Jul 21, 1993 - Transposable elements are valuable tools for the genetic analysis of bacteria ... an antibiotic resistance gene, su13. ..... In J. J. McFadden (ed.) ...
JOURNAL
OF
Vol. 176, No. 2
BACTERIOLOGY, Jan. 1994, p. 535-539
0021-9193/94/$04.00+0 Copyright © 1994, American Society for Microbiology
Efficient Transposition in Mycobacteria: Construction of Mycobacterium smegmatis Insertional Mutant Libraries CHRISTOPHE GUILHOT,* ISABEL OTAL,t INGRID VAN ROMPAEY,t CARLOS MARTIN,t AND BRIGITTE GICQUEL
Unite de Genetique Mycobacterienne, Centre National de la Recherche Scientifique URA 1300, Institut Pasteur, F-75015 Paris, France Received 21 July 1993/Accepted
1
November 1993
The Tn6ll transposon was inserted into pCG63, a temperature-sensitive plasmid isolated from an Escherichia coli-mycobacterial shuttle vector which contains the pAL5000 and pUC18 replicons. The resulting plasmid, pCG79, was used to generate a large number of insertional mutations in Mycobacterium smegmatis. These are the first mycobacterial insertional mutant libraries to be constructed by transposition directly into a mycobacterium. No highly preferential insertion sites were detected by Southern blot analysis of the chromosomal DNAs isolated from the insertion mutants. Auxotrophic mutants with various phenotypes were isolated at a frequency ranging from 0.1 to 0.4%, suggesting that the libraries are representative. The pCG79 system thus seems to be a useful tool for the study of M. smegmatis genetics and may be applicable to other mycobacteria, such as the M. tuberculosis complex.
is incubated at 39°C (7). In this article, we describe the use of these plasmids for Tn611 transposon mutagenesis. Construction of pCG79. Plasmid pCG63 contains the entirety of pAL5000 and pUC18 and the kanamycin resistance (Kmr) gene from Tn903 (7, 13) (Table 1). It also contains a mutation(s) rendering it thermosensitive for replication or maintenance. The KMr gene was replaced with the pHP45fQ cassette containing a streptomycin-spectinomycin resistance (Str-Spcr) gene to give pCG76. The Tn6JJ transposon was then inserted at the BamHI site in pCG76 to give pCG79 (Fig.
Transposable elements are valuable tools for the genetic analysis of bacteria (1) and for the study of pathogenesis (5, 8, 17). The development of a transposon mutagenesis system for Mycobacterium spp. would facilitate the analysis of these organisms. A better understanding of this group of species is important because they include Mycobacterium tuberculosis and Mycobacterium leprae, which are the causative agents of infectious diseases found worldwide. Several transposable elements have been isolated from mycobacteria in the last few years (3, 4, 6, 10). One of them, Tn610, was isolated from Mycobacterium fortuitum (11). It contains two 880-bp-long IS6100 insertion sequences flanking an antibiotic resistance gene, su13. A derivative of Tn610, Tn611, in which sul3 is replaced by the kanamycin resistance gene of Tn903, has been constructed (11). By using a nonreplicative plasmid as the vector for transposon delivery, Tn611 has been shown to transpose in Mycobacterium smegmatis. However, only a few transposition events have been obtained with this method (1 to 20 transposition events per Rg of DNA transformed). The efficiency of transformation (105 M. smegmatis or Mycobacterium bovis BCG transformants per ,ug of DNA under optimal conditions by electroporation of a replicative plasmid) and the rapid loss of the plasmid allowed us to demonstrate the ability of Tn611 to transpose, but this method is not practical for obtaining a large number of transposition
1).
