Genome Sequence of the Methanotrophic Poly-β-Hydroxybutyrate Producer Methylocystis parvus OBBP

Abstract

Methylocystis parvus OBBP is an obligate methylotroph considered the type species of the genus Methylocystis. Two pmoCAB particulate methane monooxygenase operons and one additional singleton pmoC paralog were identified in the sequence. No evidence of genes encoding soluble methane monooxygenase was found. Comparison of M. parvus OBBP and Methylocystis sp. strain Rockwell (ATCC 49242) suggests that both species should be taxonomically classified in different genera.

GENOME ANNOUNCEMENT

Methane is a greenhouse gas that accumulates in the atmosphere and contributes to climate change and global warming (7). Methane-oxidizing bacteria (methanotrophs), a subset of methylotrophs, are a group of bacteria with a specialized metabolism able to utilize C₁ compounds (2, 5). Methanotrophs are essential for the global methane cycle since they are capable of oxidizing methane (or methanol) to formaldehyde, which can be transformed into biomass or oxidized to carbon dioxide (3, 4). Type II methanotrophs belong to Alphaproteobacteria. There are four genera, Methylosinus, Methylocystis, Methylocella, and Methylocapsa (13). Methylocystis parvus OBBP (NCIMB 11129) is an obligate methylotroph originally isolated as a methane-utilizing bacterium and is considered the type species of the genus Methylocystis (16). This strain has industrial interest because it is able to produce poly-β-hydroxybutyrate (PHB) from methane (9).

The genome of M. parvus OBBP was sequenced using the Titanium kit and GS-FLX pyrosequencing equipment from Roche. Preliminary assembly of raw reads was performed using Newbler software from Roche. In order to improve the assembly quality in-house, Perl shell scripts were designed. A quality draft of 108 contigs was obtained. The genome was structurally and functionally annotated using RAST (1), an automated genome annotation system, and functions, names, and general properties of the gene products were predicted using this method. The M. parvus OBBP genome is 4.5 Mbp in size, contains 63.4% GC, encodes 49 RNAs, and contains 4,594 coding sequences.

Two pmoCAB particulate methane monooxygenase operons (8, 12) and one additional singleton pmoC paralog were identified in the sequence, in contrast with the genome of Methylocystis sp. strain Rockwell (ATCC 49242), where only a single pmoCAB particulate methane monooxygenase operon and four singleton pmoC paralogs were recognized (15). Genes encoding soluble methane monooxygenase were absent in both cases.

Other proteins involved in methane oxidation were identified; these include a methanol dehydrogenase, a set of enzymes that are involved in pyrroloquinoline quinone synthesis and tetrahydrofolate- and tetrahydromethanopterin-linked pathways, and NAD-linked formate dehydrogenase. Enzymes required for the serine cycle were identified, confirming that M. parvus OBBP possess a methane assimilation pathway linked to the central metabolite pathways via this cycle (6).

Enzymes involved in the ethylmalonyl-coenzyme A (ethylmalonyl-CoA) pathway for glyoxylate regeneration were detected in M. parvus OBBP. This strain contains the genes encoding enzymes for the tricarboxylic acid cycle and glycolysis.

The enzymes responsible for PHB metabolism were identified in the M. parvus OBBP genome, i.e., two poly-β-hydroxybutyrate polymerases (PhbCI and PhbCII), two poly-β-hydroxybutyrate depolymerases (DepA and DepB), an acetyl-CoA acetyltransferase (PhbA), one acetoacetyl-CoA reductase (PhbB), one polyhydroxyalkonate synthesis repressor (PhbR), and a phasin (10). The phbA, phbB, and phbR genes form a cluster similar to that we have found in the genome of Methylosinus trichosporium OB3b (14). None of these genes were detected in Methylocystis sp. strain Rockwell (ATCC 49242).

JSpecies (11) comparison of M. parvus OBBP and Methylocystis sp. strain Rockwell (ATCC 49242) gives an average nucleotide identity (ANI) based on BLAST (ANIb) of 78.78% (ANIb aligned, 44.35%) and an ANI based on MUMmer (ANIm) of 85.82% (ANIm aligned, 28.09%). These results suggest that the species should be taxonomically classified in different genera.

Nucleotide sequence accession number.

The M. parvus OBBP (NCIMB 11129) genome sequence has been submitted to GenBank under the accession number AJTV00000000.