Proc. Nat. Acad. Sci. USA
Vol. 72, No. 1, pp. 6-10, January 1975
Specialized Transducing Phages for Ribosomal Protein Genes of Escherichia coli* (bacteriophage X/in vitro protein synthesis/protein identification)
S. RICHARD JASKUNAS, LASSE LINDAHL, AND MASAYASU NOMURA Institute for Enzyme Research, and Departments of Biochemistry and Genetics, University of Wisconsin, Madison, Wis. 53706
Communicated by Henry Lardy, September 13, 1974 K12 strain, EC-2, in which lac had been transposed close to aroE (12). A lacZ- mutant (NO1180) was isolated and attX was deleted (NO1324) (13). (See Table 1.) Phage xCI857S7plac5 (14, 16) was integrated into the lac genes near aroE (NO1325). A low-frequency transducing lysate (LFT) was obtained by thermal induction of NO1325 following ultraviolet (UV) irradiation. A XdaroE phage was isolated from the lysate using XCI857S7 as helper and an oroE- strain (NO 1223) as a recipient. This XdaroE was subsequently integrated into the aroE locus of another strain (NO1257) that was deleted for attX and was aroE- spcs strr fus8. An LFT for trkA was obtained by thermal induction of the resultant lysogen (NO1326) following superinfection by XC1857S7. The trkA mutant allele confers a requirement for a high concentration of potassium for growth, approximately 0.1 M, when it is coupled with the kdpABC5 mutation (15). The selection for trkA + is for growth on a low concentration of potassium. Phages transducing trkA were isolated in a strain (NO1230) with the following
Specialized lambda transducing phages ABSTRACT have been isolated carrying approximately half the ribosomal protein genes of E. coli. These phages carry regions of the bacterial chromosome between aroE and fus. The ribosomal protein genes on these phages have been identified by the stimulation of ribosomal protein synthesis in ultraviolet-irradiated bacteria following infection by the transducing phage, and by the in vitro synthesis of ribosomal proteins in a DNA-dependent protein synthesizing system. The results indicate Xdspcl probably carries at least 22 ribosomal protein genes and Xdspc2 at least 26 genes. All these genes are clustered between trkA and strA. At least 13 of them have not been previously mapped.
Many of the genes for the ribosomal proteins (r-proteins) of Escherichia coli are clustered near the strA locus (for a review, see ref. 1). Intergeneric crosses have suggested that at least 22 out of approximately 50 r-protein genes are in this region of the chromosome (2-5). At present not all the r-protein genes can be distinguished in intergeneric crosses. Thus, some of the other r-protein genes may also be in this cluster. Only one structural gene has been unambiguously mapped in another region of the chromosome, that is, the gene for the 30S protein S18 (6, 7). The clustering of these genes near the str locus suggests they may be organized into transcriptional units or operons (compare refs. 1 and 8). Such genetic organization would provide a mechanism for the coordinate expression of the genes in these genetic units. Furthermore, the coordinate expression of unlinked ribosome transcriptional units, such as r-protein genes with rRNA, could result from regulation of the transcriptional units as a whole. Synthesis of all r-proteins and rRNAs is coordinately regulated in E. coli during steady state
relevant markers: trkA401 kdpABC5 spcr strr fusr. We then
screened for transductants that were sensitive to spectinomycin (Spc-S). Since sensitive alleles at spc, str, and fus are dominant over corresponding resistant alleles (1, 8), the Spc-S phenotype is an indication of the presence of the spcB gene on transducing phages. Xdtrk was obtained from a transductant (NO1275) that remained resistant to Spc, streptomycin (Str), and fusidic acid (Fus). Xdspcl came from transductant (NO1267) that was Spc-S, Str-R, and Fus-R. We also obtained an LFT by thermal induction from a XCI857S7 lysogen into which the Xdaro phage had been inserted at aroE by P1 transduction from NO1326. This strain (NO1327) was spc8 str8 fuss. Phages transducing trkA were selected as above using the strain N01230 as a recipient, and Xdspc2 was obtained from a transductant (NO1328) that was Spc-S, Str-R, and Fus-R. Since the recipient remained Str-R when Xdspc2 was integrated we conclude this phage does not carry the str8 allele of the donor in a functioning state. Table 1 lists some of the above strains and their phenotypes. Analysis of phage DNAs with restriction endonucleases (F. Blattner and S. R. Jaskunas, unpublished experiments) and electron microscopic examination of heteroduplexes (M. Fiandt, W. Szybalski and M. Nomura, unpublished experiments) have indicated that the bacterial DNA ini Xdtrk, Xdspcl, and Xdspc2 is substituted into the left arm of lambda. All three carry the C1857 and S7 genes of the starting Xplac5. All strains we used were derivatives of E. coli K12. Transduction of trkA was done as described by Epstein and Kim (15) except we used plates with 2.5 mM potassium instead of
growth (9-11). Further investigation of the genetic organization and physiological regulation of r-protein genes would be greatly aided by the isolation of specialized transducing phages carrying r-protein genes. This communication describes the isolation of lambda phages carrying many of the r-protein genes of E. coli near strA, the identification of the r-protein genes on the phages, and the expression of some of these genes in an in vitro DNA-dependent protein synthesizing system. MATERIALS AND METHODS
Isolation of Transducing Phages. We started with an E. coli Abbreviations: r-protein, ribosomal protein; Spc, spectinomycin; Str, streptomycin; Fus, fusidic acid; R, resistant; S, sensitive; UV, ultraviolet; LFT, low frequency transducing lysate. * This is paper no. 1784 from the Laboratory of Genetics.
6
Proc. Nat. Acad. Sci. USA 72
Transducing Phages for Ribosomal Protein Genes
(1975)
7
TABLE 1. Sone bacterial strains used in the present study
Strain
N01324 N01325 N01257 N01326 N01327 N01230 N01275 N01267 N01328
Synonym and relevant genotype EC-2lac-AattX N01324 (Xplac) aroE- strr AattX Alac,pro
N01257 (Xdaro) aroE- str' (Xdaro) (XCI857S7) trkA401 kdpABC5 spcr strr fusr N01230 (Xdtrk) (XC1857S7) N01230 (Xdspcl) (XCI857S7) N01230 (Xdspc2) (XC1857S7)
Lac + -
Aro
+ -
+ + + +
-
+
Phenotype* Trk Spc S + S + S + S + S + R
+ + -
+
+ +
R S
+
S
Str S S
Fus S S S
R R S
S S R R R R
R R R R
* Lac is ability to grow on lactose; Aro is ability to grow without exogenous aromatic amino acids (and shikimic acid); other symbols are explained in the text.
0.1 mM and enriched with 0.1% vitamin-free casamino acids. Plates for testing sensitivity to Spc, Str, and Fus have been described (8). The defective transducing phages were separated from helper on CsCl gradients as described by Miller (16). In Vivo Experiments. Synthesis of r-proteins was examined after infection of UV-irradiated bacteria by transducing phages. The method was adapted from those described by previous workers (17, 18) (see legend to Table 2). The strain used as a host was NO1261, a uvrB mutant that was obtained by deleting attX.
