Two novel methanotrophic strains, R-49797T and OS501, were isolated from pond water in South Africa and Japan, respectively. Strains R-49797T and OS501 shared 99.7 % 16S rRNA gene sequence similarity. Cells were Gram-stain-negative, non-motile cocci with a diplococcoid tendency and contained type I methanotroph intracytoplasmic membranes. The pmoA gene encoding particulate methane monooxygenase was present. Soluble methane monoooxygenase (sMMO) activity, the mmoX gene encoding sMMO and the nifH gene encoding nitrogenase were not detected. Methane and methanol were utilized as sole carbon source. The strains grew optimally at 25–33 °C (range 20–37 °C) and at pH 6.3–6.8 (range 5.8–9.0). The strains did not support growth in media supplemented with 1 % (w/v) NaCl. For both strains, the two major fatty acids were C16 : 1ω7c and C16 : 0 and the DNA G+C content was 65.6 mol%. The isolates belong to the family Methylococcaceae of the class Gammaproteobacteria and cluster most closely among the genera Methylocaldum, Methylococcus and Methylogaea, with a 16S rRNA gene sequence similarity of 94.2 % between strain R-49797T and its closest related type strain (Methylocaldum gracile VKM 14LT). Based on the low 16S rRNA gene sequence similarities with its nearest phylogenetic neighbouring genera, the formation of a separate lineage based on 16S rRNA and pmoA gene phylogenetic analysis, and the unique combination of phenotypic characteristics of the two isolated strains compared with the genera Methylocaldum, Methylococcus and Methylogaea, we propose to classify these strains as representing a novel species of a new genus, Methyloparacoccus murrellii gen. nov., sp. nov., within the family Methylococcaceae. The type strain of Methyloparacoccus murrellii is R-49797T ( = LMG 27482T = JCM 19379T).
Published Online:
Funding
This study was supported by the:
Geconcerteerde Onderzoeksactie (GOA) of Ghent University
(Award BOF09/GOA/005)
Flemish Fund for Scientific Research
(Award FWO11/PDO/084)
Japan Society for the Promotion of Science
(Award 22380052)
AumanA. J.,
StolyarS.,
CostelloA. M.,
LidstromM. E.(2000). Molecular characterization of methanotrophic isolates from freshwater lake sediment. . Appl Environ Microbiol66, 5259–5266. [View Article][PubMed]
BodrossyL.,
HolmesE. M.,
HolmesA. J.,
KovácsK. L.,
MurrellJ. C.(1997). Analysis of 16S rRNA and methane monooxygenase gene sequences reveals a novel group of thermotolerant and thermophilic methanotrophs, Methylocaldum gen. nov.. Arch Microbiol168, 493–503. [View Article][PubMed]
BowmanJ.(2006). The methanotrophs – the families Methylococcaceae and Methylocystaceae
. . In The Prokaryotes: a Handbook on the Biology of Bacteria, , 3rd edn., pp. 266–289. Edited by
DworkinM.,
FalkowS.,
RosenbergE.,
SchleiferK.-H.,
StackebrandtE.
. New York:: Springer;. [View Article]
BowmanJ. P.,
SlyL. I.,
NicholsP. D.,
HaywardA. C.(1993). Revised taxonomy of the methanotrophs: description of Methylobacter gen. Nov., emendation of Methylococcus, validation of Methylosinus and Methylocystis species, and a proposal that the family Methylococcaceae includes only the group-I methanotrophs. . Int J Syst Bacteriol43, 735–753. [View Article]
CleenwerckI.,
VandemeulebroeckeK.,
JanssensD.,
SwingsJ.(2002). Re-examination of the genus Acetobacter, with descriptions of Acetobacter cerevisiae sp. nov. and Acetobacter malorum sp. nov.. Int J Syst Evol Microbiol52, 1551–1558. [View Article][PubMed]
ColeJ. R.,
ChaiB.,
FarrisR. J.,
WangQ.,
KulamS. A.,
McGarrellD. M.,
GarrityG. M.,
TiedjeJ. M.(2005). The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis. . Nucleic Acids Res33, D294–D296. [View Article][PubMed]
CostelloA. M.,
LidstromM. E.(1999). Molecular characterization of functional and phylogenetic genes from natural populations of methanotrophs in lake sediments. . Appl Environ Microbiol65, 5066–5074.[PubMed]
De MeyerS. E.,
van HoordeK.,
VekemanB.,
BraeckmanT.,
WillemsA.(2011). Genetic diversity of rhizobia associated with indigenous legumes in different regions of Flanders (Belgium). . Soil Biol Biochem43, 2384–2396. [View Article]
