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Development of a conditional lethal system for a Streptomyces lividans strain and its use to investigate conjugative transfer in soil
FEMS Microbiology Ecology 38 (2001) 115^121
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Development of a conditional lethal system for a Streptomyces lividans strain and its use to investigate conjugative transfer in soil Sylvie Clerc-Bardin a , Fatma Karray b , Asa Frostegard a , Jean-Luc Pernodet b , Pascal Simonet a; * a
Laboratoire d'Ecologie Microbienne, UMR CNRS 5557, Baªt. Gre¨gor Mendel/La Doua Universite¨ Lyon I, 43 Boulevard du 11 novembre 1918, 69622 Villeurbanne Cedex, France b Laboratoire de Biologie et Ge¨ne¨tique Mole¨culaire, Institut de Ge¨ne¨tique et Microbiologie, UMR CNRS 8621, Baªt. 400, Universite¨ Paris-Sud, 91405 Orsay Cedex, France Received 21 May 2001 ; received in revised form 1 October 2001; accepted 3 October 2001 First published online 30 October 2001
Abstract In Streptomyces lividans, a conditional lethal mechanism was obtained by inactivation of gylB in the glycerol operon. The resulting strain was efficiently counter-selected on glycerol-containing medium but the mutation had no effect on the capacity of the strain to be maintained in non-sterile soil samples and to transfer derivatives of the genetic element pSAM2. However, despite the efficiency of the donor counterselection system, the transfer of a pSAM2 derivative from S. lividans to indigenous soil bacteria was not detected. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Lethal system; pSAM2; Streptomyces; Conjugation
1. Introduction Gram-positive bacteria belonging to actinomycetes and particularly to the genus Streptomyces are among the main natural producers of economically important secondary metabolites [1]. As a result, they were also among the ¢rst to bene¢t from recent advances in genetic engineering [2]. However, developing new and genetically modi¢ed strains (GMM) that could yield novel compounds in a more or less predictable way requires Streptomyces strains to be able to be transformed with e¤cient shuttle vectors that have as broad a host range as possible. For this purpose, the Streptomyces ambofaciens genetic element pSAM2 [3], which has the ability to replicate and integrate site-specifically [4] in various bacteria [5^7], was used extensively to develop a series of vectors adapted for engineering of industrial Streptomyces strains [8]. However, questions remain concerning the fate of such industrially exploited GMM if they were to be released
* Corresponding author. Tel. : +33 (4) 72 44 82 89; Fax: +33 (4) 72 43 12 23. E-mail address : [email protected] (P. Simonet).
into the environment [9]. The main concern is about their ability to spread recombinant DNA sequences to indigenous micro-organisms via gene transfer mechanisms mainly by conjugative transfer [10] when vectors such as pSAM2 derivatives are used. The conjugative properties of this element led to questions about the potential for its transfer under natural conditions. This potential transfer was supported by previous data on the e¤ciency of the conjugative transfer from donor to recipient strains co-inoculated into soil microcosms [11]. While strategies have been developed for some proteobacteria [12], similar mechanisms causing the controlled death of Gram-positive bacteria are less developed, or do not apply to Streptomyces [13], preventing any investigation of transfer of mobile genetic elements from Streptomyces to indigenous bacteria. In order to address the question of a potential transfer to the indigenous micro£ora, we developed a conditional lethal system for a Streptomyces lividans strain based on glycerol sensitivity obtained by inactivation of one of the genes involved in glycerol utilisation. The pathway for glycerol catabolism in Streptomyces coelicolor is determined by the glycerol-inducible, glucose-repressible glycerol operon (gyl CABX) [14]. The soluble glycerol kinase
0168-6496 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 9 6 ( 0 1 ) 0 0 1 8 3 - 0
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(GK) encoded by the gylA gene and the membrane-associated glycerol-3-phosphate dehydrogenase (G3PDH) encoded by the gylB gene are essential for growth on media containing glycerol as the sole carbon source [15,16]. The absence of G3PDH activity in mutant strains presenting a normal GK activity leads to glycerol sensitivity probably due to the lethal e¡ect of glycerol-3-phosphate accumulation [15,16]. In S. lividans, the organisation and regulation of the gyl operon are essentially the same as those in S. coelicolor [17]. In this article, we describe the inactivation of the gylB gene of S. lividans TK21, various controls to check the e¤ciency of the conditional lethal mechanism, and preliminary results about e¤ciency of the transfer of a pSAM2 derivative element to the indigenous soil micro£ora. 2. Materials and methods 2.1. Bacterial strains, plasmids, growth conditions and DNA manipulations The bacterial strains and plasmids used in this study are listed in Table 1. Molecular biology techniques for Streptomyces spp. and Escherichia coli were as described respectively by Hopwood et al. [18] and