J. W. Wilson Laboratory, Department of
Molecular and Cell Biology and Biochemistry, Brown University,
Providence, Rhode Island 02912,1 and
Whitehead Institute for Biomedical Research and
Massachusetts Institute of Technology, Cambridge, Massachusetts
021422
 |
TEXT |
Genetic analysis of rRNAs and
ribosomal proteins has been an essential tool in elucidating the
mechanism of protein synthesis. While numerous ribosomal protein and
rRNA mutants have been isolated, none has been examined structurally in
atomic detail. This is primarily due to the inadequacy of ribosomes
from genetically tractable organisms such as Escherichia
coli for forming crystals that diffract X-rays at sufficient
resolution. Ribosomes suitable for X-ray crystallographic analysis have
thus far been obtained exclusively from thermophiles (4, 23,
33) or halophiles (1). Regrettably, only limited
effort has been devoted to studying the ribosomes of these organisms
using genetic approaches (see, however, reference 26).
Determining the structures of mutant ribosomes of such an organism (an
organism producing ribosomes suitable for X-ray crystallographic
analysis) should define conformational perturbations that result
in specific functional consequences. We have, therefore, initiated a
genetic dissection of ribosomes of the extreme thermophile
Thermus thermophilus. Despite the promise of
Thermus as a genetic system (16, 21),
the lack of selectable genetic markers such as transposons or
antibiotic resistance mutations makes the mapping of mutations to
ribosomal protein and RNA genes problematic. Here we report the
isolation of a 23S rRNA mutation conferring resistance to the
antibiotics erythromycin and lincomycin. This selectable genetic marker
will facilitate the identification, by transformation, of 23S rRNA
mutations affecting 50S subunit structure and function.
Thermus spp. have an unusual rRNA gene composition of two
16S rRNA genes unlinked from two 23S rRNA-5S
rRNA-tRNAGly operons (3, 12, 13).
Thus, rRNA mutations are potentially obtainable by direct selection if
they exhibit a dominant or codominant phenotype or if gene conversion
occurs at a sufficiently high frequency. For these experiments we chose
the halotolerant strain T. thermophilus IB-21 (ATCC 43615).
Although the strain was originally described as Thermus sp.
strain IB-21 (17), the designation T. thermophilus is indicated by DNA-DNA hybridization experiments (32) and our own DNA sequence analysis (S. Gregory,
J. H. D. Cate, and A. E. Dahlberg, unpublished results).
Isolation of spontaneous erythromycin-resistant mutants.
We
conducted a search for erythromycin resistance mutations in 23S rRNA
which have been isolated for a number of organisms with multiple rRNA
operons (reviewed in reference 30). In several instances,
including in the cases of Streptomyces ambofaciens (22), Helicobacter pylori (29),
and Streptococcus pneumoniae (27), heterozygous
mutants indicate that this mutation is dominant or codominant, as has
been previously demonstrated using multicopy plasmid-encoded
rrn operons in E. coli (9, 24, 31).
Spontaneous, erythromycin-resistant mutants were selected by plating
approximately 109 cells from an overnight culture
grown at 72.5°C in medium 162 (7) (supplemented with
0.25% tryptone and 0.25% yeast extract) onto plates of the same
medium solidified with 2.8% agar (Difco) and containing 100 µg of
erythromycin per ml. After 3 days of incubation at 72.5°C, mutants
appeared at a frequency of approximately 10
7
and were purified by restreaking once onto medium 162-erythromycin plates and a second time onto antibiotic-free plates. Six resistant isolates, designated HG48, HG49, HG50, HG51, HG52, and HG53,
respectively, were kept for analysis.
Identification of a 23S rRNA mutation.
The most frequently
occurring macrolide resistance mutations occur at A2058 of 23S rRNA
(30), which is footprinted by macrolide, lincosamide, and
streptogramin B antibiotics in chemical probing experiments (10,
20). We therefore amplified a segment of the T. thermophilus 23S rRNA genes extending from positions 1660 to 2293 (according to E. coli 23S rRNA numbering) by PCR using Taq DNA polymerase (Promega) and oligonucleotide primers
Tth23SD (5'-GGACCTTTGGGCGCCTCCGTTACC-3') and
Tth23SE (5'-CGCCAAGGAACTCTGCAAGTTGGC-3') (both
primers from Operon Technologies) based on the T. thermophilus HB8 23S rRNA sequence (14). Direct
sequencing of PCR products revealed that three of the six isolates
(HG48, HG50, and HG53) carry the mutation A2058G (numbered according to
E. coli 23S rRNA numbering). Moreover, concurrent
terminations in the A and G lanes at the position corresponding to
position 2058 indicated that all three isolates are heterozygous (Fig.
