Vol. 127, No. 2 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Aug. 1976, P. 917-922
Copyright © 1976 American Society for Microbiology
Isolation of a Lambda Transducing Bacteriophage Carrying the relA Gene of Escherichia coli JAMES D. FRIESEN,* JACK PARKER, ROBERT J. WATSON, NIELS P. FIIL, STEEN PEDERSEN, AND FINN S. PEDERSEN Department of Biology, York University, Downsview, Ontario M3J 1P3, Canada*; University Institute of Microbiology, 0ster Farimagsgade 2A, Copenhagen K, DK 1353, Denmark; and Department of Molecular Biology, University of Aarhus, Aarhus C, DK 8000, Denmark Received for publication 2 February 1976
In Escherichia cali the relA and pyrG loci are 99% cotransducible. On the basis of this knowledge, we have isolated XcI857S7dpyrG transducing bacteriophages carrying both the pyrG and relA genes. Single lysogens of this bacteriophage show basal levels of ppGpp that are 10-fold higher than normal. Stringent factor is present among the gene products synthesized by XdpyrG relA after infection of ultraviolet-killed cells, as analyzed by polyacrylamide gel electrophoresis. The intracellular content of stringent factor, as determined by enzymatic activity, rises 20-fold after induction of a single lysogen of XdpyrG relA. As measured by two-dimensional gel electrophoresis, the amount of stringent factor in an exponentially growing strain carrying a pyrG relA plasmid is at least 10fold greater than in a normal strain. These data constitute strong evidence that stringent factor is the relA gene product. In a manner that is not yet entirely clear, the Muller-Hill (7). Bacteriophage P1 transductions relA gene product in Escherichia coli is in- were carried out as described by Lennox (11). volved with the regulation of ribonucleic acid Tris(hydroxymethyl)aminomethane (Tris)-glucose growth medium was described previously (1), as was synthesis, perhaps through the action of L-broth (11) in which lysogens were grown for largeppGpp, whose production is dependent upon scale virus production. Mutagenesis with N-methylthis gene (3, 4, 6, 13). It would be of great value N'-nitro-N-nitrosoguanidine was carried out as deto isolate a transducing phage carrying the scribed elsewhere (5). relA gene since this ought to be useful in the Radioactive labeling. Methods for the assay of purification of large amounts of stringent fac- intracellular nucleotide pools were described previtor, which appears to be the relA gene product ously (4). Nucleotide pools were measured as fol(2), for carrying out further genetic experi- lows. Strain NF952 was grown at 30°C in Tris-gluments with the relA gene, and for studies of in cose medium supplemented with 0.35 mM phosphate, 50 jig of growth requirements per ml, and vitro synthesis of the relA gene product. Until methionine. One generation before time zero, now, attempts to obtain such a transducing [32P]orthophosphate (50 ,uCi/ml) was added. At time phage have not succeeded because of the lack of zero, a portion of the culture was starved for isoleuany selectable gene linked closely enough to cine by the addition of 250 ,ug of L-valine per ml. At relA to afford a reasonable chance of incorpo- 30 min the main portion of the culture was shifted to rating into the lambda genome a portion of the 42°C, and at 50 min it was shifted to 37°C. Portions of E. coli chromosome with both genes on it. We this culture were subjected to isoleucine starvation report here that pyrG is closely linked to relA at 90 and 150 min. Samples withdrawn at the indiand have used this fact to obtain a relA X cated times were analyzed for guanosine 5'-triphosphate (GTP), ppGpp, and pppGpp pools as described transducing bacteriophage. by Fiil et al. (4). Polyacrylamide gel electrophoresis. '4C-labeled MATERIALS AND METHODS Bacteria, bacteriophage, and culture conditions. Bacterial strains used in this work are described in Table 1. Bacteriophage XcI857S7 were used; lysogens of this phage were prepared by Poul Jorgensen in the attachment deletion strain NF637 given to us by Jorgen Schrenk. The methods used for preparing the lysogens for growth and purification of the bacteriophage stocks were those described by Haseltine and
proteins were prepared from lysates of X-infected, ultraviolet-irradiated cells as described by Watson et al. (14). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was by the method of Laemmli (9). Two-dimensional gel electrophoresis was carried out as described by O'Farrell (12), except that 2% ampholytes (pH 3.5 to 10) only were used in the first dimension and 7.5% acrylamide was used in the second dimension. Stringent factor used as a marker 917
918
FRIESEN ET AL.
