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The Escherichia coli MutH protein is not the repressor of the bacteriophage Mu mom operon
381
Gene. 79 (1989) 381-383 Elsevier GEN 03014
coli MutH protein is not the repressor of the bacteriophage Mu mom operon
The Escherichia
(Dam methylation; mom operator; prophage induction; transcription control)
Stanley Hattman Department of Biology, Universityof Rochester, Rochester, NY 14627 (U.S.A.) Received
by R.M. Harshey:
Revised:
6 February
Accepted:
23 July 1988
1989
28 February
1989
SUMMARY
Expression of the bacteriophage Mu mom-operon is under tight regulatory control. One of the factors required for transcription of the operon is the host Escherichiu coli Dam activity. It was proposed that DNA methylation by this enzyme prevents the binding of a cellular repressor to an operator site containing three 5’-GATC-3’ sequences, the known target site of Dam methylation. Support for this model came from the observation of others that the requirement for Dam was almost completely suppressed in a mutH-IysA deletion mutant, suggesting that the MutH protein is the postulated transcriptional repressor. In this communication, however, I show that the Dam requirement is not effectively relieved in this deletion mutant; therefore, the MutH protein alone is not the mom repressor.
INTRODUCTION
The temperate phage Mu specifies an unusual DNA-modification function encoded by the mom gene (for a recent review, see Kahmann and Hattman, 1987). Expression of Mom modification is Correspondenceto: Dr. S. Hattman, University
of
Rochester,
Tel. (716)275-8046; Abbreviations: amphenicol; ferase;
NY
of Biology,
14627
(U.S.A.)
Fax (716)473-6889.
Ap, ampicillin; A, deletion;
EOP, efficiency
resistance;
Department
Rochester,
AP, 2-aminopurine;
Cm, chlor-
Dam, DNA-(adenine-Are’)-methyltransof plating;
Tn, transposon;
m6A, N6-methyladenine;
( ), designates
Mu prophage
R, (lyso-
genie state).
0378-l 119/89/$03.50
0
1989 Elsevier
Science Publishers
B.V. (Biomedical
under tight control, both at the transcriptional and translational levels (Hattman et al., 1987; Wulczyn and Kahmann, 1987). One of the positive regulatory factors is the host E. coli Dam enzyme, which methylates the sequence GATC to produce Gm6ATC (Hattman et al., 1978; Lacks and Greenberg, 1977). The site of Dam function was localized to a region 5’ to the mom-operon promoter. Deletions and other mutations in this target region relieve the requirement for Dam (Kahmann and Hattman, 1987; Seiler et al., 1986). It was postulated that the role of the Dam function is to methylate the GATC sequences in an operator locus, thereby preventing the binding of a repressor protein (Hattman and Ives, 1984; Hattman et al., 1985). Division)
382
into RH72 and RH724, selecting for CmR clones. The Darn phenotype was screened by analyzing cellular DNA for susceptib~ity to cleavage by MboI. E. co& strain MH4272(Mucts62pApl) was from M.M. Howe. Mucts62pApl (Leach and Symonds, 1979) is phenotypically Mom+ (Toussaint et al., 1980); it contains pApI, a Tn3 transposon fragment that confers resistance to Ap.
Seiler et al. (1986) reported that the Dam requirement is relieved only in a certain mutH deletion mutt, and they suggested that the MutH protein is the postulated repressor. Recently, I began a reinvestigation of the role of MutH in regulating mom expression. In agreement with Seiler et al. (1986), I found that mutH point mutants did not suppress the Dam requirement (unpublished observation). However, in contrast to their results, a mutH deletion (the same one as used in their study) did not effectively relieve the Dam requirement; therefore, the MutH protein cannot be the mom repressor.
