It was 5000 bp in length with a G+C content of 66 mol%. The plasmid pPRH was predicted to encode six putative open reading frames (ORFs), in which ORF2 and ORF3 formed the minimal replicon of plasmid pPRH and shared 55–61% and 60–69% homology, respectively, with the RepA and RepB proteins of reported Bortezomib rhodococcal plasmids. Sequence analysis revealed a typical ColE2-type ori located 45 bp upstream of the gene repA. Sequence and phylogenetic analysis led to the conclusion that pPRH is a representative of a novel group of pAL5000 subfamily of ColE2 family plasmids. Three shuttle vectors pRMU824, pRMU824Km and pRMU824Tc, encoding chloramphenicol resistance, were constructed.
The latter two harboured additional antibiotic resistance genes kan and tet, respectively. All vectors successfully replicated in Escherichia coli, Arthrobacter and Rhodococcus
spp. The vector pRMU824Km was employed for functional screening of 2-hydroxypyridine catabolism encoding genes from Arthrobacter sp. PY22. Sequence analysis of the cloned 6-kb DNA fragment revealed eight putative ORFs, among which hpyB gene encoded a putative monooxygenase. Escherichia coli is the dominant screening host for functional metagenomics (Taupp et al., 2011). Although E. coli can support the expression of genes from numerous donor genomes, the main limitations in using E. coli cells for functional selleck chemical screens are recognition of promoters, protein maturation and cofactor requirements in heterologous genes and proteins. One of the opportunities to expand the diversity of the expression Rebamipide systems is to create new ones based on different microorganisms and intrinsic genetic elements such as phages,
plasmids or transposons (Uchiyama & Miyazaki, 2009). The bacteria of genus Arthrobacter are Gram-positive, nonmotile obligate aerobes that belong to class Actinobacteria (Zhi et al., 2009). Arthrobacter species are very common in soils and often constitute an important or even dominant culturable fraction of the microbial communities. A main feature of arthrobacters is their nutritional versatility coupled with the ability to grow in simple media utilizing a wide range of compounds as a source of carbon and nitrogen (Cacciari & Lippi, 1987; Jones & Keddie, 1992). Recently, these microorganisms have received considerable attention because of their potential use in detoxification of xenobiotics (Eaton, 2001; Brandsch, 2006; Shapir et al., 2007), while the genetic tools applicable for manipulation of cells of Arthrobacter spp. are not well developed. Different strains of the genus Arthrobacter harbour plasmids varying in size from 41 to 380 kb (Igloi & Brandsch, 2003; Mongodin et al., 2006; Jerke et al.