_Archives
Vi rology
Arch Virol (1992) 127:65-74
© Springer-Verlag 1992 Printed in Austria
Cloning of the thymidylate synthetase gene (thyPIG 3) from the Bacillus subtilis temperate phage IG 3 Ilda Santos and Herminiade Lencastre Centro de Tecnologia Quimica e Biot6gica (CTQB), Oeiras, and Faculdade de Ci~ncias e Tecnologia, Universidade Nova de Lisboa, Portugal Accepted April 24, 1992
Summary. The thyPIG3 gene from Bacillus subtilis bacteriophage IG3 was cloned in the plasmid pHV 33. Two recombinant plasmids, plSL 61 and plSL 62 carrying that gene are effective in transforming to thymine prototrophy both Escherichia coli (by complementation) and B. subtilis (by complementation and recombination). The comparison of cloned fragment containing the thyPIG 3 gene and the thyP 3 gene from phage (1)3T, by restriction analysis and DNA hybridization, suggests a strong homology between the two. The thyPIG 3 gene was mapped in this study in the central region of the IG 3 genome. Introduction The group III temperate phages of Bacillus subtilis ((P3T, p 11, SPR, H 2, IG 1, IG 3, IG4 and E) carry a thymidilate synthetase gene (thyP) being able to convert T h y - auxotrophs to prototrophy upon lysogeny. From the whole group III only SPI3 does not share this property (for a review, see [24]. The thyP genes from the group III phages have been cloned, so far, from ~p3T- gene thyP3 1-8, 9, 12, 19-21] and from H2 gene thyPH2 [-23]. The genes are functional in Escherichia coli and B. subtilis since they convert T h y - auxotrophs of these bacteria to prototrophy. The thyP 3 as well as the thyP from IG 3 (gene thyPIG 3) has a strong homology to the B. subtilis thyA gene. It can be distinguished phenotypically from the B. subtilis thyB gene, since the lysogens grow well at 46 °C, like the ThyA + T h y B - mutants, and not like the T h y A - ThyB + strains that do not grow at this high temperature [11, 16, 20, 21]. The IG 3 prophage, like the prophage ~p3T, can be integrated in the thy A region of the host which is distinct from the normal attachment site, at the terminus of the chromosome, between ilvA and kauA [17, 20, 21; I. Santos, unpubl, obs.]. The integration of the thyA locus is due to the homology between thyP and thyA.
66
Ilda Santos and Herminia de Lencastre
In the present paper we report the cloning of the IG 3 genome in the chimeric plasmid pHV 33 [ 18] and studies of the complementation and/or recombination of the cloned thyPIG3 gene. Comparison of the thyPIG3 and thyP3 genes was also carried out, as well as the mapping of the gene on the central region of the IG 3 genome. Materials and methods Bacteria and plasmids The bacterial strains and plasmids are listed in Table 1.
Media and methods Large scale plasmid preparation was carried out by standard methods [14]. Small scale DNA plasmid preparations were made as described [2]. The digestion of DNAs with restriction enzymes, ligation with T 4 ligase, phage DNA preparation and transformation of E. coli strains were according to standard procedures [14]. Transformation of B. subtilis strains Rec + and R e c - was by the Bott and Wilson method [3]. B. subtilis DNA was isolated by the Marmur method [15]. The selection for tetracycline resistance, ampicillin sensitivity was made on LA media [14] containing 25 ~tg tetracycline/ml or 100 gg ampicillin/ ml. Selection for thymine prototrophy was carried out in minimal medium, M 9 [14] for E. coli, and SP 1 [1] for B. subtilis. The restriction fragments were isolated from the agarose gels and purified with the Gene Clean-Bit 101 System, according to the manufacturer's instructions. The radioactive probes were prepared using the Multiprime DNA Labelling System (Amersham) and the
Table 1. Strains used in this work
Strain
B. subtilis 168T + QB 943 SL 1060 IGCg 316b E. eoli W3110 IGCg 6104 W3110 IGCg6106 IGCg6107
Genotype (ptasmid)
Plasmid marker a
Source and/or reference
prototrophic trpC2 ilvA2 thyB5 thyA pyrD 1 trpC 2 recE 4 spolIA 69 trpC2 thyB5 thyA pyrD 1 recE4
F. E. Young R.E. Dedonder [6] P.J. Piggot [13] this work
thyA F - recA 1 endA 1 gyrA96 thy-1 hsdR 17 (rk- mk +) supE44 retA 1 (pHV 33) thyA (pCAT 12) thyA (plSL61) thyA (plSL62
[14] TcrAprCm r
our lab. collection
thyP3 AprCm r [12] thyPIG3 TcrCm ~ this work thyPIG3 Tc~Cm~ this work
~r resistant to; Tc tetracycline, Ap ampicillin; Cm chloramphenicol b Strain IGCg 316 was constructed using strain QB 943 as recipient in transformation with DNA extracted from strain SL 1060. The transformants were selected for Ilv + R e c - . The recE 4 colonies were scored streaking them onto TBAB plates supplemented with 0.1 ~tg of mitomycin C/ml. They were sensitive to the drug [7]
thyPIG 3 from Bacillus subtilis phage IG 3
67
nucleotide [0~-32 P]dATP (Amersham) according to manufacturer's instructions. The transfer of DNA to the membranes (Gene Screen, NEN, DuPont) and hybridization were all standard procedures [14]. Results
Pst I segments of IG 3 DNA The size of the IG3 genome was estimated to be 132.8kb (--~ 88.5MDa) [4, 5]. The PstI cleavage pattern of IG 3 observed in the present work agrees with the one reported before [ 4 ] - over 30 fragments could be resolved. Cleavage of IG 3 DNA with Pst I does not abolish transforming activity of the thyPIG 3 gene (results not shown).
