Molecular Microbiology (2002) 43(1), 173–186 Cryptic plasmids of Mycobacterium avium: Tn552 to the rescue Carolyn Kirby,1 Al Waring,2 Thomas J. Griffin IV,3 with other members of the M. avium complex (MAC), is Joseph O. Falkinham III,4 Nigel D. F. Grindley3 an opportunistic pathogen that causes a variety of and Keith M. Derbyshire1,2* diseases in animals and humans (Inderlied et al., 1993). 1Department of Biomedical Sciences, State University of MAC isolates from both clinical and environmental New York at Albany, NY, USA. sources often contain naturally occurring plasmids of 2Division of Infectious Disease, Wadsworth Center, varying size (10 to >100 kb) (Crawford and Falkinham, NYS Department of Health, NY, USA. 1990; Falkinham and Crawford, 1994; Pashley and 3Department of Molecular Biophysics and Biochemistry, Stoker, 2000). Southern hybridization studies on crude Yale University, New Haven, CT, USA. extracts of plasmid DNA from independent isolates 4Department of Biology, Virginia Polytechnic Institute, have indicated that some of these plasmids are related Blacksburg, VA, USA. (Jucker and Falkinham, 1990). Based on these studies, plasmids from M. avium, Mycobacterium intracellulare and Mycobacterium scrofulaceum isolates have been Summary assigned to at least four related groups (Jucker, 1991). Plasmids have been described in almost all bacterial The widespread occurrence of related plasmids from species analysed and have proven to be essential different isolates suggests that the plasmids have the genetic tools. In many bacteria these extrachro- ability to transfer between mycobacterial species in the mosomal DNAs are cryptic with no known markers environment. In addition, multiple plasmids are often or function, which makes their characterization and found in the same isolate, showing that these plasmids genetic exploitation extremely difficult. Here we can stably coexist and, therefore, that they represent describe a system that will allow the rescue of any cir- different plasmid incompatibility groups. Little is known cular DNA (plasmid or phage) using an in vitro trans- about these plasmids, their mechanism of replication and position system to deliver both a selectable marker coexistence, the genes that they encode or whether the (kanamycin) and an Escherichia coli plasmid origin of plasmids play any role in virulence. Various reports have replication. In this study, we demonstrate the rescue of speculated that they may contribute to virulence and also four cryptic plasmids from the opportunistic pathogen may encode resistance to metals, restriction modification Mycobacterium avium. To evaluate the host range of systems and metabolic enzymes (Crawford et al., 1981; the rescued plasmids, we have examined their ability Meissner and Falkinham, 1984; Erardi et al., 1987). to be propagated in Mycobacterium smegmatis and However, the inability to cure plasmids from these iso- Mycobacterium bovis BCG, and their compatibility lates, as well as the lack of selectable markers and an with other mycobacterial plasmids. In addition, we use understanding of the genetic makeup of these MAC a library of transposon insertions to sequence one plasmids, have prevented both rigorous analysis of plasmid, pVT2, and to begin a genetic analysis of their genetic composition and confirmation of plasmid- plasmid genes. Using this approach, we identified a associated functions (Falkinham and Crawford, 1994; putative conjugative relaxase, suggesting this myco- Pashley and Stoker, 2000). bacterial plasmid is transferable, and three genes Plasmids are important tools for the genetic analysis of required for plasmid establishment and replication. any organism. They provide a means to clone, comple- ment and express host genes, and to introduce selected genes into different genetic backgrounds. The mycobac- Introduction teria lack fully characterized, compatible plasmid systems Mycobacterium avium is a naturally occurring, slow- to facilitate genetic manipulation. Most mycobacterial growing mycobacterium. Mycobacterium avium, along plasmids are based on the low copy number vector, pAL5000 (Labidi et al., 1985; Pashley and Stoker, 2000). More recently, a second family of plasmids, the pMSC262 Accepted 27 September, 2001. *For correspondence. David Axelrod or pLR7 family, has been described (Guilhot et al., 1999; Institute, NYS Department of Health, PO Box 22002, Albany, NY 12201–2002, USA; E-mail [email protected]; Tel. Pashley and Stoker, 2000). These plasmids (pMSC262, (+1) 518 473 6079; Fax (+1) 518 486 7971. pLR7, pJAZ38, pMF1 and pCLP) are all compatible with © 2002 US Government 174 C. Kirby et al. pAL5000 (Qin et al., 1994; Gavigan et al., 1997; Bachrach The in vitro transposition reactions were electroporated et al., 2000; Picardeau