Proc. NatI. Acad. Sci. USA Vol. 75, No. 12, pp. 6163-6167, December 1978 Genetics Cluster of ribosomal protein genes in Egcherichia coli containing genes for proteins S6, S18, and L9 (temperature-sensitive mutants/two-dimensional gel electrophoresis/Plkc-mediated transduction) KATSUMI ISONO AND MADOKA KITAKAWA Max-Planck-Institut fur Molekulare Genetik, Abteilung Wittmann, Ihnestrasse 63-73 D-1000 Berlin 33, Germany Communicated by W. Beerman, September 25, 1978

ABSTRACT A mutant of Escbenchia coli K-12 isolated for A medium, on minimal A agar plates supplemented with all the temperature-sensitive growth was found to harbor an alteration requirements described above except that thymine was at 2 in ribosomal protein L9. Because the chromosomal location of Plates were incubated at 300C for 4-5 days, and colonies the structural gene for this protein (rpll) was not known, we ,gg/ml. mapped the mutation by using various Hfr strains. The fine were purified on the same plates. Purified clones were tested mapping of this gene by Plkc phage-mediated transductions for growth on suitably supplemented minimal A agar plates has revealed that it forms a gene cluster at 94 min on the E coli containing no thymine or thymine at 2 or 20 /ig/ml; those genetic map together with the genes coding for two other ribo- growing only on the two thymine-supplemented plates were somal proteins, S6 (rps1;) and S18 (QpsR). Furthermore, the re- classified as deo mutants (15). gion of the E coli genetic map containing this cluster was found to be shorter than previously estimated by approximately 2 min. RESULTS Mapping of uxuB. In our previous report (8), we placed the Many of the genes coding for ribosomal components [5S, 16S, gene coding for r-protein S6 (rpsF) and an unidentified tem- and 23S rRNAs and 53 ribosomal proteins (r-proteins)] have perature-sensitive mutation (ts-210) of mutant JE210 (Fig. lb) been localized on the Escherichia coii genetic map (for review, at 97 min and 98 min, respectively. Later we found that, al- see ref. 1). They either form gene clusters together with the though they were quite away from each other, they were co- genes coding for RNA polymerase subunits and protein syn- transducible in P1 phage-mediated transduction. Unfortu- thesis elongation factors (at 72 and 88 min) or most likely exist nately, the region of the E. coil genetic map in which these as single units [for example, the genes for r-proteins S2 (2, 3) and mutations fell was not well characterized and the only markers S20 (4, 5)]. The genes for 43 r-proteins of E. coli have thus far available were uxuA and uxuB (12). If our mapping, which been mapped at seven different loci, which leaves the genes for relied on the gradient transmission of markers from Hfr to F- 10 more r-proteins to be localized. bacteria (16, 17), were correct, then it is conceivable that either By utilizing mutants, isolated for temperature-sensitive the rpsF or ts-210 mutation of JE210 may be cotransducible growth, that harbored structurally altered r-proteins (6, 7), we with uxu. could place the genes for protein S6 (8) and protein L19 (9) at To begin with, we constructed a strain that contained rpsF, 97 and 56.4 