Methanotrophy in Movile Cave

Movile Cave is an isolated cave ecosystem that receives no input of photosynthetically fixed carbon. Instead, carbon is primarily fixed through light- independent bacterial processes such as chemolithoautotrophy and methanotrophy. Distinctive microbial floating mats appear at the surface of groundwater flooding the cave, at the redox interface between the oxygenated air above (7-10%) and the anaerobic water below. Methane, of geological origin, bubbles up into the cave and is present in the cave atmosphere (0.5-1%). The in situ methanotroph community of Movile Cave microbial floating mat was determined by examination of metagenomic sequencing and pmoA gene microarray data sets. The metagenonomic sequencing approach indicated a Methylococcus capsulatus -like organism to be the most abundant methanotroph in Movile Cave. pmoA microarray analysis indicated a high abundance of Methylocystis pmoA gene sequences with Methylococcus capsulatus-like pmoA gene sequences being relatively abundant. The methane oxidising bacterium Methylomonas strain LWB was isolated from a sample of lake water from Movile Cave. Phylogenetic analysis of the genes encoding 16S rRNA and the soluble and particulate methane monooxygenase functional gene markers pmoA and mmoX, respectively, confirmed that strain LWB belongs to the genus Methylomonas. Methylomonas LWB has a second putative copy of the particulate methane monooxygenase pXM which displays an unusual gene orientation. The Methylomonas LWB genome contains all genes encoding the typical Type I methanotroph ribulose monophosphate pathway for formaldehyde assimilation and all genes required for a complete TCA cycle. Active methane oxidisers in Movile Cave were identified by DNA Stable-isotope probing. Organisms belonging to the genera Methylomonas, Methylocystis, Methylococcus and Methylobacter- were identified from 13C-enriched DNA. Cross-feeding of the 13C label into non-methanotrophic organisms identified from the 13C-enriched DNA indicated that methanotrophs provide a carbon source for other microorganisms in Movile Cave