Development of the Micromonospora carbonacea var. africana ATCC 39149 bacteriophage pMLP1 integrase for site-specific integration in Micromonospora spp.

Dylan C. Alexander{dagger}, David J. Devlin, Duane D. Hewitt, Ann C. Horan and Thomas J. Hosted

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Micromonospora carbonacea var. africana ATCC 39149 contains a temperate bacteriophage, pMLP1, that is present both as a replicative element and integrated into the chromosome. Sequence analysis of a 4·4 kb KpnI fragment revealed pMLP1 att/int functions consisting of an integrase, an excisionase and the phage attachment site (attP). Plasmids pSPRH840 and pSPRH910, containing the pMLP1 att/int region, were introduced into Micromonospora spp. by conjugation from Escherichia coli. Sequence analysis of DNA flanking the integration site confirmed site-specific integration into a tRNAHis gene in the chromosome. The pMLP1 attP element and chromosomal bacterial attachment (attB) site contain a 24 bp region of sequence identity located at the 3' end of the tRNA. Integration of pMLP1-based plasmids in M. carbonacea var. africana caused a loss of the pMLP1 phage. Placement of an additional attB site into the chromosome allowed integration of pSPRH840 into the alternate attB site. Plasmids containing the site-specific att/int functions of pMLP1 can be used to integrate genes into the chromosome.


Abbreviations: AmR, ampicillin resistance; ApR, apramycin resistance; apr, apramycin resistance gene; att/int, region containing the excisionase, integrase gene and attP site; attB, bacterial attachment site; attP, phage attachment site; HmR, hygromycin resistance; hyg, hygromycin resistance gene; MCS, multiple cloning site; oriT, origin of transfer; PKS, polyketide synthase

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).

{dagger}Present address: Cubist Pharmaceuticals, 65 Hayden Avenue, Lexington, MA 02421, USA.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Many actinomycetes harbour plasmids or bacteriophages that contain regions capable of directing site-specific integration into the chromosome. Actinomycete plasmids capable of integration include pSAM2 from Streptomyces ambofaciens (Pernodet et al., 1984), pSG1 from Streptomyces griseus (Cohen et al., 1985), pSE101 (Brown et al., 1988) and pSE211 from Saccharopolyspora erythraea (Brown et al., 1994), pMEA100 from Amycolatopsis mediterranei (Moretti et al., 1985), pMEA300 from Amycolatopsis methanolica (Vrijbloed et al., 1994), pIJ408 from Streptomyces glaucescens (Hopwood et al., 1984; Sosio et al., 1989) and SLP1 from Streptomyces coelicolor A3(2) (Bibb et al., 1981). Actinomycete integrative bacteriophages include phiC31, a temperate phage from S. coelicolor (Lomovskaya et al., 1972), RP2 and RP3 temperate phages from Streptomyces rimosus (Rausch et al., 1993) and the VWB temperate phage from Streptomyces venezuelae (Van Mellaert et al., 1998).

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 58–112 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 coli–actinomycete shuttle vectors containing the pMLP1 integrase were constructed and their ability to direct site-specific integration into Micromonospora spp. was determined.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, media and culture conditions.
Bacterial strains used in this study are listed in Table 1. Micromonosporae cultures were grown in trypticase soy broth (TSB; BRL) or SIM-1 (Hosted et al., 2001) at 28 °C with shaking at 250 r.p.m. E. coli cultures were maintained on LB or 2YT agar and grown in 2YT broth at 37 °C (Sambrook et al., 1989). Bacteria containing plasmids were routinely grown in media supplemented with 30 µg ampicillin ml-1, 30 µg apramycin ml-1, 30 µg nalidixic acid ml-1, 25 µg kanamycin ml-1, 12·5 µg chloramphenicol ml-1 or 200 µg hygromycin ml-1 as appropriate.


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Table 1. Bacterial strains and plasmids used in this study

 
Transformation and conjugation.
Plasmids were introduced into E. coli strains by chemical transformation or electroporation. Intergeneric conjugation from E. coli ET12567(pUB307) (Flett et al., 1997) or E. coli S17-1 (Mazodier et al., 1989) into Micromonospora strains was performed in a manner similar to the method of Bierman et al. (1992) on AS1 plates (Baltz & Matsushima, 1983) supplemented with 10 mM MgCl2, 25 mM TES, pH 7·2 and a trace element solution (Hosted et al., 2001). Micromonospora strains were grown in TSB or SIM-1, homogenized, harvested by centrifugation, washed and resuspended in a half volume of TSB. E. coli donor cultures were grown overnight, diluted into fresh media and grown for 2–4 h before being harvested by centrifugation, washed and resuspended in a half volume of Luria broth. Donor and recipient cultures were mixed, plated onto AS1 plates and incubated at 28 °C for 16–20 h before overlaying with soft nutrient agar containing the appropriate antibiotic selection. Exconjugants on the AS1 plates were patched onto AS1 plates or grown directly in liquid medium.

