Mol Gen Genet (1992) 234:14-21 © Springer-Verlag 1992
Cloning and nucleotide sequence determination of twelve mutant dnaA genes of Escherichia coli Flemming G. Hansen, Sos Koefoed, and Tove Atlung Department of Microbiology, The Technical University of Denmark, DK-2800 Lyngby, Denmark Received November 19, 1991
Summary. Plasmids carrying different regions of the wild-type dnaA gene were used for marker rescue analysis of the temperature sensitivity of twelve strains carrying dnaA mutations. The different dnaA(Ts) mutations could be unambiguously located within specific regions of the dnaA gene. The mutant dnaA genes were cloned on pBR322-derived plasmids and on nucleotide sequencing by dideoxy chain termination the respective mutations were determined using M13 clones carrying the relevant parts of the mutant dnaA gene. Several of the mutant dnaA genes were found to have two mutations. The dnaA5, dnaA46, dnaA601, dnaA602, dnaA604, and dnaA606 genes all had identical mutations corresponding to an amino acid change from alanine to valine at amino acid 184 in the DnaA protein, close to the proposed ATP binding site, but all carried one further mutation giving rise to an amino acid substitution. The dnaA508 gene also had two mutations, whereas dnaA167, dnaA203, dnaA204, dnaA205, and dnaA21! each had only one. The pairs dnaA601/602, dnaA604/606, and dnaA203/204 were each found to have identical mutations. Plasmids carrying the different dnaA mutant genes intact were introduced into the respective dnaA mutant strains. Surprisingly, these homopolyploid mutant strains were found to be temperature resistant in most cases, indicating that a high intracellular concentration of the mutant DnaA protein can compensate for the decreased activity of the protein. Key words: dnaA mutations - Nucleotide sequencing Autosuppression
Introduction The dnaA gene was defined by the isolation and characterization of temperature-sensitive mutants that are defective specifically in the initiation of D N A replication in Correspondence to: F.G. Hansen
Escheriehia coli (Abe and Tomizawa 1971; Kohiyama et al. 1966; Carl 1970; Wechsler and Gross 1971 ; Beyersmann et al. 1974; Sevastopoulos et al. 1977). Some of these mutants, in particular strains carrying the dnaA46 allele, have been the subject of several studies, which have clearly indicated the importance of the dnaA gene product in the initiation of replication (e.g. Hirota et al. 1970). A possible involvement of the dnaA gene product in the regulation of replication has also been suggested (Hansen and Rasmussen 1977; Atlung et al. 1987; yon Meyenburg and Hansen 1987; Lobner-Olesen et al. 1989; Mahaffy and Zyskind 1990; Hansen et al. 1991b). The dnaA gene was precisely mapped by Hansen and von Meyenburg (1979), and the gene itself has been sequenced (E.B. Hansen et al. 1982, corrected by Ohmori et al. 1984) and found to encode a basic protein with a molecular mass of 52 500 daltons. The dnaA gene is autoregulated (Atlung et al. 1985; Braun et al. 1985; Kiicherer et al. 1986) and a DnaA-box situated between the two promoters was suggested to play a role in autoregulation (F.G. Hansen et al. 1982; Atlung et al. 1985). The concentration of DnaA protein is constant a different growth rates (Hansen et al. 1991a). The DnaA protein has been demonstrated to bind to the DnaA-boxes in the dnaA promoter region and to the four DnaA boxes in oriC in vitro (Fuller et al. 1984). In vivo footprinting experiments have demonstrated that three of the four DnaA-boxes in oriC on a plasmid are protected from methylation and thus occupied by DnaA protein during most of the cell cycle (Samitt et al. 1989). Mutations that suppress the temperature sensitivity of dnaA mutants, both intergenic (Atlung 1981, 1984) and intragenic (Kellenberger-Gujer et al. 1978; Eberle et al. 1989) have been isolated and characterized, and the intragenic suppressor mutants have been sequenced. The dnaA46(Cs) gene was found to carry four point mutations, all of which resulted in amino acid changes (Braun et al. 1987). The dnaA508(Cs) gene exhibited three point mutations, two of which resulted in amino acid changes. The third mutation in the dnaA508(Cs) mutant is of interest and reveals a change of the G U G start codon to
