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 A mutant of Escbenchia coli K-12 isolated for ABSTRACT temperature-sensitive growth was found to harbor an alteration in ribosomal protein L9. Because the chromosomal location of the structural gene for this protein (rpll) was not known, we mapped the mutation by using various Hfr strains. The fine mapping of this gene by Plkc phage-mediated transductions has revealed that it forms a gene cluster at 94 min on the E coli genetic map together with the genes coding for two other ribosomal proteins, S6 (rps1;) and S18 (QpsR). Furthermore, the region of the E coli genetic map containing this cluster was found to be shorter than previously estimated by approximately 2 min.
Many of the genes coding for ribosomal components [5S, 16S, and 23S rRNAs and 53 ribosomal proteins (r-proteins)] have been localized on the Escherichia coii genetic map (for review, see ref. 1). They either form gene clusters together with the genes coding for RNA polymerase subunits and protein synthesis elongation factors (at 72 and 88 min) or most likely exist as single units [for example, the genes for r-proteins S2 (2, 3) and S20 (4, 5)]. The genes for 43 r-proteins of E. coli have thus far been mapped at seven different loci, which leaves the genes for 10 more r-proteins to be localized. By utilizing mutants, isolated for temperature-sensitive growth, that harbored structurally altered r-proteins (6, 7), we could place the genes for protein S6 (8) and protein L19 (9) at 97 and 56.4 min, respectively, on the E. coli genetic map. More recently, we have isolated another mutant that contained an alteration in protein L9 (10). Because the gene for this protein (rplI) had not yet been mapped, we performed mapping of this mutation. During the course of this work, we found that the region of the published E. colt genetic map (11) that contained the genes for proteins S6 and S18 should be shortened by about 2 min and that, when this was done, the two genes could be brought together. Furthermore, we discovered that the genes for these two proteins as well as the gene for protein L9 formed a cluster near purA.
MATERIALS AND METHODS Bacterial strains used in this work are listed in Table 1. Plkc phage-mediated transduction and analysis of transductants for r-proteins by two-dimensional gel electrophoresis and for other markers were performed as previously reported (8). Uxu phenotypes were tested on minimal A (14) agar plates containing all the requirements and D-glucuronic acid (Sigma) added in place of glucose. Amino acids were added to the minimal A medium at 60,qg/ml each, and guanine and thymine were added at 20 jg/ml each. Spontaneous deo derivatives were obtained from MB2014 by spreading an overnight culture, washed once with minimal The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
A medium, on minimal A agar plates supplemented with all the requirements described above except that thymine was at 2 ,gg/ml. Plates were incubated at 300C for 4-5 days, and colonies were purified on the same plates. Purified clones were tested for growth on suitably supplemented minimal A agar plates containing no thymine or thymine at 2 or 20 /ig/ml; those growing only on the two thymine-supplemented plates were classified as deo mutants (15).
RESULTS Mapping of uxuB. In our previous report (8), we placed the gene coding for r-protein S6 (rpsF) and an unidentified temperature-sensitive mutation (ts-210) of mutant JE210 (Fig. lb) at 97 min and 98 min, respectively. Later we found that, although they were quite away from each other, they were cotransducible in P1 phage-mediated transduction. Unfortunately, the region of the E. coil genetic map in which these mutations fell was not well characterized and the only markers available were uxuA and uxuB (12). If our mapping, which relied on the gradient transmission of markers from Hfr to Fbacteria (16, 17), were correct, then it is conceivable that either the rpsF or ts-210 mutation of JE210 may be cotransducible with uxu. To begin with, we constructed a strain that contained rpsF, ts-210, and uxu mutations. The uxuB mutant CM8 was found to maintain the maleness of its parental Hfr P4X and we made use of this character. Strain CM8 was crossed with mutant JE210, and ArgH+ streptomycin-resistant (Str-R) recombinants were selected. They were then tested for rpsF, ts-210, thr, leu, and uxuB. One of the resultant recombinants was termed MB2014 and was used in transductions in which UxuB+ was selected for. As shown in Table 2, about 3% of the UxuB+ transductants became Ts+, indicating that they were away from each other by 1.4 min (18). Furthermore, because uxuB is transferred earliest during conjugation with an HfrH (ref. 14; unpublished data) but ts-210 is not, the gene order in this region is most likely ts-210, uxuB, thr, although the reported gene location for uxuB is at 97 min and that for ts-210 at 98 min. If uxuB maps at 97 min and if the above gene order is correct, then ts-210 may be cotransducible with pyrB which is situated at 94.7 min (11). Conversely, if the location of ts-210 at 98 min is correct, then uxuB may be cotransducible with deo, one of the nearest markers situated at 99.5 min (11). A spontaneous deo mutation was therefore introduced into MB2014 and one of the resultant derivatives, termed MB2015, and several derivatives thereof (Table 1) were subsequently analyzed. The results of various transductions carried out using MB2015 and its derivatives also are summarized in Table 2. UxuB was found to be cotransducible with deo at the frequency of 5%, and, surprisingly, ts-210 and pyrB were also cotransducible at Abbreviations: r-proteins, ribosomal proteins; Str-R,
streptomycin; Nal-R, resistance to nalidixic acid.
6163
resistance to
6164
_.
Genetics: Isono and Kitakawa
Proc. Natl. Acad. Sci. USA 75 (1978)
a
0
9.
L9
S6
_1
._
S18
a
::'rsl;:
S6
4
a
S18--
pi b
c
o
L9
~~0
a
.4
9 d
akk
ie,:,,
FIG. 1. Two-dimensional gel electropherograms of parental strain PA3092 (a) and mutants JE210 (b), JE1677 (c), and JE1997 (d). Only the portion of the gels containing r-proteins S6, S18, and L9 are shown.