EshinimaevB. Ts.,
MedvedkovaK. A.,
KhmeleninaV. N.,
SuzinaN. E.,
OsipovG. A.,
LysenkoA. M.,
TrotsenkoIuA.(2004). [New thermophilic methanotrophs of the genus Methylocaldum]. . Mikrobiologiia73, 530–539 (in Russian).[PubMed]
EttwigK. F.,
ButlerM. K.,
Le PaslierD.,
PelletierE.,
MangenotS.,
KuypersM. M.,
SchreiberF.,
DutilhB. E.,
ZedeliusJ.& other authors (2010). Nitrite-driven anaerobic methane oxidation by oxygenic bacteria. . Nature464, 543–548. [View Article][PubMed]
FosterJ. W.,
DavisR. H.(1966). A methane-dependent coccus, with notes on classification and nomenclature of obligate, methane-utilizing bacteria. . J Bacteriol91, 1924–1931.[PubMed]
HelmJ.,
WendlandtK. D.,
RoggeG.,
KappelmeyerU.(2006). Characterizing a stable methane-utilizing mixed culture used in the synthesis of a high-quality biopolymer in an open system. . J Appl Microbiol101, 387–395. [View Article][PubMed]
HoefmanS.,
van der HaD.,
De VosP.,
BoonN.,
HeylenK.(2012a). Miniaturized extinction culturing is the preferred strategy for rapid isolation of fast-growing methane-oxidizing bacteria. . Microb Biotechnol5, 368–378. [View Article][PubMed]
HoefmanS.,
Van HoordeK.,
BoonN.,
VandammeP.,
De VosP.,
HeylenK.(2012b). Survival or revival: long-term preservation induces a reversible viable but non-culturable state in methane-oxidizing bacteria. . PLoS ONE7, e34196. [View Article][PubMed]
HoefmanS.,
HeylenK.,
de VosP.(2014).Methylomonas lenta sp. nov., a methanotroph isolated from manure and a denitrification tank in Belgium. . Int J Syst Evol Microbiol61, in press. [View Article][PubMed]
HorzH. P.,
YimgaM. T.,
LiesackW.(2001). Detection of methanotroph diversity on roots of submerged rice plants by molecular retrieval of pmoA, mmoX, mxaF, and 16S rRNA and ribosomal DNA, including pmoA-based terminal restriction fragment length polymorphism profiling. . Appl Environ Microbiol67, 4177–4185. [View Article][PubMed]
HutchensE.,
RadajewskiS.,
DumontM. G.,
McDonaldI. R.,
MurrellJ. C.(2004). Analysis of methanotrophic bacteria in Movile Cave by stable isotope probing. . Environ Microbiol6, 111–120. [View Article][PubMed]
IguchiH.,
YurimotoH.,
SakaiY.(2011). Stimulation of methanotrophic growth in cocultures by cobalamin excreted by rhizobia. . Appl Environ Microbiol77, 8509–8515. [View Article][PubMed]
LoganN. A.,
LebbeL.,
HosteB.,
GorisJ.,
ForsythG.,
HeyndrickxM.,
MurrayB. L.,
SymeN.,
Wynn-WilliamsD. D.,
De VosP.(2000). Aerobic endospore-forming bacteria from geothermal environments in northern Victoria Land, Antarctica, and Candlemas Island, South Sandwich archipelago, with the proposal of Bacillus fumarioli sp. nov.. Int J Syst Evol Microbiol50, 1741–1753.[PubMed]
McDonaldI. R.,
KennaE. M.,
MurrellJ. C.(1995). Detection of methanotrophic bacteria in environmental samples with the PCR. . Appl Environ Microbiol61, 116–121.[PubMed]
MesbahM.,
PremachandranU.,
WhitmanW. B.(1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. . Int J Syst Bacteriol39, 159–167. [View Article]
Op den CampH. J. M.,
IslamT.,
StottM. B.,
HarhangiH. R.,
HynesA.,
SchoutenS.,
JettenM. S. M.,
BirkelandN. K.,
PolA.,
DunfieldP. F.(2009). Environmental, genomic and taxonomic perspectives on methanotrophic Verrucomicrobia
. . Environ Microbiol Rep1, 293–306. [View Article][PubMed]
SpieckE.,
LipskiA.(2011). Cultivation, growth physiology, and chemotaxonomy of nitrite-oxidizing bacteria. . In Methods in Enzymology: Research on Nitrification and Related Processes, Vol. 486, Part A, pp. 109–130. Edited by
KlotzM. G.
. San Diego, CA:: Academic Press;. [View Article]
TavorminaP. L.,
UsslerW.III,
OrphanV. J.(2008). Planktonic and sediment-associated aerobic methanotrophs in two seep systems along the North American margin. . Appl Environ Microbiol74, 3985–3995. [View Article][PubMed]
WhittenburyR.,
DaviesS. L.,
DaveyJ. F.(1970a). Exospores and cysts formed by methane-utilizing bacteria. . J Gen Microbiol61, 219–226. [View Article][PubMed]
WhittenburyR.,
PhillipsK. C.,
WilkinsonJ. F.(1970b). Enrichment, isolation and some properties of methane-utilizing bacteria. . J Gen Microbiol61, 205–218. [View Article][PubMed]