Vol. 173, No. 11
JOURNAL OF BACTERIOLOGY, June 1991, p. 3559-3563
0021-9193/91/113559-05$02.00/0
Chloramphenicol Resistance Mutations in the Single 23S rRNA Gene of the Archaeon Halobacterium halobium ALEXANDER S. MANKIN1* AND ROGER A. GARRETT2 Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, 119899 Moscow,
USSR,1 and Institute of Biological Chemistry B, University of Copenhagen, Solvgade 83, DK-1307 Copenhagen K, Denmark2 Received 16 November 1990/Accepted 25 March 1991
A broad range of antibiotics affecting protein biosynthesis were screened for their ability to inhibit growth of the archaeon Halobacterium halobium. Only a few drugs, including chloramphenicol, produced inhibitory effects. Mutants which showed increased resistance to chloramphenicol were isolated; of the nine tested, eight exhibited a C->U transition at position 2471 and the ninth had an A-IC transversion at position 2088 of 23S rRNA. A double mutant containing both C->U (position 2471) and A->C (position 2088) mutations was isolated, but the level of its chloramphenicol resistance did not exceed that of either single-point mutant. Inferences are made concerning the functional significance of the conserved nucleotides in rRNAs.
Mutations within rRNA genes which confer resistance to drugs are of interest for several reasons. They highlight rRNA regions of potential functional importance, and they may yield insight into the mode of drug action. Moreover, nucleotide substitutions in rRNAs could be efficiently used as selective and/or traceable markers in vector constructs. The redundancy of rRNA genes in bacteria and eukaryotes has retarded studies of mutations therein, since if a mutation occurs in one of the gene copies it is generally recessive to the remaining unmutated genes. However, this problem may be avoided for some halophilic and perhaps all thermophilic archaea (21) which exhibit a single set of rRNA genes in the chromosome. Advantages of using extreme halophiles for such studies were demonstrated by Hummel and Bock, who mapped nucleotide substitutions conferring resistance to anisomycin and thiostrepton (11, 12) in the single rRNA operon of Halobacterium halobium (9). Recently, we developed a vector transformation system for extreme halophiles by using a mutated rRNA operon bearing resistance to anisomycin and thiostrepton as a selective marker (15). During these studies, we became interested in other traceable or selectable markers in rRNA genes which would render a vector system more versatile. To achieve this, we screened a collection of ribosomal antibiotics for their potential use in selecting for mutations in rRNA genes. Chloramphenicol was identified as a good candidate, chloramphenicol-resistant mutants were selected, and the nucleotide substitutions in their 23S rRNA genes were characterized.
ments. Agar plates were prepared by adding 1/5 volume of water to the medium and solidified by adding 1.5% agar.
Testing sensitivity to ribosomal antibiotics. About 106 H. halobium cells were inoculated into 2-ml cultures containing different drugs at a concentration of 50 ,uM. Optical density of the cultures at 550 nm (OD550) was measured after 3 days of growth at 37°C. Results were normalized to the OD of a culture grown under the same conditions with no added antibiotics. Selection for chloramphenicol-resistant mutants. H. halobium cells were grown to an OD550 of -1.2, and 1 ml of culture (about 109 cells) was plated onto agar medium containing 100 ,ug of chloramphenicol per ml. Plates were sealed with adhesive tape to prevent desiccation, inverted, and incubated at 37°C. Individual colonies that appeared on plates after 2 weeks were purified by suspension in the liquid medium and replating in the presence of 100 p.g of chloramphenicol per ml. Chloramphenicol sensitivity testing. The sensitivity of wildtype cells and resistant mutants to chloramphenicol was tested by inoculating about 106 cells of each strain into 2-ml cultures containing different drug concentrations. Growth was monitored by OD550 after 3 days of incubation with constant shaking at 37°C. Results of measurements in each series were normalized to the OD of a culture incubated without antibiotics. Isolation and sequencing of rRNA. Cells were grown in 5-ml cultures in the presence of 100 ,ug of chloramphenicol per ml, pelleted, and resuspended in 400 p.l of lysis buffer (10 mM Tris-HCl [pH 7.5], 1 mM EDTA, 10 mM MgCl2) containing 20 U of RNase-free DNase I (Boehringer). After a 15-min incubation at room temperature with shaking, the water phase was extracted with phenol saturated with TE buffer, phenol-chloroform, and chloroform. Sodium acetate, pH 5.5, was added to 0.3 M, and RNA was precipitated by adding 3 volumes of ethanol. The RNA pellet was washed with 70% ethanol, dissolved in 80 ,ul of H20, and stored at -700C. Sequencing was performed by the method of Egebjerg et al. (7), by using 10 1.l (about 0.2 A260 units) of total RNA and 0.5 pM 32P-5'-end-labeled oligonucleotide primers.
MATERIALS AND METHODS Cells and growth conditions. H. halobium RI was kindly provided by F. Pfeifer. Cells were grown in complex medium (4) containing (per liter) 50 ml of 1 M Tris-HCl (pH 7.5), 250 g of NaCl, 20 g of MgSO4 7H20, 3 g of trisodium citrate 2H20, 2 g of KCI, 0.2 g of CaCl2 2H20, 3 g of yeast extract (Difco), and 5 g of Bacto-tryptone (Difco). Chloramphenicol or other antibiotics were added in certain experi-
*
Corresponding author. 3559
3560
J. BACTERIOL.
MANKIN AND GARRETT
TABLE 1. Influence of protein synthesis antibiotics on the growth of H. halobium Degree of inhibition'
Antibiotica
Anthelmycin ........... Apramycin ...........