1).
View larger version (58K):
[in this window]
[in a new window]
|
FIG. 1.
Sequencing of PCR-amplified 23S rRNA operons in the
region of A2058. Sequences from wild-type T.
thermophilus IB-21 and from the rrlA1 mutant
HG48. The presence of mixed termination products at position A2058
indicates that this mutant is heterozygous for the
rrlA1 mutation.
|
|
Strong-stop primer extension analysis (25) of total RNA
from the A2058G mutants using the oligonucleotide primer
Tth2058RT (5'-CAGTAAAGCTCCACGGGGTC-3'),
complementary to positions G2061 to G2080, confirmed the presence of
both mutant and wild-type 23S rRNA (Fig.
2), with mutant RNA comprising between 50 and 60% of the total 23S rRNA in the three mutants. We have designated the 23S rRNA gene bearing the A2058G mutation from one of the isolates
(HG48) rrlA and the wild-type 23S rRNA gene rrlB
(these designations, though conforming to standard nomenclature
[2, 8], were assigned arbitrarily). The A2058G
allele has been designated rrlA1. The site of this mutation
in the T. thermophilus 23S rRNA secondary structure
(11) is shown in Fig. 3.
View larger version (46K):
[in this window]
[in a new window]
|
FIG. 2.
Primer extension analysis of total RNA from wild-type
T. thermophilus IB-21 and from the rrlA1
mutant HG48 (25). Lane 1, extension from wild-type RNA
using dATP, dGTP, dTTP, and ddCTP; lane 2, extension from RNA from HG48
using dATP, dGTP, dTTP, and ddCTP; lane 3, extension from wild-type RNA
using four deoxynucleoside triphosphates (dNTPs) and no ddNTPs;
lane 4, extension without RNA using four dNTPs. The positions of G2056
and A2058 and of an unextended primer are indicated. The presence of
A2058G mutant 23S rRNA is apparent in lane 2.
|
|
View larger version (17K):
[in this window]
[in a new window]
|
FIG. 3.
Secondary structure of the central loop of domain V of
T. thermophilus HB8 23S rRNA (11) and the
site of the A2058G erythromycin resistance mutation.
|
|
The doubling times of both wild-type IB-21 and the rrlA1
mutant HG48 were determined to be approximately 48 min, whereas the doubling time of HG48 in the presence of 100 µg of erythromycin per
ml was about 81 min. This increase in doubling time is consistent with
roughly 60% of the ribosomes carrying the resistance mutation. Some
excess of mutant ribosomes in cells grown in erythromycin is not
unexpected given the drug's property of inhibiting assembly of
sensitive 50S subunits (6).
Macrolide-resistant mutants of H. pylori have been shown to
contain A2058G in one or both 23S rRNA-5S rRNA operons
(29), indicating that homozygosity can result from either
gene conversion or an independent mutational event at A2058 in the
second operon. Attempts to obtain second-step homozygous rrlA
rrlB double mutants of T. thermophilus by a second
round of selection on higher levels of erythromycin (400, 600, and 800 µg/ml) were unsuccessful. This suggests either that gene conversion
to homozygosity is rare or that such events do not generate
significantly higher levels of resistance. We prefer the latter
explanation, since Thermus spp. are proficient at homologous
recombination (16) and because rrlB mutations
are expected to appear spontaneously (at a frequency similar to that at
which rrlA mutations appear) in a second round of selection.
Cross-resistance to lincomycin.