Maniatis et al. [19]. Restriction enzymes, T4 DNA ligase and DNA polymerase I Klenow fragment were purchased from Roche Diagnostics (Meylan, France) and were used according to the manufacturer's recommendations. PCR ampli¢cations were conducted according to standard procedures with primers P1 (5P-GGTGACGGCGGCCTCCACTGGGACG-3P) and P2 (5P-GGTGGGTGCAGTTCTCGACGGTGCG-3P) complementary to part of the int gene encoding the integrase of pSAM2 and DNA extracted from growing colonies. Hybridisation conditions were as described previously [14] with radioactive labeling of the plasmid probe using [32 P]dCTP (3000 Ci mmol31 , Amersham, Les Ulis, France) and a random primed kit (Boehringer Mannheim, Meylan, France). After hybridisation of ¢lters at 65³C for 15 h the washing conditions included two changes of 2USSC solution at room temperature for 5 min, then two changes of 2USSC, 1% SDS solution at 65³C for 20 min. Standard culture techniques were as described by Hopwood et al. [18] for Streptomyces spp. strains which were cultured in liquid yeast extract-malt extract sucrose [20] or tryptic soy broth (Difco) media and on plates with the R2YE [21] or tryptic soy agar (Difco) solid media. The glycerol-arginine (GA) medium [22] containing 1.25% glycerol was used to counter-select the strain OS48.3 (see below) and to isolate actinomycetes from soil. In order to verify that glycerol had a toxic e¡ect on S. lividans OS48.3, media similar to GA were used in which glycerol was replaced by arabinose (1%) or in which these two carbon
sources were both present. The glycerol-sensitive OS48.3 strain was frozen in a solution containing 20% glycerol. Before utilisation of frozen OS48.3 spores, they were centrifuged and the pellet washed once with water to eliminate glycerol. Concentrations of antibiotics in selective media were as follows: ampicillin, 100 Wg ml31 ; gentamicin, 20 Wg ml31 for E. coli and 50 Wg ml31 for S. lividans; hygromycin, 50 Wg ml31 ; nosiheptide, 25 Wg ml31 ; spectinomycin, 50 Wg ml31 ; streptomycin, 50 Wg ml31 . Fungicides were also added in the media to inhibit the growth of fungi when soil suspensions were plated : cycloheximide, 200 Wg ml31 and benomyl, 50 Wg ml31 . 2.2. In vitro conjugation experiments In vitro conjugation experiments were as described by Clerc and Simonet [11]. Brie£y, an inoculum containing 107 spores of both the S. lividans OS48.3 donor strain carrying pTS130 and the S. lividans TK23 recipient strain was plated onto R2YE medium and incubated for 4 days at 28³C. The spores were then harvested and spread onto selective media containing gentamicin, spectinomycin or spectinomycin and nosiheptide for the selection of respectively the donor, the recipient and the transconjugant strains. 2.3. Soil microcosm experiments A silt loam soil collected at Montrond (France) was airdried, sieved at 2 mm and stored at 4³C until subsequent use. Soil characteristics were as follows : clay 31.4%, silt 36.4%, sand 32.2%, C/N 7.6, pH (water) 6.7 and a ¢eld capacity of 40 g of water for 100 g of dry soil. Soil microcosms consisted of 5 g equivalent dry soil in 10-ml polypropylene tubes. The soil moisture was adjusted to that of the ¢eld capacity and soil microcosms were incubated at 28³C for 5^7 days before inoculation. Experiments to establish population dynamics in soil of the various Streptomyces spp. strains were done by inoculating the soil microcosms with 107 spores per g of dry soil, establishing a ¢nal moisture content equivalent to the ¢eld capacity [11]. Then, the soil microcosms were incubated at 28³C in darkness and three of them were periodically taken for enumeration of the targeted bacteria [11]. In experiments to investigate the in situ transfer of pTS135 from the S. lividans OS48.3 donor strain to the indigenous soil micro£ora, a similar protocol was applied, the only exception being that the level of the inoculum reached 108 spores per g of dry soil and uninoculated microcosms were used as controls. Experiments were conducted in triplicate with disruption of the microcosms 0, 7 and 14 days after inoculation for analyses of donor, actinomycete and transconjugant population levels on the media de¢ned previously.
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Table 1 Bacterial strains and plasmids Strain or plasmid Strains E. coli DH5K S. lividans TK21 S. lividans TK23 S. lividans TK24 Plasmids pIJ2204 pHP456aac pUC4 pOS48.1 pOS48.2 pGM160v
Descriptiona
Reference
SpcR StrR
[32] [32] [32]
ApR , 5.7-kb BamHI-BamHI fragment of glycerol operon carrying gylA, gylB and gylX genes cloned into the BamHI site of pBR322 ApR , GenR ApR ApR , 5.7-kb BamHI-BamHI fragment of pIJ2204 cloned into BamHI site of pUC4 ApR , GenR ; 6aac cassette encoding Gen resistance cloned into PstI-SphI-digested plasmid pOS48.1 Temperature-sensitive replicon derived from pGM160; AmpR in E. coli and NosR in S. lividans
pTS130
ApR , GenR in E. coli and NosR , GenR in S. lividans; EcoRI-EcoRI insert of pOS48.2 cloned into EcoRI site of pGM160v NosR
pTS135
HygR
pTS39
NosR
pOS48.3
a
[33] [25] [24] This study This study [26] and this study This study Sezonov (unpublished) Sezonov (unpublished) [34]
Ap: ampicillin ; Gen : gentamicin; Hyg: hygromycin; Nos : nosiheptide; Spc: spectinomycin ; Str: streptomycin.