Transposition mediated by pCG79. pCG79 and pCG76 were separately introduced into M. smegmatis mc2155 by electroporation (7). Transformants were selected at 30°C on L agar plates containing kanamycin (20 [L/ml) for mc2155(pCG79) or streptomycin (20 ,ug/ml) for mc 155(pCG76). Independent KMr or Strr transformants were grown in 5 ml of 7H9 growth medium supplemented with the appropriate antibiotic (20 p.g/ml) for 72 h at 30°C. Cultures were then diluted 1:100 in antibiotic-free medium and incubated for 24 h at 39°C. The cultures were plated at various dilutions on L agar in the presence and absence of antibiotics, and Kmr and Strr colonies were scored after 5 days at 39°C. Cultures of strains containing pCG79 and pCG76 gave 3 x 103 and 9 x 102 colonies per ml, respectively, on selective plates and 7 x 10' and 4 x 108 colonies per ml, respectively, on nonselective plates. In this experiment, the plasmid containing the transposon is lost twofold less often than its parent, pCG76, from the M. smegmatis cells. The end product of Tn611 transposition was previously shown to be a cointegrate molecule resulting from the integration into the chromosome of the vector containing the transposon and one additional copy of the IS6100 sequence (Fig. 2) (11). To demonstrate that the KMr thermoresistant colonies obtained after transformation with pCG79 were the result of transposition events, they were examined by genomic Southern hybridization with an internal fragment of IS6100 as the probe (Fig. 3A). As expected, in 17 of 20 Kmr thermoresistant colonies analyzed, three fragments hybridizing with IS6100 were detected: two of them were the same size as the IS6100-
events.
We recently isolated derivatives of an Escherichia colimycobacteria shuttle vector derived from pAL5000, pCG59 and pCG63 (7). They are stably maintained in an M. smegmatis culture incubated at 30°C but are rapidly lost when the culture * Corresponding author. Mailing address: Unite de G6netique Mycobacterienne, URA1300 du CNRS, Institut Pasteur, 25 rue du Dr Roux, F-75015 Paris, France. Phone: (33) 1 45 68 88 77. Fax: (33) 1 45
68 88 43. t Present address: Departamento de Microbiologia, Facultad de Medicina, Universidad de Zaragoza, Domingo Miral s/n. 50009,
Zaragoza, Spain. t Present address: Laboratoire de Microbiologie, Universite Libre de Brusells, 1070 Brusells, Belgium. 535
536
NOTES
J. BACTERIOL.
TABLE 1. Strains, plasmids, and transposon Strain or genetic
Relevant characteristics
Reference
element
M. smegmatis mc2155 E. coli XL1-Blue
Plasmids pUC18
pHP45fl pIPC17 pCG63
pCG76 pCG79 Transposon Tn611
Efficient transformation supE44 hsdR17 recA1 endAl gyrA46 thi reLA1 lac F' [proAB+ lacIq lacZAM15
16 15
TnlO (Tetr)] ColEl replicon, Ampr ColEl replicon, Ampr Str-Spcr Derivative of pUC18 containing Tn611 Temperature-sensitive derivative of pAL5000, Kmr Derivative of pCG63, Str-Spcr Derivative of pCG76 containing Tn61, Str-Spcr Kmr Derivative of Tn6WO, Kmr
18 12 11 7
This work This work 11
containing fragments of pCG79, and the third was of variable size and corresponded to the duplication of an IS6100 element. The size of the variable IS6100-containing fragment was different for 11 of the 17 mutants. This suggests that the 17 mutants are the results of at least 11 independent transposition events. Hybridization of the 17 Kmr thermoresistant clones with pCG79 as the probe confirmed that the entire plasmid sequence was present in the chromosome in all the isolates. Several of the clones appeared to contain identical insertions. They could have resulted either from independent transposition events into a hot spot or more simply from division of cells in which transposition had occurred before plating. To determine whether there are hot spots for IS6100 transpositions, the transposition protocol described above was repeated several times. The location of the transposon differed from experiment to experiment; there was no evidence of a hot spot. Therefore, different isolates with the transposon in the same location were presumably siblings. In 3 of the 20 Kmr thermoresistant clones, only two copies of IS6100 were detected. This suggests that either (i) pCG79 had integrated into the chromosome by a mechanism different from that characterizing transposition of Tn611, (ii) pCG79 remained in the cells as extrachromosomal replicons, or (iii) deletions occurred after cointegrate formation. DNA from these clones was probed for hybridization with pCG79, and hybridizing sequences were detected in the chromosome. In one clone, a plasmid fragment of 12.3 kb was replaced by a single smaller fragment. This suggests that there had been a deletion(s) in the region containing the thermosensitive mycobacterial replicon of pCG79. This deletion could have occurred after cointegrate formation, leading to elimination of the third IS6100 copy and of a part of the 12.3-kb fragment. In the other two clones (one is shown in Fig. 3B), the plasmid fragment of 12.3 kb was replaced by two new fragments. This suggests that these two fragments are the junctions within the chromosome. Thus, in these two cases, plasmid pCG79 integrated into the chromosome by an unknown mechanism. This kind of recombination could be related to illegitimate recombination, which