Proc. Natl. Acad. Sci. USA 75 (1978)
Genetics: Isono and Kitakawa Table 1. Bacterial strains used Source or
Strain
Relevant phenotype*
referencet
JE 210 CM 8 MB2014
F- rpsF (S6), ts-210 Hfr uxuB CM 8 X JE210, rpsF (S6), ts-210, uxuB deo derivative of MB2014 P1s derivative of MB2015 serB derivative of MB2015 ts-210+ revertant of MB2018 F- aroE F- serB Hfr purA argI+ derivative of PC0950 F- pyrB purA derivative of AT2535 F- rpsR (S18) MB360, X JE1677, purA, rpsR, cycB,
2, 4 12 This paper
MB2015 MB2016 MB2018 MB2019 AB2834 AT2459 PC0950 MB360 AT2535 MB365 JE1677 MB2203
This paper This paper This paper This paper CGSC CGSC CGSC This paper CGSC This paper 3 This paper
P1s
JE1997 F- rplI (L9) 10 Hfr PK191 CGSC MB2251 PK191 X JE1997, rpII (L9), P1s This paper F- cycA 13 X316 * Only those markers relevant to this work are listed. t CGSC, E. coli Genetic Stock Center at Yale University (through the courtesy of B. Bachmann).
a high frequency (60-70%). Thus, the region of the E. coli genetic map that has not been covered by P1 phage-mediated transduction can now be covered by it and markers in this region can be connected. When these cotransduction frequencies were converted into map units in minutes according to Wu's equation (18) and as discussed by Bachmann et al. (11), the cumulative distance between pyrB and deo (4.8 min according to ref. 11) was found in fact to be much shorter (approximately 3 min). UxuB+ transductants were then purified and their rprotein 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 cotransductants among 10 Ts+ transductants examined for rpsF (8). If rpsF and ts-210 are located on the opposite sides of uxuB-that is, if uxuB is located between rpsF and ts-210then 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 between uxuB and deo is only 1.3 min (5% cotransduction) as opposed to the previous computation (12). This clearly indicates that uxuB cannot be located at 96.8 min and that the order of
these four genes most likely would be rpsF, ts-210, uxuB, deo. T~hImodso consistentwith the fact that uxuB is one of the earliest markers transferred by Hfr H (12), whereas neither ts-210 nor rpsF is (8). In other words, the point of entry of the chromosome of an HfrH strain must fall somewhere between ts-210 and uxuB. Therefore, the next step was to correlate, by Plkc transduction, the genes described above with those flanking this region. For this purpose we chose purA, pyrB, serB, and thr and performed various combinations of transductions. The results are summarized in Table 2 and Fig. 2. The most important feature of the results obtained is that we can now cover this region by Plkc transductions. The ts-210 mutation is away from pyrB by only 0.3 min. UxuB and ts-210 are away from each other by r1.3 min. The data for the cotransduction frequency between uxuB and deo had a rather large fluctuation with the average value for the distance between them of 1.1 min. Taking all these results into account, the distance between pyrB and serB, which are situated at 94.7 and 99.6 min, respectively (11), is in fact much shorter: instead of 4.9 min, it should be only 2.9 min. Therefore, this region of the E. coli genetic map should be shortened by 2 min. Fine Mapping of rpsF(S6) and rpsR (SiS). Because it has become evident that the distance between pyrB and serB (and hence between argH and leu) is --2 min shorter than previously reported, we adopted the new distance between argH and letu (12 min instead of 14 min) to correct our gradient transmission data for rpsF and ts-210 (figure 4 of ref. 8). This gave new estimates of 95.7 ± 1.2 min for the former and 95.9 + 1.2 min for the latter. These corrected values are now in good agreement with the transduction data for ts-210 as described above. Therefore, it is very likely that rpsF should actually be situated between purA and pyrB. If this is the case, then it is also interesting to determine if rpsF in fact maps close to rpsR, the structural gene for protein S18 that was the first r-protein gene mapped outside the strA cluster at 72 min (13, 19).
/
Selected Unselected Cotransduction Recipient marker marker frequency, %* MB2014 UxuB+ Ts+ 3.0 (18/592) MB2015 UxuB+ Deo+ 5.0 (27/544) MB2018 SerB+ Deo+ 86.3 (511/592) MB2018 SerB+ UxuB+ 1.0 (6/592) MB2016 AT2535 PyrB+ Ts71 (17/24) MB360 PurA+ Ts0.2 (1/592) MB365 PurA+ PyrB+ 1.7 (3/181) MB365 PyrB+ PurA+ 11.7 (16/137) MB365 PyrB+ Ts60.6 (83/137) MB365 PyrB+ UxuB2.9 (4/137) * Values in parentheses are no. transductants/total no.
75
strA /
/
/ /
/
/ //I rpsF, rpsR,rplI pyrB ts-210 purA' cyB lb I I r-cycA I -
9/1318(2.8%)
Table 2. Mapping of uxuB and ts-210 by transduction Donor AB2834
6165
841343
94 0 /61
uxuB
deo serB thr ,I ~11 4
~~~~~~~i
73t1/1170
29713P) 13711616 185%)
162%l 3/24 (13%) (245%) *21126 ,j3_1868 (02%)_ .