3. Results and discussion 3.1. Inactivation of the gylB gene of S. lividans TK21 The 5.7-kb BamHI insert of plasmid pIJ2204 [23] contains most of the gylCABX operon but lacks the gyl promoter region. This insert was cloned as a BamHI fragment into pUC4 [24], digested with BamHI, generating plasmid pOS48.1 (Fig. 1). This plasmid possesses two PstI sites, both located in the 5P end of gylB, and a single SphI site located in the 3P end of gylB. The pOS48.1 vector was digested with PstI and SphI and the extremities were blunted with the Klenow enzyme. The two fragments internal to gylB and released by this digestion were replaced by a HindIII fragment blunt-ended with the Klenow enzyme which contained the 6aac cassette [25]. The 6aac cassette confers resistance to gentamicin in E. coli and in S. lividans. Digestion of pOS48.2 with EcoRI released the complete insert, which was cloned into the EcoRI site of pGM160v, a shuttle vector whose replication is temperature-sensitive in Streptomyces spp., to generate pOS48.3. The pGM160 vector which confers nosiheptide resistance was in turn derived from pGM160 [26] by removing the HindIII fragment containing the aacC1 gene. The pOS48.3 vector was used to transform protoplasts of S. lividans TK21. One clone which harboured pOS48.3, and was therefore resistant to nosiheptide and gentamicin, was grown in TSB with nosiheptide for 2 days at 30³C (permissive temperature for pOS48.3 replication) and then used to inoculate drug-free TSB. This culture was grown for 3 days at 39³C (non-permissive for pOS48.3 replication) before samples were diluted and plated on TSB with and without gentamicin. About 20% of the colony-form-
ing units (CFU) were resistant to gentamicin. Among about 100 such clones which were tested for resistance to nosiheptide, ¢ve were found to be sensitive to nosiheptide. These clones had presumably lost the pGM160v moiety of pOS48.3 via a double crossover recombination event. This was con¢rmed in hybridisation analyses, when Southern blots of total genomic DNA from one of these clones and from strain TK21 were probed with the pOS48.3 plasmid or the 6aac cassette, and compared (results not shown). Among the ¢ve clones in which a double homologous recombination event had resulted in replacement of the gylB gene of the S. lividans chromosome by its disrupted counterpart, one was chosen for further study and designated S. lividans OS48.3. 3.2. E¤ciency of the killing system The mutant OS48.3 and parental TK21 S. lividans strains were plated on GA medium [22] containing 1.25% glycerol. This medium is the one used routinely for the isolation of actinomycetes from soil samples. Clearly, S. lividans OS48.3, harbouring the disrupted gylB gene, was unable to grow on GA medium (only a very thin transparent overlay was observed) whereas the wild-type strain displayed normal growth on the same medium (Fig. 2A). In order to determine if the mutant strain OS48.3 was unable to utilise glycerol as a carbon source or whether it was really sensitive to glycerol, the same strains were plated on media containing an alternative carbon source (arabinose) with or without glycerol. On a medium identical to GA, except for replacement of glycerol with arabinose, both strains presented similar growth kinetics and development (Fig. 2B). On GA medium containing
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Fig. 1. Construction of S. lividans OS48.3. The plasmids pOS48.1, pOS48.2 and pOS48.3 are presented. Only the insert, not the vector part, of these plasmids is drawn to scale. Double homologous recombination between pOS48.3 and the chromosome of S. lividans TK21, generating strain OS48.3, is schematically represented. The sizes of the XhoI fragments revealed in hybridisation experiments whose sizes di¡er in both S. lividans strains are indicated.
arabinose in addition to glycerol, the mutant strain OS48.3 was unable to grow when the parental strain grew normally. This demonstrated that strain OS48.3 was not only unable to utilise glycerol but was also sensitive to glycerol. Activation of the glycerol operon in the absence of the gylB gene product was lethal to S. lividans OS48.3. Interestingly, for all the experiments conducted with inocula containing 108 spores or mycelial fragments
from this strain, we never observed the development of an OS48.3 colony on the GA medium. This indicates that the probability of escaping the counter-selection system is far below 1038 . This result makes the lethal system very e¤cient compared to other methods previously described for some proteobacteria [12]. For instance, various plasmidbearing killing genes including hok, gef and rel cloned under inducible promoters lacked e¤ciency due to the
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this study does not depend on stable maintenance of plasmids or expression of foreign genes. Moreover, the fact that the lethal activity was obtained by replacement of a gene by its disrupted and deleted homologue prevented inactivation of the killing mechanism by a simple reversion event. Any mutation in other genes of the glycerol operon, which would prevent accumulation of glycerol-3-phosphate, would abolish the lethal e¡ect of glycerol. However, as glycerol is the major carbon source in the GA medium, such a mutant would not grow or would only grow very poorly on this medium and this would therefore have no consequences for the e¤ciency of the counter-selection. 3.3. Conjugation and soil colonisation properties of the mutant strain Controls were performed to demonstrate that the mutation in the gylB gene had not a¡ected two important properties of the strain which are essential for future studies of gene transfer to indigenous soil micro£ora: conjugation capacities and non-sterile soil colonisation ability. In vitro transfer rates, expressed as the number of transconjugants per potential recipient genotype (U100), were similar to those observed with the wild-type strain [11] reaching 90^ 100% whereas the S. lividans TK23 spontaneous mutation frequency to nosiheptide resistance was estimated to be 5U1037 . In non-sterile soil microcosms, similar colonisation dynamics were observed between the wild-type and the gylB mutant strains. From a 107 -spore inoculum the number of recovered CFU decreased rapidly to about 5U106 þ 2U106 per g of dry soil and remained stable at this level (Fig. 3). This result indicates that neither inactivation of this gene nor the additional metabolic load
Fig. 2. Growth of the parental (TK21) and mutant (OS48.3) S. lividans strains on media with and without glycerol: (A) GA medium with 1.25% glycerol; (B) similar medium in which glycerol was replaced by arabinose (1%); (C) GA medium containing glycerol and arabinose.
loss of the plasmid, to the insu¤cient production of the lethal protein, to chromosomal mutations leading to resistance to its action, or to mutational inactivation of the lethal gene itself. Only duplication of the suicide functions arranged on a plasmid in a way to prevent inactivation by deletion and recombination permitted a decrease in the mutation rate to about 1038 [27]. The e¤ciency of the gylB-based system developed for the S. lividans strain in
Fig. 3. Survival of the wild-type (F) (S. lividans TK21) and mutant (a) (S. lividans OS48.3) strains in non-sterile soil microcosms. Error bars represent the standard deviation of triplicate analyses.
