[GG
Mol Gen Genet (1991) 226:297-304 002689259100105J
© Springer-Verlag 1991
The 41 carboxy-terminal residues of the miniF plasmid CcdA protein are sufficient to antagonize the killer activity of the CcdB protein Philippe Bernard and Martine Couturier Laboratoire de G~n~tique, D6partement de BiologieMol6culaire, Universit~ libre de Bruxelles, rue des Chevaux, 67, B-1640 Rhode Saint Gen~se, Belgium Received May 10, 1990 / November 9, 1990 Summary. The ccd operon of plasmid F encodes two genes, ccdA and ccdB, which contribute to the high stability of the plasmid by post-segregational killing of plasmid-free bacteria. The CcdB protein is lethal to bacteria and the CcdA protein is an antagonist of this lethal action. A 520 bp fragment containing the terminal part of the ccdA gene and the entire ccdB gene of plasmid F was cloned downstream of the tac promoter. Although the CcdB protein was expressed from this fragment, no killing of host bacteria was observed. We found that the absence of killing was due to the presence of a small polypeptide, CcdA41, composed of the 41 C-terminal residues of the CcdA protein. This polypeptide has retained the ability to regulate negatively the lethal activity of the CcdB protein. Key words: F plasmid- Stability functions - ccd mechanism
Introduction Bacterial low copy number plasmids are generally very stably inherited. This implies the existence of very precise mechanisms ensuring their stable maintenance. Mechanisms controlling plasmid replication (copy number), partition of replicas into daughter cells and resolution of multimers prevent plasmid loss. Low copy number plasmids often possess a fourth stability improving mechanism that involves killing of plasmid-free bacteria rather than prevention of plasmid loss. Loci responsible for killing bacteria that have lost a plasmid include the ccd (Jaff6 et al. 1985) and stm or Flm (Golub and Panzer 1988; Loh etal. 1988) loci of plasmid F, the parB (Gerdes et al. 1986) and parD (Bravo et al. 1988) loci of the R1 plasmid and the pem loci of the R100 plasmid (Tsuchimoto and Ohtsubo 1989). Offprint requests to: P. Bernard
The ccd locus has been shown to contribute to the high stability of plasmid F (Ogura and Hiraga 1983). This locus contains two genes, H and G, also called ccdA and ccdB, which encode proteins o f 72 and 101 residues, respectively (Bex et al. 1983 ; Miki et al. 1984a). These genes are organized in an operon together with the resolvase-encoding gene D (O'Connor et al. 1986; Lane et al. 1986). The ccdAam22 mutation, a miniF amber mutation causing the disappearance of the CcdA protein, renders miniF hybrids lethal to sup ° host cells. When an additional mutation prevents synthesis of the CcdB protein, the lethal phenotype is abolished (Karoui et al. 1983). These results and those of other laboratories (Ogura and Hiraga 1983; Miki etal. 1984b) have led to the conclusion that the CcdB protein is responsible for the killing of bacteria and that the CcdA protein negatively controls CcdB activity (directly or indirectly). In recA + bacteria, the CcdB protein causes the induction of the SOS functions and of lambdoid prophages (Karoui et al. 1983; Mori et al. 1984; Bai!one et al. 1984; Sommer et al. 1985). However, the CcdB protein exhibits killing activity in both recA + and r e c A - bacteria (Karoui etal. 1983; Ogura and Hiraga 1983). Miki etal. (1988) have shown that the product of the heat shock gene groES is required for CcdB protein-mediated killing. In this paper, we show that the 41 residue carboxyterminal portion of the CcdA protein is sufficient to antagonize the lethal activity of the CcdB protein. Materials and methods Bacterial strains, phages and plasmids. Escherichia coli
