Functional analysis of long-chain n-alkane degradation by Dietzia spp.

Abstract

The genetic background of long-chain n-alkane degradation was investigated in detail in strain E1, a member of the genetically unexplored Dietzia genus. A suicide vector carrying a 518-bp alkB fragment was site-specifically integrated into the E1 chromosome, and the full alkB, as well as its chromosomal environment was sequenced after plasmid rescue experiments. Four out of the nine putative genes were strongly induced by long-chain n-alkanes in wild-type E1. ORF4 encoded a natural fusion protein consisting of an integral membrane alkane hydroxylase and a rubredoxin domain. The significance of the alkB-rub gene in n-alkane degradation was investigated in phenotypic tests, and the disruption mutant strain exhibited severely impaired growth on n-C(20) alkane carbon source. The mutation was successfully complemented with the expression of intact AlkB-Rub protein, the full-length form of which was detected by simultaneous immunoblotting. The presented data furnish the first experimental evidence of the in vivo existence of an AlkB-Rub natural fusion protein, which plays a major role in long-chain n-alkane degradation.

Two Novel Alkane Hydroxylase-Rubredoxin Fusion Genes Isolated from a Dietzia Bacterium and the Functions of Fused Rubredoxin Domains in Long-Chain n -Alkane Degradation

“…CF8, AlkB in Prauserella rugosa NRRL B-2295, and Dietzia sp. E1Ј, as well as in AlkW1 in the present report) (3,11,28). Among these forms, the AlkB-Rd fusion proteins are less frequently found and their functions have not been defined.…”

“…In addition, the gene encoding the AlkB-Rd fusion protein was also cloned from Dietzia sp. E1, which has confirmed functions in the degradation of C 20 n-alkanes (3). Previously, we also isolated Dietzia sp.…”

“…strain CF8 (11), Prauserella rugosa NRRL B-2295 (27), and Dietzia sp. E1 (3). Whether this kind of fusion is beneficial for the hydroxylation of long-chain alkanes by Grampositive bacteria is still unknown.…”

High efficiency degradation of alkanes and crude oil by a salt-tolerant bacterium Dietzia species CN-3

“…Furthermore, this result was similar to Dietzia sp. E1, suggesting that the transcript levels of the alkB gene increased with the increasing length of the n-alkane chain, including C 12 , C 16 , C 18 and C 20 (Bihari et al, 2011). Although the alkB and CYP153 genes are frequently reported to be related to the degradation of small-and medium-chain-length alkanes (C 5 eC 17 ) (Rojo, 2009), Dietzia sp.…”

“…Dietzia sp. E1 utilizes C 6 eC 36 alkanes as the carbon source (Bihari et al, 2011) and D. cinnamea strain P4 can consume C 11 eC 36 alkanes (Von der Weid et al, 2007). Moreover, Dietzia strains have the capacity to degrade aromatic hydrocarbons, including naphthalene (Von der Weid et al, 2007), phenanthrene (Al-Awadhi et al, 2007) and fluoranthene (Kumar et al, 2011).…”