In the genome of Rhodococcus erythropolis NRRL B-16531, two CYP153 homologues
were recently detected (van Beilen et al., 2006), and the presence of CYP153 alkane hydroxylase was also proved in Dietzia sp. E1 (Bihari et al., 2010). The complementation study not only proved the physiological significance of the expressed alkane hydroxylases, but the presence of the presumed fusion forms of AlkB-Rubs could be investigated simultaneously. Use of the FLAG-tagged AlkB-Rubs in phenotypic tests allowed direct detection of the putative fusion of AlkB and www.selleckchem.com/products/Rapamycin.html Rub domains by immunoblotting. Although the FLAG sequences were fused to the C-termini of the approximately 6-kDa Rub domains, only large, 59–68-kDa proteins were detected in cell lines carrying the plasmids pNV18Sm-E1BRF, pNV18Sm-DpBRF, pNV18Sm-DmBRF, pNV18Sm-DcBRF and pNV18Sm-DnBRF (Fig. 3b). While a nonspecific signal also appeared in the blot, it did not complicate the interpretation. The FLAG-tagged proteins were clearly expressed in all desired cell lines, and their size verified the natural fusion of AlkB and Rub domains in five Dietzia spp. In most cases, the observed differences in the mobilities of AlkB-Rubs were in good correlation with the expected protein sizes; however, further analysis is necessary for the exact identification of N-terminal regions (Fig. 4). Available DNA sequence data suggest the presence of AlkB-Rubs in other actinomycetes strains as well. Alkane hydroxylase
activity encoded by alkB-rub has been described only for Prauserella rugosa NRRL B-2295 p38 MAPK activity (Smits et al., 2002),
although it is also likely in Nocardioides sp. CF8 (Hamamura et al., 2001). Nonetheless, the authors only annotated the alkB-rub genes, but the putative natural fusion proteins were not investigated at a translational level. Therefore, to our best knowledge, the data of the present report provide the first direct in vivo evidence for the existence of AlkB-Rub fusion proteins, which play a major role in long-chain n-alkane degradation. Concerning the genetic arrangement of the alkB region, Dietzia sp. E1 displays high similarity to that of the two strains Galeterone mentioned above. A single alkB homologue and a downstream ORF encoding its putative TetR-type regulator were detected in the chromosome of all three strains. In spite of the similarities, there are marked differences in substrate preference. While putative AlkB-Rubs of P. rugosa NRRL B-2295 and Nocardioides sp. CF8 are responsible for the initial hydroxylation of n-C10–n-C16 and n-C6–n-C8 alkanes, respectively, the AlkB-Rub of Dietzia spp. E1 acts on the n-C20 alkane. In contrast to P. rugosa NRRL B-2295 and Nocardioides sp. CF8, the examined five Dietzia spp. and R. erythropolis NRRL B-16531 (van Beilen et al., 2002b) can deplete >n-C16 alkanes (Table 2). Nevertheless, strain NRRL B-16531 harbours four alkB and rub homologues in the chromosome, which hinders a clear-cut identification of the genes responsible for alkane degradation.