Gene, 110 (1992) 115-118 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00
115
GENE 06274
An Escherichia coli-Mycobacterium Shuttle cosmid vector, p M S C 1 (Recombinant DNA; Lawrist4; bacteriophage ~. or/; kanamycin resistance; Mycobacterium smegmatis; BCG; electroporation; genomic library)
Stephen Hinshelwood and Neil G. Stoker Bacterial Molecular Genetics Unit, Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, London WCI E 7HT (U.K.) Tel. (44-71)6368636 Received by K.F. Chater: 23 July 1991 Revised/Accepted: 6 October/7 October 1991 Received at publishers: 7 November 1991
SUMMARY
A shuttle cosmid vector, pMSC1, has been constructed which replicates in Escher/chia coli and Mycobacter/um smegmatis. The vector was mainly derived from the A ori cosmid, Lawrist4, and the Mycobacter/umfortuitum cryptic plasmid, pAL5000, which replicates in M. smegmatis and Mycobacter/um boris BCG. The vector contains two cos sites which facilitates library construction, unique BamHI and HindIII sites for cloning, and a kanamycin-resistance-encoding gene for selection in mycobacteria. After packaging, the vector sequences comprise 10.3 kb, so that the theoretical size limits for inserts are 30-42 kb. A genomic library from M. smegmatis was constructed in E. coil; clones from this library were transferred into M. smegmatis by electroporation, and back again to E. coli, without any apparent rearrangements. This vector will be useful in cloning genes encoding complex pathways in mycobacteria.
INTRODUCTION
The mycobacteria are Gram + bacteria characterized by their thick lipid-rich cell wall. A number of mycobacterial species have been associated with human disease; many, such as Mycobacter/um avium, are opportunistic pathogens which are generally problematical only in immunocompromised individuals. However, M. tuberculosis and M. leprae, the causative agents of tuberculosis and leprosy, respecCorrespondence to: Dr. N.G. Stoker, Bacterial Molecular Genetics Unit, Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, Keppel St., London WCIE 7HT (U.K.) Tel. (44-71)927 2425; Fax (44-71) 6374314. Abbreviations: BCG, Bacille Calmette-Gu~rin (Mycobacter/um); bp, base pair(s); efu, colony-forming unit(s); EtdBr, ethidium bromide; kb, kilobase(s) or 1000 bp; Km, kanamycin; M., Mycobacterium;nt, nucleotide(s); ori, orion of DNA replication; R, resistance/resistant; TES buffer, 1 M NaCl/20 mM "Iris- HC! pH 8.0/20 mM EDTA.
tively, are major pathogens. Indeed, with the spread of AIDS, the incidence of tuberculosis is rising (Elliott et al., 1990), and there is an urgent need for a greater understanding of the biology of these organisms. The study of mycobacterial molecular genetics has been revolutionized by the availability of plasmid cloning vectors. These have largely been based on replicons from the M. fortuitum plasmid pAL5000 (Snapper et al., 1988; Ranes et al., 1990; Stover et al., 1991), although vectors containing or/from the mycobacteriophage D29 (Lazraq et al., 1991) and the Corynebacter/um diphther/ae plasmid pNG2 (Radford and Hodgson, 1991) have been described. Another major step forward has been the use of electroporation, and the isolation of high frequency transforming strains of M. smegmatis (Snapper etal., 1990),. a fastgrowing nonpathogenic species which has been widely used as a model mycobacterium. We have constructed a shuttle cosmid vector to allow production of libraries containing large segments of cloned
116 DNA which can be introduced into mycobacteria. This will facilitate the cloning of complex pathways involving many closely-linked genes, such as may exist in siderophore synthesis or the synthesis of cell wall components. In addition, complementation of mutants for which there is no strong selection is a sh~pler process with large inserts, in that relatively few clones need to be screened. The phage ~, or/has been uscd in constructing a series of E. coli cosmid vectors, as yields of DNA from recombinant cosmids based on this or/are higher and more consistent than has been found with ColE1 or/-based vectors (Little and Cross, 1985). Improvements which have been described for these vectors include the insertion of transcriptional terminators on either side of the cloning sites to insulate the vector sequences from cloned DNA (Gibson et al., 1987), removal of DNA which cross-hybridizes to pBR322 (LoristX) and the insertion of an extra cos site to simplify the cloning protocol (Lawrist4). All these vectors carry the Km R gene from Tn5 which can be used as a selectable marker in mycobacteria.