In Vitro Experiments. The in vitro synthesis and detection of r-proteins in a DNA-dependent protein synthesizing system was done as previously described (19) (see legends to Figs. 2 and 3). RESULTS
Genetics. The regions of the E. coli chromosome carried by the various lambda transducing phages that we have isolated are illustrated schematically in Fig. 1. As described in the Materials and Methods section the presence of the spc5 allele on Xdspcl and Xdspc2 has been shown by the Spc-S phenotype of the strain N01230 (trkA - spcr strrfusr) lysogenized with these transducing phages (see Table 1). The Xdtrk phage does not carry the spc locus by this test. Since the product of the spcA gene is r-protein S5 (1), we conclude that both Xdspcl and Xdspc2 express this r-protein gene in stable lysogens. The presence of at least a part of the str gene on Xdspcl has been suggested from several genetic experiments. For example, this phage could transduce a strd strain to Str-independence with the frequency of about 10-4, presumably using a recombination mechanism. Neither the Xdtrk nor the Xdspc2 phages can transduce the strd strain to Str-independence. Expression of the Phage-Coded Ribosomal Protein 'Genes In Vivo. In order to identify other r-protein genes on the phages we have measured the stimulation of r-protein synthesis in E. coli cells following infection by the transducing phages. The expression of the host r-protein genes was reduced by prior UV irradiation of the host. The experimental procedures are described in the legend to Table 2. It should be noted that the measurements were done on the total r-proteins including
those which may not have incorporated into ribosome particles. The stimulation of the synthesis of individual r-proteins by Xdtrk, Xdspcl, and Xdspc2 relative to cells infected with XCI857S7 is shown in Table 2. There are 26 proteins whose synthesis was stimulated 4-fold or greater by Xdspc2 and 22 for Xdspcl. This stimulation probably occurred because the structural genes for these proteins are on the phages. Xdtrk does not carry any r-protein genes that can be detected with this method. The synthesis of most r-proteins was stimulated to approximately the same extent by Xdspcl and Xdspc2. However, the synthesis of S3, S17, S19, L2, L16, and L29 was stimulated to a significantly greater extent by Xdspc2 than by Xdspcl. The genes for S17 and L29 seem to be on Xdspc2 but not Xdspcl. The genes for S3 and L16 seem to be on both phages but expressed to a different extent. Finally, while the genes for S19 and L2 seem to be on Xdspc2, it is not clear whether they are present on Xdspcl. We have not observed any stimulation of the synthesis of the product of the strA gene, S12, with either phage significantly greater than with Xdtrk. The data shown in Table 2 (and other experiments not shown) suggest that the expression of the various r-protein genes on the transducing phages in UV-irradiated cells is not coordinate. The reason for this apparent noncoordinate expression is not known. We have studied stability of rproteins synthesized under these conditions by pulse-labeling for 1 min followed by chasing with nonradioactive amino acids. It was found that several r-proteins synthesized are somewhat unstable. However, the instabilities of the r-proteins in these cells did not differ sufficiently to account for the observed variation in the net synthesis rate. aroE
trkA
spcA
strA
fus
Adaro
Adtrk Adspc2 Adspcl
FIG. 1. The str-spc region on the E. coli genetic map and the genes incorporated into various X transducing phages. The "end points" of the various phages are not 'exactly known. Xdspcl ap~pears to carry at least a part of the str gene. However, no additional r-protein genes have been detected on Xdspcl relative to )kdspc2 (see the text).
8
Biochemistry: Jaskunas et al.
Proc. Nat. Acad. Sci. USA 72 (1976)
TABLE 2. Stimulation of the synthesis of individual ribosomal proteins by Xdtrk, Xdspcl, and Xdspc2 Protein
Si S2 S3 S4 S5 S6 S7 S8 S9 | S115 S10 S12 S13 S14 S15 S16 S17 S18
Xdtrk
Xdspc2
0.9 1.5 1.2 1.3 1.0 1.1 1.1
Xdspcl 0.5 4.3 35.7 40.2 0.8 8.8 6.8
1.1
14.7
15.0
* 1.0 2.0 1.0 1.1 1.0 1.3 1.1 0.8
* 1.5 3.2 22.2 15.6 1.2 1.1 1.2 1.1
0.5 17.3 29.0 45.0 0.8 6.5 7.0 * 0.9 1.9 18.7 15.4 3.0 1.7 6.0 0.7
Protein
Xdtrk
Xdspcl
Xdspc2
Protein
Xdtrk
Xdspcl
Xdspc2
S19 S20 S21
0.9
2.1 2.1 1.5 1.8 3.4 2.3 0.5 26.2 22.0 0.7 0.8 0.7 0.6 14.6 1.1 5.1 47.3 58.0
4.3 0.8 0.9 0.9 17.2 1.4 1.2 25.7 21.0 0.6 0.7 0.5 0.6 15.6 0.8 4.5 47.2 75.4
L16 L17 L18 L19 L20 L21 L22 L23 L24 L25 L27 L28 L29 L30 L31 L32 L33
1.0 1.3 0.9 1.0 1.2 1.0 1.1 1.0 1.0 1.0 1.0 1.0 0.9 0.9
7.9 59.7 23.2 6.7
26.7 50.9 23.6 6.0
Li L2 L3 L4 L5 L6 L7 L8 L9 L10
Lii L12 L13 L14 L15
0.7 1.0 1.1 1.0 1.3 1.0 1.0 1.1 1.0
1.0 1.0 1.0 1.2 1.1 1.1 1.1 1.0
1.4 1.0
1.7 1.0 9.8 4.0 37.9 2.1 1.7 2.0 0.9 33.0 2.0 1.2
1.6 0.8
14.3 4.8 37.6 2.0 1.0 1.4 15.7 33.9 2.0 1.0
Cells exponentially growing in minimal maltose medium were labeled with [14C]leucine (0.75 MCi/ml, 281 Ci/mol) for at least three generations during which the label was completely incorporated. When the culture reached 2 X 108/ml it was concentrated to 1 X 109/ml in the same medium and irradiated for 12 min, 30 cm from a germicidal lamp (4 ml of cell suspension per 100-mm diameter glass petri dish). Magnesium sulfate was added to 0.02 M and the cells were infected with phages at a multiplicity of infection of 5. The transducing phages used in these experiments were purifiod on CsCl gradients and contained approximately 1% contaminating helper phage. Control cells were infected with XCI857S7. Protein synthesis in the noninfected irradiated cells was approximately 0.4% of that of the nonirradiated cells and all phages stimulated protein synthesis approximately 10-fold. Following a 15-min incubation at 370 to allow phage absorption, the culture was diluted 5-fold with prewarmed minimal maltose media. The [3H]leucine (15 1Ci/ml, 55 Ci/mmol) was added 15 min later. The incorporation was stopped after 10 min by adding excess nonradioactive leucine, incubating for 30 sec, and pouring the cells on ice. Samples of the cells were precipitated on Whatman 3MM paper with 7% trichloroacetic acid + nonradioactive leucine and worked up essentially as previously described (22). These filters were later oxidized to measure the aH/14C ratio in total protein. The remaining cells were lysed by the lysozyme freeze-thaw technique (9), carrier ribosomes were added, and proteins were extracted with acetic acid and separated by electrophoresis on a two-dimensional gel (23). The 3H/14C ratio of each r-protein spot, except Si and L31, was determined after oxidizing the stained spot in a Packard oxidizer. (S1 was not separated from non-ribosomal proteins and no stained spot was seen at the position of L31.) The numbers shown in the table for r-protein i are the ratios of Ai for bacteria infected with a transducing phage to Ai for bacteria infected with XCI857S7. Ai is a measure of the differential synthesis rate of the ith r-protein after infection relative to that in normal cells and is defined: (8H/14C) in ith r-protein 3H in ith r-protein/8H in total protein = (SH/14C) in total protein 14C in ith r-protein/14C in total protein. Results of two experiments have been averaged. Not all the proteins are completely resolved from other r-proteins in the two-dimensional gel. Those proteins where there is significant overlap include: S9 and S11, S5 and Lii, L8 and L9, L14 and L15, and S15 and S17. Proteins L26 and S20 are the same protein (24).