-
1022/2484 (411%)
(33%) 2/8012.5%)388 % 23/221 IMM) 6761887
176.2%)
11296(03%)
13/1329 (1.0%)
FIG. 2. Summary of transduction experiments and alignment of genes on the E. coli genetic map (circle) between purA and thr. All the genes are placed on the map according to their relative distances from purA as calculated from the cotransduction frequencies as described by Wu (17).. Markers at arrowtails are those selected for in transductions, and those at arrowheads are unselected ones examined afterward. When arrows point in both directions, they indicate that reciprocal transductions were performed. Numbers indicate cotransductants among total tranaductants tested with the percentage given in brackets.
6166
Genetics: Isono and Kitakawa
Proc. Nati. Acad. Sci. USA 75 (1978)
Table 3. Fine mapping of rpsF, rpsR, and rplI by transduction
Selected Unselected Cotransduction marker frequency, %* Recipient marker
Donor AB2834 MB2016 MB2016 MB2019t
JE210 AT2535 MB360 MB2203
Ts+ PyrB+ PurA+ PurA+
RpsF+ RpsFRpsFRpsR+
MB2251t
MB2203
PurA+
RpsR+
+
a ----r-
+
+
I
I
cycA
I
3 (1/40) 13 (3/24) 39 (9/23) 39 (14/36)
i
I
I
purA
I +
rpsR cycB
(RpsF-) 25 (12/48)
b
cycA
+
+
+
(RplI-) .,
* Values in parentheses are no. transductants/total no. t For detail of these transductions, see Table 4.
I
First, we transduced purA + of MB2016 into MB360 purA, purified 24 transductants to single colonies, and analyzed them for the coinheritance of rpsF (the S6 mutation) by two-dimensional electrophoresis. As shown in Table 3, 9 of 23 (39%) PurA+ transductants received the mutant allele for rpsF of MB2016. Accordingly, we performed transductions of pyrB and ts-210 and analyzed the unselected marker rpsF of the transductants. The results (Table 3) indicate that the order of genes irrthis region is purA, rpsF, pyrB, ts-210. This strongly suggests that the two r-protein genes, rpsF and rpsR, may indeed be close to each other. Among the mutants we have characterized for r-protein alterations, there were two mutants with altered S18 protein (10). One of them, JE1677 (Fig. ic) was chosen for the fine mapping of rpsR. It was first made sensitive to P1kc as described (9) and then made resistant to D-cycloserine (cyc). The resultant derivative was crossed with MB360 purA, and ArgH+ nalidixic acid-resistant (Nal-R) recombinants were selected. One of them, which maintained the S18 alteration of JE1677 and cyc and had, in addition, acquired the purA of MB360, was termed MB2203 and used in subsequent experiments. PurA+ was transduced from MB2019 into MB2203 and the unselected markers cyc, rpsF, and rpsR were accordingly examined. The Table 4. Three-point cross transductions of purA, cycB, and r-protein mutations Recombination* TransB ductants, no.t Donor r-protein cycB A
rpsF
MB2019
+ -
rpsF rpsR rpsR
+ + -
rplI
MB2251
rplI rpsR rpsR
+ -
ad ac abcd ab ad ac abcd ab
ad abcd ac ab ad
10 4 1 21 11
abcd
1 0 36
ac ab
B
A
rpsF (rplI) I
a
b
i I
i c II d ~~ ~~~~I i
I
I I
a II I I
.
I I
I I
rpsF (rDlI)
i
I
I I
+
+
b I
II
I
I
c
~~I I
~
II I
d
I-
purA cycB rpsR rpsR cyd3 In all cases the recipient was MB2203 purA. * Necessary recombinational event according to either model A or model B is shown. t No transductants that had become either double mutants (rpsR + rpsF or rpfI) or wild type were obtained. purA
purA
I~~~~~~~~~~~ +
rpsR cycB
FIG. 3. Two possible orientations of genes cycA and cycB with 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 of four crossovers (thin broken lines) whereas, in a, two crossovers suffice to produce such recombinants. Therefore, both cycA and cycB genes are on the far side of rpsR (and hence the new r-protein gene cluster) from purA.
results (Tables 3 and 4) show that the two r-protein genes are indeed very close to each other, most likely forming a hitherto unknown r-protein gene cluster, because there were no segregants with respect to rpsF and rpsR. Discovery of New r-Protein Gene Cluster. We have recently isolated a mutant (JE1997; Fig. ld) with altered protein L9 (10). The gene coding for this protein (rplI) had not yet been localized and, therefore, we undertook mapping of the mutation. A preliminary mapping placed the mutation between purA and pyrB, thus raising the possibility of the gene rplI being in the neighborhood of rpsF and rpsR. P1 phage-mediated transductions were then carried out (Table 3). It was discovered not only that rpll was cotransducible with purA as were rpsF and rpsR but also that the segregations between rpsF and rpsR and between rplI and rpsR were less than 3% (no segregants among 36 and 48 transductants tested, respectively). This strongly indicates these three genes form a gene cluster. Results of three-point cross experiments by Plkc transduction of purA, cyc, and either rpsF or rpsl (Table 4) favor the gene order in this region as depicted in model A (purA, rpsR, cyc). This is not in agreement with the data presented by de Wilde et al. (19), in which the order of the genes was reported to be purA, cycA, rpsR. Because the cyc mutation we introduced into MB2203 (selected for resistance to 25 ,uM D-cycloserine; see below) was independent of the cycA mutation originally reported (20), we tested the possibility that the two cyc mutations are different. The cycA of X316 (20) was therefore transduced into MB2203 and MB360 by selecting in each case for PurA+. Using MB360 as recipient, we found that the cycA of X316 was cotransducible with purA at a frequency of 10.3% (23 of 224) in contrast to 24.5% in the case of the cyc of MB2203 (Fig. 2). Furthermore, we found that 24% (54 of 229) of the PurA+ transductants derived from MB2203 were Cyc+ (cycloserinesensitive). Although we can not rule out the possibility that the cyc mutation in MB2203 is in fact caused by double mutations, at least we can conclude from these results that it is different from the cycA mutation of X316 because otherwise the occurrence of Cyc+ transductants at a high frequency can not be explained. Hence, we termed the cyc mutation of MB2203 cycB. The levels of resistance to D-cycloserine of cycA and cycB mutations are slightly but significantly different, even though the latter was isolated in the same condition as the former (20): whereas the latter (cycB) confers resistance to up to 0.1 mM
Genetics: Isono and Kitakawa
D-cycloserine, the former (cycA) does so only up to 0.05 mM (data not shown). We therefore further investigated the gene order including cycA and cycB by analyzing the r-proteins of PurA+ transductants obtained from the cross X316 X MB2203 as described above. It was found that all 17 PurA+ transductants (35%) that were Cyc+ possessed the wild-type form of S18. Because it has been established (Table 4) that the cycB mutation of MB2203 is situated on the far side of the new ribosomal protein gene cluster from purA and because the distance between them is small (about 0.1 min), this result strongly indicates that the four genes concerned reside in the E. coli chromosome as depicted in Fig. Sa. If cycA is situated between purA and rpsR as shown in Fig. 3b, then PurA+ CycA+ RpsR+ CycB+ transductants could arise only as a result of four crossovers, a rather rare event. Thus, we conclude that both cycA and cycB are on the far side of the new r-protein gene cluster from purA.