BamHI BamHI | Hindlll
C08
N
h
e
~ /leo
pYUB12 oe
cat
BamH'l Hindlll
Hind~ll ) pal III t Ligase ©oe
N
h
e
~
EXPERIMENTAL AND DISCUSSION (a) Construction of pMSCI The basis for the E. coil replicon was the cosmid vector Lawrist4. This is a cointegrate of LoristX (a slight modification of Lorist2; Gibson et al., 1987; P.F.R, Little, personal communication) and pUCcos 1, a plasmid based on the pUC series (Vieira and Messing, 1982) in which the polylinker-containing PvuII fragment has been replaced by the ). cos region (PJ. de Jong, personal communication). In Lawrist4, pUCcosl has been integrated by homologous recombination at the single cos site of LoristX to produce a chimeric vector containing two cos sites (P.J. de Jong, personal communication; Fig. 1). The introduction of the second cos site facilitates cloning (see legend to Fig. 2). After packaging, the pUC sequences are lost, and the vector is identical to LoristX. The mycobacterial replicon was pAL5000 (Rauzier et 8.1., 1988) which was present in a shuttle plasmid, pYUBI2 (Snapper et al., 1988), and has been shown to replicate in M. smegmatis and M. bovis BCG (Lugosi et al., 1989). The strategy used is shown in Fig. 1. A H i n d l l l fragment from pYUBI2, containing all the pALS000-derived sequence, was cloned into the unique H i n d l l l site in Lawrist4 to produce pSRV2. Plasmid pMSC1 was generated by deleting the two small B a m H I fragments in pSRV2 by digestion followed by religation. Plasmid pMSCI contains unique B a m H l and H i n d I l l sites suitable for cloning, and is 13.1 kb. After linearization at the S e a l site, only the 10.3 kb of DNA between the two cos sites is packaged, so
Fig.1, Constructionof cosmid pMSCI, Plasmid pYUBI2(Snapper et al., 1988)was digested with Hindlll, and the fragmentcontaining
/~ _.
BamHl~
BamHI
Ligase foe
N h e ~
oo.4
~
BamH1"~
,.c,
13,1kb Z _ ~ . indll' ,-coRV
pAL5000 sequenceflanked by fd terminator sequences from plJ666 was purified by agarose gel electrophoresis. This fragment was ligated with Hindlll-digested Lawrist4, resultingin pSRV2, in which the asymmetric BaraHI site in the pYUBI2 fragment was close to the BamHl site in Lawrist4. Digestion of pSRV2 with BamHl followed by religation removedthe smallBamHl fragments,producingpMSCI. The derivationof the DNA is shown as follows: open, LoristX; shaded, pUCcosl; blackened, plJ666; thin line, pALS000.Open trianglesindicate transoriptional terminators which have been deliberately inserted to isolate vectors from transcription in cloned inserts. The terminators originated from E. coil trpA and rrnC genes (Lawrist4), and fd (pYUBI2). Small open circles, E. coil on'; small filled circles, mycobacterialon.. The phage Aere, ell, O and P genes and the PR promoter lie between the Nhel and BamHI sites in pMSCI. Their organization in the Lorist vectors has been shown in Gibson et al. (1987). All restriction enzymes were obtained from GibcoBRL (Paisley, Scotland).