In Vitro Synthesis of Ribosomal Proteins with Xdspcl DNA as Template. The ability of DNA from Xdspcl to code for several 30S r-proteins was also demonstrated in a DNAdependent in vitro protein synthesizing system. Proteins were synthesized in the presence of ['5S]methionine and the 30S r-proteins were recovered by reconstitution into 30S particles (19). As shown in Fig. 2, some of the proteins made with E. coli DNA and Xdspcl DNA as templates were found in the reconstituted particles. By contrast there was no significant amount of radioactive proteins that cosedimented with the reconstituted particles when the template was XDNA. The identity of the radioactive r-proteins in the reconstituted particles was determined immunologically with antisera to purified 30S proteins as previously described (19). Examples are shown in Fig. 3 and the results are summarized in Table 3. With E. coli DNA we detected the synthesis of 13 30S proteins and with Xdspcl DNA we detected 6. None were
detected when XDNA was used. We conclude that Xdspcl carries the structural genes for these six r-proteins. The genes for the proteins made with E. coli DNA but not with Xdspcl DNA may not be carried by this phage. These conclusions are consistent with the results of the in vivo experiments described previously and summarized in Table 3. In vitro synthesis of several 30S proteins could not be detected with either E. coli DNA or Xdspcl DNA as template. We have not scored these proteins in Table 3 since we cannot say whether these structural genes are present on Xdspcl, without a positive control. There are several possible explanations for our inability to detect these proteins. Three of them, S12, S18, and S21, do not have any methionine in the mature protein (ref. 20, and unpublished experiments in this laboratory). Thus, unless a methionine-containing precursor, e.g., a precursor with a formylmethionyl group at the N-terminal, could incorporate
Proc. Nat. Acad. Sci. USA 72
(1975)
Transducing Phages for Ribosomal Protein Genes
a
9
c E.COLI
E-C4;i 0O
_.
o10
_X I xI 10
20
10 20 FRACTION NUMBER
10
20
FIG. 2. Ability of various types of DNA to stimulate synthesis of 30S r-proteins in vitro. [35S]Methionine-labeled protein was synthesized in a DNA-dependent cell-free system, using E. coli DNA [prepared as described previously (19)], Xdspcl, or XCI857S7 DNA [prepared as described by Zubay, et al. (25)] as template. Radioactive 30S r-proteins were purified by passing the in vitro products through a DEAE-cellulose column and reconstituting 30S ribosomal subunits in the presence of carrier nonradioactive 16S RNA and 30S r-proteins (19). After reconstitution the samples were centrifuged through a 5-20% sucrose gradient in 30 mM Tris- HC1 (pH 7.4), 20 mM MgC12, and 0.33 AM KCl using a Spinco SW 27 rotor. After the run the gradients were pumped through a continuous-flow photometer monitoring A260nm and were fractionated; a 50 ul (a and c) or 25 ,ul (b) aliquot of each fraction was analyzed for radioactivity. Sedimentation was from left to right. Total incorporation per 10 1AI of the in vitro system
was:
E. coli DNA: 1.5 X 105
cpm;
b
d
FIG. 3. Identification by radioimmunodiffusion of 30S rproteins synthesized in vitro. Proteins were extracted from the reconstituted 30S particles (Fig. 2) and tested by radioimmunodiffusion assay (19). Each set of pictures shows a gel (left) and an autoradiogram of the gel (right). The center wells of gels (a) and (b) received proteins synthesized with E. coli (a) or Xdspcl DNA (b) as templates, whereas the outer wells received antisera against the indicated proteins. The center wells of gels (c) and (d) received antiserum against S13 (c) or S20 (d), whereas the outer wells received protein synthesized with DNA of the indicated origins as templates. The autoradiograms were exposed for 3 weeks (a), 6 days (b), or 4 days (c) and (d).
Xdspcl
DNA: 2.8 X 105 cpm; XDNA: 1.8 X 105 cpm. Radioactive methionine giving 105 cpm corresponds to about 3.5 pmol. The fractions of total radioactivity which were recovered in the reconstituted particles were (after corrections for recovery during the purification): E. coli DNA: 1.5%; Xdspcl DNA: 2.6%; XDNA: 0.16%.
into 30S subunits in reconstitution, we could not detect them. Also S1 does not incorporate well under the conditions used. Other proteins may not be detected because of improper posttranslational modification in the in vitro system. DISCUSSION
From the stimulation of r-protein synthesis in irradiated bacteria following infection by the transducing phages we conclude that \d8pc2 probably carries at least 26 genes and Xdspcl at least 22. In addition, genetic experiments have suggested that Xdspcl carries at least a part of the 8tr gene whose expression has not been detected in these experiments. We cannot eliminate the possibility that some of the stimulation in the irradiated cells was a secondary effect of some phage-coded proteins on residual host r-protein synthesis. Also, the r-proteins synthesized in these cells were identified only by co-electrophoresis with mature proteins. It is conceivable that the radioactivity in some of these spots was from precursors or breakdown products of other proteins. However, the conclusions are strengthened by the consistency of the in vivo and in vitro experiments (see Table 3). All the proteins that were synthesized in vitro were also synthesized in vivo and the proteins not synthesized in vivo were not synthesized in vitro. Previously 22 r-proteins genes have been reported to be near 8trA (1-5). Many of these genes are on Xd8pcl or Xdspc2. These include: S3, S4, S5, S7, S8, S12, S17, L6, L13, and L22. In addition these phages carry several genes which
TABLE 3. 30S ribosomal protein genes of Xdspcl In vitro
Protein Si S2 S3 S4 S5 S6 S7 S8 S9
Sil S10 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21
In vivo
E. coli
Xdspcl
Xdspcl
+
_
+ + +
+ +
+ + +
+ _ + _
_ + + + + +
+
+ + + +
+ + _ -
+ + _ -
+ +
-
-
-
Proteins synthesized in vitro using DNA from E. coli and Xdspcl as templates were analyzed as described in Figs. 2 and 3. Positive detection is indicated by +. Failure to do so is indicated by -, if synthesis of the protein could be demonstrated with the other template. Blanks indicate that the protein could not be detected in reconstituted particles using either E. coli or Xdspcl DNA as template in the in vitro system. In the case of S17, we did not perform the assay due to unavailability of antisera. The third column is qualitative interpretation of the results in Table 2.
