Molecular Microbiology (2003) 47(5), 1385–1393

Conjugal immunity of Streptomyces strains carrying the integrative element pSAM2 is due to the pif gene (pSAM2 immunity factor) Christophe Possoz,* Josette Gagnat, Guennadi Sezonov,† Michel Guérineau and Jean-luc Pernodet Institut de Génétique et Microbiologie, UMR CNRS 8621, Université Paris-Sud, Orsay, France.

Summary Mechanisms of conjugal immunity preventing redundant exchange between two cells harbouring the same conjugative element have been reported in diverse bacteria. Such a system does exist for pSAM2, a conjugative and integrative element of Streptomyces. The apparition of the conjugative free form of pSAM2 in the donor strain during mating can be considered as the initial step of transfer. We analysed the genes involved in transfer inhibition by mating donors harbouring pSAM2 with recipient strains containing different regions of pSAM2. The conjugal immunity was previously thought to be mediated by the transcriptional repressor KorSA. Although the transfer efficiency is reduced by its presence in the recipient, the initiation of the transfer process is not affected. In contrast, the presence in the recipient strain of a single pSAM2 gene, pif (pSAM2 immunity factor), was sufficient to abolish both transfer and initiation of transfer. Thus, the clustered genes korSA and pif act complementarily to maintain pSAM2 in a ‘prophage’ state under non-conjugal conditions. KorSA is involved in intracellular signalling, whereas Pif participates in intercellular signalling. The Pif nudix motif is essential for its activity. This is the first protein of the nudix family shown to be involved in bacterial conjugation. Introduction Bacterial conjugation is the process whereby a DNA molecule is transferred from a donor to a recipient bacterium Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Publishing Ltd, 200347Original ArticleC. Possoz et al.pSAM2 conjugal immunity

Accepted 15 November, 2002. *For correspondence. E-mail [email protected]; Tel. (+33) 169154640; Fax (+33) 1 69 15 45 44. †Present address: Institut Jacques Monod, Laboratoire de Génétique Microbienne; 4 place Jussieu, 75252 Paris cedex 5, France.

© 2003 Blackwell Publishing Ltd

via a specialized protein complex. Regarding the presence of the two main elements involved in DNA processing, the relaxase and the origin of transfer, oriT (reviewed in Lanka and Wilkins, 1995; Byrd and Matson, 1997), the mechanistic principle of conjugative transfer seemed to be conserved in most bacterial systems studied based on singlestranded DNA processing. In the first step of this common process, specific intercellular communication, specified by various mechanisms, guides the donor and recipient bacteria into a paired formation. Streptomyces has developed a conjugation mechanism, completely different from all others studied, occurring through transfer of doublestranded DNA (Possoz et al., 2001), which appears to be related to chromosome partitioning during sporulation or cell division (Wu et al., 1995). As Streptomyces is not motile but grows as branched mycelium, there is no obvious need for a specific aggregation system. Regulations preventing redundant plasmid exchange between donor cells have been demonstrated in Grampositive (Weidlich et al., 1992; for a review, see Dunny, 1990) and Gram-negative (reviewed by Frost et al., 1994) bacteria. For instance, the transfer region of the F plasmid carries two exclusion genes, traS (Minkley and IppenIhler, 1977) and traT (Achtman et al., 1977), whose phenotypes are additive. TraT, an outer cell membrane protein, is thought to interfere with the formation or stabilization of mating contacts (surface exclusion) and TraS specifies an inner membrane protein blocking DNA synthesis (entry exclusion). Most of the Streptomyces plasmids and integrative elements are conjugative and can promote both pock formation and the mobilization of chromosomal segments (Hopwood and Kieser, 1993). A pock corresponds to a local inhibition of growth and development of the recipient strain in contact with the donor strain. The only example of a putative exclusion system, preventing redundant exchange between Streptomyces harbouring the same conjugative element, was proposed for the integrative element SLP1 of Streptomyces coelicolor (Hagège et al., 1999). The characterization of this type of mechanism could make a large contribution to understanding horizontal gene transfer in Streptomyces. Streptomycetes are the most important antibiotic-producing bacteria and, furthermore, it has been suggested that resistance genes are transferred from this species to

