JOURNAL OF BACTERIOLOGY, Nov. 1980, p. 603-6070021-9193/80/11-0603/05$02.00/0

Vol. 144, No. 2

The Gene for Ribosomal Protein S21, rpsU, Maps Close todnaG at 66.5 mm on the Escherichia coli Chromosomal

Linkage MapERIC R. DABBS

Max-Planck-Institut fur Molekulare Genetik, Abt. Wittmann, Berlin, Germany

A series of mutations causing alterations in ribosomal protein S21 was mappedclose to dnaG, around 66.5 min on the Escherichia coli. chromosomal linkagemap. They cotransduced about 95% with this marker. These mutations definegene rpsU, which is very likely the structural gene for protein S21.

The chromosomal map locations of most ofthe genes for ribosomal proteins in Escherichiacoli are now known. Apart from the main str-spc cluster at 72 min (14), the genes are scatteredover the chromosome (for a recent review, seereference 9); however the genetic location ofrpsU (coding for protein S21 from the smallribosomal subunit) has not yet been pinpointed.An approximate location for rpsU has been

determined using intergeneric heteromerozy-gotes of Enterobacteriaceae (18). These experi-ments indicated that the S21 gene was locatedbetween metC and argG (64 to 68 min on the E.coli chromosomal linkage map). A selection pro-ducing mutants with alterations in many ribo-somal proteins has been described (5). Amongmutants isolated in this selection were severalwith alterations in protein S21 (4). The purposeof this work was to use the mutants to map thegene for protein S21, rpsU, more precisely thanhas been possible so far.

MATERIALS AND METHODSCell growth, ribosomal protein preparation, and

two-dimensional polyacrylamide gel electrophoresiswere as described before (4). Mutagenesis and genetictechniques were also done esssentially as before (3).Strains used in this work are listed in Table 1. Plvirwas used in all transductions. The auxotrophic enrich-ment technique (for argG and uxa mutants) used amixture of ampicillin (50 ,ug/ml) and D-cycloserine(200 ,ug/ml) instead of penicillin. argG mutants weredistinguished from other arginine mutants by theirability to grow on arginine but not citrulline, and bythe locus being transferred very early by Hfr KL14.Galacturonic acid as sole carbon source was used at0.2%, as was glucose.

Strain TAl was constructed as follows. Strain JR27(see Table 1) was used as recipient in a transductionin which strain PC3 (Table 1) was a donor of uxa+.Transductants were screened at 42°C to determinewhich ones had also acquired the temperature-sensi-tive dnaG mutation. A dnaG transductant was thenused to select galacturonic acid-requiring (uxa) mu-tants by auxotroph enrichment. Since genes for the

enzymes involved in galacturonic acid utilization arescattered around the chromosome (13), candidatestrains were checked for cotransduction of the utili-zation mutation with dnaG. Strain TAI was chosen.It gave similar cotransduction frequencies of uxa withdnaG and rpsU (15 to 20%) as did strain JR27, andshowed a low reversion rate (below 10-8). To checkthat the uxa mutation was on the same side of dnaGas uxaA of JR27, P1 lysates of TA1, JR27, and A19were used as donors of uxa+ into TAl. If the uxamutation of TAl was allelic with the uxaA of JR27,then the only uxa+ colonies to grow up would berevertants. If the uxa mutation of TAl were on theother side of dnaG compared to uxaA, then the num-ber of uxa+ colonies would be similar to that obtainedwhen the donor phage was grown on strain A19. Theratio of uxa+ colony numbers (normalized by checkingselection of leu+ and arg+ into strain JC411) for phagegrown on strains TAI, JR27, and A19 was approxi-mately 1:4:100, respectively.

Therefore, the uxa mutation of strain TAl wasclose to, but probably not allelic with, the uxaA mu-tation of strain JR27. It is probably uxaC, which hasbeen reported to cotransduce about 97% with uxaA(13). Strains TA2 and TA3 were also derived from thednaG transductant of strain JR27. Lysates of strainsVT442 and VT547 were used as donors of dnaG+;transductants which were rpsU metC his thyA rpsLwere made argG as described above. Hfr JC5072 wasthen used as donor of thyA+, and recombinants werescreened by UV sensitivity for the presence of recAgene.To allow phenotypic expression of temperature re-

sistance when this was the marker being selected forin a transduction using a temperature-sensitive dnaGrecipient, cells were spread on plates and incubated at30°C for 2 h before shifting to 42°C.