was purified as will be described later (F. S. Pederson, in Alfred Benzon Symposium IX, Control of Ribosome Synthesis, in press). Gels were dried and autoradiographed. For the experiment reported in Table 2, cells were labeled with 35SO42- (2 mCi/ml, 0.03 mM). After electrophoresis of the cell sonic extracts, the appropriate spots were cut out and counted as described elsewhere (S. Pederson, S. V. Reeh, J. Parker, R. J. Watson, J. D. Friesen, and N. P. Fiil, Mol. Gen. Genet., in press). Nonribosomal stringent factor assay. Portions containing an amount of cells corresponding to 0.05 to 1.5 ml of a culture having an absorbancy at 450 nm of 1.0 were withdrawn from the culture at 20min intervals. The cells were washed in buffer I (5 mM ethylenediaminetetraacetate, 10 mM Tris-chloride [pH 8.5]) and suspended in 30 ,ul of the same buffer containing 1 mg of lysozyme per ml. The cell suspension was incubated for 5 min at 25°C. At this time, 50 ,ul of buffer II (300 mM NH4CI, 5 mM dithiothreitol, 10 mM Tris-chloride [pH 8.5], 40% ethanol, 1% Brij-58, 100 ,ug of ribonuclease per ml, and 20 ,ug of thiostrepton per ml) was added. One minute after the addition of buffer II, 20 ,ul of buffer III (50 mM adenosine 5'-triphosphate, 2.5 mM GTP, and a-[32P] GTP) was added, and incubation was continued for 60 min at 25°C. The reaction was terminated by the addition of 100 pLI of 10% trichloroacetic acid, and the amount of 32P in pppGpp and ppGpp was determined as described previously (4). Units of stringent factor activity were calculated from the slope of the linear part of the curve in a plot of pppGpp plus ppGpp formation as a function of the amount of cells added. One unit of stringent factor converts 1 ,umol of GTP to pppGpp and ppGpp per min at 25°C under conditions for nonribosomal
synthesis.
J. BACTERIOL.
RESULTS AND DISCUSSION In E. coli the pyrG locus is 99% cotransducible with the relA gene (J. Neuhard, personal communication; our unpublished data). This suggested that isolation of a XdpyrG specialized transducing phage might be a good method of obtaining a phage carrying the relA gene since the latter is not directly selectable. We had available apyrG mutant ofE. coli (S0535 given to us by J. Neuhard) from which we were unable to select a X-sensitive derivative; therefore it was necessary first to transfer pyrG to a Xsensitive strain. Selection for pyrG (a requirement for cytidine) can only take place in a cdd background (inability to convert cytidine to uridine) (8). As has been shown by Karlstrom (8), cdd mutants can be recognized by their ability to grow in the presence of fluorodeoxycytidine and their inability to use deoxycytidine in place of uridine as a growth factor (note that strain AT2538 is pyrE). Three such mutants were selected from strain AT2538 after nitrosoguanidine mutagenesis and selection that made use of the growth characteristics described above. These were found to lack cytidine deaminase enzymatic activity when tested in cell extracts (kindly done for us by A. Munch-Petersen). One of these cdd mutants was transduced to pyrG as described in Table 1. The remainder of the steps in the construction of strain 627 (pyrG relA) are described in Table 1. A X bacteriophage capable of transducing
NF162 JF411 NF637 JF611 JF618
TABLE 1. Strains used Genotype thi-1 pyrE60 argE3 his-4 proA2 thr-1 leu-6 mtl-i xyl-5 ara-14 galK2 lacYl str-31 X- supE44 metB leu-6 his-1 argG6 lacYl malAl xyl-7 mtl-2 gal-6 Astr-104 sup-59 cdd pyrG metB argA relAl spoT As AT2538, but argE+ relC del (gal attX bio uvrB) thi As AT2538, but cdd As JF611, but pyrG
JF622 JF624
As JF618, but spontaneous nalA As JF622, but pyrG+ relAl
JF627
As JF624, but pyrG
JF629 JF630 NF952 JF762
As JF627, but XcI857S7dpyrG relAl Same as JF629, independent As 627, but XcI857S7dpyrG relAl (single lysogen) As NF952, but argE+ relC
CP78 NF987
thi-1 thr leu argH, his JF622 carrying a plasmid containing pyrG+ relA +
Strain AT2538
S0535
Source B. Bachmann
J. Neuhard
J. Schrenk Described in text
P1 transduction from S0535 and penicillin enrichment P1 transduction from NF162 By nitrosoguanidine mutagenesis and penicillin enrichment Described in text Described in text P1 transduction from strain JF411 Described in text Constructed by J. Collins
V VOL. TRANSDUCING PHAGE CARRYING relA GENE 127, 1976
pyrG was isolated from an induced lysogen of strain NF637, an attX deletion strain. Approximately 1011 cells of strain JF627 were infected with a multiplicity of 10 of these X phages and were then plated on agar lacking cytidine. Approximately 70 colonies appeared; these were tested for temperature sensitivity, ability to produce X plaque formers, and ability to trans-
919
duce the pyrG gene. Six of the original colonies had the characteristics expected of cells doubly lysogenic for XdpyrG and X helper; two of these strains, JF629 and JF630, were also capable of transducing the relA gene. Lysates of XdpyrG relA were prepared by heat induction of strain JF630 and purified as described in Materials and Methods. The trans-
0
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a'
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20
40
60
80
100
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140
160
180
TIME (minutes) FIG. 1. ppGpp (0, 0), pppGpp (A, A), and GTP (+) synthesis in an induced single lysogen of XdpyrG relA (strain NF952). The arrows indicate the time of onset of isoleucine starvation. Filled symbols indicate isoleucine-starved culture; open symbols indicate unstarved culture.