1386 C. Possoz et al. pathogens (Davies, 1994). To study the mechanism for avoiding redundant exchange, also called self-transfer, we used pSAM2, an integrative and conjugative element from Streptomyces ambofaciens (Pernodet et al., 1984). Its integration system is very similar to that of temperate phages (Boccard et al., 1989). It can replicate like typical plasmids (Smokvina et al., 1991; Hagège et al., 1994). The repSA, xis and int genes, encoding the replicase, the excisionase and the integrase, respectively, are organized as an operon activated by the pra gene product (Sezonov et al., 1998). In absence of a recipient strain (lacking a related conjugative element), these functions are not expressed by the wild-type pSAM2 element as KorSA represses pra transcription, maintaining pSAM2 integrated in the chromosome (Sezonov et al., 2000). The analysis of the kinetics and the localization of pSAM2 status (free or integrated) revealed that pSAM2 is excised and replicates in the donor a few hours after mating with a recipient strain. After transfer as doublestranded DNA to the recipient strain, pSAM2 replicates, spreads within the mycelium and then integrates into the chromosome of the recipient cells (Possoz et al., 2001). pSAM2 is normally integrated and the appearance of the free forms indicates, and can be used as a reporter of, the onset of transfer. There is presumably a signal provoking transfer initiation because no free forms are detected in mixed cultures of two strains harbouring pSAM2, and transfer is at least three orders of magnitude lower than when one strain lacked pSAM2 (Possoz et al., 2001). This indicates that there is a mechanism for avoiding conjugation with a strain already containing pSAM2 and that transfer is blocked before the initial step of excision as a circular form. We investigated whether there are pSAM2 gene(s) conferring to the host a donor identity? If such genes do exist, a host devoid of pSAM2 but harbouring these genes should no longer be recognized as a recipient for conjugation. The involvement of KorSA was strongly suspected. This protein of the GntR repressor family controls the expression of the replication and integration/excision functions of pSAM2 via the repression of the activator pra (Sezonov et al., 1998; 2000). In SLP1, an integrative element of Streptomyces coelicolor, ImpA and ImpC which also belong to the GntR repressor family are believed to constitute a central repressor. Hagège et al. (1999) suggested that their dilution or inactivation activates the transfer process after cell to cell contact and fusion. Thus, the conjugative process in mating between two pSAM2-donor strains could be prevented by the presence of KorSA in both compartments, maintaining pSAM2 in a constant chromosomally integrated state. In this paper, we demonstrate that neither the main transfer protein, TraSA, nor the transcriptional repressor,

KorSA, are involved in the inhibition of transfer initiation. A unique pSAM2 gene, orf131, was shown to have this role. It was called pif for pSAM2 immunity factor. It encodes a protein of 131 aa, which is a member of the nudix (nucleotide diphosphate linked to some other moiety, X) hydrolase family.

Results Rationale of the experiments pSAM2 transfer between two donor strains is inefficient (Possoz et al., 2001). During such ‘mating’ between strains, pSAM2 is continually detected integrated into the chromosome and never as the conjugative, free form observed in efficient mating with a recipient strain lacking pSAM2. Therefore, it appears that the initiation of the transfer process (excision of pSAM2 in the donor strain) is a regulated step, presumably involving at least one gene carried by pSAM2. To identify the genes involved in this regulation, strains derived from Streptomyces lividans TK23, lacking the complete form of pSAM2, but containing different regions of pSAM2 were constructed. They contained either pSAM2 derivatives with various deletions or pSAM2 fragments carried by a fC31-derived integrative vector. The efficiency of transfer of marked pSAM2 derivatives from the donor strain S. lividans TK24 (streptomycin resistant) to these various different S. lividans TK23 (spectinomycin resistant) recipient strains was determined. The S. lividans TK24 donor strain transferred the pSAM2 marked derivatives used with an efficiency of about one to S. lividans TK23. Transfer efficiency is expressed as the fraction of the spores from the recipient strain harbouring pSAM2 after mating. Thus, measuring the transfer efficiency to S. lividans TK23 harbouring different pSAM2 regions will reflect their capacity to receive and to conserve pSAM2 upon sporulation. To ensure that the decreased transfer efficiency is due to a defect in the initiation step, the pSAM2 status (free or integrated) was analysed in the same experiment. Only decreased transfer efficiency correlated with an absence of the free form of pSAM2 in the donor strain during mating, was considered as reflecting defective transfer initiation.

A unique BamHI fragment carrying korSA and orf131 greatly reduces pSAM2 transfer efficiency The transfer efficiency of pTS135, a pSAM2 derivative conferring resistance to hygromycin, to a recipient strain harbouring pTS89 (Smokvina et al., 1991) was measured. pTS89 is a conjugative-deficient pSAM2 derivative, conferring resistance to nosiheptide, deleted of the 2 kb Asp718I fragment containing the 5¢-part of the activator pra gene, the entire korSA and orf131 genes (Fig. 1). The © 2003 Blackwell Publishing Ltd, Molecular Microbiology, 47, 1385–1393

pSAM2 conjugal immunity 1387 Fig. 1. Map of the pSAM2 Asp718I–Asp718I fragment conferring immunity to conjugal transfer. All restriction sites are numbered according to the sequence submitted previously to the EMBL data bank under accession no. AJ005260. The thin bars below the map correspond to the pSAM2 fragments inserted into an integrative vector giving pOS555, pOS556 and pOS680. a, transfer efficiency of pOS923, a conjugative pSAM2B2 derivative, in mating experiments with recipient strains harbouring different pSAM2 fragments.