DISCUSSION The data presented in this paper show that the region of the E. coli genetic map containing pyrB, uxuB, and serB can now be covered by Plkc-mediated transduction and the overall map distance in this region should in fact be shorter by approximately 2 min than that previously published (11). An extensive genetic analysis of this region utilizing r-protein mutants has revealed the existence of a new cluster of genes for r-proteins which contains at least the genes for proteins S6, S18, and L9. It would be of immediate interest to construct a specialized X transducing phage covering this region in order to investigate whether this cluster contains genes for some other r-proteins as well. Because neither of the r-protein gene clusters at 72 and 88 min contains the genes coding for, for example, r-proteins S1, S9, L13, etc. or for protein synthesis initiation factors IF-1 and IF-2, whose gene locations are as yet not known, it would be worthwhile to explore this region. A recent report by Takata (21) showed that there seemed to be another r-protein gene cluster near argG at 68 min which contains the genes for proteins S15, L21, L27, and S21. He reported that the gene for S21 seemed a little away from the former three. If the genes for the former three proteins form
Proc. Natl. Acad. Sci. USA 75 (1978)
6167
a cluster, it indicates that there are at least four r-protein gene clusters in E. coil. We are grateful to Dr. H. G. Wittmann for discussion and to Dr. B. Bachmann for providing us with various E. coli strains. 1. Nomura, M., Morgan, E. A. & Jaskunas, S. R. (1977) Annu. Rev. Genet. 11, 297-347. 2. Nashimoto, H. & Uchida, H. (1975) J. Mol. Biol. 96,443-453. 3. Yamamoto, M., Strycharz, W. A. & Nomura, M. (1976) Cell 8, 129-138. 4. Bock, A., Ruffler, D., Piepersberg, W. & Wittmann, H. G. (1974)
Mol. Gen. Genet. 134, 325-332. 5. Friesen, J. D., Parker, J., Watson, R. J., Fiil, N. P. & Pederson, S. (1976) Mol. Gen. Genet. 144, 115-118. 6. Isono, K., Krauss, J. & Hirota, Y. (1976) Mol. Gen. Genet. 149, 297-302. 7. Isono, K., Cumberlidge, A. G., Isono, S. & Hirota, Y. (1977) Mol. Gen. Genet. 152,239-243. 8. Isono, K. & Kitakawa, M. (1977) Mol. Gen. Genet. 153, 115120. 9. Kitakawa, M. & Isono, K. (1977) Mol. Gen. Genet. 158, 149155. 10. Isono, S., Isono, K. & Hirota, Y. (1978) Mol. Gen. Genet. 165, 15-20. 11. Bachmann, B. J., Low, K. B. & Taylor, A. L. (1976) Bacteriol. Rev. 40, 116-167. 12. Robert-Baudouy, J. M. & Portalier, R. C. (1974) Mol. Gen. Genet. 131,31-46. 13. Bollen, A., Faelen, M., Lecopq, J. P., Herzog, A., Zengel, J., Kahan, L. & Nomura, M. (1973) J. Mol. Biol. 76,463-472. 14. Davis, B. D. & Mingioli, E. S. (1950) J. Bactersol. 60, 17-28. 15. Lomax, M. S. & Greenberg, G. R. (1968) J. Bacteriol. 96,501514. 16. de Haan, P. G., Hoekstra, W. P. M., Verhoef, C. & Felix, H. S. (1969) Mutat. Res. 8, 505-512. 17. Taylor, A. L. (1972) in Experiments in Molecular Genetics, ed. Miller, J. H. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), pp. 451-454. 18. Wu, T. T. (1966) Genetics 54,405-410. 19. de Wilde, M., Michel, F. & Broman, K. (1974) Mol. Gen. Genet. 133,329-3. 20. Curtiss, R., III, Charamella, L. J., Berg, C. M. & Harris, P. E. (1965) J. Bacteriol. 90, 1238-1250. 21. Takata, R. (1978) Mol. Gen. Genet. 160, 151-155.