K12 strains, phages and plasmids are described in Table 1. Construction by in vitro recombination techniques of plasmid pULB2208, which overproduces the CcdB protein, is described in Fig. 1. Plasmid pULB2210 was constructed by digesting pULB2208 with Sinai and
298 Table 1. Bacterial strains, phages and plasmids
Description
Source or reference
Strains C600 594 N100 (MMt 52) QR48
thr-l, thi-1, leuB6, lacY1, tonA21, supE44 lac-3350, galK2, galT22, rpsL179, IN(rrnD-rrnE) l galk2, rpsL200, IN(rrnD-rrnE) l, recA3 recA, supE
Appleyard (1954) Campbell (1965) Gottesman and Yarmolinsky (1968) Signer and Weil (t 968)
Phages )~pSC138 2pSC138ccdAam22 2pSC138cop5 2pSC 138ccdAam22cop5
MiniF Apr plasmid cloned in the 2631 vector 2pSC138 with an amber mutation in the ccdA gene 2pSC138 copy number mutant 2pSC138 double mutant
Couturier et al. (1979) Karoui et al. (1983) Bex et al. (1986) This work
Apr expression vector with the strong tac promoter pBR322 Tcr cloning vector Tcr and Cmr cloning vector derived from plasmid P15A EcoRt f5 fragment deleted of the 43.61-46.9 kb KpnI miniF sequence and cloned into the EcoRI site of pKT279 EcoR1 f5 ccdAam22 fragment cloned into the EcoRI site of pKT279 EcoRI f5 ccdAam22 fragment deleted of the 43.6146.9 kb KpnI miniF sequence and cloned into the EcoRI site of pKT279 NdeI 43.1 to BglII 46.92 kb fragment (0.52 kb) ofpULB2015 cloned into the Sinai site of pKK223-3 43.1 kb NdeI to 46.92 kb BglII fragment (0.52 kb) of pULB2215 cloned into the SmaI site of pKK223-3 pULB2208 deleted of the 43.35 kb Sinai to 4.55 kb HindIII miniF/pKK223-3 sequence EcoRI f5cop5 fragment of 2pSC138cop5 cloned into the EcoRI site of the pKT279 cloning vector HincII 43.07 to PstI 43.6 kb fragment of pULB2015 cloned into the SmaI-PstI sites of pKK223-3 HincII 43.07 to PstI 43.6 kb fragment of pULB2215 cloned into the SmaI-PstI sites of pKK223-3 BamHI 42.84 to SalI 49 kb fragment ofpULB2015 cloned into the BamHI-SalI sites of pACYC184
Brosius and Holy (1984) Talmadge and Gilbert (1980) Chang and Cohen (1978) Karoui et al. (1983)
Plasmids pKK223-3 pKT279 pACYCI84 pULB2015 pULB2007 pULB2215 pULB2208 pULB2212 pULB2210 pULB2006 pULB2216 pULB2218 pULB2230
Karoui et al. (1983) This work This work This work This work Bex et al. (1986) This work This work This work
Ap, ampicillin; Tc, tetracycline; Cm, chloramphenicol
HindIII endonucleases, filling-in sticky ends with the Klenow fragment of D N A polymerase I and ligating with T4 D N A ligase. Plasmid pULB2215 was constructed by digestion of pULB2207 with KpnI and ligation with T4 D N A ligase. Phasmid 2pSC138ccdAam22cop5 was constructed by in vivo recombination between plasmid pULB2006 (pKTf5cop5, Bex et al. 1986) and phasmid 2pSC138ccdAam22 (Karoui et al. 1983). C600/ pULB2006 was infected at a multiplicity of 5 with 2pSC138ccdAam22. After 2 h, the bacterial lysate was centrifuged, its supernatant constituting a phasmid stock with some recombinant phasmid. The 2pSC138ccdAam22cop5 recombinant phasmids were selected on L broth supplemented with 150 gg of ampicillin after infection of the recA - supE strain QR48(2). Media, antibiotics and enzymes. L broth contains 10 g tryptone (Gibco, UK), 5 g yeast extract (Gibco, U K ) and 5 g NaC1 per 1. Antibiotics were used at the following concentrations to select for plasmid-carrying bacteria: 50 gg/ml ampicillin, 10 gg/ml tetracycline.