min, respectively, on the E. coli genetic map. More ts-210, and uxu mutations. The uxuB mutant CM8 was found recently, we have isolated another mutant that contained an to maintain the maleness of its parental Hfr P4X and we made alteration in protein L9 (10). Because the gene for this protein use of this character. Strain CM8 was crossed with mutant (rplI) had not yet been mapped, we performed mapping of this JE210, and ArgH+ streptomycin-resistant (Str-R) recombinants mutation. During the course of this work, we found that the were selected. They were then tested for rpsF, ts-210, thr, leu, region of the published E. colt genetic map (11) that contained and uxuB. One of the resultant recombinants was termed the genes for proteins S6 and S18 should be shortened by about MB2014 and was used in transductions in which UxuB+ was 2 min and that, when this was done, the two genes could be selected for. As shown in Table 2, about 3% of the UxuB+ brought together. Furthermore, we discovered that the genes transductants became Ts+, indicating that they were away from for these two proteins as well as the gene for protein L9 formed each other by 1.4 min (18). Furthermore, because uxuB is a cluster near purA. transferred earliest during conjugation with an HfrH (ref. 14; unpublished data) but ts-210 is not, the gene order in this region MATERIALS AND METHODS is most likely ts-210, uxuB, thr, although the reported gene Bacterial strains used in this work are listed in Table 1. Plkc location for uxuB is at 97 min and that for ts-210 at 98 min. If phage-mediated transduction and analysis of transductants for uxuB maps at 97 min and if the above gene order is correct, r-proteins by two-dimensional gel electrophoresis and for other then ts-210 may be cotransducible with pyrB which is situated markers were performed as previously reported (8). Uxu phe- at 94.7 min (11). Conversely, if the location of ts-210 at 98 min notypes were tested on minimal A (14) agar plates containing is correct, then uxuB may be cotransducible with deo, one of all the requirements and D-glucuronic acid (Sigma) added in the nearest markers situated at 99.5 min (11). A spontaneous place of glucose. Amino acids were added to the minimal A deo mutation was therefore introduced into MB2014 and one medium at 60,qg/ml each, and guanine and thymine were of the resultant derivatives, termed MB2015, and several de- added at 20 jg/ml each. rivatives thereof (Table 1) were subsequently analyzed. Spontaneous deo derivatives were obtained from MB2014 The results of various transductions carried out using MB2015 by spreading an overnight culture, washed once with minimal and its derivatives also are summarized in Table 2. UxuB was found to be cotransducible with deo at the frequency of 5%, The publication costs of this article were defrayed in part by page and, surprisingly, ts-210 and pyrB were also cotransducible at charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviations: r-proteins, ribosomal proteins; Str-R, resistance to this fact. streptomycin; Nal-R, resistance to nalidixic acid. 6163 Downloaded by guest on October 3, 2021 6164 Genetics: Isono and Kitakawa Proc. Natl. Acad. Sci. USA 75 (1978)