et al., 2000). Except for one into E. coli and kanamycin-resistant (Kmr) transformants member, pCLP, these plasmids have not been completely selected. Restriction analysis of plasmid DNA from these sequenced or characterized, and only regions associated transformants revealed that a significant proportion of with replication have been identified (Le Dantec et al., these were circular transposons, in which Tn552 had 2001). Thus, there is still a clear demand for additional, inserted into itself close to one end, but outside regions well-characterized mycobacterial plasmids. The develop- necessary for replication and kanamycin resistance. ment and characterization of additional plasmid vectors These transposon circles are most probably a conse- will not only provide significant new insight into plasmid quence of the low amounts of target DNA in the reaction biology in this species, but it will also facilitate, inevitably, and are not normally seen under standard reaction the genetic analysis of all mycobacteria, including the conditions (Griffin et al., 1999). To enrich for insertions major human pathogen Mycobacterium tuberculosis. into the M. avium plasmids, all the colonies from a A major hurdle in characterizing plasmids from organ- single electroporation were pooled and plasmid DNA isms for which there exist only a limited number of genetic was isolated. This DNA was then subjected to gel elec- tools is the inability to purify plasmid DNA and to intro- trophoresis, without digestion, to separate large plasmid duce it into more genetically amenable organisms such DNA from the smaller transposon DNA circles. The larger as Escherichia coli. Here we describe a novel way to circular species were purified from the agarose gel and rescue cryptic plasmids from crude DNA extracts using retransformed into E. coli. This enrichment step ensured an in vitro transposition system. A derivative of Tn552, that all the plasmid DNAs screened were plasmids Tn552kan∑ori, has been constructed which contains a rescued from the M. avium extract rather than transposon selectable marker and an E. coli plasmid origin of repli- circles. cation (Griffin et al., 1999). In vitro transposition of this element into a plasmid DNA confers on the plasmid the Restriction and Southern analysis of rescued ability to replicate, and be selected, in E. coli. We show plasmid DNAs that this system is very robust and can be used to rescue different coexisting plasmids from a single extract. A Plasmid DNAs of Kmr transformants from each extract library of transposon insertions was also used to deter- were analysed by SalI restriction digestion. Analysis of the mine the DNA sequence of one of the plasmids, and restriction patterns suggested that at least four plasmid allowed regions required for plasmid replication to be types had been rescued from the crude extracts, which is genetically defined. entirely consistent with the number of plasmids originally observed in each isolate by Jucker and Falkinham (1990). All the plasmids rescued from the MD1 extract had a Results similar profile (Fig. 1, left); consistent with rescue of a single plasmid and with the prediction that MD1 contained Rescue of cryptic plasmids only one plasmid, pVT2. Single-fragment differences in Crude DNA extracts from two M. avium isolates were individual plasmid profiles are a result of insertion of the used as a substrate in an in vitro transposition reaction 2 kb Tn552 transposon. Plasmids with three different using Tn552kan∑ori. One isolate, MD1, was thought to restriction patterns were rescued from MD22, consistent contain a single plasmid, pVT2 (Jucker and Falkinham, with this strain carrying more than one plasmid (Fig. 1, 1990). The second strain, MD22, had been predicted right). The three profiles were classed as types A, B and to contain up to four different plasmids based on Sou- C, with 26, 14 and 8 plasmids assigned to each class thern hybridization analyses (Jucker and Falkinham, respectively. Southern hybridization confirmed that all 1990). Samples of these DNA extracts were examined by plasmids within a group show complete cross-hybridiza- agarose gel electrophoresis and ethidium bromide stain- tion and, therefore, are independent insertions into the ing, which identified a potential plasmid species in the same plasmid (data not shown). There were also several MD1 extract, in addition to chromosomal DNA (data plasmids that had unique profiles, which did not belong to not shown). By contrast, only chromosomal DNA was any of the four classes, and they have not been studied detected in the MD22 extract. This may be a consequence further. of the difficulty in purifying plasmid DNA from this species, To confirm the assignment of these plasmids to differ- and/or that these