Molecular Microbiology (1995) 17(3), 533-544
Characterization of pra, a gene for replication control in pSAM2, the integrating element of Streptomyces
ambofaciens Guennadi Sezonov, Juliette Hag6ge, t Jean-Luc Pernodet, * Annick Friedmann and Michel Gu~rineau Laboratoire de Biologie et G~n~tique Moleculaire, Institut de G~n~tique et Microbiologie, URA CNRS 1354, B~timent 400, Universite Paris-Sud, F-91405 Orsay Cedex, France. Summary pSAM2 is a genetic element found integrated in Streptomyces ambofaciens (B2) and additionally in a replicating form in two mutants B3 and B4. The presence of the pSAM2 replicating form in these mutants was the result of mutations located on pSAM2 in the pra locus, named pra3 and pra4, respectively. The pra gene is not directly involved in replication, but its inactivation led to the disappearance of the pSAM2 free form; therefore, it was considered as a replication regulator. The pra3 and pra4 mutations were located in the pra promoter and were shown to be point substitutions that increase the promoter strength. The replication regulator role of pra was demonstrated by the fact that its constitutive expression in cells harbouring pSAM2B2, which is normally only integrated, led to the appearance of the pSAM2 replicating form. Northern analysis showed that the pra gene transcript can be detected only for the replicating mutants B3 and B4 and that the three adjacent genes korSA, pra and traSA were transcribed separately. As replication of pSAM2 is not needed for its maintenance but is an indispensable stage of its transfer, the pra gene, described formally as an activator of pSAM2 replication, is patently involved in pSAM2 transfer. Introduction
Streptomyces spp. are Gram-positive mycelial soil bacteria notable for their capacity to synthesize a large variety of secondary metabolites, for undergoing a complex life Received 23 February, 1995; revised 31 March, 1995; accepted 5 April, 1995. tPresent address: Departmentof Genetics, Stanford University of Medicine, Stanford, California 94305, USA. *For correspondence. E-mail
[email protected]; Tel. (1) 69 41 69 13; Fax (1) 69 41 66 78. © 1995BlackwellScienceLtd
cycle of morphological differentiation and for having DNA with a high (about 73%) G + C content. pSAM2 is an integrating genetic element from Streptomyces ambofaciens (Pernodet et aL, 1984) showing an integration system very similar to that of temperate phages (Boccard et aL, 1989) and able to integrate into the host chromosome at the attB site that overlaps a putative pro-tRNA gene (Mazodier et aL, 1990). Its sitespecific recombination system consists of the attachment site attP and the int and xis genes responsible for the integration and excision at the attB site. It also possesses functions common to Streptomyces plasmids (reviewed by Hopwood et aL, 1987; Hopwood and Kieser, 1993; Wohlleben and Muth, 1993) such as replication, transfer, pock formation and mobilization of chromosomal markers (Smokvina et aL, 1991). The major gene for the transfer, traSA, the genes spdA, B, C and D possibly involved in intramycelial transfer and the gene korSA, involved in a kil-korsystem associated with transfer, have been identified (Hag~ge et al., 1993a). All known integrating genetic elements of actinomycetes are stably maintained in the chromosome after site-specific integration (reviewed by Omer and Cohen, 1989). The free form of these elements may co-exist together with the integrated form (pSAM2, Pernodet etaL, 1984; pSG1, Cohen etaL, 1985; pMEA100, Moretti etaL, 1985; pSE101, Brown etaL, 1988; plJ408, Sosio etaL, 1989; pSE211, Brown et aL, 1990; SLP1, Brasch et aL, 1993). In contrast to their integration functions which have been well studied, the role of the replication and the mechanism of its control remain obscure. S. ambofaciens JI3212 (strain B3) harbours a mutant form of pSAM2 named pSAM2B3, which is present in both the free and integrated forms (Pernodet et aL, 1984). The existence of the free form should result from replication since pSAM2B3 derivatives that have lost the integrating functions can replicate as covalently closed circular molecules in the absence of the integrated form (Smokvina et aL, 1991). Hag~ge et aL (1993b; 1994) demonstrated that it replicates by a rolling-circle replication (RCR) mechanism (Gruss and Ehrlich, 1989). Two separate regions involved in pSAM2 replication have previously been characterized. One contains the gene repSA encoding the replication initiator protein (Hagege etaL, 1994) and the other one contains the plus origin of replication (ori)
534
G. Sezonov et al.
Table 1. Plasmids.
