Its termini contain the inverted repeat sequence 5′-CCTGC … GCAGG-3′, and its 5′-ends are covalently capped with protein (Overhage et al., 2005; Parschat et al., 2007). Our previous sequence analysis of pAL1 and predictions of possible secondary structures formed by potential telomeric 3′-overhangs indicated significant differences of the ‘left’ and ‘right’ terminus of pAL1, raising the question of whether each terminus of pAL1 is recognized, or even capped, by a specific protein (Parschat et al., Selleckchem Bafilomycin A1 2007). Rhodococcal plasmids pHG201 and pHG205 are other examples of actinomycete
linear plasmids that do not show striking homology between their ‘left’ and ‘right’ telomere sequences (Kalkus et al., 1998), but their TPs have not been described. In contrast, the
ends of Streptomyces CYC202 manufacturer linear replicons usually contain well-conserved terminal palindromic sequences (Zhang et al., 2006). The gene product of pAL1.102 is the only protein exhibiting a weak similarity to known (Streptomyces) TPs; however, due to the low sequence similarity, its annotation as a ‘putative terminal protein’ was tentative (Parschat et al., 2007). As a first step toward characterizing the telomere complex of pAL1, we identified the protein attached to both termini of pAL1 and demonstrated its specific deoxynucleotidylation in vitro. The strains and plasmids used in this study are listed in Table 1. For isolation of total DNA, A. nitroguajacolicus Rü61a [pAL1] was grown in a mineral salts medium (Parschat et al., 2003) on 8 mM sodium benzoate at 30 °C. Arthrobacter nitroguajacolicus Rü61a [pAL1, pART2malE-ORF102 or pART2malE-ORF103] was cultivated in a mineral salts medium supplemented with 4 mM 4-hydroxyquinaldine Ribose-5-phosphate isomerase and 140 μg mL−1 kanamycin. Cells were harvested by centrifugation at an OD600 nm of approximately 2.5. Escherichia coli DH5α clones containing derivatives of pMal-c2x or pART2 were grown in lysogeny broth (LB) (Sambrook & Russell, 2001) at 37 °C in the presence of 100 μg mL−1 ampicillin or 50 μg mL−1 kanamycin, respectively. For the synthesis of fusion
proteins of maltose-binding protein (MBP) and the protein encoded by pAL1.102 (termed pORF102), E. coli K12 ER2508 [pLysSRARE] harboring pMal-c2x-ORF102 was grown in LB with ampicillin (100 μg mL−1), chloramphenicol (34 μg mL−1), and auto induction solutions ‘5052’ and ‘M’ (Studier, 2005) at 30 °C. Cells were harvested by centrifugation at an OD600 nm of ∼5 and stored at −80 °C before use. Total DNA of A. nitroguajacolicus Rü61a [pAL1] was isolated according to Rainey et al. (1996). Plasmid DNA was isolated using the EZNA Plasmid Miniprep kit (Peqlab, Erlangen, Germany). Gel extraction of DNA fragments from agarose gels was performed with the Perfectprep gel cleanup kit (Eppendorf, Hamburg, Germany). For cloning purposes, DNA fragments were purified using the High Pure PCR Product Purification kit (Roche Diagnostics GmbH, Mannheim, Germany).