10
Biochemistry: Jaskunas et al.
had not been previously located. These include: S11, S13, S14, S19, L2, L5, Lii, L14, L15, L16, L17, L18, L19, L23, L24, L29, and L30. This list of "new" 50S genes may include the four 50S genes that have been previously mapped to this region but identified by a different nomenclature (2-5). Thus, the available data indicate that at least 35 to 39 r-protein genes are clustered near strA. Twenty-six of these genes are on Xdspc2. Since Xdtrk does not code for any of these proteins we can conclude that all 26 of these genes are almost certainly between trkA and strA. Not all the genes suggested to be linked to strA could be detected on Xdspcl or Xdspc2. These include genes for S9, S10, S15, S16, Li, L3, L4, and L25. Since neither phage covers fus, which may be in a transcriptional unit with r-proteins (8), some of these missing genes may be located beyond the "end points" of the phages (see Fig. 1). The stimulation of the synthesis of L1, L3, and L4 in the irradiated cells may have been obscured by co-electrophoresis of the r-proteins with lambda-coded proteins in the two-dimensional gel. One of these proteins, L4, appears to be the product of the eryA locus, which has been mapped close to spcA (1, 21) and would be expected to be on either Xdspcl or Xdspc2. It is also possible that some genes on the phages are not expressed. Thus, the list of r-protein genes on these phages may be incomplete. Hybridization studies have shown that neither Xdspcl nor Xdspc2 contains genes for 16S or 23S rRNA (J. Ingversen and M. Nomura, unpublished experiments). This eliminates many r-proteins as potential translational products of 16S and 23S rRNAs. It also suggests that r-protein genes can be expressed in vitro without simultaneous transcription of rRNA genes. This observation is relevant to the expression of E. coli r-protein genes in vivo, since experiments with fragments of Xdspcl DNA generated by digestion with restriction endonucleases show that the synthesis of r-proteins in vitro is due to a bacterial promoter and not to a lambda promoter (L. Lindahl, unpublished experiments). Finally, we note that specialized transducing phages for r-protein genes described in this paper will be valuable tools for further investigation of the organization of these important genes as well as the regulation of their expression. Note Added in Proof. A Xdfus phage has been isolated that carries the genes aroE, trkA, spcA, strA, and fus. It stimulates the synthesis of 30 ribosomal proteins in UV-irradiated bacteria. We wish to thank M. Stroud, L. Sadowski, K. Ryan, and K. Oakden for their technical assistance, Drs. J. R. Beckwith, W. Reznikoff, and W. Epstein for providing us with bacterial strains,
and Drs. F. Blattner and L. Kahan for useful discussion. This work was supported in part by the College of Agriculture and Life Sciences, University of Wisconsin, and by grants from the
Proc. Nat. Acad. Sci. USA 72
(1976)
National Science Foundation (GB-31086X2) and the National Institute of General Medical Sciences (GM-20427). S.R.J. was a recipient of a National Institutes of Health special postdoctoral fellowship and L.L. is a recipient of a postdoctoral fellowship from European Molecular Biology Organization. 1. Jaskunas, S. R., Nomura, M. & Davies, J. (1974) in Ribosomes, eds. Nomura, M., Tissieres, A. & Lengyel, P. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), pp. 333-368. 2. Takata, R. (1972) Mol. Gen. Genet. 118, 363-371. 3. Dekio, S., Takata, R. & Osawa, S. (1970) Mol. Gen. Genet. 109, 131-141. 4. Dekio, S. (1971) Mol. Gen. Genet. 113, 20-30. 5. Sypherd, P. S. & Osawa, S. (1974) in Ribosomes, eds. Nomura, M., Tissieres, A. & Lengyel, P. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), pp. 669-678. 6. Bollen, A., Faelen, M., Lecocq, J. P., Herzog, A., Zengel, J., Kahan, L. & Nomura, M. (1973) J. MGI. Biol. 76, 463-472. 7. Kahan, L., Zengel, J., Nomura, M., Bollen, A. & Herzog, A. (1973) J. Mol. Biol. 76, 473-483. 8. Nomura, M. & Engbaek, F. (1972) Proc. Nat. Acad. Sci. USA 69, 1526-1530. 9. Dennis, P. (1974) J. Mol. Biol. 88, 25-41. 10. Dennis, P. & Nomura, M. (1974) Proc. Nat. Acad. Sci. USA 71, 3819-3823. 11. Kjeldgaard, N. 0. & Gausing, K. (1974) in Ribosomes, eds. Nomura, M., Tissieres, A. & Lengyel, P. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), pp. 369392. 12. Beckwith, J. R., Signer, E. R. & Epstein, W. (1966) Cold Spring Harbor Symp. Quant. Biol. 31, 393-401. 13. Shimada, K., Weisberg, R. A. & Gottesman, M. E. (1972) J. Mol. Biol. 63, 483-503. 14. Ippen, K., Shapiro, J. & Beckwith, J. (1971) J. Bacteriol. 108, 5-9. 15. Epstein, W. & Kim, B. S. (1971) J. Bacteriol. 108, 639-644. 16. Miller, J. H. (1972) Experiments in Molecular Genetics (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). 17. Ptashne, M. (1967) Proc. Nat. Acad. Sci. USA 57, 306-313. 18. Murialdo, H. & Siminovitch, L. (1971) in The Bacteriophage Lambda, ed. Hershey, A. D. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), pp. 711-723. 19. Kaltschmidt, E., Kahan, L. & Nomura, M. (1974) Proc. Nat. Acad. Sci. USA 71, 446-450. 20. Kaltschmidt, E., Dzionara, M. & Wittmann, H. G. (1970) Mol. Gen. Genet. 109, 292-297. 21. Wittmann, H. G., Stoffler, G., Apirion, D., Rosen, L., Tanaka, K., Otaka, E. & Osawa, S. (1973) Mol. Gen. Genet. 127, 175-189. 22. Mans, R. J. & Novelli, G. D. (1961) Arch. Biochem. Biophys. 94, 48-53. 23. Kaltschmidt, E. & Wittmann, H. G. (1970) Anal. Biochem. 36, 401-412. 24. Wittmann, H. G. (1974) in Ribosomes, eds. Nomura, M., Tissieres, A. & Lengyel, P. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) pp. 93-114. 25. Zubay, G., Chambers, D. A. & Cheong, L. C. (1970) in The Lactose Operon, eds. Beckwith, J. R. & Zipser, D. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), pp. 375-391.