117 1
2
3
4
5
6
7
8
9
10
kb --23 --9.6 --6.7 --4.5 --2.3 --2.0
-0.5
Fig. 2. Construction of an M. smegmatis genomic library in pMSCI. Lanes !-I0: BamHl digests of cosmid DNA from ten randomly-picked colonies electrophoresed on a 0.8% agarose gel stained with EtdBr. Methods. DNA was purified from M. smegmatis as described previously (ClarkCurtiss et al., 1985), except that the CsCl/EtdBr density gradient step was replaced by extraction with phenol/chloroform/isoamyl alcohol (100:100:4), followed by extraction with chloroform/isoamyl alcohol (100:4) and ethanol precipitation. The DNA was partially digested with Sau3A, size fractionated on 10-40% sucrose gradients in TES buffer, and centrifuged (25000 rpm for 17 h in a Beckman SW55Ti rotor). Fractions containing high molecular weight DNA were ethanol precipitated. Hasmid pMSCI was digested with Seal, treated with calf intestinal alkaline phosphatase (Boehringer-Mannheim, Lewes, England), and redigested with BamHl. M. smegmatis and pMSCI DNA were ligated together, packaged in vitro and an aliquot was plated in E. coli 1046 (Cami and Kourilsky, 1978) on Luria agar containing 40 pg Km/ml. A library of several thousand colonies was obtained. Packaging reagents were purchased from Amersham International pie (Amersham, England). Luria broth base was purchased from Gibco-BRL (Paisley, Scotland).
theoretically fragments of DNA between 30 and 42 kb can be cloned. The trpA and rrnC transcriptional terminators from Lawrist4 are intact, and protect the Aori from adventitious transcription from cloned inserts which might affect replication in E. coll. One fd-derived terminator from pYUB 12 remains adjacent to the HindIII cloning site, while that next to the BamHI site has been lost. Thus it is possible that transcription from sequences cloned into the BamHI site could affect replication in mycobacteria. The likelihood of this happening may be low, as sequencing of pAL5000 (Rauzier et ai., 1988) suggests that an open reading frame (ORF4) leads away from the BamHI site, and transcriptional terminator sequences at the end of this gene might reduce any potential problems. To test how well pMSCI functioned as a cosmid vector, a genomic library of several thousand clones was con-
structed from M. smegmatis DNA. Seventy-two clones were picked, and cosmid DNA was prepared, digested with BamHl and analysed by agarose gel electrophoresis. All clones contained high molecular weight inserts, and all but two yielded different restriction patterns (Fig. 2). The two which appeared to be identical may have resulted from inadvertently picking the same colony twice.
(b) Shuttling cosmids between Mycobacteriumsmegmatis and Escherichia coli To confirm that the cosmid would replicate in M. smegmatis, pMSC1 and two cosmids were electroporated into M. smegmatis mc2155 (Snapper etal., 1990) (Table I). Cosmid DNA was purified from Km R M. smegmatis colonies as described (Kado and Liu, 1981), and then reintroduced into E. coil Restriction digest patterns of the cosmids were identical to those seen before transfer to M. smegmatis (not shown). (c) Conclusions Plasmid pMSCI has the following useful properties. (1) It contains or/s from bacteriophage 2 and pAL5000, so that it will replicate in E. coli and in mycobacterial species including M. smegmatis and M. boris BCG. (2) It carries the Tn5 Km n gene, which functions in E. coli and mycobacte'ria. TABLE I Electroporation of pMSCI and cosmid clones into Mycobacterium smegmatis a Vectorb (ng)
Km a coloniesc
CfU/pgd
Relative efficiency of transformationc
no DNA a pMSC1 a (50) cos33 ~ (250) cos34 a (50)
0 181 39 4
7.2 x 103 3.2 × 102 1.6 × 102
1 0.18 0.09
M. smegmatis mc2 155 was grown in tryptone soya broth (Oxoid, Basingstoke, England) + 0.05% Tween-80 to A6000.8. The cells were washed in 10% glycerol, and resuspended in 10% glycerol in 1/200 of the original volume. The 200-pl aiiquots were pulsed at 12.5 kV/cm using a BioRad Gene Pulser in cuvettes with 0.2 cm electrode separation. The cells were diluted into 5 ml Luria broth and incubated at 37°C for 1 h with shaking, pelleted and resuspended in 300 #1 Luria broth. Then 150/~! was plated out on Luria agar containing 40/~g Km/ml. b Plasmid pMSCI is shown in Fig. 1. Clones cos33 and cos34 were constructed by inserting DNA from M. smegmatis which had been partially digested with Sau3A into the BamHI site of pMSCI, followed by in vitro packaging. c Total number of Km R colonies obtained from 150/~1 of culture. d Efficiency of transformation expressed as the number of Km R cfu per pg DNA. e Efficiency of transformation of cos33 and cos34 relative to pMSCI, taking their larger size (approximately fourfold) into consideration.