0
a) 10 0
a)
c
a)
E0 ._x Q
80
100
120
TIME (minutes)
FIG. 2. ppGpp, pppGpp, and GTP synthesis in an induced single lysogen of XdpyrG relA relC (strain JF762). The experiment was performed as described in the legend to Fig. 1. Symbols are the same as in Fig. 1.
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FRIESEN ET AL.
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4.
_S
A B C D FIG. 3. Autoradiogram of a sodium dodecyl sulfate-polyacrylamide gel showing '4C-labeled proteins synthesized in ultraviolet-killed cells under the direction of various bacteriophages. '4C-labeled proteins were prepared as described in the text and subjected to gel electrophoresis in a 9% polyacrylamide gel in the presence of 0.1 % sodium dodecyl sulfate. Protein synthesized under direction of (A) XcI857S7 (20 p,l containing 55,000 cpm), (B) XcI857S7dpyrG (20 pi; 55,000 cpm), (C) XcI857S7dpyrG relA (20 pi; 60,000
ducing and helper phage bands were approximately the same size in equilibrium CsCl gradients, with the former phage being slightly lighter than the latter. The two bands were separated by only 2 mm in the CsCl equilibrium gradient so it was not possible to obtain a high purification of either phage from its companion. The XdpyrG relA phage appears to be of the Xdgal type based on the restriction endonuclease cutting pattern of the phage deoxyribonucleic acid (J. Collins, N. P. Fiil, P. Jorgensen, and J. D. Friesen, in Alfred Benzon Symposium IX, Control of Ribosome Synthesis, in press). A single lysogen of the XdpyrG relA phage was obtained by infecting strain JF627 with a very low multiplicity (0.01) of the semipurified transducing phage. The single lysogen (strain NF952) was characterized by its inability to grow at 42°C or to produce plaque-forming units and its ability to produce XdpyrG relA transducing phage only when induced in the presence of superinfecting purified helper phage; strain NF952, which is phenotypically stringent, reverted to the relaxed phenotype when heat pulse-cured to the pyrG state. Strain NF952, the single lysogen, was induced and examined for its ability to produce ppGpp and pppGpp (Fig. 1). The striking result obtained is that the basal (i.e., non-amino acidstarved) level of both of these nucleotides rose dramatically shortly after the initiation of phage induction and thereafter increased more than 10-fold to levels comparable to those present in amino acid-starved normal cells. However, the rise in the pool levels of ppGpp and pppGpp when the induced lysogen was subjected to amino acid starvation is no greater than before induction, especially when the increase in the optical density of the culture is taken into account. Induction of a XcI857S7 lysogen or single lysogens of a variety of X transducing phage carrying other E. coli genes did not result in any significant increase of basal level ppGpp or pppGpp (unpublished data). The increase of basal level ppGpp after induction of the XdpyrG relA single lysogen is most likely due to an increase in relA gene product. A relC derivative of strain NF952 was constructed by P1 transduction. This strain (JF762) was heat induced and subjected to amino acid starvation to measure ppGpp synthesis (Fig. 2). Before induction the relC single lysogen has a typical relaxed phenotype. Howcpm), and (D) uninfected, ultraviolet-irradiated E. coli. The arrow is drawn to indicate the position occupied by purified stringent factor.
VOL. 127, 1976
921
TRANSDUCING PHAGE CARRYING relA GENE
reiC strain from relaxed to stringent. This in turn might indicate that the relC lesion results in a decreased affinity of the ribosome for the stringent factor.
0
,t I.C 4
. I
I
0
o I
0
0
,L.
4%
ID 1.
N
z
us
I
z
i-
.
co
on
i ISO 120 60 TIME (MINUTES) FIG. 5. Increase in amount of stringent factor after heat induction of a single lysogen of XdpyrG relA (NF952). A culture growing in M9 plus glucose at 32°C was shifted to 43°C and then to 37°C at the times indicated by the arrows. At intervals, volumes of 0.05 to 1.5 ml were withdrawn and assayed for stringent factor as described in the text. (A) Absorbancy at 450 nm. (B) Stringent factor.