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brought by the gentamicin resistance a¡ected the capacities of the mutant bacteria to survive in non-sterile soil microcosms. 3.4. Potential for conjugation in non-sterile soils In this set of experiments, spores from the S. lividans strain OS48.3 used as donor of pTS135 (conferring hygromycin resistance) were inoculated into non-sterile soil microcosms at a level of 108 spores per g of dry soil. In the non-sterile soil samples used in this study, the number of hygromycin-resistant actinomycetes reached 5.43U 104 þ 4.86U103 CFU per g of dry soil with a total number of culturable actinomycetes of about 3.07U107 þ 4.7U106 . This led us to select the pSAM2 derivative plasmid pTS135 conferring hygromycin resistance for further experiments (rather than the other pSAM2 derivative plasmid pTS130 which confers resistance to nosiheptide). In order to counter-select the donor strain, which is required for the actual detection of transconjugants, the soil suspensions were plated on the GA medium. Incubation of the donor strain for 7 or 14 days in soil did not signi¢cantly increase the number of culturable actinomycetes exhibiting resistance to hygromycin compared to the control soil (P 6 0.05). In order to detect among the indigenous colonies those which could actually be recipients of pTS135, pTS135 DNA was used as a probe in colony hybridisations with 200 hygromycin-resistant clones isolated from each soil sample. Surprisingly, we detected positive hybridisation signals for colonies isolated from both inoculated and control soil samples. In order to overcome problems of speci¢city due to the use of probes consisting of various sequences and genes, the oligonucleotides P1 and P2 , complementary to the part of the int gene encoding the integrase of pSAM2 derivatives, were designed and used in PCR reactions. In this case, DNA from the hygromycin-resistant clones that hybridised with pTS135 did not amplify with the P1 and P2 primers whatever the stringency of the PCR annealing step. These results indicate that these bacteria did not contain the int gene and, thus, could not be considered to be recipients of the pSAM2 derivative element. Transfer of this conjugative element from a donor strain to the indigenous soil micro£ora failed to be detected indicating that transfer frequencies remained below 5.9U1034 (the detection limit). This low frequency, which does not con¢rm previous results when both donor and recipient strains were inoculated into non-sterile soils [11], could be due to a genetic incompatibility between the donor and a su¤cient quantity of indigenous recipient strains preventing replication and/or integration of the pSAM2 derivative element, or its degradation by restriction systems. Other hypotheses could deal with physiological and morphological incompatibilities due to the resistant forms the actinomycetes develop in soil since a mycelial state is required to allow conjugation to proceed [28]. Finally, speci¢c localisation
in soil micro-aggregates of the indigenous actinomycetes could prevent the donor strain's physical contact with the recipient cells [29]. However, in other ecosystems such as activated sludge [30] or marine environments [31] transfer frequencies of conjugative plasmids from an inoculated donor strain to indigenous bacteria were in a range between 1034 and 1036 . This would mean that if the pSAM2 derivative plasmids had been transferred with the same e¤ciency to the indigenous soil actinomycetes, detection of transconjugants would logically remain improbable with respect to the detection limit of the plating approach presented here. However, what is striking in our study and deserves further investigation is the inability of actinomycetes in the indigenous micro£ora to become recipients of the pSAM2 element at frequencies similar to those of an inoculated Streptomyces strain. The e¤ciency of the counter-selection developed in this study will be of great help to address these fundamental questions. Acknowledgements We thank Dr Colin Smith for the gift of pIJ2204 and for helpful suggestions and Dr Guennadi Sezonov for the gift of pTS130 and pTS135. This work was supported by a grant received from the `Ministe©re Franc°ais de l'Enseignement Supe¨rieur et de la Recherche' to S.C.B. F.K. was supported by the CNRS PICS programme of scienti¢c collaboration and A.F. by the Swedish Council for Forestry and Agricultural Research. References [1] Theilleux, J. (1993) The actinomycetes. In: Industrial Microbiology : Microorganisms of Industrial Interest (Leveau, J.-Y. and Bouix, M., Eds.), pp. 468^488. TecpDoc Lavoisier, Paris. [2] Baltz, R.H. (1998) Genetic manipulation of antibiotic-producing Streptomyces. Trends Microbiol. 6, 76^83. [3] Pernodet, J.L., Simonet, J.M. and Guerineau, M. (1984) Plasmids in di¡erent strains of Streptomyces ambofaciens: free and integrated form of plasmid pSAM2. Mol. Gen. Genet. 198, 35^41. [4] Boccard, F., Smokvina, T., Pernodet, J.L., Friedmann, A. and Guerineau, M. (1989) The integrated conjugative plasmid pSAM2 of Streptomyces ambofaciens is related to temperate bacteriophages. EMBO J. 8, 973^980. [5] Boccard, F., Smokvina, T., Pernodet, J.L., Friedmann, A. and Guerineau, M. (1989) Structural analysis of loci involved in pSAM2 sitespeci¢c integration in Streptomyces. Plasmid 21, 59^70. [6] Martin, C., Mazodier, P., Mediola, M.V., Gicquel, B., Smokvina, T., Thompson, C.J. and Davies, J. (1991) Site-speci¢c integration of the Streptomyces plasmid pSAM2 in Mycobacterium smegmatis. Mol. Microbiol. 5, 2499^2502. [7] Mazodier, P., Thompson, C. and Boccard, F. (1990) The chromosomal integration site of the Streptomyces element pSAM2 overlaps a putative tRNA gene conserved among actinomycetes. Mol. Gen. Genet. 222, 431^434. [8] Sezonov, G., Blanc, V., Bamas-Jacques, N., Friedmann, A., Pernodet, J.L. and Guerineau, M. (1997) Complete conversion of antibiotic precursor to pristinamycin IIA by overexpression of Streptomyces pristinaespiralis biosynthetic genes. Nature Biotechnol. 15, 349^353.
