Essential Genes of Escherichia coli - Temperature-Sensitive Nonsense Mutations in
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JOURNAL OF BACTERIOLOGY, Dec. 1973, P. 1336-1342 Vol. 116, No. 3
Copyright 0 1973 American Society for Microbiology Printed in U.S.A.
Temperature-Sensitive Nonsense Mutations in
Essential Genes of Escherichia coli
DAVID BECKMAN1 AND STEPHEN COOPER
Department of Microbiology, University of Michigan Medical School, Ann Arbor, Michigan 48104
Received for publication 24 September 1973
Cells containing nonsense mutations in essential genes have been isolated in a
strain of Escherichia coli that carried the su4ts gene which specifies a tempera-
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ture-sensitive tyrosine transfer ribonucleic acid. Such cells are unable to form
colonies at temperatures which inactivate this suppressor transfer ribonu-
cleic acid. A screening procedure for the identification of mutants that carry
temperature-sensitive nonsense mutations in essential genes is described, and
certain properties of two such mutants are reported.
We wish to report the isolation of a new class type with respect to that oligomer. TSS mu-
of conditional lethal mutations of bacteria, tants of the lac repressor have been described by
temperature-sensitive nonsense (TSN) muta- Sadler and Novick (10).
tions. These mutations have unique features Suppressor-sensitive (nonsense) mutations
which are of considerable genetical and bio- arise from an alteration in a nucleotide triplet
chemical interest. coding for a given amino acid (sense) so that it
Conditional lethal mutations are mutations specifies polypeptide chain termination (non-
in which a particular cellular function is defec- sense). This results in the synthesis of incom-
tive only under certain conditions. Two major plete polypeptide chains. These incomplete
types of conditional lethal mutations have been polypeptide chains generally have no biological
described: (i) temperature-sensitive missense activity. The effects of a given nonsense muta-
(TSM) mutations in which a complete polypep- tion can be suppressed by a second mutation
tide chain that is biologically inactive at ele- which results in the synthesis of an altered
vated temperatures is synthesized, and (ii) transfer ribonucleic acid (tRNA) capable of
suppressor-sensitive (nonsense) mutations' in recognizing the nonsense codon and inserting a
which a polypeptide chain is prematurely ter- specific amino acid at the corresponding point
minated in the absence of an active suppressor, in the polypeptide chain. If the inserted amino
and the incomplete polypeptide chain is biologi- acid is different from the amino acid at this
cally inactive. position in the wild-type protein, the sup-
TSM mutations arise from an alteration in a pressed protein may be temperature sensitive.
nucleotide triplet coding for a given amino acid However, if the suppressor tRNA molecule itself
(sense) so that it specifies another amino acid is temperature sensitive, suppression will be
(missense). If this amino acid substitution temperature sensitive and the mutation will be
renders the mutant polypeptide inactive at a TSN mutation. It should be noted that
elevated temperatures, but functional at some proteins formed by suppression of a nonsense
lower temperatures, the mutant cell will have a codon may also have either a TL or TSS
thermolabile (TL) phenotype with respect to phenotype.
that polypeptide. Altemately, the amino acid It is theoretically possible to get TSM muta-
substitution may render the mutant polypep- tions in bacteria for all functions-those which
tide incapable of being assembled into an oligo- can be replaced by medium supplements
mer at elevated temperatures. If the oligomer (nonessential functions) as well as those which
formed at lower temperatures is stable to ele- cannot be replaced by medium supplements
vated temperatures, the mutant cell will have a (essential functions)-because cells containing
temperature-sensitive synthesis (TSS) pheno- TSM mutations can be grown at the permissive
temperature. Suppressor-sensitive (nonsense)
' Present address: Department of Microbiology, Albany mutations in bacteria, however, are known only
Medical College, Albany, N.Y. 12208. for nonessential functions (e.g., tryptophan syn-
1336VOL. 116, 1973 TEMPERATURE-SENSITIVE NONSENSE MUTATIONS 1337
thetase) because, if the nonsense mutation were buffer (pH 6.0) containing 500 ug of N-methyl-N'-
in an essential function (e.g., RNA polymerase), nitro-N-nitrosoguanidine (NG) per ml at 37 C for 30
the cell would not be recognized as a mutant if min. The cells were then washed and inoculated into
it contained a suppressor and could not grow if LB broth. This culture was grown overnight at 25 C
it did not contain a suppressor. For this reason, before being plated.