0
TABLE 2. Amount of stringent factor and elongation factor Ga Strain
FIG. 4. Autoradiogram of a two-dimensional electropherogram of '4C-labeled proteins synthesized in ultraviolet-killed cells under the direction of AdpyrG relA. The proteins were prepared and analyzed as described in the text. Stringent factor is circled. ever, after heat induction this strain accumulates a significant amount of ppGpp and pppGpp, indicating that the increased amount of relA gene product in the cell can change the
Stringent factor G (cpm) factor (cpm) Elongation 50 16,775 953 23,100
Ratio
1/330 CP78 1/25 NF987 a Exponentially growing cells of strain CP78 or NF987 were labeled with 3SO42- and subjected to electrophoresis as described in the text. Stringent factor and G factor spots were cut and counted. A background of 30 cpm, obtained by counting a comparable size of gel without any spots, was subtracted.
922
FRIESEN ET AL.
Semipurified XdpyrG relA phage was adsorbed to a culture of ultraviolet-irradiated strain NF637, the infected cells were labeled with [14C]leucine and ['4C]lysine, and the crude cell lysate was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography (Fig. 3). The gel patterns show a protein band from the XdpyrG relA lysate, which is at the position occupied by partially purified stringent factor. XdpyrG or X helper lysates showed no corresponding band (Fig. 3). When analyzed by the two-dimensional electrophoretic method of O'Farrell (12) and autoradiography (Fig. 4), a spot exactly corresponding to that of stringent factor was seen. This is a strong indication that stringent factor is the relA gene product. Further indication of this comes from the observation that an induced XdpyrG relA single lysogen contains at least 20 times as much stringent factor as normal E. coli (Fig. 5). This is also true of a strain carrying a pyrG relA plasmid (Table 2), where the ratio of stringent factor to G factor in the plasmid-carrying cells is some 13-fold higher than in normal cells. However, in our hands the use of the plasmid strain as a source of stringent factor for protein purification was much more erratic than was the induced single lysogen.
ACKNOWLEDGMENTS We thank Isobel Matthews, Joe Motsch, Hanne WormLeonhard, and Ulla Pedersen for technical assistance and Gordon Temple for photography. This research was supported by the National Research Council of Canada (grant 5734), the National Cancer Institute of Canada, the Danish Science Research Council, and a NATO International Research Grant. S.P. is the holder of a NATO Fellowship, and R.J.W. is the recipient of a National Research Council of Canada Postgraduate Scholarship.
J. BACTERIOL. LITERATURE CITED 1. Anderson, E. H. 1946. Growth requirements of virus resistant mutants of Escherichia coli strain B. Proc. Natl. Acad. Sci. U.S.A. 32:120-126. 2. Block, R., and W. A. Haseltine. 1973. Thenrolability of the stringent factor in rel mutants of E. coli. J. Mol. Biol. 77:625-628. 3. Cashel, M., and J. Gallant. 1969. Two compounds implicated in the function of the RC gene of Escherichia coli. Nature (London) 221:838-841. 4. Fiil, N. P., K. von Meyenburg, and J. D. Friesen. 1972. Accumulation and turnover of guanosine tetraphosphate inEscherichia coli. J. Mol. Biol. 71:769-783. 5. Guerola, N., J. L. Ingraham, and E. Cerdk-Olmedo. 1971. Induction of closely linked multiple mutations by nitrosoguanidine. Nature (London) New Biol. 230:122-125. 6. Haseltine, W. A., R. Block, K. Weber, and W. Gilbert. 1972. MSI and MSII made on the ribosome in idling step of protein synthesis. Nature (London) 238:381385. 7. Haseltint, W. A., and B. Muller-Hill. 1972. Transformation with xhSOdlac DNA and measurement of lac messenger RNA, p. 319-327. In J. F. Miller (ed.), Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 8. Karlstrom, 0. 1968. Mutants of Escherichia coli defective in ribonucleoside and deoxyribonucleoside catabolism. J. Bacteriol. 95:1069-1077. 9. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 10. Lederberg, J., and N. Zinder. 1948. Concentration of biochemical mutants of bacteria with penicillin. J. Am. Chem. Soc. 70:4267-4268. 11. Lennox, E. S. 1955. Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1:190-206. 12. O'Farrell, P. H. 1975. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250:40074021. 13. Pederson, F. S., E. Lund, and N. 0. Kjeldgaard. 1973. Codon specific tRNA dependent in uitro synthesis of ppGpp and pppGpp. Nature (London) New Biol. 243:13-15. 14. Watson, R. J., J. Parker, N. P. Fiil, J. G. Flaks, and J. D. Friesen. 1975. New chromosomal location for structural genes of ribosomal proteins. Proc. Natl. Acad. Sci. U.S.A. 72:2765-2769.