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Development of a conditional lethal system for a Streptomyces lividans strain and its use to investigate conjugative transfer in soil Sylvie Clerc-Bardin a , Fatma Karray b , Asa Frostegard a , Jean-Luc Pernodet b , Pascal Simonet a; * a
Laboratoire d'Ecologie Microbienne, UMR CNRS 5557, Baªt. Gre¨gor Mendel/La Doua Universite¨ Lyon I, 43 Boulevard du 11 novembre 1918, 69622 Villeurbanne Cedex, France b Laboratoire de Biologie et Ge¨ne¨tique Mole¨culaire, Institut de Ge¨ne¨tique et Microbiologie, UMR CNRS 8621, Baªt. 400, Universite¨ Paris-Sud, 91405 Orsay Cedex, France Received 21 May 2001 ; received in revised form 1 October 2001; accepted 3 October 2001 First published online 30 October 2001
Abstract In Streptomyces lividans, a conditional lethal mechanism was obtained by inactivation of gylB in the glycerol operon. The resulting strain was efficiently counter-selected on glycerol-containing medium but the mutation had no effect on the capacity of the strain to be maintained in non-sterile soil samples and to transfer derivatives of the genetic element pSAM2. However, despite the efficiency of the donor counterselection system, the transfer of a pSAM2 derivative from S. lividans to indigenous soil bacteria was not detected. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Lethal system; pSAM2; Streptomyces; Conjugation
1. Introduction Gram-positive bacteria belonging to actinomycetes and particularly to the genus Streptomyces are among the main natural producers of economically important secondary metabolites [1]. As a result, they were also among the ¢rst to bene¢t from recent advances in genetic engineering [2]. However, developing new and genetically modi¢ed strains (GMM) that could yield novel compounds in a more or less predictable way requires Streptomyces strains to be able to be transformed with e¤cient shuttle vectors that have as broad a host range as possible. For this purpose, the Streptomyces ambofaciens genetic element pSAM2 [3], which has the ability to replicate and integrate site-specifically [4] in various bacteria [5^7], was used extensively to develop a series of vectors adapted for engineering of industrial Streptomyces strains [8]. However, questions remain concerning the fate of such industrially exploited GMM if they were to be released
* Corresponding author. Tel. : +33 (4) 72 44 82 89; Fax: +33 (4) 72 43 12 23. E-mail address : [email protected] (P. Simonet).
into the environment [9]. The main concern is about their ability to spread recombinant DNA sequences to indigenous micro-organisms via gene transfer mechanisms mainly by conjugative transfer [10] when vectors such as pSAM2 derivatives are used. The conjugative properties of this element led to questions about the potential for its transfer under natural conditions. This potential transfer was supported by previous data on the e¤ciency of the conjugative transfer from donor to recipient strains co-inoculated into soil microcosms [11]. While strategies have been developed for some proteobacteria [12], similar mechanisms causing the controlled death of Gram-positive bacteria are less developed, or do not apply to Streptomyces [13], preventing any investigation of transfer of mobile genetic elements from Streptomyces to indigenous bacteria. In order to address the question of a potential transfer to the indigenous micro£ora, we developed a conditional lethal system for a Streptomyces lividans strain based on glycerol sensitivity obtained by inactivation of one of the genes involved in glycerol utilisation. The pathway for glycerol catabolism in Streptomyces coelicolor is determined by the glycerol-inducible, glucose-repressible glycerol operon (gyl CABX) [14]. The soluble glycerol kinase
0168-6496 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 9 6 ( 0 1 ) 0 0 1 8 3 - 0
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(GK) encoded by the gylA gene and the membrane-associated glycerol-3-phosphate dehydrogenase (G3PDH) encoded by the gylB gene are essential for growth on media containing glycerol as the sole carbon source [15,16]. The absence of G3PDH activity in mutant strains presenting a normal GK activity leads to glycerol sensitivity probably due to the lethal e¡ect of glycerol-3-phosphate accumulation [15,16]. In S. lividans, the organisation and regulation of the gyl operon are essentially the same as those in S. coelicolor [17]. In this article, we describe the inactivation of the gylB gene of S. lividans TK21, various controls to check the e¤ciency of the conditional lethal mechanism, and preliminary results about e¤ciency of the transfer of a pSAM2 derivative element to the indigenous soil micro£ora. 2. Materials and methods 2.1. Bacterial strains, plasmids, growth conditions and DNA manipulations The bacterial strains and plasmids used in this study are listed in Table 1. Molecular biology techniques for Streptomyces spp. and Escherichia coli were as described respectively by Hopwood et al. [18] and Maniatis et al. [19]. Restriction enzymes, T4 DNA ligase and DNA polymerase I Klenow fragment were purchased from Roche