has already been described for M. bovis BCG and M. smegmatis (9, 11). For all the Kmr clones from this experiment that were analyzed by Southern blot, no case of conservative transposition of Tn611 was observed. Construction of M. smegmatis insertion mutant libraries. To evaluate the randomness of Tn611 transposition into the chromosome of M. smegmatis, four independent insertional libraries were constructed as described above. A total of 26,700 Kmr thermoresistant clones randomly selected from these libraries were tested for auxotrophy (Table 2). They were replica plated at 39°C on minimal medium containing 0.2% glucose, 10 mM ammonia, and kanamycin (20 ,ug/ml). Seventyeight clones were unable to grow on this minimal medium, although they grew on rich medium. These clones were analyzed as described by Carlton and Brown (2) to determine their specific auxotrophic requirements (Table 2). Among the auxotrophs isolated, the following requirements were found: 15 for leucine, 9 for histidine, 3 for valine plus isoleucine, 5 for adenine, 1 for alanine, 4 for tryptophan, 2 for aspartic acid, 11 for vitamins (see Table 2), 11 for purines, 2 for tyrosine, 1 for pyrimidines, 1 for methionine, 1 for phenylalanine, and 1 for threonine. Hence, 14 different phenotypes were found among the 25 requirements tested, showing that the randomness of Tn611 insertion is high. Further analysis of the Ala - mutant showed that it displayed a leaky phenotype; after replating, it grew on minimal medium but more slowly than on the same medium supplemented with alanine (20 ,ug/ml). Several biosynthetic pathways for the synthesis of alanine in E. coli have been described (14). M. smegmatis may thus also have more than one alanine-biosynthetic pathway. If this is the case, the Ala- mutant described above could be affected in one of these pathways (maybe the most efficient), resulting in growth on minimal medium but at a much slower rate than the wild-type strain. An alternative explanation for this phenotype is that this mutant is affected in the regulation of alanine biosynthesis; the underexpression of a biosynthetic gene would explain the slow growth of this mutant on minimal medium. Fifteen auxotrophic mutants of library 1 were analyzed by genomic Southern blot analysis. All of these mutants contained three copies of IS6100, suggesting that all of them resulted from insertional mutations. The transposon seems to have inserted at the same location in four of the five Leu - mutants. Similarly, the three Val - Ile - mutants seem to have resulted from the same insertion event. The three His - and the three Ade - mutants have different insertions (data not shown). These results suggest that the 16 auxotrophic mutants identified in library 1 resulted from at least 11 different transposition events. The isolation of siblings is therefore not a major problem in this mutagenesis protocol. To assess the stability of insertion, we measured the reversion frequency for three auxotrophic mutants. These clones were cultivated in antibiotic-free medium until saturation. Dilutions of these cultures were then plated on minimal and rich media. After 5 days of growth, the number of colonies was scored. In each case, the reversion frequency was lower than 2 x 10-6 per cell. The reversion could be due to either (i) excision of the transposon or (ii) a suppressive mutation.
FIG. 1. Construction of plasmids pCG76 and pCG79. Transposon Tn611 was cloned into a derivative of pCG63 (7) containing an Str-Spcr gene. Amp, ampicillin resistance gene; Km, kanamycin resistance gene; Str/Sp, streptomycin-spectinomycin resistance gene; ori-myco, mycobacterial origin of replication; ori-E. coli, E. coli origin of replication. Only relevant restriction sites are shown.
Pstl digestion +
/
\
T4 poigmerese treatment
\
/Purified
2 kb
EcoRI-PstI fragment
T4 polgmerase treatment
\Xbal
Pstl
Pstl
BamHI digestion
Klenow treatment
8.6 kb EcoRi fragment
Kienow treatment
,nyco
ori-E.coli Str/Sp
'79
tkb
Psti
XbalI-
537
538
NOTES
J. BACTERIOL.
A
P 1 2 3 4 5 6 7 8 9 10
kb .12 .-a5 ...