JOURNAL OF BACTERIOLOGY, Aug. 1976, P. 917-922
Copyright © 1976 American Society for Microbiology
Isolation of a Lambda Transducing Bacteriophage Carrying the relA Gene of Escherichia coli JAMES D. FRIESEN,* JACK PARKER, ROBERT J. WATSON, NIELS P. FIIL, STEEN PEDERSEN, AND FINN S. PEDERSEN Department of Biology, York University, Downsview, Ontario M3J 1P3, Canada*; University Institute of Microbiology, 0ster Farimagsgade 2A, Copenhagen K, DK 1353, Denmark; and Department of Molecular Biology, University of Aarhus, Aarhus C, DK 8000, Denmark Received for publication 2 February 1976
In Escherichia cali the relA and pyrG loci are 99% cotransducible. On the basis of this knowledge, we have isolated XcI857S7dpyrG transducing bacteriophages carrying both the pyrG and relA genes. Single lysogens of this bacteriophage show basal levels of ppGpp that are 10-fold higher than normal. Stringent factor is present among the gene products synthesized by XdpyrG relA after infection of ultraviolet-killed cells, as analyzed by polyacrylamide gel electrophoresis. The intracellular content of stringent factor, as determined by enzymatic activity, rises 20-fold after induction of a single lysogen of XdpyrG relA. As measured by two-dimensional gel electrophoresis, the amount of stringent factor in an exponentially growing strain carrying a pyrG relA plasmid is at least 10fold greater than in a normal strain. These data constitute strong evidence that stringent factor is the relA gene product. In a manner that is not yet entirely clear, the Muller-Hill (7). Bacteriophage P1 transductions relA gene product in Escherichia coli is in- were carried out as described by Lennox (11). volved with the regulation of ribonucleic acid Tris(hydroxymethyl)aminomethane (Tris)-glucose growth medium was described previously (1), as was synthesis, perhaps through the action of L-broth (11) in which lysogens were grown for largeppGpp, whose production is dependent upon scale virus production. Mutagenesis with N-methylthis gene (3, 4, 6, 13). It would be of great value N'-nitro-N-nitrosoguanidine was carried out as deto isolate a transducing phage carrying the scribed elsewhere (5). relA gene since this ought to be useful in the Radioactive labeling. Methods for the assay of purification of large amounts of stringent fac- intracellular nucleotide pools were described previtor, which appears to be the relA gene product ously (4). Nucleotide pools were measured as fol(2), for carrying out further genetic experi- lows. Strain NF952 was grown at 30°C in Tris-gluments with the relA gene, and for studies of in cose medium supplemented with 0.35 mM phosphate, 50 jig of growth requirements per ml, and vitro synthesis of the relA gene product. Until methionine. One generation before time zero, now, attempts to obtain such a transducing [32P]orthophosphate (50 ,uCi/ml) was added. At time phage have not succeeded because of the lack of zero, a portion of the culture was starved for isoleuany selectable gene linked closely enough to cine by the addition of 250 ,ug of L-valine per ml. At relA to afford a reasonable chance of incorpo- 30 min the main portion of the culture was shifted to rating into the lambda genome a portion of the 42°C, and at 50 min it was shifted to 37°C. Portions of E. coli chromosome with both genes on it. We this culture were subjected to isoleucine starvation report here that pyrG is closely linked to relA at 90 and 150 min. Samples withdrawn at the indiand have used this fact to obtain a relA X cated times were analyzed for guanosine 5'-triphosphate (GTP), ppGpp, and pppGpp pools as described transducing bacteriophage. by Fiil et al. (4). Polyacrylamide gel electrophoresis. '4C-labeled MATERIALS AND METHODS Bacteria, bacteriophage, and culture conditions. Bacterial strains used in this work are described in Table 1. Bacteriophage XcI857S7 were used; lysogens of this phage were prepared by Poul Jorgensen in the attachment deletion strain NF637 given to us by Jorgen Schrenk. The methods used for preparing the lysogens for growth and purification of the bacteriophage stocks were those described by Haseltine and
proteins were prepared from lysates of X-infected, ultraviolet-irradiated cells as described by Watson et al. (14). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was by the method of Laemmli (9). Two-dimensional gel electrophoresis was carried out as described by O'Farrell (12), except that 2% ampholytes (pH 3.5 to 10) only were used in the first dimension and 7.5% acrylamide was used in the second dimension. Stringent factor used as a marker 917
918
FRIESEN ET AL.