Name
Construction
Form
Reference
pSAM2B2
pSAM2 from S. ambofaciens ATCC15154 (B2)
Int
pSAM2B3
A mutant of pSAM2 from S. ambofaciens JI3212 (B3)
Free, int
pSAM2B4
A mutant of pSAM2 from S. ambofaciens RP181110(B4)
Free, int
pTS39
plJ39 inserted in the EcoRl(6.66) site of pSAM2B3
Free, int
pTS42
~ interposon in Bglll(5.11) of pTS39
Free, int
pTS65
Deletion of Kpnl(5.29)EcoRl(6.66) of pTS39
Free, int
pTS84
The Kpnl(3.30) site digested by Klenow polymerase in pTS65
Int
pTS89
Deletion of the Kpnl(3.30)Kpnl(5.29) of pTS39
Int
pTS129
The EcoRl(17.02) site of pTS39 was filled by Klenow polymerase and the hyg gene (Zalacain et aL, 1986) fragment filled also by Klenow was cloned instead of plJ39 in the EcoRl(6.66) site Deletion of the BspHl(3.16)BspHl(3.39) fragment of pTS129 and cloning of pBR329+ tsr gene in the EcoRI site of the hyg gene (Zalacain et aL, 1986) Deletion variant of pOS11 (Simonet et aL, 1987) regularly loosing pBR329 and about 600 bp of pOS7 (Simonet et aL, 1987) in S. fividans. High copy number in Streptomyces Cloned from the chromosome of S. lividans TK24[pSAM2•2] the 18.5 kb PstI-Pstl fragment containing pSAM2B2 was inserted in the Pstl site of pBR329 The EcoRI-EcoRI fragment of pOS221 cloned instead of EcoRl(10.86)-EcoRl(6.66) fragment of pTS42 The Kpnl(3.3O)-Kpnl(5.29)
Free, int
Pernodet et al. (1984) Pernodet et aL (1984) Pernodet et aL (1984) Smokvina et aL (1991) Smokvina et aL (1991) Smokvina et aL (1991) Smokvina et aL (1991) Smokvina et aL (1991 ) This work
pTS136
pOS11A
pOS221
pOS521
pOS522
pOS524
pOS525
pOS526
pOS527
pOS531
Int
This work pOS532
Free, int
Smokvina et aL (1991)
Int
Boccard et aL, (1988)
pOS539
pOS541 pOS543
Int
This work, Fig. 3
fragment of pOS221 cloned in the Kpnl(3.30) site of pTS89 The Kpnl(3.30)-Kpnl(5.29) 'mosaic' fragment cloned in pTS89, consists of the Kpnl(3.30)-Nrul(3.63) fragment from pSAM2B2 and Nrul(3.63)-Kpnl(5.29) from pSAM2B3 The Kpnl(3.30)-Kpnl(5.29) 'mosaic' fragment cloned in pTS89, consists of the Kpnl(3.30)-Nrul(3.63) fragment from pSAM2~ and Nrul(3.63)-Kpnl(5.29) from pSAM2B2 Fragment of pSAM2B2 (nt 21 to 159 in Fig. 2) cloned after PCR amplification as a BamHI-EcoRI fragment in the BamHI/EcoRI sites of plJ487. Contains the pSAM2B2 pra gene promoter Fragment of pSAM2e3 (nt 21 to 159 in Fig. 2) cloned after PCR amplification as a BamHI-EcoRI fragment in the BamHI/EcoRI sites of plJ487. Contains the pSAM2B3 pra gene promoter Coding part of the pra gene with a possible transcription terminator region (nt 160 to 669 in Fig. 2) cloned after a PCR amplification in the BamHI/Hindlll sites of pIJ487 ermE* promoter cloned as an EcoRI-BamHI fragment upstream of the pra gene in pOS531 Complete sequence of the pra gene with the first 52 transcribed but not translated nucleotides and possible transcription terminator region (nt 107 to 669 in Fig. 2) cloned in the BamHI/Hindlll sites of plJ487 ermE* promoter cloned as an EcoRI-BamHI fragment in pOS539 Fragment of pSAM2B4 (nt 21 to 159 in Fig. 2) cloned after PCR amplification as a BamHI-EcoRI fragment in the BamHI/EcoRI sites of plJ487. Contains the pSAM2B4 pra gene promoter
Int Int
This work Fig. 3 This work, Fig. 3
Free, int This work Fig. 3
Free
This work
Free
This work
Free
This work, Fig. 7A
Free
This work, Fig. 7A
Free
This work, Fig. 7A
Free
This work, Fig, 7A
Free
This work
Int, integrated; nt, nucleotide.
(Hag~ge et aL, 1993b). off is the site used to start the synthesis of a single-stranded DNA (ssDNA) replication intermediate corresponding to one strand of pSAM2. Derivatives affected either in repSA function or in or/are unable to transfer (Smokvina et aL, 1991; Hag~ge et aL, 1994). This shows that replication (which is not needed for pSAM2 maintenance) is absolutely required for transfer. Therefore replication, which provides the unique
mode of plasmid maintenance, plays a different role for integrating genetic elements. After transfer, pSAM2 is found integrated in both the donor and recipient chromosomes. This requires a transient activation of the replication together with the site-specific integration/excision functions and supposes specific regulation mechanisms. In this study, we have studied the mutations in two independent mutants of pSAM2, pSAM283 and pSAM2B4, ~) 1995 Blackwell Science Ltd, Molecular Microbiology, 17, 533-544
Regulation of pSAM2 replication 535 PvuII 0.00 Eco RI 10.86 SmaI 10.59 / | SmaI 0.:
Replication
SmaI 0.53 Bst Eli 0.63
_ / ApaLI 0.95
•
~o
~ sixm I spdA
r~pSA
SmaI 1.85 NruI 1.95
err84 Nru I8.62 ~
mrxis pSAM2
int
Bg1I/7.77
traSA
10.92 kb
. ApaLI 2.68
Replication NruI 3.07 pra 1 ~ - BspHI3.16 ~-,,,,,~" Kpn 13.30 ,g~ - BspHI 3.39 ~" ApaLl(only in B2) 3.57 korSA " Nru 1 3.63 BamHI 3.67
att7.58
Bam HI 7.35 Barn HI 7.27
orf154 o r i ~ ~ \
SmaI 6.93 Eco RI 6.66 Barn HI 6.26 ~B ApaLI 5.87 BamHI 5.41 Kpn 15.29
SacI 4.31 SacI 4.58
SacI 4.68 gllI 5.11
4 7
Replication
~
BamHI 5.26 Fig. 1. Map of pSAM2 showing the ORFs with identified or unknown functions and the attP site. The regions involved in replication are indicated. Only the restriction sites mentioned in the text and the Bglll, BstEII, Pvull, Sacl and Smal sites are shown. The restriction site ApaLl(3.57) was found only in pSAM2s2. The numbers indicate the position (in kilobases) of the restriction sites in the sequence of the whole plasmid.