None None Bruceantin ........... None Celesticetin ........... None None Geneticin ........... Gentamicin ........... None None Hygromycin ........... None Kanamycin ........... None Kasugamycin ........... None Micrococcin ........... None Neomycin ........... None Paromomycin ........... None Spectinomycin ........... None Spiromycin ........... None Streptomycin ........... None Tetracycline ........... Althiomycin ........... Moderate Moderate Amicetin ........... Moderate Blasticitin ........... Moderate Carbomycin ........... Erythromycin ........... Moderate Narciclasin ...........Moderate
Strong Anisomycin ........... Strong Bottromycin ........... Chloramphenicol ........... Strong Strong Pactamycin ........... Strong Puromycin ........... Strong Sparsomycin ........... Strong Thiostrepton ........... a All antibiotics were tested at 50 ,uM concentrations, except for thiostrepton, which was tested at 10 ,uM. b Inhibition of growth in the presence of drugs was graded as follows: none, growth at 80 to 100% that of the control; moderate, growth at 25 to 80% that of the control; and strong, U at position 2471 and A->C
CTAG
C T AG CTAO
A A
CTAG
U
_~~~~ _ ~~~~
_
G A A
G AC C
C C
CA W.T.
RIAC
28
A A 2471U A G A G
A A C
.f
A
G _n '..
.A.
A
G
RICU
W.T.
FIG. 1. cDNA sequencing gels showing regions of the 23S rRNA from the parental wild-type (W.T.) and mutant strains of H. halobium where the mutations conferring chloramphenicol resistance occur. The sequence of the template RNA is indicated, and the mutated nucleotides are enlarged and numbered.
VOL. 173, 1991
MUTATIONS IN 23S rRNA
G- C-2460 A- U U A-U U-A C-G
C 2080
3561
C-G
GACG A
U
AO
A
Oe
CCAAGGGG AAGC. I II lI I II *I
G G U U AU C U A U C G U C G G
*A
GG
A AG A
A
A G U CGUC A C UC G
IJ I
I
Gc
I UI I I I I
U lUC G CGG U G AGCC UuU A r G CU A .
UC
AGG \\
C A
.
2500
AcG \\ AGAC A G\\\ \C AU 0 G 0 A UC~~ C UUUG \ AG~~~~~ U 260 G.UGU UG
UG~~~~~~
C-G G .U A- U G- C FIG. 2. Secondary structure of the peptidyl transferase center in domain V in H. halobium 23S rRNA (14). 0, nucleotides whose alterations can confer chloramphenicol resistance in eukaryotic mitochondria or E. coli (20); 0, positions which are protected from chemical modification by chloramphenicol bound to the E. coli ribosome (16); -*, mutations mapped in the present study. at 2088) in the same ribosome would yield even higher resistance. In selecting for a double mutant, advantage was taken of the fact that the C-*U 2471 mutation coincided precisely with a mutation conferring anisomycin resistance in halobacteria (12). Thus, RlAC cells were grown in liquid culture containing chloramphenicol and then 108 cells were plated onto agar medium containing 50 ,ug of chloramphenicol per ml and 5 ,g of anisomycin per ml. After 10 days, about 20 colonies appeared on the plate. Two of these were transferred to liquid culture containing 100 ,ug of chloramphenicol per ml, and RNA from the cultured cells was subjected to sequence analysis. Sequencing results showed retention of the original A-*C mutation at position 2088 and the appearance of a second mutation, C-*U, at position 2471, conferring resistance to both anisomycin and chloramphenicol. The chloramphenicol resistance of the double mutant, designated RlACCU, did not, however, exceed that of the individual mutants; growth rates of the double and single-point mutants were similar at all tested concentrations of the drug (Fig. 3).
DISCUSSION General tolerance of halophilic archaea to protein synthesis antibiotics. A collection of antibiotics that inhibit bacterial and eukaryotic ribosomes were screened for their ability to inhibit the growth of an extreme halophile. The results extend previous observations (3, 17) in showing that extreme halophiles are resistant to many of these drugs. Thus, even at a relatively high concentration (50 ,uM), few antibiotics were inhibitory and, of these, only anisomycin, puromycin,
sparsomycin, pactamycin, and thiostrepton exhibited effects at concentrations below 5 puM. Several factors may contribute to this general resistance: the impermeability of the cell wall or membrane, the fact that the ribosomes may lack antibiotic binding sites, and the possibility that the chemical reactivities of the drugs could be altered at high cellular salt concentrations. However, this last factor is unlikely to provide a general explanation, since methanogens and extreme thermophiles are also resistant to many of these drugs (3). The effect of cell wall permeability on antibiotic resistance was tested in experiments with spheroplasts of H. halobium deprived of their cell wall by EDTA treatment (data not shown). These were exposed to several 30S subunit-targeting antibiotics, including streptomycin, kanamycin, hygromycin, paromomycin, apramycin, gentamicin, and spectinomycin. We reasoned that if spheroplasts were sensitive to the antibiotics and protein synthesis was blocked, then they would be unable to regenerate their cell wall and resume growth. The observation that the antibiotic-treated spheroplasts resumed growth at the same rate and efficiency as the untreated control spheroplasts indicated that it was not the impermeability of the cell wall that caused drug resistance. It is known that many ribosomal antibiotics which are ineffective against archaea in vivo also have no effect on in vitro translation systems (2), and it is likely, therefore, that the predominant reason for the resistance of H. halobium is the lack of antibiotic binding sites on the ribosome. This explanation may result from many archaeon-specific structural features in both ribosomal RNAs and proteins (13, 14, 19). One example is the presence of a U or G at position 2084
3562
J. BACTERIOL.
MANKIN AND GARRETT
120
100
Ao
80 60
a:
CD
40
20O
i
5__
l
0
1 00 1 50 75 CHLORAMPHENICOL CONCENTRATION [ug/ml]
25
50
200
FIG. 3. Growth of wild-type and mutant H. halobium strains in the presence of chloramphenicol. Growth of the strains in the presence of a different concentration of chloramphenicol was normalized to growth in the absence of the drug (see Materials and Methods).