was purified as will be described later (F. S. Pederson, in Alfred Benzon Symposium IX, Control of Ribosome Synthesis, in press). Gels were dried and autoradiographed. For the experiment reported in Table 2, cells were labeled with 35SO42- (2 mCi/ml, 0.03 mM). After electrophoresis of the cell sonic extracts, the appropriate spots were cut out and counted as described elsewhere (S. Pederson, S. V. Reeh, J. Parker, R. J. Watson, J. D. Friesen, and N. P. Fiil, Mol. Gen. Genet., in press). Nonribosomal stringent factor assay. Portions containing an amount of cells corresponding to 0.05 to 1.5 ml of a culture having an absorbancy at 450 nm of 1.0 were withdrawn from the culture at 20min intervals. The cells were washed in buffer I (5 mM ethylenediaminetetraacetate, 10 mM Tris-chloride [pH 8.5]) and suspended in 30 ,ul of the same buffer containing 1 mg of lysozyme per ml. The cell suspension was incubated for 5 min at 25°C. At this time, 50 ,ul of buffer II (300 mM NH4CI, 5 mM dithiothreitol, 10 mM Tris-chloride [pH 8.5], 40% ethanol, 1% Brij-58, 100 ,ug of ribonuclease per ml, and 20 ,ug of thiostrepton per ml) was added. One minute after the addition of buffer II, 20 ,ul of buffer III (50 mM adenosine 5'-triphosphate, 2.5 mM GTP, and a-[32P] GTP) was added, and incubation was continued for 60 min at 25°C. The reaction was terminated by the addition of 100 pLI of 10% trichloroacetic acid, and the amount of 32P in pppGpp and ppGpp was determined as described previously (4). Units of stringent factor activity were calculated from the slope of the linear part of the curve in a plot of pppGpp plus ppGpp formation as a function of the amount of cells added. One unit of stringent factor converts 1 ,umol of GTP to pppGpp and ppGpp per min at 25°C under conditions for nonribosomal
synthesis.
J. BACTERIOL.
RESULTS AND DISCUSSION In E. coli the pyrG locus is 99% cotransducible with the relA gene (J. Neuhard, personal communication; our unpublished data). This suggested that isolation of a XdpyrG specialized transducing phage might be a good method of obtaining a phage carrying the relA gene since the latter is not directly selectable. We had available apyrG mutant ofE. coli (S0535 given to us by J. Neuhard) from which we were unable to select a X-sensitive derivative; therefore it was necessary first to transfer pyrG to a Xsensitive strain. Selection for pyrG (a requirement for cytidine) can only take place in a cdd background (inability to convert cytidine to uridine) (8). As has been shown by Karlstrom (8), cdd mutants can be recognized by their ability to grow in the presence of fluorodeoxycytidine and their inability to use deoxycytidine in place of uridine as a growth factor (note that strain AT2538 is pyrE). Three such mutants were selected from strain AT2538 after nitrosoguanidine mutagenesis and selection that made use of the growth characteristics described above. These were found to lack cytidine deaminase enzymatic activity when tested in cell extracts (kindly done for us by A. Munch-Petersen). One of these cdd mutants was transduced to pyrG as described in Table 1. The remainder of the steps in the construction of strain 627 (pyrG relA) are described in Table 1. A X bacteriophage capable of transducing
NF162 JF411 NF637 JF611 JF618
TABLE 1. Strains used Genotype thi-1 pyrE60 argE3 his-4 proA2 thr-1 leu-6 mtl-i xyl-5 ara-14 galK2 lacYl str-31 X- supE44 metB leu-6 his-1 argG6 lacYl malAl xyl-7 mtl-2 gal-6 Astr-104 sup-59 cdd pyrG metB argA relAl spoT As AT2538, but argE+ relC del (gal attX bio uvrB) thi As AT2538, but cdd As JF611, but pyrG
JF622 JF624
As JF618, but spontaneous nalA As JF622, but pyrG+ relAl
JF627
As JF624, but pyrG
JF629 JF630 NF952 JF762
As JF627, but XcI857S7dpyrG relAl Same as JF629, independent As 627, but XcI857S7dpyrG relAl (single lysogen) As NF952, but argE+ relC
CP78 NF987
thi-1 thr leu argH, his JF622 carrying a plasmid containing pyrG+ relA +
Strain AT2538
S0535
Source B. Bachmann
J. Neuhard
J. Schrenk Described in text
P1 transduction from S0535 and penicillin enrichment P1 transduction from NF162 By nitrosoguanidine mutagenesis and penicillin enrichment Described in text Described in text P1 transduction from strain JF411 Described in text Constructed by J. Collins
V VOL. TRANSDUCING PHAGE CARRYING relA GENE 127, 1976
pyrG was isolated from an induced lysogen of strain NF637, an attX deletion strain. Approximately 1011 cells of strain JF627 were infected with a multiplicity of 10 of these X phages and were then plated on agar lacking cytidine. Approximately 70 colonies appeared; these were tested for temperature sensitivity, ability to produce X plaque formers, and ability to trans-
919
duce the pyrG gene. Six of the original colonies had the characteristics expected of cells doubly lysogenic for XdpyrG and X helper; two of these strains, JF629 and JF630, were also capable of transducing the relA gene. Lysates of XdpyrG relA were prepared by heat induction of strain JF630 and purified as described in Materials and Methods. The trans-
0
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a) 500
V
0
o 400 c
a'
300
2E
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.6
2001
0
20
40
60
80
100
120
140
160
180
TIME (minutes) FIG. 1. ppGpp (0, 0), pppGpp (A, A), and GTP (+) synthesis in an induced single lysogen of XdpyrG relA (strain NF952). The arrows indicate the time of onset of isoleucine starvation. Filled symbols indicate isoleucine-starved culture; open symbols indicate unstarved culture.