RESULTSMutant strains with altered protein S21.

Four mutants with alterations in ribosomal pro-tein S21 were used in this work. They wereVT442, VT490, VT547, and VT608. These wereisolated in separate selections and therefore hadindependent mutational origins. In strainsVT442 (Fig. Lb) and VT608, protein S21 was

603

on February 11, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

604 DABBS

TABLE 1. Strains used

Strain

A19VT442VT490VT547VT608JR27

PC3JC411

KL14KL209JC5072

TAl

TA2

TA3

Relevant phenotype

Hfr met rna (ArpsUI derivative of A19rpsU2 derivative of A19rpsU3 derivative of A19rpsU4 derivative of A19F- uxaA metC his thyArpsL

Hfr dnaG leu thy rpsLF- leu-6 his-I argG6 metBilacYI gal-6 xyl- 7 mtl-2maLAl rpsL104 tonA2tsx-1 supE44

Hfr- thi-l rel-IHfr malB16 supE44Hfr thr-300 ike-318 spc.300recA67

F- dnaG uxa metC histhyA rpsL

F- rpsUl argG metC hisrecA rpsL

F- rpsU3 argG metC hisrecA rpsL

Source or

reference

Gorini4444Isono

IsonoIsono

IsonoIsonoIsono

This work

This work

This work

a The A19 strain used in this work had lost its abilityto transfer markers, but retained its surface exclusionproperties.

more basic than that of wild type; in strainsVT490 and VT547 (Fig. lc), protein S21 was lessbasic than wild type. Although the alteration inelectrophoretic mobility of protein S21 in eachpair of strains was similar, it has not yet beendetermined if the amino acid substitution was

the same in each of the pairs.These mutants had other ribosomal protein

alterations in addition to the altered proteins S8and S12 of strain VT from which they were

derived (5). VT442 and VT608 also had an al-tered, more basic, protein L25 (Fig. lb). VT490had an alteration in proteins L7 and L12, andVT547 had alterations in proteins S4 and S7(Fig. lc). None of the mutations responsible forthe alterations in ribosomal proteins (other thanS21) detected in these mutants mapped in theregion of the chromosome where the gene forprotein S21 was located. This is consistent withthe previously determined locations for thestructural genes for these proteins (1).Mapping ofmutant loci. Previous work (18)

suggested that the gene for protein S21 was

located between metC and argG (64 min and 68min, respectively). I found that none of themutations responsible for the altered S21 proteinin the four mutant strains mentioned above co-transduced with argG.A marker was then chosen about 2 min away

from argG, towards metC; this was uxaA,C.uxaA,C genes code for enzymes in the pathway

for utilization of galacturonic acid (13). Crosseswere set up where the donors were three S21mutants and the recipient was strain JR27(uxaA his metC thyA rpsL). Selection was foruxa+, and transductants were analyzed on two-dimensional gels. With donors VT442, VT547,and VT608, 6 of 38, 7 of 40, and 7 of 39 trans-ductants, respectively, had acquired the muta-tion responsible for altered protein S21. Overall,these mutant loci cotransduced 17% with uxaA(range 16 to 18%).Gel analysis also revealed that strain JR27

lacked ribosomal protein L7, the acetylated formof ribosomal protein L12. Such mutants havebeen found before (4, 9), and the lack of proteinL7 may be due to a mutation either in the L12structural gene rpIL (E. Dabbs, unpublisheddata), or in the L12 acetylase gene rimK (9). Inno case did a strain lacking protein L7 showdetectable deleterious effects in its growth.Therefore, the use of such a strain is unlikely tohave an effect on the results of the transductionexperiments described above.Cotransduction with dnaG. To map more

precisely the mutations responsible for alteredprotein S21, it was necessary to determine onwhich side of uxaA they were. Therefore, strainPC3 (dnaG leu thy rpsL) was used as a donor ofuxaA+ to strain JR27. Of 28 transductants, 6(21%) acquired the temperature-sensitive dnaGmutation. This cotransduction frequency wascomparable to that obtained between uxaA andrpsU (see above). To deternine ifdnaG and therpsU mutations were on the same side of uxaA,lysates of the four mutants were used as donorsof temperature resistance to strain PC3 (dnaG).About 96% of the dnaG+ transductants alsoshowed an altered S21 protein (26 of 26, 24 of24, 31 of 34, and 24 of 28 transductants, respec-tiyely). Since spontaneous temperature-resist-ant revertants occurred on some control platesin these transductions, the cotransduction fig-ures were considered to be a minimum, andthese results did not exclude the possibility thatmutations in dnaG and mutations altering pro-tein S21 were allelic. The following experimentwas done to clarify this point and also to orderdnaG, uxa, and the mutations affecting proteinS21.