Vol. 72, No. 1, pp. 6-10, January 1975
Specialized Transducing Phages for Ribosomal Protein Genes of Escherichia coli* (bacteriophage X/in vitro protein synthesis/protein identification)
S. RICHARD JASKUNAS, LASSE LINDAHL, AND MASAYASU NOMURA Institute for Enzyme Research, and Departments of Biochemistry and Genetics, University of Wisconsin, Madison, Wis. 53706
Communicated by Henry Lardy, September 13, 1974 K12 strain, EC-2, in which lac had been transposed close to aroE (12). A lacZ- mutant (NO1180) was isolated and attX was deleted (NO1324) (13). (See Table 1.) Phage xCI857S7plac5 (14, 16) was integrated into the lac genes near aroE (NO1325). A low-frequency transducing lysate (LFT) was obtained by thermal induction of NO1325 following ultraviolet (UV) irradiation. A XdaroE phage was isolated from the lysate using XCI857S7 as helper and an oroE- strain (NO 1223) as a recipient. This XdaroE was subsequently integrated into the aroE locus of another strain (NO1257) that was deleted for attX and was aroE- spcs strr fus8. An LFT for trkA was obtained by thermal induction of the resultant lysogen (NO1326) following superinfection by XC1857S7. The trkA mutant allele confers a requirement for a high concentration of potassium for growth, approximately 0.1 M, when it is coupled with the kdpABC5 mutation (15). The selection for trkA + is for growth on a low concentration of potassium. Phages transducing trkA were isolated in a strain (NO1230) with the following
Specialized lambda transducing phages ABSTRACT have been isolated carrying approximately half the ribosomal protein genes of E. coli. These phages carry regions of the bacterial chromosome between aroE and fus. The ribosomal protein genes on these phages have been identified by the stimulation of ribosomal protein synthesis in ultraviolet-irradiated bacteria following infection by the transducing phage, and by the in vitro synthesis of ribosomal proteins in a DNA-dependent protein synthesizing system. The results indicate Xdspcl probably carries at least 22 ribosomal protein genes and Xdspc2 at least 26 genes. All these genes are clustered between trkA and strA. At least 13 of them have not been previously mapped.
Many of the genes for the ribosomal proteins (r-proteins) of Escherichia coli are clustered near the strA locus (for a review, see ref. 1). Intergeneric crosses have suggested that at least 22 out of approximately 50 r-protein genes are in this region of the chromosome (2-5). At present not all the r-protein genes can be distinguished in intergeneric crosses. Thus, some of the other r-protein genes may also be in this cluster. Only one structural gene has been unambiguously mapped in another region of the chromosome, that is, the gene for the 30S protein S18 (6, 7). The clustering of these genes near the str locus suggests they may be organized into transcriptional units or operons (compare refs. 1 and 8). Such genetic organization would provide a mechanism for the coordinate expression of the genes in these genetic units. Furthermore, the coordinate expression of unlinked ribosome transcriptional units, such as r-protein genes with rRNA, could result from regulation of the transcriptional units as a whole. Synthesis of all r-proteins and rRNAs is coordinately regulated in E. coli during steady state
relevant markers: trkA401 kdpABC5 spcr strr fusr. We then
screened for transductants that were sensitive to spectinomycin (Spc-S). Since sensitive alleles at spc, str, and fus are dominant over corresponding resistant alleles (1, 8), the Spc-S phenotype is an indication of the presence of the spcB gene on transducing phages. Xdtrk was obtained from a transductant (NO1275) that remained resistant to Spc, streptomycin (Str), and fusidic acid (Fus). Xdspcl came from transductant (NO1267) that was Spc-S, Str-R, and Fus-R. We also obtained an LFT by thermal induction from a XCI857S7 lysogen into which the Xdaro phage had been inserted at aroE by P1 transduction from NO1326. This strain (NO1327) was spc8 str8 fuss. Phages transducing trkA were selected as above using the strain N01230 as a recipient, and Xdspc2 was obtained from a transductant (NO1328) that was Spc-S, Str-R, and Fus-R. Since the recipient remained Str-R when Xdspc2 was integrated we conclude this phage does not carry the str8 allele of the donor in a functioning state. Table 1 lists some of the above strains and their phenotypes. Analysis of phage DNAs with restriction endonucleases (F. Blattner and S. R. Jaskunas, unpublished experiments) and electron microscopic examination of heteroduplexes (M. Fiandt, W. Szybalski and M. Nomura, unpublished experiments) have indicated that the bacterial DNA ini Xdtrk, Xdspcl, and Xdspc2 is substituted into the left arm of lambda. All three carry the C1857 and S7 genes of the starting Xplac5. All strains we used were derivatives of E. coli K12. Transduction of trkA was done as described by Epstein and Kim (15) except we used plates with 2.5 mM potassium instead of
growth (9-11). Further investigation of the genetic organization and physiological regulation of r-protein genes would be greatly aided by the isolation of specialized transducing phages carrying r-protein genes. This communication describes the isolation of lambda phages carrying many of the r-protein genes of E. coli near strA, the identification of the r-protein genes on the phages, and the expression of some of these genes in an in vitro DNA-dependent protein synthesizing system. MATERIALS AND METHODS
Isolation of Transducing Phages. We started with an E. coli Abbreviations: r-protein, ribosomal protein; Spc, spectinomycin; Str, streptomycin; Fus, fusidic acid; R, resistant; S, sensitive; UV, ultraviolet; LFT, low frequency transducing lysate. * This is paper no. 1784 from the Laboratory of Genetics.
6
Proc. Nat. Acad. Sci. USA 72
Transducing Phages for Ribosomal Protein Genes
(1975)
7
TABLE 1. Sone bacterial strains used in the present study
Strain
N01324 N01325 N01257 N01326 N01327 N01230 N01275 N01267 N01328
Synonym and relevant genotype EC-2lac-AattX N01324 (Xplac) aroE- strr AattX Alac,pro
N01257 (Xdaro) aroE- str' (Xdaro) (XCI857S7) trkA401 kdpABC5 spcr strr fusr N01230 (Xdtrk) (XC1857S7) N01230 (Xdspcl) (XCI857S7) N01230 (Xdspc2) (XC1857S7)
Lac + -
Aro
+ -
+ + + +
-
+
Phenotype* Trk Spc S + S + S + S + S + R
+ + -
+
+ +
R S
+
S
Str S S
Fus S S S
R R S
S S R R R R
R R R R
* Lac is ability to grow on lactose; Aro is ability to grow without exogenous aromatic amino acids (and shikimic acid); other symbols are explained in the text.
0.1 mM and enriched with 0.1% vitamin-free casamino acids. Plates for testing sensitivity to Spc, Str, and Fus have been described (8). The defective transducing phages were separated from helper on CsCl gradients as described by Miller (16). In Vivo Experiments. Synthesis of r-proteins was examined after infection of UV-irradiated bacteria by transducing phages. The method was adapted from those described by previous workers (17, 18) (see legend to Table 2). The strain used as a host was NO1261, a uvrB mutant that was obtained by deleting attX.
