UPR9073 du CNRS, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, F-75005 Paris, France1
Aventis Pharma SA, 13 Quai Jules Guesde, 94403 Vitry sur Seine Cedex, France2
Author for correspondence: Richard H. Buckingham. Tel: +33 1 5841 5120. Fax: +33 1 5841 5020. e-mail: rhb{at}ibpc.fr
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ABSTRACT |
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Keywords: translation, ribosome, termination
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INTRODUCTION |
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Studies of premature peptidyl-tRNA dissociation (or drop-off) have been greatly facilitated as a result of the isolation of a conditional lethal temperature-sensitive mutant affecting pth (Atherly & Menninger, 1972 ). Bacteria harbouring this mutation stop growing after a shift to non-permissive temperatures, following the accumulation of peptidyl-tRNA and the inhibition of protein synthesis. The mutation was identified as a single nucleotide change leading to an amino-acid substitution Gly100Asp (De La Vega et al., 1996
). Though distant from the active site proposed in the three-dimensional structure (Schmitt et al., 1997
), this Gly residue is highly conserved in Pth from many different organisms. Recent work has shown that the mutation leads to a Pth protein that is unstable in vivo, at both permissive and non-permissive temperatures, but which shows a specific activity comparable to that of the wild-type enzyme (Cruz-Vera et al., 2000
). The mutant enzyme seems not to be correctly folded and is subject to degradation by ClpP and Lon proteases (Cruz-Vera et al., 2000
).
In addition to allowing studies of peptidyl-tRNA accumulation, the thermosensitive Pth mutant has led to improved understanding of the drop-off process as a result of the isolation and characterization of extragenic revertants and multicopy suppressors of the pth(ts) mutation. Thus, overproduction of tRNALys raises considerably the threshold for thermosensitive growth by promoting more synthesis of the tRNA for which starvation occurs first (Heurgué-Hamard et al., 1996 ). GroESL overproduction, presumably by improving the efficiency of mutant Pth protein folding, increases Pth activity. Apart from revertants directly affecting the pth gene, mutations inactivating termination factor RF3, or reducing the intracellular concentration of ribosome recycling factor RRF, suppress the pth(ts) mutation. In vitro experiments show that suppression is a result of a reduced frequency of peptidyl-tRNA drop-off in the cell (Heurgué-Hamard et al., 1998
). The studies that led to the isolation of these and other suppressors of pth(ts) showed that under some conditions of selection and in some strain backgrounds high rates of reversion to thermoresistance were observed. Here we examine this finding in more detail and show that the high rates of reversion are due to duplication of the pth(ts) gene.
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METHODS |
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Growth conditions.
LuriaBertani broth (LB) was employed as rich medium. Antibiotics were added as necessary at the following final concentrations: 50 µg kanamycin ml-1, 200 µg ampicillin ml-1. Growth experiments at different temperatures were performed on agar plates. For pBAD30-derived plasmids, arabinose was added for induction at 0·02% for expression of pth at about chromosomal level or at 0·2% for maximum promoter activity.
Determination of reversion rates.
Mutation rates were measured as described by Luria & Delbrück (1943) . Serial dilutions (typically from 10-7 to 10-12) of an overnight culture of the pth(ts) mutant strains were made and grown overnight at 30 °C. The last dilution that grew was used to prepare 30 independent cultures each containing 2050 bacterial cells in 200 µl rich medium. These were grown separately at permissive temperature (30 °C) for several hours, the period being determined by preliminary experiments such that about half the cultures acquired no revertants. Each culture was then completely plated on preheated agar plates and grown at non-permissive temperature. The number of plates on which no clones appeared allows determination of the mutation rate according to the equation P=-(1/N)ln F, where P is the mutation rate per cell per generation, N the total number of bacterial cells per culture, and F the fraction of cultures acquiring no revertants (Luria & Delbrück, 1943
).
PCR amplification of pth and pth::Kn.
The presence of the intact and interrupted pth gene was verified by PCR using the pairs of oligonucleotide primers pt1-pt5, pt1-mk6 and mk2-pt5 shown in Fig. 1. Primer sequences were: mk2, TGATGTTACAGATGAGATGGTC; mk6, CGCCTGAGCGAGACGA; pt1, GAATTCAATGGCACCGACGAAAATAC; pt5, GGGACTAACAGGCGGACA.
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Southern blotting.
Following restriction enzyme digestion, DNA fragments were separated by gel electrophoresis in 0·7% agarose gels. Phage DNA digested with BstEII and labelled with [
-32P]ATP was used as molecular mass marker. A labelled probe for the pth gene was prepared by PCR amplification of the complete gene followed by labelling with a Ready Prime II labelling kit (Pharmacia) according to the manufacturers instructions.