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 A mutant of Escbenchia coli K-12 isolated for ABSTRACT temperature-sensitive growth was found to harbor an alteration in ribosomal protein L9. Because the chromosomal location of the structural gene for this protein (rpll) was not known, we mapped the mutation by using various Hfr strains. The fine mapping of this gene by Plkc phage-mediated transductions has revealed that it forms a gene cluster at 94 min on the E coli genetic map together with the genes coding for two other ribosomal proteins, S6 (rps1;) and S18 (QpsR). Furthermore, the region of the E coli genetic map containing this cluster was found to be shorter than previously estimated by approximately 2 min.
Many of the genes coding for ribosomal components [5S, 16S, and 23S rRNAs and 53 ribosomal proteins (r-proteins)] have been localized on the Escherichia coii genetic map (for review, see ref. 1). They either form gene clusters together with the genes coding for RNA polymerase subunits and protein synthesis elongation factors (at 72 and 88 min) or most likely exist as single units [for example, the genes for r-proteins S2 (2, 3) and S20 (4, 5)]. The genes for 43 r-proteins of E. coli have thus far been mapped at seven different loci, which leaves the genes for 10 more r-proteins to be localized. By utilizing mutants, isolated for temperature-sensitive growth, that harbored structurally altered r-proteins (6, 7), we could place the genes for protein S6 (8) and protein L19 (9) at 97 and 56.4 min, respectively, on the E. coli genetic map. More recently, we have isolated another mutant that contained an alteration in protein L9 (10). Because the gene for this protein (rplI) had not yet been mapped, we performed mapping of this mutation. During the course of this work, we found that the region of the published E. colt genetic map (11) that contained the genes for proteins S6 and S18 should be shortened by about 2 min and that, when this was done, the two genes could be brought together. Furthermore, we discovered that the genes for these two proteins as well as the gene for protein L9 formed a cluster near purA.
MATERIALS AND METHODS Bacterial strains used in this work are listed in Table 1. Plkc phage-mediated transduction and analysis of transductants for r-proteins by two-dimensional gel electrophoresis and for other markers were performed as previously reported (8). Uxu phenotypes were tested on minimal A (14) agar plates containing all the requirements and D-glucuronic acid (Sigma) added in place of glucose. Amino acids were added to the minimal A medium at 60,qg/ml each, and guanine and thymine were added at 20 jg/ml each. Spontaneous deo derivatives were obtained from MB2014 by spreading an overnight culture, washed once with minimal The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
A medium, on minimal A agar plates supplemented with all the requirements described above except that thymine was at 2 ,gg/ml. Plates were incubated at 300C for 4-5 days, and colonies were purified on the same plates. Purified clones were tested for growth on suitably supplemented minimal A agar plates containing no thymine or thymine at 2 or 20 /ig/ml; those growing only on the two thymine-supplemented plates were classified as deo mutants (15).
RESULTS Mapping of uxuB. In our previous report (8), we placed the gene coding for r-protein S6 (rpsF) and an unidentified temperature-sensitive mutation (ts-210) of mutant JE210 (Fig. lb) at 97 min and 98 min, respectively. Later we found that, although they were quite away from each other, they were cotransducible in P1 phage-mediated transduction. Unfortunately, the region of the E. coil genetic map in which these mutations fell was not well characterized and the only markers available were uxuA and uxuB (12). If our mapping, which relied on the gradient transmission of markers from Hfr to Fbacteria (16, 17), were correct, then it is conceivable that either the rpsF or ts-210 mutation of JE210 may be cotransducible with uxu. To begin with, we constructed a strain that contained rpsF, ts-210, and uxu mutations. The uxuB mutant CM8 was found to maintain the maleness of its parental Hfr P4X and we made use of this character. Strain CM8 was crossed with mutant JE210, and ArgH+ streptomycin-resistant (Str-R) recombinants were selected. They were then tested for rpsF, ts-210, thr, leu, and uxuB. One of the resultant recombinants was termed MB2014 and was used in transductions in which UxuB+ was selected for. As shown in Table 2, about 3% of the UxuB+ transductants became Ts+, indicating that they were away from each other by 1.4 min (18). Furthermore, because uxuB is transferred earliest during conjugation with an HfrH (ref. 14; unpublished data) but ts-210 is not, the gene order in this region is most likely ts-210, uxuB, thr, although the reported gene location for uxuB is at 97 min and that for ts-210 at 98 min. If uxuB maps at 97 min and if the above gene order is correct, then ts-210 may be cotransducible with pyrB which is situated at 94.7 min (11). Conversely, if the location of ts-210 at 98 min is correct, then uxuB may be cotransducible with deo, one of the nearest markers situated at 99.5 min (11). A spontaneous deo mutation was therefore introduced into MB2014 and one of the resultant derivatives, termed MB2015, and several derivatives thereof (Table 1) were subsequently analyzed. The results of various transductions carried out using MB2015 and its derivatives also are summarized in Table 2. UxuB was found to be cotransducible with deo at the frequency of 5%, and, surprisingly, ts-210 and pyrB were also cotransducible at Abbreviations: r-proteins, ribosomal proteins; Str-R,
streptomycin; Nal-R, resistance to nalidixic acid.
6163
resistance to
6164
_.
Genetics: Isono and Kitakawa
Proc. Natl. Acad. Sci. USA 75 (1978)
a
0
9.
L9
S6
_1
._
S18
a
::'rsl;:
S6
4
a
S18--
pi b
c
o
L9
~~0
a
.4
9 d
akk
ie,:,,
FIG. 1. Two-dimensional gel electropherograms of parental strain PA3092 (a) and mutants JE210 (b), JE1677 (c), and JE1997 (d). Only the portion of the gels containing r-proteins S6, S18, and L9 are shown.