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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Table 2. Oligonucleotide primers used in this study

 
Molecular genetic techniques.
The methods used for plasmid isolation, restriction enzyme digestion, DNA modification and E. coli transformation were all done according to the suppliers' recommendations. Micromonospora genomic DNA was isolated as described previously (Hosted et al., 1993). PCR amplifications were performed using the Advantage GC DNA polymerase and reagents (Clontech) and PCR products were cloned utilizing Topo TA cloning kits (Invitrogen) or the pNOTA vector (5'–3'). DNA fragment ends were made blunt using T4 DNA polymerase.

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 NruI–NotI 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 NruI–XhoI fragment, treated with T4 DNA polymerase to create blunt fragment ends and inserted into pCRTopo 2.1 to create pSPRH853. A 2·6 kb KpnI–PstI fragment from pSPRH853 containing xisM, intM and attP was inserted into KpnI/PstI-digested pSPRH826 to create pSPRH840 (Fig. 1a).



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Fig. 1. Schematic of pSPRH840 and pSET152 integration into the M. carbonacea var. africana chromosome. (a) pSPRH840 and the M. carbonacea var. africana attB site. (b) pSPRH840 integrated into the M. carbonacea var. africana attB site. (c) pSET152 integrated into the M. carbonacea var. africana pSET152 attB site. Restriction fragments described in the text and in Figs 2, 3 and 6 are depicted by the horizontal lines with their sizes (in kb). Chromosomal DNA (hatched box), tRNA-His (black arrow and box) and the attP region (open arrow and box) are shown. Coding regions of xis, int, hyg, apr and lacZ are indicated by arrows. The oriT region is indicated by a solid black line.

 
(iii) pSPRH900 conjugation vector.
A 2·6 kb region containing the pUC replicon, the multiple cloning site (MCS) and oriT was amplified from pSPRH826 using the oligonucleotide primers PRD5 and PRD6 and the Seamless cloning kit (Stratagene). A 1·3 kb fragment containing the apramycin resistance (ApR) gene (apr) was amplified from pJR225 (Paget & Davies, 1996) using the oligonucleotide primers PRD11 and PRD12. PCR products were digested with Eam1104I and ligated together to create pSPRH900.

(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 [{alpha}-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.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Isolation and characterization of the pMLP1 integrase region
The cosmid pSPR150, containing pMLP1 DNA (Table 1), was originally isolated utilizing degenerate PCR primers for type I PKS genes. Sequence analysis of a 0·8 kb BamHI fragment subclone demonstrated an internal segment containing an ORF with similarity to a family of phage integrases, such as the Mycobacterium phage Ms6 integrase (Freitas-Vieira et al., 1998). Closer examination of the sequence indicated strong similarity to the C-terminal conserved catalytic domain found within integrases (Argos et al., 1986; Esposito & Scocca, 1997; Nunes-Duby et al., 1998). Sequence analysis of a 4·4 kb KpnI fragment, identified by hybridization, showed the presence of putative att/int functions consisting of an integrase (intM), an excisionase (xisM) and the attP element. Further sequence analysis of pSPR150 revealed a major portion of a 45–50 kb bacteriophage we have named pMLP1 temperate bacteriophage (data not shown). In addition to phage-related genes a short incomplete coding region with similarity to PKS type I genes was located within the cosmid. The pMLP1 bacteriophage was also observed as a 45–50 kb extrachromosomal element on PFGE analysis (data not shown).

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 helix–turn–helix motif (amino acid residues 16–63) 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. 2a) 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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Fig. 2. Confirmation of pSPRH840 integration in Micromonospora spp. (a) Ethidium-bromide-stained agarose gel of KpnI-digested genomic DNA. Lanes: 1, M. carbonacea var. africana; 2 and 3, MC840 isolates. (a) Southern blot analysis of KpnI-digested genomic DNA hybridized with the intM gene fragment from pSPRH840. Lanes: 1, M. carbonacea var. africana; 2 and 3, MC840 isolates. (b) Southern analysis of PstI-digested genomic DNA hybridized with the hyg gene fragment. Lanes: 1, M. halophytica var. nigra; 2 and 3, MH840 isolates. Arrows indicate hybridizing fragments discussed in the text.

 
The single 3·1 kb hybridizing fragment in the MC840 lanes indicates that pSPRH840 has integrated into the chromosome (Fig. 2b). The MC840 exconjugants lacked the 4·4 and 3·5 kb hybridizing KpnI fragments, corresponding to free and integrated pMLP1 and also lacked the set of brightly staining pMLP1 bands (Fig. 2a). This indicates that integration of pSPRH840 results in a loss of the pMLP1 phage from M. carbonacea var. africana. Loss of pMLP1 phage is also seen for pSPRH910 integrants.