spores resulting from this mating were tested on selective media: about 10% of the spores resistant to nosiheptide were also resistant to hygromycin. Thus, the presence of pTS89 affected but did not abolish transfer of pTS135. Therefore, the deletion of pra, korSA and orf131 affected the immunity to pSAM2 conjugation, as transfer efficiency in their absence was two orders of magnitude higher than that in their presence (transfer efficiency with the complete pSAM2 present in the recipient is about 10-3). A BamHI fragment of 1592 bp, containing the genes korSA, orf131 and the double-stranded origin for rolling circle replication (dso) (Hagège et al., 1993), was inserted into an integrative vector, and introduced into S. lividans TK23, yielding TK23(pOS555) (Fig. 1). This dramatically reduced transfer efficiency, lowering it to a level similar to that between two donor strains containing complete pSAM2. This BamHI fragment may therefore contain all the elements necessary for the inhibition of transfer. Two additional recipient strains were constructed, one containing korSA and the other orf131. KorSA does not modify the recipient status The BglII (297)–BsrGI (955) fragment was deleted from the BamHI fragment containing both orf131 and korSA, yielding pOS556. pOS556 thus contained only the korSA gene under the control of its own promoter but not the KorSA-binding site necessary to repress its own expression (as shown on Fig. 5, the BsrGI site is located in the middle of this operator locus). The strain S. lividans TK23(pOS556) was constructed with the pOS556 integrated in the chromosome through an integrative vector derived from the fC31 bacteriophage. In this strain, korSA was transcribed, as verified by slot blot experiments (data not shown) with total RNA using the BsrGI (955)–BamHI (1746) fragment described in Fig. 1 as a probe. The transfer efficiency from a hygromycin-marked pSAM2 derivative (pOS923) to this recipient strain S. lividans TK23(pOS556) during mating was 0.1, to be compared with 10-3 with a recipient strain containing the complete pSAM2. Thus, a strain containing KorSA retained the ability to behave as a recipient. The initiation step of transfer, detected as the apparition of free pSAM2 copies, was analysed in a mating between © 2003 Blackwell Publishing Ltd, Molecular Microbiology, 47, 1385–1393

the donor strain S. lividans TK24(pOS947 + pOS923) expressing SalM methyltransferase and the recipient strain S. lividans TK23(pOS556) expressing korSA but not the SalM methyltransferase. The strains were mixed on solid medium, in an equal proportion, and samples collected after various times of mating. The integrated or free state of pSAM2 was determined for each sample by Southern blotting using total DNA extracted from the mixture of the two strains digested with EcoRI. A 4.2 kb EcoRI pSAM2 fragment containing attP was used as the probe. pSAM2 integrates via site specific recombination between attP and attB in the host chromosome (Boccard et al., 1989; Raynal et al., 1998), leading to formation of attL and attR. For the integrated copy, the probe reveals two fragments, one of 6.7 containing attL and one of 5.2 kb containing attR; for the free copy, a fragment of 4.2 kb corresponding to the entire probe fragment is detected. Resistance (the donor strain) or sensitivity (the recipient strain) to SalI restriction indicates the strain in which the pSAM2 is present (Possoz et al., 2001). pSAM2 molecules sensitive to SalI are obtained only after transfer and replication in the recipient strain. SalI digestion of these integrated or free molecules generates numerous small fragments and the presence of the largest one (2.3 kb) was used as a marker for sensitivity to SalI. After 15 h of mating, a 4.2 kb band corresponding to the free form of pSAM2 was detected (Fig. 2, lane 1). It was as intense as that of a recipient strain without KorSA (Possoz et al., 2001). Thus, the presence of korSA in the recipient strain did not affect the initiation of the transfer process. SalI digestion resulted in a pSAM2-derived band of 2.3 kb (Fig. 2, lane 2). Thus, the presence of korSA in the recipient prevent neither transfer nor replication of pSAM2 in the recipient. The only particularity of the kinetics of the pSAM2 status in this mating was the persistence of the free forms even after 72 h: in mating with a recipient strain lacking korSA, the free forms almost disappeared after 48 h. pif (orf131) confers immunity to pSAM2 conjugation Previous DNA sequence analysis based on the highly biased codon usage of Streptomyces genes predicted an open reading frame (ORF) within the BamHI fragment

1388 C. Possoz et al.

Fig. 2. Determination of pSAM2 status during mating on solid medium between Streptomyces lividans TK24(pOS923 + pOS947) expressing SalM and the recipient strain S. lividans TK23(pOS556) expressing KorSA. Total DNA of the mixed culture was extracted after 15 h (lanes 1 and 2) and 72 h (lanes 3 and 4) and digested with EcoRI (lanes 1 and 3) or EcoRI + SalI (lanes 2 and 4). An a-32P-labelled EcoRI– EcoRI 4.2 kb pSAM2 fragment containing the attP site was used as the probe in Southern blotting analysis.

inhibiting pSAM2 transfer. This orf131 (formerly orf154) is upstream of korSA but transcribed in the opposite direction (Hagège et al., 1993). We named it pif for pSAM2 immunity factor because its expression triggers immunity to pSAM2 conjugation (see below). A S. lividans TK23 recipient strain containing pif was constructed by transformation with pOS680, an integrative vector derived from bacteriophage fC31 carrying the BglII(297)–NcoI(1152) fragment, shown in Fig. 1. The transfer efficiency of pOS923 to S. lividans TK23(pOS680), the recipient strain expressing pif, was 2.10-3, similar to the transfer efficiency between two donor strains carrying the entire pSAM2 element (Fig. 1). Thus the pif gene is a good candidate for the immunity factor of pSAM2, preventing the initiation of the transfer process. To confirm its involvement, the initiation of transfer was assessed, as described above, in a mating between strain S. lividans TK24(pOS923) as the donor and a recipient strain, TK23(pOS680), expressing pif. Only bands corresponding to the integrated state of pSAM2 were detected, and the 4.2 kb band corresponding to the conjugative free forms was absent (Fig. 3). The presence of pif in the recipient strain thus reduced the transfer efficiency by preventing initiation of the transfer process, as pSAM2 did not excise. Thus, pif is sufficient to confer donor status to a strain. pif is transcribed even in the absence of conjugation We analysed the transcriptional activity of pif in strains containing pSAM2, either pSAM2B2 or pSAM2B3. pSAM2B2 is the wild-type form, present only integrated at the attB

