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 ϳ4-Mb circular chromosome and an ϳ600-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 (ϳ99% 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.Methylibium petroleiphilum strain PM1, which belongs to a newly described genus and species (57), is a motile bacterium belonging to the Comamonadaceae family of the Betaproteobacteria and is an important member of subsurface microbial communities in many gasoline-contaminated aquifers. Furthermore, PM1 is a methylotroph that can grow aerobically on the fuel oxygenate methyl tert-butyl ether (MTBE) and oxidize it completely to carbon dioxide (9, 34). MTBE is a suspected carcinogen that has contaminated drinking water wells throughout the United States due to the preponderance of underground leaking storage tanks, the widespread usage of MTBE, and its recalcitrance and mobility in groundwater. PM1 can also oxidize aromatic hydrocarbons (toluene, benzene, oxylene, and phenol) (20) and n-alkanes (C 5 to C 12 ) (57; K. Hristova, unpublished data) and has been used in two bioaugmentation field trials in gasoline-contaminated aquifers in California (67) and Montana (18,73). In contaminated sites amended with oxygen, in situ MTBE degradation was observed and corresponded to increases in native populations of Methylibium sp. (ϳ99% similarity to PM1, based on the 16S rRNA gene) (38,67,82). PM1-l...

Methylobacterium Genome Sequences: A Reference Blueprint to Investigate Microbial Metabolism of C1 Compounds from Natural and Industrial Sources

“…As exemplified here for M. petroleiphilum [54], Beta-proteobacterial methylotrophs may also employ the serine cycle for C1 carbon assimilation[54], [74]. As that of the Alpha-proteobacterium S. pomeroyi , the genome of M. petroleiphilum contains a single gene cluster encoding all required functions of the serine cycle (Fig.…”

Structural and kinetic characterization of two 4-oxalocrotonate tautomerases in Methylibium petroleiphilum strain PM1

“…M. petroleiphilum is unusual in that it has two 4-oxalocrotonate tautomerases, encoded in two distinct operons ( dmpI and dmpII , where dmp stands for dimethylphenol) that provide the enzymes for a pair of meta -fission pathways [18,19]. Presumably, the presence of these parallel catabolic pathways imparts an ability to use a wider variety of aromatic molecules as carbon and energy sources.…”

“…Methylibium petroleiphilum strain PM1 is one such organism [18]. The organism was originally isolated from a biofilter that was used at a sewage treatment plant for treating oil refinery discharges.…”

Gene mdpC plays a regulatory role in the methyl-tert-butyl ether degradation pathway of Methylibium petroleiphilum strain PM1

“…A putative ATP-dependent transcriptional regulator has been identified within the MTBE-degradation gene cluster, designated Mpe_B0601 or mdpC (Kane et al , 2007). To examine the role of mdpC as a regulator of the MTBE degradation pathway of strain PM1, we developed an mdpC insertion-mutant strain of PM1 and designed an RT-qPCR assay to quantify mdpC transcription in the presence or absence of MTBE and TBA.…”