The A2058G mutation has been
shown to confer resistance to clindamycin (a lincosamide) in vitro to
reconstituted 50S subunits of Thermus aquaticus
(15) and has been demonstrated to confer resistance to
macrolide, lincosamide, and streptogramin B antibiotics in vivo to
several organisms (30). Not unexpectedly, we found that it
confers high-level cross-resistance to lincomycin in vivo to T. thermophilus. Levels of drug resistance were determined by
streaking overnight cultures for single colonies on medium 162 plates containing closely spaced concentrations of either erythromycin
or lincomycin. Wild-type T. thermophilus IB-21 is not highly
sensitive to erythromycin and was inhibited at an antibiotic concentration of only 50 µg/ml. The rrlA1 mutants are only
weakly resistant to erythromycin and were inhibited by an antibiotic concentration of 400 µg/ml. In contrast, these mutants exhibited high-level resistance to lincomycin. The MIC for the wild-type strain
was found to be only 2 µg/ml, whereas rrlA1 mutants were resistant to lincomycin at concentrations higher than 800 µg/ml.
The three remaining mutants (HG49, HG51, and HG52), with an
unidentified mutation of a gene tentatively designated
eryA, behaved uniformly with regard to antibiotic resistance
phenotypes. They are weakly resistant to erythromycin (completely
inhibited by an antibiotic concentration of 400 µg/ml) and only very
weakly resistant to lincomycin, showing marginal inhibition at an
antibiotic concentration of 10 µg/ml and complete inhibition at a
antibiotic concentration of 40 µg/ml. They are thus phenotypically
distinct from the rrlA1 mutants. Sequencing of several
candidate sites within rrlA(B) and within
rplD and rplV (encoding ribosomal proteins L4 and
L22, respectively [see reference 5]) did not reveal the
nature of the mutation. While a ribosomal mutation has not been
excluded, it is also quite possible that eryA affects
antibiotic uptake.
Utility of the A2058G mutation.
We have demonstrated that it
is possible to obtain 23S rRNA mutants of T. thermophilus
exhibiting a dominant or codominant antibiotic-resistance phenotype,
despite the presence of two 23S rRNA gene copies. The principle value
of this mutation as a selectable genetic marker is as a tool for the
identification of other 23S rRNA mutations of interest, such as
ram mutations, or other mutations producing a functional
defect for which there is a genetic selection. Such mutations could be
mapped genetically to rrlA by transformation to erythromycin
resistance (using DNA from the erythromycin-resistant mutant) and
screening for loss of the original phenotype. Alternatively, mutations
in cis to A2058G could be enriched by transforming a wild-type recipient with DNA from pooled mutants selected in the rrlA1 background, simultaneously selecting for both
phenotypes. Finally, site-directed mutations may be introduced into the
chromosomal rRNA operons by homologous recombination using the
rrlA1 mutation as a selectable marker. Such an approach has
been used with Halobacterium halobium (19). To
facilitate such experiments as well as to obtain strains producing
uniform populations of mutant ribosomes for structural studies, a
strain of T. thermophilus bearing a deletion of one of the
23S rRNA-5S rRNA-tRNAGly operons can be
constructed using the same methodology employed to delete the
Thermus genes leuB (28),
slpA (18), and rpsQ encoding
ribosomal protein S17 (26).
This work was supported by a grant (GM19756) from the National
Institutes of Health to A.E.D. and a grant from the Whitehead Institute
and a Searle Scholar Award to J.H.D.C.
The authors are grateful to Michael O'Connor, Jill Thompson, and Mark
Bayfield for critical reading of the manuscript, to Sun-Thorn Pond-Tor
for technical assistance, and to George Q. Pennabble for insight.
S.T.G. gives special thanks to Elaine Fredrick and Hanako Gregory.
| 1.
|
Ban, N.,
P. Nissen,
J. Hansen,
P. B. Moore, and T. A. Steitz.
2000.
The complete atomic structure of the large ribosomal subunit at 2.4 A resolution.
Science
289:905-920[Abstract/Free Full Text].
|
| 2.
|
Berlyn, M. K.
1998.
Linkage map of Escherichia coli K-12, edition 10: the traditional map.
Microbiol. Mol. Biol. Rev.
62:814-984[Abstract/Free Full Text].
|
| 3.
|
Borges, K. M., and P. L. Berquist.
1993.
Genomic restriction map of the extreme thermophilic bacterium Thermus thermophilus HB8.
J. Bacteriol.
175:103-110[Abstract/Free Full Text].
|
| 4.
|
Cate, J. H.,
M. M. Yusopov,
G. Z. Yusopova,
T. N. Earnest, and H. F. Noller.
1999.
X-ray crystal structures of 70S ribosome functional complexes.
Science
285:2095-2104[Abstract/Free Full Text].
|
| 5.
|
Chittum, H. S., and W. S. Champney.