EXPERIMENTAL
(b) Biological assay for mom expression To test for expression of the mom function in various genetic backgrounds, the Mucts62pApl mom + prophage was theory induced and progeny phage were assayed for their ability to grow on strains K704 and K704(Pl). Because expression of the mom modification function protects Mu DNA against Pl-directed restriction, Mu forms plaques with almost equal efficiency on the two strains. If the mom modification function is poorly expressed then Mu forms plaques at greatly reduced efficiency on K704(Pl), i.e., loo-fold lower or less. The results of such a study are summarized in Table I. It is evident that the mutH deletion mutation did not effectively suppress the Dam requirement for efficient expression of mom modification; i.e., after prophage induction in AmutH dam - cells, progeny phage had relative EOPs on K704(Pl) ranging from 2 x 10W3to 2 x 10m2. Although thesevalues are IOto loo-fold higher than for phage grown in dam cells, they still reflect poor expression of mom; they
AND DISCUSSION
(a) Strains E. coti K704 and K704fPl) were from our collection E. coli strain RH72(kI857), with the I prophage integrated near the mutH locus was obtained from R. Hoess. This strain is the parent of both RH724~[mutH-~~~~] (Hoess and Fan, 1975, obtained from R. Grafstrom) and RH72 (cured of I in my laboratory). RH724 A[mutH-lysA ] and RH72 were made by curing the cells of the prophage and screening survivors of growth at 42°C for their MutH phenotype. GM2972 dam- 13 : : Tn9 CmR (Marinus et al., 1983) was from M.G. Marinus; the dam- 13 : : Tn9 CmR marker was transduced by P 1vir
TABLE I Biological assay of Mom function in various Mu lysogens a Mu lysogen b
Relevant genotype
Relative EOPO
RH72 RH724 RH72 dam::Tn9 CmR RH724 dam : : Tn.9 CmR
mutH + dam +
0.5-0.7
km&i dam +
OS-O.6
mutH+ dam-
1-3 x 1o-4
AmutH dam _
2 x 10-3-2
x 1o-2
a Overnight saturated cultures were grown at 32°C in broth plus 50 pg Ap/ml(20 @gCm/ml were included for the CmR strains). After dilution and growth in fresh medium, exponentially growing Mucts62pAplmom + lysogens were induced for 30 min at 42°C and shifted to 37°C until lysis was complete. Phage lysates were assayed on K704 and K704(Pl). ’ Strains are described in section a E Relative EOP = [titer on K7~Pl)/titer on K704]. The values represent the ranges from three independent experiments, in which a total of six independent, freshly isolated RH724 dam::Tn9 CmR lysogens were used. It should be noted that the high value (2 x lo- 2, was from a culture that lysed poorly and had a low yield of phage. In general, the RH724 dum::Tn9 CmR lysogen was delayed in its lysis compared to the other strains (120 min vs. 90 min).
383
are similar to the values observed when Mu is grown lyticahy (Toussaint, 1976), a condition in which the mom m~i~cation is inefficiently expressed. Similar data were also obtained with phage Mucts62mom + (not shown).
ACKNOWLEDGEMENTS
(c) Conclusions and discussion
REFERENCES
In a previous study Seiler et al. (1986) screened a number of mutH mutations for their ability to suppress the Dam requirement for mom modification. Only in the AmutH deletion strain, RH724, did the mutation appear to give suppression. However, using the same deletion I did not confm their observation. In the experiments summarized in Table I, the bacterial strains did not contain plasmid pBR322, in contrast to those used by SeiIer and coworkers. However, in separate experiments where pBR322 was present, the same results were obtained as in Table I (data not shown). Therefore, the presence/ absence of pBR322 was not a factor contributing to the differences in our observed relative EOPs. Because my results show that the d mutH mutant does not suppress the Dam requirement, I conclude that the MutH protein alone cannot be the postulated Mu mom repressor. Admittedly, mom expression was not assayed directly, for example, by measuring mom mRNA, and thus several processes intervene between the primary mom-operon expression and the plaque assay. However, this caveat also applies to the Seiler et al. (1986) experiments; the purpose of my study was to see if using their experimental approach one could provide evidence that MutH is the mom-controlling repressor. However, because the AmutH mutation did partially suppress the requirement for Dam (Table I), it is possible that the MutH protein may be involved in the repression process as an accessory factor which potentiates the activity or specificity of another protein, whose identity remains unknown. Alternatively, if the delay in lysis of the AmutH dam--induced lysogen also involves a reduced rate of DNA packaging, then this might allow additional time for the (low level) Mom function to be expressed.
