) MC106 (Comer and B6ck, 1976) a strain which contains the fi mutation of J P l l l 6 in addition to a mutation in the ~ gene rendering the mutant resistant to p-fluorophenylalanine (Hennecke and B6ck, 1975). Selection was for growth on LB plates at 40 ° C. With all three strains and with all the lysates used thermoresistant transductants arose with high frequency. In addition, the thermoresistant transductants of MC 106 were sensitive to p-fluorophenylalanine. The pps- marker (inability to utilize pyruvate as sole carbon source) of MC106, which is located counterclockwise in the near neighborhood of the PRSgenes (Brice and Kornberg, 1968), was also tested for being complemented by the transducing phages. However, none of the thermoresistant transductants of MC106 were able to grow on pyruvate as sole carbon source.
PRS-activity of the Thermoresistant Tranductants Next we determined the PRS activities in crude extracts of the thermoresistant transductants described above in order to see whether the phenotypic reversion of temperature-sensitivity is correlated with a regenerated PRS activity. The results obtained are summarized in Table 3. The PRS assays were carried
Table 3. PRS activity in crude extracts" of thermoresistant transductants Receptor strains
Transducing phage
AB1360 AB1361 HH1 JP1116 MC106
Specific PRS-activity b using the substrate 1~C Phenylalanine
14C p-Fluorophenylalanine
at 28 ° C
at 37 ° C
at 28 ° C
at 37 ° C
2105 550 70 t015 918
2903 0 0 249 237
1063 n.d. n.d. 421 0
1596 n.d. n.d. 107 0
AB1361
HFTL-2 HFTL-7 HFTL-8 HFTL-13
2984 2627 4238 3457
3244 2985 4436 4843
n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d.
HH1 JPlll6 MC106
HFTL-8 HFTL-8 HFTL-8
3133 4745 5076
3576 5122 5466
n.d. n.d. 1555
n.d. n.d. 1699
" Crude extracts were prepared as described by C o m e r and B6ck (1976) b Expressed as cpm/5 gg crude extract protein determined in the aminoacylation assay of Kosakowski and B6ck (1970) n.d. = n o t determined
~--~20-
~15o
.~ 10i
5-
ncl_
2'2
2'g
33
37
42
Incubation temperature (°C) Fig. 1. Temperature-activity relationship of the PRS-activity in crude extracts of E. coli strains. ([]) AB1361, (©) AB1360, (e) thermoresistant transductant of AB1361 obtained with HFTL-8
out both at 28 ° C and 37 ° C in order to differentiate between thermostabile and thermolabile PRS species. In contrast to the PRS activities of the four recipient strains the specific activities of the enzymes of the thermoresistant transductants are greatly increased and, in addition, comparatively thermostabile. The transducing phage is able to complement the defective enzymes of both ct and fi temperature-sensitive mutants. In addition, the p-fluorophenylalanine-resistant trait of strain MC106 is abolished in the thermoresistant transductants of strain MC106 and their PRS is able to use p-fluorophenylalanine as a substrate. However, the specific attachment activity of p-fluorophenylalanine to t R N A is about 30% of the value obtained with phenylalanine as substrate, whereas the PRS of strains with wild-type e genes (AB1360, J P l l l 6 ) show a value of 50% (Table 3; Hennecke and B6ck, 1975). This result and the finding that thermoresistant transductants exhibit about twice the specific PRS-activity of control strain AB1360 (Table 3) favour the conclusion that the transductants have regained their active PRS via the formation of hybrid enzymes containing both wild-type and mutant enzyme subunits. Supporting evidence comes from the experiment of Figure 1 which shows the temperature-activity relationship of the PRS-activity of a thermoresistant transductant derived from AB1361 and those of its parental strains. Here the PRS of the transductant is only partially thermostabile, a behaviour which was recently observed in the case of hybrid phenylalanyl-tRNA synthetases present in merodiploid strains (Hennecke, 1976) and which points to the presence of the temperature-sensitive c~ subunit in the hybrid enzyme. The fact that about double the amount of PRSactivity is generated in the transductants as compared
H. Hennecke et al. : A 2 Phage Transducing the PRS Genes
209