118 (3) Two convenient cloning sites are present: BamHl
and Hindlll. (4) Transcriptional terminators are present on both sides of the Hindlll site, and on the 2 ori side of the BamHI site to protect the or/s from adventitious transcription originating in cloned sequences. (5) It contains two cos sites, allowing simple preparation of vector arms. (6) When packaged, the vector is 10.3 kb, thus allowing cloning of fragments from 30 to 42 kb. (7) Cosmid clones have been shuttled from E. coil to M. smegmatis and back to E. coli without apparent rearrangement. (8) The ability to transfer large fragments of D N A into mycobacteria will help analysis of biosynthetic pathways which may involve large numbers of genes, not only from M. smegmatis, but also from species which grow slowly (e.g., M. tuberculosis), or are as yet uncultivable (e.g., M. leprae). It will also aid the complementation of mutants where there is no strong selection. (9) The nt sequences of all parts of the vector have been reported, and the predicted sequence of pMSC1 is available upon request.
ACKNOWLEDGEMENTS S.H. was supported by MRC studentship No. G77/9529, and by the British Leprosy Relief Association, LEPRA. N.G.S. was partly supported by the Overseas Development Administration. We would like to thank Jenny Thompson, Pieter de Jong, Peter Little and Koen de Smet for helpful discussions.
REFERENCES Cami, B. and Kourilsky, P.: Screeningof cloned recombinant DNA in bacteria by in situ colony hybridization.Nucleic Acids Res. 5 (1978) 2381-2390.
Clark-Curtiss, J.E., Jacobs, W.R., Docherty, M.A., Ritchie, L.R. and Curtiss III, R.: Molecular analysis of DNA and construction of genomic libraries of Mycobacterium leprae. J. Bacteriol. 161 (1985) 1093-1102. Elliott, A.M., Luo, N., Tembo, G., Halwiindi, B., Steenbergen, G., Machiels, L., Pobee, J., Nunn, P., Hayes, R.J. and McAdam, K.P.WJ.: Impact of HIV on tuberculosisin Zambia: a cross sectional study. Br. Med. J. 301 (1990) 412-415. Gibson, T.J., Coulson, A.R., Sulston, J.E. and Little, P.F.R.: Lorist2, a cosmid with transcriptionalterminators insulatingvector genes from interference by promoters within the insert: effecton DNA yield and cloned insert frequency. Gene 53 (1987) 275-281. Kado, C.I. and Liu, S.-T.: Rapid procedure for detection and isolation of large and smallplasmids. J. Bacteriol. 145 (1981) 1365-1373. Lazraq, R., Houssainiiraqui,M., Clavelseres,S. and David, H.L.: Cloning and expressionof the origin of replicationof mycobacteriophageD29 in Mycobacterium smegmatis. FEMS Microbiol. Lett. 80 (1991) 117-120.