Isolation of temperature-sensitive mutants. Two
nonsense mutations in essential functions have separate methods were used to identify temperature-
heretofore been restricted to bacterial viruses. sensitive cells. In the replica plating method, cells
In this instance, the mutant virus can be grown from a mutagenized culture were plated on LB agar
in a permissive suppressor-containing strain and incubated at 30 C for 48 h. These master plates
and studied in a nonpermissive strain. were then replicated to LB agar, and the replica plates
Gallucci et al. (4) have isolated a strain of were incubated at 42 C overnight. The master plates
Escherichia coli, PNG46, that carries a muta- were then compared to the replica plates, and colonies
tion in the su4 gene, a structural gene for a which grew at 30 C but did not grow at 42 C were
picked into isotonic saline and streaked on LB agar at
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tyrosine tRNA that leads to the synthesis of a 30 and 42 C to confirm their temperature-sensitive
temperature-sensitive suppressor tRNA. If cells phenotype. TSN mutant G100 was isolated by the
which carry this su4P gene are grown at 30 C, replica plating method. In the microcolony method,
they have 21% of the suppressor activity of cells cells from a mutagenized culture were plated on LB
which carry the su4+ gene. If cells which carry agar, and the plates were incubated at 30 C until
the su4ta gene are grown at 42 C, however, they microcolonies were visible (usually 24 h). The plates
have no detectable suppressor activity. were then incubated at 42 C for 24 h. Microcolonies
We have isolated a number of temperature- were picked from these plates and retested as de-
sensitive mutants of PNG46 that are defective scribed above. TSN mutant C45 was isolated by the
in essential functions. Two of these mutants are microcolony method.
Colony staining for alkaline phosphatase. Colo-
temperature sensitive due to the presence of nies were tested for alkaline phosphatase (AP; EC
nonsense mutations in essential genes and, 3.1.3.1) activity by the method of Messer and Viel-
therefore, can form colonies at 30 C due to the metter (7). This consisted of treating the colonies on
presence of an active suppressor tRNA, but the plate with 1.0 M Tris buffer (pH 8.0) containing 5
cannot form colonies at 42 C due to the absence mg of Fast Blue RR salt (Sigma Chemical Co.) per ml
of an active suppressor tRNA at this tempera- and 2 mg of naphthol AS-MX phosphate (Sigma
ture. In this paper we describe methods for the Chemical Co.) per ml. Alkaline phosphatase-positive
identification and isolation of mutants carrying (AP+) colonies turned purple within 1 to 2 min.
TSN mutations and report certain properties of Reversion Tests. Samples of LB broth-grown cul-
tures of each temperature-sensitive mutant to be
two such mutants. tested were spotted onto TG-LP-CAA agar in a
MATERIALS AND METHODS circular array. After these spots dried, one drop of
diethylsulfate (DES), a mutagen, was spotted in the
Bacteria and bacteriophage. Bacterial strains center of the array. After incubation at 42 C for 48 h,
H12 (Phoam-) and H12R7a (su4+, pho.m-), a deriva- individual colonies were visible within the spots of the
tive of H12, were obtained from B. Bachman of the E. array and these were tested for AP activity as de-
coli genetic stock center (2). Strain PNG46 (su4ts, scribed above. Mutants which gave AP42+ and APR,-
phoami), a derivative of H12R7a (4), was obtained revertants were retested by spreading a sample of an
from S. Zangrossi. Some experiments were performed LB broth-grown culture on TG-LP-CAA agar, spot-
with a derivative of PNG46, PNG468, which was ting a drop of DES in the center, incubating at 42 C
selected for streptomycin resistance. These strains are for 48 h, and testing for AP activity.