Hattman, S. and Ives, J.: Sl nuclease mapping of the phage Mu mom gene promoter: a model for the regulation of mom expression. Gene 29 (1984) 185-198. Hattman, S., Brooks, J.E. and Masurekar, M.: Sequence speciflcity of the Pl modification methylase (M . Ew Pl) and the DNA methylase (M . Ecodmz) controlled by the E. coli dam gene. J. Mol. Biol. 126 (1978) 367-380. Hattman, S., Ives, J., Margolin, W. and Howe, M.M.: Regulation and expression of the bacteriophage Mu mom gene: mapping of the transactivation (Dad) function to the C region. Gene 39 (1985) 71-76. Hattman, S., Ives, J., Wall, L. and MariC, S.: The bacteriophage Mu corn gene appears to specify a translation factor required for mom gene expression. Gene 55 (1987) 345-351. Hoess, R.H. and Fan, D.P.: Further characterization of a nonessential mutator gene in Escherichiu coli K-12. J. Bacterial. 124 (1975) 650-660. Kahmann, R. and Hattman, S.: Regulation and expression ofthe mom gene. In Symonds, N., Toussaint, A., Van de Putte, P. and Howe, M.M. (Eds.), Phage Mu. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1987, pp. 93-109. Lacks, S. and Greenberg, B.: Complementary specificity of restriction endonucleases of Diplococcus pneumoniae with respect to DNA methylation. J. Mol. Biol. 144 (1977) 153-168. Leach, D.L. and Symonds, N.: The isolation and characterisation of a plaque-forming derivative of bacteriophage Mu carrying a fragment ofTn3 conferring ampicillin resistance. Mol. Gen. Genet. 172 (1979) 179-184. Marinus, M.G., Carraway, M., Frey, AZ., Brown, L. and Arraj, J.A.: Insertion mutations in the dnm gene of ,%c/rerichiu coli K-12. Mol. Gen. Genet. 192 (1983) 288-289. Seiler, A., Blocker, H., Frank, R. and Kahmann, R.: The mom gene of bacteriophage Mu: the mechanism of methylationdependent expression. EMBO J. 5 (1986) 2719-2728. Toussaint, A.: The DNA modification function of temperate phage Mu-l. Virology 70 (1976) 17-27. Toussaint, A., Desmet, L. and Faelen, M.: Mapping of the m~i~cation function of temperate phage Mu-l. Mol. Gen. Genet. 177 (1980) 351-353. Wulczyn, F.G. and Kahmann, R.: Post-transcriptional regulation of the bacteriophage Mu mom gene by the corn gene product. Gene 51 (1987) 139-147.
This work was supported by PHS Grant No. GM-29227.
Gene. 79 (1989) 381-383 Elsevier GEN 03014
coli MutH protein is not the repressor of the bacteriophage Mu mom operon
The Escherichia
(Dam methylation; mom operator; prophage induction; transcription control)
Stanley Hattman Department of Biology, Universityof Rochester, Rochester, NY 14627 (U.S.A.) Received
by R.M. Harshey:
Revised:
6 February
Accepted:
23 July 1988
1989
28 February
1989
SUMMARY
Expression of the bacteriophage Mu mom-operon is under tight regulatory control. One of the factors required for transcription of the operon is the host Escherichiu coli Dam activity. It was proposed that DNA methylation by this enzyme prevents the binding of a cellular repressor to an operator site containing three 5’-GATC-3’ sequences, the known target site of Dam methylation. Support for this model came from the observation of others that the requirement for Dam was almost completely suppressed in a mutH-IysA deletion mutant, suggesting that the MutH protein is the postulated transcriptional repressor. In this communication, however, I show that the Dam requirement is not effectively relieved in this deletion mutant; therefore, the MutH protein alone is not the mom repressor.
INTRODUCTION
The temperate phage Mu specifies an unusual DNA-modification function encoded by the mom gene (for a recent review, see Kahmann and Hattman, 1987). Expression of Mom modification is Correspondenceto: Dr. S. Hattman, University
of
Rochester,
Tel. (716)275-8046; Abbreviations: amphenicol; ferase;
NY
of Biology,
14627
(U.S.A.)
Fax (716)473-6889.
Ap, ampicillin; A, deletion;
EOP, efficiency
resistance;
Department
Rochester,
AP, 2-aminopurine;
Cm, chlor-
Dam, DNA-(adenine-Are’)-methyltransof plating;
Tn, transposon;
m6A, N6-methyladenine;
( ), designates
Mu prophage
R, (lyso-
genie state).
