Genome Sequence of the Methanotrophic Alphaproteobacterium Methylocystis sp. Strain Rockwell (ATCC 49242)

Abstract

Methylocystis sp. strain Rockwell (ATCC 49242) is an aerobic methane-oxidizing alphaproteobacterium isolated from an aquifer in southern California. Unlike most methanotrophs in the Methylocystaceae family, this strain has a single pmo operon encoding particulate methane monooxygenase but no evidence of the genes encoding soluble methane monooxygenase. This is the first reported genome sequence of a member of the Methylocystis species of the Methylocystaceae family in the order Rhizobiales.

TEXT

Methylocystis spp. are ubiquitous methanotrophic inhabitants of many ecosystems (3, 4, 7, 8, 12), likely due to metabolic versatility within this genus. For instance, Methylocystis parvus ferments poly-beta-hydroxybutyrate anaerobically (16), some isolates grow on multicarbon compounds (2, 6), and Methylocystis sp. strain SC2 expresses two particulate methane monooxygenases (pMMO) with different methane oxidation kinetics (1). Methylocystis sp. strain Rockwell has a high capacity to oxidize ammonia and hydroxylamine to nitrite (11) and is highly tolerant of ammonium but strongly inhibited by nitrite (10).

The genome of Methylocystis sp. strain Rockwell (ATCC 49242) was sequenced, assembled, and annotated by the U.S. Department of Energy (DOE) Joint Genome Institute. A quality draft of 79 contigs and 6 scaffolds was assembled from Roche 454 Titanium and Illumina sequence reads. Illumina reads were assembled using Velvet (18), and contigs larger than 800 bp were shredded into 1-kb pseudoreads with 200 bp of overlap. Pseudoreads were combined with 454 reads into a hybrid 454/Illumina hybrid assembly using Newbler. Automatic annotation was performed using the Prokaryotic Dynamic Programming Genefinding Algorithm (5). The Methylocystis sp. strain Rockwell draft genome is 4.6 Mbp in size and 63% G+C and contains a single rRNA operon, a full complement of tRNA genes, and 4,637 predicted protein-encoding genes. Genome closure, manual annotation, and comparative analysis are under way with assistance from the Joint Genome Institute and the MicroScope annotation platform of bacterial genomes at Genoscope (15).

A single particulate methane monooxygenase operon (pmoCAB) and four singleton pmoC paralogs were identified in the draft sequence, but genes encoding soluble methane monooxygenase were absent. The copper homeostasis genes copCD are located downstream and in the same orientation as pmoCAB. This arrangement has not been observed in other methanotroph genomes but is identical to that of ammonia monooxygenase operons in ammonia-oxidizing Nitrosomonas strains (13). The structural gene for methanobactin (9), a component of the pMMO-associated copper acquisition system, was not observed, suggesting its absence or significant sequence differences from characterized genes. Genes encoding methanol dehydrogenase, pyrroloquinoline quinone cofactor biosynthesis proteins, tetrahydromethanopterin-linked and tetrahydrofolate-mediated pathways, NAD-linked formate dehydrogenase, serine cycle enzymes for formaldehyde assimilation, and an ethylmalonyl-coenzyme A pathway for glyoxylate regeneration were found. As in the genome sequence of Methylosinus trichosporium OB3b (14), genes encoding marker enzymes for the Calvin cycle and ribulose monophosphate pathways were absent. However, genes encoding a system for acetate activation were found, suggesting a strategy for survival in the absence of methane (2).

A full complement of genes for N₂ fixation, as well as ammonia and nitrate/nitrite transport and assimilation, was present. Genes encoding enzymes for the oxidation of hydroxylamine to nitrite (haoAB) and for reduction of nitrite to nitric oxide (nirK) were identified, even though nitrous oxide was not produced by this strain when it was grown with excess ammonium or nitrite (10). Genes encoding a conjugative transfer system similar to that of other Rhizobiales were identified and may contribute to genome plasticity (17).

This first reported genome of the methanotrophic species Methylocystis suggests substantial structural and metabolic diversity within the methanotroph canon.

Nucleotide sequence accession number.

The Methylocystis sp. strain Rockwell genome nucleotide sequence has been submitted to GenBank and assigned accession number AEVM00000000.