15 A U G , thus possibly affecting translational efficiency (Eberle et al. 1989). Furthermore, some dnaA(Ts) alleles have been sequenced. The dnaA46 gene was reported to carry one mutation, which was identical to one of those found in dnaA46(Cs); the dnaA167 gene also carried one mutation (Ohmori et al. 1984). Sequence determination of mutations affecting the activity of a protein provides a molecular approach to determine regions of functional importance. Another approach has been to determine the dnaA gene sequences from several different species, including several gramnegative (Skovgaard and Hansen 1987; Skovgaard 1990; Fujita et al. 1989) and gram-positive bacteria (Fujita et al. 1990). This analysis has demonstrated that the D n a A protein is conserved in large parts of the eubacterial kingdom (Ogasawara et al. 1990). DnaA proteins from the different bacterial species exhibit two highly conserved domains that are separated by a variable region. A comparison between the sequences from the closely related bacteria E. eoli, Salmonella typhimurium and Serratia marcescens shows that a N-terminal domain of 63 amino acids (aa) and an C-terminal domain of 333 aa, which are virtually identical, in all three species, are separated by regions of 71 aa, 70 aa and 69 aa, respectively, that show little conservation (Skovgaard and Hansen 1987). We have previously mapped various dnaA mutations to different domains of the gene (Hansen et al. 1984). In the present study we have extended this analysis by sequencing twelve different dnaA mutants in order to obtain more precise molecular insight into the nature of the mutations.
Table 1. Escheriehia coli strains
Materials and methods Bacterial strains and growth conditions. Bacterial strains used are listed in Table 1. LB medium (Miller 1972) was used routinely for growth and maintenance of bacteria. Construction o f plasmids carrying mutant dnaA genes. Plasmids carrying the wild-type and twelve mutant dnaA genes were constructed by two different methods as follows. The dnaA +, dnaA5, dnaA46, dnaA167, dnaA203, dnaA204, and dnaA205 genes were isolated from transducing k-phages carrying the respective alleles (Hansen et al. 1984). )~ D N A was cut with HindIII and XhoI and ligated to pBR322 cut with HindIII and SalI. The ligated D N A was used to transform strain CM987 and clones carrying the fragment with the dnaA gene were identified by restriction analysis using appropriate restriction enzymes. The HindIII-XhoI fragment bearing the dnaA gene also carries the r p m H gene and part of the rnpA gene. Therefore, the original plasmids were cut with ClaI to produce plasmids that carry only the dnaA gene and its intact promoter region. The plasmid carrying the dnaA + gene was designated pFHC539 (Fig. 1A). The dnaA211, dnaA508, dnaA601, dnaA602, dnaA604, and dnaA606 genes were isolated directly from EcoRI-NcoI restricted D N A from the mutant strains. As vector we used plasmid pFHC265 (Hansen et al. 1981), a pBR322 derivative carrying the asnA gene, which contains EcoRI and NcoI sites. After ligation the D N A was used to transform strain HC120, which carries the dnaN59(Ts) mutation. Temperature-resistant and ampicillin-resistant transformants were subjected to analysis with restriction enzymes. Positive clones carried a 2.5 kb EcoRI-NcoI fragment containing the intact dnaN gene and the 444 Cterminal amino acids of the dnaA gene. These clones were
Strain
Genotypea
Source, reference or construction
CM987
asnA31asnB32 thi-1 relA1 spoT1 recA 1 [K12metE46 trp-3 hiss4 thi-1 9alK2 lacY1 or lacZ4 mtl-1 ara-9 tsx-3 ton-1 rpsL8 or 9 supE44 ()~c+)] [b] dnaA5 [b] dnaA46 [b] dnaA205 [b] dnaA204 [b] dnaA203 [b] dnaA508 [b] dnaA167 [b] dnaA601 [b] dnaA606 [b] dnaA601 )~[b] dnaA602 )~[b] dnaA604 )~[b] dnaA606 )~K12 dnaN59 pyrE ilv met his rpsL8 or 9 B/r leu pro lac gal trp his at9 thyA Strr drm hss-K12 met dnaA211
von Meyenburg et al. 1982
CM906 CM75l CM752 CM753 CM754 CM755 CM903 CM905 CM2500 CM2507 CM2733 CM2735 CM2738 CM2740 HC120 WM440
a For genetic symbols see Bachmann (1990) b Genotype of strain CM906 given in brackets
Ilv + derivative of CM750, Hansen and von Meyenburg 1979 Hansen and yon Meyenburg 1979 Hansen and von Meyenburg 1979 Hansen and von Meyenburg 1979 Hansen and von Meyenburg 1979 Hansen and von Meyenburg 1979 Hansen and von Meyenburg 1979 Hansen and von Meyenburg 1979 Hansen et al. 1984 Hansen et al. 1984 Hansen et al. 1984 Hansen et al. 1984 Hansen et al. 1984 Hansen et al. 1984 Sakakibara and Mizukami 1980 Beyersmann et al. 1974
16
A
EC
P1
P2
E
Sp
PI
D
P1
Sc
EV
X/S
pFHC539 1000
dnaA
B
pTAC850 pTAC3306
tet
.............................................................................