Isolation of TC r Thy + E. coli transformants Since the Pst I cloning site in the pHV 33 vector is in the ampicilfin resistance gene [18], a genomic bank of phage IG 3 was constructed in the shuttle vector pHV 33 [18] by ligation of the phage DNA restricted with PstI and the DNA vector also restricted with the same enzyme. The ligation samples were used to transform an E. coli thymine-requiring strain (W 3110) by selecting for tetracycline resistance and then for ampicillin sensitivity to score the clones. Colonies were also selected for thymine independence. The results are summarized in Table 2. The vector DNA was treated with alkaline phosphatase (AP) prior to ligation and the transformation data showed that this treatment was effective in increasing the number of clones containing inserts (50%) as compared to the ligation mixture prepared with the untreated vector (4%). By replica plating we observed that 2% of the TcrAp S recombinants showed a Thy + phenotype while the TcrApSThy - clones probably contained different sequences of the IG 3 genome. All the T h y - recombinant clones were kept for other cloning studies. When the primary selection was done to thymine prototrophy again by replica plating we found that almost 80% of the Thy + clones were also Tc r while the Thy +Tc S transformants probably have segregated the Tc r character or have lost the plasmid. Still by replica plating we verified that 99% o f the TcrApSThy + transformants were Cm r but when the selective pressure was done to Thy + only 25% of the Thy+TcrAp ~ were also Cm ~ (results not shown), meaning that the Cm ~ gene and the pC 194 origin of replication retained in the parental plasmid pHV 33 [18] was probably lost on the majority of the Thy + transformants. Since according to transformation data [11, 20, 21] the thyP 3 gene of phage
Vi rology
Arch Virol (1992) 127:65-74
© Springer-Verlag 1992 Printed in Austria
Cloning of the thymidylate synthetase gene (thyPIG 3) from the Bacillus subtilis temperate phage IG 3 Ilda Santos and Herminiade Lencastre Centro de Tecnologia Quimica e Biot6gica (CTQB), Oeiras, and Faculdade de Ci~ncias e Tecnologia, Universidade Nova de Lisboa, Portugal Accepted April 24, 1992
Summary. The thyPIG3 gene from Bacillus subtilis bacteriophage IG3 was cloned in the plasmid pHV 33. Two recombinant plasmids, plSL 61 and plSL 62 carrying that gene are effective in transforming to thymine prototrophy both Escherichia coli (by complementation) and B. subtilis (by complementation and recombination). The comparison of cloned fragment containing the thyPIG 3 gene and the thyP 3 gene from phage (1)3T, by restriction analysis and DNA hybridization, suggests a strong homology between the two. The thyPIG 3 gene was mapped in this study in the central region of the IG 3 genome. Introduction The group III temperate phages of Bacillus subtilis ((P3T, p 11, SPR, H 2, IG 1, IG 3, IG4 and E) carry a thymidilate synthetase gene (thyP) being able to convert T h y - auxotrophs to prototrophy upon lysogeny. From the whole group III only SPI3 does not share this property (for a review, see [24]. The thyP genes from the group III phages have been cloned, so far, from ~p3T- gene thyP3 1-8, 9, 12, 19-21] and from H2 gene thyPH2 [-23]. The genes are functional in Escherichia coli and B. subtilis since they convert T h y - auxotrophs of these bacteria to prototrophy. The thyP 3 as well as the thyP from IG 3 (gene thyPIG 3) has a strong homology to the B. subtilis thyA gene. It can be distinguished phenotypically from the B. subtilis thyB gene, since the lysogens grow well at 46 °C, like the ThyA + T h y B - mutants, and not like the T h y A - ThyB + strains that do not grow at this high temperature [11, 16, 20, 21]. The IG 3 prophage, like the prophage ~p3T, can be integrated in the thy A region of the host which is distinct from the normal attachment site, at the terminus of the chromosome, between ilvA and kauA [17, 20, 21; I. Santos, unpubl, obs.]. The integration of the thyA locus is due to the homology between thyP and thyA.