1994.
Ribosomal protein gene sequence changes in erythromycin-resistant mutants of Escherichia coli.
J. Bacteriol.
176:6192-6198[Abstract/Free Full Text].
|
| 6.
|
Chittum, H. S., and W. S. Champney.
1995.
Erythromycin inhibits the assembly of the large ribosomal subunit in growing Escherichia coli cells.
Curr. Microbiol.
30:273-279[CrossRef][Medline].
|
| 7.
|
Degryse, E.,
N. Glansdorff, and A. Pierard.
1978.
A comparative analysis of extreme thermophilic bacteria belonging to the genus Thermus.
Arch. Microbiol.
117:189-196[CrossRef][Medline].
|
| 8.
|
Demerec, M.,
E. A. Adelberg,
A. J. Clark, and P. E. Hartman.
1966.
A proposal for a uniform nomenclature in bacterial genetics.
Genetics
54:61-76[Free Full Text].
|
| 9.
|
Douthwaite, S.
1992.
Functional interactions within 23S rRNA involving the peptidyl transferase center.
J. Bacteriol.
174:1333-1338[Abstract/Free Full Text].
|
| 10.
|
Douthwaite, S.
1992.
Interaction of the antibiotics clindamycin and lincomycin with Escherichia coli 23S ribosomal RNA.
Nucleic Acids Res.
20:4717-4720[Abstract/Free Full Text].
|
| 11.
|
Gutell, R. R.,
M. W. Gray, and M. N. Schnare.
1993.
A compilation of large subunit (23S and 23S-like) ribosomal RNA structures: 1993.
Nucleic Acids Res.
21:3055-3074[Free Full Text].
|
| 12.
|
Hartmann, R. K.,
N. Ulbrich, and V. A. Erdmann.
1987.
An unusual rRNA operon constellation: in Thermus thermophilus HB8 the 23S/5S rRNA operon is a separate entity from the 16S rRNA operon.
Biochimie
69:1097-1104[Medline].
|
| 13.
|
Hartmann, R. K., and V. A. Erdman.
1989.
Thermus thermophilus 16S rRNA is transcribed from an isolated transcription unit.
J. Bacteriol.
171:2933-2941[Abstract/Free Full Text].
|
| 14.
|
Hopfl, P.,
N. Ulrich,
R. K. Hartmann,
W. Ludwig, and K. H. Schleifer.
1988.
Complete nucleotide sequence of a 23S ribosomal RNA gene from Thermus thermophilus HB8.
Nucleic Acids Res.
16:9043[Free Full Text].
|
| 15.
|
Khaitovich, P.,
T. Tenson,
P. Kloss, and A. S. Mankin.
1999.
Reconstitution of functionally active Thermus aquaticus large ribosomal subunits with in vitro-transcribed rRNA.
Biochemistry
38:1780-1788[CrossRef][Medline].
|
| 16.
|
Koyama, Y.,
T. Hoshino,
N. Tomizuka, and K. Furukawa.
1986.
Genetic transformation of the extreme thermophile Thermus thermophilus and of other Thermus spp.
J. Bacteriol.
166:338-340[Abstract/Free Full Text].
|
| 17.
|
Kristjansson, J. K.,
G. O. Hreggvidsson, and G. A. Alfredsson.
1986.
Isolation of halotolerant Thermus spp. from submarine hot springs in Iceland.
Appl. Environ. Microbiol.
52:1313-1316[Abstract/Free Full Text].
|
| 18.
|
Lasa, I.,
J. R. Caston,
L. A. Fernandez-Herrero,
M. A. de Pedro, and J. Berenguer.
1992.
Insertional mutagenesis in the extreme thermophilic eubacteria Thermus thermophilus HB8.
Mol. Microbiol.
6:1555-1564[CrossRef][Medline].
|
| 19.
|
Mankin, A. S.,
I. M. Zyrianova,
V. K. Kagramanova, and R. A. Garrett.
1992.
Introducing mutations into the single-copy chromosomal 23S rRNA gene of the archaeon Halobacterium halobium by using an rRNA operon-based transformation system.
Proc. Natl. Acad. Sci. USA
89:6535-6539[Abstract/Free Full Text].
|
| 20.
|
Moazed, D., and H. F. Noller.
1987.