Little, P.F.R. and Cross, S.H.: A cosmid vector that facilitatesrestriction enzyme mapping. Prec. Natl. Acad. Sci. USA 82 (1985) 3159-3163. Lugosi, L., Jacobs, W.R. and Bloom, B.R.: Genetic transformation of BCG. Tubercle 70 (1989) 159-170. Radford, A.J. and Hodgson, A.L.M.: Construction and characterization of a Mycobacterium-Escherichiacoli shuttle vector. Plasmid 25 (1991) 149-153. Ranes, M.G., Rauzier,J., Lagranderie, M., Gheorghiu, M. and Gicquel, B.: Functional analysis of pAL5000, a plasmid from Mycobacterium fortuitum: construction of a 'mini' mycobacterium-Escherichia coil shuttle vector. J. Bacteriol. 172 (1990) 2793-2797. Rauzier, J., Moniz-Pereira,J. and GicqueI-Sanzey, B.: Complete nucleotide sequenceof pAL5000, a plasmid from Mycobacterimnfortuitum. Gene 71 (1988) 315-321. Snapper, S.B., Lugosi, L., Jekkel, A., Melton, R.E., Kieser, T., Bloom, B.R. and Jacobs, W.R.: Lysogeny and transformation in mycobaeteria: stable expressionof foreign genes. Prec. Natl. Acad. Sci. USA 85 (1988) 6987-6991. Snapper, S.B., Melton, R.E., Mustafa, S., Kieser,T. and Jacobs, W.R.: Isolation and characterizationof efficientplasmidtransformationmutants of Mycobacterium smegmaas. Mol. Microbiol. 4 (1990) 19111919. Stover, C.K., de la Cruz, V.F., Fuerst, T.R., Budein, L.A., Bennett, L.T., Bansal, G.P., Young, J.F., Lee, M.H., Hatfull, G.F., Snapper, S.B., Barletta, R.G., Jacobs, W.R. and Bloom, B.R.: New use of BCG for recombinant vaccines. Nature 351 (1991) 456-460. Vieira, J. and Messing,J.: The pUC plasmids, an M 13mp7-derivedsystem for insertion mutagenesis and sequencingwith synthetic universal primers. Genc 19 (1982) 259-268.
115
GENE 06274
An Escherichia coli-Mycobacterium Shuttle cosmid vector, p M S C 1 (Recombinant DNA; Lawrist4; bacteriophage ~. or/; kanamycin resistance; Mycobacterium smegmatis; BCG; electroporation; genomic library)
Stephen Hinshelwood and Neil G. Stoker Bacterial Molecular Genetics Unit, Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, London WCI E 7HT (U.K.) Tel. (44-71)6368636 Received by K.F. Chater: 23 July 1991 Revised/Accepted: 6 October/7 October 1991 Received at publishers: 7 November 1991
SUMMARY
A shuttle cosmid vector, pMSC1, has been constructed which replicates in Escher/chia coli and Mycobacter/um smegmatis. The vector was mainly derived from the A ori cosmid, Lawrist4, and the Mycobacter/umfortuitum cryptic plasmid, pAL5000, which replicates in M. smegmatis and Mycobacter/um boris BCG. The vector contains two cos sites which facilitates library construction, unique BamHI and HindIII sites for cloning, and a kanamycin-resistance-encoding gene for selection in mycobacteria. After packaging, the vector sequences comprise 10.3 kb, so that the theoretical size limits for inserts are 30-42 kb. A genomic library from M. smegmatis was constructed in E. coil; clones from this library were transferred into M. smegmatis by electroporation, and back again to E. coli, without any apparent rearrangements. This vector will be useful in cloning genes encoding complex pathways in mycobacteria.