RESULTS
Reversion rate of the thermosensitive pth mutation depends strongly on selection temperature and genetic background
Previous studies have shown that the apparent frequency of reversion of the pth(ts) mutation isolated by Atherly & Menninger (1972) varies widely according to the non-permissive temperature employed, the plating medium and the strain background (Heurgué-Hamard et al., 1996
; V. Heurgué-Hamard & L. Mora, unpublished observations). Extragenic suppressor mutations such as the tRNA missense suppressor affecting the glyW gene were selected at temperatures of 810 °C above the threshold for thermosensitive growth, in strains with low apparent rates of reversion, consistent with point mutation rates (Heurgué-Hamard et al., 1996
). To obtain more quantitative data on mutation rates under different conditions, we determined the proportion of small independent cultures that acquired no revertants over a period of growth, as described by Luria & Delbrück (1943)
. These measurements allowed calculation of the reversion probability per cell per generation. Two pth(ts) strains were studied, VH733 and VH4, with very different threshold temperatures for thermosensitivity (Table 2
). In both cases, however, when the reversion rate was measured at 4 °C above this threshold, high rates of reversion were observed, in the range 10-310-5. The more thermosensitive strain, VH4, allowed the study of reversion rates over a range of temperature, and showed a striking decrease in reversion rate to 10-8 at 42 °C (Table 2
).
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Revertants selected under conditions of high reversion rate are due to duplication of the pth gene
A collection of pth(ts) revertants was made from a large number of independent cultures of the pth(ts) strain VH733 under conditions of high reversion rate, conserving one revertant only per culture. To determine whether the pth gene was still necessary for the growth of these revertants, a strain (JMM35) was constructed in which the chromosomal pth locus is interrupted by a cassette conferring kanamycin resistance, with the requirement for Pth met by a plasmid (pBADPth) carrying a wild-type pth gene expressed from an arabinose-inducible promoter. Each of the thermoresistant revertant strains, either before or after transformation with pBADPth, was transduced with a P1 lysate made on JMM35, selecting for kanamycin resistance in the presence of arabinose. Transductants of the thermosensitive control strain VH733 could only be obtained if the strain carried the plasmid pBADPth prior to transduction, as expected in view of the essential nature of the pth gene. In contrast, each of the thermoresistant revertant strains yielded about 200 kanamycin-resistant transductants whether or not the plasmid was present. Each of the plasmid-free transduced revertants became once more thermosensitive, except in one case (numbered rev5). Similar experiments were performed with the more thermosensitive strain VH4, selecting thermoresistant revertants at 38 °C. In this case also the revertants, but not the parent strain, could be transduced to kanamycin resistance with pth::Kn.
These observations suggested that thermoresistance had been acquired by duplication of the mutant pth gene. Three types of experiment were performed to test this hypothesis, by PCR amplification of the pth gene, Southern blot analysis, and Western blot analysis using anti-Pth sera. First, PCR amplifications were performed on revertants and transduced revertants. A pair of primers was chosen in the region neighbouring pth (see Fig. 1a), one upstream of the pth gene (pt1) and one within the coding sequence (pt5). In the case of both the transduced and non-transduced revertants, a fragment of the size corresponding to the normal pth gene was amplified, confirming the existence of a non-interrupted locus (Fig. 1b
d
). A further specific primer (mk6) was chosen in the middle of the kanamycin-resistance gene introduced into pth which, in combination with pt1, amplified a fragment of the expected length (1·6 kb) only in the case of the transduced revertants, confirming the presence of the interrupted pth gene (Fig. 1b
d
). Thus, it is clear that the revertants harbour at least two pth loci, one of which is interrupted on transduction with a P1 phage lysate on strain JMM35. The transduced revertants thus showed both the presence of a normal pth gene and a further interrupted copy of the gene (Fig. 1b
).
Southern blot analysis was performed with probes specific to pth and to the kanamycin-resistance cassette on DNA extracted from FTP4993 and the 12 thermoresistant revertants after digestion with EcoRV. A single fragment was seen in each case (see Fig. 2b, c
), of the expected size, 6·6 kb, as in the case of the VH733 parent strain (Fig. 2b
, lane 2) and the Pth wild-type strain Xac (Fig. 2c
, lane 2). After the transduction of the revertants with pth::Kn described above, a second fragment appeared in addition to that of 6·6 kb, of the size expected (11·6 kb) in the case of insertion of the kanamycin-resistance cassette into pth. Analysis of DNA from JMM35 showed no 6·6 kb fragment, but only the chromosomal inactivated fragment and a 4·8 kb molecule from the linearized pBADPth plasmid (Fig. 2b
, lane 3). The identity of the more slowly migrating fragments was confirmed by hybridization first with the anti-Kn probe, followed by partial dehybridization and subsequent hybridization with the anti-pth probe (Fig. 2b
, c
). No new fragments were visible in the case of the revertants, suggesting that the region of the chromosome containing pth and undergoing duplication is probably larger than the region of about 6 kb covered by the restriction fragments.