Ti resistant, Hfr, and lysogenic for bacteriophage Phage P1 transduction. Transducing lysates were
lambda. prepared by the method of Caro and Berg (3) and used
Bacteriophage Plkc (3) was used for transduction. within 2 h of preparation. Transduction experiments
Suppressor-sensitive mutants of phage T4, amB22 (9) were performed by the method of Miller (8). Trans-
and amPS292 (9) were used to test the suppressor ductants were plated on TG-LP-CAA agar.
phenotype of temperature-resistant revertants. Temperature shift experiments. Temperature
Media. LB broth was adjusted to pH 7.2 with 1 N shift experiments were performed as described in the
NaOH before use and contained 1% tryptone, 0.5% legends to the individual figures. The optical density
yeast extract, 0.1% glucose, and 1% NaCl. The (OD) was followed with a Zeiss PMQII spectropho-
tris (hydroxymethyl) aminomethane (Tris)!- glucose-low tometer.
phosphate medium described by Garen and Garen (5) Mapping of TSN mutations. The TSN mutations
was supplemented with 0.2% Casamino Acids (TG- were mapped by using the set of Hfr strains developed
LP-CAA). by Low (6). This method allows a general location to
Mutagenesis. Essentially, the procedure of Adel- be assigned to a mutation, depending on whether or
berg et al. (1) was followed. Log-phase cells were not recombination occurs with a given Hfr. All mat-
harvested, suspended at a concentration of approxi- ings were carried out in LB broth. The temperature-
mately 10' per ml, and incubated in Tris-maleate sensitive recipients were grown overnight in LB broth1338 BECKMAN AND COOPER J. BACTERIOL.
with shaking to produce F- phenocopies. of revertants will depend on the reversion fre-
quency of the particular TSN mutation.
RESULTS Cells containing TSM mutations, on the
The main problem in the identification of other hand, should yield only one type of
cells which carry TSN mutations is how to single-step, temperature-resistant revertant.
distinguish them from cells which carry TSM This type of revertant represents a missense-to-
mutations. We have used two approaches to this sense change within the defective gene and is
problem: (i) the analysis of temperature-resist- analogous to TRR-1 described above.
ant revertants (TRR), and (ii) the analysis of Since TRR-2 (AP.2+) represents a mutation
temperature-resistant transductants (TRT). from temperature-sensitive to temperature-
Analysis of temperature-resistant revert- resistant suppression, whereas TRR-1 (AP42-)
ants. The pattern with respect to the alkaline represents a mutation within the defective gene
phosphatase (AP) phenotypes of temperature- itself, there should be a correlation between the
resistant revertants expected to arise from cells alkaline phosphatase phenotype of tempera-
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containing TSN and TSM mutations is shown ture-resistant revertants of cells containing
in Fig. 1. Cells containing TSN mutations TSN mutations and the ability of these revert-
should yield two types of single-step tempera- ants to support the growth of suppressor-sensi-
ture-resistant revertants. TRR-1 represents a tive bacteriophage.