INTRODUCTION
The mycobacteria are Gram + bacteria characterized by their thick lipid-rich cell wall. A number of mycobacterial species have been associated with human disease; many, such as Mycobacter/um avium, are opportunistic pathogens which are generally problematical only in immunocompromised individuals. However, M. tuberculosis and M. leprae, the causative agents of tuberculosis and leprosy, respecCorrespondence to: Dr. N.G. Stoker, Bacterial Molecular Genetics Unit, Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, Keppel St., London WCIE 7HT (U.K.) Tel. (44-71)927 2425; Fax (44-71) 6374314. Abbreviations: BCG, Bacille Calmette-Gu~rin (Mycobacter/um); bp, base pair(s); efu, colony-forming unit(s); EtdBr, ethidium bromide; kb, kilobase(s) or 1000 bp; Km, kanamycin; M., Mycobacterium;nt, nucleotide(s); ori, orion of DNA replication; R, resistance/resistant; TES buffer, 1 M NaCl/20 mM "Iris- HC! pH 8.0/20 mM EDTA.
tively, are major pathogens. Indeed, with the spread of AIDS, the incidence of tuberculosis is rising (Elliott et al., 1990), and there is an urgent need for a greater understanding of the biology of these organisms. The study of mycobacterial molecular genetics has been revolutionized by the availability of plasmid cloning vectors. These have largely been based on replicons from the M. fortuitum plasmid pAL5000 (Snapper et al., 1988; Ranes et al., 1990; Stover et al., 1991), although vectors containing or/from the mycobacteriophage D29 (Lazraq et al., 1991) and the Corynebacter/um diphther/ae plasmid pNG2 (Radford and Hodgson, 1991) have been described. Another major step forward has been the use of electroporation, and the isolation of high frequency transforming strains of M. smegmatis (Snapper etal., 1990),. a fastgrowing nonpathogenic species which has been widely used as a model mycobacterium. We have constructed a shuttle cosmid vector to allow production of libraries containing large segments of cloned
116 DNA which can be introduced into mycobacteria. This will facilitate the cloning of complex pathways involving many closely-linked genes, such as may exist in siderophore synthesis or the synthesis of cell wall components. In addition, complementation of mutants for which there is no strong selection is a sh~pler process with large inserts, in that relatively few clones need to be screened. The phage ~, or/has been uscd in constructing a series of E. coli cosmid vectors, as yields of DNA from recombinant cosmids based on this or/are higher and more consistent than has been found with ColE1 or/-based vectors (Little and Cross, 1985). Improvements which have been described for these vectors include the insertion of transcriptional terminators on either side of the cloning sites to insulate the vector sequences from cloned DNA (Gibson et al., 1987), removal of DNA which cross-hybridizes to pBR322 (LoristX) and the insertion of an extra cos site to simplify the cloning protocol (Lawrist4). All these vectors carry the Km R gene from Tn5 which can be used as a selectable marker in mycobacteria.
BamHI BamHI | Hindlll
C08
N
h
e
~ /leo
pYUB12 oe
cat
BamH'l Hindlll
Hind~ll ) pal III t Ligase ©oe
N
h
e
~
EXPERIMENTAL AND DISCUSSION (a) Construction of pMSCI The basis for the E. coil replicon was the cosmid vector Lawrist4. This is a cointegrate of LoristX (a slight modification of Lorist2; Gibson et al., 1987; P.F.R, Little, personal communication) and pUCcos 1, a plasmid based on the pUC series (Vieira and Messing, 1982) in which the polylinker-containing PvuII fragment has been replaced by the ). cos region (PJ. de Jong, personal communication). In Lawrist4, pUCcosl has been integrated by homologous recombination at the single cos site of LoristX to produce a chimeric vector containing two cos sites (P.J. de Jong, personal communication; Fig. 1). The introduction of the second cos site facilitates cloning (see legend to Fig. 2). After packaging, the pUC sequences are lost, and the vector is identical to LoristX. The mycobacterial replicon was pAL5000 (Rauzier et 8.1., 1988) which was present in a shuttle plasmid, pYUBI2 (Snapper et al., 1988), and has been shown to replicate in M. smegmatis and M. bovis BCG (Lugosi et al., 1989). The strategy used is shown in Fig. 1. A H i n d l l l fragment from pYUBI2, containing all the pALS000-derived sequence, was cloned into the unique H i n d l l l site in Lawrist4 to produce pSRV2. Plasmid pMSC1 was generated by deleting the two small B a m H I fragments in pSRV2 by digestion followed by religation. Plasmid pMSCI contains unique B a m H l and H i n d I l l sites suitable for cloning, and is 13.1 kb. After linearization at the S e a l site, only the 10.3 kb of DNA between the two cos sites is packaged, so
Fig.1, Constructionof cosmid pMSCI, Plasmid pYUBI2(Snapper et al., 1988)was digested with Hindlll, and the fragmentcontaining
/~ _.