site and pSAM2B3 is a mutant form, present both integrated and free, owing to overexpression of pra (Sezonov et al., 1995). There are two potential translation start codons in the DNA sequence, one encoding a form of 154 aa (hence, the former name of orf154) and one a form of 131 aa (orf131). It could be noticed that the second GTG start corresponding to orf131 is immediately followed by two GTG codons. S1 mapping with total RNA extracted from S. lividans strains containing pSAM2B2 or pSAM2B3 revealed three transcription start points (Fig. 4), all downstream from the start codon of the longer form. The encoded protein is thus the shorter form of 131 aa. All three pif transcription start points are near the sequence recognized by KorSA, suggesting coregulation of the two divergently transcribed genes (Fig. 5). As korSA, pif is transcribed in the absence of Pra activator expression (as pra transcription is not detected in pSAM2B2), and even in the absence of conjugation. Pif activity required its nudix motif The only feature found in the Pif protein by sequence comparisons was a nudix motif (position 24–47). Nudix motifs are defined by the consensus sequence GX5EX7REUXEEXGU where U is usually I,L or V (Bessman et al., 1996) and are found in a large number of proteins in diverse species from viruses to humans. They are hydrolases, whose substrates are nucleotide diphosphates linked to one of various other moieties (Bessman et al., 1996), but the biological functions of most of them are unknown. The involvement of this nudix motif in Pif function was investigated by site-directed mutagenesis. Saturation mutagenesis of the 10 con-

Fig. 3. Determination of pSAM2 status during mating on solid medium between S. lividans TK24(pOS923) and the recipient strain S. lividans TK23(pOS680) expressing pif (orf131). Total DNA of the mixed culture was extracted after 15 h (lane 1) and 72 h (lane 2) and digested with EcoRI. An a-32P-labelled EcoRI–EcoRI 4.2 kb pSAM2 fragment containing the attP site was used as the probe for Southern blotting. © 2003 Blackwell Publishing Ltd, Molecular Microbiology, 47, 1385–1393

pSAM2 conjugal immunity 1389 served residues in the module of the phosphorylase MutT homologues (GGX5EX7REUXEEXGU, consisting of the nudix motif consensus and one additional G residue) revealed only four essential residues (Shimokawa et al., 2000). We constructed the double mutant G48A, G49D (Fig. 6) described to inactivate the nudix motif. Transfer efficiency of pOS923 was measured in matings with TK23(pOS960); this recipient strain contains the BglII– NcoI fragment, as in pOS680, carrying the G48A G49D double mutation of the nudix motif (the encoded protein was named Pif-1). The transfer efficiency was approximately 0.25 to be compared with 10-3 with a strain containing pif (Fig. 6). Therefore, the nudix motif of Pif was required for immunity to pSAM2 conjugation. The inactivation of pif provokes apparition of pSAM2 free forms

Fig. 4. High-resolution S1 mapping of the pif gene transcriptional start. The 440 bp DNA probe (see Experimental procedures) was obtained by polymerization using labelled K4 opp oligonucleotide.Lane 1, DNA probe treated with S1 enzyme in absence of RNA; lane 2, DNA probe treated with S1 enzyme in the presence of total RNA from S. lividans TK24(pSAM2B2); lane 3, DNA probe treated with S1 enzyme in the presence of total RNA from S. lividans TK24(pSAM2B3). The sizes of the protected DNA fragments were determined by comparison with the sequence of the Asp718I(129)–Asp718I(2121) fragment in M13mp18 using the K4 opp oligonucleotide.

To determine the effect of the inactivation of pif within a donor strain, the wild-type pif allele was replaced by pif-1 in the pSAM2B2 derivative pOS923, yielding pOS961. The transformation efficiency of S. lividans TK24 protoplasts with pOS961 was similar to that with pOS923. However, the transformants exhibited a particular phenotype: a blood-red colour and poor sporulation (data not shown). The status of pOS961 was then analysed by Southern blotting (as described above). Interestingly, free forms of pOS961 were detected, whereas pOS923, the parent of pOS961, is only integrated (Fig. 7). Thus, the inactivation

Fig. 5. Transcriptional start points of the pif gene. The sequence upstream and downstream from the presumed pif gene start codon (the most upstream start codon compatible with transcriptional start points was chosen) is presented (numbered according to the sequence in EMBL data bank under accession no. AJ005260). The positions of the three transcriptional start points are indicated by +1, followed by the number corresponding to the signal numbers in Fig. 4 and marked by vertical arrows. The boxed sequence indicates the KorSA DNA binding site (Sezonov et al., 2000); the transcriptional start of korSA gene is marked as +1korSA and with a vertical arrow. The various oligonucleotides used are shown by horizontal arrows; nucleotide changes included in oligo MD-2 and oligo MD-3 are indicated. © 2003 Blackwell Publishing Ltd, Molecular Microbiology, 47, 1385–1393

1390 C. Possoz et al.

Fig. 6. Description of the nudix motif. The consensus sequence of the nudix motif conserved in all nudix proteins is indicated (U indicates I, L or V) and the essential residues, identified by Shimokawa et al. (2000) in a subgroup of the nudix hydrolase family, are highlighted in black (only three out of the four essential residues are present in the consensus sequence of the global nudix hydrolase family). Alignment of the nudix motif of Pif and Pif-1 reveals that the only differences are AD instead of GG. a, transfer efficiency of pOS923 in mating with recipient strains harbouring either pOS680 (pif) or pOS960 (pif-1).

