Whole-genome analysis of the methyl tert-butyl ether-degrading beta-proteobacterium Methylibium petroleiphilum PM1

Abstract

Methylibium petroleiphilum PM1 is a methylotroph distinguished by its ability to completely metabolize the fuel oxygenate methyl tert-butyl ether (MTBE). Strain PM1 also degrades aromatic (benzene, toluene, and xylene) and straight-chain (C(5) to C(12)) hydrocarbons present in petroleum products. Whole-genome analysis of PM1 revealed an approximately 4-Mb circular chromosome and an approximately 600-kb megaplasmid, containing 3,831 and 646 genes, respectively. Aromatic hydrocarbon and alkane degradation, metal resistance, and methylotrophy are encoded on the chromosome. The megaplasmid contains an unusual t-RNA island, numerous insertion sequences, and large repeated elements, including a 40-kb region also present on the chromosome and a 29-kb tandem repeat encoding phosphonate transport and cobalamin biosynthesis. The megaplasmid also codes for alkane degradation and was shown to play an essential role in MTBE degradation through plasmid-curing experiments. Discrepancies between the insertion sequence element distribution patterns, the distributions of best BLASTP hits among major phylogenetic groups, and the G+C contents of the chromosome (69.2%) and plasmid (66%), together with comparative genome hybridization experiments, suggest that the plasmid was recently acquired and apparently carries the genetic information responsible for PM1's ability to degrade MTBE. Comparative genomic hybridization analysis with two PM1-like MTBE-degrading environmental isolates (approximately 99% identical 16S rRNA gene sequences) showed that the plasmid was highly conserved (ca. 99% identical), whereas the chromosomes were too diverse to conduct resequencing analysis. PM1's genome sequence provides a foundation for investigating MTBE biodegradation and exploring the genetic regulation of multiple biodegradation pathways in M. petroleiphilum and other MTBE-degrading beta-proteobacteria.

FIG. 1.

Circular maps of the chromosome (A) and megaplasmid pPM1 (B) of M. petroleiphilum PM1. Outer rings 1 and 2 show all CDSs, colored by functional category (dark gray, hypothetical proteins; light gray, conserved hypothetical and unknown function; brown, general function prediction; red, replication and repair; green, energy metabolism; blue, carbon and carbohydrate metabolism; cyan, lipid metabolism; magenta, transcription; yellow, translation; orange, amino acid metabolism; pink, metabolism of cofactors and vitamins; light red, purine and pyrimidine metabolism; lavender, signal transduction; sky blue, cellular processes; and pale green, structural RNAs). Genes coding for major metabolic features (green, methylotrophy; red, aromatic hydrocarbon degradation; and light blue, alkane degradation) are shown on rings 3 and 4. Large repeat regions are indicated on rings 5 and 6. The larger repeated regions colored light gray (29-kb repeat) and dark gray (40-kb repeat) are described in the text, while the other colors represent repeated IS elements, ISmp1-8. Ring 7 shows the deviation from the average G+C, and the innermost ring, ring 8, shows the GC skew (G−C)/(G+C). The plasmid and chromosome are not drawn to scale.

FIG. 2.

Schematic diagram of an M. petroleiphilum PM1 cell showing structural features and cellular processes, including predicted methylotrophy and fuel hydrocarbon degradation pathways. Two adjacent arrows imply multiple steps. Abbreviations: MTBE, methyl tert-butyl ether; HMTBE, hydroxymethyl tert-butyl ether; TBF, tert-butyl formate; TBA, tert-butyl alcohol; HIBA, hydroxyisobutyric acid. Systems involved in metal resistance and transport, secretion, motility, chemotaxis, and electron transport are shown within or associated with the cell membrane. Cofactors are labeled green, and electron donors are labeled red in the cytosol. The figure is not drawn to scale.

FIG. 3.

Phylogenetic tree of MpeA3393, which putatively codes for the large subunit of methanol dehydrogenase. The protein product from MpeA3393 is most similar to glucose dehydrogenase (GluDH) from B. fungorum (81% similar; GenBank accession no. ZP_00283396) and the methanol dehydrogenase large subunit (Mdh large) from M. capsulatus Bath (75% similar; accession no. AAU90462). Protein names are shown in square brackets.

FIG. 4.

MTBE and TBA degradation by M. petroleiphilum PM1. The graph shows data for the parent strain MP0005, which carries the <SmO> marker on the megaplasmid (•, MTBE; ○, TBA), and the megaplasmid-free mutant MP0007 (▪, MTBE; □, TBA).