Genome and proteome of long-chain alkane degrading Geobacillus thermodenitrificans NG80-2 isolated from a deep-subsurface oil reservoir

Abstract

The complete genome sequence of Geobacillus thermodenitrificans NG80-2, a thermophilic bacillus isolated from a deep oil reservoir in Northern China, consists of a 3,550,319-bp chromosome and a 57,693-bp plasmid. The genome reveals that NG80-2 is well equipped for adaptation into a wide variety of environmental niches, including oil reservoirs, by possessing genes for utilization of a broad range of energy sources, genes encoding various transporters for efficient nutrient uptake and detoxification, and genes for a flexible respiration system including an aerobic branch comprising five terminal oxidases and an anaerobic branch comprising a complete denitrification pathway for quick response to dissolved oxygen fluctuation. The identification of a nitrous oxide reductase gene has not been previously described in Gram-positive bacteria. The proteome further reveals the presence of a long-chain alkane degradation pathway; and the function of the key enzyme in the pathway, the long-chain alkane monooxygenase LadA, is confirmed by in vivo and in vitro experiments. The thermophilic soluble monomeric LadA is an ideal candidate for treatment of environmental oil pollutions and biosynthesis of complex molecules.

Fig. 1.

Circular maps of the G. thermodenitrificans NG80-2 chromosome and plasmid pWL1071. (a) The chromosome map from the outside inward: the first and second circles show predicted protein-coding regions on the plus and minus strands, respectively (colors were assigned according to the color code of the COG functional classes; see key); the third circle shows transposase genes and insertion sequence elements in red; the fourth and fifth circles show tRNAs and rRNAs in dark slate blue and dark golden red, respectively; the sixth and seventh circles show percentage G + C in relation to the mean G + C and GC skew, respectively. (b) The plasmid map shows ORFs color-coded according to their assigned functions (see key).

Fig. 2.

Proteomic characterization of pathways involved in hexadecane metabolism in G. thermodenitrificans NG80-2. Differentially expressed proteins in hexadecane-grown cells, in comparison with sucrose-grown cells, were investigated by 2-D electrophoresis (2-DE)/MALDI-TOF MS analysis. The predicted metabolic pathways for sucrose and hexadecane are shown. The enzymes of the pathways and corresponding genes in NG80-2 (as gene ID numbers) are noted. Proteins detected by 2-DE/MALDI-TOF MS are highlighted. Blue, induced; red up-regulated; yellow, down-regulated; green, unchanged. Proteins with pI values outside the range of pH 4 to 7 were not investigated. Analogs are in brackets. P, phosphate; DH, dehydrogenase.

Fig. 3.

Characterization of long-chain alkane monooxygenase LadA. (a) Induced expression of LadA as an extracellular protein in hexadecane-grown NG80-2 cells. Sucrose-grown cells were used as the reference state. Sections of 2-D PAGE gels stained with colloidal CBB G-250 are shown. The spot in circle was identified as LadA by MALDI-TOF MS analysis. (b) Expression and purification of LadA in E. coli as shown by SDS/PAGE. Lane 1, crude extract of E. coli BL21 (pET-ladA) before IPTG induction; lane 2, crude extract induced with IPTG; lane 3, fractions eluted from Ni²⁺ column; lane 4, molecular size markers. (c) Hydroxylation of hexadecane by purified LadA. GC chromatographs show conversion of hexadecane to 1-hexadecanol. IS, internal standard (squalane). (d) Specific activity of purified LadA on alkanes with different chain lengths.