a

_1

0 9. L9 S6

_.._

S18

a

::'rsl;:

S6

a 4 S18--

pi b c

o ~~0

L9 a

FIG. 1. Two-dimensional gel electropherograms .4 of parental strain PA3092 (a) and mutants JE210 JE1997 (d). Only the portion 9 ie,:,, (b), JE1677 (c), and d of the gels containing r-proteins S6, S18, and L9 are akk shown. Downloaded by guest on October 3, 2021 Genetics: Isono and Kitakawa Proc. Natl. Acad. Sci. USA 75 (1978) 6165

Table 1. Bacterial strains used these four genes most likely would be rpsF, ts-210, uxuB, deo. T~hImodso consistentwith the fact that uxuB is one of the ear- Source or liest markers transferred by Hfr H (12), whereas neither ts-210 Strain Relevant phenotype* referencet nor rpsF is (8). In other words, the point of entry of the chro- JE 210 F- rpsF (S6), ts-210 2, 4 mosome of an HfrH strain must fall somewhere between ts-210 CM 8 Hfr uxuB 12 and uxuB. MB2014 CM 8 X JE210, rpsF (S6), ts-210, This paper Therefore, the next step was to correlate, by Plkc trans- uxuB duction, the genes described above with those flanking this MB2015 deo derivative of MB2014 This paper region. For this purpose we chose purA, pyrB, serB, and thr and MB2016 P1s derivative of MB2015 This paper performed various combinations of transductions. The results MB2018 serB derivative of MB2015 This paper are summarized in Table 2 and Fig. 2. The most important MB2019 ts-210+ revertant of MB2018 This paper feature of the results obtained is that we can now cover this AB2834 F- aroE CGSC region by Plkc transductions. The ts-210 mutation is away from AT2459 F- serB CGSC pyrB by only 0.3 min. UxuB and ts-210 are away from each PC0950 Hfr purA CGSC other by r1.3 min. The data for the cotransduction frequency MB360 argI+ derivative of PC0950 This paper between uxuB and deo had a rather large fluctuation with the AT2535 F- pyrB CGSC average value for the distance between them of 1.1 min. MB365 purA derivative of AT2535 This paper Taking all these results into account, the distance between JE1677 F- rpsR (S18) 3 pyrB and serB, which are situated at 94.7 and 99.6 min, re- MB2203 MB360, X JE1677, purA, rpsR, cycB, This paper spectively (11), is in fact much shorter: instead of 4.9 min, it P1s should be only 2.9 min. Therefore, this region of the E. coli JE1997 F- rplI (L9) 10 genetic map should be shortened by 2 min. PK191 Hfr CGSC Fine Mapping of rpsF(S6) and rpsR (SiS). Because it has MB2251 PK191 X JE1997, rpII (L9), P1s This paper become evident that the distance between pyrB and serB (and X316 F- cycA 13 hence between argH and leu) is --2 min shorter than previously * Only those markers relevant to this work are listed. reported, we adopted the new distance between argH and letu t CGSC, E. coli Genetic Stock Center at Yale University (through the (12 min instead of 14 min) to correct our gradient transmission courtesy of B. Bachmann). data for rpsF and ts-210 (figure 4 of ref. 8). This gave new es- timates of 95.7 ± 1.2 min for the former and 95.9 + 1.2 min for a high frequency (60-70%). Thus, the region of the E. coli ge- the latter. These corrected values are now in good agreement netic map that has not been covered by P1 phage-mediated with the transduction data for ts-210 as described above. transduction can now be covered by it and markers in this re- Therefore, it is very likely that rpsF should actually be situated gion can be connected. When these cotransduction frequencies between purA and pyrB. If this is the case, then it is also inter- were converted into map units in minutes according to Wu's esting to determine if rpsF in fact maps close to rpsR, the equation (18) and as discussed by Bachmann et al. (11), the structural gene for protein S18 that was the first r-protein gene cumulative distance between pyrB and deo (4.8 min according mapped outside the strA cluster at 72 min (13, 19). to ref. 11) was found in fact to be much shorter (approximately 3 min). UxuB+ transductants were then purified and their r- protein S6 was examined as described (8). None of the 24 transductants tested showed wild-type S6, however. The two genes, rpsF and ts-210, were placed away from each other by 0.3 + 1.2 min (mean + SD) and there were no co- transductants among 10 Ts+ transductants examined for rpsF / 75 (8). If rpsF and ts-210 are located on the opposite sides of strA uxuB-that is, if uxuB is located between rpsF and ts-210- / then the cotransduction frequency between uxuB and rpsF / / would be -50%. The data we obtained above indicate that this / / was not the case. Moreover, as described above, the distance //I/ between uxuB and deo is only 1.3 min (5% cotransduction) as rpsF,rpsR,rplI opposed to the previous computation (12). This clearly indicates purA' cyB pyrB ts-210 uxuB deo serB thr that uxuB cannot be located at 96.8 min and that the order of lb I Ir-cycA-I ~11 ,I ~~~~~~~~~~~~~~i4 - Table 2. Mapping of uxuB and ts-210 by transduction 9/1318(2.8%) 94 29713P) 73t1/1170