In Vitro Experiments. The in vitro synthesis and detection of r-proteins in a DNA-dependent protein synthesizing system was done as previously described (19) (see legends to Figs. 2 and 3). RESULTS
Genetics. The regions of the E. coli chromosome carried by the various lambda transducing phages that we have isolated are illustrated schematically in Fig. 1. As described in the Materials and Methods section the presence of the spc5 allele on Xdspcl and Xdspc2 has been shown by the Spc-S phenotype of the strain N01230 (trkA - spcr strrfusr) lysogenized with these transducing phages (see Table 1). The Xdtrk phage does not carry the spc locus by this test. Since the product of the spcA gene is r-protein S5 (1), we conclude that both Xdspcl and Xdspc2 express this r-protein gene in stable lysogens. The presence of at least a part of the str gene on Xdspcl has been suggested from several genetic experiments. For example, this phage could transduce a strd strain to Str-independence with the frequency of about 10-4, presumably using a recombination mechanism. Neither the Xdtrk nor the Xdspc2 phages can transduce the strd strain to Str-independence. Expression of the Phage-Coded Ribosomal Protein 'Genes In Vivo. In order to identify other r-protein genes on the phages we have measured the stimulation of r-protein synthesis in E. coli cells following infection by the transducing phages. The expression of the host r-protein genes was reduced by prior UV irradiation of the host. The experimental procedures are described in the legend to Table 2. It should be noted that the measurements were done on the total r-proteins including
those which may not have incorporated into ribosome particles. The stimulation of the synthesis of individual r-proteins by Xdtrk, Xdspcl, and Xdspc2 relative to cells infected with XCI857S7 is shown in Table 2. There are 26 proteins whose synthesis was stimulated 4-fold or greater by Xdspc2 and 22 for Xdspcl. This stimulation probably occurred because the structural genes for these proteins are on the phages. Xdtrk does not carry any r-protein genes that can be detected with this method. The synthesis of most r-proteins was stimulated to approximately the same extent by Xdspcl and Xdspc2. However, the synthesis of S3, S17, S19, L2, L16, and L29 was stimulated to a significantly greater extent by Xdspc2 than by Xdspcl. The genes for S17 and L29 seem to be on Xdspc2 but not Xdspcl. The genes for S3 and L16 seem to be on both phages but expressed to a different extent. Finally, while the genes for S19 and L2 seem to be on Xdspc2, it is not clear whether they are present on Xdspcl. We have not observed any stimulation of the synthesis of the product of the strA gene, S12, with either phage significantly greater than with Xdtrk. The data shown in Table 2 (and other experiments not shown) suggest that the expression of the various r-protein genes on the transducing phages in UV-irradiated cells is not coordinate. The reason for this apparent noncoordinate expression is not known. We have studied stability of rproteins synthesized under these conditions by pulse-labeling for 1 min followed by chasing with nonradioactive amino acids. It was found that several r-proteins synthesized are somewhat unstable. However, the instabilities of the r-proteins in these cells did not differ sufficiently to account for the observed variation in the net synthesis rate. aroE
trkA
spcA
strA
fus
Adaro
Adtrk Adspc2 Adspcl
FIG. 1. The str-spc region on the E. coli genetic map and the genes incorporated into various X transducing phages. The "end points" of the various phages are not 'exactly known. Xdspcl ap~pears to carry at least a part of the str gene. However, no additional r-protein genes have been detected on Xdspcl relative to )kdspc2 (see the text).
8
Biochemistry: Jaskunas et al.
Proc. Nat. Acad. Sci. USA 72 (1976)
TABLE 2. Stimulation of the synthesis of individual ribosomal proteins by Xdtrk, Xdspcl, and Xdspc2 Protein
Si S2 S3 S4 S5 S6 S7 S8 S9 | S115 S10 S12 S13 S14 S15 S16 S17 S18
Xdtrk
Xdspc2
0.9 1.5 1.2 1.3 1.0 1.1 1.1
Xdspcl 0.5 4.3 35.7 40.2 0.8 8.8 6.8
1.1
14.7
15.0
* 1.0 2.0 1.0 1.1 1.0 1.3 1.1 0.8
* 1.5 3.2 22.2 15.6 1.2 1.1 1.2 1.1
0.5 17.3 29.0 45.0 0.8 6.5 7.0 * 0.9 1.9 18.7 15.4 3.0 1.7 6.0 0.7
Protein
Xdtrk
Xdspcl
Xdspc2
Protein
Xdtrk
Xdspcl
Xdspc2
S19 S20 S21
0.9
2.1 2.1 1.5 1.8 3.4 2.3 0.5 26.2 22.0 0.7 0.8 0.7 0.6 14.6 1.1 5.1 47.3 58.0
4.3 0.8 0.9 0.9 17.2 1.4 1.2 25.7 21.0 0.6 0.7 0.5 0.6 15.6 0.8 4.5 47.2 75.4
L16 L17 L18 L19 L20 L21 L22 L23 L24 L25 L27 L28 L29 L30 L31 L32 L33
1.0 1.3 0.9 1.0 1.2 1.0 1.1 1.0 1.0 1.0 1.0 1.0 0.9 0.9
7.9 59.7 23.2 6.7
26.7 50.9 23.6 6.0
Li L2 L3 L4 L5 L6 L7 L8 L9 L10
Lii L12 L13 L14 L15
0.7 1.0 1.1 1.0 1.3 1.0 1.0 1.1 1.0
1.0 1.0 1.0 1.2 1.1 1.1 1.1 1.0
1.4 1.0
1.7 1.0 9.8 4.0 37.9 2.1 1.7 2.0 0.9 33.0 2.0 1.2
1.6 0.8
14.3 4.8 37.6 2.0 1.0 1.4 15.7 33.9 2.0 1.0
Cells exponentially growing in minimal maltose medium were labeled with [14C]leucine (0.75 MCi/ml, 281 Ci/mol) for at least three generations during which the label was completely incorporated. When the culture reached 2 X 108/ml it was concentrated to 1 X 109/ml in the same medium and irradiated for 12 min, 30 cm from a germicidal lamp (4 ml of cell suspension per 100-mm diameter glass petri dish). Magnesium sulfate was added to 0.02 M and the cells were infected with phages at a multiplicity of infection of 5. The transducing phages used in these experiments were purifiod on CsCl gradients and contained approximately 1% contaminating helper phage. Control cells were infected with XCI857S7. Protein synthesis in the noninfected irradiated cells was approximately 0.4% of that of the nonirradiated cells and all phages stimulated protein synthesis approximately 10-fold. Following a 15-min incubation at 370 to allow phage absorption, the culture was diluted 5-fold with prewarmed minimal maltose media. The [3H]leucine (15 1Ci/ml, 55 Ci/mmol) was added 15 min later. The incorporation was stopped after 10 min by adding excess nonradioactive leucine, incubating for 30 sec, and pouring the cells on ice. Samples of the cells were precipitated on Whatman 3MM paper with 7% trichloroacetic acid + nonradioactive leucine and worked up essentially as previously described (22). These filters were later oxidized to measure the aH/14C ratio in total protein. The remaining cells were lysed by the lysozyme freeze-thaw technique (9), carrier ribosomes were added, and proteins were extracted with acetic acid and separated by electrophoresis on a two-dimensional gel (23). The 3H/14C ratio of each r-protein spot, except Si and L31, was determined after oxidizing the stained spot in a Packard oxidizer. (S1 was not separated from non-ribosomal proteins and no stained spot was seen at the position of L31.) The numbers shown in the table for r-protein i are the ratios of Ai for bacteria infected with a transducing phage to Ai for bacteria infected with XCI857S7. Ai is a measure of the differential synthesis rate of the ith r-protein after infection relative to that in normal cells and is defined: (8H/14C) in ith r-protein 3H in ith r-protein/8H in total protein = (SH/14C) in total protein 14C in ith r-protein/14C in total protein. Results of two experiments have been averaged. Not all the proteins are completely resolved from other r-proteins in the two-dimensional gel. Those proteins where there is significant overlap include: S9 and S11, S5 and Lii, L8 and L9, L14 and L15, and S15 and S17. Proteins L26 and S20 are the same protein (24).