Proc. Natl. Acad. Sci. USA 75 (1978)
Genetics: Isono and Kitakawa Table 1. Bacterial strains used Source or
Strain
Relevant phenotype*
referencet
JE 210 CM 8 MB2014
F- rpsF (S6), ts-210 Hfr uxuB CM 8 X JE210, rpsF (S6), ts-210, uxuB deo derivative of MB2014 P1s derivative of MB2015 serB derivative of MB2015 ts-210+ revertant of MB2018 F- aroE F- serB Hfr purA argI+ derivative of PC0950 F- pyrB purA derivative of AT2535 F- rpsR (S18) MB360, X JE1677, purA, rpsR, cycB,
2, 4 12 This paper
MB2015 MB2016 MB2018 MB2019 AB2834 AT2459 PC0950 MB360 AT2535 MB365 JE1677 MB2203
This paper This paper This paper This paper CGSC CGSC CGSC This paper CGSC This paper 3 This paper
P1s
JE1997 F- rplI (L9) 10 Hfr PK191 CGSC MB2251 PK191 X JE1997, rpII (L9), P1s This paper F- cycA 13 X316 * Only those markers relevant to this work are listed. t CGSC, E. coli Genetic Stock Center at Yale University (through the courtesy of B. Bachmann).
a high frequency (60-70%). Thus, the region of the E. coli genetic map that has not been covered by P1 phage-mediated transduction can now be covered by it and markers in this region can be connected. When these cotransduction frequencies were converted into map units in minutes according to Wu's equation (18) and as discussed by Bachmann et al. (11), the cumulative distance between pyrB and deo (4.8 min according to ref. 11) was found in fact to be much shorter (approximately 3 min). UxuB+ transductants were then purified and their rprotein 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 cotransductants among 10 Ts+ transductants examined for rpsF (8). If rpsF and ts-210 are located on the opposite sides of uxuB-that is, if uxuB is located between rpsF and ts-210then 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 between uxuB and deo is only 1.3 min (5% cotransduction) as opposed to the previous computation (12). This clearly indicates that uxuB cannot be located at 96.8 min and that the order of
these four genes most likely would be rpsF, ts-210, uxuB, deo. T~hImodso consistentwith the fact that uxuB is one of the earliest markers transferred by Hfr H (12), whereas neither ts-210 nor rpsF is (8). In other words, the point of entry of the chromosome of an HfrH strain must fall somewhere between ts-210 and uxuB. Therefore, the next step was to correlate, by Plkc transduction, the genes described above with those flanking this region. For this purpose we chose purA, pyrB, serB, and thr and performed various combinations of transductions. The results are summarized in Table 2 and Fig. 2. The most important feature of the results obtained is that we can now cover this region by Plkc transductions. The ts-210 mutation is away from pyrB by only 0.3 min. UxuB and ts-210 are away from each other by r1.3 min. The data for the cotransduction frequency between uxuB and deo had a rather large fluctuation with the average value for the distance between them of 1.1 min. Taking all these results into account, the distance between pyrB and serB, which are situated at 94.7 and 99.6 min, respectively (11), is in fact much shorter: instead of 4.9 min, it should be only 2.9 min. Therefore, this region of the E. coli genetic map should be shortened by 2 min. Fine Mapping of rpsF(S6) and rpsR (SiS). Because it has become evident that the distance between pyrB and serB (and hence between argH and leu) is --2 min shorter than previously reported, we adopted the new distance between argH and letu (12 min instead of 14 min) to correct our gradient transmission data for rpsF and ts-210 (figure 4 of ref. 8). This gave new estimates of 95.7 ± 1.2 min for the former and 95.9 + 1.2 min for the latter. These corrected values are now in good agreement with the transduction data for ts-210 as described above. Therefore, it is very likely that rpsF should actually be situated between purA and pyrB. If this is the case, then it is also interesting to determine if rpsF in fact maps close to rpsR, the structural gene for protein S18 that was the first r-protein gene mapped outside the strA cluster at 72 min (13, 19).
/
Selected Unselected Cotransduction Recipient marker marker frequency, %* MB2014 UxuB+ Ts+ 3.0 (18/592) MB2015 UxuB+ Deo+ 5.0 (27/544) MB2018 SerB+ Deo+ 86.3 (511/592) MB2018 SerB+ UxuB+ 1.0 (6/592) MB2016 AT2535 PyrB+ Ts71 (17/24) MB360 PurA+ Ts0.2 (1/592) MB365 PurA+ PyrB+ 1.7 (3/181) MB365 PyrB+ PurA+ 11.7 (16/137) MB365 PyrB+ Ts60.6 (83/137) MB365 PyrB+ UxuB2.9 (4/137) * Values in parentheses are no. transductants/total no.
75
strA /
/
/ /
/
/ //I rpsF, rpsR,rplI pyrB ts-210 purA' cyB lb I I r-cycA I -
9/1318(2.8%)
Table 2. Mapping of uxuB and ts-210 by transduction Donor AB2834
6165
841343
94 0 /61
uxuB
deo serB thr ,I ~11 4
~~~~~~~i
73t1/1170
29713P) 13711616 185%)
162%l 3/24 (13%) (245%) *21126 ,j3_1868 (02%)_ .