with a wild-type PRS strain, indicates that the transductants are merodiploid for the PRS genes (Piepersberg etal., 1975; Hennecke, 1976; Springer etal., 1977a). However, there is no genetic evidence at the moment which could decide between an integration of the transducing phage DNA into the PRS region or into the normal attachment site (att2) of the host genome. IF3 activity is also reverted in the thermoresistant transductants of AB1361; the detailed results are reported by Springer et al. (1977b). Analysis of Proteins Synthesized in UVL-irradiated Cells The results presented above have indicated that the transducing 2 phages might carry the structural genes for the c~ and fl subunits of phenylalanyl-tRNA synthetase. Final proof for this inference was brought about by the SDS gel electrophoretic analysis of radioactively labelled proteins synthesized in UVL-irradiated cells (Murialdo and Siminovitch, 1971) upon infection with a purified 2p2 lysate. The infection stimulates [14C]-L-leucine incorporation into proteins several fold over the low background of the host. Two different E. coli strains were used as hosts: (i) a UV sensitive strain 159, and (ii) strain 159 lysogenized with a 2ind- bacteriophage. Since 2p2 contains E. coli DNA in the b2 region, we used Y199, a phage carrying several deletions in the b2 region, as control. After labelling, the extracts of infected and non-infected strains were analyzed by SDS slab gel electrophoresis and by subsequent autoradiography (Fig. 2). Lane 3 of Figure 2 demonstrates the residual background protein synthesis of the extract of the non-infected strain 159 2ind . Likewise, when control phage Y199 infects strain 159 2ind- only background protein synthesis is visible (Fig. 2 lane 5) indicating that the repressor of 2ind- is still active after the UVL-irradiation. Infection of strain 159 by )@2, on the other hand, results in the synthesis of several proteins, among them 2 coded proteins as well as proteins coded by the E. coli DNA which is integrated in the specialized transducing phage (Fig. 2, lane 6). When 2 p2 infects strain 159 2ind , four bands appear (Fig. 2, lane 4) which are not repressible by the cI gene product of 2ind . The expression of these proteins is therefore assumed to be under the control of E. coli promoters and genes. The molecular weights of these four proteins from top to bottom are 94,000, 78,000, 38,000 and 22,000. The molecular weight values of the first and the third proteins are identical with those of the fl and c~subunits of PRS, respectively (Hanke et al., 1974; Fayat et al., 1974). Furthermore, the positions of the first and third proteins coincide
Fig. 2. Scintillation autograph (fluorograph) of the SDS gel electrophoretic analysis of radioactively labelled crude extracts from E. coli strains infected with 2 strains. Samples 1, 2, 11, and 12 contain the non-labelled marker proteins bovine serum albumin (Mr 69,000), alcohol dehydrogenase (M r 35,000), chymotrypsinogen A (Mr 25,000), and cytochrome c (M s 12,500), which have been used for molecular weight determination (not shown). Samples 7 and 8 contain non-labelled purified c~ and /3 subunits of PRS, respectively. Their position on the Coomassie blue-stained gel (not shown) is given by the arrows. Samples 3 6 contain the following crude extracts: (3) non-infected strain 1592ind(3480 cpm on the gel), (4) 1592ind- infected with 2p2 (3200 cpm), (5) 159)0ind- infected with Y199 (3200 cpm), (6) strain 159 infected with 2p2 (5800 cpm). Sample 9 contains the anti-PRS cross-reacting material from the high speed supernatant (S100) of the extract of strain 159 infected with Y199 (521 cpm), and sample 10 shows the anti-PRS cross-reacting material from the SI00 of the extract of 159 infected with ,~p2
exactly with the positions of purified non-labelled fi and c~ subunits, respectively, that have been applied on lanes 8 and 7 (Fig. 2) and localized by staining the gel with Coomassie blue (not shown). Finally, the electrophoretic analysis of the immunoprecipitated anti-PRS cross reacting material in the crude extracts of the UVL-irradiated strain 159 infected with 2p2 clearly revealed two protein bands coinciding with the position of the fi and c~ subunits of PRS (Fig. 2, lane 10). These proteins are not precipitated by PRS specific antibodies from crude extracts of strain 159 infected with phage Y199 (Fig. 2, lane 9). For the appearance of the low molecular weight bands in samples 9 and 10 of Figure 2 we have no other explanation at the moment except that they could represent unspecifically precipitated proteins or degradation products.