of the 23S rRNA (Fig. 2) in the archaeal kingdom in contrast an A in bacteria and chloroplasts (10), which correlates with the resistance of the former and sensitivity of the latter to erythromycin, given that this position was shown to be involved in antibiotic binding in Escherichia coli. Chloramphenicol resistance of H. halobium is conferred by mutations in the 23S rRNA gene. Despite the considerable evolutionary divergence of archaea from bacteria and eukaryotes, functionally important regions of rRNAs, and possibly also the proteins, are highly conserved (6, 8). Consequently, antibiotics acting in such regions often have interkingdom specificity. Our results show that chloramphenicol belongs to a limited group of such antibiotics which affect the growth of extreme halophiles (Table 1 and Fig. 3), albeit at much higher concentrations than those which inhibit E. coli (20). However, even at high drug concentrations (100 ,ug/ml), some residual growth of H. halobium, which may correlate with weaker binding of the drug to the halobacterial ribosomes in vitro (17), occurs. In this paper, we have described H. halobium mutants which are much more tolerant to chloramphenicol than wild-type cells. Though, in principle, chloramphenicol resistance could derive from alterations in various cellular components (5), the following lines of evidence indicate that the resistant phenotype is conferred by single-base mutations in the 23S rRNA gene. (i) All nine randomly picked chloramphenicol-resistant mutants had a single nucleotide substitution in domain X of the 23S rRNA (C->U at position 2471 or A--C at position 2088); none of the six colonies picked from nonselective plates showed deviations from the wild type. (ii) Transformation of the H. halobium wild-type strain with a vector based on the complete rRNA operon cloned from the anisomycin-resistant mutant exhibiting a C-+U transition at position 2471 induced an anisomycin- and chloramphenicol-resistant phenotype in the transformed cells (15). (iii) The locations of the nucleotide substitutions coincide with mutations correlated previously with chloramphenicol resistance in eukaryotic mitochondria and in E. coli; all such to
mutations have been mapped to the peptidyl transferase center (Fig. 1) of domain V (reference 20 and references
therein). (iv) Direct footprinting of the chloramphenicol binding site on the E. coli ribosome revealed protection at four positions, which included the A at position 2088 (16)
(Fig. 1).
The presence of only one rRNA operon in the H. halobium chromosome provided an opportunity to select for a mutant bearing two chloramphenicol resistance mutations. This was possible because one of the mutations mapped (C-*>U at position 2471) also conferred resistance to anisomycin (12). The result demonstrated that resistance was not additive, which implies that maximum ribosome-mediated resistance was achieved by each mutation. Residual chloramphenicol sensitivity observed at very high antibiotic concentrations may reflect another drug target in the halobacterial cell. Are invariant nucleotides in rRNA indispensable for ribosomal functions? A limited number of invariant positions in the large rRNAs of different kingdoms are strictly conserved in all the sequences studied so far (6). It is generally assumed that these nucleotides are directly or indirectly involved in ribosomal functions such that their mutations will be either lethal or highly disadvantageous for the cell and will, therefore, be selected against during evolution. One of the invariant positions corresponds to the C at position 2471 in the H. halobium 23S rRNA (6, 14). Surprisingly, when U was substituted for this base in the RlCU mutant, the growth rate of the mutated strain was unaltered in the absence of chloramphenicol. The same mutation also arose when another halobacterial strain, SB3, was cultivated in the laboratory with no selection (1). Thus, the mutant strain was quite competitive with an "orthodox" strain having a C at position 2471. This ease of substituting a U for a C at position 2471 in the laboratory contrasts with its strict evolutionary conservation and raises the question of whether this reflects a difference between cell growth conditions in the laboratory and natural
MUTATIONS IN 23S rRNA
VOL. 173, 1991
environments. The most obvious distinction is that cells are normally grown in the laboratory in rich media, while natural conditions are significantly tougher, with cells often facing starvation. Thus, it could be that some of the conserved nucleotides, including the C at position 2471, are essential only at near-starvation conditions. Frequency of spontaneous mutations in the rRNA operon of H. halobium. Spontaneous single-point mutants with a chloramphenicol-resistant phenotype arose in halobacterial cultures with a frequency of about 10-7. This is close to the frequencies observed for thiostrepton- or anisomycin-resistant mutants, which are also characterized by single-nucleotide substitutions in the 23S rRNA gene (11, 12), and this probably corresponds to the mutation rate of the H. halobium genome. The total length of the rRNA coding