Diagnostics (Meylan, France) and were used according to the manufacturer's recommendations. PCR ampli¢cations were conducted according to standard procedures with primers P1 (5P-GGTGACGGCGGCCTCCACTGGGACG-3P) and P2 (5P-GGTGGGTGCAGTTCTCGACGGTGCG-3P) complementary to part of the int gene encoding the integrase of pSAM2 and DNA extracted from growing colonies. Hybridisation conditions were as described previously [14] with radioactive labeling of the plasmid probe using [32 P]dCTP (3000 Ci mmol31 , Amersham, Les Ulis, France) and a random primed kit (Boehringer Mannheim, Meylan, France). After hybridisation of ¢lters at 65³C for 15 h the washing conditions included two changes of 2USSC solution at room temperature for 5 min, then two changes of 2USSC, 1% SDS solution at 65³C for 20 min. Standard culture techniques were as described by Hopwood et al. [18] for Streptomyces spp. strains which were cultured in liquid yeast extract-malt extract sucrose [20] or tryptic soy broth (Difco) media and on plates with the R2YE [21] or tryptic soy agar (Difco) solid media. The glycerol-arginine (GA) medium [22] containing 1.25% glycerol was used to counter-select the strain OS48.3 (see below) and to isolate actinomycetes from soil. In order to verify that glycerol had a toxic e¡ect on S. lividans OS48.3, media similar to GA were used in which glycerol was replaced by arabinose (1%) or in which these two carbon
sources were both present. The glycerol-sensitive OS48.3 strain was frozen in a solution containing 20% glycerol. Before utilisation of frozen OS48.3 spores, they were centrifuged and the pellet washed once with water to eliminate glycerol. Concentrations of antibiotics in selective media were as follows: ampicillin, 100 Wg ml31 ; gentamicin, 20 Wg ml31 for E. coli and 50 Wg ml31 for S. lividans; hygromycin, 50 Wg ml31 ; nosiheptide, 25 Wg ml31 ; spectinomycin, 50 Wg ml31 ; streptomycin, 50 Wg ml31 . Fungicides were also added in the media to inhibit the growth of fungi when soil suspensions were plated : cycloheximide, 200 Wg ml31 and benomyl, 50 Wg ml31 . 2.2. In vitro conjugation experiments In vitro conjugation experiments were as described by Clerc and Simonet [11]. Brie£y, an inoculum containing 107 spores of both the S. lividans OS48.3 donor strain carrying pTS130 and the S. lividans TK23 recipient strain was plated onto R2YE medium and incubated for 4 days at 28³C. The spores were then harvested and spread onto selective media containing gentamicin, spectinomycin or spectinomycin and nosiheptide for the selection of respectively the donor, the recipient and the transconjugant strains. 2.3. Soil microcosm experiments A silt loam soil collected at Montrond (France) was airdried, sieved at 2 mm and stored at 4³C until subsequent use. Soil characteristics were as follows : clay 31.4%, silt 36.4%, sand 32.2%, C/N 7.6, pH (water) 6.7 and a ¢eld capacity of 40 g of water for 100 g of dry soil. Soil microcosms consisted of 5 g equivalent dry soil in 10-ml polypropylene tubes. The soil moisture was adjusted to that of the ¢eld capacity and soil microcosms were incubated at 28³C for 5^7 days before inoculation. Experiments to establish population dynamics in soil of the various Streptomyces spp. strains were done by inoculating the soil microcosms with 107 spores per g of dry soil, establishing a ¢nal moisture content equivalent to the ¢eld capacity [11]. Then, the soil microcosms were incubated at 28³C in darkness and three of them were periodically taken for enumeration of the targeted bacteria [11]. In experiments to investigate the in situ transfer of pTS135 from the S. lividans OS48.3 donor strain to the indigenous soil micro£ora, a similar protocol was applied, the only exception being that the level of the inoculum reached 108 spores per g of dry soil and uninoculated microcosms were used as controls. Experiments were conducted in triplicate with disruption of the microcosms 0, 7 and 14 days after inoculation for analyses of donor, actinomycete and transconjugant population levels on the media de¢ned previously.
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Table 1 Bacterial strains and plasmids Strain or plasmid Strains E. coli DH5K S. lividans TK21 S. lividans TK23 S. lividans TK24 Plasmids pIJ2204 pHP456aac pUC4 pOS48.1 pOS48.2 pGM160v
Descriptiona
Reference
SpcR StrR
[32] [32] [32]
ApR , 5.7-kb BamHI-BamHI fragment of glycerol operon carrying gylA, gylB and gylX genes cloned into the BamHI site of pBR322 ApR , GenR ApR ApR , 5.7-kb BamHI-BamHI fragment of pIJ2204 cloned into BamHI site of pUC4 ApR , GenR ; 6aac cassette encoding Gen resistance cloned into PstI-SphI-digested plasmid pOS48.1 Temperature-sensitive replicon derived from pGM160; AmpR in E. coli and NosR in S. lividans
pTS130
ApR , GenR in E. coli and NosR , GenR in S. lividans; EcoRI-EcoRI insert of pOS48.2 cloned into EcoRI site of pGM160v NosR
pTS135
HygR
pTS39
NosR
pOS48.3
a
[33] [25] [24] This study This study [26] and this study This study Sezonov (unpublished) Sezonov (unpublished) [34]
Ap: ampicillin ; Gen : gentamicin; Hyg: hygromycin; Nos : nosiheptide; Spc: spectinomycin ; Str: streptomycin.
