 |
TEXT |
The miaA gene encodes a
tRNA prenyltransferase that catalyzes the addition of a
2-isopentenyl group from dimethylallyl diphosphate to
the N6-nitrogen of adenosine adjacent to the
anticodon at position 37 of 10 of 46 Escherichia coli tRNA
species that read codons starting with U residues (i6A-37
in Fig. 1) (3, 5, 19, 26,
35). In E. coli, the i6A-37 tRNA
modification is further methylthiolated by the action of the
miaB gene product, which is dependent on iron, and possibly another enzyme activity (MiaC) to form
ms2i6A-37 (Fig. 1), except in
tRNASec (11, 12). Methylthiolation is
dependent on prior formation of i6A-37 by the MiaA tRNA
prenyltransferase (3), and miaA mutants contain
fully unmodified A-37 residues in their tRNA.
View larger version (22K):
[in this window]
[in a new window]
|
FIG. 1.
Formation of the ms2i6A-37
modification in E. coli tRNA by the MiaA and MiaBC enzyme
activities. See the text for details. Ubi, ubiquinone; IPP, isopentenyl
diphosphate; DMAPP, dimethylallyl diphosphate; Met, methionine; Cys,
cysteine; SAM, S-adenosylmethionine; SAH,
S-adenosylhomocysteine.
|
|
The presence of A-37 instead of the ms2i6A-37
tRNA modification in miaA mutants of E. coli and
Salmonella enterica serovar Typhimurium results in multiple
defects in translation efficiency, codon context sensitivity, and
fidelity (8, 20). These translation defects impart broadly
pleiotropic phenotypes to miaA mutants, including decreased
growth rate and yield (10), altered sensitivity to amino
acid analogs (10), increased oxidation of certain amino acids and tricarboxylic acid cycle intermediates (32),
altered utilization of primary carbon sources (32), and
suppression of Tet(M) protein-induced tetracycline resistance
(4). In contrast, the presence of i6A-37
instead of ms2i6A-37 in miaB mutants
leads to milder defects in translation than those of miaA
mutants (8, 11).
We demonstrated previously that miaA mutants exhibit a
moderate mutator phenotype that results in GC
TA transversion
mutations (5, 6). The genetic basis for this
miaA mutator phenotype has not been determined, nor has it
been determined whether miaB mutants show a mutator
phenotype. However, it was shown that the miaA mutator
phenotype was not due to polarity on the expression of the downstream
hfq gene, which encodes a pleiotropic regulator, or
hflA-region genes, which encode a protease
(32). Recently, Humayun and coworkers described a new
pathway of mutagenesis that results in increased transversion mutations
in response to translation stress (1, 17, 28, 29). This
translation stress-induced mutagenesis (TSM) pathway is induced in
mutA mutants, which contain an anticodon mutation in the
glyV tRNA gene that causes insertion of glycine instead of
aspartic acid in proteins, such as the proofreading subunit of DNA
polymerase (30, 31). Unexpectedly, the TSM pathway was
recently shown to depend on recombination functions (1, 28,
29). In reviewing inducible mutagenic pathways in E. coli, Humayun hypothesized that the miaA mutator
phenotype may be a manifestation of the TSM pathway (17).
In this report, we show that the miaA mutator phenotype is
dependent on recombination functions similar to, but not exactly the
same as, those required for the TSM pathway. We also demonstrate a
correlation between the GCTA mutator phenotype and the severity of
defects in translation in different tRNA modification-deficient mutants.
miaA mutator phenotype depends on recombination
functions.
Previously, we detected the miaA mutator
phenotype in the Cupples-Miller mutation tester strain CC104, which can
revert to lacZ+ only by a specific GCTA
transversion, but not in the five other tester strains for the
remaining transversion and transition mutations (6). To
determine the genetic requirements of the miaA mutator phenotype, we constructed strains in which the miaA tRNA
modification mutation was combined with mutations defective in
recombination, the SOS response, or some pathways of DNA repair (Table
1).