mutation from nonsense to sense within the Out of 310 mutants with temperature-sensi-
defective gene itself. This type of revertant will tive phenotypes on LB agar that we isolated,
be alkaline phosphatase negative when grown two, C45 and G100, gave AP,2+ and AP42-
and be derepressed at 42 C (AP42-). TRR-2 revertants. The pattern of temperature-resist-
represents a mutation from temperature-sensi- ant revertants obtained from three mutants
tive to temperature-resistant suppression. This with temperature-sensitive phenotypes (C45,
type of revertant, therefore, will be AP42+. For a G100, and G71) with and without DES is shown
given TSN mutation, the ratio of the two types in Table 1. It is clear from this table that
TRR-1 TRR-1 TRR-2
L:I LiU1S:
iSU4'
ess )(s4Sd
,phoam
AP-
pho a.ess
AP
(su4+, pho
AP+
ess
FIG. 1. Pattern of temperature-resistant revertants (TRR) expected from two kinds of temperature-sensitive
cells: (i) cells containing temperature-sensitive missense (TSM) mutations and (ii) cells containing tempera-
ture-sensitive nonsense (TSN) mutations.VOL. 116, 1973 TEMPERATURE-SENSITIVE NONSENSE MUTATIONS 1339
mutant G71, even in the presence of DES, support the growth of suppressor-sensitive bac-
yields only AP,2- revertants. In contrast, how- teriophage (T4amB22 and T4amPS292). That
ever, mutants C45 and G100 both product is, AP42+ revertants support the growth of
AP,2+ and AP,2- revertants. Similar results T4amB22 and T4amPS292 at 42 C, whereas
were obtained with a number of single-colony AP42- revertants do not support the growth of
isolates of G100 and C45. Therefore, by this T4amB22 or T4amPS292 at 42 C. In control
criterion, mutants C45 and G100 carry TSN experiments, we showed that both AP42+ and
mutations, whereas mutant G71 carries a TSM AP42- revertants support the growth of wild-
mutation. The reversion frequencies for the type T4 at 42 C. These experiments support the
temperature-resistant revertants are of the hypothesis that the AP42+ revertants are resist-
order of magnitude expected for single-step ant to elevated temperatures due to the pres-
revertants. A photograph of plates from an ence of a temperature-resistant suppressor.
experiment similar to the one described above is Analysis of temperature-resistant trans-
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shown in Fig. 2. ductants. The expected pattern with respect to
There was a perfect correlation between the alkaline phosphatase phenotype when cells con-
alkaline phosphatase phenotype of tempera- taining TSN and TSM mutations are trans-
ture-resistant revertants of mutants C45, G100, duced to temperature resistance with phage P1
and G71 and the ability of these revertants to previously grown on H12R7a (su4+) and H12
TABLE 1. Alkaline phosphatase phenotypes of temperature-resistant revertants (TRR) of
temperature-sensitive mutants
-DES + DES
Temperature-sensitive
mutant Frequency of Relative no. of TRRa Frequency of Relative no. of TRR"
TRR AP42+' AP42- TRR AP,+ | AP,-
C45 5.5 x 10-l 73 27 2.5 x 10-' 91 9
G100 3.1 x 10-6 68 32 7.8 x 10-6 87 13
G71 1.2 x 10-4 0 100 9.3 x 10-5 0 100
a Calculated from an experiment in which the total number of temperature-resistant revertants was: C45,
688; G100, 580; and G71, 4468.
b Calculated from an experiment in which the total number of temperature-resistant revertants was: C45,
3488; G100, 1430; and G71, 4092.
FIG. 2. Pattern of temperature-resistant revertants, obtained with and without diethylsulfate (DES), from
three mutants with temperature-sensitive phenotypes. The dark colonies are AP+ and the light colonies are
AP. Experimental details are given in Materials and Methods.1340 BECKMAN AND COOPER J. BACTRIOL.
phenotypes (C45, G100, and L33) are trans-
duced with phage P1 previously grown on H12
(su4-) and H12R7a (su4+) is shown in Table 2.
Mutant L33 was presumed to carry a TSM
mutation because it gave only AP,2- revertants
and was used as a control for the transduction
experiments in place of mutant G71 because
L33 has a much lower reversion frequency than
G71 and, therefore, was a more suitable recipi-
TRT-1 TRT- 1 TRT-2 ent for transduction. It is clear from Table 2
(Su4 phoam,ess+I isu4S,phoam,ess+ ph am essnon that mutant L33 yields AP42- transductants
AP' AP42 AP+4 exclusively when transduced with PlH12 (su4-)
or P1H12R7a (su4+). In contrast, however, mu-
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FIG. 3. Pattern of temperature-resistant transduc- tants C45 and G100 both yield AP,2+ as well as
tants (TRT) expected when cells containing tempera- AP42 transductants when transduced with
ture-sensitive missense (TSM) or temperature-sensi- P1H12R7a (su4+), but yield AP42- transduc-
tive nonsense (TSN) mutations are transduced to tants exclusively when transduced with PlH12
temperature-resistance with phage Pl previously
grown on H12 (P1H12 [su4- ]) or H12R7a (P1H12R7a
(su4-). These transduction results support the
[su4+ ]). idea that mutants C45 and G100 carry TSN
mutations, whereas mutant L33 carries a TSM
(su4-) is shown in Fig. 3. Cells containing TSN mutation.
