New Lead Discovery, Schering Plough Research Institute, 2015 Galloping Hill Road, K15-B425-MS4800, Kenilworth, NJ 07033, USA
Correspondence
Thomas Hosted
thomas.hosted{at}spcorp.com
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ABSTRACT |
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The GenBank accession numbers for the sequences reported in this paper are AY150027 (M. carbonacea var. africana pMLP1 att/int region), AY150029, AY150033 and AY150028 (M. carbonacea var. africana pSPRH840 attL, attR and attB sites, respectively), and AY150031, AY150032 and AY150030 (M. halophytica var. nigra pSPRH840 attL, attR and attB sites, respectively).
Present address: Cubist Pharmaceuticals, 65 Hayden Avenue, Lexington, MA 02421, USA.
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INTRODUCTION |
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The att/int functions necessary for integration consist of an integrase gene and a short DNA recognition region designated the phage attachment site or attP element. The integrase acts as a site-specific DNA recombinase directing recombination between the attP element and a specific chromosomal location designated the attB site (Boccard et al., 1989b). Often actinomycete attB sites are tRNA genes that share a region of sequence identity with the attP element designated the segment of identity (Reiter et al., 1989
). Recombination between the attP element and the chromosomal attB site creates an attP/attB juncture, designated attL, which regenerates a functional tRNA gene and an attB/attP juncture, designated attR, that generates a truncated tRNA. The attB sites for pSAM2, pSE211, pSG1, SLP1, RP3 and VWB integration are tRNA genes, which share a 58112 bp segment of identity with the corresponding attP element (Bar-Nir et al., 1992
; Boccard et al., 1989b
; Brown et al., 1990
; Gabriel et al., 1995
; Omer & Cohen, 1986
; Van Mellaert et al., 1998
). Since tRNA genes in actinomycetes are often conserved at the sequence level, att/int functions can often direct integration into genetically diverse hosts. For example, pSAM2-derived plasmids integrate into numerous Streptomyces species (Boccard et al., 1989a
; Kuhstoss et al., 1989
; Simonet et al., 1987
) and Mycobacterium smegmatis (Martin et al., 1991
). Plasmids containing att/int functions can direct integration of a single copy of genes into the chromosome. Often the integration event is neutral with respect to secondary metabolite production and has the advantage of being maintained without antibiotic selection (Baltz & Hosted, 1996
).
Micromonospora carbonacea var. africana ATCC 39149 produces three antibiotics that inhibit function of the ribosome. These include the oligosaccharide antibiotic evernimicin, chloramphenicol and a novel glycosylated thiostrepton (Hosted et al., 2001; Puar et al., 1998
). Evernimicin exhibits a broad spectrum of biological activity against Gram-positive and some Gram-negative bacteria, including the clinically important Gram-positive pathogens: glycopeptide-resistant enterococci, methicillin-resistant staphylococci and penicillin-resistant streptococci (Foster & Rybak, 1999
). In an effort to gain further understanding of the roles individual genes in the evernimicin cluster play in the biosynthesis of evernimicin, methods have been developed for insertional mutagenesis in M. carbonacea var. africana (Hosted et al., 2001
).
The potential utility for cloning vectors capable of site-specific integration in actinomycetes was recognized early by the groups characterizing the pSLP1 (Omer et al., 1988), pSAM2 (Smokvina et al., 1990
) and phiC31 (Kuhstoss et al., 1991
) integration systems. There are many examples in which integration vectors have been directly applied to studies on antibiotic biosynthesis. They have been used for simply determining the function of potential regulatory genes in a deletion mutant of the cephamycin biosynthetic cluster by complementation (Alexander & Jensen, 1998
) or to direct production of a preferential metabolite by inserting an additional copy of a pristinamycin biosynthetic gene (Sezonov et al., 1997
). The availability of vectors utilizing these integration systems or novel integration systems in Micromonospora spp. would greatly further our ability to understand how antibiotic biosynthesis occurs in these organisms.