regions in the single rRNA operon of H. halobium is 4,654 bp. If the frequency of spontaneous mutations is even along the genome, then about 1 in 2,000 cells will have a mutation in the rRNA. An abundance of such mutants might be increased even further if a negative selection (18) utilizing amino acid analogs is applied. Cells having fully active wild-type ribosomes would incorporate more of the amino acid analog and thus would accumulate a greater amount of anemic proteins than those having a less efficient mutant ribosome. Several cycles of such selection would eventually enrich cell populations in the translational mutants. This approach applied to a singlerRNA-operon organism, like H. halobium, would be extremely useful for studying rRNA functions. ACKNOWLEDGMENTS We are grateful to E. Cundliffe, R. Amils, and J.-L. Pernodet for making antibiotics available from their collections. We thank A. Bogdanov, E. Cundliffe, L. Johansen, and V. Kagramanova for helpful discussions and critical reading of the manuscript. A.S.M. thanks FEBS, the Danish Center of Microbiology, and the Plasmid Foundation, Denmark, for support during his stay at R.A.G.'s laboratory. The research was supported by grants from the Danish Science Research Council. REFERENCES 1. Akhmanova, A. S., and A. S. Mankin. Unpublished results. 2. Amils, R., L. Ramirez, J. L. Sanz, I. Marin, A. G. Pisabarro, and D. Urena. 1989. The use of functional analysis of the ribosome as a tool to determine archaebacterial phylogeny. Can. J. Microbiol. 35:141-147. 3. Bock, A., and 0. Kandler. 1985. Antibiotic sensitivity of archaebacteria, p. 525-544. In C. R. Woese and R. S. Wolfe (ed.), The bacteria, vol. VIII. Academic Press, Inc., New York. 4. Cline, S. W., W. L. Lam, R. L. Charlebois, L. C. Schalkwyk, and W. F. Doolittle. 1989. Transformation methods for halophilic archaebacteria. Can. J. Biochem. 35:148-152. 5. Cundliffe, E. 1981. In E. F. Gale, E. Cundliffe, P. E. Reynolds, M. H. Richmond, and M. J. Waring (ed.), Mechanisms of the
3563
antibiotic action, p. 402-547. John Wiley & Sons, Inc., London. 6. Egebjerg, J., N. Larsen, and R. A. Garrett. 1990. Structural map of 23S rRNA, p. 168-179. In W. A. Hill, A. Dahlberg, R. A. Garrett, P. B. Moore, D. Schlessinger, and J. R. Warner (ed.), The ribosome: structure, function, & evolution. American Society for Microbiology, Washington, D.C. 7. Egebjerg, J., H. Leffers, A. Christiansen, H. Andersen, and R. A. Garrett. 1987. Domain VI of E. coli 23S rRNA: structure, assembly and function. J. Mol. Biol. 196:125-136. 8. Gutell, R. R., B. Weiser, C. R. Woese, and H. F. Noller. 1985. Comparative anatomy of 16S-like rRNA. Prog. Nucleic Acid Res. Mol. Biol. 32:155-216. 9. Hofman, J. D., R. H. Lau, and W. F. Doolittle. 1979. The number, physical organization and transcription of ribosomal RNA cistrons in an archaebacterium: Halobacterium halobium. Nucleic Acids Res. 7:1321-1333. 10. Hummel, H., U. Bar, G. Heller, and A. Bock. 1985. Antibiotic sensitivity pattern of in vitro polypeptide synthesis systems from Methanosarcina barkeri. Syst. Appl. Microbiol. 6:125131. 11. Hummel, H., and A. Bock. 1987. Thiostrepton resistance mutations in the gene for 23S ribosomal RNA of halobacteria. Biochimie 69:857-861. 12. Hummel, H., and A. Bock. 1987. 23S ribosomal RNA mutations in halobacteria conferring resistance to the anti-80S ribosome targeted antibiotic anisomycin. Nucleic Acids Res. 15:24312443. 13. Leffers, H., J. Kjems, L. Ostergaard, N. Larsen, and R. A. Garrett. 1987. Evolutionary relationships amongst archaebacteria. A comparative study of 23S rRNAs of a sulphur-dependent extreme thermophile, an extreme halophile and a thermophilic methanogen. J. Mol. Biol. 195:43-61. 14. Mankin, A. S., and V. K. Kagramanova. 1986. Complete nucleotide sequence of the single ribosomal RNA operon of Halobacterium halobium; secondary structure of the archaebacterial 23S rRNA. Mol. Gen. Genet. 202:152-161. 15. Mankin, A. S., et al. Unpublished results. 16. Moazed, D., and H. F. Noller. 1987. Chloramphenicol, erythromycin, carbomycin and vernamycin B protect overlapping sites in the peptidyltransferase region of 23S ribosomal RNA. Biochimie 69:879-884. 17. Schmid, G., T. Pecher, and A. Bock. 1982. Properties of the translational apparatus of archaebacteria. Zentralbl. Bakteriol. Mikrobiol. Hyg. Abt. 1 Orig. C3:209-217. 18. Soppa, J., and D. Oesterhelt. 1989. Halobacterium sp. GRB: a species to work with!? Can. J. Microbiol. 35:205-209. 19. Spiridonova, V. A., A. S. Akhmanova, V. K. Kagramanova, A. K. E. Kopke, and A. S. Mankin. 1989. Ribosomal protein gene cluster of Halobacterium halobium: nucleotide sequence of the genes coding for S3 and L29 equivalent ribosomal proteins. Can. J. Microbiol. 35:153-159. 20. Vester, B., and R. A. Garrett. 1988. The importance of highly conserved nucleotides in the binding region of chloramphenicol at the peptidyl transfer centre of Escherichia coli 23S ribosomal RNA. EMBO J. 7:3577-3587. 21. Woese, C. R., 0. Kandler, and M. L. Wheelis. Towards a natural system of organisms: proposal for the domain archaea, bacteria and eukarya. 1990. Proc. Natl. Acad. Sci. USA 87:4576-4579.