pTAC3310
...............................
pTAC3337
.................................
,
...........................................................
pTAC3336
........................................................................................
pTAC3308
.................................................................
marker rescue have the following structure (see Fig. 1). Plasmid pTAC850: a 1492 bp EcoRI-XhoI fragment, carrying the dnaA gene except for 21 N-terminal codons, is positioned between the EcoRI and SalI sites of pBR322, p T A C 3 3 0 6 : p F H C 5 3 9 was resected with PvuII. pTAC3308: pFHC539 was resected with PvuI. pTAC3310: an 835 bp RsaI fragment was cloned in the EeoRV site of pBR322; a similar plasmid, pTAC3309, has the same RsaI fragment in the opposite orientation. pTAC3336 and pTAC3337 are derivatives of pTAC3309 and pTAC3310, respectively, which were resected with
SphI. C
dnaa5o8iZZZZZZZ~ZZZZZI~!ZEZZZZZZZZZZZZZ3 ..............................
dnaA5 dnaA46 dnaAO01
F(
I............................................................................................
',
',
"
Cloning and nucleotide sequence determination of twelve mutant dnaA genes of Escherichia coli Flemming G. Hansen, Sos Koefoed, and Tove Atlung Department of Microbiology, The Technical University of Denmark, DK-2800 Lyngby, Denmark Received November 19, 1991
Summary. Plasmids carrying different regions of the wild-type dnaA gene were used for marker rescue analysis of the temperature sensitivity of twelve strains carrying dnaA mutations. The different dnaA(Ts) mutations could be unambiguously located within specific regions of the dnaA gene. The mutant dnaA genes were cloned on pBR322-derived plasmids and on nucleotide sequencing by dideoxy chain termination the respective mutations were determined using M13 clones carrying the relevant parts of the mutant dnaA gene. Several of the mutant dnaA genes were found to have two mutations. The dnaA5, dnaA46, dnaA601, dnaA602, dnaA604, and dnaA606 genes all had identical mutations corresponding to an amino acid change from alanine to valine at amino acid 184 in the DnaA protein, close to the proposed ATP binding site, but all carried one further mutation giving rise to an amino acid substitution. The dnaA508 gene also had two mutations, whereas dnaA167, dnaA203, dnaA204, dnaA205, and dnaA21! each had only one. The pairs dnaA601/602, dnaA604/606, and dnaA203/204 were each found to have identical mutations. Plasmids carrying the different dnaA mutant genes intact were introduced into the respective dnaA mutant strains. Surprisingly, these homopolyploid mutant strains were found to be temperature resistant in most cases, indicating that a high intracellular concentration of the mutant DnaA protein can compensate for the decreased activity of the protein. Key words: dnaA mutations - Nucleotide sequencing Autosuppression
Introduction The dnaA gene was defined by the isolation and characterization of temperature-sensitive mutants that are defective specifically in the initiation of D N A replication in Correspondence to: F.G. Hansen
