Although representatives with spiral-shaped cells are described for many functional groups of bacteria, this cell morphotype has never been observed among methanotrophs. Here, we show that spiral-shaped methanotrophic bacteria do exist in nature but elude isolation by conventional approaches due to the preference for growth under micro-oxic conditions. The helical cell shape may enable rapid motility of these bacteria in water-saturated, heterogeneous environments with high microbial biofilm content, therefore offering an advantage of fast cell positioning under desired high methane/low oxygen conditions. The pmoA genes encoding a subunit of particulate methane monooxygenase from these methanotrophs form a new genus-level lineage within the family Methylococcaceae, type Ib methanotrophs. Application of a pmoA-based microarray detected these bacteria in a variety of high-latitude freshwater environments including wetlands and lake sediments. As revealed by the environmental pmoA distribution analysis, type Ib methanotrophs tend to live very near the methane source, where oxygen is scarce. The former perception of type Ib methanotrophs as being typical for thermal habitats appears to be incorrect because only a minor proportion of pmoA sequences from these bacteria originated from environments with elevated temperatures.

Methylomonas paludis sp. nov., the first acidtolerant member of the genus Methylomonas, from an acidic wetland et al., 2012). Members of these species are Gram-negative, rod-shaped or coccobacillary cells that produce pink or orange carotenoid pigments, are motile by means of a single flagellum, and grow between pH 5.0 and 9.0, with an optimum at pH 6.5-7.0.Representatives of the genus Methylomonas are especially abundant in various semi-neutral or slightly alkaline environments, such as rice paddies, water-saturated soils, volcanic areas, fresh and marine waters, lakes and sediments (Auman et al., 2000;Auman & Lidstrom, 2002; Hutchens et al., 2004; Lin et al., 2004; Lüke et al., 2010;Dianou et al., 2012). Recently, however, evidence for the presence of Methylomonas-like bacteria in acidic boreal forest soils and Sphagnum-dominated wetlands was obtained in several cultivation-independent studies (Morris et al., 2002; JaatinenAbbreviations: ICMs, intracytoplasmic membranes; MMO, methane monooxygenase; PFK, 6-phosphofructokinase; PLFA, polar lipid fatty acid; RuBP, ribulose-1,5-bisphosphate; RuMP, ribulose monophosphate.The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the partial sequences of the pmoA, mxaF and nifH genes of strain MG30 T are HE801216-HE801219, respectively.

An aerobic methanotrophic bacterium was isolated from a collapsed palsa soil in northern Norway and designated strain NE2 T . Cells of this strain were Gram-stain-negative, non-motile, non-pigmented, slightly curved thick rods that multiplied by normal cell division. The cells possessed a particulate methane monooxygenase enzyme (pMMO) and utilized methane and methanol. Strain NE2 T grew in a wide pH range of 4.1-8.0 (optimum pH 5.2-6.5) at temperatures between 6 and 32 8C (optimum 18-25 8C), and was capable of atmospheric nitrogen fixation under reduced oxygen tension. The major cellular fatty acids were C 18 : 1 v7c, C 16 : 0 and C 16 : 1 v7c, and the DNA G+C content was 61.7 mol%. The isolate belonged to the family Beijerinckiaceae of the class Alphaproteobacteria and was most closely related to the facultative methanotroph Methylocapsa aurea KYG T (98.3 % 16S rRNA gene sequence similarity and 84 % PmoA sequence identity). However, strain NE2 T differed from Methylocapsa aurea KYG T by cell morphology, the absence of pigmentation, inability to grow on acetate, broader pH growth range, and higher tolerance to NaCl. Therefore, strain NE2 T represents a novel species of the genus Methylocapsa, for which we propose the name Methylocapsa palsarum sp. nov. The type strain is NE2 T (5LMG 28715 T 5VKM B-2945 T ).

Two isolates of aerobic methanotrophic bacteria, strains Sph1T and Sph2, were obtained from cold methane seeps in a floodplain of the river Mukhrinskaya, Irtysh basin, West Siberia. Another morphologically and phenotypically similar methanotroph, strain OZ2, was isolated from a sediment of a subarctic freshwater lake, Archangelsk region, northern Russia. Cells of these three strains were Gram-stain-negative, light-pink-pigmented, non-motile, encapsulated, large cocci that contained an intracytoplasmic membrane system typical of type I methanotrophs. They possessed a particulate methane monooxygenase enzyme and utilized only methane and methanol. Strains Sph1 T , Sph2 and OZ2 were able to grow at a pH range of 4.0-8.9 (optimum at pH 6.0-7.0) and at temperatures between 2 and 36 C. Although their temperature optimum was at 20-25 C, these methanotrophs grew well at lower temperatures, down to 4 C. The major cellular fatty acids were C 16 : 1 !5c, C 16 : 1 !6c, C 16 : 1 !7c, C 16 : 1 !8c, C 16 : 0 and C 14 : 0 ; the DNA G+C content was 51.4-51.9 mol%. Strains Sph1 T , Sph2 and OZ2 displayed nearly identical (99.1-99.7 % similarity) 16S rRNA gene sequences and belonged to the family Methylococcaceae of the class Gammaproteobacteria. The most closely related organism was Methylovulum miyakonense HT12 T (96.0-96.5 % 16S rRNA gene sequence similarity and 90 % pmoA sequence similarity). The novel isolates, however, differed from Methylovulum miyakonense HT12 T by cell morphology, pigmentation, absence of soluble methane monooxygenase, more active growth at low temperatures, growth over a broader pH range and higher DNA G+C content. On the basis of these differences, we propose a novel species, Methylovulum psychrotolerans sp. nov., to accommodate these methanotrophs. Strain Sph1 The genus Methylovulum belongs to the class Gammaproteobacteria, family Methylococcaceae, and is so far represented by the only species, Methylovulum miyakonense, which accommodates strictly aerobic, neutrophilic, obligateAbbreviations: ICM, intracytoplasmic membrane; pMMO, particulate methane monooxygenase; sMMO, soluble methane monooxygenase.The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences and the partial sequences of the pmoA gene of Methylovulum psychrotolerans strains Sph1 T , Sph2 and OZ2 are KT381578-KT381583, respectively.