JOURNAL OF BACTERIOLOGY, June 1991, p. 3559-3563
0021-9193/91/113559-05$02.00/0
Chloramphenicol Resistance Mutations in the Single 23S rRNA Gene of the Archaeon Halobacterium halobium ALEXANDER S. MANKIN1* AND ROGER A. GARRETT2 Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, 119899 Moscow,
USSR,1 and Institute of Biological Chemistry B, University of Copenhagen, Solvgade 83, DK-1307 Copenhagen K, Denmark2 Received 16 November 1990/Accepted 25 March 1991
A broad range of antibiotics affecting protein biosynthesis were screened for their ability to inhibit growth of the archaeon Halobacterium halobium. Only a few drugs, including chloramphenicol, produced inhibitory effects. Mutants which showed increased resistance to chloramphenicol were isolated; of the nine tested, eight exhibited a C->U transition at position 2471 and the ninth had an A-IC transversion at position 2088 of 23S rRNA. A double mutant containing both C->U (position 2471) and A->C (position 2088) mutations was isolated, but the level of its chloramphenicol resistance did not exceed that of either single-point mutant. Inferences are made concerning the functional significance of the conserved nucleotides in rRNAs.
Mutations within rRNA genes which confer resistance to drugs are of interest for several reasons. They highlight rRNA regions of potential functional importance, and they may yield insight into the mode of drug action. Moreover, nucleotide substitutions in rRNAs could be efficiently used as selective and/or traceable markers in vector constructs. The redundancy of rRNA genes in bacteria and eukaryotes has retarded studies of mutations therein, since if a mutation occurs in one of the gene copies it is generally recessive to the remaining unmutated genes. However, this problem may be avoided for some halophilic and perhaps all thermophilic archaea (21) which exhibit a single set of rRNA genes in the chromosome. Advantages of using extreme halophiles for such studies were demonstrated by Hummel and Bock, who mapped nucleotide substitutions conferring resistance to anisomycin and thiostrepton (11, 12) in the single rRNA operon of Halobacterium halobium (9). Recently, we developed a vector transformation system for extreme halophiles by using a mutated rRNA operon bearing resistance to anisomycin and thiostrepton as a selective marker (15). During these studies, we became interested in other traceable or selectable markers in rRNA genes which would render a vector system more versatile. To achieve this, we screened a collection of ribosomal antibiotics for their potential use in selecting for mutations in rRNA genes. Chloramphenicol was identified as a good candidate, chloramphenicol-resistant mutants were selected, and the nucleotide substitutions in their 23S rRNA genes were characterized.
ments. Agar plates were prepared by adding 1/5 volume of water to the medium and solidified by adding 1.5% agar.
Testing sensitivity to ribosomal antibiotics. About 106 H. halobium cells were inoculated into 2-ml cultures containing different drugs at a concentration of 50 ,uM. Optical density of the cultures at 550 nm (OD550) was measured after 3 days of growth at 37°C. Results were normalized to the OD of a culture grown under the same conditions with no added antibiotics. Selection for chloramphenicol-resistant mutants. H. halobium cells were grown to an OD550 of -1.2, and 1 ml of culture (about 109 cells) was plated onto agar medium containing 100 ,ug of chloramphenicol per ml. Plates were sealed with adhesive tape to prevent desiccation, inverted, and incubated at 37°C. Individual colonies that appeared on plates after 2 weeks were purified by suspension in the liquid medium and replating in the presence of 100 p.g of chloramphenicol per ml. Chloramphenicol sensitivity testing. The sensitivity of wildtype cells and resistant mutants to chloramphenicol was tested by inoculating about 106 cells of each strain into 2-ml cultures containing different drug concentrations. Growth was monitored by OD550 after 3 days of incubation with constant shaking at 37°C. Results of measurements in each series were normalized to the OD of a culture incubated without antibiotics. Isolation and sequencing of rRNA. Cells were grown in 5-ml cultures in the presence of 100 ,ug of chloramphenicol per ml, pelleted, and resuspended in 400 p.l of lysis buffer (10 mM Tris-HCl [pH 7.5], 1 mM EDTA, 10 mM MgCl2) containing 20 U of RNase-free DNase I (Boehringer). After a 15-min incubation at room temperature with shaking, the water phase was extracted with phenol saturated with TE buffer, phenol-chloroform, and chloroform. Sodium acetate, pH 5.5, was added to 0.3 M, and RNA was precipitated by adding 3 volumes of ethanol. The RNA pellet was washed with 70% ethanol, dissolved in 80 ,ul of H20, and stored at -700C. Sequencing was performed by the method of Egebjerg et al. (7), by using 10 1.l (about 0.2 A260 units) of total RNA and 0.5 pM 32P-5'-end-labeled oligonucleotide primers.
MATERIALS AND METHODS Cells and growth conditions. H. halobium RI was kindly provided by F. Pfeifer. Cells were grown in complex medium (4) containing (per liter) 50 ml of 1 M Tris-HCl (pH 7.5), 250 g of NaCl, 20 g of MgSO4 7H20, 3 g of trisodium citrate 2H20, 2 g of KCI, 0.2 g of CaCl2 2H20, 3 g of yeast extract (Difco), and 5 g of Bacto-tryptone (Difco). Chloramphenicol or other antibiotics were added in certain experi-
*
Corresponding author. 3559
3560
J. BACTERIOL.
MANKIN AND GARRETT
TABLE 1. Influence of protein synthesis antibiotics on the growth of H. halobium Degree of inhibition'
Antibiotica
Anthelmycin ........... Apramycin ...........