Mutation frequencies were determined essentially as described by
Cupples and Miller (see Fig. 2) (7). Briefly, strains were
grown overnight to saturation at 37°C with shaking in 5 ml of
Luria-Bertani broth (10 g of NaCl per liter) supplemented with 30 mg of
L-cysteine per liter. Bacterial cells were washed twice by
collection by low-spread centrifugation (
4,000 × g)
and resuspension in 5 ml of minimal (E) salts (9) lacking
a carbon source. Bacterial pellets were resuspended in 0.5 ml of
minimal (E) salts. Resuspended cells (0.1 ml) were spread onto plates
containing minimal (E) salts, 0.4% (wt/vol) lactose, 0.01 M
FeSO4, and 1.5% (wt/vol) Bacto agar or serially diluted in
minimal (E) salts and spread onto plates containing 0.4% (wt/vol)
glucose instead of lactose. Plates containing lactose or glucose were
incubated at 37°C for 3 days or 1 day before colonies were counted to
determine the number of lacZ+ revertants or
viable F' episome-containing bacteria, respectively. Reconstitution
experiments in which fixed numbers of lacZ+
revertants of CC104 miaA+ and CC104
miaA were purposefully added to cultures before plating indicated that CC104 miaA+
lacZ+ colonies began to appear after 1 day and
reached a maximum number that remained constant on days 2 and 3 (data
not shown). In contrast, CC104 miaA lacZ+
colonies did not appear until after 2 days and reached a maximum number
by 3 days (data not shown); therefore, mutation frequencies were
determined after 3 days of incubation at 37°C.
We could not detect the miaA mutator phenotype in CC104
miaA containing mutations in recA, recB, or
recD, which are defective in homologous recombination
functions (1, 17, 29) or in CC105 (ATTA tester) as
reported previously (6) (Fig.
2). These patterns were confirmed in
qualitative experiments in which blue lacZ+
papillae were observed in lawns of bacteria on plates containing minimal medium and
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-Gal)
(data not shown). In these experiments, we used the recA430 allele, which is partially defective in homologous recombination and
SOS induction (18), because strains containing this
mutation grow faster than recA null mutants. Still, this
leaky recA allele prevented the miaA mutator
phenotype. The recB or recD transposon insertion
mutations abolish homologous recombination (18) or result
in hyper-recombination in some cases (18) and increased F'
episome replication (15), respectively. In contrast to
recA, recB, and recD mutants, the miaA
mutator phenotype was still observed to varying extents in CC104
miaA (GCTA tester) containing
lexA(Ind
), polB, uvrA, umuDC, and
mutY mutations, which confer defects in SOS induction, DNA
polymerase II, nucleotide excision repair, SOS mutagenesis, and GO
repair, respectively (Fig. 2) (1, 13, 17, 24, 29, 37).
Although the miaA mutator phenotype was evident, its extent
was somewhat reduced in the lexA(Ind)
(2.3-fold) and mutY (1.8-fold) mutants compared to that of
the CC104 miaA+ and CC104 miaA pair
(3.7-fold) (Fig. 2).
View larger version (22K):
[in this window]
[in a new window]
|
FIG. 2.
Mutation frequencies of Cupples-Miller tester strains
CC104 and CC105, which can revert to lacZ+ only
by GCTA and ATTA transversions, respectively (7),
containing a miaA mutation combined with mutations defective
in recombination, the SOS pathway, or DNA repair. Mutation frequencies
were determined as described in the text. Experiments were performed
independently at least three times, and standard errors of the means
are shown.