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Reversion rate following shut-off of Pth synthesis
In the pth(ts) mutant the capacity of Pth to hydrolyse the peptidyl-tRNA dissociating from the ribosome diminishes with increasing temperature, until a threshold temperature is reached at which Pth activity becomes inadequate to sustain cell growth. This may be due to a reduction in Pth activity with temperature or to an increase in the rate of formation of cytoplasmic peptidyl-tRNA, the observations of Cruz-Vera et al. (2000) suggesting that the latter is of greater importance. In either case, the experiments described above show that the cell is able to counter an inadequate level of Pth activity within a certain range of temperatures above the threshold temperature for growth by multiplication of the number of copies of the mutant pth gene. This interpretation predicts that if the intracellular level of Pth is reduced to a small fraction of its value in the parent cell, this mechanism will no longer function. We therefore constructed strains in which Pth synthesis is under the control of a tight inducible promoter, and measured reversion frequencies after shut-off of pth expression.
Preliminary experiments with strain JMM35, in which wild-type Pth is expressed exclusively from the arabinose-inducible pBAD promoter on a low-copy-number plasmid, showed that many hours of growth were necessary after arabinose elimination to dilute the intracellular Pth. A similar result was obtained with the arabinose-inducible pth system recombined with the chromosome at the maltose operon locus (data not shown). A plasmid was therefore constructed equivalent to pBADpth but encoding the thermosensitive enzyme, and used to transform FTP4993, in which the chromosomal pth gene was then inactivated as in JMM35. In the presence of 0·02% arabinose, the resulting strain is similar in behaviour to a chromosomal pth(ts) strain. This construction has the advantage that upon elimination of arabinose not only is the synthesis of new Pth molecules arrested, but the short half-life of the thermosensitive Pth mutant results in a rapid decrease in the intracellular concentration of the enzyme. The reversion rate at 43 °C was studied as described above, and there was a total absence of revertants. We estimate that this corresponds to a reversion rate lower than 3x10-9.
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DISCUSSION |
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At first sight, it might seem surprising that there should be such a critical level of Pth activity required for growth, such that a mere doubling in level of the enzyme at a given temperature should suffice to restore normal or near normal growth. A likely explanation for this is suggested by a consideration of the mechanism of peptidyl-tRNA drop-off from the ribosome. It was shown by Menninger (1978) that different families of peptidyl-tRNAs accumulate at rates differing by as much as 200-fold after shift of a thermosensitive Pth strain to non-permissive temperature. Studies of the kinetics of drop-off of different peptidyl-tRNAs from the cognate codon on the ribosome have failed to show large differences in rate (Dinçbas et al., 1999
; Dinçbas-Renqvist et al., 2000
; V. Heurgué-Hamard, unpublished data). Likewise, the specificity of Pth towards different peptidyl-tRNA substrates does not show variations sufficient to account for the range of rates of peptidyl-tRNA accumulation (Dinçbas et al., 1999
; Dinçbas-Renqvist et al., 2000
; V. Heurgué-Hamard, unpublished data). These observations support the conclusion of Menninger (1978)
that a substantial part of peptidyl-tRNA drop-off arises from translational errors; thus, drop-off occurs as a result of a weak semi-cognate codonanticodon interaction rather than a cognate interaction. In this case, it is readily understandable that conditions under which Pth is rather limiting in the cell, leading to partial starvation for some species of aminoacyl-tRNA, will favour translational errors and increase yet further peptidyl-tRNA drop-off, imposing a greater substrate load on the available Pth. This may suffice to explain the need for a critical level of the enzyme.
Gene amplification is an extensively documented phenomenon in prokaryotic cells and can extend from simple duplication of a gene to amplification of more than 100-fold (for recent reviews, see Bachellier et al., 1966 ; Hughes, 1999
). Several examples in E. coli and Salmonella typhimurium have been described, such as the duplication at rates of 10-5 or more per cell per generation of a mutant Gly-tRNA synthetase gene (Folk & Berg, 1971
), amplification of the argF region in some Hfr strains of E. coli (Jessop & Clugston, 1985
) and amplification of the lac region (Tlsty et al., 1984
). Amplification is generally dependent on repeated sequences within the genome, such as the rrn genes (Anderson & Roth, 1981
), insertion elements (Jessop & Clugston, 1985
), REP sequences (Shyamala et al., 1990
) or rhs repeated sequence elements (Lin et al., 1984
).
Although this has not been conclusively shown, it is probable that all currently identified mechanisms of suppression of the thermoresistance of pth(ts) depend on continued Pth activity in the cell. It is also clear that with the exception of the duplication mechanism shown here, reversion occurs at low frequency, of the order of 10-8 per cell per generation or lower, consistent with point-mutation events (Drake, 1969 ).
Saccharomyces cerevisiae chromosome VIII (Ouzounis et al., 1995 ) and fragments of mammalian and mouse origin available in public databases reveal the existence of pth genes of the prokaryotic type. Biochemical studies of rabbit reticulocytes, however, have revealed another type of Pth activity, due to a phosphodiesterase which removes the terminal adenosine of tRNA together with the peptide, which appear then to be cleaved in a subsequent step (Gross et al., 1992a
, b
). Recycling of the tRNA then requires repair of the 3' terminus by tRNA-CCA nucleotidyl transferase activity. It remains to be seen whether either the prokaryotic-type Pth activity or the phosphodiesterase-type Pth activity is essential to cell viability in cells of higher organisms.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 2 February 2001;
accepted 2 March 2001.
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