841343. 0 /61 162%l 13711616 185%) 1022/2484 Selected Unselected Cotransduction (245%) 3/24 (13%) (411%) *21126 ,j3_1868(02%)_ Donor Recipient marker marker frequency, %* (33%) 2/8012.5%)388 % 23/221 IMM) 6761887 AB2834 MB2014 UxuB+ Ts+ 3.0 (18/592) 176.2%) MB2015 UxuB+ Deo+ 5.0 (27/544) 11296(03%) 13/1329(1.0%) MB2018 SerB+ Deo+ 86.3 (511/592) MB2018 SerB+ UxuB+ 1.0 (6/592) FIG. 2. Summary of transduction experiments and alignment ofgenes on the E. coli genetic map (circle) between purA and thr. All MB2016 AT2535 PyrB+ Ts- 71 (17/24) the genes are placed on the map according to their relative distances MB360 PurA+ Ts- 0.2 (1/592) from purA as calculated from the cotransduction frequencies as de- MB365 PurA+ PyrB+ 1.7 (3/181) scribed by Wu (17).. Markers at arrowtails are those selected for in MB365 PyrB+ PurA+ 11.7 (16/137) transductions, and those at arrowheads are unselected ones examined MB365 PyrB+ Ts- 60.6 (83/137) afterward. When arrows point in both directions, they indicate that MB365 PyrB+ UxuB- 2.9 (4/137) reciprocal transductions were performed. Numbers indicate co- transductants among total tranaductants tested with the percentage * Values in parentheses are no. transductants/total no. given in brackets. Downloaded by guest on October 3, 2021 6166 Genetics: Isono and Kitakawa Proc. Nati. Acad. Sci. USA 75 (1978)

Table 3. Fine mapping of rpsF, rpsR, and rplI by transduction a + + + cycA I I Selected Unselected Cotransduction ----r- Donor Recipient marker marker frequency, %* II AB2834 JE210 Ts+ RpsF+ 3 (1/40) i MB2016 AT2535 PyrB+ RpsF- 13 (3/24) I I I MB2016 MB360 PurA+ RpsF- 39 (9/23) purA rpsR cycB + MB2019t MB2203 PurA+ RpsR+ 39 (14/36) (RpsF-) MB2251t MB2203 PurA+ RpsR+ 25 (12/48) b + cycA + + (RplI-) * ., Values in parentheses are no. transductants/total no. I t For detail of these transductions, see Table 4. I~~~~~~~~~~~~~~

First, we transduced purA + of MB2016 into MB360 purA, purA + rpsR cycB purified 24 transductants to single colonies, and analyzed them for the coinheritance of rpsF (the S6 mutation) by two-di- FIG. 3. Two possible orientations of genes cycA and cycB with mensional electrophoresis. As shown in Table 3, 9 of 23 (39%) respect to purA and rpsR. Our data (see text) favor a because, in b, ++++ (wild type for all four markers) can arise only as a result offour PurA+ transductants received the mutant allele for rpsF of crossovers (thin broken lines) whereas, in a, two crossovers suffice to MB2016. Accordingly, we performed transductions of pyrB and produce such recombinants. Therefore, both cycA and cycB genes ts-210 and analyzed the unselected marker rpsF of the trans- are on the far side of rpsR (and hence the new r-protein gene cluster) ductants. The results (Table 3) indicate that the order of genes from purA. irrthis region is purA, rpsF, pyrB, ts-210. This strongly suggests that the two r-protein genes, rpsF and rpsR, may indeed be results (Tables 3 and 4) show that the two r-protein genes are