Molecular Genetic and Functional Analysis of the Genes Encoding Alkane 1‑Monoozygenase Synthesis in Members of the Genus <i>Rhodococcus</i>

Abstract

Abstract—Organization and localization of the alkB genes and of alkane 1-monooxygenases they encode in members of the genus Rhodococcus was investigated. All members of a phylogenetic group were found to possess specific types of the alkB genes (alkB1-type in the operons containing rubredoxin-coding genes, rubredoxin reductase, and a regulatory protein and/or alkB2-type in the operons lacking the rubredoxin-reductase-coding gene, and supplementary, separately located determinants of the alkB3−alkB8 type). The alkB1-type genes were present in the chromosomes of bacteria of groups B1 (R. aetherivorans, R. ruber), С (R. opacus, R. jostii, R. wratislaviensis, R. koreensis), D (R. erythropolis, R. qingshengii), G (R. triatomae), and E (R. fascians). The alkB2-type genes occurred in strictly specified loci of the chromosomes of members of the phylogenetic groups А (R. hoagii/R. equi), В2 (R. coprophilus, R. pyridinivorans, R. rhodochrous), and D (R. erythropolis, R. qingshengii). The separately located alkB3−alkB5 were revealed in the chromosomes of members of the D group D (alkB5 was found only in R. qingshengii), the alkB6 genes occurred in members of the groups B1 and В2, and alkB7−alkB8 were present in members of group Е. The proteins coded by the genes alkB1 and alkB2 belonged to three phylogenetic groups. The first one comprised AlkB1-type proteins of members of groups В1 and С and the AlkB2-type proteins of members of groups D and A. The second one was represented by AlkB2-type proteins of bacteria of group В2. The third group comprised AlkB1-type proteins of members of groups G and D. Alkane 1-monooxygenases encoded by separately localized genes alkB3−alkB8 were represented by three phylogenetic lineages: AlkB3−AlkB5, AlkB6, and AlkB7 and AlkB8. In the genome of R. pyridinivorans 5Ар, the genes alkB2 and alkB6 characteristic of group В1 were revealed. This determinants were shown to be required for biosurfactant synthesis. Emulsifying activity of the mutant with impaired alkB2 gene at 28 and 42°С was 16 and 3 times lower, respectively, while the amount of trehalose-containing surfactants decreased 7 and 3 times, respectively. Independent on the cultivation temperature, the mutant with impaired alkB6 gene exhibited 1.2 times lower emulsifying activity and more than twofold lower decrease of the synthesis of trehalose-containing surfactants. These results indicated the key role of alkB2 in biosurfactant synthesis at different cultivation temperatures. The role of alkB6 increased at 42°С, probably due to its elevated transcriptional activity.