Strain TAl (dnaG uxa metC his thyA rpsL,see above) was constructed and used as a recip-ient. Strains VT442 (altered protein S21 morebasic) and VT547 (altered protein S2.1 less basic)were used as donors. Transductants selected foruxa+ were scored for the dnaG phenotype andpresence or absence of altered protein S21. Withstrain VT442 as donor, 7 of 39 transductantswere temperature resistant, and these same 7had an altered protein S21 (see Table 2). With

J. BACTERIOL.

on February 11, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

RIBOSOMAL PROTEIN S21 GENE LOCATION 605

,j

S4

S1_s..>~~ ~ ~ ~ ~

*:*~~~_~~~2

a

_.sfl.

- a-

-

_

S...* 0__._1._0

40, A

S21

c

__:.J_.,.

,i...

AS21

*I 41

b

Fs a

Z

3* t

AAS21

d

FIG. 1. Two-dimensional electropherograms of 70S (a-c) or 30S (d) ribosomal proteins. (a) A19 (wild type),(b) VT442, (c) VT547, and (d) merodiploid with rpsU mutation from VT442. First dimension runs from left toright, second dimension runs from top to bottom of page. Position of protein S21 is indicated. Positioh ofproteins L25, S4, and S7 (see text) is also indicated (a to c).

strain VT547 as donor, 21 of 97 (22%) werednaG+, whereas 22 of 97 (23%) had an alteredprotein S21 (Table 2). All dnaG+ transductantshad altered protein S21, as did an additionaltransductant that had retained the dnaG mu-tation of the recipient. Therefore, these data arecompatible with the mutation in strain VT547responsible for the alteration in protein S21being closer to uxa than dnaG (Fig. 2). If themutation was an rpsU mutation, (see below),then the order was uxaA,C-rpsU-dnaG (Fig. 2).Of 22 of the transductants that were rpsU, 21(95%) were dnaG+; this is close to the 94%

observed (see above) when transductants se-lected for dnaG+ were scored for alteration inprotein S21.Mutations are in the structural gene

rpsU. Strains TA2 and TA3 (rpsU argG metChis recA rpsL) were constructed as describedabove. One had the altered, more basic, proteinS21 of strain VT442; the other had the altered,less basic, protein S21 of strain VT547. Thestrains were crossed with Hfr KL209, with selec-tion for argG+ metC+ recombinants. Plating wasdone in the presence of streptomycin to selectagainst the Hfr strain, and recombinants were

VOL. 144, 1980

on February 11, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

TABLE 2. P1-mediated cross between uxa, rpsU, and dnaG

Selected marker Resulting characteristics Relative fre-Cross Donor/recipient (no.scored) quency (% of to-

uxa rpsU dnaG tal)

1 VT442 uxa rpsU uxa+ (39) + + + 0 (0)dnaG+/TAl uxa + + - 32 (82)rpsU+dnaG + - + 7(18)

+ _ 0 (0)

2 VT547 uxa+ rpsU uxa+ (97) + + + 0 (0)dnaG+/TAl uxa + + - 75 (77)rpsU dnaG + - + 21 (22)

+ - - 1 (I)

argG uxaA,C rpsU dnaG

I ~~~~~~~~~~~~~~I140/560 22/97 '105/11217% ' 23% 94%

21/9722%

0/5600%

FIG. 2. Map ofregion around rpsU, as determinedby transduction. Arrow points to marker beingscored.

screened by UV sensitivity for continued pres-ence of recA. A recombinant derived from TA2and one derived from TA3 were grown in mini-mal medium supplemented with histidine. Res-olution of protein S21 into a double spot wasclearly seen when 30S subunit proteins fromthese strains were run on two-dimensional gels(recombinant of TA2 shown in Fig. ld). Thisindicated that the mutations causing the alter-ations in protein S21 which mapped close todnaG were in the structural gene for proteinS21, rpsU.