2.8
-.42 .-
target DNA
-_0.9
A B
x
1.7
x
4
IS66 00a is duplicated
Postl
Psti Psti
Pstl PstI
Pst6l ==4
x
IS6 iI Ob
IS6 1 00a Km
vector
*X
Str/Sp IS6 100a
kb
B
probe IS6100
5.-
[IS6I Ob Is
duplicated ,,x
Psti A E
Pstl
IL.
IS6100b 2 kb
stl! stl
Km IS6100B
0.9 kb
Pti
IPti -W
NMI
Str/Sp
2.8 s
A
vector
IS6 IOOb
2mm1.7m-
varlable
FIG. 2. Replicative transposition of Tn611 from pCG79. The sequence of IS6100 is similar to those of elements of the IS6 family (11). In common with other members of this family, IS6100 has been shown to transpose by a replicative mechanism (11). One of the two copies of IS6100 is duplicated during the transposition process of Tn61l. It generates a cointegrate with three copies of IS6100. The two insertion sequences resulting from the duplication of one element are in the same orientation. The two possible results of transposition, depending on which IS6100 copy is duplicated, are shown. The dark line indicates the internal IS6100 fragment used as a probe in hybridization with genomic DNA of the insertional mutants. In PstI-digested DNA, three hybridizing bands are expected: two identical to those in the plasmid and a third one of different size in each independent clone. The diagram below shows these hybridizing bands as well as their respective sizes. They correspond to the bands shown in Fig. 3A. The use of pCG79 in its entirety as a probe reveals bands identical to the plasmid bands plus an extra band corresponding to the duplication of an insertion sequence. See the legend to Fig. 1 for abbreviations. Only the restriction sites used for the Southern analysis are indicated.
However, the frequency of reversion is very low, demonstrating that Tn611 is stable after transposition into the chromosome. In conclusion, a temperature-sensitive plasmid can be used to deliver Tn611 to M. smegmatis mc2155 and efficiently obtain a large number of different stable insertion mutants. Our data suggest that Tn611 inserts into the chromosome of M. smegmatis at many different locations, with an apparently high
1.2m0.9_-
FIG. 3. Southern blot hybridization analysis of 10 Tn6ll insertions in M. smegmatis mc2155. Chromosomal DNA was isolated from Kmr colonies at 39°C. DNA was digested with PstI, and the fragments were separated by agarose gel electrophoresis and probed With the radiolabeled HindIII-PstI internal fragment of IS6100 (A) or radiolabeled pCG79 (B). Neither PCG79 nor IS6100 hybridized to DNA from M. smegmatis mc2155 (data not shown). Lane P, pCG79 digested with PstI; lanes 1 to 10, DNA from 10 Km' colonies grown at 39°C.
degree of randomness. These libraries include numerous auxotrophic mutants. This temperature-sensitive plasmid combined with the Tn611 transposon allows transposon mutagenesis, which is a powerful tool for genetic analysis, in M. smegmatis. The value of this system for slow-growing mycobacteria such as M. bovis BCG and M. tuberculosis is under investigation. We hope that it can be used for the study of mycobacterial virulence factors and provide information for the design of new vaccines and new drugs.
NOTES
VOL. 176, 1994
TABLE 2. Auxotrophic types Transposon cl of % Auxotested trophs library
1
4,500
0.4
2 3
4,200 7,300
0.1 0.2
4
10,700
0.4
Auxotroph phenotypes identified' (no. of clones)
7.
5 Leu-, 3 His-, 3 Val- Ile-, 3 Ade1 Ala-, I Trp2 His-, 1 Asp5 Vit-, 3 Leu-, 3 Pur-, 2 Trp-, 2 His-, 1 Ade-, 2 ND 8 Pur-, 7 Leu-, 6 Vit-, 2 Tyr-, 2 His-, 1 Asp-, 1 Pyr-, 1 Met-, 1 Phe-, 1 Thr-, 1 Trp-, 1 Ade-, 9 ND
8.
aAbbreviations: Pur, purines (adenine and guanine); Pyr, pyrimidines (thymidine, uracil, and cytidine); Vit, vitamins (biotin, thiamine, inositol, calcium pantothene, and pyridoxine); ND, undetermined requirement.