|
|
The absence of the miaA mutator phenotype in both
recA and recB mutants is consistent with the
interpretation that this phenomenon depends on recombination functions,
similar to TSM (1, 17, 28, 29). Likewise, the
miaA mutator phenotype and TSM were not dependent on
polB and umuDC functions (Fig. 2) (1,
27). However, there are noteworthy differences between the
miaA mutator phenotype and TSM. Only GCTA
transversion was increased in miaA mutants, whereas the
mutA mutator phenotype, which has been hypothesized to
induce TSM (17), led primarily to an increase in ATTA
transversions in tester strain CC105 (7.8-fold) as well as ATCG and
GCTA transversions in tester strains CC101 and CC104 (5.1-fold and
3.7-fold, respectively) (23). The recD mutation
abolished the miaA mutator phenotype, whereas a
recD mutation failed to affect induction of TSM in
mutA mutants (29). Finally, the miaA
mutator phenotype was reduced, but not abolished, by the
lexA(Ind) mutation, whereas TSM was not
reduced in cells unable to induce the SOS response (28).
Nonetheless, the striking dependence of both the miaA
mutator phenotype and TSM on recombination functions suggests that
miaA mutations may partly induce the TSM response, albeit
more weakly than mutA mutations. Testing of this hypothesis will require a much deeper understanding of the mechanism of the TSM
response and of the reasons why it depends on recombination functions,
especially in cells lacking preformed DNA lesions (28).
miaB and hisT tRNA modification mutants
lack a mutator phenotype.
We tested whether other tRNA
modification mutations induced GCTA transversion mutations (Fig.
3). We found that the hisT mutant, which lacks pseudouridine residues at positions 38, 39, and 40 in the anticodon stem-loop of tRNAs (3, 8, 33, 35), has a
barely detectable mutator phenotype. For reasons that are not
understood, there was more variation in lacZ+
reversion of hisT mutants than in that of miaA
mutants (Fig. 3). Mutation of miaB did not cause a mutator
phenotype.
View larger version (20K):
[in this window]
[in a new window]
|
FIG. 3.
Mutation frequencies of strain CC104 (GCTA tester)
containing a miaA or miaB mutation or a
hisT mutation and thus defective in
ms2i6A-37 or pseudouridine tRNA modification,
respectively. Experiments were performed as described for Fig. 2.
|
|
These results have three implications. First, induction of GCTA
transversion, and possibly TSM, seems to be correlated with the
severity of translation defects and pleiotropic phenotypes caused by
tRNA undermodification, where miaA > hisT > miaB (3, 8, 33).
Second, undermodification of ms2i6A-37 to
i6A-37 in miaB mutants does not induce
mutagenesis. The i6A-37 tRNA undermodification also occurs
in miaA+ bacteria that are subjected to mild
limitation for iron, and it has been suggested that this
undermodification acts as a physiological switch in response to this
stress (3). Our results show that this undermodification
likely does not act as a switch to increase GCTA transversion.
Third, tRNA modifications in response to other stress conditions have
been reported (3). With the exception of oxygen-dependent
hydroxylation of ms2i6A-37 to form
ms2io6A-37 in Salmonella
(3), the mechanisms and physiological implications of
these tRNA undermodifications remain largely unexplored
(3). It is possible that other stress conditions may
trigger GCTA transversion, and possibly the TSM pathway, through
tRNA undermodification in wild-type bacteria.
Overexpression of MutS mismatch repair protein suppresses the
miaA mutator phenotype.
Recently we reported that
overexpression of the MutS DNA mismatch binding protein leads to a
recA-independent decrease in GCTA transversion mutations
in wild-type E. coli and mutY mutants (37). Therefore, we tested whether overexpression of the
MutS and MutL mismatch repair proteins decreases GCTA transversion in miaA mutants (Fig. 4). We
found that overexpression of MutS or MutL suppressed the
miaA mutator phenotype, with MutS eliciting the stronger
effect (Fig. 4). This result suggests that the miaA mutator
phenotype, and possibly part of the TSM response, may involve a
mismatch recognizable by the MutS protein, which is present at
just-sufficient levels in exponentially growing E. coli
cells and is strongly down-regulated in bacterial cells that are in
stationary phase, such as those used in these experiments (14,
34).