characterized by the co-presence of replicating and integrated forms. This led to the identification of a gene involved in pSAM2 replication activation and called for this reason pra. This gene was previously designated rmf (Hag6ge et aL, 1993a). This represents the first step in characterizing and dissecting the complex regulatory mechanisms allowing co-ordinated expression of pSAM2 functions. Results
Identification of the pra locus between the genes korSA and traSA Smokvina et aL (1991) demonstrated that digesting by Klenow polymerase of the Kpnl(3.30) site (pTS84, Table 1) located in the pSAM2 region between the genes korSA and traSA (Fig. 1), which are involved in pSAM2 transfer (Hag6ge et aL, 1993a), resulted in the disappearance of the replicating form without affecting the integration. This showed that this region was involved in the replication of pSAM2B3. Its sequence was determined (Fig. 2A) and analysed using a program similar to FRAME (Bibb et aL, 1984), based on the biased codon usage in © 1995 Blackwell Science Ltd, Molecular Microbiology, 17, 533-544
organisms having a high G + C content (Wright and Bibb, 1992). Analysis revealed an open reading frame (ORF) with the expected Streptomyces codon usage and this ORF was named pra. To confirm the rote of pra in replication, the BspHl(3.16)-BspHl(3.39) fragment (Fig. 2A), internal to the pra coding region, was deleted giving pTS136. As expected if pra was involved in replication, this deletion variant pTS136 derived from the replicative pTS129 (Table 1) was only integrative. The derivatives pTS84 and pTS136 have both lost the ability to form pocks and to transfer. The pra gene is the third element involved in pSAM2 replication. As it has been possible to build an artificial minimal replicon devoid of pra (Hag6ge et aL, 1994), it is not directly implicated in the machinery of replication. However in this minimal replicon, the sequence upstream of repSA was changed and the repSA gene was probably not under its normal expression control. This suggests a regulatory role for the pra gene. Analysis of the pra gene sequence The 840bp sequence starting at the site BamHI (3.67) towards the traSA gene (Fig. 1) is presented (Fig. 2A).
536
G. Sezonovetal.
A stop codon of the korSA gene
BamHI(3.~)
Nr~(3.63)
1GGATCCCAGCAGCCGACTGACGACCGCTCAACTCCTCACAGCCCGTCGCGAGTTCTCTGT
. . . .
. . . . . . . . . .
÷,
61CGCGGCGGGTTGACTCATGTATAG~kGTGGT(~ACTCTTCTTCATGTCACTCATATACAT GCGCCGCCCAACTGAGTACATATCcTCACCACqTGAGAAGAAGTACAGTGAGTATATGTA
-----"A~U(3.57)
_.~
RBS fM R Q I P V 121GAGTGACGGAGTCCAGCCTCTATAGAGGAGTGATCCGCTGTGCGTCAGATCCCCGTCGAC CTCACTGCCTCAGGTCGGAGATATCTCCTCACTAGGCGACACGCAGTCTAGGGGCAGCTG
D
T S A A T V M V A K T P E P K V K D R ]81ACCTCCGCCGCAACCGTGATGGTCGCCAAGACTCCGGAGCCGAAGGTGAAGGACCGCCGG TGGAGGCGGCGTTGGCACTACCAGCGGTTCTGAGGCCTCGGCTTCCACTTCCTGGCGGCC B~HI(3.39) T G E L A V D A E T G A K L M T V N V 241ACCGGTGAGCTGGCCGTCGACGCCGAGACCGGTGCCAAGCTCATGACCGTGAACGTGATG TGGCCACTCGACCGGCAGCTGCGGCTCTGGCCACGGTTCGAGTACTGGCACTTGCACTAC
R
]R
F A A N D E V E I L S V T V P E T G I 301TTCGCGGCCAACGACGAAGTCGAGATTCTGTCCGTGACCGTCCCGGAGACCGGTATCTCC AAGCGCCGGTTGCTGCTTCAGCTCTAAGACAGGCACTGGCAGGGCCTCTGGCCATAGAGG
M
S
Kp~(3.30) G E L A M G T P V A L T G L I A R P W 361 G G T G A A C T G G C C A T G G G T A C C C C G G T C G C G C T G A C G G G G C T C A T C G C C C G G C C G T G G G A G CCACTTGACCGGTACCCATGGGGCCAGCGCGACTGCCCCGAGTAGCGGGCCGGCACCCTC N
E
F
N
G
Q
K
R
H
G
I
A
F
R
A
V
A
V
T
E
S
421AACGAGTTCAACGGCCAGAAGCGGCACGGCATCGCGTTCCGCGCGGTCGCGGTCACGTCG TTGCTCAAGTTGCCGGTCTTCGCCGTGCCGTAGCGCAAGGCGCGCCAGCGCCAGTGCAGC
Fig. 2. Nucleotide sequences. A. Nucleotide sequence of the pra gene from pSAM2Ba. The numbers on the left indicate nucleotide positions. Some restriction sites are shown, the corresponding numbers in parentheses indicate the position (in kilobases) of the restriction sites in the sequence of the whole plasmid (Fig. 1). The predicted amino acid sequence encoded by the pra gene is shown above the nucleotide sequence, fM indicates the first predicted translated amino acid from the GTG codon and the potential RBS. The inverted repeat (IR) is indicated by arrows. The common sequence found upstream of korSA, pra and traSA genes is indicated by a dotted line. The position of the first transcribed nucleotide is indicated by +1. The positions of the predicted - 1 0 and - 3 5 boxes of the pra gene promoter are underlined. The point substitutions found in the pra gene from pSAM2B3 and pSAM2B4 are boxed and indicated above the sequence and their origins are shown in parentheses. The stop codon TGA of the korSA gene corresponds to the nucleotides 1043-1045 of Fig. 2B in Hagege et aL (1993a). The traSA gene putative start codons compatible with pra and traSA transcription analysis are boxed. The start codon at the positions 822-824 was the proposed start in Hag~ge et aL (1993a). B. Alignment of the sequences found upstream of the genes korSA, pra and traSA. The numbers indicate the distance between the end of the sequence and the first proposed translation start codon of the genes.