0
a) 10 0
a)
c
a)
E0 ._x Q
80
100
120
TIME (minutes)
FIG. 2. ppGpp, pppGpp, and GTP synthesis in an induced single lysogen of XdpyrG relA relC (strain JF762). The experiment was performed as described in the legend to Fig. 1. Symbols are the same as in Fig. 1.
920
FRIESEN ET AL.
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4.
_S
A B C D FIG. 3. Autoradiogram of a sodium dodecyl sulfate-polyacrylamide gel showing '4C-labeled proteins synthesized in ultraviolet-killed cells under the direction of various bacteriophages. '4C-labeled proteins were prepared as described in the text and subjected to gel electrophoresis in a 9% polyacrylamide gel in the presence of 0.1 % sodium dodecyl sulfate. Protein synthesized under direction of (A) XcI857S7 (20 p,l containing 55,000 cpm), (B) XcI857S7dpyrG (20 pi; 55,000 cpm), (C) XcI857S7dpyrG relA (20 pi; 60,000
ducing and helper phage bands were approximately the same size in equilibrium CsCl gradients, with the former phage being slightly lighter than the latter. The two bands were separated by only 2 mm in the CsCl equilibrium gradient so it was not possible to obtain a high purification of either phage from its companion. The XdpyrG relA phage appears to be of the Xdgal type based on the restriction endonuclease cutting pattern of the phage deoxyribonucleic acid (J. Collins, N. P. Fiil, P. Jorgensen, and J. D. Friesen, in Alfred Benzon Symposium IX, Control of Ribosome Synthesis, in press). A single lysogen of the XdpyrG relA phage was obtained by infecting strain JF627 with a very low multiplicity (0.01) of the semipurified transducing phage. The single lysogen (strain NF952) was characterized by its inability to grow at 42°C or to produce plaque-forming units and its ability to produce XdpyrG relA transducing phage only when induced in the presence of superinfecting purified helper phage; strain NF952, which is phenotypically stringent, reverted to the relaxed phenotype when heat pulse-cured to the pyrG state. Strain NF952, the single lysogen, was induced and examined for its ability to produce ppGpp and pppGpp (Fig. 1). The striking result obtained is that the basal (i.e., non-amino acidstarved) level of both of these nucleotides rose dramatically shortly after the initiation of phage induction and thereafter increased more than 10-fold to levels comparable to those present in amino acid-starved normal cells. However, the rise in the pool levels of ppGpp and pppGpp when the induced lysogen was subjected to amino acid starvation is no greater than before induction, especially when the increase in the optical density of the culture is taken into account. Induction of a XcI857S7 lysogen or single lysogens of a variety of X transducing phage carrying other E. coli genes did not result in any significant increase of basal level ppGpp or pppGpp (unpublished data). The increase of basal level ppGpp after induction of the XdpyrG relA single lysogen is most likely due to an increase in relA gene product. A relC derivative of strain NF952 was constructed by P1 transduction. This strain (JF762) was heat induced and subjected to amino acid starvation to measure ppGpp synthesis (Fig. 2). Before induction the relC single lysogen has a typical relaxed phenotype. Howcpm), and (D) uninfected, ultraviolet-irradiated E. coli. The arrow is drawn to indicate the position occupied by purified stringent factor.
VOL. 127, 1976
921
TRANSDUCING PHAGE CARRYING relA GENE
reiC strain from relaxed to stringent. This in turn might indicate that the relC lesion results in a decreased affinity of the ribosome for the stringent factor.
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i ISO 120 60 TIME (MINUTES) FIG. 5. Increase in amount of stringent factor after heat induction of a single lysogen of XdpyrG relA (NF952). A culture growing in M9 plus glucose at 32°C was shifted to 43°C and then to 37°C at the times indicated by the arrows. At intervals, volumes of 0.05 to 1.5 ml were withdrawn and assayed for stringent factor as described in the text. (A) Absorbancy at 450 nm. (B) Stringent factor.