of Pif, provokes the constant presence of the free forms of pSAM2 usually detected only transiently during mating. Discussion The integrative mobile genetic element pSAM2, normally integrated in the chromosome of Streptomyces, is capable of conjugal transfer. This transfer is similar to that of conjugative Streptomyces plasmids both in its visual manifestation (pock formation) and in the major transfer protein involved. The existence of a specific system preventing initiation of transfer between mycelia carrying pSAM2, when they are in contact was postulated from the observation of the absence of excision. The appearance of the free circular forms of pSAM2 during mating with a recipient strain indicates the onset of transfer initiation and this phenomenon was exploited as in previous work (Possoz et al., 2001). Here, the status of pSAM2 was analysed during mating with recipient strains harbouring different regions of pSAM2. This led to the identification of a single gene, called pif for pSAM2 immunity factor, conferring the conjugal immunity to pSAM2. The candidates for the genes encoding the pSAM2 immunity factor included: (i) the genes involved in the translocation of pSAM2 from the donor to the recipient (traSA) and in its diffusion inside the recipient mycelium (spread gene spdA, B, C, D); and (ii) korSA repressing the expression of the activator Pra required for induction of pSAM2 excision and replication (Sezonov et al., 1998). KorSA, like ImpA–ImpC proteins of the S. coelicolor integrative element SLP1, belongs to the GntR repressor family. Hagège et al. (1999) have shown that introduction of the SLP1 impA–impC genes into a recipient strain reduces transfer efficiency to 2.10-4, the same effect could be postulated for KorSA. We investigated the effect of KorSA on pSAM2 transfer by mating with a recipient strain in which it was expressed constitutively. The transfer efficiency was 0.1; 100 times higher than that with a recipient strain containing pSAM2 (10-3). Thus, the strain containing KorSA behaved as a recipient. Moreover, it exhibited no

conjugal immunity: the pSAM2 conjugative free form was observed in the donor as during normal mating (Possoz et al., 2001). The only difference to controls was the prolonged persistence of the free forms, which seemed to be due to a dysfunction in the establishment of pSAM2 in the recipient. Indeed, such persistence has been described previously (Possoz et al., 2001) in mating with a recipient strain expressing a restriction–modification system. In this case, the transfer process was expected to be efficient but the establishment of pSAM2 in the new host was impaired, due to digestion by the restriction enzyme expressed in the recipient. Thus, the free forms persisted because recipient mycelium not harbouring pSAM2 was still present in the vicinity. The transcriptional repressor KorSA negatively controls expression of the pra gene. Pra is an activator of the replication and integration–excision functions of pSAM2 and absolutely required during mating (Sezonov et al., 1995). Thus, initiation of transfer must result in the derepression of the activator Pra most probably by relieving the action of the repressor KorSA. However, this repressor expressed both in the donor and in the recipient did not prevent transfer initiation. So, unlike the model proposed for SLP1, our findings imply that the derepression of the functions necessary for transfer are not due to the dilution of the central transcriptional repressor after fusion between a donor and a recipient cell. It is more likely that a signal transiently inactivates KorSA. We showed that a single pSAM2 gene called pif confers immunity to a recipient strain, and does so as effectively as the complete pSAM2 element. Therefore, the pif gene alone confers donor identity to a recipient strain. Reciprocally, several observations indicate that a strain containing

Fig. 7. Effect of the pif mutation on pSAM2 status. Southern blotting was done on EcoRI-digested DNA from S. lividans TK24 containing either pOS923, a wild-type derivative of pSAM2B2 (lanes 1 and 2) or pOS961, a pSAM2B2 derivative containing the inactive pif-1 allele (lanes 3 and 4), using an a-32P-labelled EcoRI 4.2 kb pSAM2 fragment containing the attP site as the probe. © 2003 Blackwell Publishing Ltd, Molecular Microbiology, 47, 1385–1393

pSAM2 conjugal immunity 1391 pSAM2 with an inactivated pif gene behaves as a recipient strain (without making presumptions about its capacity to transfer pSAM2). This is the case for S. lividans (pTS89) in which pra and korSA are also deleted. This could also be the case for S. lividans harbouring pSAM2 with an inactivated pif gene in which the constant presence of the free form of pSAM2 could be interpreted as the continuous activation of the transfer process. This activation could be due to the fact that within a colony, the mycelium hyphae are in contact and because of pif inactivation, they recognize each other as a possible recipient. Isolated colonies of S. lividans harbouring a pSAM2 with an inactivated pif gene exhibit a particular phenotype (blood-red colour and poor sporulation), which is reminiscent of the pocks associated with conjugative transfer. For this reason, this phenotype was named ‘self-pocks’ to evoke the possible constitutive activation of the self-transfer process. There is no known DNA binding motif or a membrane domain in the Pif protein sequence. Thus, it is unlikely that Pif is a repressor like KorSA or localized at the bacterial surface to interfere directly with the putative pSAM2 products of a neighbouring donor strain. Moreover, Pif does not require other pSAM2 elements for activity, but requires its nudix motif. The nature of the initiation process is not known but the nudix protein Pif may act by modifying a host component in the donor strain, thus preventing recognition between donors’ strains. An example of such a modification has been described by Kloosterman et al. (2002) as a novel mechanism for enzyme activity regulation. In Bacillus methanolicus, the nudix hydrolase protein ACT stimulates the activity of the alcohol (methanol) dehydogenase (MDH) by hydrolytic removal of the nicotinamide mononucleotide NMN(H) moiety of the NAD(H) cofactor of MDH.