DISCUSSIONThe work reported here utilized several mu-

tants with alterations in ribosomal protein S21to map the gene for this protein, rpsU, very nearto dnaG. The gene order was dnaG-rpsU-uxaA,C-argG (Fig. 2). It was shown that thiswas the structural gene, rather than a modifiergene. The latter was a rather less likely possibil-ity since the mutations at this locus caused morethan one type of alteration in the protein; also,the amino acid sequence reveals no modifiedresidues in this protein (20).

rpoD, the gene for the sigma factor of RNApolymerase, also maps near dnaG (7, 8) whoseproduct is the primase enzyme ofDNA synthesis

(16). The reported order of genes is dnaG-rpoD-uxaA,C (7), so rpsU and rpoD are both locatedon the same side of dnaG. The reported cotrans-duction frequency between dnaG and rpoD is 80to 90% (6, 7) compared with a frequency foundin this work of 94 to 95% for dnaG and rpsU.Although the certain order must await crossesbetween rpsU and rpoD mutant strains, thecotransduction data would suggest the orderdnaG-rpsU-rpoD-uxaA,C. This clustering ofgenes coding for components ofDNA, RNA, andprotein biosynthesizing machinery has parallels.Thus, the genes for the ,B and ff subunits ofRNA polymerase are adjacent to a cluster ofgenes coding for ribosomal components at 82min (11), and the gene for the a subunit isembedded among the ribosomal protein genes ofthe str-spc cluster (10). Also, the rpsB gene, forribosomal protein S2, is near the dnaE (polC)gene at 4 min (12).One unexplained observation was that co-

transduction between the uxaA mutation ofJR27 and dnaG was found to be about 20%.Previously, Chen and Carl (2) reported a uxaC-dnaG figure of 49%. They and Nemoz et al. (13)also found uxaC-argG cotransduction to be 2%.An argG derivative of strain TA1, which wastherefore dnaG uxa argG, was constructed. Itwas used as recipient in a selection for arginineprototrophy. Of 560 transductants, 40 (7%) wereuxa+. In agreement with the work cited above(2), and also with an earlier report (19), nocotransduction (0 of 560) was found betweenargG and dnaG. In the work described in thispaper, uxaA,C appeared to be located moreclockwise on the chromosome than would be thecase from previous work (2). The reason for thediscrepancy was not clear.The mapping studies also indicated that there

was not a scorable phenotype associated withrpsU mutations; transductants with such muta-tions were indistinguishable in growth rate fromrpsU+ transductants, and showed no tempera-

606 DABBS J. BACTERIOL.

on February 11, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

RIBOSOMAL PROTEIN S21 GENE LOCATION 607

ture or cold sensitivity. However, one propertyof mutAnts VT442 and VT608 was found to befunction of the rpsU mutation. Strains with analtered protein S21 which was more basic thanwild type were found to lose all of this proteinfrom the ribosome when ribosomes were washedin 0.5 M NH4Cl buffer. Although protein S21has been found to be a protein which exchangesin vivo (15), ribosomes with wild-type (or al-tered, less basic) protein S21 were not signifi-cantly depleted by washing in 0.5 M NH4Clbuffer, as assayed by two-dimensional gels. Anal-ysis of transductants derived from strains VT442and VT608 indicated that the less-tight bindingof protein S21 to the ribosome in these mutantswas the result of the mutational alteration inprotein S21.There was another interesting property ofmu-

tants with a more basic protein S21. In mutantswith a less basic protein S21, changes in otherribosomal proteins showed no systematic pat-tern. However, all five mutants isolated in theselection described elsewhere (5) that possesseda more basic protein S21 (including the two usedin the genetic studies, plus three more isolatedrecently), also had an alteration in large subunitprotein L25 (Fig. lb), the gene for which mapsat 47 min on the chromosomal linkage map (17).These five had independent mutational origins,but not only was the sort of alteration in proteinL25 always the same, no other of the approxi-mately 600 mutants isolated in the selection (5)had an alteration in protein L25. This suggestsa close interaction at some level between pro-teins S21 and L25.

ACKNOWLEDGMENTSI thank K. Isono for useful discussion during this work, H.

G. Wittmann for support and reading the manuscript, and B.Schroeter for excellent technical assistance.

LITERATURE CITED1. Bachmann, B. J., and K. B. Low. 1980. Linkage map of

Escherichia coli K-12, edition 6. Microbiol. Rev. 44:1-56.