View larger version (19K):
[in this window]
[in a new window]
|
FIG. 4.
Mutation frequencies of strain CC104 miaA
(GCTA tester) containing an empty vector plasmid (pControl) or
plasmids that overexpress the MutL (pMutL) or MutS (pMutS) mismatch
repair proteins by about 20- and 60-fold, respectively
(16). Experiments were performed as described for Fig. 2,
except that 50 µg of kanamycin per ml was added to growth media and
plates to maintain the plasmids.
|
|
We thank Tiffany Tsui and Gang Feng for helpful information and
critical discussions of this work; M. Goodman, Susan Lovett, Jeffrey
Miller, B. Wanner, G. Weinstock, and R. Woodgate for strains; and Susan
Rosenberg for plasmids.
This work was supported by NIH grant RO1-CA77193 and by resources of
the Lilly Research Laboratories.
| 1.
|
Al Mamun, A. A.,
M. S. Rahman, and M. Z. Humayun.
1999.
Escherichia coli cells bearing mutA, a mutant glyV tRNA gene, express a recA-dependent error-prone DNA replication activity.
Mol. Microbiol.
33:732-740[CrossRef][Medline].
|
| 2.
|
Arps, P. J., and M. E. Winkler.
1987.
Structural analysis of the Escherichia coli K-12 hisT operon by using a kanamycin resistance cassette.
J. Bacteriol.
169:1061-1070[Abstract/Free Full Text].
|
| 3.
|
Björk, G. R., and T. Rasmuson.
1998.
Links between tRNA modification and metabolism and modified nucleosides as tumor markers, p. 471-491.
In
H. Grosjean, and R. Benne (ed.), Modification and editing of RNA. ASM Press, Washington, D.C.
|
| 4.
|
Burdett, V.
1993.
tRNA modification activity is necessary for Tet(M)-mediated tetracycline resistance.
J. Bacteriol.
175:7209-7215[Abstract/Free Full Text].
|
| 5.
|
Connolly, D. M., and M. E. Winkler.
1989.
Genetic and physiological relationships among the miaA gene, 2-methylthio-N6-(2-isopentenyl)-adenosine tRNA modification, and spontaneous mutagenesis in Escherichia coli K-12.
J. Bacteriol.
171:3233-3246[Abstract/Free Full Text].
|
| 6.
|
Connolly, D. M., and M. E. Winkler.
1991.
Structure of Escherichia coli K-12 miaA and characterization of the mutator phenotype caused by miaA insertion mutations.
J. Bacteriol.
173:1711-1721[Abstract/Free Full Text].
|
| 7.
|
Cupples, C. G., and J. H. Miller.
1989.
A set of lacZ mutations in Escherichia coli that allow rapid detection of each of the six base substitutions.
Proc. Natl. Acad. Sci. USA
86:5345-5349[Abstract/Free Full Text].
|
| 8.
|
Curran, J. F.
1998.
Modified nucleosides in translation, p. 493-516.
In
H. Grosjean, and R. Benne (ed.), Modification and editing of RNA. ASM Press, Washington, D.C.
|
| 9.
|
Davis, R. W.,
D. Botstein, and J. R. Roth.
1980.
Advanced bacterial genetics.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 10.
|
Ericson, J. U., and G. R. Björk.
1986.
Pleiotropic effects induced by modification deficiency next to the anticodon of tRNA from Salmonella typhimurium LT2.
J. Bacteriol.
166:1013-1021[Abstract/Free Full Text].
|
| 11.
|
Esberg, B., and G. R. Björk.
1995.
The methylthio group (ms2) of N6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A) present next to the anticodon contributes to the decoding efficiency of the tRNA.
J. Bacteriol.
177:1967-1975[Abstract/Free Full Text].
|
| 12.
|
Esberg, B.,
H.-C. E. Leung,
H.-C. T. Tsui,
G. R. Björk, and M. E. Winkler.
1999.
