Draft Genome Sequence of Methylovulum psychrotolerans Sph1T, an Obligate Methanotroph from Low-Temperature Environments

ABSTRACT Methylovulum psychrotolerans Sph1T is an aerobic, obligate methanotroph, which was isolated from cold methane seeps in West Siberia. This bacterium possesses only a particulate methane monooxygenase and is widely distributed in low-temperature environments. Strain Sph1T has the genomic potential for biosynthesis of hopanoids required for the maintenance of intracytoplasmic membranes.

M ethylovulum psychrotolerans Sph1 T is a neutrophilic aerobic methanotroph of the class Gammaproteobacteria, family Methylococcaceae. It was isolated from a cold methane seep in the Irtysh basin, West Siberia (1). This methanotroph grows well at low temperatures and is commonly detected in various low-temperature environments, such as arctic lakes, glaciers, and northern peatlands (2)(3)(4). Cells of M. psychrotolerans Sph1 T are Gram-negative, nonmotile, encapsulated, large cocci that multiply by binary fission and utilize methane and methanol.
For the genome sequencing of strain Sph1 T , a combination of fragment and mate-paired library approaches was used. Both libraries were sequenced with the Illumina MiSeq platform using 2 ϫ 250-bp paired-end sequencing reagents. Matepaired reads were treated with the NextClip tool (5), resulting in 1,082,354 read pairs with a mean insert size of 3,183 bp. Paired-end reads were filtered and trimmed by quality with CLC Genomics Workbench (Qiagen, Germany) using recommended parameters, resulting in 431,801 read pairs. De novo assembly was performed with the SPAdes version 3.11.0 assembler (6). The total length of the final assembly was 5,189,806 bp; it consisted of 97 genomic scaffolds with an N 50 value of 334,445 bp. Final genome coverage was 40ϫ for the mate-paired library and 23ϫ for the paired-end library. The estimated size of the M. psychrotolerans Sph1 T genome is 5.2 Mb (coverage, 63ϫ), with an average GϩC content of 50.8%. In total, 4,577 predicted protein-coding genes were identified.
The average nucleotide identity between the genomes of M. psychrotolerans Sph1 T and another described member of this genus, M. miyakonense HT12 T (7), is only 79%, which confirms their classification as two different species. The genome of M. psychrotolerans Sph1 T contains a single pmoCAB operon for particulate methane monooxygenase (MMO). The presence of the homologous pxmABC operon, which is characteristic of many gammaproteobacterial methanotrophs (7,8), was not detected. In contrast to M. miyakonense HT12 T , an operon encoding soluble MMO (mmoXYBZDC) is lacking in M. psychrotolerans Sph1 T . The ability to grow on methanol is explained by the presence of the gene operons encoding MxaFI-and XoxF-methanol dehydrogenases (9). Genes involved in tetrahydromethanopterin (H 4 MTP), tetrahydrofolate (H 4 -folate)-linked C1 transfer, and formate oxidation were identified. A complete set of genes encoding formaldehyde assimilation in the ribulose monophosphate pathway was present, while the serine cycle was incomplete.
The genome includes all genes required for glycogen biosynthesis (glgA, glgB, and glgC) (10) and nitrogen metabolism, including genes for nitrate/nitrite reduction, ammonium and urea uptake and assimilation, as well as key genes for nitrogen fixation.
Strain Sph1 T has the genomic potential for the biosynthesis of hopanoids, which is required for the maintenance of intracytoplasmic membranes (11). A complete set of genes for the nonmevalonate pathway and the ispA gene encoding farnesyl diphosphate synthase were identified. The genes responsible for squalene synthesis (12), two copies of the shc gene encoding the enzyme responsible for the cyclization of squalene into diploptene (13), as well as the hpnH and hpnG genes involved in the conversion of diploptene to bacteriohopanetetrol (14) were also present.
Accession number(s). The M. psychrotolerans Sph1 T genome sequence was deposited in GenBank under the accession no. PGFZ00000000.

ACKNOWLEDGMENT
This genome analysis was performed within the Russian state budgetary support framework of research topic no. 01201350930.