In vitro enzyme reactions with EcoRI, PstI, BamHI, NdeI, HindIII, KpnI, BgIII, SmaI, T4 D N A ligase and the Klenow fragment of D N A polymerase I were carried out according to manufacturers' instructions.
Preparation ofplasmid DNA. Plasmid D N A was purified as described by Clewell and Helinski (1969). For quick preparation of plasmid D N A , the method described by Birnboim and Doly (1979) was used. Transformation. Competent cells and transformation were performed as described by Lederberg and Cohen (1974). D N A sequencing. The EcoRI-PstI fragments of pULB2208 (ccdA) and pULB2212 (ccdAam22) were cloned into the polylinker of M 1 3 m p l I (Messing 1983) and sequenced by the dideoxy chain-termination procedure (Sanger et al. 1977), using c~[35S]dATP, a synthetic 17 nucleotide primer and a Sequenase kit obtained from the US Biochemical Corporation.
299
Results Cloning o f the ccdB gene
Sail
~ . BamHI \~Sall \Pstl \Hindlll
pULB2208
Fig. 1. Plasmid pULB2208 was constructed by cloning a 520 bp NdeI-BglII fragment of plasmid pULB2015 into the SmaI site of
the pKK223-3 expression vector. Plasmid pULB2015 is an hybrid between vector pKT279 and the EcoRI f5 fragment of the F plasmid (miniF) deleted of its central KpnI region. Plasmid pULB2015 was digested by NdeI and BgllI restriction endonucleases and a 520 bp fragment including the terminal part of the ccdA gene (A 1ccdA) and the entire ccdB gene was purified by electrophoresis in an agarose gel. The 520 bp fragment was blunt-ended with DNA polymerase I and cloned into the SmaI site of plasmid pKK223-3. The pKK223-3 expression vector, containing the strong tac promoter and the rrnBT1T2 ribosomal terminator allows strong expression of cloned genes without affecting the stability of the hostvector system
Table 2. Transformation rate of C600/ pULB2230, C600 and 594 strains by plasmids pKK223-3, pULB2208, pULB2212, pULB2216 and pULB2218
Plasmids
pKK223-3 pULB2208 pULB2212 pULB2216 pULB2218
The 520 bp NdeI-BglII fragment of miniF containing the carboxy-terminal part of the ccdA gene (ccdA gene deleted at the 5' region: deletion A 1) and the entire ccdB gene was cloned downstream from the tac promoter (DeBoer et al. 1983) in the pKK223-3 expression vector (Fig. 1). It was shown that the resulting pULB2208 recombinant plasmid synthesizes large amounts of a protein with the same molecular weight as the CcdB protein (tested in a mini-cell system, data not shown). However, when introduced by transformation into 2-sensitive or 2-1ysogenic strains C600 and 594, the plasmid does not kill transformed bacteria and fails to express the CcdB protein phenotype, including inhibition of cell division (filamentation) and induction of lambdoid prophage. Table 2 shows the rate of transformation of strains C600/pULB2230, C600 and 594 by plasmid pULB2208. Nucleotide sequence analysis of the ccdA gene (Fig. 2) reveals an open reading frame for a putative polypeptide (CcdA41) composed of the 41 carboxy-terminal amino acid residues of the CcdA protein. We considered two hypotheses which might explain the fact that pULB2208 is not lethal for host bacteria: (1) the plasmid pULB2208 expresses a polypeptide,
Relevant characteristics
Vector AlccdA+ccdB + AlccdAam22ccdB + A2ccdA+ccdB + zt2cedAam22ccdB +
Rate of transformation C600/pULB2230
C600
594
supE, ccd +
supE, ccd
sup°, ced-
1 1 1 1 1
1.1 1.3 1.2
Mol Gen Genet (1991) 226:297-304 002689259100105J
© Springer-Verlag 1991