B~HI(3.16) L T A A G S K A A . ........ 481 C T G A C C G C T G C G G G C T C G A A G G C T G C C T G A T C A T G A C G T G G T T C A T G G T C G C T G T G G T T G GACTGGCGACGCCCGAGCTTCCGACGGACTAGTACTGCACCAAGTACCAGCGACACCAAC
541 T G G T C G T C G C T G C T G C G G G T C T C C T G C G G T G G C G G C G C C C C G C C T G G T A C T G G C T C A C C T ACCAGCAGCGACGACGCCCAGAGGACGCCACCGCCGCGGGGCGGACCATGACCGAGTGGA
Nr~(3.07) 601TCGGGGCCCTGGTCGCGAC~-~CGGGTCCTGGTCCGCTACGCCTCGGTC~'~-~AAGCGT AGCCCCGGGACCAGCGCTGCCACGCCCAGGACCAGGCGATGCGGAGCCAGTACCTTCGCA
661GCGGGCTGACGGTCCCGCCCTCACGCTGGCGGCTGGCTCTGGCCCG~GCGAATCGGC CGCCCGACTGCCAGGGCGGGAGTGCGACCGCCGACCGAGACCGGGCCTACCGCTTAGCCG
721 C G G C G C C T G A G T C C C G G C C G C C G C G C A T C T T G C G G T T A C G T C C C A C T C G T A C C G G C C T G G GCCGCGGACTCAGGGCCGGCGGCGCGTAGAACGCCAATGCAGGGTGAGCATGGCCGGACC
781 T C C T G C G G C T C A A G C T C C G G C C G G G A C A G G A T G C C T T C G A C ~ - ~ C C G C C A C C A C C G A C C AGGACGCCGAGTTCGAGGCCGGCCCTGTCCTACGGAAGCTGCACCGGCGGTGGTGGCTGG
B
CTGTGGTTGACTCATGTAcAGGAG-GGTTGACTCATGTAtAGGAG~ CTGTGGTTG
2 1 bp--
startcodon of korSA
7 2 bp
startcodonofpra
80 b p
startcodon of traSA
© 1995 Blackwell Science Ltd, Molecular Microbiology, 17, 533-544
Regulation of pSAM2 replication 537 This sequence displays one ORF with typical Streptomyces codon usage, which may encode a 116 amino acid protein with a predicted M r of 12119. Among several possible translation start codons, we have chosen as the most probable that located 9bp downstream of the gAGGAG sequence (Fig. 2A), corresponding to the consensus streptomycete ribosome-binding site (RBS) (Strohl, 1992). In addition to the already described inverted repeat (IR) adjacent to the pra gene (Hag~ge et aL, 1993a), a new one upstream of the pra coding region is indicated in Fig. 2A. Direct repeats of 20bp with only one mismatch were found upstream of the korSA and pra genes, and part of this sequence is also present upstream of traSA (Fig. 2B). During determination of the pra gene sequence, the sequence of the traSA gene located downstream was corrected. With this corrected sequence, the traSA coding part could begin up to 201 bp upstream of the previously published initiation codon (Hag~ge et aL, 1993a). The predicted Pra sequence did not show any significant similarities with proteins in the data bases.
ambofaciens ATCC15154 (B2) (Pernodet et aL, 1984). The inability to detect the replicating form of pSAM2B2 does not mean that it is not able to replicate since it can transfer, a process that strongly depends on replication (Smokvina et aL, 1991). For pSAM2B3 and pSAM2B4, the presence of the replicating form is the result of mutations located in the pSAM2 sequence. This was shown by the fact that the status (integrated only or integrated and free) of pSAM2 depends on the pSAM2 sequence (pSAM2B2, pSAM2B3 or pSAM2B4, and not on the host strain (Pernodet et aL, 1984; Boccard et aL, 1988). With the mutations carried by pSAM2B3 and pSAM2B4, the replicating form co-exists with the integrated one. These mutations do not correspond to extensive deletions or rearrangements since no change in the restriction maps of pSAM2B2, pSAM2B3 or pSAM2B4 has been detected (Boccard et aL, 1988; Smokvina et aL, 1991). To locate the mutation in pSAM2B3, w e constructed a series of 'mosaic' plasmids composed of DNA fragments of pSAM2B2 and pSAM2B3 (Fig. 3) (see the details of the 'mosaic' plasmid constructions in the Experimental procedures). The replacement in pTS42 (a pTS39 replicating derivative; Table 1) of the EcoRl(10.86)-EcoRl(6.66) fragment by the corresponding one from integrating pSAM2B2 led to the variant pOS521, which was only present in the
Presence of pSAM2B3 replicating form is the result of a mutation in the pra locus pSAM2 was found as an integrated element in the strain S. spdD spdC spdB spdA traSA .~--- ,~-- .~- ~ ~ I EcoRI( l O.86)
I
~
pra
korSA
I I I I KpnI(3.30) NruI(3.63 ) KpnI(5.29) EcoRI(6.66)
!
I
I
I
I
I
EcoRI
KpnI NruI
KpnI
EcoRI
I EcoRI
I I KpnI NruI
I KpnI
I EcoRI
pSAM2(B3) or pTS39 replicating+integrated pSAM2(B2)
I
[ EcoRI
I
[ EcoRI
['----~
I
I
KpnI NruI
I
I
KpnI NruI
~
EcoRI
Kp~I NruI
EcoRI
KpnI NruI
[ KpnI
I
pOSS2~ integrated
EcoRI
I KpnI
I
integrated
I
I
pOS522 integrated
EcoRI
T - - ~ KpnI
EcoRI
KpnI
EcoRI
~
p0S524
integrated
pOS525 replicating+integrated
Fig. 3. Structure and status of the 'mosaic' variants of pTS39 containing the DNA fragments from integrative pSAM2B2. The positions of ORFs in the EcoRl(10.86)-EcoRl(6.66) fragment of pSAM2 are shown by arrows. Only the restriction sites used for cloning are indicated. The white boxes correspond to the DNA of pTS39 (pSAM2B3) and the black ones to the DNA from integrated pSAM2B2. The name and the status of the recombinant plasmids are indicated on the right. © 1995 Blackwell Science Ltd, Molecular Microbiology, 17, 533-544