-
1022/2484 (411%)
(33%) 2/8012.5%)388 % 23/221 IMM) 6761887
176.2%)
11296(03%)
13/1329 (1.0%)
FIG. 2. Summary of transduction experiments and alignment of genes on the E. coli genetic map (circle) between purA and thr. All the genes are placed on the map according to their relative distances from purA as calculated from the cotransduction frequencies as described by Wu (17).. Markers at arrowtails are those selected for in transductions, and those at arrowheads are unselected ones examined afterward. When arrows point in both directions, they indicate that reciprocal transductions were performed. Numbers indicate cotransductants among total tranaductants tested with the percentage given in brackets.
6166
Genetics: Isono and Kitakawa
Proc. Nati. Acad. Sci. USA 75 (1978)
Table 3. Fine mapping of rpsF, rpsR, and rplI by transduction
Selected Unselected Cotransduction marker frequency, %* Recipient marker
Donor AB2834 MB2016 MB2016 MB2019t
JE210 AT2535 MB360 MB2203
Ts+ PyrB+ PurA+ PurA+
RpsF+ RpsFRpsFRpsR+
MB2251t
MB2203
PurA+
RpsR+
+
a ----r-
+
+
I
I
cycA
I
3 (1/40) 13 (3/24) 39 (9/23) 39 (14/36)
i
I
I
purA
I +
rpsR cycB
(RpsF-) 25 (12/48)
b
cycA
+
+
+
(RplI-) .,
* Values in parentheses are no. transductants/total no. t For detail of these transductions, see Table 4.
I
First, we transduced purA + of MB2016 into MB360 purA, purified 24 transductants to single colonies, and analyzed them for the coinheritance of rpsF (the S6 mutation) by two-dimensional electrophoresis. As shown in Table 3, 9 of 23 (39%) PurA+ transductants received the mutant allele for rpsF of MB2016. Accordingly, we performed transductions of pyrB and ts-210 and analyzed the unselected marker rpsF of the transductants. The results (Table 3) indicate that the order of genes irrthis region is purA, rpsF, pyrB, ts-210. This strongly suggests that the two r-protein genes, rpsF and rpsR, may indeed be close to each other. Among the mutants we have characterized for r-protein alterations, there were two mutants with altered S18 protein (10). One of them, JE1677 (Fig. ic) was chosen for the fine mapping of rpsR. It was first made sensitive to P1kc as described (9) and then made resistant to D-cycloserine (cyc). The resultant derivative was crossed with MB360 purA, and ArgH+ nalidixic acid-resistant (Nal-R) recombinants were selected. One of them, which maintained the S18 alteration of JE1677 and cyc and had, in addition, acquired the purA of MB360, was termed MB2203 and used in subsequent experiments. PurA+ was transduced from MB2019 into MB2203 and the unselected markers cyc, rpsF, and rpsR were accordingly examined. The Table 4. Three-point cross transductions of purA, cycB, and r-protein mutations Recombination* TransB ductants, no.t Donor r-protein cycB A
rpsF
MB2019
+ -
rpsF rpsR rpsR
+ + -
rplI
MB2251
rplI rpsR rpsR
+ -
ad ac abcd ab ad ac abcd ab
ad abcd ac ab ad
10 4 1 21 11
abcd
1 0 36
ac ab
B
A
rpsF (rplI) I
a
b
i I
i c II d ~~ ~~~~I i
I
I I
a II I I
.
I I
I I
rpsF (rDlI)
i
I
I I
+
+
b I
II
I
I
c
~~I I
~
II I
d
I-
purA cycB rpsR rpsR cyd3 In all cases the recipient was MB2203 purA. * Necessary recombinational event according to either model A or model B is shown. t No transductants that had become either double mutants (rpsR + rpsF or rpfI) or wild type were obtained. purA
purA
I~~~~~~~~~~~ +
rpsR cycB
FIG. 3. Two possible orientations of genes cycA and cycB with 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 of four crossovers (thin broken lines) whereas, in a, two crossovers suffice to produce such recombinants. Therefore, both cycA and cycB genes are on the far side of rpsR (and hence the new r-protein gene cluster) from purA.
results (Tables 3 and 4) show that the two r-protein genes are indeed very close to each other, most likely forming a hitherto unknown r-protein gene cluster, because there were no segregants with respect to rpsF and rpsR. Discovery of New r-Protein Gene Cluster. We have recently isolated a mutant (JE1997; Fig. ld) with altered protein L9 (10). The gene coding for this protein (rplI) had not yet been localized and, therefore, we undertook mapping of the mutation. A preliminary mapping placed the mutation between purA and pyrB, thus raising the possibility of the gene rplI being in the neighborhood of rpsF and rpsR. P1 phage-mediated transductions were then carried out (Table 3). It was discovered not only that rpll was cotransducible with purA as were rpsF and rpsR but also that the segregations between rpsF and rpsR and between rplI and rpsR were less than 3% (no segregants among 36 and 48 transductants tested, respectively). This strongly indicates these three genes form a gene cluster. Results of three-point cross experiments by Plkc transduction of purA, cyc, and either rpsF or rpsl (Table 4) favor the gene order in this region as depicted in model A (purA, rpsR, cyc). This is not in agreement with the data presented by de Wilde et al. (19), in which the order of the genes was reported to be purA, cycA, rpsR. Because the cyc mutation we introduced into MB2203 (selected for resistance to 25 ,uM D-cycloserine; see below) was independent of the cycA mutation originally reported (20), we tested the possibility that the two cyc mutations are different. The cycA of X316 (20) was therefore transduced into MB2203 and MB360 by selecting in each case for PurA+. Using MB360 as recipient, we found that the cycA of X316 was cotransducible with purA at a frequency of 10.3% (23 of 224) in contrast to 24.5% in the case of the cyc of MB2203 (Fig. 2). Furthermore, we found that 24% (54 of 229) of the PurA+ transductants derived from MB2203 were Cyc+ (cycloserinesensitive). Although we can not rule out the possibility that the cyc mutation in MB2203 is in fact caused by double mutations, at least we can conclude from these results that it is different from the cycA mutation of X316 because otherwise the occurrence of Cyc+ transductants at a high frequency can not be explained. Hence, we termed the cyc mutation of MB2203 cycB. The levels of resistance to D-cycloserine of cycA and cycB mutations are slightly but significantly different, even though the latter was isolated in the same condition as the former (20): whereas the latter (cycB) confers resistance to up to 0.1 mM
Genetics: Isono and Kitakawa
D-cycloserine, the former (cycA) does so only up to 0.05 mM (data not shown). We therefore further investigated the gene order including cycA and cycB by analyzing the r-proteins of PurA+ transductants obtained from the cross X316 X MB2203 as described above. It was found that all 17 PurA+ transductants (35%) that were Cyc+ possessed the wild-type form of S18. Because it has been established (Table 4) that the cycB mutation of MB2203 is situated on the far side of the new ribosomal protein gene cluster from purA and because the distance between them is small (about 0.1 min), this result strongly indicates that the four genes concerned reside in the E. coli chromosome as depicted in Fig. Sa. If cycA is situated between purA and rpsR as shown in Fig. 3b, then PurA+ CycA+ RpsR+ CycB+ transductants could arise only as a result of four crossovers, a rather rare event. Thus, we conclude that both cycA and cycB are on the far side of the new r-protein gene cluster from purA.