mutations should yield two types of tempera- Mapping of TSN mutations. Mutants C45
ture-resistant transductants when infected with and G100 were mapped in the 0- to 10-min and
phage P1 previously grown on H12R7a (su4+). 40- to 50-min regions, respectively, of the E. coli
TRT-1 results from transduction of the func- genetic map as described in Materials and
tional allele of the defective gene and will be Methods.
Temperature shift experiments. A tempera-
AP,2-. TRT-2 results from transduction of the ture shift experiment with mutant C45 is shown
su4+ gene and will, therefore, be AP,2+. For a in Fig. 4. After the shift, the OD increased for 4
given TSN mutation, the ratio of the two types h before beginning to level off at a value which
of transductants will depend on the transduc- was approximately 20 times the starting OD at
tion frequency of the functional allele of the 42 C (culture A in Fig. 4). A temperature shift
defective gene. Cells containing TSN mutations experiment with mutant G100 is shown in Fig.
should yield only one type of temperature- 5. In this case, the OD continued to increase for
resistant transductant, TRT-1, when infected about 2 h after the shift, but then began to
with phage P1 previously grown on H12 (su4-). decrease dramatically before leveling off.
In contrast, cells containing TSM mutations
should yield only one type of temperature-
resistant transductant, TRT-1, when infected DISCUSSION
with phage P1 previously grown on either Clearly the most useful property of a TSN
H12R7a (su4+) or H12 (su4-. In this case, the mutation in a given gene is the defective synthe-
defective function is not suppressor sensitive sis of the polypeptide product of that gene. In
and, therefore, temperature-resistant transduc- contrast, a TSM mutation in the same gene will
tants can arise only from transduction of the result in the synthesis of a polypeptide that is
functional allele of the defective gene. identical to the wild-type polypeptide with the
The strength of the transduction method for exception of one amino acid. Therefore, al-
distinguishing TSN from TSM mutations lies in though it may be extremely difficult to identify
the fact that the donor strains (H12 and the polypeptide product of a gene that carries a
H12R7a) are isogenic with the exception of the TSM mutation, it may be possible by standard
su4 gene. biochemical techniques to identify the polypep-
We found that mutants C45 and G100 gave tide product of a gene that carries a TSN
AP42+ and AP2- transductants when trans- mutation. The value of this approach has long
duced to temperature resistance with phage P1 been recognized in the study of bacterial vi-
previously grown on H12R7a (su4+), but gave ruses. In this instance, the polypeptide product
AP42- transductants exclusively when trans- of a given gene may often be identified by
duced to temperature resistance with phage P1 comparing the polypeptides induced by phage
previously grown on H12 (su4-). The pattern of containing a suppressor-sensitive mutation in
temperature-resistant transductants obtained permissive and nonpermissive hosts. In a simi-
when three mutants with temperature-sensitive lar manner, it should be possible to identify theVOL. 116, 1973 TEMPERATURE-SENSITIVE NONSENSE MUTATIONS 1341
TABLE 2. Alkaline phosphatase phenotypes of temperature-resistant transductants (TRT) of
temperature-sensitive mutants
P1H12 (su4-) P1H12R7a (su4+)
Temperature-sensitive
mutant TRT/PFU Relative no. of TRT' TRT/PFU Relative no. of TRT5
AP42+ AP42- AP42+ AP42-
C45 9.4 x 10-4 0 100 1.1 X 10-3 23 77
G100 1.7 x 10-4 0 100 1.2 x 10-' 52 48
L33c 1.4 x 10-' 0 100 8.3 x 10-4 0 100
a Calculated from an experiment in which the actual number of temperature-resistant transductants was:
C45, 1411; G100, 242; and L33, 1872.
b Calculated from an experiment in which the actual number of temperature-resistant transductants was:
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C45, 1072; G100, 120; and L33, 1036.
c Temperature-sensitive mutant L33 was identified as carrying a TSM mutation by reversion analysis as
described in the text.