538
G. Sezonov et al.
integrated form (Fig. 3). For a more precise location, smaller DNA fragments were replaced. With pOS522, it was shown that the pSAM2B3 mutation is located in the Kpnl(3.30)-Kpnl(5.29) DNA fragment (Fig. 3). Finally, using the 'mosaic' variants pOS524 and pOS525, the mutation of pSAM2B3 was located inside the 324bp Kpnl(3.30)-Nrul(3.63) fragment (Fig. 3). Thus, the mutation carried by pSAM2B3 is located in the DNA fragment that includes 108bp upstream of the proposed pra gene translation start codon and a part of the pra gene coding region. This mutation was called pra3. The pra3 and pra4 mutations are point substitutions upstream of the pra gene coding region To localize the pra3 mutation more precisely, the DNA sequences of a region encompassing the Kpnl(3.30)Nrul(3.63) fragment from pSAM2B2 and pSAM2B3 were determined and compared. The only mutation found was a C to T transition located 67 bp upstream of the pra start codon (position 93 in Fig. 2A), in the Kpnl(3.30)Nrul(3.63) fragment. This base substitution is located in the recognition site for ApaLI (GTGCAC) (Fig, 2A), an enzyme that had never been previously used for pSAM2 restriction analysis. There are three ApaLI sites in pSAM2B3 and four in pSAM2B2, which allowed us to check the presence of pra3 by simple restriction analysis. We assumed that the independent mutant pSAM2B4, which exhibits the same phenotype as pSAM2B3 (coexistence of the integrated and replicating forms), would also carry the mutation in the pra locus. A derivative of pSAM2B4, pOS11A (Smokvina et aL, 1991), was sequenced in this locus. DNA sequence analysis around the Kpnl(3.30) restriction site of pOS11A revealed the presence of a G to T transversion, located 8 bp upstream of the position of the pra3 mutation (Fig. 2A). Therefore, the presence of the pSAM2B3 replicating form, and most likely the presence of the pSAM2B4 replicating form, results from point mutations upstream of the pra gene, probably in its promoter region. The mutations pra3 and pra4 are located in the pra gene promoter To locate the position of the mutations relative to the transcription start point, we performed primer-extension reactions with a 40-mer oligonucleotide hybridizing within the coding part of the pra gene (Fig. 4). Similar results were obtained irrespective of whether the RNA had been isolated from Streptomyces fividans TK24 harbouring pSAM2B3 or pOS11A (a derivative of pSAM2B4) plasmids. The transcription initiation nucleotide T (position 107,
Fig. 4. High-resolution mapping of the transcription start point of the pra gene by primer extension analysis. Primer extension reaction with 401~gRNA extractedfrom: lane 1, S. fividans TK24(pSAM2B3);lane 2, S. lividansTK24(pOS11A) (a derivative of pSAM2B4).The primer used was the 40-mer oligonucleotide 5'GGTCCTTCACC'F-rCGGCTCCGGAGTC'I-rGGCGACCATCAC-3', which is complementary to a sequencewithin the pra gene coding part, 36 bp downstream of the predicted pra gene translation start codon GTG. Each primer extension product was run on polyacrylamide gel together with the sequencing reactions of the pra gene promoter region. The same primer was used for the sequencing reactions.
indicated +1) is located 53 bp upstream from the proposed GTG-translation initiation codon (Fig. 2A). On the basis of the consensus sequence proposed for Streptomyces promoters that are similar to those recognized by Escherichia coil RNA polymerases containing the (~7osubunit ( - 10 as TAgPuPuT and - 3 5 as I-FGACPu, Strohl, 1992), we suggest that the sequence CB2/TB3ACtcT corresponds to the region - 1 0 and "I-I'GACt corresponds to the region - 3 5 (Fig. 2A). The distance between the - 1 0 and the - 3 5 boxes is 17 bp (consensus 18 bp) and between - 10 and +1 is 8bp (consensus 6bp). The mutation in pSAM2B3 modified the first nucleotide of the - 1 0 region and could influence promoter strength. The mutation in pSAM2B4 is situated in the spacer region between the - 1 0 and - 3 5 boxes. It could also alter the configuration of a secondary structure formed by inverted repeats located upstream of the coding part of the pra gene. It should be noted that the mutation in pSAM2E4 is located inside the 20bp sequence (Fig. 2A, dotted line) which is also present upstream of the coding part of the korSA gene (Fig. 2B), and part of this sequence is present upstream of the traSA gene (Fig. 2A, dotted line; Fig. 2B). The presence of similar DNA sequences © 1995BlackwellScienceLtd, MolecularMicrobiology,17, 533-544
Regulation of pSAM2 replication
539
detected only for the pSAM2B4 derivative pOS11A (data not shown). It is concluded that all three genes, korSA, pra and traSA, are transcribed independently, at least in the mycelium that is 24 h old. Comparative study of the wild-type and mutated pra promoters
Fig. 5. Northern blot of total RNA using the pra gene coding part as a hybridizationprobe. The RNAs were isolatedfrom the strains S. ambofaciens (lanes 2, 3) and S. lividans TK24 (lanes4, 5, 6) containing the followingplasmids:lanes2 and 4, pSAM2B2; lanes3 and 5, pSAM2B3; lane 6, pOSl 1A (pSAM2B4derivative).Lane 1 contains total RNA from control strain S. lividans TK24. The positions of 1 kb ladderDNA fragmentsare indicated. upstream of the korSA, pra and traSA genes may indicate a common regulation of their expression.
The DNA fragment extending from the stop codon of the korSA gene up to the pra translation start codon was considered to contain the pra gene promoter. This fragment was cloned upstream of the promoterless aph gene conferring kanamycin (Km) resistance in the promoter-probe vector plJ487 (Ward et aL, 1986) in order to estimate the relative strengths of the pra promoters. The level of the Km resistance is proportional to the strength of the transcriptional start signals preceding the aph gene (Beck et aL, 1982). Three strains of S. lividans TK24 containing the recombinant plasmids pOS526, pOS527 and pOS543, having the pra gene promoters from pSAM2B2, pSAM2B3 or pSAM2B4, respectively, were examined for their level of Km resistance. The results of the spore suspension titration are presented in Fig. 6. The two mutated promoters provided considerably higher levels of Km resistance than the pra gene promoter from pSAM2B2. Thus, the pra3 and pra4 mutations increased the strength of pra Surviving Fraction
Transcriptional analysis of the pra gene To determine the presence and size of the pra gene transcripts, Northern hybridization of the total mycelial RNA of strains S. ambofaciens and S. fividans TK24 containing different derivatives of pSAM2 was performed using the complete coding sequence of the pra gene as a probe. The results of this hybridization are presented in Fig. 5. A mRNA transcript of about 0.5kb was detected in the strains harbouring pSAM2B3 and pSAM2B4. No signal was detected for the strains harbouring pSAM2B2. The intensity of the hybridization in the case of the derivative of pSAM2B4 - pOS11A - was higher, but it must be noted that the copy number of pOS11A is higher than that of pSAM2B3 (Smokvina et aL, 1991). The size of the mRNA, about 0.5 kb, corresponds to the size of the pra gene (0.4 kb). The pra gene and the two genes flanking it on the physical map of pSAM2 (korSA and traSA; Fig. 1) are transcribed in the same direction, from korSA to traSA. Northern hybridization allowed a unique transcript to be detected for each gene. The 32p-labelled DNA fragment corresponding to the korSA gene hybridized with a 1 kb mRNA for all pSAM2-containing strains tested (data not shown). The DNA probe, corresponding to the coding part of the traSA gene, hybridized with a 1.8kb mRNA © 1995BlackwellScienceLtd, MolecularMicrobiology,17, 533-544
B3 i~ I -
102
B4
"~ B2 I
0
15
50
100 Km ~g/ml
Fig. 6. Sensitivityto kanamycinof S. lividans TK2.4 harbouring different plasmids.Control, S. lividans TK24(plJ487); B2, S. lividans TK24(pOS526) (pra promoter from pSAM282); B4 S. lividans TK24(pOS543) (pra promoter from pSAM2B4);B3 S. fividans TK24(pOS527) (pra promoter from pSAM2B3).