The 41 carboxy-terminal residues of the miniF plasmid CcdA protein are sufficient to antagonize the killer activity of the CcdB protein Philippe Bernard and Martine Couturier Laboratoire de G~n~tique, D6partement de BiologieMol6culaire, Universit~ libre de Bruxelles, rue des Chevaux, 67, B-1640 Rhode Saint Gen~se, Belgium Received May 10, 1990 / November 9, 1990 Summary. The ccd operon of plasmid F encodes two genes, ccdA and ccdB, which contribute to the high stability of the plasmid by post-segregational killing of plasmid-free bacteria. The CcdB protein is lethal to bacteria and the CcdA protein is an antagonist of this lethal action. A 520 bp fragment containing the terminal part of the ccdA gene and the entire ccdB gene of plasmid F was cloned downstream of the tac promoter. Although the CcdB protein was expressed from this fragment, no killing of host bacteria was observed. We found that the absence of killing was due to the presence of a small polypeptide, CcdA41, composed of the 41 C-terminal residues of the CcdA protein. This polypeptide has retained the ability to regulate negatively the lethal activity of the CcdB protein. Key words: F plasmid- Stability functions - ccd mechanism
Introduction Bacterial low copy number plasmids are generally very stably inherited. This implies the existence of very precise mechanisms ensuring their stable maintenance. Mechanisms controlling plasmid replication (copy number), partition of replicas into daughter cells and resolution of multimers prevent plasmid loss. Low copy number plasmids often possess a fourth stability improving mechanism that involves killing of plasmid-free bacteria rather than prevention of plasmid loss. Loci responsible for killing bacteria that have lost a plasmid include the ccd (Jaff6 et al. 1985) and stm or Flm (Golub and Panzer 1988; Loh etal. 1988) loci of plasmid F, the parB (Gerdes et al. 1986) and parD (Bravo et al. 1988) loci of the R1 plasmid and the pem loci of the R100 plasmid (Tsuchimoto and Ohtsubo 1989). Offprint requests to: P. Bernard
The ccd locus has been shown to contribute to the high stability of plasmid F (Ogura and Hiraga 1983). This locus contains two genes, H and G, also called ccdA and ccdB, which encode proteins o f 72 and 101 residues, respectively (Bex et al. 1983 ; Miki et al. 1984a). These genes are organized in an operon together with the resolvase-encoding gene D (O'Connor et al. 1986; Lane et al. 1986). The ccdAam22 mutation, a miniF amber mutation causing the disappearance of the CcdA protein, renders miniF hybrids lethal to sup ° host cells. When an additional mutation prevents synthesis of the CcdB protein, the lethal phenotype is abolished (Karoui et al. 1983). These results and those of other laboratories (Ogura and Hiraga 1983; Miki etal. 1984b) have led to the conclusion that the CcdB protein is responsible for the killing of bacteria and that the CcdA protein negatively controls CcdB activity (directly or indirectly). In recA + bacteria, the CcdB protein causes the induction of the SOS functions and of lambdoid prophages (Karoui et al. 1983; Mori et al. 1984; Bai!one et al. 1984; Sommer et al. 1985). However, the CcdB protein exhibits killing activity in both recA + and r e c A - bacteria (Karoui etal. 1983; Ogura and Hiraga 1983). Miki etal. (1988) have shown that the product of the heat shock gene groES is required for CcdB protein-mediated killing. In this paper, we show that the 41 residue carboxyterminal portion of the CcdA protein is sufficient to antagonize the lethal activity of the CcdB protein. Materials and methods Bacterial strains, phages and plasmids. Escherichia coli