0378-l 119/89/$03.50
0
1989 Elsevier
Science Publishers
B.V. (Biomedical
under tight control, both at the transcriptional and translational levels (Hattman et al., 1987; Wulczyn and Kahmann, 1987). One of the positive regulatory factors is the host E. coli Dam enzyme, which methylates the sequence GATC to produce Gm6ATC (Hattman et al., 1978; Lacks and Greenberg, 1977). The site of Dam function was localized to a region 5’ to the mom-operon promoter. Deletions and other mutations in this target region relieve the requirement for Dam (Kahmann and Hattman, 1987; Seiler et al., 1986). It was postulated that the role of the Dam function is to methylate the GATC sequences in an operator locus, thereby preventing the binding of a repressor protein (Hattman and Ives, 1984; Hattman et al., 1985). Division)
382
into RH72 and RH724, selecting for CmR clones. The Darn phenotype was screened by analyzing cellular DNA for susceptib~ity to cleavage by MboI. E. co& strain MH4272(Mucts62pApl) was from M.M. Howe. Mucts62pApl (Leach and Symonds, 1979) is phenotypically Mom+ (Toussaint et al., 1980); it contains pApI, a Tn3 transposon fragment that confers resistance to Ap.
Seiler et al. (1986) reported that the Dam requirement is relieved only in a certain mutH deletion mutt, and they suggested that the MutH protein is the postulated repressor. Recently, I began a reinvestigation of the role of MutH in regulating mom expression. In agreement with Seiler et al. (1986), I found that mutH point mutants did not suppress the Dam requirement (unpublished observation). However, in contrast to their results, a mutH deletion (the same one as used in their study) did not effectively relieve the Dam requirement; therefore, the MutH protein cannot be the mom repressor.
EXPERIMENTAL
(b) Biological assay for mom expression To test for expression of the mom function in various genetic backgrounds, the Mucts62pApl mom + prophage was theory induced and progeny phage were assayed for their ability to grow on strains K704 and K704(Pl). Because expression of the mom modification function protects Mu DNA against Pl-directed restriction, Mu forms plaques with almost equal efficiency on the two strains. If the mom modification function is poorly expressed then Mu forms plaques at greatly reduced efficiency on K704(Pl), i.e., loo-fold lower or less. The results of such a study are summarized in Table I. It is evident that the mutH deletion mutation did not effectively suppress the Dam requirement for efficient expression of mom modification; i.e., after prophage induction in AmutH dam - cells, progeny phage had relative EOPs on K704(Pl) ranging from 2 x 10W3to 2 x 10m2. Although thesevalues are IOto loo-fold higher than for phage grown in dam cells, they still reflect poor expression of mom; they
AND DISCUSSION
(a) Strains E. coti K704 and K704fPl) were from our collection E. coli strain RH72(kI857), with the I prophage integrated near the mutH locus was obtained from R. Hoess. This strain is the parent of both RH724~[mutH-~~~~] (Hoess and Fan, 1975, obtained from R. Grafstrom) and RH72 (cured of I in my laboratory). RH724 A[mutH-lysA ] and RH72 were made by curing the cells of the prophage and screening survivors of growth at 42°C for their MutH phenotype. GM2972 dam- 13 : : Tn9 CmR (Marinus et al., 1983) was from M.G. Marinus; the dam- 13 : : Tn9 CmR marker was transduced by P 1vir
TABLE I Biological assay of Mom function in various Mu lysogens a Mu lysogen b
Relevant genotype
Relative EOPO
RH72 RH724 RH72 dam::Tn9 CmR RH724 dam : : Tn.9 CmR
mutH + dam +
0.5-0.7
km&i dam +
OS-O.6
mutH+ dam-
1-3 x 1o-4
AmutH dam _
2 x 10-3-2
x 1o-2
a Overnight saturated cultures were grown at 32°C in broth plus 50 pg Ap/ml(20 @gCm/ml were included for the CmR strains). After dilution and growth in fresh medium, exponentially growing Mucts62pAplmom + lysogens were induced for 30 min at 42°C and shifted to 37°C until lysis was complete. Phage lysates were assayed on K704 and K704(Pl). ’ Strains are described in section a E Relative EOP = [titer on K7~Pl)/titer on K704]. The values represent the ranges from three independent experiments, in which a total of six independent, freshly isolated RH724 dam::Tn9 CmR lysogens were used. It should be noted that the high value (2 x lo- 2, was from a culture that lysed poorly and had a low yield of phage. In general, the RH724 dum::Tn9 CmR lysogen was delayed in its lysis compared to the other strains (120 min vs. 90 min).