210
The E. coli DNA-coded protein with the molecular weight of 22,000 daltons was identified as the protein synthesis initiation factor IF3 (Springer et al., 1977b). The protein with the molecular weight of 78,000 daltons has not yet been identified. It will be interesting to see whether it is also a component of the protein synthesizing system. Acknowledgements. We are greatly indebted to Dr. Max Gottesman
for his generous gift of the LFT lysates which have made possible this study. We also wish to thank Monique Graffe and Heidi Franz for their expert assistance. This work has been supported by a grant from the Deutsche Forschungsgemeinschaft and by an EMBO short-term fellowship.
References Bachmann, B.J., Low, K.B., Taylor, A.L.: Recalibrated linkage map of Escherichia coli K-12. Bact. Rev. 40, 116-167 (1976) B6ck, A., Neidhardt, F.C.: Genetic mapping of phenylalanylsRNA synthetase in Escherichia eoli. Science (Wash.) 157, 7 8 3 9 (1967) Bonner, W.M., Laskey, R.A. : A film detection method for tritiumlabelled proteins and nucleic acids in polyacrylamide gels. Europ. J. Biochem. 46, 83-88 (1974) Brice, C.B., Kornberg, H.L.: Genetic control of isocitrate lyase activity in Escheriehia coli. J. Bact. 96, 2t85-2186 (1968) Cameron, J.R., Panasenko, S.M., Lehman, I.R., Davis, R.W.: In vitro construction of bacteriophage 2 carrying segments of the Eseheriehia coli chromosome: Selection of hybrids containing the gene for DNA ligase. Proc. nat. Acad. Sci. (Wash.) 72, 3416-3420 (1975) Campbell, D.H., Garvey, J.S., Cremer, N.E., Sussdorf, D.H.: Methods in immunology, 2nd ed. NewYork: W.A. Benjamin Inc. 1970 Comer, M.M., B6ck, A.: Genes for the e and fl subunits of the phenylalanyl-transfer ribonucleic acid synthetase of Eseherichia coli. J. Bact. 127, 923-933 (1976) Ehrenstein, G. von, Lipmann, F.: Experiments on hemoglobin biosynthesis. Proc. nat. Acad. Sci. (Wash.) 47, 941 950 (1961) Fayat, G., Blanquet, S., Dessen, P., Batelier, G., Waller, J.-P.: The molecular weight and subunit composition of phenylalanyltRNA synthetase from Escherichia coli K12. Biochimie 56, 3541 (1974) Gottesman, M.E., Yarmolinsky, M.B.: Integration-negative mutantsofbacteriophage lambda. J. molec. Biol. 31,487 505 (1968) Hanke, T., Bartmann, P., Hennecke, H., Kosakowski, H.M., Jaenicke, R., Holler, E., B6ck, A. : L-phenylalanyl-tRNA synthetase of Escherichia eoli K10. A reinvestigation of molecular weight and subunit structure. Europ. J. Biochem. 43, 601-607 (1974) Hennecke, H. : Use of mutant enzymes to demonstrate the presence of two active sites on phenytalanyl-tRNA synthetase from E. coli. FEBS Lett. 72, 182=186 (1976) Hennecke H., B6ck, A. : Altered c~subunits in phenylalanyl-tRNA syiithetases from p-fluorophenylalanine-resistant strains of Escherichia eoli. Europ. J. Biochem. 55, 431 437 (1975) Hennecke, H., Walther, I., Franz, H.: Immunochemical studies on phenylalanyl-tRNA synthetase from Escherichia eoli. Hoppe-Seylers Z. physiol. Chem. 358, 197 208 (1977) Jacob, F., Campbell, A.: Sur le syst6me de r6pression assurant l'immunit6 chez les bact6ries lysog6nes. C. R. Acad. Sci. (Paris) 248, 3219-3221 (1959)