Descriptions of Roseiarcus fermentans gen. nov., sp. nov., a bacteriochlorophyll a-containing fermentative bacterium related phylogenetically to alphaproteobacterial methanotrophs, and of the family Roseiarcaceae fam. nov. A light-pink-pigmented, microaerophilic bacterium was obtained from a methanotrophic consortium enriched from acidic Sphagnum peat and designated strain Pf56 T . Cells of this bacterium were Gram-negative, non-motile, thick curved rods that contained a vesicular intracytoplasmic membrane system characteristic of some purple non-sulfur alphaproteobacteria. The absorption spectrum of acetone/methanol extracts of cells grown in the light showed maxima at 363, 475, 505, 601 and 770 nm; the peaks at 363 and 770 nm are characteristic of bacteriochlorophyll a. However, in contrast to purple non-sulfur bacteria, strain Pf56 T was unable to grow phototrophically under anoxic conditions in the light. Best growth occurred on some sugars and organic acids under micro-oxic conditions by means of fermentation. The fermentation products were propionate, acetate and hydrogen. Slow chemo-organotrophic growth was also observed under fully oxic conditions. Light stimulated growth. C 1 substrates were not utilized. Strain Pf56 T grew at pH 4.0-7.0 (optimum pH 5.5-6.5) and at 15-30 6C (optimum 22-28 6C). The major cellular fatty acids were 19 : 0 cyclo v8c and 18 : 1v7c; quinones were represented by ubiquinone Q-10. The G+C content of the DNA was 70.0 mol%. Strain Pf56 displays 93.6-94.7 and 92.7-93.7 % 16S rRNA gene sequence similarity to members of the families Methylocystaceae and Beijerinckiaceae, respectively, and belongs to a large cluster of environmental sequences retrieved from various wetlands and forest soils in cultivationindependent studies. Phenotypic, genotypic and chemotaxonomic characteristics of strain Pf56 T suggest that it represents a novel genus and species of bacteriochlorophyll a-containing fermentative bacteria, for which the name Roseiarcus fermentans gen. nov., sp. nov. is proposed. Strain Pf56 T (5DSM 24875 T 5VKM B-2876 T ) is the type strain of Roseiarcus fermentans, and is also the first characterized member of a novel family within the class Alphaproteobacteria, Roseiarcaceae fam. nov.

The bacterial genus Methylococcus, which comprises aerobic thermotolerant methanotrophic cocci, was described half-a-century ago. Over the years, a member of this genus, Methylococcus capsulatus Bath, has become a major model organism to study genomic and metabolic basis of obligate methanotrophy. High biotechnological potential of fast-growing Methylococcus species, mainly as a promising source of feed protein, has also been recognized. Despite this big research attention, the currently cultured Methylococcus diversity is represented by members of the two species, M. capsulatus and M. geothermalis, while finished genome sequences are available only for two strains of these methanotrophs. This study extends the pool of phenotypically characterized Methylococcus strains with good-quality genome sequences by contributing four novel isolates of these bacteria from activated sludge, landfill cover soil, and freshwater sediments. The determined genome sizes of novel isolates varied between 3.2 and 4.0Mb. As revealed by the phylogenomic analysis, strains IO1, BH, and KN2 affiliate with M. capsulatus, while strain Mc7 may potentially represent a novel species. Highest temperature optima (45–50°C) and highest growth rates in bioreactor cultures (up to 0.3h−1) were recorded for strains obtained from activated sludge. The comparative analysis of all complete genomes of Methylococcus species revealed 4,485 gene clusters. Of these, pan-genome core comprised 2,331 genes (on average 51.9% of each genome), with the accessory genome containing 846 and 1,308 genes in the shell and the cloud, respectively. Independently of the isolation source, all strains of M. capsulatus displayed surprisingly high genome synteny and a striking similarity in gene content. Strain Mc7 from a landfill cover soil differed from other isolates by the high content of mobile genetic elements in the genome and a number of genome-encoded features missing in M. capsulatus, such as sucrose biosynthesis and the ability to scavenge phosphorus and sulfur from the environment.

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