In this study, we have isolated and characterized the att/int functions from the M. carbonacea var. africana pMLP1 phage. Escherichia coliactinomycete shuttle vectors containing the pMLP1 integrase were constructed and their ability to direct site-specific integration into Micromonospora spp. was determined.
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METHODS |
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Cosmid library preparation and isolation of a pMLP1 integrase containing cosmid.
A M. carbonacea var. africana genomic DNA library was prepared in SuperCos I (Stratagene) as described previously (Hosted et al., 2001). Degenerate PCR primers PR144 and PR145, designed to amplify polyketide synthase (PKS) type I genes (Hutchinson & Fujii, 1995
), were used to screen the cosmid library and isolate cosmid pSPR150. Description of the oligonucleotide primers used in this study are listed in Table 2
. A 0·8 kb BamHI subclone from pSPR150 with BLAST homology to the Mycobacterium phage Ms6 integrase (Freitas-Vieira et al., 1998
) was used as a hybridization probe to identify a 4·4 kb pSPR150 KpnI fragment containing the entire att/int region which was subcloned to yield pSPRH819.
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Nucleotide sequence analysis.
DNA sequence was determined using Taq dideoxy terminator cycle sequencing (SeqWright). Amino acid and sequence homology searches were performed using BLASTN and BLASTX sequence homology searches (Altschul et al., 1990). Plasmid constructions, analysis of primary DNA sequence and determination of protein coding regions were performed using the Gene Constructor and Gene Inspector Kit software package (Textco). Alignment of DNA and protein sequences was performed using GeneWorks (IntelliGenetics). PCR primers were designed using OLIGO PRIMER analysis software (National Biosciences).
Construction of conjugation and integration vectors
(i) pSPRH826 conjugation vector.
The hygromycin resistance (HmR) gene (hyg) was removed from p16R1 (Garbe et al., 1994) as a 1·1 kb NruINotI fragment, treated with T4 DNA polymerase to create blunt fragment ends and inserted into SspI-digested pUC19 to create pSPRH825. The RK2 origin of transfer (oriT) was removed from pRL1058 (Wolk et al., 1991
) by digestion with PstI, treated with T4 DNA polymerase and inserted into NdeI-digested and T4 DNA polymerase-treated pSPRH825 to create pSPRH826.
(ii) pSPRH840 integration vector.
The pMLP1 xisM, intM and attP region was removed from pSPRH819 as a 2·5 kb NruIXhoI fragment, treated with T4 DNA polymerase to create blunt fragment ends and inserted into pCRTopo 2.1 to create pSPRH853. A 2·6 kb KpnIPstI fragment from pSPRH853 containing xisM, intM and attP was inserted into KpnI/PstI-digested pSPRH826 to create pSPRH840 (Fig. 1a).
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(iv) pSPRH910 integration vector.
Using the seamless cloning kit and the oligonucleotide primers PRD14 and PRD15, a 3·9 kb region containing the pUC replicon, the MCS, apr and oriT was amplified from pSPRH900. A 2·1 kb fragment containing the pMLP1 xisM, intM and attP region was amplified from pSPRH840 using the oligonucleotide primers PRD16 and PRD17. Both PCR products were digested with Eam1104I and ligated together to create pSPRH910.
(v) Integration vectors containing alternate attB sites.
A 0·5 kb EcoRI fragment containing the M. halophytica var. nigra ATCC 33088 pMLP1 chromosomal attB (see below) was removed from pSPRH892 and inserted into EcoRI-digested pSET152 to create pSPRA2301. A 0·8 kb EcoRI fragment containing the M. carbonacea var. africana pMLP1 chromosomal attB site was removed from pSPRH890 and inserted into EcoRI-digested pSET152 to generate pSPRA2303.
Hybridization and Southern blot analysis.
Digested genomic DNA was separated via electrophoresis with 0·8 % (w/v) agarose gels containing running buffer and ethidium bromide. DNA in the gel was transferred to Zeta-Probe GT nylon membrane (Bio-Rad) using a Turboblotter transfer system (Schleicher & Schuell). Radiolabelled DNA probes were generated with [-32P]dCTP and the DECAprime II labelling kit, and ULTRAhyb (Ambion) solution was used for hybridization of Southern blots.