Chloramphenicol, erythromycin, carbomycin and vernamycin B protect overlapping sites in the peptidyl transferase region of 23S ribosomal RNA.
Biochimie
69:879-884[Medline].
|
| 21.
|
Oshima, T.
1995.
Genes and genetic manipulation in Thermus thermophilus, p. 185-205.
In
R. Sharp, and R. Williams (ed.), Thermus species. Plenum Press, New York, N.Y.
|
| 22.
|
Pernodet, J.,
F. Boccard,
M. Alegre,
M. Blondelet-Rouault, and M. Guerineau.
1988.
Resistance to macrolides, lincosamides and streptogramin B antibiotics due to a mutation in an rRNA operon of Streptomyces ambofaciens.
EMBO J.
7:277-282[Medline].
|
| 23.
|
Schluenzen, F.,
A. Tocilj,
R. Zarivach,
J. Harms,
M. Gluehmann,
D. Janell,
A. Bashan,
H. Bartels,
I. Agmon,
F. Franceschi, and A. Yonath.
2000.
Structure of functionally activated small ribosomal subunit at 3.3 angstroms resolution.
Cell
102:615-623[CrossRef][Medline].
|
| 24.
|
Sigmund, C. D.,
M. Ettayebi, and E. A. Morgan.
1984.
Antibiotic resistance mutations in 16S and 23S ribosomal RNA genes of Escherichia coli.
Nucleic Acids Res.
12:4653-4663[Abstract/Free Full Text].
|
| 25.
|
Sigmund, C. D.,
M. Ettayebi,
A. Borden, and E. A. Morgan.
1988.
Antibiotic resistance mutations in ribosomal RNA genes of Escherichia coli.
Methods Enzymol.
164:673-690[Medline].
|
| 26.
|
Simitsopoulou, M.,
H. Avila, and F. Franceschi.
1999.
Ribosomal gene disruption in the extreme thermophile Thermus thermophilus HB8.
Eur. J. Biochem.
266:524-532[Medline].
|
| 27.
|
Tait-Kamradt, A.,
T. Davies,
M. Cronan,
M. R. Jacobs,
P. C. Appelbaum, and J. Sutcliffe.
2000.
Mutations in 23S rRNA and ribosomal protein L4 account for resistance in pneumococcal strains selected in vitro by macrolide passage.
Antimicrob. Agents Chemother.
44:2118-2125[Abstract/Free Full Text].
|
| 28.
|
Tamakoshi, M.,
T. Yaoi,
T. Oshima, and A. Yamagishi.
1999.
An efficient gene replacement and deletion system for an extreme thermophile, Thermus thermophilus.
FEMS Microbiol. Lett.
173:431-437[CrossRef][Medline].
|
| 29.
|
Versalovic, J.,
D. Shortridge,
K. Kibler,
M. V. Griffy,
J. Beyer,
R. K. Flamm,
S. K. Tanaka,
D. Y. Graham, and M. F. Go.
1996.
Mutations in 23S rRNA are associated with clarithromycin resistance in Helicobacter pylori.
Antimicrob. Agents Chemother.
40:477-480[Abstract].
|
| 30.
|
Vester, B., and S. Douthwaite.
2001.
Macrolide resistance conferred by base substitutions in 23S rRNA.
Antimicrob. Agents Chemother.
45:1-12[Free Full Text].
|
| 31.
|
Vester, B., and R. A. Garrett.
1987.
A plasmid-encoded and site-directed mutation in Escherichia coli 23S RNA that confers resistance to erythromycin: implications for the mechanism of action of erythromycin.
Biochimie
69:891-900[Medline].
|
| 32.
|
Williams, R. A.,
K. E. Smith,
S. G. Welch,
J. Micallef, and R. J. Sharp.
1995.
DNA relatedness of Thermus strains, description of Thermus brockianus sp. nov., and proposal to reestablish Thermus thermophilus (Oshima and Imahori).
Int. J. Syst. Bacteriol.
45:495-499[CrossRef][Medline].
|
| 33.
|
Wimberly, B. T.,
D. E. Brodersen,
W. M. Clemons,
R. J. Morgan-Warren,
A. P. Carter,
C. Vonrhein,
T. Hartsch, and V. Ramakrishnan.
2000.
The structure of the 30S ribosomal subunit.
Nature
407:327-339[CrossRef][Medline].
|
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Text