0
TABLE 2. Amount of stringent factor and elongation factor Ga Strain
FIG. 4. Autoradiogram of a two-dimensional electropherogram of '4C-labeled proteins synthesized in ultraviolet-killed cells under the direction of AdpyrG relA. The proteins were prepared and analyzed as described in the text. Stringent factor is circled. ever, after heat induction this strain accumulates a significant amount of ppGpp and pppGpp, indicating that the increased amount of relA gene product in the cell can change the
Stringent factor G (cpm) factor (cpm) Elongation 50 16,775 953 23,100
Ratio
1/330 CP78 1/25 NF987 a Exponentially growing cells of strain CP78 or NF987 were labeled with 3SO42- and subjected to electrophoresis as described in the text. Stringent factor and G factor spots were cut and counted. A background of 30 cpm, obtained by counting a comparable size of gel without any spots, was subtracted.
922
FRIESEN ET AL.
Semipurified XdpyrG relA phage was adsorbed to a culture of ultraviolet-irradiated strain NF637, the infected cells were labeled with [14C]leucine and ['4C]lysine, and the crude cell lysate was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography (Fig. 3). The gel patterns show a protein band from the XdpyrG relA lysate, which is at the position occupied by partially purified stringent factor. XdpyrG or X helper lysates showed no corresponding band (Fig. 3). When analyzed by the two-dimensional electrophoretic method of O'Farrell (12) and autoradiography (Fig. 4), a spot exactly corresponding to that of stringent factor was seen. This is a strong indication that stringent factor is the relA gene product. Further indication of this comes from the observation that an induced XdpyrG relA single lysogen contains at least 20 times as much stringent factor as normal E. coli (Fig. 5). This is also true of a strain carrying a pyrG relA plasmid (Table 2), where the ratio of stringent factor to G factor in the plasmid-carrying cells is some 13-fold higher than in normal cells. However, in our hands the use of the plasmid strain as a source of stringent factor for protein purification was much more erratic than was the induced single lysogen.
ACKNOWLEDGMENTS We thank Isobel Matthews, Joe Motsch, Hanne WormLeonhard, and Ulla Pedersen for technical assistance and Gordon Temple for photography. This research was supported by the National Research Council of Canada (grant 5734), the National Cancer Institute of Canada, the Danish Science Research Council, and a NATO International Research Grant. S.P. is the holder of a NATO Fellowship, and R.J.W. is the recipient of a National Research Council of Canada Postgraduate Scholarship.
J. BACTERIOL. LITERATURE CITED 1. Anderson, E. H. 1946. Growth requirements of virus resistant mutants of Escherichia coli strain B. Proc. Natl. Acad. Sci. U.S.A. 32:120-126. 2. Block, R., and W. A. Haseltine. 1973. Thenrolability of the stringent factor in rel mutants of E. coli. J. Mol. Biol. 77:625-628. 3. Cashel, M., and J. Gallant. 1969. Two compounds implicated in the function of the RC gene of Escherichia coli. Nature (London) 221:838-841. 4. Fiil, N. P., K. von Meyenburg, and J. D. Friesen. 1972. Accumulation and turnover of guanosine tetraphosphate inEscherichia coli. J. Mol. Biol. 71:769-783. 5. Guerola, N., J. L. Ingraham, and E. Cerdk-Olmedo. 1971. Induction of closely linked multiple mutations by nitrosoguanidine. Nature (London) New Biol. 230:122-125. 6. Haseltine, W. A., R. Block, K. Weber, and W. Gilbert. 1972. MSI and MSII made on the ribosome in idling step of protein synthesis. Nature (London) 238:381385. 7. Haseltint, W. A., and B. Muller-Hill. 1972. Transformation with xhSOdlac DNA and measurement of lac messenger RNA, p. 319-327. In J. F. Miller (ed.), Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 8. Karlstrom, 0. 1968. Mutants of Escherichia coli defective in ribonucleoside and deoxyribonucleoside catabolism. J. Bacteriol. 95:1069-1077. 9. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 10. Lederberg, J., and N. Zinder. 1948. Concentration of biochemical mutants of bacteria with penicillin. J. Am. Chem. Soc. 70:4267-4268. 11. Lennox, E. S. 1955. Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1:190-206. 12. O'Farrell, P. H. 1975. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250:40074021. 13. Pederson, F. S., E. Lund, and N. 0. Kjeldgaard. 1973. Codon specific tRNA dependent in uitro synthesis of ppGpp and pppGpp. Nature (London) New Biol. 243:13-15. 14. Watson, R. J., J. Parker, N. P. Fiil, J. G. Flaks, and J. D. Friesen. 1975. New chromosomal location for structural genes of ribosomal proteins. Proc. Natl. Acad. Sci. U.S.A. 72:2765-2769.