Escheriehia coli (Abe and Tomizawa 1971; Kohiyama et al. 1966; Carl 1970; Wechsler and Gross 1971 ; Beyersmann et al. 1974; Sevastopoulos et al. 1977). Some of these mutants, in particular strains carrying the dnaA46 allele, have been the subject of several studies, which have clearly indicated the importance of the dnaA gene product in the initiation of replication (e.g. Hirota et al. 1970). A possible involvement of the dnaA gene product in the regulation of replication has also been suggested (Hansen and Rasmussen 1977; Atlung et al. 1987; yon Meyenburg and Hansen 1987; Lobner-Olesen et al. 1989; Mahaffy and Zyskind 1990; Hansen et al. 1991b). The dnaA gene was precisely mapped by Hansen and von Meyenburg (1979), and the gene itself has been sequenced (E.B. Hansen et al. 1982, corrected by Ohmori et al. 1984) and found to encode a basic protein with a molecular mass of 52 500 daltons. The dnaA gene is autoregulated (Atlung et al. 1985; Braun et al. 1985; Kiicherer et al. 1986) and a DnaA-box situated between the two promoters was suggested to play a role in autoregulation (F.G. Hansen et al. 1982; Atlung et al. 1985). The concentration of DnaA protein is constant a different growth rates (Hansen et al. 1991a). The DnaA protein has been demonstrated to bind to the DnaA-boxes in the dnaA promoter region and to the four DnaA boxes in oriC in vitro (Fuller et al. 1984). In vivo footprinting experiments have demonstrated that three of the four DnaA-boxes in oriC on a plasmid are protected from methylation and thus occupied by DnaA protein during most of the cell cycle (Samitt et al. 1989). Mutations that suppress the temperature sensitivity of dnaA mutants, both intergenic (Atlung 1981, 1984) and intragenic (Kellenberger-Gujer et al. 1978; Eberle et al. 1989) have been isolated and characterized, and the intragenic suppressor mutants have been sequenced. The dnaA46(Cs) gene was found to carry four point mutations, all of which resulted in amino acid changes (Braun et al. 1987). The dnaA508(Cs) gene exhibited three point mutations, two of which resulted in amino acid changes. The third mutation in the dnaA508(Cs) mutant is of interest and reveals a change of the G U G start codon to
15 A U G , thus possibly affecting translational efficiency (Eberle et al. 1989). Furthermore, some dnaA(Ts) alleles have been sequenced. The dnaA46 gene was reported to carry one mutation, which was identical to one of those found in dnaA46(Cs); the dnaA167 gene also carried one mutation (Ohmori et al. 1984). Sequence determination of mutations affecting the activity of a protein provides a molecular approach to determine regions of functional importance. Another approach has been to determine the dnaA gene sequences from several different species, including several gramnegative (Skovgaard and Hansen 1987; Skovgaard 1990; Fujita et al. 1989) and gram-positive bacteria (Fujita et al. 1990). This analysis has demonstrated that the D n a A protein is conserved in large parts of the eubacterial kingdom (Ogasawara et al. 1990). DnaA proteins from the different bacterial species exhibit two highly conserved domains that are separated by a variable region. A comparison between the sequences from the closely related bacteria E. eoli, Salmonella typhimurium and Serratia marcescens shows that a N-terminal domain of 63 amino acids (aa) and an C-terminal domain of 333 aa, which are virtually identical, in all three species, are separated by regions of 71 aa, 70 aa and 69 aa, respectively, that show little conservation (Skovgaard and Hansen 1987). We have previously mapped various dnaA mutations to different domains of the gene (Hansen et al. 1984). In the present study we have extended this analysis by sequencing twelve different dnaA mutants in order to obtain more precise molecular insight into the nature of the mutations.