9.
10.
11. 12.
We thank Shamila Nair and Alex Edelman for critical reading of the manuscript. I. Van Rompaey and C. Guilhot are recipients of IRSIA and Fondation Merieux fellowships, respectively. This work was supported by the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases.
13.
14.
REFERENCES
1. Berg, C. M., D. E. Berg, and E. A. Groisman. 1989. Transposable elements and the genetic engineering of bacteria, p. 879-925. In D. E. Berg and M. M. Howe (ed.), Mobile DNA. American Society for Microbiology, Washington, D.C. 2. Carlton, B. C., and B. J. Brown. 1981. Gene mutation, p. 222-242. In P. Gerhardt (ed.), Manual of methods for general bacteriology. American Society for Microbiology, Washington, D.C. 3. Cirillo, J. D., R. G. Barletta, B. R. Bloom, and W. R. J. Jacobs. 1991. A novel transposon trap for mycobacteria: isolation and characterization of IS1096. J. Bacteriol. 173:7772-7780. 4. Collins, C. M., and D. M. Stephens. 1991. Identification of an insertion sequence, IS1081, in Mycobacterium bovis. FEMS Microbiol. Lett. 83:11-16. 5. Fields, P. I., R. V. Swamsom, C. G. Haidiris, and F. Heifron. 1986. Mutants of Salmonella typhimurium that cannot survive within the macrophage are avirulent. Proc. Natl. Acad. Sci. USA 83:51895193. 6. Guilhot, C., B. Gicquel, J. Davies, and C. Martin. 1992. Isolation
15. 16.
17.
18.
539
and analysis of IS6120, a new insertion sequence from Mycobacterium smegmatis. Mol. Microbiol. 6:107-113. Guilhot, C., B. Gicquel, and C. Martin. 1992. Temperaturesensitive mutants of the Mycobacterium plasmid pAL5000. FEMS Microbiol. Lett. 98:181-186. Isberg, R. R., and S. Falkow. 1985. Genetic analysis of bacterial virulence determinants in Bordetella pertussis and the pathogenic yersinia. Curr. Top. Microbiol. Immunol. 118:1-11. Kanalpa, G. V., B. R. Boom, and W. R. J. Jacobs. 1991. Insertional mutagenesis and illegitimate recombination in mycobacteria. Proc. Natl. Acad. Sci. USA 88:5433-5437. Martin, C., M. Ranes, and B. Gicquel. 1990. Plasmids, antibiotic resistance, and mobile genetic elements in mycobacteria, p. 121138. In J. J. McFadden (ed.), Molecular biology of the mycobacteria. Academic Press, London. Martin, C., J. Timm, J. Rauzier, R. G6mez-Lus, J. Davies, and B. Gicquel. 1990. Transposition of an antibiotic resistance element in mycobacteria. Nature (London) 345:739-743. Prentki, P., and H. M. Krisch. 1984. In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29:303-313. Ranes, M. G., J. Rauzier, M. Lagranderie, M. Gheorghiu, and B. Gicquel. 1990. Functional analysis of pAL5000, a plasmid from Mycobacterium fortuitum: constrtiction of a "mini" Mycobacterium-Escherichia coli shuttle vector. J. Bacteriol. 172:2793-2797. Reitzer, L. J., and B. Magasanik. 1987. Ammonia assimilation and the biosynthesis of glutamine, glutamate, aspartate, asparagine, L-alanine, and D-alanine, p. 302-320. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington, D.C. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Snapper, S. B., R. E. Melton, S. Mustapha, T. Kieser, and W. R. J. Jacobs. 1990. Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol. Microbiol. 4:1911-1919. Taylor, R. K., C. Manoil, and J. J. Mekalanos. 1989. Broad-hostrange vectors for delivery of TnphoA: use in genetic analysis of secreted virulence determinants of Vibrio cholerae. J. Bacteriol. 173:7772-7780. Yanisch-Perron, C. J., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33:103-119.