DISCUSSION The data presented in this paper show that the region of the E. coli genetic map containing pyrB, uxuB, and serB can now be covered by Plkc-mediated transduction and the overall map distance in this region should in fact be shorter by approximately 2 min than that previously published (11). An extensive genetic analysis of this region utilizing r-protein mutants has revealed the existence of a new cluster of genes for r-proteins which contains at least the genes for proteins S6, S18, and L9. It would be of immediate interest to construct a specialized X transducing phage covering this region in order to investigate whether this cluster contains genes for some other r-proteins as well. Because neither of the r-protein gene clusters at 72 and 88 min contains the genes coding for, for example, r-proteins S1, S9, L13, etc. or for protein synthesis initiation factors IF-1 and IF-2, whose gene locations are as yet not known, it would be worthwhile to explore this region. A recent report by Takata (21) showed that there seemed to be another r-protein gene cluster near argG at 68 min which contains the genes for proteins S15, L21, L27, and S21. He reported that the gene for S21 seemed a little away from the former three. If the genes for the former three proteins form
Proc. Natl. Acad. Sci. USA 75 (1978)
6167
a cluster, it indicates that there are at least four r-protein gene clusters in E. coil. We are grateful to Dr. H. G. Wittmann for discussion and to Dr. B. Bachmann for providing us with various E. coli strains. 1. Nomura, M., Morgan, E. A. & Jaskunas, S. R. (1977) Annu. Rev. Genet. 11, 297-347. 2. Nashimoto, H. & Uchida, H. (1975) J. Mol. Biol. 96,443-453. 3. Yamamoto, M., Strycharz, W. A. & Nomura, M. (1976) Cell 8, 129-138. 4. Bock, A., Ruffler, D., Piepersberg, W. & Wittmann, H. G. (1974)
Mol. Gen. Genet. 134, 325-332. 5. Friesen, J. D., Parker, J., Watson, R. J., Fiil, N. P. & Pederson, S. (1976) Mol. Gen. Genet. 144, 115-118. 6. Isono, K., Krauss, J. & Hirota, Y. (1976) Mol. Gen. Genet. 149, 297-302. 7. Isono, K., Cumberlidge, A. G., Isono, S. & Hirota, Y. (1977) Mol. Gen. Genet. 152,239-243. 8. Isono, K. & Kitakawa, M. (1977) Mol. Gen. Genet. 153, 115120. 9. Kitakawa, M. & Isono, K. (1977) Mol. Gen. Genet. 158, 149155. 10. Isono, S., Isono, K. & Hirota, Y. (1978) Mol. Gen. Genet. 165, 15-20. 11. Bachmann, B. J., Low, K. B. & Taylor, A. L. (1976) Bacteriol. Rev. 40, 116-167. 12. Robert-Baudouy, J. M. & Portalier, R. C. (1974) Mol. Gen. Genet. 131,31-46. 13. Bollen, A., Faelen, M., Lecopq, J. P., Herzog, A., Zengel, J., Kahan, L. & Nomura, M. (1973) J. Mol. Biol. 76,463-472. 14. Davis, B. D. & Mingioli, E. S. (1950) J. Bactersol. 60, 17-28. 15. Lomax, M. S. & Greenberg, G. R. (1968) J. Bacteriol. 96,501514. 16. de Haan, P. G., Hoekstra, W. P. M., Verhoef, C. & Felix, H. S. (1969) Mutat. Res. 8, 505-512. 17. Taylor, A. L. (1972) in Experiments in Molecular Genetics, ed. Miller, J. H. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), pp. 451-454. 18. Wu, T. T. (1966) Genetics 54,405-410. 19. de Wilde, M., Michel, F. & Broman, K. (1974) Mol. Gen. Genet. 133,329-3. 20. Curtiss, R., III, Charamella, L. J., Berg, C. M. & Harris, P. E. (1965) J. Bacteriol. 90, 1238-1250. 21. Takata, R. (1978) Mol. Gen. Genet. 160, 151-155.
