2c); however, neither resolvases from Rhodococcus nor Corynebacterium spp. were related to the arthrobacterial counterparts. A 23-nt site (1090–1067 bp) showing 60% similarity to ColE2 ori (Yagura et al., 2006) was found on the complementary DNA strand at 45 nt upstream of the repA gene (Fig. 1b). Specific combinations of genes were tested to determine the minimal region required for autonomous replication of pPRH. Plasmids pAPrepAB4 containing repAB MS-275 genes and pAPrepA2 harbouring the repA gene only were constructed. pAPrepAB4 transformed Arthrobacter oxydans PY21, A. rhombi VP3, Arthrobacter sp. 68b and Rhodococcus sp. SQ1. By using a second derivative, pAPrepA2,
no transformants were obtained in all Arthrobacter and Rhodococcus spp. strains tested. The Escherichia coli–Arthrobacter–Rhodococcus shuttle vector pRMU824 conferring resistance to chloramphenicol was constructed as described in ‘’Materials and methods’’ (Fig. 3). In addition, the tetracycline or kanamycin resistance gene was inserted into the plasmid pRMU824 to expand the applicability of the vector. Thus,
two shuttle vectors pRMU824Km and pRMU824Tc were obtained (Fig. 3). All shuttle vectors successfully replicated in Arthrobacter sp. 68b, 83, 85, A. oxydans PY21, Rhodococcus sp. SQ1 and E. coli. Approximately nine copies of the pRMU824Km vector per Arthrobacter sp. 68b cell were found. The analysis of www.selleckchem.com/products/torin-1.html plasmid loss, during cultivation in rich medium without antibiotic pressure, showed that segregational stability depended on the tested strain: 37 ± 3% of A. oxydans PY21 cells retained the plasmid Celecoxib after 40 generations, and under the same conditions, only 6 ± 0.7% of Arthrobacter sp. 68b cells contained the vector. To analyse the compatibility of the developed
vectors, A. oxydans PY21 harbouring the pRMU824Tc plasmid was additionally transformed with pART2gfp (Sandu et al., 2005). The clones simultaneously resistant to kanamycin and tetracycline and producing a green fluorescent protein were easily screened. Both recombinant plasmids were isolated from A. oxydans PY21 and used to transform E. coli in the presence of appropriate antibiotic. The restriction analysis of the isolated individual plasmids confirmed that the pRMU824Tc and pART2gfp plasmids were compatible with each other in the Arthrobacter spp. cells. To test the applicability of the developed vectors for functional screening, the genes encoding the initial steps of 2-hydroxypyridine biodegradation in Arthrobacter sp. PY22 were chosen as a target. It was proposed that catabolism of 2-hydroxypyridine proceeds via formation of 2,3,6-trihydroxypyridine, which could spontaneously oxidize and dimerize to blue pigment, 4,5,4′,5′-tetrahydroxy-3,3′-diazadiphenoquinone-(2,2′) (for review, Kaiser et al., 1996). Total DNA from Arthrobacter sp. PY22, the strain degrading 2-hydroxypyridine and forming a blue pigment (Semėnaitė et al.