Identification of the miaB gene, involved in methylthiolation of isopentenylated A37 derivatives in the tRNA of Salmonella typhimurium and Escherichia coli.
J. Bacteriol.
181:7256-7265[Abstract/Free Full Text].
|
| 13.
|
Escarceller, M.,
J. Hicks,
G. Gudmundsson,
G. Trump,
D. Touati,
S. Lovett,
P. L. Foster,
K. McEntee, and M. F. Goodman.
1994.
Involvement of Escherichia coli DNA polymerase II in response to oxidative damage and adaptive mutation.
J. Bacteriol.
176:6221-6228[Abstract/Free Full Text].
|
| 14.
|
Feng, G.,
H.-C. T. Tsui, and M. E. Winkler.
1996.
Depletion of the cellular amounts of the MutS and MutH methyl-directed mismatch repair proteins in stationary-phase Escherichia coli K-12 cells.
J. Bacteriol.
178:2388-2396[Abstract/Free Full Text].
|
| 15.
|
Foster, P. L., and W. A. Rosche.
1999.
Increased episomal replication accounts for the high rate of adaptive mutation in recD mutants of Escherichia coli.
Genetics
152:15-30[Abstract/Free Full Text].
|
| 16.
|
Harris, R. S.,
G. Feng,
K. J. Ross,
R. Sidhu,
C. Thulin,
S. Longerich,
S. K. Szigety,
M. E. Winkler, and S. M. Rosenberg.
1997.
Mismatch repair protein MutL becomes limiting during stationary-phase mutation.
Genes Dev.
11:2426-2437[Abstract/Free Full Text].
|
| 17.
|
Humayun, M. Z.
1998.
SOS and Mayday: multiple inducible mutagenic pathways in Escherichia coli.
Mol. Microbiol.
30:905-910[CrossRef][Medline].
|
| 18.
|
Kowalczykowski, S. C.,
D. A. Dixon,
A. K. Eggleston,
S. D. Lauder, and W. M. Rehrauer.
1994.
Biochemistry of homologous recombination in Escherichia coli.
Microbiol. Rev.
58:401-465[Abstract/Free Full Text].
|
| 19.
|
Leung, H. C.,
Y. Chen, and M. E. Winkler.
1997.
Regulation of substrate recognition by the MiaA tRNA prenyltransferase modification enzyme of Escherichia coli K-12.
J. Biol. Chem.
272:13073-13083[Abstract/Free Full Text].
|
| 20.
|
Li, J.,
B. Esberg,
J. F. Curran, and G. R. Björk.
1997.
Three modified nucleosides present in the anticodon stem and loop influence the in vivo aa-tRNA selection in a tRNA-dependent manner.
J. Mol. Biol.
271:209-221[CrossRef][Medline].
|
| 21.
|
Lloyd, R. G.,
C. Buckman, and F. E. Benson.
1987.
Genetic analysis of conjugational recombination in Escherichia coli K12 strains deficient in RecBCD enzyme.
J. Gen. Microbiol.
133:2531-2538[Medline].
|
| 22.
|
Mahdi, A. A., and R. G. Lloyd.
1989.
Identification of the recR locus of Escherichia coli K-12 and analysis of its role in recombination and DNA repair.
Mol. Gen. Genet.
216:503-510[CrossRef][Medline].
|
| 23.
|
Michaels, M. L.,
C. Cruz, and J. H. Miller.
1990.
mutA and mutC: two mutator loci in Escherichia coli that stimulate transversions.
Proc. Natl. Acad. Sci. USA
87:9211-9215[Abstract/Free Full Text].
|
| 24.
|
Michaels, M. L., and J. H. Miller.
1992.
The GO system protects organisms from the mutagenic effect of the spontaneous lesion 8-hydroxyguanine (7,8-dihydro-8-oxoguanine).