Isolation of plasmid integration sites and chromosomal attB sites.
To isolate chromosomal DNA flanking integration sites, genomic DNA from pSPRH840 or pSPRH910 exconjugants was digested with BamHI, BsiWI, KpnI, PstI or XmnI, self-ligated and transformed into E. coli to identify attL- or attR-containing plasmids. Based on sequence analysis of the attL and attR regions, PCR primers PDH504 and PDH505 and primers PDH502 and PDH503 were designed to amplify parental attB regions from M. carbonacea var. africana and M. halophytica var. nigra, respectively.
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RESULTS |
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The putative integrase gene, designated intM, encodes a putative protein of 392 aa with a deduced molecular mass of 43·1 kDa. IntM shows similarity to the integrase family of proteins, particularly to integrases from bacteriophage. Alignment of IntM with the VWB (Van Mellaert et al., 1998) and Ms6 phage integrases (Freitas-Vieira et al., 1998
) identified three highly conserved regions, Boxes A, B and C (Esposito & Scocca, 1997
). BoxA contains the highly conserved residues TGLRxGExxxL, considered the hallmark of the tyrosine recombinases. BoxB contains the HxLRHxxAxxL consensus motif, which includes an invariant arginine residue. BoxC contains the LGH motif and the highly conserved tyrosine residue involved in the formation of the phosphotyrosyl bond to the 3' end of the DNA during integration (Yang & Mizuuchi, 1997
). There is a 12 bp imperfect inverted repeat sequence located 77 bp downstream of intM that may function as a transcriptional terminator. Downstream of the putative terminator is a putative attP element with homology to tRNA genes, which is followed by a 13 bp inverted repeat sequence separated by five bases.
The xisM gene and its product which encodes a putative protein with 141 aa with a deduced molecular mass of 15·8 kDa, is located directly upstream of intM and shows significant similarity to proteins believed to function as excisionases. A similar gene organization has been described for other actinomycete integrative elements (Boccard et al., 1989a; Brown et al., 1994
; Gabriel et al., 1995
). XisM contains an N-terminal helixturnhelix motif (amino acid residues 1663) and a highly basic pI, both of which are characteristic of DNA-binding proteins.
Site-specific integration of pSPRH840 and pSPRH910 integration vectors
MC840 exconjugants were generated by conjugation of the pSPRH840 integration vector into M. carbonacea var. africana (Table 1). When a Southern blot of parental and MC840 genomic DNA digested with KpnI (Fig. 2
a) was hybridized with an intM gene fragment, 4·4 and 3·5 kb hybridizing fragments were observed in the parental lane and a single 3·1 kb hybridizing fragment was found in the MC840 lanes (Fig. 2b
). The 4·4 kb hybridizing KpnI fragment in the parental lane would be expected from the free form of the pMLP1 phage and represents the sequenced 4·4 kb KpnI fragment from pSPR150. The 3·5 kb hybridizing fragment in the parental lane indicates that pMLP1 also exists as an integrated form in the chromosome. The hybridization intensity of the 4·4 kb fragment is significantly greater than the 3·5 kb fragment, suggesting that the free form of pMLP1 is at a high copy number relative to the integrated copy (Fig. 2b
). Freely replicating pMLP1 DNA is observed as a set of brightly staining bands (banding pattern predicted from pSPR150 digestions) in an ethidium-bromide-stained agarose gel, also indicating that pMLP1 exists at a high copy number (Fig. 2a
).
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MC910 exconjugants were generated by conjugation of the pSPRH910 integration vector into M. carbonacea var. africana (Table 1). Southern blot analysis of several MC840 and MC910 exconjugants revealed that the pMLP1 intM consistently directed integration into a site-specific locus (data not shown). The frequency of pSPRH910 integration into M. carbonacea var. africana was 1x10-3 per recipient c.f.u.