K12 strains, phages and plasmids are described in Table 1. Construction by in vitro recombination techniques of plasmid pULB2208, which overproduces the CcdB protein, is described in Fig. 1. Plasmid pULB2210 was constructed by digesting pULB2208 with Sinai and
298 Table 1. Bacterial strains, phages and plasmids
Description
Source or reference
Strains C600 594 N100 (MMt 52) QR48
thr-l, thi-1, leuB6, lacY1, tonA21, supE44 lac-3350, galK2, galT22, rpsL179, IN(rrnD-rrnE) l galk2, rpsL200, IN(rrnD-rrnE) l, recA3 recA, supE
Appleyard (1954) Campbell (1965) Gottesman and Yarmolinsky (1968) Signer and Weil (t 968)
Phages )~pSC138 2pSC138ccdAam22 2pSC138cop5 2pSC 138ccdAam22cop5
MiniF Apr plasmid cloned in the 2631 vector 2pSC138 with an amber mutation in the ccdA gene 2pSC138 copy number mutant 2pSC138 double mutant
Couturier et al. (1979) Karoui et al. (1983) Bex et al. (1986) This work
Apr expression vector with the strong tac promoter pBR322 Tcr cloning vector Tcr and Cmr cloning vector derived from plasmid P15A EcoRt f5 fragment deleted of the 43.61-46.9 kb KpnI miniF sequence and cloned into the EcoRI site of pKT279 EcoR1 f5 ccdAam22 fragment cloned into the EcoRI site of pKT279 EcoRI f5 ccdAam22 fragment deleted of the 43.6146.9 kb KpnI miniF sequence and cloned into the EcoRI site of pKT279 NdeI 43.1 to BglII 46.92 kb fragment (0.52 kb) ofpULB2015 cloned into the Sinai site of pKK223-3 43.1 kb NdeI to 46.92 kb BglII fragment (0.52 kb) of pULB2215 cloned into the SmaI site of pKK223-3 pULB2208 deleted of the 43.35 kb Sinai to 4.55 kb HindIII miniF/pKK223-3 sequence EcoRI f5cop5 fragment of 2pSC138cop5 cloned into the EcoRI site of the pKT279 cloning vector HincII 43.07 to PstI 43.6 kb fragment of pULB2015 cloned into the SmaI-PstI sites of pKK223-3 HincII 43.07 to PstI 43.6 kb fragment of pULB2215 cloned into the SmaI-PstI sites of pKK223-3 BamHI 42.84 to SalI 49 kb fragment ofpULB2015 cloned into the BamHI-SalI sites of pACYC184
Brosius and Holy (1984) Talmadge and Gilbert (1980) Chang and Cohen (1978) Karoui et al. (1983)
Plasmids pKK223-3 pKT279 pACYCI84 pULB2015 pULB2007 pULB2215 pULB2208 pULB2212 pULB2210 pULB2006 pULB2216 pULB2218 pULB2230
Karoui et al. (1983) This work This work This work This work Bex et al. (1986) This work This work This work
Ap, ampicillin; Tc, tetracycline; Cm, chloramphenicol
HindIII endonucleases, filling-in sticky ends with the Klenow fragment of D N A polymerase I and ligating with T4 D N A ligase. Plasmid pULB2215 was constructed by digestion of pULB2207 with KpnI and ligation with T4 D N A ligase. Phasmid 2pSC138ccdAam22cop5 was constructed by in vivo recombination between plasmid pULB2006 (pKTf5cop5, Bex et al. 1986) and phasmid 2pSC138ccdAam22 (Karoui et al. 1983). C600/ pULB2006 was infected at a multiplicity of 5 with 2pSC138ccdAam22. After 2 h, the bacterial lysate was centrifuged, its supernatant constituting a phasmid stock with some recombinant phasmid. The 2pSC138ccdAam22cop5 recombinant phasmids were selected on L broth supplemented with 150 gg of ampicillin after infection of the recA - supE strain QR48(2). Media, antibiotics and enzymes. L broth contains 10 g tryptone (Gibco, UK), 5 g yeast extract (Gibco, U K ) and 5 g NaC1 per 1. Antibiotics were used at the following concentrations to select for plasmid-carrying bacteria: 50 gg/ml ampicillin, 10 gg/ml tetracycline.