None None Bruceantin ........... None Celesticetin ........... None None Geneticin ........... Gentamicin ........... None None Hygromycin ........... None Kanamycin ........... None Kasugamycin ........... None Micrococcin ........... None Neomycin ........... None Paromomycin ........... None Spectinomycin ........... None Spiromycin ........... None Streptomycin ........... None Tetracycline ........... Althiomycin ........... Moderate Moderate Amicetin ........... Moderate Blasticitin ........... Moderate Carbomycin ........... Erythromycin ........... Moderate Narciclasin ...........Moderate
Strong Anisomycin ........... Strong Bottromycin ........... Chloramphenicol ........... Strong Strong Pactamycin ........... Strong Puromycin ........... Strong Sparsomycin ........... Strong Thiostrepton ........... a All antibiotics were tested at 50 ,uM concentrations, except for thiostrepton, which was tested at 10 ,uM. b Inhibition of growth in the presence of drugs was graded as follows: none, growth at 80 to 100% that of the control; moderate, growth at 25 to 80% that of the control; and strong, U at position 2471 and A->C
CTAG
C T AG CTAO
A A
CTAG
U
_~~~~ _ ~~~~
_
G A A
G AC C
C C
CA W.T.
RIAC
28
A A 2471U A G A G
A A C
.f
A
G _n '..
.A.
A
G
RICU
W.T.
FIG. 1. cDNA sequencing gels showing regions of the 23S rRNA from the parental wild-type (W.T.) and mutant strains of H. halobium where the mutations conferring chloramphenicol resistance occur. The sequence of the template RNA is indicated, and the mutated nucleotides are enlarged and numbered.
VOL. 173, 1991
MUTATIONS IN 23S rRNA
G- C-2460 A- U U A-U U-A C-G
C 2080
3561
C-G
GACG A
U
AO
A
Oe
CCAAGGGG AAGC. I II lI I II *I
G G U U AU C U A U C G U C G G
*A
GG
A AG A
A
A G U CGUC A C UC G
IJ I
I
Gc
I UI I I I I
U lUC G CGG U G AGCC UuU A r G CU A .
UC
AGG \\
C A
.
2500
AcG \\ AGAC A G\\\ \C AU 0 G 0 A UC~~ C UUUG \ AG~~~~~ U 260 G.UGU UG
UG~~~~~~
C-G G .U A- U G- C FIG. 2. Secondary structure of the peptidyl transferase center in domain V in H. halobium 23S rRNA (14). 0, nucleotides whose alterations can confer chloramphenicol resistance in eukaryotic mitochondria or E. coli (20); 0, positions which are protected from chemical modification by chloramphenicol bound to the E. coli ribosome (16); -*, mutations mapped in the present study. at 2088) in the same ribosome would yield even higher resistance. In selecting for a double mutant, advantage was taken of the fact that the C-*U 2471 mutation coincided precisely with a mutation conferring anisomycin resistance in halobacteria (12). Thus, RlAC cells were grown in liquid culture containing chloramphenicol and then 108 cells were plated onto agar medium containing 50 ,ug of chloramphenicol per ml and 5 ,g of anisomycin per ml. After 10 days, about 20 colonies appeared on the plate. Two of these were transferred to liquid culture containing 100 ,ug of chloramphenicol per ml, and RNA from the cultured cells was subjected to sequence analysis. Sequencing results showed retention of the original A-*C mutation at position 2088 and the appearance of a second mutation, C-*U, at position 2471, conferring resistance to both anisomycin and chloramphenicol. The chloramphenicol resistance of the double mutant, designated RlACCU, did not, however, exceed that of the individual mutants; growth rates of the double and single-point mutants were similar at all tested concentrations of the drug (Fig. 3).
DISCUSSION General tolerance of halophilic archaea to protein synthesis antibiotics. A collection of antibiotics that inhibit bacterial and eukaryotic ribosomes were screened for their ability to inhibit the growth of an extreme halophile. The results extend previous observations (3, 17) in showing that extreme halophiles are resistant to many of these drugs. Thus, even at a relatively high concentration (50 ,uM), few antibiotics were inhibitory and, of these, only anisomycin, puromycin,
sparsomycin, pactamycin, and thiostrepton exhibited effects at concentrations below 5 puM. Several factors may contribute to this general resistance: the impermeability of the cell wall or membrane, the fact that the ribosomes may lack antibiotic binding sites, and the possibility that the chemical reactivities of the drugs could be altered at high cellular salt concentrations. However, this last factor is unlikely to provide a general explanation, since methanogens and extreme thermophiles are also resistant to many of these drugs (3). The effect of cell wall permeability on antibiotic resistance was tested in experiments with spheroplasts of H. halobium deprived of their cell wall by EDTA treatment (data not shown). These were exposed to several 30S subunit-targeting antibiotics, including streptomycin, kanamycin, hygromycin, paromomycin, apramycin, gentamicin, and spectinomycin. We reasoned that if spheroplasts were sensitive to the antibiotics and protein synthesis was blocked, then they would be unable to regenerate their cell wall and resume growth. The observation that the antibiotic-treated spheroplasts resumed growth at the same rate and efficiency as the untreated control spheroplasts indicated that it was not the impermeability of the cell wall that caused drug resistance. It is known that many ribosomal antibiotics which are ineffective against archaea in vivo also have no effect on in vitro translation systems (2), and it is likely, therefore, that the predominant reason for the resistance of H. halobium is the lack of antibiotic binding sites on the ribosome. This explanation may result from many archaeon-specific structural features in both ribosomal RNAs and proteins (13, 14, 19). One example is the presence of a U or G at position 2084
3562
J. BACTERIOL.
MANKIN AND GARRETT
120
100
Ao
80 60
a:
CD
40
20O
i
5__
l
0
1 00 1 50 75 CHLORAMPHENICOL CONCENTRATION [ug/ml]
25
50
200
FIG. 3. Growth of wild-type and mutant H. halobium strains in the presence of chloramphenicol. Growth of the strains in the presence of a different concentration of chloramphenicol was normalized to growth in the absence of the drug (see Materials and Methods).