The expression of pif, like that of korSA, is not induced in the conjugative condition as transcription is detected in monoculture of pSAM2 donor strains. These clustered genes constitute a functional module, maintaining pSAM2 in a ‘prophage’ state under non-conjugal conditions. However, KorSA acts on pSAM2 elements present in the mycelium in which it is produced whereas Pif participates in intercellular signalling, rendering its host strain unable to induce the transfer process in neighbouring donor strains. Another integrative element, pMEA300 from Amycolatopsis methanolica, contains a gene called mut which encodes a protein similar to Pif and is also a member of the nudix family. A stimulation of spontaneous mutagenesis under stress conditions has been associated with the product of this gene (Vrijbloed, 1996) but its effect on pMEA300 transfer was not investigated. The small conserved nudix motif is widespread in species as diverse as virus and humans. Few functions have been attributed to this family, which includes invasive protein (Bessman et al., 2001), but many are involved in cell detoxification (Bessman et al., 1996). However, Pif is the first nudix protein shown to be involved in a bacterial conjugation process.

Experimental procedures Bacterial stains and plasmids The Streptomyces strains used were S. lividans 66 derivatives: TK23 (spc-1), spectinomycin resistant, and TK24 (str-6), streptomycin resistant (Hopwood et al., 1983). Escherichia coli DH5a (Hanahan, 1983) was used for cloning experiments and plasmid propagation. Plasmids used in this study are listed in Table 1.

Table 1. List of the plasmids used in this study. Plasmids

Description

pSAM2B2 pSAM2B3 pOS923 pTS89 pTS135 pOS947 pOS555

pOS961 pTO1*

pSAM2 present only integrated at the attB site pSAM2 present both integrated and free, due to over expression of pra pOS536, whole pSAM2B2 derivative, containing Ωhyg at the HindIII site Deletion derivative of pOS923 in which orf131 (pif), korSA and pra are inactivated pSAM2B3 containing Ωhyg in the unique AvrII site salM gene cloned in the integrative, non-conjugative vector pTO1* (Sezonov et al., 1998) BamHI(154)–BamHI(1746) fragment from pSAM2 containing korSA and orf131 (pif) cloned in the integrative vector pTO1* (Sezonov et al., 1998) BamHI(154)–BamHI(1746) fragment from pSAM2 containing korSA, cloned in the integrative vector pTO1* (Sezonov et al., 1998); orf131 (pif) present in the fragment was inactivated by deletion of the BglII(297)–BsrGI(955) fragment BamHI(154)–NcoI(1152) fragment from pSAM2 containing orf131 cloned in the integrative vector pTO1* (Sezonov et al., 1998) BamHI(154)–NcoI(1152) fragment from pOS961 containing the mutant pif-1, cloned in the integrative vector pSET152 (Bierman et al., 1992) pif-1 mutant derivative of pOS923 Shuttle φC31 derivative vector, replicative in E. coli, integrative and non-conjugative in Streptomyces

pSET152

Shuttle φC31 derivative vector, replicative in E. coli, integrative in Streptomyces.

pOS556

pOS680 pOS960

© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 47, 1385–1393

Source Pernodet et al. (1984) Sezonov et al. (1995) Possoz et al. (2001) Smokvina et al. (1991) This work Possoz et al. (2001) This work Sezonov et al. (2000)

This work This work This work Sezonov et al. (1995) Tabakov et al. (1994) Bierman et al. (1992)

1392 C. Possoz et al. Culture, transformation conditions and DNA isolation Culture and other techniques for Streptomyces subsp. were as described by Kieser et al. (2000) and for E. coli as described by Sambrook et al. (1989). Hickey Tressner (HT) medium (Pridham et al., 1957) was used for Streptomyces cultures and mating. Streptomyces transformants carrying the thiostrepton resistance gene (tsr) (Thompson et al., 1980) were selected using 50 mg ml-1 of thiostrepton. Transformants carrying the Whyg cassette (Blondelet-Rouault et al., 1997), conferring hygromycin resistance were selected using 200 mg ml-1 of hygromycin B.

Status of pSAM2 and transfer efficiency To analyse pSAM2 status, total DNA was extracted from mixed cultures on solid medium. Mycelium was obtained from separate HT liquid cultures of the two strains after 48 h. Donor and recipient mycelium were fragmented, mixed and then spread on HT agar plates covered with cellophane membranes. To calculate transfer efficiency the spores of two S. lividans strains were mixed on HT solid medium, in equal proportion. After sporulation, transfer efficiency was calculated as the ratio of recipient cells containing pSAM2 to total recipient cells using appropriate selective medium. Experiments were repeated at least three times.