3. Results and discussion 3.1. Inactivation of the gylB gene of S. lividans TK21 The 5.7-kb BamHI insert of plasmid pIJ2204 [23] contains most of the gylCABX operon but lacks the gyl promoter region. This insert was cloned as a BamHI fragment into pUC4 [24], digested with BamHI, generating plasmid pOS48.1 (Fig. 1). This plasmid possesses two PstI sites, both located in the 5P end of gylB, and a single SphI site located in the 3P end of gylB. The pOS48.1 vector was digested with PstI and SphI and the extremities were blunted with the Klenow enzyme. The two fragments internal to gylB and released by this digestion were replaced by a HindIII fragment blunt-ended with the Klenow enzyme which contained the 6aac cassette [25]. The 6aac cassette confers resistance to gentamicin in E. coli and in S. lividans. Digestion of pOS48.2 with EcoRI released the complete insert, which was cloned into the EcoRI site of pGM160v, a shuttle vector whose replication is temperature-sensitive in Streptomyces spp., to generate pOS48.3. The pGM160 vector which confers nosiheptide resistance was in turn derived from pGM160 [26] by removing the HindIII fragment containing the aacC1 gene. The pOS48.3 vector was used to transform protoplasts of S. lividans TK21. One clone which harboured pOS48.3, and was therefore resistant to nosiheptide and gentamicin, was grown in TSB with nosiheptide for 2 days at 30³C (permissive temperature for pOS48.3 replication) and then used to inoculate drug-free TSB. This culture was grown for 3 days at 39³C (non-permissive for pOS48.3 replication) before samples were diluted and plated on TSB with and without gentamicin. About 20% of the colony-form-
ing units (CFU) were resistant to gentamicin. Among about 100 such clones which were tested for resistance to nosiheptide, ¢ve were found to be sensitive to nosiheptide. These clones had presumably lost the pGM160v moiety of pOS48.3 via a double crossover recombination event. This was con¢rmed in hybridisation analyses, when Southern blots of total genomic DNA from one of these clones and from strain TK21 were probed with the pOS48.3 plasmid or the 6aac cassette, and compared (results not shown). Among the ¢ve clones in which a double homologous recombination event had resulted in replacement of the gylB gene of the S. lividans chromosome by its disrupted counterpart, one was chosen for further study and designated S. lividans OS48.3. 3.2. E¤ciency of the killing system The mutant OS48.3 and parental TK21 S. lividans strains were plated on GA medium [22] containing 1.25% glycerol. This medium is the one used routinely for the isolation of actinomycetes from soil samples. Clearly, S. lividans OS48.3, harbouring the disrupted gylB gene, was unable to grow on GA medium (only a very thin transparent overlay was observed) whereas the wild-type strain displayed normal growth on the same medium (Fig. 2A). In order to determine if the mutant strain OS48.3 was unable to utilise glycerol as a carbon source or whether it was really sensitive to glycerol, the same strains were plated on media containing an alternative carbon source (arabinose) with or without glycerol. On a medium identical to GA, except for replacement of glycerol with arabinose, both strains presented similar growth kinetics and development (Fig. 2B). On GA medium containing
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Fig. 1. Construction of S. lividans OS48.3. The plasmids pOS48.1, pOS48.2 and pOS48.3 are presented. Only the insert, not the vector part, of these plasmids is drawn to scale. Double homologous recombination between pOS48.3 and the chromosome of S. lividans TK21, generating strain OS48.3, is schematically represented. The sizes of the XhoI fragments revealed in hybridisation experiments whose sizes di¡er in both S. lividans strains are indicated.
arabinose in addition to glycerol, the mutant strain OS48.3 was unable to grow when the parental strain grew normally. This demonstrated that strain OS48.3 was not only unable to utilise glycerol but was also sensitive to glycerol. Activation of the glycerol operon in the absence of the gylB gene product was lethal to S. lividans OS48.3. Interestingly, for all the experiments conducted with inocula containing 108 spores or mycelial fragments
from this strain, we never observed the development of an OS48.3 colony on the GA medium. This indicates that the probability of escaping the counter-selection system is far below 1038 . This result makes the lethal system very e¤cient compared to other methods previously described for some proteobacteria [12]. For instance, various plasmidbearing killing genes including hok, gef and rel cloned under inducible promoters lacked e¤ciency due to the
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this study does not depend on stable maintenance of plasmids or expression of foreign genes. Moreover, the fact that the lethal activity was obtained by replacement of a gene by its disrupted and deleted homologue prevented inactivation of the killing mechanism by a simple reversion event. Any mutation in other genes of the glycerol operon, which would prevent accumulation of glycerol-3-phosphate, would abolish the lethal e¡ect of glycerol. However, as glycerol is the major carbon source in the GA medium, such a mutant would not grow or would only grow very poorly on this medium and this would therefore have no consequences for the e¤ciency of the counter-selection. 3.3. Conjugation and soil colonisation properties of the mutant strain Controls were performed to demonstrate that the mutation in the gylB gene had not a¡ected two important properties of the strain which are essential for future studies of gene transfer to indigenous soil micro£ora: conjugation capacities and non-sterile soil colonisation ability. In vitro transfer rates, expressed as the number of transconjugants per potential recipient genotype (U100), were similar to those observed with the wild-type strain [11] reaching 90^ 100% whereas the S. lividans TK23 spontaneous mutation frequency to nosiheptide resistance was estimated to be 5U1037 . In non-sterile soil microcosms, similar colonisation dynamics were observed between the wild-type and the gylB mutant strains. From a 107 -spore inoculum the number of recovered CFU decreased rapidly to about 5U106 þ 2U106 per g of dry soil and remained stable at this level (Fig. 3). This result indicates that neither inactivation of this gene nor the additional metabolic load
Fig. 2. Growth of the parental (TK21) and mutant (OS48.3) S. lividans strains on media with and without glycerol: (A) GA medium with 1.25% glycerol; (B) similar medium in which glycerol was replaced by arabinose (1%); (C) GA medium containing glycerol and arabinose.
loss of the plasmid, to the insu¤cient production of the lethal protein, to chromosomal mutations leading to resistance to its action, or to mutational inactivation of the lethal gene itself. Only duplication of the suicide functions arranged on a plasmid in a way to prevent inactivation by deletion and recombination permitted a decrease in the mutation rate to about 1038 [27]. The e¤ciency of the gylB-based system developed for the S. lividans strain in
Fig. 3. Survival of the wild-type (F) (S. lividans TK21) and mutant (a) (S. lividans OS48.3) strains in non-sterile soil microcosms. Error bars represent the standard deviation of triplicate analyses.