H. Hennecke et al. : A ,t Phage Transducing the PRS Genes Jacob, F., Wollmann, W.L.: Etude g6n+tique d'un bact6riophage temper+ d'Escherichia coli. I. Le systbme g6n6tique du bact6riophage 2. Ann. Inst. Pasteur 87, 653~690 (1954) Kosakowski, H.M., B6ck, A.: The subunit structure of phenylalanyl-tRNA synthetase of Escheriehia eoli. Europ. J. Biochem. 12, 67-73 (1970) Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.) 227, 680-685 (1970) Laskey, R.A., Mills, A.D.: Quantitative film detection of aH and 14C in polyacrylamide gels by fluorography. Europ. J. Biochem. 56, 335-341 (1975) Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. : Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275 (1951) Miller, J.H. : Experiments in molecular genetics. New York: Cold Spring Harbor Laboratory 1972 Muench, K.H., Berg, P. : Preparation of aminoacyl-ribonucleic acid synthetases from Escheriehia eoli. In: Procedures in nucleic acid research, Vol. 1, pp. 375-383 (Cantoni, G.L. and Davis, D.R., eds.). New York: Harper and Row, Publishers 1966 Murialdo, H., Siminovitch, L.: The morphogenesis of bacteriophage lambda. III. Identification of genes specifying morphogenetic proteins. In: The bacteriophage lambda, pp. 711-723 (Hershey, A.D., ed.). New York: Cold Spring Harbor Laboratory 1971 Perricaudet, M., Tiollais, P. : Defective bacteriophage chromosome, potential vector for DNA fragments obtained after cleavage by Bacillus amyloliquefaciens endonuclease (BAM I). FEBS Lett. 56, 7-11 (1975) Piepersberg, A., Hennecke, H., Engelhard, M., Nass, G., B6ck, A.: Cross-reactivity of phenylalanyl-transfer ribonucleic acid ligases from different microorganisms. J. Bact. 124, 1482-1488 (1975) Patshne, M. : Isolation of the 2 phage repressor. Proc. nat. Acad. Sci. (Wash.) 57, 306~13 (1967) Rambach, A., Tiollais, P. : Bacteriophage 2 having EcoRI endonuclease sites only in the nonessential region of the genome. Proc. nat. Acad. Sci. (Wash.) 71, 3927 3930 (1974) Russell, R.R.B., Pittard, A.J.: Mutants of Escherichia coli unable to make protein at 42 ° C. J. Bact. 108, 790 798 (1971) Schrenk, W.J., Weisberg, R.A. : A simple method for making new transducing lines of coliphage 2. Molec. gen. Genet. 137, 101-107 (1975) Shimada, K., Weisberg, R., Gottesman, M.: Prophage lambda at unusual chromosomal locations. I. Location of the secondary attachment sites and the properties of the lysogens. J. molec. Biol. 63, 483-503 (1972) Springer, M., Graffe, M., Grunberg-Manago, M. : Characterization of an E. coli mutant with a thermolabile initiation factor IF3 activity. Molec. gen. Genet. 151, 17 26 (1977a) Springer, M., Graffe, M., Hennecke, H.: Specialized transducing phage for initiation factor IF3 gene in E. coli. Proc. nat. Acad. Sci. (Wash.), in press (1977b) Studier, F.W.: Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J. molec. Biol. 79, 2/37 248 (1973) Taylor, A.L., Thoman, M.S.: The genetic map of Escherichia coli K-12. Genetics 50, 659-677 (1964) Zubay, G.J. : The isolation and fractionation of soluble ribonucleic acid. J. molec. Biol. 4, 347-356 (1962)
Communicated by H.G. Wittmann
Received January 20 / February 14, 1977