383
are similar to the values observed when Mu is grown lyticahy (Toussaint, 1976), a condition in which the mom m~i~cation is inefficiently expressed. Similar data were also obtained with phage Mucts62mom + (not shown).
ACKNOWLEDGEMENTS
(c) Conclusions and discussion
REFERENCES
In a previous study Seiler et al. (1986) screened a number of mutH mutations for their ability to suppress the Dam requirement for mom modification. Only in the AmutH deletion strain, RH724, did the mutation appear to give suppression. However, using the same deletion I did not confm their observation. In the experiments summarized in Table I, the bacterial strains did not contain plasmid pBR322, in contrast to those used by SeiIer and coworkers. However, in separate experiments where pBR322 was present, the same results were obtained as in Table I (data not shown). Therefore, the presence/ absence of pBR322 was not a factor contributing to the differences in our observed relative EOPs. Because my results show that the d mutH mutant does not suppress the Dam requirement, I conclude that the MutH protein alone cannot be the postulated Mu mom repressor. Admittedly, mom expression was not assayed directly, for example, by measuring mom mRNA, and thus several processes intervene between the primary mom-operon expression and the plaque assay. However, this caveat also applies to the Seiler et al. (1986) experiments; the purpose of my study was to see if using their experimental approach one could provide evidence that MutH is the mom-controlling repressor. However, because the AmutH mutation did partially suppress the requirement for Dam (Table I), it is possible that the MutH protein may be involved in the repression process as an accessory factor which potentiates the activity or specificity of another protein, whose identity remains unknown. Alternatively, if the delay in lysis of the AmutH dam--induced lysogen also involves a reduced rate of DNA packaging, then this might allow additional time for the (low level) Mom function to be expressed.
Hattman, S. and Ives, J.: Sl nuclease mapping of the phage Mu mom gene promoter: a model for the regulation of mom expression. Gene 29 (1984) 185-198. Hattman, S., Brooks, J.E. and Masurekar, M.: Sequence speciflcity of the Pl modification methylase (M . Ew Pl) and the DNA methylase (M . Ecodmz) controlled by the E. coli dam gene. J. Mol. Biol. 126 (1978) 367-380. Hattman, S., Ives, J., Margolin, W. and Howe, M.M.: Regulation and expression of the bacteriophage Mu mom gene: mapping of the transactivation (Dad) function to the C region. Gene 39 (1985) 71-76. Hattman, S., Ives, J., Wall, L. and MariC, S.: The bacteriophage Mu corn gene appears to specify a translation factor required for mom gene expression. Gene 55 (1987) 345-351. Hoess, R.H. and Fan, D.P.: Further characterization of a nonessential mutator gene in Escherichiu coli K-12. J. Bacterial. 124 (1975) 650-660. Kahmann, R. and Hattman, S.: Regulation and expression ofthe mom gene. In Symonds, N., Toussaint, A., Van de Putte, P. and Howe, M.M. (Eds.), Phage Mu. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1987, pp. 93-109. Lacks, S. and Greenberg, B.: Complementary specificity of restriction endonucleases of Diplococcus pneumoniae with respect to DNA methylation. J. Mol. Biol. 144 (1977) 153-168. Leach, D.L. and Symonds, N.: The isolation and characterisation of a plaque-forming derivative of bacteriophage Mu carrying a fragment ofTn3 conferring ampicillin resistance. Mol. Gen. Genet. 172 (1979) 179-184. Marinus, M.G., Carraway, M., Frey, AZ., Brown, L. and Arraj, J.A.: Insertion mutations in the dnm gene of ,%c/rerichiu coli K-12. Mol. Gen. Genet. 192 (1983) 288-289. Seiler, A., Blocker, H., Frank, R. and Kahmann, R.: The mom gene of bacteriophage Mu: the mechanism of methylationdependent expression. EMBO J. 5 (1986) 2719-2728. Toussaint, A.: The DNA modification function of temperate phage Mu-l. Virology 70 (1976) 17-27. Toussaint, A., Desmet, L. and Faelen, M.: Mapping of the m~i~cation function of temperate phage Mu-l. Mol. Gen. Genet. 177 (1980) 351-353. Wulczyn, F.G. and Kahmann, R.: Post-transcriptional regulation of the bacteriophage Mu mom gene by the corn gene product. Gene 51 (1987) 139-147.
This work was supported by PHS Grant No. GM-29227.