In Vitro Synthesis of Ribosomal Proteins with Xdspcl DNA as Template. The ability of DNA from Xdspcl to code for several 30S r-proteins was also demonstrated in a DNAdependent in vitro protein synthesizing system. Proteins were synthesized in the presence of ['5S]methionine and the 30S r-proteins were recovered by reconstitution into 30S particles (19). As shown in Fig. 2, some of the proteins made with E. coli DNA and Xdspcl DNA as templates were found in the reconstituted particles. By contrast there was no significant amount of radioactive proteins that cosedimented with the reconstituted particles when the template was XDNA. The identity of the radioactive r-proteins in the reconstituted particles was determined immunologically with antisera to purified 30S proteins as previously described (19). Examples are shown in Fig. 3 and the results are summarized in Table 3. With E. coli DNA we detected the synthesis of 13 30S proteins and with Xdspcl DNA we detected 6. None were
detected when XDNA was used. We conclude that Xdspcl carries the structural genes for these six r-proteins. The genes for the proteins made with E. coli DNA but not with Xdspcl DNA may not be carried by this phage. These conclusions are consistent with the results of the in vivo experiments described previously and summarized in Table 3. In vitro synthesis of several 30S proteins could not be detected with either E. coli DNA or Xdspcl DNA as template. We have not scored these proteins in Table 3 since we cannot say whether these structural genes are present on Xdspcl, without a positive control. There are several possible explanations for our inability to detect these proteins. Three of them, S12, S18, and S21, do not have any methionine in the mature protein (ref. 20, and unpublished experiments in this laboratory). Thus, unless a methionine-containing precursor, e.g., a precursor with a formylmethionyl group at the N-terminal, could incorporate
Proc. Nat. Acad. Sci. USA 72
(1975)
Transducing Phages for Ribosomal Protein Genes
a
9
c E.COLI
E-C4;i 0O
_.
o10
_X I xI 10
20
10 20 FRACTION NUMBER
10
20
FIG. 2. Ability of various types of DNA to stimulate synthesis of 30S r-proteins in vitro. [35S]Methionine-labeled protein was synthesized in a DNA-dependent cell-free system, using E. coli DNA [prepared as described previously (19)], Xdspcl, or XCI857S7 DNA [prepared as described by Zubay, et al. (25)] as template. Radioactive 30S r-proteins were purified by passing the in vitro products through a DEAE-cellulose column and reconstituting 30S ribosomal subunits in the presence of carrier nonradioactive 16S RNA and 30S r-proteins (19). After reconstitution the samples were centrifuged through a 5-20% sucrose gradient in 30 mM Tris- HC1 (pH 7.4), 20 mM MgC12, and 0.33 AM KCl using a Spinco SW 27 rotor. After the run the gradients were pumped through a continuous-flow photometer monitoring A260nm and were fractionated; a 50 ul (a and c) or 25 ,ul (b) aliquot of each fraction was analyzed for radioactivity. Sedimentation was from left to right. Total incorporation per 10 1AI of the in vitro system
was:
E. coli DNA: 1.5 X 105
cpm;
b
d
FIG. 3. Identification by radioimmunodiffusion of 30S rproteins synthesized in vitro. Proteins were extracted from the reconstituted 30S particles (Fig. 2) and tested by radioimmunodiffusion assay (19). Each set of pictures shows a gel (left) and an autoradiogram of the gel (right). The center wells of gels (a) and (b) received proteins synthesized with E. coli (a) or Xdspcl DNA (b) as templates, whereas the outer wells received antisera against the indicated proteins. The center wells of gels (c) and (d) received antiserum against S13 (c) or S20 (d), whereas the outer wells received protein synthesized with DNA of the indicated origins as templates. The autoradiograms were exposed for 3 weeks (a), 6 days (b), or 4 days (c) and (d).
Xdspcl
DNA: 2.8 X 105 cpm; XDNA: 1.8 X 105 cpm. Radioactive methionine giving 105 cpm corresponds to about 3.5 pmol. The fractions of total radioactivity which were recovered in the reconstituted particles were (after corrections for recovery during the purification): E. coli DNA: 1.5%; Xdspcl DNA: 2.6%; XDNA: 0.16%.
into 30S subunits in reconstitution, we could not detect them. Also S1 does not incorporate well under the conditions used. Other proteins may not be detected because of improper posttranslational modification in the in vitro system. DISCUSSION
From the stimulation of r-protein synthesis in irradiated bacteria following infection by the transducing phages we conclude that \d8pc2 probably carries at least 26 genes and Xdspcl at least 22. In addition, genetic experiments have suggested that Xdspcl carries at least a part of the 8tr gene whose expression has not been detected in these experiments. We cannot eliminate the possibility that some of the stimulation in the irradiated cells was a secondary effect of some phage-coded proteins on residual host r-protein synthesis. Also, the r-proteins synthesized in these cells were identified only by co-electrophoresis with mature proteins. It is conceivable that the radioactivity in some of these spots was from precursors or breakdown products of other proteins. However, the conclusions are strengthened by the consistency of the in vivo and in vitro experiments (see Table 3). All the proteins that were synthesized in vitro were also synthesized in vivo and the proteins not synthesized in vivo were not synthesized in vitro. Previously 22 r-proteins genes have been reported to be near 8trA (1-5). Many of these genes are on Xd8pcl or Xdspc2. These include: S3, S4, S5, S7, S8, S12, S17, L6, L13, and L22. In addition these phages carry several genes which
TABLE 3. 30S ribosomal protein genes of Xdspcl In vitro
Protein Si S2 S3 S4 S5 S6 S7 S8 S9
Sil S10 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21
In vivo
E. coli
Xdspcl
Xdspcl
+
_
+ + +
+ +
+ + +
+ _ + _
_ + + + + +
+
+ + + +
+ + _ -
+ + _ -
+ +
-
-
-
Proteins synthesized in vitro using DNA from E. coli and Xdspcl as templates were analyzed as described in Figs. 2 and 3. Positive detection is indicated by +. Failure to do so is indicated by -, if synthesis of the protein could be demonstrated with the other template. Blanks indicate that the protein could not be detected in reconstituted particles using either E. coli or Xdspcl DNA as template in the in vitro system. In the case of S17, we did not perform the assay due to unavailability of antisera. The third column is qualitative interpretation of the results in Table 2.
