Genome Sequence of the Haloalkaliphilic Methanotrophic Bacterium Methylomicrobium alcaliphilum 20Z

GENOME ANNOUNCEMENT

Aerobic methanotrophy, widespread within the bacterial world, is commonly found in Alphaproteobacteria, Gammaproteobacteria, and Verrucomicrobia (3, 8, 10). Methylomicrobium alcaliphilum 20Z represents a methanotrophic clade adapted to high-pH and high-salinity ecosystems (4, 6, 13).

The assembled genome sequence of M. alcaliphilum 20Z was determined at Genoscope. A mate-paired GSflx 454 library with 3-kb insert size was constructed (22 Mbp; ≈4.6-fold coverage) and combined with 454 Titanium reads (71 Mbp; ≈15-fold coverage) for assembly using Newbler (version 2.0.01.14) with validation via the Consed interface (www.phrap.org). For gap closure, 1,163 PCRs between contigs were performed and sequenced. Potential base errors were corrected by mapping a total of 1,3497,122 Illumina 36-bp reads onto the whole genome sequence using SOAP (http://soap.genomics.org.cn) as described previously (1) to finalize the 4.8-Mb assembly. Sequence annotation and comparative genome analysis are under way using the MicroScope platform at Genoscope (14).

M. alcaliphilum 20Z features a relatively large circular chromosome of 4.67 Mb, with an average GC content of 48.75%, three complete rRNA operons, 44 tRNAs, and 4,083 predicted protein-coding sequences in the initial annotation. The genome also features a circular plasmid of 128 kb of very similar GC content (48.70%) encoding putative systems for replication and conjugation transfer. To our knowledge, this is the first reported plasmid for a methanotrophic bacterium with a known genome sequence.

The C₁ metabolism gene inventory in M. alcaliphilum 20Z includes a single copy of the membrane-associated methane monooxygenase gene cluster (pmoCAB) (12), pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (mxaFI) and an associated cytochrome c, genes for MDH assembly and PQQ biosynthesis, tetrahydromethanopterin- and tetrahydrofolate-linked C₁ transfer pathways, aldehyde ferredoxin oxidoreductase (aorAB), membrane-associated quinoprotein formaldehyde dehydrogenase (adh), two formate dehydrogenases, and the ribulose monophosphate (RuMP) pathway. Although serine cycle enzyme activities have not been previously detected in strain 20Z (6), genes encoding glyA, sga, hpr, gck, mclA, and mtkAB are present. Key enzymes of the Calvin-Benson-Bassham cycle (5) are missing. Complete sets of genes for function of the Embden-Meyerhof-Parnas pathway, the Entner-Doudoroff pathway, and the pentose phosphate pathway were identified. Similar to other type I methanotrophs (11, 15), the genome encodes all enzymes of the citric acid cycle.

Nitrogen metabolism involves the gene inventory for urea uptake and hydrolysis, nitrate/nitrite reduction, and direct ammonium uptake, as well as glutamine synthetase/glutamate synthase and glutamate and alanine dehydrogenases for ammonium assimilation (7). A gene homologous to hydroxylamine oxidoreductase (hao), possibly handling hydroxylamine toxicity arising from oxidation of ammonia by pMMO, is present (2).

The genome sequence confirms that M. alcaliphilum 20Z has an ectoine biosynthesis ectABCask operon (9). Accumulation of sucrose is encoded by an sps-spp-fruK-amy gene cluster, for sucrose phosphate synthase/phosphatase, fructokinase, and a specific amylosucrase, respectively.

Availability of the genome of M. alcaliphilum 20Z greatly expands the use of molecular approaches for experimental and biotechnological investigations of this strain.