Table 1. Escheriehia coli strains
Materials and methods Bacterial strains and growth conditions. Bacterial strains used are listed in Table 1. LB medium (Miller 1972) was used routinely for growth and maintenance of bacteria. Construction o f plasmids carrying mutant dnaA genes. Plasmids carrying the wild-type and twelve mutant dnaA genes were constructed by two different methods as follows. The dnaA +, dnaA5, dnaA46, dnaA167, dnaA203, dnaA204, and dnaA205 genes were isolated from transducing k-phages carrying the respective alleles (Hansen et al. 1984). )~ D N A was cut with HindIII and XhoI and ligated to pBR322 cut with HindIII and SalI. The ligated D N A was used to transform strain CM987 and clones carrying the fragment with the dnaA gene were identified by restriction analysis using appropriate restriction enzymes. The HindIII-XhoI fragment bearing the dnaA gene also carries the r p m H gene and part of the rnpA gene. Therefore, the original plasmids were cut with ClaI to produce plasmids that carry only the dnaA gene and its intact promoter region. The plasmid carrying the dnaA + gene was designated pFHC539 (Fig. 1A). The dnaA211, dnaA508, dnaA601, dnaA602, dnaA604, and dnaA606 genes were isolated directly from EcoRI-NcoI restricted D N A from the mutant strains. As vector we used plasmid pFHC265 (Hansen et al. 1981), a pBR322 derivative carrying the asnA gene, which contains EcoRI and NcoI sites. After ligation the D N A was used to transform strain HC120, which carries the dnaN59(Ts) mutation. Temperature-resistant and ampicillin-resistant transformants were subjected to analysis with restriction enzymes. Positive clones carried a 2.5 kb EcoRI-NcoI fragment containing the intact dnaN gene and the 444 Cterminal amino acids of the dnaA gene. These clones were
Strain
Genotypea
Source, reference or construction
CM987
asnA31asnB32 thi-1 relA1 spoT1 recA 1 [K12metE46 trp-3 hiss4 thi-1 9alK2 lacY1 or lacZ4 mtl-1 ara-9 tsx-3 ton-1 rpsL8 or 9 supE44 ()~c+)] [b] dnaA5 [b] dnaA46 [b] dnaA205 [b] dnaA204 [b] dnaA203 [b] dnaA508 [b] dnaA167 [b] dnaA601 [b] dnaA606 [b] dnaA601 )~[b] dnaA602 )~[b] dnaA604 )~[b] dnaA606 )~K12 dnaN59 pyrE ilv met his rpsL8 or 9 B/r leu pro lac gal trp his at9 thyA Strr drm hss-K12 met dnaA211
von Meyenburg et al. 1982
CM906 CM75l CM752 CM753 CM754 CM755 CM903 CM905 CM2500 CM2507 CM2733 CM2735 CM2738 CM2740 HC120 WM440
a For genetic symbols see Bachmann (1990) b Genotype of strain CM906 given in brackets
Ilv + derivative of CM750, Hansen and von Meyenburg 1979 Hansen and yon Meyenburg 1979 Hansen and von Meyenburg 1979 Hansen and von Meyenburg 1979 Hansen and von Meyenburg 1979 Hansen and von Meyenburg 1979 Hansen and von Meyenburg 1979 Hansen and von Meyenburg 1979 Hansen et al. 1984 Hansen et al. 1984 Hansen et al. 1984 Hansen et al. 1984 Hansen et al. 1984 Hansen et al. 1984 Sakakibara and Mizukami 1980 Beyersmann et al. 1974
16
A
EC
P1
P2
E
Sp
PI
D
P1
Sc
EV
X/S
pFHC539 1000
dnaA
B
pTAC850 pTAC3306
tet
.............................................................................
pTAC3310
...............................
pTAC3337
.................................
,
...........................................................
pTAC3336
........................................................................................
pTAC3308
.................................................................
marker rescue have the following structure (see Fig. 1). Plasmid pTAC850: a 1492 bp EcoRI-XhoI fragment, carrying the dnaA gene except for 21 N-terminal codons, is positioned between the EcoRI and SalI sites of pBR322, p T A C 3 3 0 6 : p F H C 5 3 9 was resected with PvuII. pTAC3308: pFHC539 was resected with PvuI. pTAC3310: an 835 bp RsaI fragment was cloned in the EeoRV site of pBR322; a similar plasmid, pTAC3309, has the same RsaI fragment in the opposite orientation. pTAC3336 and pTAC3337 are derivatives of pTAC3309 and pTAC3310, respectively, which were resected with
SphI. C
dnaa5o8iZZZZZZZ~ZZZZZI~!ZEZZZZZZZZZZZZZ3 ..............................
dnaA5 dnaA46 dnaAO01
F(
I............................................................................................
',
',
"