BamHl~
BamHI
Ligase foe
N h e ~
oo.4
~
BamH1"~
,.c,
13,1kb Z _ ~ . indll' ,-coRV
pAL5000 sequenceflanked by fd terminator sequences from plJ666 was purified by agarose gel electrophoresis. This fragment was ligated with Hindlll-digested Lawrist4, resultingin pSRV2, in which the asymmetric BaraHI site in the pYUBI2 fragment was close to the BamHl site in Lawrist4. Digestion of pSRV2 with BamHl followed by religation removedthe smallBamHl fragments,producingpMSCI. The derivationof the DNA is shown as follows: open, LoristX; shaded, pUCcosl; blackened, plJ666; thin line, pALS000.Open trianglesindicate transoriptional terminators which have been deliberately inserted to isolate vectors from transcription in cloned inserts. The terminators originated from E. coil trpA and rrnC genes (Lawrist4), and fd (pYUBI2). Small open circles, E. coil on'; small filled circles, mycobacterialon.. The phage Aere, ell, O and P genes and the PR promoter lie between the Nhel and BamHI sites in pMSCI. Their organization in the Lorist vectors has been shown in Gibson et al. (1987). All restriction enzymes were obtained from GibcoBRL (Paisley, Scotland).
117 1
2
3
4
5
6
7
8
9
10
kb --23 --9.6 --6.7 --4.5 --2.3 --2.0
-0.5
Fig. 2. Construction of an M. smegmatis genomic library in pMSCI. Lanes !-I0: BamHl digests of cosmid DNA from ten randomly-picked colonies electrophoresed on a 0.8% agarose gel stained with EtdBr. Methods. DNA was purified from M. smegmatis as described previously (ClarkCurtiss et al., 1985), except that the CsCl/EtdBr density gradient step was replaced by extraction with phenol/chloroform/isoamyl alcohol (100:100:4), followed by extraction with chloroform/isoamyl alcohol (100:4) and ethanol precipitation. The DNA was partially digested with Sau3A, size fractionated on 10-40% sucrose gradients in TES buffer, and centrifuged (25000 rpm for 17 h in a Beckman SW55Ti rotor). Fractions containing high molecular weight DNA were ethanol precipitated. Hasmid pMSCI was digested with Seal, treated with calf intestinal alkaline phosphatase (Boehringer-Mannheim, Lewes, England), and redigested with BamHl. M. smegmatis and pMSCI DNA were ligated together, packaged in vitro and an aliquot was plated in E. coli 1046 (Cami and Kourilsky, 1978) on Luria agar containing 40 pg Km/ml. A library of several thousand colonies was obtained. Packaging reagents were purchased from Amersham International pie (Amersham, England). Luria broth base was purchased from Gibco-BRL (Paisley, Scotland).
theoretically fragments of DNA between 30 and 42 kb can be cloned. The trpA and rrnC transcriptional terminators from Lawrist4 are intact, and protect the Aori from adventitious transcription from cloned inserts which might affect replication in E. coll. One fd-derived terminator from pYUB 12 remains adjacent to the HindIII cloning site, while that next to the BamHI site has been lost. Thus it is possible that transcription from sequences cloned into the BamHI site could affect replication in mycobacteria. The likelihood of this happening may be low, as sequencing of pAL5000 (Rauzier et ai., 1988) suggests that an open reading frame (ORF4) leads away from the BamHI site, and transcriptional terminator sequences at the end of this gene might reduce any potential problems. To test how well pMSCI functioned as a cosmid vector, a genomic library of several thousand clones was con-
structed from M. smegmatis DNA. Seventy-two clones were picked, and cosmid DNA was prepared, digested with BamHl and analysed by agarose gel electrophoresis. All clones contained high molecular weight inserts, and all but two yielded different restriction patterns (Fig. 2). The two which appeared to be identical may have resulted from inadvertently picking the same colony twice.
