Evolutionary relationship between (putative) replication proteins of previously characterised AICEs (bold, underlined), newly found AICEs (boxed), actinomycete plasmids (bold grey) and other chromosome encoded homologues with more than 35% identity to AICE-encoded Rep proteins. Abbreviations: SACE, Sac. erythraea NRRL23338; SCO/Sco, S. coelicolor A3(2); SAV/Sav, S. avermitilis MA-4680; Sare, Sal. arenicola CNS205; Strop, Sal. tropica CNB-440; FRAAL, Frankia alni strain ACN14a; Franean, Frankia sp. strain EAN1pec; Francci3, Frankia sp. strain CcI3; Mflv, Myc. gilvum; Mmcs, Mycobacterium sp. MCS; ΦRv2, prophage of Mycobacterium tuberculosis H37Rv; Mvan, Mycobacterium vanbaalenii PYR-1; MAV, Mycobacterium avium 104; nfa, Nocardia farcinica IFM 10152; PTH, Pelotomaculum thermopropionicum SI. Plasmids: pSLS of Streptomyces laurentii; pSG5 of S. ghanaensis; pSVH1 of Streptomyces venezuelae; pSA1.1, Streptomyces cyaneus. The phylogenetic tree was constructed using the neighbour-joining algorithm of Mega version 4.0 (Tamura et al. 2007). The scale bar represents 0.2 substitutions per site

Comparison of the phylogenetic relationship, as shown by connecting lines, of the Amycolatopsis 16S rDNA sequences (~1,250 bps) with that of the RepAM and TraJ sequences reveals co-evolution of the pMEA-elements with their Amycolatopsis host strains. Phylogenetic trees were reconstructed by neighbour-joining with Mega version 4.0 using CLUSTALW alignment and by calculating evolutionary distances by the Kimura-2 parameter method. Bootstrap values were calculated from 1,000 replicate trees. Bootstrap values over 50% are shown. The scale bar represents 0.1, 0.01, and 0.05 substitutions per nucleotide position for RepAM, 16S rDNA and TraJ, respectively. Figure 4 was taken from te Poele et al. (2007)

Structural organisation of (a) previously characterised and sequenced AICEs: pMEA100 of A. mediterranei; pMEA300 of A. methanolica; pSE211 of Sac. erythraea NRRL23338; pSAM2 of S. ambofaciens; pMR2 of M. rosaria; SLP1 of S. coelicolor A3(2); pSE101 of Sac. erythraea NRRL23338 (b) newly found AICEs: pSE101 and pSE222 of Sac. erythraea NRRL23338; AICESav3728 and AICESav3708 of S. avermitilis MA-4680; AICESco3250 and AICESco5349 of S. coelicolor A3(2); AICEMflv3036 of Myc. gilvum PYR-GCK; AICEFranean5323 and AICEFranean6303 of Frankia sp. strain EAN1pec; AICEFraal5456 of F. alni ACN14a; AICESare1562 and AICESare1922 of Sal. arenicola CNS205; AICEStrop0058 of Sal. tropica CNB-440. (c) AICE-remnants: Sare1208 of Sal. arenicola CNS205; Sco3997 of S. coelicolor A3(2); insertion Sco3937 of S. coelicolor A3(2). All newly found elements were named after the locus tag of their integrase gene. The prefixes of locus tags of the AICEs of Sac. erythraea NRRL23338 (SACE), S. coelicolor A3(2) (SCO), S. avermitilis MA-4680 (SAV), Frankia sp. strain EAN1pec (Franean), F. alni ACN14a (FRAAL), Sal. arenicola CNS205 (Sare), Sal. tropica CNB-440 (Strop), and Myc. gilvum PYR-GCK (Mflv) were left out for clarity. The size of the elements and the tRNA gene in which the elements are inserted are indicated below the element name at the right. Colour coding: orange, genes and sites involved in excision/integration; dark yellow, genes most likely involved in replication and its control; dark yellow with vertical black lines, repAM genes; dark yellow with vertical red lines, repSA genes; red bar, pMEA-specific hairpin structure; blue, putative conjugation genes; dark blue, putative main transfer genes; lime, putative regulatory genes; dark green, Nudix hydrolase genes; white, orfs with unknown functions; arrows with diagonal black lines, orfs with G + C content <55%; pink, transposons (tn), IS-elements (IS), and pseudogenes (ps); lavender with white diagonal lines, genes encoding DNA primase/polymerase (Prim-pol) proteins; red, genes with annotated function: gltA, glycosyltransferase; hnh, HNH-endonuclease signature; aph, aminoglycoside phosphotransferase; flav, flavoprotein; re, predicted restriction endonuclease; phd, encoding prevent-host-death family protein; thio, thioesterase superfamily protein; exp, predicted RND superfamily drug exporter; pept, peptidase C14 caspase catalytic subunit p20; HAD, HAD-superfamily hydrolase subfamily IA; ich, isochorismatase hydrolase; dprA; encoding DNA-protecting protein; rni, ribonuclease inhibitor; thiC, thiamine biosynthetic gene. The following colours correspond to genes shared by two or more elements, lavender, pMEA100, pSE211, SLP1, AICESco5349, AICESare1922, AICEStrop0058, Sare1208; grey, pSE222, AICESare1562, AICESare1922, AICEStrop0058; bright yellow (GGDEF-domain), pSE211, pSAM2, AICESco5349, AICESare1562; light blue, pSE101, pSE102, pSE222; bright green (single-stranded binding protein), pMEA100 and pSE222; plum, pSE211, pSE222; black, AICESare1562, AICESare1922, Sare1208; arrows with black confetti; highly similar protein clusters on AICESco3250 and Sco3997. Grey band between pSE101 and pSE102 indicates a highly similar DNA region. Black arrow head indicates partial protein coding sequence and back diagonal bar indicates a frameshift. The shared att site located between AICESco3708 and AICESco3728 is highlighted in both elements. Figure 1 was taken from te Poele et al. (2008)

(a) Alignment of the putative nicking sites and the flanking 8 bp repeats (underlined) of pMEA300, pMEA100, pSE211, pSE101, pSE102, and the AICE remnant Sare1208 (b) Secondary structures of the hairpins of pMEA300, pMEA100, pSE211, pSE101, pSE102, and the AICE remnant Sare1208, as predicted by mfold (Zuker 2003). The conserved 8 bp repeats are outlined by bars. The black arrows indicate the putative nicking sites, which are located in ssDNA regions. Black star indicates 1 bp mismatch in 8 bp repeat of Sare1208. Figure 3 was adapted from te Poele et al. (2006)