Draft Genome Sequence of Uncultured Upland Soil Cluster Gammaproteobacteria Gives Molecular Insights into High-Affinity Methanotrophy

ABSTRACT Aerated soils form the second largest sink for atmospheric CH4. A near-complete genome of uncultured upland soil cluster Gammaproteobacteria that oxidize CH4 at <2.5 ppmv was obtained from incubated Antarctic mineral cryosols. This first genome of high-affinity methanotrophs can help resolve the mysteries about their phylogenetic affiliation and metabolic potential.

A erated soils remove 4 to 6% of CH 4 from the atmosphere (1) as a result of high-affinity methanotrophy that has been demonstrated by the detection of the high-affinity form of particulate methane monooxygenase, encoded by pmo genes (2)(3)(4)(5)(6). In the absence of isolates and genomes, phylogenetic identity and physiology of atmospheric methane-oxidizing bacteria have remained a mystery (7). Here, we report a near-complete genome of uncultured upland soil cluster Gammaproteobacteria (USC␥) obtained from incubated mineral cryosols (pH 8.51) collected from Taylor Dry Valley, Antarctica. These samples exhibited CH 4 oxidation when incubated with 2.5 ppmv at 4°C and 10°C, and were found to contain USC␥-like pmoA genes (8).
A high-quality genome bin containing a pmoA gene 100% identical to one of our USC␥-like pmoA clone sequences was identified. Reads mapped to the scaffolds in this genome bin were reassembled using IDBA_UD (default settings). CheckM (14) determined that it was 89.99 to 91.9% complete with 0 to 0.09% contamination and 0% strain heterogeneity. Annotation was performed using Prokka v1.12-beta (15), BLAST (16), and KEGG Automatic Annotation Server v2.1 (17). A single copy of twenty 30S and thirty-one 50S ribosomal proteins, one 5S rRNA operon, a single copy of 16S and 23S rRNA gene fragments, 38 tRNA genes, and 3,012 coding sequences were retrieved. The 488 nt-long 16S rRNA gene shared 99% identity to uncultured bacteria (JQ684308, HM445440, and DQ823229) and 94% identity to Thioalkalivibrio (NR_074692) and Ectothiorhodospira (NR_125567) of Chromatiales, which is phylogenetically closely related to Methylococcales but includes no methanotrophs.
The USC␥ draft genome contained the complete operon of pmoCAB. The pmoA gene shared 72% identity with Methylocaldum szegediense (Methylococcales). Genes encoding methanol dehydrogenase and accessory proteins, enzymes for the tetrahydrofolate and tetrahydromethanopterin-linked C1 transfer pathways, and two formate dehydrogenases were identified. Although gene encoding formaldehyde dehydrogenase (FALDH) was missing, genes for pyrroloquinoline quinone (PQQ) synthesis were identified, indicative of the potential use of PQQ-dependent FALDH. As with the genomes of other gammaproteobacterial methanotrophs, the USC␥ draft genome encodes all essential genes for a complete serine biosynthesis pathway for formaldehyde assimilation. Genes encoding enzymes for nitrogen metabolism were also identified. Manual annotation and phylogenetic and comparative analyses are in progress to pinpoint the phylogenetic affiliation of USC␥ and to elucidate differences in the metabolic potential between high-and low-affinity methanotrophs.
Accession number(s). This whole-genome shotgun project and the USC␥ draft genome have been deposited at DDBJ/ENA/GenBank under the accession no. MUGK00000000. The version described in this paper is version MUGK01000000.

ACKNOWLEDGMENTS
Field expedition and logistics were funded by grants from the New Zealand Ministry of Business, Innovation and Employment (UOWX1401) and the New Zealand Antarctic Research Institute (NZARI2013-7) to S.C.C. and C.K.L. The Institute for Applied Ecology New Zealand (https://aenz.aut.ac.nz/) supported M.C.Y.L.'s travel to and from New Zealand. This research was supported by a National Science Foundation grant (DEB-1442059) to T.C.O. and grants from Department of Geosciences, Princeton Environmental Institute and Council on Science and Technology (CST) of Princeton University to C.R.E.
We thank the staff at Research Computing, Office of Information Technology, Princeton University for their technical support with the computational analyses. We also thank the staff at the International Centre for Terrestrial Antarctic Research, Antarctic New Zealand and Scott Base for their administrative and logistical assistance.