of the 23S rRNA (Fig. 2) in the archaeal kingdom in contrast an A in bacteria and chloroplasts (10), which correlates with the resistance of the former and sensitivity of the latter to erythromycin, given that this position was shown to be involved in antibiotic binding in Escherichia coli. Chloramphenicol resistance of H. halobium is conferred by mutations in the 23S rRNA gene. Despite the considerable evolutionary divergence of archaea from bacteria and eukaryotes, functionally important regions of rRNAs, and possibly also the proteins, are highly conserved (6, 8). Consequently, antibiotics acting in such regions often have interkingdom specificity. Our results show that chloramphenicol belongs to a limited group of such antibiotics which affect the growth of extreme halophiles (Table 1 and Fig. 3), albeit at much higher concentrations than those which inhibit E. coli (20). However, even at high drug concentrations (100 ,ug/ml), some residual growth of H. halobium, which may correlate with weaker binding of the drug to the halobacterial ribosomes in vitro (17), occurs. In this paper, we have described H. halobium mutants which are much more tolerant to chloramphenicol than wild-type cells. Though, in principle, chloramphenicol resistance could derive from alterations in various cellular components (5), the following lines of evidence indicate that the resistant phenotype is conferred by single-base mutations in the 23S rRNA gene. (i) All nine randomly picked chloramphenicol-resistant mutants had a single nucleotide substitution in domain X of the 23S rRNA (C->U at position 2471 or A--C at position 2088); none of the six colonies picked from nonselective plates showed deviations from the wild type. (ii) Transformation of the H. halobium wild-type strain with a vector based on the complete rRNA operon cloned from the anisomycin-resistant mutant exhibiting a C-+U transition at position 2471 induced an anisomycin- and chloramphenicol-resistant phenotype in the transformed cells (15). (iii) The locations of the nucleotide substitutions coincide with mutations correlated previously with chloramphenicol resistance in eukaryotic mitochondria and in E. coli; all such to
mutations have been mapped to the peptidyl transferase center (Fig. 1) of domain V (reference 20 and references
therein). (iv) Direct footprinting of the chloramphenicol binding site on the E. coli ribosome revealed protection at four positions, which included the A at position 2088 (16)
(Fig. 1).
The presence of only one rRNA operon in the H. halobium chromosome provided an opportunity to select for a mutant bearing two chloramphenicol resistance mutations. This was possible because one of the mutations mapped (C-*>U at position 2471) also conferred resistance to anisomycin (12). The result demonstrated that resistance was not additive, which implies that maximum ribosome-mediated resistance was achieved by each mutation. Residual chloramphenicol sensitivity observed at very high antibiotic concentrations may reflect another drug target in the halobacterial cell. Are invariant nucleotides in rRNA indispensable for ribosomal functions? A limited number of invariant positions in the large rRNAs of different kingdoms are strictly conserved in all the sequences studied so far (6). It is generally assumed that these nucleotides are directly or indirectly involved in ribosomal functions such that their mutations will be either lethal or highly disadvantageous for the cell and will, therefore, be selected against during evolution. One of the invariant positions corresponds to the C at position 2471 in the H. halobium 23S rRNA (6, 14). Surprisingly, when U was substituted for this base in the RlCU mutant, the growth rate of the mutated strain was unaltered in the absence of chloramphenicol. The same mutation also arose when another halobacterial strain, SB3, was cultivated in the laboratory with no selection (1). Thus, the mutant strain was quite competitive with an "orthodox" strain having a C at position 2471. This ease of substituting a U for a C at position 2471 in the laboratory contrasts with its strict evolutionary conservation and raises the question of whether this reflects a difference between cell growth conditions in the laboratory and natural
MUTATIONS IN 23S rRNA
VOL. 173, 1991
environments. The most obvious distinction is that cells are normally grown in the laboratory in rich media, while natural conditions are significantly tougher, with cells often facing starvation. Thus, it could be that some of the conserved nucleotides, including the C at position 2471, are essential only at near-starvation conditions. Frequency of spontaneous mutations in the rRNA operon of H. halobium. Spontaneous single-point mutants with a chloramphenicol-resistant phenotype arose in halobacterial cultures with a frequency of about 10-7. This is close to the frequencies observed for thiostrepton- or anisomycin-resistant mutants, which are also characterized by single-nucleotide substitutions in the 23S rRNA gene (11, 12), and this probably corresponds to the mutation rate of the H. halobium genome. The total length of the rRNA coding