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A
appearance of replicating pSAM2 promoter RBS
p0S541 ~ pOS539
pOS531 pOS532
~
l +1
pra coding termination part region
~
~
~
9bp
~
yes [pSAM2B2] yes [pSAM2B2]
~N~'~X,'~ [e r m E ' ~ ~ ' ~ ~ ]
pIJ487
no no no
Fig. 7. Functional analysis of pra. A. Schematic map of the different constructions used. S. hvidansTK24(pSAM2B2)was transformed by the recombinant plasmids listed on the left. In the middle, the structure of the plasmids is presented. The white boxes with internal inscriptions indicate the promoters used, the black boxes indicate the RBS site, and the shaded boxes indicate the pra gene coding part and the transcription termination region. The numbers refer to the nucleotide positions in Fig. 2. The appearance of the pSAM2B2free form was tested both by isolation of plasmid DNA and by Southern hybridization. B. Appearance of the pSAM2 free form in S. lividans TK24(pSAM2B2)as a result of pra expression. Total DNA, restricted by EcoRI, was analysed by Southern hybridization with the a2p-labelled EcoRl(6.66)-EcoRl(10.86) pSAM2 fragment. DNA was extracted from S. lividans TK24 containing: lane 1, pSAM2B2+ plJ487; lane 2, pSAM2B2+ pOS541 ; lane 3, pSAM2B2+ pOS539; lane 4, pSAM2B2+ pOS531 ; lane 5, pSAM2B2+ pOS532; lane 6, pSAM2B2. Two DNA fragments (6.7 and 5.2 kb) of integrated pSAM2 digested by EcoRI hybridize with the probe, while EcoRI-digested free form of pSAM2 gives the hybridizing band of 4.2 kb. This band, indicating the presence of the free form of pSAM2, was detected only for the lanes 2 and 3. The diffuse band about 3.5 kb at the bottom of lane 2 corresponds to pSAM2 single-stranded DNA. The positions of 1 kb ladder DNA fragments are indicated.
gene promoter. Therefore, the replication of pSAM2B3 and pSAM2B4 is a consequence of pra gene overexpression, suggesting a role of the pra gene in pSAM2 replication activation. This conclusion fits well with the previous observation that the inactivation of the pra gene in pSAM2B3 led to the disappearance of the replicating form.
Expression in trans of the pra gene provokes the appearance of the pSAM2 replicating form If the pra gene is involved in pSAM2 replication activation, its expression in trans should provoke the appearance of the free form of pSAM2B2. To check this, the pra gene was placed under the control of the constitutive and strong foreign ermE* promoter, which is derived from ermE (Bibb et aL, 1986) by removal of three nucleotides (M. J. Bibb, personal communication). The various constructions carried by the multicopy vector plJ487 are presented in Fig. 7A. These constructions were introduced by protoplast transformation in S. fividans TK24(pSAM282) and their effect on the status of pSAM282 was studied (Fig. 7B). plJ487 had no influence on the pSAM2 status (Fig. 7B, lane 1). The appearance of the free form of pSAM2B2 was observed when the plasmid pOS541 was introduced
in S. lividans TK24(pSAM2B2) (Fig. 7B, lane 2). In this construction, the transcribed sequence (including the proposed RBS and all the pra coding sequence) up to the possible transcription termination region was placed under the control of the ermE* promoter. With this construction, together with the replicating form of pSAM2B2, the presence of an additional band was detected. This strongly hybridizing band was attributed to ssDNA as it is the only band that could be transferred onto a nitrocellulose filter under non-denaturing conditions (data not shown; for details of method see Hag~ge et aL, 1993b). The plasmid pOS539 contains the RBS sequence and the entire pra gene, like pOS541, but not the ermE* promoter. It was also able to provoke the appearance of the free pSAM2 form (Fig. 7B, lane 3). Probably, taking into account the multicopy status of the vector plJ487, even a weak promoter activity from the RBS-containing fragment or from the upstream region of the vector would be sufficient for the pra gene activity to be manifested. Contrary to pOS541, the band corresponding to ssDNA is absent (see the Discussion). No pSAM2B2 free form was detected with plasmid pOS532 lacking the RBS-containing non-translated 53 bp DNA fragment (Fig. 7B, lane 5). This is probably because of the lack of a pra RNA translation. The same negative © 1995 BlackwellScienceLtd, MolecularMicrobiology,17, 533-544
Regulation of pSAM2 replication 541 result was obtained for plasmid pOS531, containing only the coding part of the pra gene with the putative terminator region (Fig. 7B, lane 4). The pSAM2B2 free form co-existed with an integrated copy (Fig. 7B, lanes 2, 3). To confirm that the appearance of the pSAM2B2 free form is the result of replication activation and not simply of excision, the existence of nonoccupied chromosomal attachment sites (attB) was tested. The attB site is unique. When unoccupied, it is present in a 7.5 kb Pstl chromosomal fragment in S. fividans TK24, while an occupied site corresponds to a 18.5kb Pstl fragment (Boccard et aL, 1988). The 18.5 Pstl fragment was observed, indicating that pSAM2B2 was still integrated in the presence of Pra. By overexposing the autoradiography, a faint 7.5 kb band could be detected in all strains, even in S. fividans TK24 containing only pSAM2B2 (data not shown). This band corresponds to roughly 1% of unoccupied attB sites. As almost all the attB sites are occupied, comparison of band intensity for the integrated and free forms shows that the free form is present at more than one copy per genome. Therefore, the appearance of more than one copy of the free form of pSAM2B2 per genome observed in the presence of Pra cannot be explained by excision only. These results show that the pra gene product can activate the appearance of the replicating form in trans. They agree with the hypothesis that the pra gene product activates pSAM2 replication. Discussion