10
Biochemistry: Jaskunas et al.
had not been previously located. These include: S11, S13, S14, S19, L2, L5, Lii, L14, L15, L16, L17, L18, L19, L23, L24, L29, and L30. This list of "new" 50S genes may include the four 50S genes that have been previously mapped to this region but identified by a different nomenclature (2-5). Thus, the available data indicate that at least 35 to 39 r-protein genes are clustered near strA. Twenty-six of these genes are on Xdspc2. Since Xdtrk does not code for any of these proteins we can conclude that all 26 of these genes are almost certainly between trkA and strA. Not all the genes suggested to be linked to strA could be detected on Xdspcl or Xdspc2. These include genes for S9, S10, S15, S16, Li, L3, L4, and L25. Since neither phage covers fus, which may be in a transcriptional unit with r-proteins (8), some of these missing genes may be located beyond the "end points" of the phages (see Fig. 1). The stimulation of the synthesis of L1, L3, and L4 in the irradiated cells may have been obscured by co-electrophoresis of the r-proteins with lambda-coded proteins in the two-dimensional gel. One of these proteins, L4, appears to be the product of the eryA locus, which has been mapped close to spcA (1, 21) and would be expected to be on either Xdspcl or Xdspc2. It is also possible that some genes on the phages are not expressed. Thus, the list of r-protein genes on these phages may be incomplete. Hybridization studies have shown that neither Xdspcl nor Xdspc2 contains genes for 16S or 23S rRNA (J. Ingversen and M. Nomura, unpublished experiments). This eliminates many r-proteins as potential translational products of 16S and 23S rRNAs. It also suggests that r-protein genes can be expressed in vitro without simultaneous transcription of rRNA genes. This observation is relevant to the expression of E. coli r-protein genes in vivo, since experiments with fragments of Xdspcl DNA generated by digestion with restriction endonucleases show that the synthesis of r-proteins in vitro is due to a bacterial promoter and not to a lambda promoter (L. Lindahl, unpublished experiments). Finally, we note that specialized transducing phages for r-protein genes described in this paper will be valuable tools for further investigation of the organization of these important genes as well as the regulation of their expression. Note Added in Proof. A Xdfus phage has been isolated that carries the genes aroE, trkA, spcA, strA, and fus. It stimulates the synthesis of 30 ribosomal proteins in UV-irradiated bacteria. We wish to thank M. Stroud, L. Sadowski, K. Ryan, and K. Oakden for their technical assistance, Drs. J. R. Beckwith, W. Reznikoff, and W. Epstein for providing us with bacterial strains,
and Drs. F. Blattner and L. Kahan for useful discussion. This work was supported in part by the College of Agriculture and Life Sciences, University of Wisconsin, and by grants from the
Proc. Nat. Acad. Sci. USA 72
(1976)
National Science Foundation (GB-31086X2) and the National Institute of General Medical Sciences (GM-20427). S.R.J. was a recipient of a National Institutes of Health special postdoctoral fellowship and L.L. is a recipient of a postdoctoral fellowship from European Molecular Biology Organization. 1. Jaskunas, S. R., Nomura, M. & Davies, J. (1974) in Ribosomes, eds. Nomura, M., Tissieres, A. & Lengyel, P. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), pp. 333-368. 2. Takata, R. (1972) Mol. Gen. Genet. 118, 363-371. 3. Dekio, S., Takata, R. & Osawa, S. (1970) Mol. Gen. Genet. 109, 131-141. 4. Dekio, S. (1971) Mol. Gen. Genet. 113, 20-30. 5. Sypherd, P. S. & Osawa, S. (1974) in Ribosomes, eds. Nomura, M., Tissieres, A. & Lengyel, P. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), pp. 669-678. 6. Bollen, A., Faelen, M., Lecocq, J. P., Herzog, A., Zengel, J., Kahan, L. & Nomura, M. (1973) J. MGI. Biol. 76, 463-472. 7. Kahan, L., Zengel, J., Nomura, M., Bollen, A. & Herzog, A. (1973) J. Mol. Biol. 76, 473-483. 8. Nomura, M. & Engbaek, F. (1972) Proc. Nat. Acad. Sci. USA 69, 1526-1530. 9. Dennis, P. (1974) J. Mol. Biol. 88, 25-41. 10. Dennis, P. & Nomura, M. (1974) Proc. Nat. Acad. Sci. USA 71, 3819-3823. 11. Kjeldgaard, N. 0. & Gausing, K. (1974) in Ribosomes, eds. Nomura, M., Tissieres, A. & Lengyel, P. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), pp. 369392. 12. Beckwith, J. R., Signer, E. R. & Epstein, W. (1966) Cold Spring Harbor Symp. Quant. Biol. 31, 393-401. 13. Shimada, K., Weisberg, R. A. & Gottesman, M. E. (1972) J. Mol. Biol. 63, 483-503. 14. Ippen, K., Shapiro, J. & Beckwith, J. (1971) J. Bacteriol. 108, 5-9. 15. Epstein, W. & Kim, B. S. (1971) J. Bacteriol. 108, 639-644. 16. Miller, J. H. (1972) Experiments in Molecular Genetics (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). 17. Ptashne, M. (1967) Proc. Nat. Acad. Sci. USA 57, 306-313. 18. Murialdo, H. & Siminovitch, L. (1971) in The Bacteriophage Lambda, ed. Hershey, A. D. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), pp. 711-723. 19. Kaltschmidt, E., Kahan, L. & Nomura, M. (1974) Proc. Nat. Acad. Sci. USA 71, 446-450. 20. Kaltschmidt, E., Dzionara, M. & Wittmann, H. G. (1970) Mol. Gen. Genet. 109, 292-297. 21. Wittmann, H. G., Stoffler, G., Apirion, D., Rosen, L., Tanaka, K., Otaka, E. & Osawa, S. (1973) Mol. Gen. Genet. 127, 175-189. 22. Mans, R. J. & Novelli, G. D. (1961) Arch. Biochem. Biophys. 94, 48-53. 23. Kaltschmidt, E. & Wittmann, H. G. (1970) Anal. Biochem. 36, 401-412. 24. Wittmann, H. G. (1974) in Ribosomes, eds. Nomura, M., Tissieres, A. & Lengyel, P. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) pp. 93-114. 25. Zubay, G., Chambers, D. A. & Cheong, L. C. (1970) in The Lactose Operon, eds. Beckwith, J. R. & Zipser, D. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), pp. 375-391.