regions in the single rRNA operon of H. halobium is 4,654 bp. If the frequency of spontaneous mutations is even along the genome, then about 1 in 2,000 cells will have a mutation in the rRNA. An abundance of such mutants might be increased even further if a negative selection (18) utilizing amino acid analogs is applied. Cells having fully active wild-type ribosomes would incorporate more of the amino acid analog and thus would accumulate a greater amount of anemic proteins than those having a less efficient mutant ribosome. Several cycles of such selection would eventually enrich cell populations in the translational mutants. This approach applied to a singlerRNA-operon organism, like H. halobium, would be extremely useful for studying rRNA functions. ACKNOWLEDGMENTS We are grateful to E. Cundliffe, R. Amils, and J.-L. Pernodet for making antibiotics available from their collections. We thank A. Bogdanov, E. Cundliffe, L. Johansen, and V. Kagramanova for helpful discussions and critical reading of the manuscript. A.S.M. thanks FEBS, the Danish Center of Microbiology, and the Plasmid Foundation, Denmark, for support during his stay at R.A.G.'s laboratory. The research was supported by grants from the Danish Science Research Council. REFERENCES 1. Akhmanova, A. S., and A. S. Mankin. Unpublished results. 2. Amils, R., L. Ramirez, J. L. Sanz, I. Marin, A. G. Pisabarro, and D. Urena. 1989. The use of functional analysis of the ribosome as a tool to determine archaebacterial phylogeny. Can. J. Microbiol. 35:141-147. 3. Bock, A., and 0. Kandler. 1985. Antibiotic sensitivity of archaebacteria, p. 525-544. In C. R. Woese and R. S. Wolfe (ed.), The bacteria, vol. VIII. Academic Press, Inc., New York. 4. Cline, S. W., W. L. Lam, R. L. Charlebois, L. C. Schalkwyk, and W. F. Doolittle. 1989. Transformation methods for halophilic archaebacteria. Can. J. Biochem. 35:148-152. 5. Cundliffe, E. 1981. In E. F. Gale, E. Cundliffe, P. E. Reynolds, M. H. Richmond, and M. J. Waring (ed.), Mechanisms of the
3563
antibiotic action, p. 402-547. John Wiley & Sons, Inc., London. 6. Egebjerg, J., N. Larsen, and R. A. Garrett. 1990. Structural map of 23S rRNA, p. 168-179. In W. A. Hill, A. Dahlberg, R. A. Garrett, P. B. Moore, D. Schlessinger, and J. R. Warner (ed.), The ribosome: structure, function, & evolution. American Society for Microbiology, Washington, D.C. 7. Egebjerg, J., H. Leffers, A. Christiansen, H. Andersen, and R. A. Garrett. 1987. Domain VI of E. coli 23S rRNA: structure, assembly and function. J. Mol. Biol. 196:125-136. 8. Gutell, R. R., B. Weiser, C. R. Woese, and H. F. Noller. 1985. Comparative anatomy of 16S-like rRNA. Prog. Nucleic Acid Res. Mol. Biol. 32:155-216. 9. Hofman, J. D., R. H. Lau, and W. F. Doolittle. 1979. The number, physical organization and transcription of ribosomal RNA cistrons in an archaebacterium: Halobacterium halobium. Nucleic Acids Res. 7:1321-1333. 10. Hummel, H., U. Bar, G. Heller, and A. Bock. 1985. Antibiotic sensitivity pattern of in vitro polypeptide synthesis systems from Methanosarcina barkeri. Syst. Appl. Microbiol. 6:125131. 11. Hummel, H., and A. Bock. 1987. Thiostrepton resistance mutations in the gene for 23S ribosomal RNA of halobacteria. Biochimie 69:857-861. 12. Hummel, H., and A. Bock. 1987. 23S ribosomal RNA mutations in halobacteria conferring resistance to the anti-80S ribosome targeted antibiotic anisomycin. Nucleic Acids Res. 15:24312443. 13. Leffers, H., J. Kjems, L. Ostergaard, N. Larsen, and R. A. Garrett. 1987. Evolutionary relationships amongst archaebacteria. A comparative study of 23S rRNAs of a sulphur-dependent extreme thermophile, an extreme halophile and a thermophilic methanogen. J. Mol. Biol. 195:43-61. 14. Mankin, A. S., and V. K. Kagramanova. 1986. Complete nucleotide sequence of the single ribosomal RNA operon of Halobacterium halobium; secondary structure of the archaebacterial 23S rRNA. Mol. Gen. Genet. 202:152-161. 15. Mankin, A. S., et al. Unpublished results. 16. Moazed, D., and H. F. Noller. 1987. Chloramphenicol, erythromycin, carbomycin and vernamycin B protect overlapping sites in the peptidyltransferase region of 23S ribosomal RNA. Biochimie 69:879-884. 17. Schmid, G., T. Pecher, and A. Bock. 1982. Properties of the translational apparatus of archaebacteria. Zentralbl. Bakteriol. Mikrobiol. Hyg. Abt. 1 Orig. C3:209-217. 18. Soppa, J., and D. Oesterhelt. 1989. Halobacterium sp. GRB: a species to work with!? Can. J. Microbiol. 35:205-209. 19. Spiridonova, V. A., A. S. Akhmanova, V. K. Kagramanova, A. K. E. Kopke, and A. S. Mankin. 1989. Ribosomal protein gene cluster of Halobacterium halobium: nucleotide sequence of the genes coding for S3 and L29 equivalent ribosomal proteins. Can. J. Microbiol. 35:153-159. 20. Vester, B., and R. A. Garrett. 1988. The importance of highly conserved nucleotides in the binding region of chloramphenicol at the peptidyl transfer centre of Escherichia coli 23S ribosomal RNA. EMBO J. 7:3577-3587. 21. Woese, C. R., 0. Kandler, and M. L. Wheelis. Towards a natural system of organisms: proposal for the domain archaea, bacteria and eukarya. 1990. Proc. Natl. Acad. Sci. USA 87:4576-4579.