MH840 exconjugant strains were obtained by conjugation of pSPRH840 into M. halophytica var. nigra (Table 1). A Southern blot of parental and MH840 PstI-digested DNA when probed with the hyg gene fragment showed no hybridization in parental lane, but a 12·5 kb hybridizing fragment in both MH840 lanes, indicating pSPRH840 integration into the chromosome of this Micromonospora species as well (Fig. 2b
).
Analysis of the attL and attR juncture regions and chromosomal attB site
Integration of pSPRH840 into the chromosome generates attL and attR juncture regions formed by site-specific recombination within the homologous regions of attP on the plasmid and attB in the genome (Fig. 1a). It was possible to get sequence information from the attL and attR junctures because genomic DNA flanking the plasmid integration site could be cloned due to its contiguous relationship with the replicon and the resistance marker of the integrating plasmid. Alignment of the sequence obtained from the attL- or attR-containing plasmids with non-coding sequence downstream of intM defined the boundaries of vector and genomic DNA. These genomic sequences were used to generate oligonucleotide primers for PCR amplification of genomic DNA spanning the attB site from a parental (non-pSPRH840 integrant) strain (Fig. 1a
).
Homology searches of the attB site sequences from M. carbonacea var. africana and M. halophytica var. nigra showed the presence of a tRNAHis gene (Fig. 1a, Fig. 3
). To localize the pMLP1 attP site the non-coding sequence downstream of intM was aligned with the corresponding attB, attL and attR sequences from each organism (Fig. 4
). The M. carbonacea var. africana attB site and the M. halophytica var. nigra attB site contain a 24 or 25 bp segment, respectively, with identity with the pMLP1 attP element. The attB site is located at the 3' end of the tRNA gene and does not extend to include the anticodon loop (GUG) of the tRNA (see Fig. 3
). In both organisms the integration regenerated a functional tRNA gene at attL and a truncated tRNA at attR (Fig. 4
). Both tRNAHis genes are followed by inverted repeat sequences that could act as transcriptional terminators. Downstream of the M. carbonacea var. africana tRNAHis gene a second segment of identity was identified with the attP element that included 11 and 10 bp regions of the inverted repeat that follows the tRNAHis gene (Fig. 4
). A similar identity region was not found downstream of the M. halophytica var. nigra tRNAHis region.
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pSPRH910 integrant strains were obtained for Micromonospora spp. SCC1047 and M. rosaria. Sequence analysis of the attR regions from M. rosaria and Micromonospora spp. SCC1047 indicated that pSPRH910 integrated into a tRNAHis gene and that the segment of identity is the same as for M. carbonacea var. africana.
M. carbonacea var. africana pSET152 and pSPR840 double integrants
pSET152 (Bierman et al., 1992), which contains phiC31 att/int functions (Kuhstoss et al., 1991
), was introduced into M. carbonacea var. africana to obtain MC152 integrants (Table 1
). Southern analysis of several MC152 exconjugants revealed a different site-specific locus for pSET152 in M. carbonacea var. africana (data not shown). Sequence analysis of attL and attR juncture clones from a MC152 integrant indicates that pSET152 integrates into the 5' end of the same ORF as has been shown in S. ambofaciens (data not shown) (Kuhstoss & Rao, 1991
).
pSPRH840 was conjugated into MC152 to obtain the double integrant strain MC152.840 (Table 1). A Southern blot of BamHI-digested genomic DNA from parental, MC840, MC152 and MC152.840 strains when hybridized with an apr gene probe (pSET152 marker) hybridized with a 3·6 kb fragment in the MC152 and MC152.840 lanes (Fig. 1c
, Fig. 5
a). A hyg gene probe (pSPRH840 marker) hybridized to a 5·6 kb fragment in the MC840 and MC152.840 lanes (Fig. 1b
, Fig. 5b
). A pUC19 probe (backbone for pSET152 and pSPRH840) hybridized with 4·5 and 3·6 kb fragments in the MC152 lanes, a 5·6 kb fragment in the MC840 lanes and all three (5·6, 4·5 and 3·6 kb) fragments in the MC152.840 lanes (Fig. 1b, c
, Fig. 5c
). The presence of both sets of hybridizing bands in the MC152.840 strains indicated that pSPRH840 and pSET152 integrate at two unique chromosomal loci and can co-exist in the M. carbonacea var. africana chromosome.