(b) Shuttling cosmids between Mycobacteriumsmegmatis and Escherichia coli To confirm that the cosmid would replicate in M. smegmatis, pMSC1 and two cosmids were electroporated into M. smegmatis mc2155 (Snapper etal., 1990) (Table I). Cosmid DNA was purified from Km R M. smegmatis colonies as described (Kado and Liu, 1981), and then reintroduced into E. coil Restriction digest patterns of the cosmids were identical to those seen before transfer to M. smegmatis (not shown). (c) Conclusions Plasmid pMSCI has the following useful properties. (1) It contains or/s from bacteriophage 2 and pAL5000, so that it will replicate in E. coli and in mycobacterial species including M. smegmatis and M. boris BCG. (2) It carries the Tn5 Km n gene, which functions in E. coli and mycobacte'ria. TABLE I Electroporation of pMSCI and cosmid clones into Mycobacterium smegmatis a Vectorb (ng)
Km a coloniesc
CfU/pgd
Relative efficiency of transformationc
no DNA a pMSC1 a (50) cos33 ~ (250) cos34 a (50)
0 181 39 4
7.2 x 103 3.2 × 102 1.6 × 102
1 0.18 0.09
M. smegmatis mc2 155 was grown in tryptone soya broth (Oxoid, Basingstoke, England) + 0.05% Tween-80 to A6000.8. The cells were washed in 10% glycerol, and resuspended in 10% glycerol in 1/200 of the original volume. The 200-pl aiiquots were pulsed at 12.5 kV/cm using a BioRad Gene Pulser in cuvettes with 0.2 cm electrode separation. The cells were diluted into 5 ml Luria broth and incubated at 37°C for 1 h with shaking, pelleted and resuspended in 300 #1 Luria broth. Then 150/~! was plated out on Luria agar containing 40/~g Km/ml. b Plasmid pMSCI is shown in Fig. 1. Clones cos33 and cos34 were constructed by inserting DNA from M. smegmatis which had been partially digested with Sau3A into the BamHI site of pMSCI, followed by in vitro packaging. c Total number of Km R colonies obtained from 150/~1 of culture. d Efficiency of transformation expressed as the number of Km R cfu per pg DNA. e Efficiency of transformation of cos33 and cos34 relative to pMSCI, taking their larger size (approximately fourfold) into consideration.
118 (3) Two convenient cloning sites are present: BamHl
and Hindlll. (4) Transcriptional terminators are present on both sides of the Hindlll site, and on the 2 ori side of the BamHI site to protect the or/s from adventitious transcription originating in cloned sequences. (5) It contains two cos sites, allowing simple preparation of vector arms. (6) When packaged, the vector is 10.3 kb, thus allowing cloning of fragments from 30 to 42 kb. (7) Cosmid clones have been shuttled from E. coil to M. smegmatis and back to E. coli without apparent rearrangement. (8) The ability to transfer large fragments of D N A into mycobacteria will help analysis of biosynthetic pathways which may involve large numbers of genes, not only from M. smegmatis, but also from species which grow slowly (e.g., M. tuberculosis), or are as yet uncultivable (e.g., M. leprae). It will also aid the complementation of mutants where there is no strong selection. (9) The nt sequences of all parts of the vector have been reported, and the predicted sequence of pMSC1 is available upon request.
ACKNOWLEDGEMENTS S.H. was supported by MRC studentship No. G77/9529, and by the British Leprosy Relief Association, LEPRA. N.G.S. was partly supported by the Overseas Development Administration. We would like to thank Jenny Thompson, Pieter de Jong, Peter Little and Koen de Smet for helpful discussions.
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