66
Ilda Santos and Herminia de Lencastre
In the present paper we report the cloning of the IG 3 genome in the chimeric plasmid pHV 33 [ 18] and studies of the complementation and/or recombination of the cloned thyPIG3 gene. Comparison of the thyPIG3 and thyP3 genes was also carried out, as well as the mapping of the gene on the central region of the IG 3 genome. Materials and methods Bacteria and plasmids The bacterial strains and plasmids are listed in Table 1.
Media and methods Large scale plasmid preparation was carried out by standard methods [14]. Small scale DNA plasmid preparations were made as described [2]. The digestion of DNAs with restriction enzymes, ligation with T 4 ligase, phage DNA preparation and transformation of E. coli strains were according to standard procedures [14]. Transformation of B. subtilis strains Rec + and R e c - was by the Bott and Wilson method [3]. B. subtilis DNA was isolated by the Marmur method [15]. The selection for tetracycline resistance, ampicillin sensitivity was made on LA media [14] containing 25 ~tg tetracycline/ml or 100 gg ampicillin/ ml. Selection for thymine prototrophy was carried out in minimal medium, M 9 [14] for E. coli, and SP 1 [1] for B. subtilis. The restriction fragments were isolated from the agarose gels and purified with the Gene Clean-Bit 101 System, according to the manufacturer's instructions. The radioactive probes were prepared using the Multiprime DNA Labelling System (Amersham) and the
Table 1. Strains used in this work
Strain
B. subtilis 168T + QB 943 SL 1060 IGCg 316b E. eoli W3110 IGCg 6104 W3110 IGCg6106 IGCg6107
Genotype (ptasmid)
Plasmid marker a
Source and/or reference
prototrophic trpC2 ilvA2 thyB5 thyA pyrD 1 trpC 2 recE 4 spolIA 69 trpC2 thyB5 thyA pyrD 1 recE4
F. E. Young R.E. Dedonder [6] P.J. Piggot [13] this work
thyA F - recA 1 endA 1 gyrA96 thy-1 hsdR 17 (rk- mk +) supE44 retA 1 (pHV 33) thyA (pCAT 12) thyA (plSL61) thyA (plSL62
[14] TcrAprCm r
our lab. collection
thyP3 AprCm r [12] thyPIG3 TcrCm ~ this work thyPIG3 Tc~Cm~ this work
~r resistant to; Tc tetracycline, Ap ampicillin; Cm chloramphenicol b Strain IGCg 316 was constructed using strain QB 943 as recipient in transformation with DNA extracted from strain SL 1060. The transformants were selected for Ilv + R e c - . The recE 4 colonies were scored streaking them onto TBAB plates supplemented with 0.1 ~tg of mitomycin C/ml. They were sensitive to the drug [7]
thyPIG 3 from Bacillus subtilis phage IG 3
67
nucleotide [0~-32 P]dATP (Amersham) according to manufacturer's instructions. The transfer of DNA to the membranes (Gene Screen, NEN, DuPont) and hybridization were all standard procedures [14]. Results
Pst I segments of IG 3 DNA The size of the IG3 genome was estimated to be 132.8kb (--~ 88.5MDa) [4, 5]. The PstI cleavage pattern of IG 3 observed in the present work agrees with the one reported before [ 4 ] - over 30 fragments could be resolved. Cleavage of IG 3 DNA with Pst I does not abolish transforming activity of the thyPIG 3 gene (results not shown).
Isolation of TC r Thy + E. coli transformants Since the Pst I cloning site in the pHV 33 vector is in the ampicilfin resistance gene [18], a genomic bank of phage IG 3 was constructed in the shuttle vector pHV 33 [18] by ligation of the phage DNA restricted with PstI and the DNA vector also restricted with the same enzyme. The ligation samples were used to transform an E. coli thymine-requiring strain (W 3110) by selecting for tetracycline resistance and then for ampicillin sensitivity to score the clones. Colonies were also selected for thymine independence. The results are summarized in Table 2. The vector DNA was treated with alkaline phosphatase (AP) prior to ligation and the transformation data showed that this treatment was effective in increasing the number of clones containing inserts (50%) as compared to the ligation mixture prepared with the untreated vector (4%). By replica plating we observed that 2% of the TcrAp S recombinants showed a Thy + phenotype while the TcrApSThy - clones probably contained different sequences of the IG 3 genome. All the T h y - recombinant clones were kept for other cloning studies. When the primary selection was done to thymine prototrophy again by replica plating we found that almost 80% of the Thy + clones were also Tc r while the Thy +Tc S transformants probably have segregated the Tc r character or have lost the plasmid. Still by replica plating we verified that 99% o f the TcrApSThy + transformants were Cm r but when the selective pressure was done to Thy + only 25% of the Thy+TcrAp ~ were also Cm ~ (results not shown), meaning that the Cm ~ gene and the pC 194 origin of replication retained in the parental plasmid pHV 33 [18] was probably lost on the majority of the Thy + transformants. Since according to transformation data [11, 20, 21] the thyP 3 gene of phage