2. Chen, L L., and P. L. Carl. 1975. Genetic map locationof the Escherichia coli dnaG gene. J. Bacteriol. 124:1613-1614.

3. Dabbs, E. R. 1978. Kasugamycin-dependent mutants ofEscherichia coli. J. Bacteriol. 136:994-1001.

4. Dabbs, E. R. 1978. Mutational alterations in 50 proteinsof the Escherichia coli ribosome. Mol. Gen. Genet. 165:73-78.

5. Dabbs, E. R., and H. G. Wittmann. 1976. A strain of

Escherichia coli which gives rise to mutations in a largenumber of ribosomal proteins. Mol. Gen. Genet. 149:303-309.

6. Gross, G., J. Hoffman, C. Ward, D. Hager, G. Bur-dick, H. Berger, and R. Burgess. 1978. Mutationaffecting thermostability of sigma subunit of Esche-richia coli RNA polymerase lies near the dnaG locusat about 66 min on the E. coli genetic map. Proc. Natl.Acad. Sci. U.S.A. 75:427-431.

7. Harris, J. D., J. S. Heilig, L. I. Martinez, R. Calendar,and L. A. Isaksson. 1978. Temperature sensitive Esch-erichia coli mutant producing a temperature sensitivea subunit of DNA-dependent RNA polymerase. Proc.Natl. Acad. Sci. U.S.A. 75:6177-6181.

8. Haris, J. D., L I. Martinez, and R. Calendar. 1977. Agene from Escherichia coli affecting the sigma subunitof RNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 74:1836-1840.

9. Isono, K. 1980. Genetics of ribosomal proteins and theirmodifying and processing enzymes in Escherichia coli,p. 641-669. In G. Chambliss, G. R. Craven, J. Davies,K. Davis, L. Kahan, and M. Nomura (ed.), Ribosomes,structure, function, and genetics, University Park Press,Baltimore.

10. Jaskunas, S. R., R. R. Burgess, and M. Nomura. 1975.Identification of a gene for the a subunit of RNApolymerase at the str-spc region of the Escherichia colichromosome. Proc. Natl. Acad. Sci. U.S.A. 72:5036-5040.

11. Lindahl, L., M. Yamamoto, M. Nomura, J. B. Kirsch-baum, B. Allet, and J. B. Rochaix. 1977. Mapping ofa cluster of genes for components of the transcriptionaland translational machineries of Escherichia coli. J.Mol. Biol. 109:23-47.

12. Nashimoto, H., and H. Uchida. 1975. Late steps in theassembly of 30S ribosomal proteins in vivo in a specti-nomycin resistant mutant of Escherichia coli. J. Mol.Biol. 96:443-453.

13. Nemoz, G., J. Robert-Baudouy, and F. Stoeber. 1976.Physiological and genetic regulation of the aldohexu-ronate transport system in Escherichia coli. J. Bacte-riol. 127:706-718.

14. Nomura, M., E. A. Morgan, and S. R. Jaskunas. 1977.Genetics of bacterial ribosomes. Annu. Rev. Genet. 11:297-347.

15. Robertson, W. R., S. J. Dowsett, and S. J. J. Hardy.1977. Exchange of ribosomal proteins among the ribo-somes of Escherichia coli. Mol. Gen. Genet. 157:205-214.

16. Rowen, L, and A. Kornberg. 1978. Primase, the dnaGprotein ofEscherichia coli. J. Biol. Chem. 253:758-764.

17. Schnier, J., and K. Isono. 1979. The gene for ribosomalprotein L25 (rplY) maps at 47.3 min, near nalA inEscherichia coli K12. Mol. Gen. Genet. 176:313-318.

18. Takata, R. 1978. Genetic studies of the ribosomal proteinsin Escherichia coli. XI. Mol. Gen. Genet. 160:151-155.

19. Wechsler, J. A., and J. D. Gross. 1971. Escherichia colimutants temperature sensitive for DNA synthesis. Mol.Gen. Genet. 113:273-284.

20. Vanderkerckhove, J., W. Rombauts, B. Peeters, andB. Wittmann-Liebold. 1975. Determination of thecomplete amino acid sequence of protein S21 from theEscherichia coli ribosome. Hoppe-Seyler's Z. Physiol.Chem. 356:1955-1976.

VOL. 144, 1980

on February 11, 2021 by guest

http://jb.asm.org/

Dow

nloaded from