lapping oligonucleotides containing the desired mismatches and oligonucleotides containing a unique restriction site in the Asp718I fragment to allow easy replacement of an internal portion. The first PCR round was carried out with oligonucleotides K4 opp and MD-2 and oligonucleotides MD-3 and MD-4 using the Asp718I- Asp718I fragment of pSAM2B2 (positions 129–2121 in the sequence accession no. AJ005260) cloned in the E. coli vector pUC18 as the template. The second round was carried out with the oligonucleotides K4 opp and MD-4 using the two PCR products of the first round as template. The final PCR product was digested with SacII and SgrAI and used to replace the corresponding fragment in the cloned Asp718I–Asp718I fragment of pSAM2B2. To construct pOS961, this mutated Asp718I fragment was used to replace the wild-type one in pOS923. The oligonucleotide MD-4 (5¢-CGTCCGCGGGGGCCTC CATGGGGCCGATGT-3¢) contains a SacII site indicated in bold. The SgrAI site, and oligonucleotides MD-2 and MD-3 are indicated in Fig. 5.

pSAM2 sequence All restriction sites and positions of the cloned fragments refer to the sequence submitted previously to the EMBL data bank under accession no. AJ005260.

Acknowledgements RNA isolation, High resolution S1 mapping Total RNA was isolated from the Streptomyces strains and high resolution S1 mapping was performed as described by Kieser et al. (2000). The probe was prepared by the method of Raibaud et al. (1991). To obtain a ssDNA corresponding to the presumed promoter region of the pif gene, the Asp718I (129)-Asp718I (2121) fragment was inserted into Asp718I site of the M13mp18 vector and its ssDNA was isolated (the orientation of the cloned fragment was checked to provide the desired strand DNA). To synthesize the second strand, the ssDNA was annealed with labelled oligonucleotide K4 opp. Synthesized double-stranded DNA was directly digested with NcoI, giving a labelled fragment of 440 bp. Total RNA was hybridized with this DNA fragment and treated with nuclease S1. The same oligonucleotide (K4opp, Fig. 5) that had been used to synthesize the labelled fragment of 440 bp was used as sequencing primer. About 40 mg of total RNA was used for each reaction.

DNA sequencing The dideoxy method of Sanger et al. (1977) as modified by Biggin et al. (1983) was used. To overcome the problems of band compression in sequencing gels due to the high stability of GC-rich structures, the Deaza T7 sequencing kit obtained from Pharmacia LKB was used. [a-35S]-dATP was obtained from Amersham.

Construction of pOS961 The directed mutagenesis of the pif gene was performed by two rounds of polymerase chain reaction (PCR) using over-

We would like to thank A. Raynal for critically reading the manuscript and for helpful discussions. C.P. received a doctoral fellowship from the Ministère de la Recherche and a postdoctoral fellowship from CNRS.

References Achtman, M., Kennedy, N., and Skurray, R. (1977) Cell–cell interactions in conjugating Escherichia coli: role of traT protein in surface exclusion. Proc Natl Acad Sci USA 74: 5104–5108. Bessman, M.J., Frick, D.N., and O'Handley, S.F. (1996) The MutT proteins or ‘Nudix’ hydrolases, a family of versatile, widely distributed, ‘housecleaning’ enzymes. J Biol Chem 271: 25059–25062. Bessman, M.J., Walsh, J.D., Dunn, C.A., Swaminathan, J., Weldon, J.E., and Shen, J. (2001) The gene, ygdP, associated with the invasiveness of Escherichia coli K1, designates a nudix hydrolase (Orf 176) active on Adenosine (5¢) pentaphospho (5¢) adenosine. J Biol Chem 30: 30. Bierman, M., Logan, R., O’Brien, K., Seno, E.T., Rao, R.N., and Schoner, B.E. (1992) Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene 116: 43–49. Biggin, M.D., Gibson, T.J., and Hong, G.F. (1983) Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci USA 80: 3963–3965. Blondelet-Rouault, M.H., Weiser, J., Lebrihi, A., Branny, P., and Pernodet, J.L. (1997) Antibiotic resistance gene cassettes derived from the omega interposon for use in E. coli and Streptomyces. Gene 190: 315–317. © 2003 Blackwell Publishing Ltd, Molecular Microbiology, 47, 1385–1393