pSAM2 is present as one integrated copy per genome in the strain S. ambofaciens B2 and can be found simultaneously as one integrated copy and as autonomously replicating (5-10 copies per genome) forms in strains B3 and B4 (Pernodet et al., 1984). Moreover, it has been proved that pTS41, a derivative of pSAM2B3 lacking a functional integration system, could replicate and exist solely as a free form (Smokvina et al., 1991). This prompted us to analyse pSAM2B3 and pSAM2B4 mutants in order to localize and characterize a locus involved in the regulation of pSAM2 status. The majority of site-specific integrating elements from Streptomyces are, like pSAM2, easily obtained in the free form without any structural rearrangement (with the exception of SLP1 (Omer and Cohen, 1984; Grant et aL, 1989)) in the original host or after conjugal transfer to another Streptomyces strain (Hopwood et al., 1984; Cohen et aL, 1985; Moretti et al., 1985; Sosio et aL, 1989). This indicates that the integrating genetic elements have a silent mechanism of autonomous replication that can be temporarily activated or be constitutively active in replicating mutants. The results obtained showed that the pra gene product © 1995 Blackwell Science Ltd, Molecular Microbiology, 17, 533-544
provokes the appearance of the free form of pSAM2B2. This could be the result of activation of replication functions or of activation of excision functions only. The latter explanation is unlikely as it was observed that pra gene expression led to the appearance of more than one copy of the pSAM2 free form per genome. It is difficult to imagine how a integrated copy of pSAM2 could generate more than one free copy by simple excision. Moreover, if the free form observed was non-replicative and resulted only from excision, some chromosomes should be devoid of the integrated copy. This is not the case, because we could hardly detect the presence of non-occupied chromosomal attB sites in the strains S. fividans TK24(pSAM2B2) containing plasmids that induce the appearance of the pSAM2B2 free form. The results that allow us to consider that the pra gene activates pSAM2 replication are: (i) the pra gene is not directly involved in the machinery of replication as it was possible to construct a pSAM2 minimal replicon devoid of pra (Hagege et aL, 1994), although in this construct the repSA gene was probably not under its normal expression control; (ii) any disruption or deletion of the pra gene gives rise to variants that contain only the integrated form which is unable to transfer; (iii) the pra3 and pra4 mutations are point substitutions in the promoter region of the pra gene that enhance the strength of the promoter; (iv) the constitutive expression of the pra gene in a multicopy plasmid provoked the appearance of the replicating form of normally integrated pSAM2B2.When pra is expressed under the control of the strong promoter ermE* (pOS541), accumulation of pSAM2 ssDNA was observed together with appearance of the free form of pSAM2. This could be the result of a strong activation of repSA expression leading to a disequilibrium between the synthesis of the plus strand and its conversion into double-stranded DNA. Accumulation of pSAM2 ssDNA was not observed with pOS539 where pra transcription was very low (unpublished data). Several models to explain how Pra acts on replication could be proposed. In the simplest one, Pra could be a direct activator of repSA gene expression. In this first model, pSAM2 replication would be positively regulated. In an alternative model, where pSAM2 replication would be negatively regulated, Pra could negatively regulate a gene whose product represses replication. As the genes repSA (involved in pSAM2 replication), xis and int (implicated in pSAM2 integration and excision) seem to be organized as an operon (Hag~ge et aL, 1994), they could be regulated together. A single mechanism could control both site-specific excision mediated by the int and xis gene products and replication, through an increase in repSA expression. The mechanisms of replication regulation for integrating elements of actinomycetes are almost unknown. In the case of SLP1, the imp locus encoding ImpA and ImpC
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proteins was purported to be a negative regulator of multiple SLP1 functions including replication and transfer (Shiffman and Cohen, 1993). For all known E. coil plasmids, as well as for intensively studied RCR plasmids from Gram-positive bacteria (see for reference Janni~)re et aL, 1993), the replication is controlled by the regulation of the level of Rep protein expression and/or origin accessibility, which together regulate the frequency of plasmid DNA synthesis initiation. Among the known mechanisms of plasmid replication regulation, negative regulation, limiting the plasmid's copy number, prevails (reviewed by Novick, 1989; Polisky, 1988; NordstrSm, 1990; Janni~re et aL, 1993; Solar et aL, 1993). It must be emphasized that, differing from plasmids, replication of pSAM2 is not required for maintenance but it is absolutely required and transiently activated during transfer. This may imply that the control of replication in integrating elements is quite different from that found in plasmids. In plasmids, the copy number has to be limited, this is generally achieved by negative control. In integrating elements, one can suppose that once transient replication is activated it should be as efficient as possible. In this context, a positive regulation might be advantageous. The regulation of pra gene expression remains unknown. In plJ101, korA (similar to korSA) regulates both its own transcription and transcription of the tra gene (similar to traSA) (Stein et aL, 1989). In the case of pSAM2, a common sequence was found in the promoter region of pra and upstream of korSA and traSA genes. This might indicate a possible role for this sequence in the binding of a common regulator protein, putatively KorSA, suggesting that pra could be regulated as a gene of the transfer system. Therefore, in the co-ordinated expression of the different functions of pSAM2 leading to transfer, pra could be a regulatory gene whose role is to activate replication when the mycelium is engaged in transfer.
Experimental procedures Bacterial strains and plasmids S. lividans TK24 is the commonly used host strain for cloning experiments (Hopwood et aL, 1983). S. ambofaciens strains are described in Pernodet et al. (1984). E. coil JM101 (Yanisch-Perron et aL, 1985) was used as a host for the M13 vectors, and E. coil DH5c( for the plasmid vectors. The plasmids used are listed in Table 1. Culture and transformation condition General culture and genetic techniques for Streptomyces spp. were as described by Hopwood et al. (1985) and for E. coil as described by Sambrook et aL (1989). Streptomyces transformants carrying the thiostrepton resistance (tsr) gene (Thompson et al., 1980) were selected using 50p.gm1-1 of nosiheptide.