J. Bacteriol.
174:6321-6325[Free Full Text].
|
| 25.
|
Miller, J. H.
1992.
A short course in bacterial genetics.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 26.
|
Moore, J. A., and C. D. Poulter.
1997.
Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: a binding mechanism for recombinant enzyme.
Biochemistry
36:604-614[CrossRef][Medline].
|
| 27.
|
Murphy, H. S., and M. Z. Humayun.
1997.
Escherichia coli cells expressing a mutant glyV (glycine tRNA) gene have a UVM-constitutive phenotype: implications for mechanisms underlying the mutA or mutC mutator effect.
J. Bacteriol.
179:7507-7514[Abstract/Free Full Text]. (Erratum, 180:2271, 1998.)
|
| 28.
|
Ren, L.,
A. A. Al Mamun, and M. Z. Humayun.
1999.
The mutA mistranslator tRNA-induced mutator phenotype requires recA and recB genes, but not the derepression of lexA-regulated functions.
Mol. Microbiol.
32:607-615[CrossRef][Medline].
|
| 29.
|
Ren, L.,
A. A. M. Al Mamun, and M. Z. Humayun.
2000.
Requirement for homologous recombination functions for expression of the mutA mistranslator tRNA-induced mutator phenotype in Escherichia coli.
J. Bacteriol.
182:1427-1431[Abstract/Free Full Text].
|
| 30.
|
Slupska, M. M.,
C. Baikalov,
R. Lloyd, and J. H. Miller.
1996.
Mutator tRNAs are encoded by the Escherichia coli mutator genes mutA and mutC: a novel pathway for mutagenesis.
Proc. Natl. Acad. Sci. USA
93:4380-4385[Abstract/Free Full Text].
|
| 31.
|
Slupska, M. M.,
A. G. King,
L. I. Lu,
R. H. Lin,
E. F. Mao,
C. A. Lackey,
J.-H. Chiang,
C. Baikalov, and J. H. Miller.
1998.
Examination of the role of DNA polymerase proofreading in the mutator effect of miscoding tRNAs.
J. Bacteriol.
180:5712-5717[Abstract/Free Full Text].
|
| 32.
|
Tsui, H.-C.,
H. C. Leung, and M. E. Winkler.
1994.
Characterization of broadly pleiotropic phenotypes caused by an hfq insertion mutation in Escherichia coli K-12.
Mol. Microbiol.
13:35-49[Medline].
|
| 33.
|
Tsui, H.-C. T.,
P. J. Arps,
D. M. Connolly, and M. E. Winkler.
1991.
Absence of hisT-mediated tRNA pseudouridylation results in a uracil requirement that interferes with Escherichia coli K-12 cell division.
J. Bacteriol.
173:7395-7400[Abstract/Free Full Text].
|
| 34.
|
Tsui, H.-C. T.,
G. Feng, and M. E. Winkler.
1997.
Negative regulation of mutS and mutH repair gene expression by the Hfq and RpoS global regulators of Escherichia coli K-12.
J. Bacteriol.
179:7476-7487[Abstract/Free Full Text].
|
| 35.
|
Winkler, M. E.
1998.
Genetics and regulation of base modification in the tRNA and rRNA of prokaryotes and eukaryotes, p. 441-469.
In
H. Grosjean, and R. Benne (ed.), Modification and editing of RNA. ASM Press, Washington, D.C.
|
| 36.
|
Woodgate, R.
1992.
Construction of a umuDC operon substitution mutation in Escherichia coli.
Mutat. Res.
281:221-225[CrossRef][Medline].
|
| 37.
|
Zhao, J., and M. E. Winkler.
2000.
Reduction of GCTA transversion mutation by overexpression of MutS in Escherichia coli K-12.
J. Bacteriol.
182:5025-5028[Abstract/Free Full Text].
|
![[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}
Houston Medical School, Houston,
Texas 77030
Top
Abstract
Text