close to each other. indeed very close to each other, most likely forming a hitherto Among the mutants we have characterized for r-protein al- unknown r-protein gene cluster, because there were no segre- terations, there were two mutants with altered S18 protein (10). gants with respect to rpsF and rpsR. One of them, JE1677 (Fig. ic) was chosen for the fine mapping Discovery of New r-Protein Gene Cluster. We have recently of rpsR. It was first made sensitive to P1kc as described (9) and isolated a mutant (JE1997; Fig. ld) with altered protein L9 (10). then made resistant to D-cycloserine (cyc). The resultant de- The gene coding for this protein (rplI) had not yet been local- rivative was crossed with MB360 purA, and ArgH+ nalidixic ized and, therefore, we undertook mapping of the mutation. acid-resistant (Nal-R) recombinants were selected. One of them, A preliminary mapping placed the mutation between purA and which maintained the S18 alteration of JE1677 and cyc and pyrB, thus raising the possibility of the gene rplI being in the had, in addition, acquired the purA of MB360, was termed neighborhood of rpsF and rpsR. P1 phage-mediated trans- MB2203 and used in subsequent experiments. PurA+ was ductions were then carried out (Table 3). It was discovered not transduced from MB2019 into MB2203 and the unselected only that rpll was cotransducible with purA as were rpsF and markers cyc, rpsF, and rpsR were accordingly examined. The rpsR but also that the segregations between rpsF and rpsR and between rplI and rpsR were less than 3% (no segregants among Table 4. Three-point cross transductions of purA, cycB, and 36 and 48 transductants tested, respectively). This strongly r-protein mutations indicates these three genes form a gene cluster. Results of three-point cross experiments by Plkc transduction Recombina- of purA, cyc, and either rpsF or rpsl (Table 4) favor the gene tion* Trans- order in this region as depicted in model A (purA, rpsR, cyc). Donor r-protein cycB A B ductants, no.t This is not in agreement with the data presented by de Wilde MB2019 rpsF + ad ad 10 et al. (19), in which the order of the genes was reported to be rpsF - ac abcd 4 purA, cycA, rpsR. Because the cyc mutation we introduced into rpsR + abcd ac 1 MB2203 (selected for resistance to 25 ,uM D-cycloserine; see rpsR - ab ab 21 below) was independent of the cycA mutation originally re- MB2251 rplI + ad ad 11 ported (20), we tested the possibility that the two cyc mutations rplI - ac abcd 1 are different. The cycA of X316 (20) was therefore transduced rpsR + abcd ac 0 into MB2203 and MB360 by selecting in each case for PurA+. rpsR - ab ab 36 Using MB360 as recipient, we found that the cycA of X316 was cotransducible with purA at a frequency of 10.3% (23 of 224) A B in contrast to 24.5% in the case of the cyc of MB2203 (Fig. 2). rpsF rpsF Furthermore, we found that 24% (54 of 229) of the PurA+ (rplI) + + (rDlI) transductants derived from MB2203 were Cyc+ (cycloserine-

