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Methylocella Palustris Gen. Nov., Sp. Nov., a New Methane-Oxidizing Acidophilic Bacterium from Peat Bogs, Representing a Novel Subtype of Serine-Pathway Methanotrophs

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Methylocella Palustris Gen. Nov., Sp. Nov., a New Methane-Oxidizing Acidophilic Bacterium from Peat Bogs, Representing a Novel Subtype of Serine-Pathway Methanotrophs International Journal of Systematic and Evolutionary Microbiology (2000), 50, 955–969 Printed in Great Britain Methylocella palustris gen. nov., sp. nov., a new methane-oxidizing acidophilic bacterium from peat bogs, representing a novel subtype of serine-pathway methanotrophs Svetlana N. Dedysh,1,5 Werner Liesack,2 Valentina N. Khmelenina,3 Natalia E. Suzina,3 Yuri A. Trotsenko,3 Jeremy D. Semrau,4 Amy M. Bares,4 Nicolai S. Panikov1 and James M. Tiedje5 Author for correspondence: Svetlana N. Dedysh. Tel: j7 95 135 0591. Fax: j7 95 135 6530. e-mail: dedysh!inmi.host.ru 1 Institute of Microbiology, A new genus, Methylocella, and a new species, Methylocella palustris, are Russian Academy of proposed for three strains of methane-oxidizing bacteria isolated from acidic Sciences, Moscow 117811, Sphagnum peat bogs. These bacteria are aerobic, Gram-negative, colourless, Russia non-motile, straight and curved rods that utilize the serine pathway for carbon 2 Max-Planck-Institut fu$ r assimilation, multiply by normal cell division and contain intracellular poly-β- Terrestrische hydroxybutyrate granules (one at each pole). These strains use methane and Mikrobiologie, D-35043 Marburg, methanol as sole sources of carbon and energy and are moderately acidophilic Germany organisms with growth between pH 45 and pH 70, the optimum being at pH 5 0–5 5. The temperature range for growth is 10–28 C with the optimum at 3 Institute of Biochemistry S and Physiology of 15–20 SC. The intracytoplasmic membrane system is different from those of Microorganisms, Russian type I and II methanotrophs. Cells contain an extensive periplasmic space and a Academy of Sciences, vesicular membrane system connected to the cytoplasmic membrane. The Pushchino, Moscow region strains grew only on media with a low salt content (02–05glN1). All three 142292, Russia strains were found to possess soluble methane monooxygenase and are able 4 Department of Civil and to fix atmospheric nitrogen via an oxygen-sensitive nitrogenase. No products Environmental were observed in a PCR with particulate methane monooxygenase-targeted Engineering, The University of Michigan, primers; hybridization with a pmoA probe was also negative. The major Ann Arbor, MI 48109-2125, phospholipid fatty acids are 18:1 acids. The GMC content of the DNA is USA 612 mol%. The three strains share identical 16S rRNA gene sequences and 5 Center for Microbial represent a novel lineage of methane-oxidizing bacteria within the α-subclass Ecology, Michigan State of the class Proteobacteria and are only moderately related to type II University, East Lansing, methanotrophs of the Methylocystis–Methylosinus group. The three strains are MI 48824-1325, USA most closely related to the acidophilic heterotrophic bacterium Beijerinckia indica subsp. indica (965% 16S rDNA sequence similarity). Collectively, these strains comprise a new species and genus Methylocella palustris gen. nov., sp. nov.; strain KT (l ATCC 700799T) is the type strain. Keywords: Methylocella palustris gen. nov., sp. nov., acidophilic methane-oxidizing bacteria, serine-pathway methanotrophs INTRODUCTION and energy. The current classification separates all the known methanotrophs into three groups (types I, II Methane-oxidizing bacteria (MOB) are a ubiquitous and X) by multiple criteria, including cell morphology, group of micro-organisms possessing the unique the arrangement of intracytoplasmic membranes, the ability to utilize methane as the sole source of carbon pathway for formaldehyde assimilation, the DNA GjC content and the major phospholipid fatty acids. ................................................................................................................................................. Both 5S rRNA and 16S rRNA sequence analyses Abbreviations: FAME, fatty acid methyl ester; ICM, intracytoplasmic indicated that all known MOB form phylogenetically membrane; MOB, methane-oxidizing bacteria; pMMO, particulate meth- ane monooxygenase; sMMO, soluble methane monooxygenase. coherent clusters within the α- and γ-subclasses of the Proteobacteria (Hanson & Hanson, 1996). The EMBL/GenBank/DDBJ accession number for the 16S rRNA gene of Methylocella palustris strain KT is Y17144. Although MOB have been isolated from diverse 01279 # 2000 IUMS 955 S. N. Dedysh and others environments including soils, sediments, wetlands, liquid cultures. In the latter case, M2 medium was supple- freshwater and marine habitats (Whittenbury et al., mented with 0n5% (v\v) of a vitamin stock solution 1970a; Hanson & Hanson, 1996; King, 1992), the containing the following (mg per 100 ml): biotin (0n4); known ecophysiological types of methanotrophs are nicotinic acid (10); riboflavin (10); and inositol (10). To quite limited. Most MOB available in pure cultures study the ultrastructure of cells grown in Cu-rich medium, copper was added aseptically as CuCl#;2H#O to M2 medium are both mesophilic and neutrophilic; however, after autoclaving to achieve a copper concentration of 5 µM. thermophilic (Bodrossy et al., 1997), psychrophilic For growth, screw-cap 500 ml serum bottles were used with (Omelchenko et al., 1993; Bowman et al., 1997) and a headspace:liquid-space ratio of 4:1. After inoculation, the alkaliphilic (Khmelenina et al., 1997) methanotrophs bottles were sealed with silicone rubber septa and methane have been described. was added aseptically by a syringe equipped with a dis- posable filter (0n22 µm) to achieve a 15–20% concentration Until recently, MOB that colonize acidic environments in the headspace. Bottles were incubated on a rotary shaker remained unknown and pure cultures of acidophilic (200 r.p.m.) at 20 mC. Methanotrophic bacteria were sub- methanotrophs were absent from culture collections. cultured at 4–5-week intervals. Last year, however, we reported on the enrichment of Reference bacterial strains. Methylococcus capsulatus (Bath) methanotrophic communities from acidic peat bogs of T T ATCC 33009 , Methylosinus sporium NCIMB 11126 , four boreal sites in West Siberia and European North Methylosinus trichosporium OB3b NCIMB 11131T, Methylo- Russia (Dedysh et al., 1998a) and on the isolation of cystis parvus OBBP NCIMB 11129T, Methylomicrobium three pure cultures of MOB from these enrichments album BG8 ATCC 33003, Beijerinckia indica subsp. indica (Dedysh et al., 1998b). These isolates are the first ATCC 9039T and Rhodopseudomonas acidophila ATCC representatives of a new group of previously uncul- 25092T were used as reference strains. The methanotrophs turable MOB and initial studies have revealed them to were cultivated on NMS medium with methane as the be considerably different from other methanotrophs. growth substrate (Whittenbury et al., 1970a), while Beijer- The 16S rRNA gene sequence data indicate that the inckia indica was grown on nitrogen-free mineral medium acidophilic strains represent a novel lineage of MOB with glucose (Becking, 1984) and Rhodopseudomonas acido- only moderately related to the known cluster of α- phila was grown in the light under anaerobic conditions on succinate\mineral medium (Pfennig, 1969). proteobacterial methanotrophs, i.e. Methylosinus– Methylocystis spp. (Dedysh et al., 1998b). Further- Verification of strain purity. Both phase-contrast and elec- more, the acidophilic strains are most closely affiliated tron microscopy were used to check the acidophilic isolates with the heterotrophic bacterium Beijerinckia indica for contamination. The absence of heterotrophic satellites subsp. indica, while no other known group of methano- was checked by plating methanotrophic isolates on several media containing different organic substrates. These media trophs has any close relatives that are not methano- were as follows: standard undiluted and 10-fold-diluted trophs (Hanson & Hanson, 1996). The nucleotide Luria–Bertani agar (1n0% tryptone, 0n5% yeast extract, sequence of the PCR-amplified fragment of the mmoX 1n0% NaCl) and M2-agar media (Noble agar) amended with gene [which encodes the α-subunit of the soluble 0n1% (w\v) glucose, fructose or sucrose. As a control, the methane monooxygenase (sMMO) hydroxylase] in same agar mineral medium was used without any organic these acidophilic methanotrophs diverges significantly substrates. To prove the homogeneity of isolated methano- from these gene fragments in known methanotrophs trophic strains, the cells were grown on methane and and forms a branch that is distinct from the two methanol with subsequent RFLP analysis of the cloned 16S known mmoX sequence groups [the Methylocystis– rRNA genes to examine possible shifts in population Methylosinus-like mmoX sequences and the Methyl- structure. For RFLP analysis, a single colony of every strain was used to inoculate a liquid culture. This culture was ococcus capsulatus (Bath) mmoX sequence]. grown on methane to the mid-exponential phase. Then cells In this paper we describe the cell ultrastructure and were collected by centrifugation and divided into two parts, physiology of these three methanotrophic isolates from one of which was again grown in liquid culture with methane, peat bogs, as well as their biochemical, chemo- while the other sample was transferred to M2 medium taxonomic and genotypic characteristics. We propose containing 0n3% (w\v) methanol. After 5 d incubation, the cells were harvested, genomic DNA was extracted from both a new genus, Methylocella gen. nov., and the species cultures by using an SDS-based method (see Methods). Methylocella palustris sp. nov. for these strains. PCR-mediated amplification of 16S rRNA genes
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