pSAM2 conjugal immunity 1393 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. Boorstein, W.R., and Craig, E.A. (1989) Primer extension analysis of RNA. Methods Enzymol 180: 347–369. Byrd, D.R., and Matson, S.W. (1997) Nicking by transesterification: the reaction catalysed by a relaxase. Mol Microbiol 25: 1011–1022. Davies, J. (1994) Inactivation of antibiotics and the dissemination of resistance genes. Science 264: 375–382. Dunny, G.M. (1990) Genetic functions and cell–cell interactions in the pheromone-inducible plasmid transfer system of Enterococcus faecalis. Mol Microbiol 4: 689–696. Frost, L.S., Ippen-Ihler, K., and Skurray, R.A. (1994) Analysis of the sequence and gene products of the transfer region of the F sex factor. Microbiol Rev 58: 162–210. Hagège, J., Pernodet, J., Friedmann, A., and Guérineau, M. (1993) Mode and origin of replication of pSAM2, a conjugative integrating element of Streptomyces ambofaciens. Mol Microbiol 10: 799–812. Hagège, J., Boccard, F., Smokvina, T., Pernodet, J.-L., Friedmann, A., and Guérineau, M. (1994) Identification of a gene encoding the replication initiator protein of the Streptomyces integrating element, pSAM2. Plasmid 31: 166–183. Hagège, J.M., Brasch, M.A., and Cohen, S.N. (1999) Regulation of transfer functions by the imp locus of the Streptomyces coelicolor plasmidogenic element SLP1. J Bacteriol 181: 5976–5983. Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166: 557–580. Hopwood, D.A., and Kieser, T. (1993) Conjugative plasmids in Streptomyces. In Bacterial Conjugation. D. B. Clewell (ed.). New York: Plenum Press, pp. 293–311. Hopwood, D.A., Kieser, T., Wright, H.M., and Bibb, M.J. (1983) Plasmids, recombination and chromosome mapping in Streptomyces lividans 66. J Gen Microbiol 129: 2257–2269. Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F., and Hopwood, D.A. (2000) Practical Streptomyces Genetics. Norwich: John Innes Foundation. Kloosterman, H., Vrijbloed, J.W., and Dijkhuizen, L. (2002) Molecular, biochemical and functional characterization of a Nudix hydrolase protein that stimulates activity of a nicotinoprotein alcohol dehydrogenase. J Biol Chem 27: 27. Lanka, E., and Wilkins, B.M. (1995) DNA processing reactions in bacterial conjugation. Annu Rev Biochem 64: 141– 169. Minkley, Jr, E.G. and Ippen-Ihler, K. (1977) Identification of a membrane protein associated with expression of the surface exclusion region of the F transfer operon. J Bacteriol 129: 1613–1622. Pernodet, J.L., Simonet, J.M., and Guérineau, M. (1984) Plasmids in different strains of Streptomyces ambofaciens: free and integrated form of plasmid pSAM2. Mol Gen Genet 198: 35–41. Possoz, C., Ribard, C., Gagnat, J., Pernodet, J.L., and Guérineau, M. (2001) The integrative element pSAM2 from Streptomyces: kinetics and mode of conjugal transfer. Mol Microbiol 42: 159–166. Pridham, T.G., Anderson, P., Foley, C., Lindenfelser, L.A., © 2003 Blackwell Publishing Ltd, Molecular Microbiology, 47, 1385–1393

Hesseltine, C.W., and B.R. (1957) A selection of media for maintenance and taxonomic study of Streptomyces. Antibiotics Annu 1956–57: 947–953. Raibaud, A., Zalacain, M., Holt, T.G., Tizard, R., and Thompson, C.J. (1991) Nucleotide sequence analysis reveals linked N-acetyl hydrolase, thioesterase, transport, and regulatory genes encoded by the bialaphos biosynthetic gene cluster of Streptomyces hygroscopicus. J Bacteriol 173: 4454–4463. Raynal, A., Tuphile, K., Gerbaud, C., Luther, T., Guérineau, M., and Pernodet, J.L. (1998) Structure of the chromosomal insertion site for pSAM2: functional analysis in Escherichia coli. Mol Microbiol 28: 333–342. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Laboratory Press. Sanger, F., Nicklen, S., and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467. Sezonov, G., Hagège, J., Pernodet, J.L., Friedmann, A., and Guérineau, M. (1995) Characterization of pra, a gene for replication control in pSAM2, the integrating element of Streptomyces ambofaciens. Mol Microbiol 17: 533–544. Sezonov, G., Duchene, A.M., Friedmann, A., Guérineau, M., and Pernodet, J.L. (1998) Replicase, excisionase, and integrase genes of the Streptomyces element pSAM2 constitute an operon positively regulated by the pra gene. J Bacteriol 180: 3056–3061. Sezonov, G., Possoz, C., Friedmann, A., Pernodet, J.L., and Guérineau, M. (2000) KorSA from the Streptomyces integrative element pSAM2 is a central transcriptional repressor: target genes and binding sites. J Bacteriol 182: 1243– 1250. Shimokawa, H., Fujii, Y., Furuichi, M., Sekiguchi, M., and Nakabeppu, Y. (2000) Functional significance of conserved residues in the phosphohydrolase module of Escherichia coli MutT protein. Nucleic Acids Res 28: 3240–3249. Smokvina, T., Boccard, F., Pernodet, J.L., Friedmann, A., and Guérineau, M. (1991) Functional analysis of the Streptomyces ambofaciens element pSAM2. Plasmid 25: 40–52. Tabakov, V., Voeikova, T.A., Tokmakova, I.L., Bolotin, A.P., Vavilova, E., Lomovskaia, N., and D. (1994) Intergeneric Escherichia coli-Streptomyces conjugation as a means for the transfer of conjugative plasmids into producers of antibiotics chlortetracycline and bialaphos. Genetika 30: 57– 61. Thompson, C.J., Ward, M.J., and Hopwood, D.A. (1980) DNA cloning in Streptomyces: resistance genes from antibioticproducing species. Nature 286: 525–527. Vrijbloed, J.W. (1996) Functional analysis of the integrative plasmid pMEA300 of the Actinomycete Amycolatopsis methanolica. PhD Thesis, Department of Microbiology, University of Groningen, Groningen. Weidlich, G., Wirth, R., and Galli, D. (1992) Sex pheromone plasmid pAD1-encoded surface exclusion protein of Enterococcus faecalis. Mol Gen Genet 233: 161–168. Wu, L.J., Lewis, P.J., Allmansberger, R., Hauser, P.M., and Errington, J. (1995) A conjugation-like mechanism for prespore chromosome partitioning during sporulation in Bacillus subtilis. Genes Dev 9: 1316–1326.