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When a Southern blot of XhoI/XbaI-digested genomic DNA was hybridized with a SacIXhoI fragment from pSET152 containing the 5' end of apr, a 2·5 kb fragment in MC2301 and a 2·2 kb fragment in MC2303 were detected (Fig. 6). The XhoIXbaI fragments contain the 5' end of apr, the pUC replicon and the alternate attB sites in the MCS. The 0·8 kb M. halophytica var. nigra and 0·5 kb M. carbonacea var. africana attB sites in the MCS would increase the size of the pSET152 XhoIXbaI 1·7 kb fragment (Fig. 1c
) to 2·5 kb for MC2301 and 2·2 kb for MC2303. The loss of the 2·2 or 2·5 kb hybridizing fragment and the appearance of a 9·4 or 9·7 kb hybridizing fragment was seen in MC2301.840 and MC2303.840 lanes, respectively (Fig. 6
). The 9·4 or 9·7 kb fragment would be expected when pSPRH840 integrated into the alternate attB site provided by pSPRA2301 or pSPRA2303 and would not occur if pSPRH840 had integrated into the native attB site. In addition, the set of brightly ethidium-bromide-staining bands corresponding to digested pMLP1 are still present in the MC2301.840 and MC2303.840 strains (data not shown), but they were lost when pSPRH840 integrates into the native attB site (see Fig. 2b
).
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DISCUSSION |
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Plasmids containing the site-specific integrase of pMLP1 can be used to integrate genes as a single copy into the chromosome. This procedure has many advantages over methods involving autonomously replicating plasmids. Plasmid integration can be maintained without antibiotic selection and self-replicating plasmids can cause a reduction in secondary metabolite yields that would negate any positive effect of the gene(s) present on the plasmid (Baltz & Hosted, 1996). Integration of pMLP1-based plasmids are neutral with respect to evernimicin and chloramphenicol production in M. carbonacea var. africana fermentations (unpublished results). This would allow pMLP1-based integration vectors to be used in studies characterizing antibiotic biosynthesis in M. carbonacea var. africana alone or in conjunction with the insertional mutagenesis vectors previously developed (Hosted et al., 2001
).
Southern blot analysis of wild-type M. carbonacea var. africana genomic DNA revealed an integrated and free form of the pMLP1 phage. The relative intensity of the band corresponding to the free form of pMLP1 suggested a high copy number. This was confirmed by the observation that the pMLP1 DNA digestion pattern could be observed as a set of brightly staining bands in an ethidium-bromide-stained agarose gel. Integration of the pMLP1-based plasmids pSPRH840 or pSPRH910 resulted in the curing of pMLP1 as judged by agarose gel electrophoresis or Southern blot analysis. When pSPRH840 integrates into the alternate attB sites in the MC2301 or MC2303 strains the pMLP1 banding patterns were present in the digested genomic DNA of the double integrants. The lack of an available pMLP1 attB site in the MC840 strains causes a curing of pMLP1, but pMLP1 remains stable in MC2301.840 and MC2303.840 when pSPRH840 integrates into the alternate attB site. This suggests that the ability to lysogenize M. carbonacea var. africana is part of the pMLP1 life cycle and integration of a vector in this site disrupts it sufficiently to cause a curing of pMLP1.
A PCR amplified version of the pMLP1 attB site was integrated into the M. carbonacea var. africana chromosome at the phiC31 attB site. When given the option of two pMLP1 attB sites, pSPRH840 integration occurred at the alternate pMLP1 attB site in all strains. Perhaps the integration of the pMLP1 phage into native attB affects targeting of the integrase and directs integration into the alternate attB site. In the absence of the alternate attB site pSPRH840 must displace pMLP1 before integration of the plasmid. The ability to place alternate attB sites on the chromosome allows for the construction of strains capable of containing multiple integration vectors and could extend the range of pMLP1-based integration vectors to additional actinomycetes.
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Received 24 February 2003;
revised 12 May 2003;
accepted 22 May 2003.