Selection of clones was performed in E. coli DH5α, Arthrobacter sp. selleck chemicals 68b and Rhodococcus sp. SQ1
bacteria, which could not utilize 2-hydroxypyridine. E. coli DH5α cells were transformed by ligation mixtures, and kanamycin-resistant clones were grown on NA plates supplemented with IPTG and 2-hydroxypyridine. As the visual inspection of plates did not reveal any coloured colonies, all clones were harvested from agar plates and pooled. The mixture of the recombinant plasmids was isolated and consequently used to transform Arthrobacter sp. 68b and Rhodococcus sp. SQ1 bacteria. A single clone (from ca 4000 clones) was selected on the NA medium supplemented with kanamycin and 2-hydroxypyridine by screening for pigment production. The pHYP1 plasmid containing a 6-kb DNA insert was isolated from the blue pigment producing clone of Rhodococcus sp. SQ1. Sequence analysis of the cloned 6-kb DNA fragment from pHYP1 revealed eight putative ORFs (Fig. 4). Six of them shared the significant (71–87%) sequence homology with hypothetical proteins of the pSI-1 plasmid from Arthrobacter sp. AK-1 (Jerke et al., 2008). Moreover, an identical arrangement of ORFs in both plasmids was observed. The predicted functions of ORFs from pHYP1 are presented in Table 2. New cryptic plasmid, not related to known arthrobacterial
ones, was isolated from A. rhombi PRH1. A conserved sequence found at 45 bp upstream of the repA gene showed remarkable homology to the typical ColE2-type ori (Leret et al., 1998; Yagura et al., 2006). The predicted minimal replication operon of pPRH consisted of repAB genes, which is in accordance with the previous findings that both repA PD0325901 clinical trial and repB are required for replication of pAL5000 (Stolt & Stoker, 1996a), pFAJ2600 (De Mot et al., 1997), pBLA8 (Leret et al., 1998) and pCASE1 (Tsuchida et al., 2009). All these results supported the conclusion that pPRH is a member of the pAL5000 subfamily of ColE2 family (Stolt & Stoker, 1996a), plasmids belonging to the theta replication C
class (Bruand et al., 1993). Usually, repB is located downstream and overlaps with repA of these plasmids, suggesting that both of these genes form an operon. Correspondingly, the start codon of repB in pPRH overlaps with the stop Carteolol HCl codon of the repA by one nucleotide. A putative rep operon in pPRH may also include ORF4, which overlaps with repB and encodes a hypothetical protein. The function of ORF4 in the plasmid replication and/or maintenance remains unclear. Phylogenetic analysis of RepA and RepB of pPRH showed that they formed a distinct branch on phylogenetic tree suggesting their evident divergence from homologous proteins (Fig. 2a,b). Two putative conserved domains related replicase and primase, respectively, were detected in RepA from pPRH, which is a common structural feature of other RepA proteins associated with theta replication (De Mot et al., 1997; Leret et al., 1998; Sekine et al., 2006; Matsui et al.