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brought by the gentamicin resistance a¡ected the capacities of the mutant bacteria to survive in non-sterile soil microcosms. 3.4. Potential for conjugation in non-sterile soils In this set of experiments, spores from the S. lividans strain OS48.3 used as donor of pTS135 (conferring hygromycin resistance) were inoculated into non-sterile soil microcosms at a level of 108 spores per g of dry soil. In the non-sterile soil samples used in this study, the number of hygromycin-resistant actinomycetes reached 5.43U 104 þ 4.86U103 CFU per g of dry soil with a total number of culturable actinomycetes of about 3.07U107 þ 4.7U106 . This led us to select the pSAM2 derivative plasmid pTS135 conferring hygromycin resistance for further experiments (rather than the other pSAM2 derivative plasmid pTS130 which confers resistance to nosiheptide). In order to counter-select the donor strain, which is required for the actual detection of transconjugants, the soil suspensions were plated on the GA medium. Incubation of the donor strain for 7 or 14 days in soil did not signi¢cantly increase the number of culturable actinomycetes exhibiting resistance to hygromycin compared to the control soil (P 6 0.05). In order to detect among the indigenous colonies those which could actually be recipients of pTS135, pTS135 DNA was used as a probe in colony hybridisations with 200 hygromycin-resistant clones isolated from each soil sample. Surprisingly, we detected positive hybridisation signals for colonies isolated from both inoculated and control soil samples. In order to overcome problems of speci¢city due to the use of probes consisting of various sequences and genes, the oligonucleotides P1 and P2 , complementary to the part of the int gene encoding the integrase of pSAM2 derivatives, were designed and used in PCR reactions. In this case, DNA from the hygromycin-resistant clones that hybridised with pTS135 did not amplify with the P1 and P2 primers whatever the stringency of the PCR annealing step. These results indicate that these bacteria did not contain the int gene and, thus, could not be considered to be recipients of the pSAM2 derivative element. Transfer of this conjugative element from a donor strain to the indigenous soil micro£ora failed to be detected indicating that transfer frequencies remained below 5.9U1034 (the detection limit). This low frequency, which does not con¢rm previous results when both donor and recipient strains were inoculated into non-sterile soils [11], could be due to a genetic incompatibility between the donor and a su¤cient quantity of indigenous recipient strains preventing replication and/or integration of the pSAM2 derivative element, or its degradation by restriction systems. Other hypotheses could deal with physiological and morphological incompatibilities due to the resistant forms the actinomycetes develop in soil since a mycelial state is required to allow conjugation to proceed [28]. Finally, speci¢c localisation
in soil micro-aggregates of the indigenous actinomycetes could prevent the donor strain's physical contact with the recipient cells [29]. However, in other ecosystems such as activated sludge [30] or marine environments [31] transfer frequencies of conjugative plasmids from an inoculated donor strain to indigenous bacteria were in a range between 1034 and 1036 . This would mean that if the pSAM2 derivative plasmids had been transferred with the same e¤ciency to the indigenous soil actinomycetes, detection of transconjugants would logically remain improbable with respect to the detection limit of the plating approach presented here. However, what is striking in our study and deserves further investigation is the inability of actinomycetes in the indigenous micro£ora to become recipients of the pSAM2 element at frequencies similar to those of an inoculated Streptomyces strain. The e¤ciency of the counter-selection developed in this study will be of great help to address these fundamental questions. Acknowledgements We thank Dr Colin Smith for the gift of pIJ2204 and for helpful suggestions and Dr Guennadi Sezonov for the gift of pTS130 and pTS135. This work was supported by a grant received from the `Ministe©re Franc°ais de l'Enseignement Supe¨rieur et de la Recherche' to S.C.B. F.K. was supported by the CNRS PICS programme of scienti¢c collaboration and A.F. by the Swedish Council for Forestry and Agricultural Research. References [1] Theilleux, J. (1993) The actinomycetes. In: Industrial Microbiology : Microorganisms of Industrial Interest (Leveau, J.-Y. and Bouix, M., Eds.), pp. 468^488. TecpDoc Lavoisier, Paris. [2] Baltz, R.H. (1998) Genetic manipulation of antibiotic-producing Streptomyces. Trends Microbiol. 6, 76^83. [3] Pernodet, J.L., Simonet, J.M. and Guerineau, M. (1984) Plasmids in di¡erent strains of Streptomyces ambofaciens: free and integrated form of plasmid pSAM2. Mol. Gen. Genet. 198, 35^41. [4] Boccard, F., Smokvina, T., Pernodet, J.L., Friedmann, A. and Guerineau, M. (1989) The integrated conjugative plasmid pSAM2 of Streptomyces ambofaciens is related to temperate bacteriophages. EMBO J. 8, 973^980. [5] Boccard, F., Smokvina, T., Pernodet, J.L., Friedmann, A. and Guerineau, M. (1989) Structural analysis of loci involved in pSAM2 sitespeci¢c integration in Streptomyces. Plasmid 21, 59^70. [6] Martin, C., Mazodier, P., Mediola, M.V., Gicquel, B., Smokvina, T., Thompson, C.J. and Davies, J. (1991) Site-speci¢c integration of the Streptomyces plasmid pSAM2 in Mycobacterium smegmatis. Mol. Microbiol. 5, 2499^2502. [7] Mazodier, P., Thompson, C. and Boccard, F. (1990) The chromosomal integration site of the Streptomyces element pSAM2 overlaps a putative tRNA gene conserved among actinomycetes. Mol. Gen. Genet. 222, 431^434. [8] Sezonov, G., Blanc, V., Bamas-Jacques, N., Friedmann, A., Pernodet, J.L. and Guerineau, M. (1997) Complete conversion of antibiotic precursor to pristinamycin IIA by overexpression of Streptomyces pristinaespiralis biosynthetic genes. Nature Biotechnol. 15, 349^353.
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