EC EC
0 0
o
cL
to
0
0.01 I I I I
0 100 200 300 400 0 100 200 300 400
MINUTES AFTER SHIFT TO 4t MINUTES AFTER SHIFT TO 420
FIG. 4. Temperature-shift experiments with C45 FIG. 5. Temperature-shift experiment with G100
(0), C45R2a (A) and PNG46 (0). Cultures growing (a), G100R2 (A), and PNG468 (0). Cultures growing
exponentially at 30 C in LB broth were shifted to 42 C exponentially at 30 C in LB broth were shifted to 42 C
by dilution into prewarmed LB broth. As indicated in by dilution into prewarmed LB broth. As indicated in
the figure, two dilutions were made from the 30 C the figure, two dilutions were made from the 30 C
culture of C45. C45R2a is a temperature-resistant culture of G100. G100R2 is a temperature-resistant
revertant (AP42-) of C45. The doubling times at 30 C revertant (AP42-) of G100. The doubling times at 30 C
of C45, PNG46, and C45R2a are 58, 46 and 59 min, of G100, PNG468, and G100R2 are 77, 50, and 53 min,
respectively. respectively.
polypeptide product of a bacterial gene if a this time there appears to be no direct method
mutation carrying a TSN mutation in that gene to select TSM mutants of the TSS type. Our
is available. This can be done by comparing the method for the isolation of cells containing TSN
polypeptides synthesized at the permissive and mutations, however, results in the direct isola-
nonpermissive temperatures. tion of mutants with a TSS phenotype.
We wish to emphasize that, although mu- We are currently carrying out studies to
tants with a TSS phenotype have been reported further characterize mutants C45 and G100. In
previously (10), these are of the TSM type. At addition, we are attempting to isolate addi-1342 BECKMAN AND COOPER J. BACTERIOL.
tional mutants of the TSN type. 4. Gallucci, E., G. Pacchetti, and S. Zangrossi. 1970.
Genetic studies on temperature sensitive nonsense
ACKNOWLEDGMENTS suppression. Mol. Gen. Genet. 106:362-370.
5. Garen, A., and S. Garen. 1963. Complementation in vivo
We thank Therese Ruettinger for excellent technical as- between structural mutants of alkaline phosphatase for
sistance. This work was supported by Public Health Service E. coli. J. Mol. Biol. 7:13-22.
grant AI10059-03 from the National Institute of Allergy and 6. Low, B. 1973. Rapid mapping of conditional and auxo-
Infectious Diseases. trophic mutations in Escherichia coli K-12. J. Bacte-
riol. 113:798-812.
LITERATURE CITED 7. Messer, W., and W. Vielmetter. 1965. High resolution
colony staining for the detection of bacterial growth
1. Adelberg, E. A., M. Mandel, and G. C. C. Chen. 1965. requirements using naphthol azo-dye techniques. Bio-
Optimal conditions for mutagenesis by N-methyl-N'- chem. Biophys. Res. Commun. 21:182-186.
nitro-N-nitrosoguanidine in Escherichia coli K12. Bio- 8. Miller, J. 1972. Experiments in molecular genetics. Cold
chem. Biophys. Res. Commun. 18:788-795. Spring Harbor Laboratory, New York.
2. Bachman, B. J. 1972. Pedigrees of some mutant strains of 9. Person, S., and M. Osborn. 1968. The conversion of
Escherichia coli K-12. Bacteriol. Rev. 36:525-557. amber suppressors to ochre suppressors. Proc. Nat.
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3. Caro, L., and C. M. Berg. 1971. P1 transduction, p. Acad. Sci. U.S.A. 60:1030-1037.
444-458. In L. Grossman and K. Moldave (ed.), 10. Sadler, J. R., and A. Novick. 1965. The properties of
Methods in enzymology, vol. 21. Academic Press Inc., repressor and the kinetics of its action. J. Mol. Biol.
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