In vitro enzyme reactions with EcoRI, PstI, BamHI, NdeI, HindIII, KpnI, BgIII, SmaI, T4 D N A ligase and the Klenow fragment of D N A polymerase I were carried out according to manufacturers' instructions.
Preparation ofplasmid DNA. Plasmid D N A was purified as described by Clewell and Helinski (1969). For quick preparation of plasmid D N A , the method described by Birnboim and Doly (1979) was used. Transformation. Competent cells and transformation were performed as described by Lederberg and Cohen (1974). D N A sequencing. The EcoRI-PstI fragments of pULB2208 (ccdA) and pULB2212 (ccdAam22) were cloned into the polylinker of M 1 3 m p l I (Messing 1983) and sequenced by the dideoxy chain-termination procedure (Sanger et al. 1977), using c~[35S]dATP, a synthetic 17 nucleotide primer and a Sequenase kit obtained from the US Biochemical Corporation.
299
Results Cloning o f the ccdB gene
Sail
~ . BamHI \~Sall \Pstl \Hindlll
pULB2208
Fig. 1. Plasmid pULB2208 was constructed by cloning a 520 bp NdeI-BglII fragment of plasmid pULB2015 into the SmaI site of
the pKK223-3 expression vector. Plasmid pULB2015 is an hybrid between vector pKT279 and the EcoRI f5 fragment of the F plasmid (miniF) deleted of its central KpnI region. Plasmid pULB2015 was digested by NdeI and BgllI restriction endonucleases and a 520 bp fragment including the terminal part of the ccdA gene (A 1ccdA) and the entire ccdB gene was purified by electrophoresis in an agarose gel. The 520 bp fragment was blunt-ended with DNA polymerase I and cloned into the SmaI site of plasmid pKK223-3. The pKK223-3 expression vector, containing the strong tac promoter and the rrnBT1T2 ribosomal terminator allows strong expression of cloned genes without affecting the stability of the hostvector system
Table 2. Transformation rate of C600/ pULB2230, C600 and 594 strains by plasmids pKK223-3, pULB2208, pULB2212, pULB2216 and pULB2218
Plasmids
pKK223-3 pULB2208 pULB2212 pULB2216 pULB2218
The 520 bp NdeI-BglII fragment of miniF containing the carboxy-terminal part of the ccdA gene (ccdA gene deleted at the 5' region: deletion A 1) and the entire ccdB gene was cloned downstream from the tac promoter (DeBoer et al. 1983) in the pKK223-3 expression vector (Fig. 1). It was shown that the resulting pULB2208 recombinant plasmid synthesizes large amounts of a protein with the same molecular weight as the CcdB protein (tested in a mini-cell system, data not shown). However, when introduced by transformation into 2-sensitive or 2-1ysogenic strains C600 and 594, the plasmid does not kill transformed bacteria and fails to express the CcdB protein phenotype, including inhibition of cell division (filamentation) and induction of lambdoid prophage. Table 2 shows the rate of transformation of strains C600/pULB2230, C600 and 594 by plasmid pULB2208. Nucleotide sequence analysis of the ccdA gene (Fig. 2) reveals an open reading frame for a putative polypeptide (CcdA41) composed of the 41 carboxy-terminal amino acid residues of the CcdA protein. We considered two hypotheses which might explain the fact that pULB2208 is not lethal for host bacteria: (1) the plasmid pULB2208 expresses a polypeptide,
Relevant characteristics
Vector AlccdA+ccdB + AlccdAam22ccdB + A2ccdA+ccdB + zt2cedAam22ccdB +
Rate of transformation C600/pULB2230
C600
594
supE, ccd +
supE, ccd
sup°, ced-
1 1 1 1 1
1.1 1.3 1.2