Recombinant clones of S. lividans TK24 containing derivatives of plJ487 (Ward et aL, 1986) carrying the aph resistance gene transcribed from a heterologous promoter were selected on minimal medium (Hopwood et aL, 1985) with 15 I~gml- 1 of Km obtained from Sigma. To determine the level of Km resistance, the spore suspensions of the recombinants clones were titrated onto 0, 15, 25, 50 and 100t~g m1-1 Km in minimal medium. DNA isolation Plasmid DNA was isolated from E. coliand from Streptomyces spp. by alkaline lysis (Hopwood et aL, 1985). M13mp18/19 phage double-stranded DNA carrying inserts as well as ssDNA sequencing templates were prepared as described by Messing (1983). Total DNAs were isolated based on Hopwood et aL (1985). RNA isolation and Northern hybridization Total RNAs from the Streptomyces strains harbouring pSAM2B2, pSAM2B3 and the pSAM2B4 derivative pOS11A (Smokvina et aL, 1991) were isolated as described by Hopwood et al. (1985). For Northern hybridization, about 4050 t~g total RNA was denatured with glyoxal and dimethyl sulphoxide (Sambrook et aL, 1989) and after electrophoresis was transferred on Hybond-N filter (Amersham). Detection of the appearance of pSAM2 free form To detect the appearance of pSAM2 free form, the plasmid fraction isolated by alkaline lysis was checked for the presence of covalently closed circular DNA. Restriction analysis was performed using ApaLI to confirm the presence of the characteristic additional ApaLI site in pSAM2B2. Total DNAs of recombinant strains digested by EcoRI were checked for the presence of free and/or integrated pSAM2 sequence by Southern hybridization with the [~-32p]-dATP-labelled EcoRl(6.66)-EcoRl(10.86) fragment of pSAM2. Construction of plasmids The derivatives of shuttle plasmid pTS39, presenting the Pra3 phenotype as pSAM2B3 (Smokvina etaL, 1991), were used as a source of the pSAM2B3 DNA. Plasmid pOS221 (Boccard et aL, 1988) was used as a source of the pSAM2B2 DNA. To obtain the 'mosaic' variant pOS521, the plasmid pTS42 was used (Smokvina et aL, 1991). As pTS42 contains the additional ~ interposon sequence (Prentki and Krisch, 1984) inside the EcoRl(10.86)-EcoRl(6.66) fragment, it allowed us to check the origin of the cloned fragment for pOS521. Plasmid pOS522 was obtained after cloning of the Kpnl(3.30)Kpnl(5.29) DNA fragment from pSAM2B2 in the unique Kpnl site of pSAM2B3 derivative pTS89 (Table 1). Plasmids pOS524 and pOS525 were constructed on the base of pTS89. At the beginning, the Kpnl(3.30)-Kpnl(5.29) DNA fragment was reconstructed in the phages M13mp18/19 using either the 336 bp Kpnl(3.30)-Nrul(3.63) sub-fragment from pSAM2B2 or that from pSAM2B3 in a combination with the 1.66kb Nrul(3.63)-Kpnl(5.29) fragments from pSAM2B3 © 1995 BlackwellScienceLtd, MolecularMicrobiology,17, 533-544
Regulation of pSAM2 replication 543 or pSAM2B2, respectively (Fig. 3). The cloning of the 'mosaic' KpnI-Kpnl fragments in pTS89 has generated two plasmids, pOS524 and pOS525 (Fig. 3). After identification of the additional ApaLl(3.57) site in the pra gene promoter of pSAM2B2, all the 'mosaic' constructions were tested for its presence by restriction analysis. To construct the plasmids pOS526, pOS527 and pOS543, the polymerase chain reaction (PCR)-generated 139 bp fragments from pSAM2B2, pSAM2B3 and pSAM2B4, corresponding to the sequence between the stop codon of the korSA gene and the start codon of the pra gene (positions 21 and 159 in Fig. 2), were cloned as a BamHI-Kpnl fragment into phage M13mp18. The absence of PCR-generated mutations in the fragments was checked by sequencing. Subsequently, such DNA fragments were recloned as EcoRI-BamHI fragments into plJ487.
DNA sequencing The dideoxy method of Sanger et aL (1977), as modified by Biggin et al. (1983), was used. Oligonucleotides were synthesized on an Applied Biosystems model 381A DNA synthesizer. To overcome the problems of band compression in sequencing gels caused by the high stability of G+C-rich structures, the Deaza T7 sequencing kit obtained from Pharmacia LKB has been used. The [=-3SS]-dATP was obtained from Amersham. Sequences were determined for both strands.
Primer extension analysis Primer extension analysis was carried out as described by Boorstein and Craig (1989), using a kit from Promega. To determinate precisely the start site of transcription, the oligonucleotide that had been used in the primer extension reaction was used as sequencing primer. About 401~g total RNA was used for each reaction.
Computer-assisted sequence analysis DNA and protein sequences were analysed by using the PC/ GENEpackage (IntelliGenetics, Inc.). The predicted Pra protein was scanned for similarities to the Swissprot data bank Version 30 using the FASTAprogram (Pearson and Lipman, 1988). The TFASTAprogram was used to compare the Pra protein sequence with the DNA sequences present in the EMBL database (Version 41) by translating the DNA sequences to proteins in all six frames. The sequence is available in the EMBL Data Library under accession number Z34987.
Acknowledgements We are grateful to M. J. Bibb for the kind gift of the ermE* promoter and M. Zalacain for the kind gift of the hyg gene. We wish to thank M. Chandler for helpful comments on the manuscript and C. Gerbaud for computer assistance. This work has been done as part of the 'Bioavenir' programme supported by RhSne-Poulenc with the participation of the French Minist~res de la Recherche, et de I'Enseignement superieur, de rlndustrie et du Commerce Exterieur. © 1995 BlackwellScience Ltd, MolecularMicrobiology,17, 533-544
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