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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Fig. 3. Cloverleaf structure of the tRNAHis genes found in Micromonospora attB sites. 3' sequences are shown in parentheses for a, M. carbonacea var. africana attB; b, MC840 attL; c, M. halophytica var. nigra attB; d, MC840 attL. Nucleotides with identity to the attP element are in bold type. The anticodon 5' GUG is boxed.

 


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Fig. 4. (a) Alignment of the nucleotide sequences of the pMLP1 attP element, M. carbonacea var. africana attB site, MC840 attL region and MC840 attR region. (b) Alignment of the nucleotide sequences of the pMLP1 attP element, M. halophytica var. nigra attB site, MH840 attL region and MH840 attR region. Identical sequences are enclosed in boxes. Heavy arrows indicate the tRNAHis-encoding region in the genome for attB and after pSPRH840 integration for attL. Arrows indicate inverted repeat sequences. Additional regions with identity outside of the box are shown in bold type.

 
Both tRNAHis genes lacked 3' CCA sequences that are absent in the majority of actinomycete tRNA genes (Fig. 1b, Fig. 4) (Plohl & Gamulin, 1990; Sedlmeier et al., 1994). Sequence analysis of the attL juncture region from the MC840 and MH840 strains showed the generation of new tRNAHis genes which also lacked 3' CCA sequences (Fig. 3). The 3' tRNA gene nucleotides were changed from 5'-GTA-3' to 5'-GGC-3' in MC840 and from 5'-CAG-3' to 5'-GGC-3' in MH840.

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. 5a). 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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Fig. 5. Southern analysis of BamHI-digested genomic DNA hybridized with (a) an apr gene fragment, (b) a hyg gene fragment or (c) pUC19 probes. Lanes: 1 and 2, MC152 isolates; 3 and 4, MC840 isolates; 5 and 6, MC152.840 isolates. Arrows indicate hybridizing fragments discussed in the text. The pUC19 probe hybridizes strongly to a 3·6 kb fragment and faintly to a 4·5 kb fragment in pSET152 lanes (Fig. 1), and a 5·6 kb fragment in pSPRH840 lanes.

 
Introduction of alternate attB sites into M. carbonacea var. africana
It was proposed that the PCR-amplified attB sites could be placed into the genome and act as alternate sites for pSPRH840 integration. To determine whether these alternate attB sites could act as an integration site the pSET152 derivatives pSPRA2301 and pSPRA2303 containing the 0·8 kb M. halophytica var. nigra and 0·5 kb M. carbonacea var. africana attB sites, respectively, were integrated into the M. carbonacea var. africana chromosome to obtain integrant strains MC2301 and MC2303, respectively (Table 1). This study has already demonstrated that pSPRH840 and pSET152 can been integrated into M. carbonacea var. africana simultaneously (see Fig. 5). The plasmid pSPRH840 was conjugated into strains MC2301 and MC2303 to obtain MC2301.840 and MC2303.840 exconjugants, respectively (Table 1).

When a Southern blot of XhoI/XbaI-digested genomic DNA was hybridized with a SacI–XhoI 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 XhoI–XbaI 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 XhoI–XbaI 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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Fig. 6. Southern analysis of XhoI/XbaI double-digested genomic DNA hybridized with the SacI and XhoI fragment probe containing the 5' end of apr. Lanes: 1, MC2301; 2–5, MC2301.840 isolates; 6, MC152; 7, MC840; 8, MC152.840; 9, MC2303; 10–13, MC2303.840 isolates.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A region containing the att/int functions from the M. carbonacea var. africana pMLP1 lysogenic phage has been shown to direct site-specific integration of plasmids into the chromosome of Micromonospora spp. Integration was determined to occur in a tRNA gene which is similar to other actinomycete integrative elements. When aligned with the chromosomal attB sites the pMLP1 attP had 24 bp of identity with M. carbonacea var. africana, M. rosaria and Micromonospora spp. SCC1047 attB sites and 25 bp of identity with the M. halophytica var. nigra attB site. This is the shortest segment of identity described for an actinomycete integrative element that integrates into a tRNA gene. Examples of other tRNA attB sites characterized include VWB bacteriophage (45 bp), pSE101 (46 bp), pMEA100 (47 bp), pSE211 (57 bp), pSAM2 (58 bp), pSG1 (60 bp) and SLP1 (112 bp). For all of the Micromonospora spp. examined the pMPL1 attB site is located at the extreme 3' end of the tRNA gene and does not include the anticodon loop. The longer attB sites characterized extend to include the anticodon loop of the tRNA. The pMLP1 attB site is novel in that it is much smaller than the other attB sites and does not include the anticodon loop. In spite of this, integration remains site-specific, targeting only the tRNAHis gene in the four Micromonospora spp. characterized.

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.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 24 February 2003; revised 12 May 2003; accepted 22 May 2003.