I I i . I I I I sensitive). Although we can not rule out the possibility that the I I cyc mutation in MB2203 is in fact caused by double mutations, I I I I a i b i c I d a I b I c I d at least we can conclude from these results that it is different i I I ~~~~~~I I ~ mutation of because otherwise the occur- I ~~I I from the cycA X316 I I I I I- rence of Cyc+ transductants at a high frequency can not be I mutation of MB2203 purA rpsR cyd3 purA cycB rpsR explained. Hence, we termed the cyc cycB. In all cases the recipient was MB2203 purA. The levels of resistance to D-cycloserine of cycA and cycB * Necessary recombinational event according to either model A or model B is shown. mutations are slightly but significantly different, even though t No transductants that had become either double mutants (rpsR + the latter was isolated in the same condition as the former (20): rpsF or rpfI) or wild type were obtained. whereas the latter (cycB) confers resistance to up to 0.1 mM Downloaded by guest on October 3, 2021 Genetics: Isono and Kitakawa Proc. Natl. Acad. Sci. USA 75 (1978) 6167

D-cycloserine, the former (cycA) does so only up to 0.05 mM a cluster, it indicates that there are at least four r-protein gene (data not shown). We therefore further investigated the gene clusters in E. coil. and the of order including cycA cycB by analyzing r-proteins We are grateful to Dr. H. G. Wittmann for discussion and to Dr. B. PurA+ transductants obtained from the cross X316 X MB2203 Bachmann for providing us with various E. coli strains. as described above. It was found that all 17 PurA+ transductants (35%) that were Cyc+ possessed the wild-type form of S18. 1. Nomura, M., Morgan, E. A. & Jaskunas, S. R. (1977) Annu. Rev. Because it has been established (Table 4) that the cycB mutation Genet. 11, 297-347. of MB2203 is situated on the far side of the new ribosomal 2. Nashimoto, H. & Uchida, H. (1975) J. Mol. Biol. 96,443-453. protein gene cluster from purA and because the distance be- 3. Yamamoto, M., Strycharz, W. A. & Nomura, M. (1976) Cell 8, tween them is small (about 0.1 min), this result strongly indi- 129-138. 4. Bock, A., Ruffler, D., Piepersberg, W. & Wittmann, H. G. (1974) cates that the four genes concerned reside in the E. coli chro- Mol. Gen. Genet. 134, 325-332. mosome as depicted in Fig. Sa. If cycA is situated between purA 5. Friesen, J. D., Parker, J., Watson, R. J., Fiil, N. P. & Pederson, and rpsR as shown in Fig. 3b, then PurA+ CycA+ RpsR+ CycB+ S. (1976) Mol. Gen. Genet. 144, 115-118. transductants could arise only as a result of four crossovers, a 6. Isono, K., Krauss, J. & Hirota, Y. (1976) Mol. Gen. Genet. 149, rather rare event. Thus, we conclude that both cycA and cycB 297-302. are on the far side of the new r-protein gene cluster from 7. Isono, K., Cumberlidge, A. G., Isono, S. & Hirota, Y. (1977) Mol. purA. Gen. Genet. 152,239-243. 8. Isono, K. & Kitakawa, M. (1977) Mol. Gen. Genet. 153, 115- DISCUSSION 120. 9. Kitakawa, M. & Isono, K. (1977) Mol. Gen. Genet. 158, 149- The data presented in this paper show that the region of the E. 155. coli genetic map containing pyrB, uxuB, and serB can now be 10. Isono, S., Isono, K. & Hirota, Y. (1978) Mol. Gen. Genet. 165, covered by Plkc-mediated transduction and the overall map 15-20. distance in this region should in fact be shorter by approxi- 11. Bachmann, B. J., Low, K. B. & Taylor, A. L. (1976) Bacteriol. Rev. mately 2 min than that previously published (11). 40, 116-167. An extensive genetic analysis of this region utilizing r-protein 12. Robert-Baudouy, J. M. & Portalier, R. C. (1974) Mol. Gen. Genet. mutants has revealed the existence of a new cluster of genes for 131,31-46. r-proteins which contains at least the genes for proteins S6, S18, 13. Bollen, A., Faelen, M., Lecopq, J. P., Herzog, A., Zengel, J., Kahan, L. & M. J. and L9. It would be of interest to construct a spe- Nomura, (1973) Mol. Biol. 76,463-472. immediate 14. Davis, B. D. & Mingioli, E. S. (1950) J. Bactersol. 60, 17-28. cialized X transducing phage covering this region in order to 15. Lomax, M. S. & Greenberg, G. R. (1968) J. Bacteriol. 96,501- investigate whether this cluster contains genes for some other 514. r-proteins as well. Because neither of the r-protein gene clusters 16. de Haan, P. G., Hoekstra, W. P. M., Verhoef, C. & Felix, H. S. at 72 and 88 min contains the genes coding for, for example, (1969) Mutat. Res. 8, 505-512. r-proteins S1, S9, L13, etc. or for protein synthesis initiation 17. Taylor, A. L. (1972) in Experiments in Molecular Genetics, ed. factors IF-1 and IF-2, whose gene locations are as yet not Miller, J. H. (Cold Spring Harbor Laboratory, Cold Spring known, it would be worthwhile to explore this region. Harbor, NY), pp. 451-454. A recent report by Takata (21) showed that there seemed to 18. Wu, T. T. (1966) Genetics 54,405-410. 19. de Wilde, M., Michel, F. & Broman, K. (1974) Mol. Gen. Genet. be another r-protein gene cluster near argG at 68 min which 133,329-3. contains the genes for proteins S15, L21, L27, and S21. He re- 20. Curtiss, R., III, Charamella, L. J., Berg, C. M. & Harris, P. E. ported that the gene for S21 seemed a little away from the (1965) J. Bacteriol. 90, 1238-1250. former three. If the genes for the former three proteins form 21. Takata, R. (1978) Mol. Gen. Genet. 160, 151-155. Downloaded by guest on October 3, 2021