Methanotrophic bacteria, and environmental genomics/transcriptomics

Our research covers topics at the molecular, genomic, cellular, and community levels. At present, we are particularly interested in [1] the molecular biology and ecology of methanotrophic bacteria, [2] soil metatranscriptomics, and [3] microbial communities in Sphagnum-dominated peatlands.

Methanotrophic bacteria

Our most recent research focused on (i) Methylocystis sp. strain SC2, (ii) composition and dynamics of atmospheric methane oxidizers in upland soils, and (iii) acetate utilization by Methylocystis spp.

(i) The key enzyme of methanotrophic bacteria is particulate methane monooxygenase (pMMO), which converts methane to methanol. pMMO is encoded by three consecutive open reading frames (pmoCAB) in both type I and type II methanotrophs. We previously showed that the type II methanotroph Methylocystis sp. strain SC2 possesses two different pMMO isozymes. The pMMO1 and pMMO2 exhibit different methane oxidation kinetics (Baani and Liesack, 2008). The conventional pMMO1 is produced and oxidizes methane only at mixing ratios >600 ppmv. In contrast, the pMMO2 is constitutively produced and oxidizes methane at lower mixing ratios, even at the trace level of atmospheric methane. Wild-type strain SC2 is able to maintain at atmospheric methane for >3 months. Growth occurs at 10-100 ppmv. The apparent Km of pMMO2 (0.11 μM) in strain SC2 corresponds well with the Km(app) values for methane oxidation in soils consuming atmospheric methane. At present, we use an integrated approach of genomics and transcriptomics to investigate the response of strain SC2 to different growth conditions.

(ii) Microbial oxidation is the only biological sink for atmospheric methane. We assessed the activity, composition and seasonal dynamics of atmospheric methane oxidizers in grassland near Giessen (Germany), along a soil moisture gradient. Net uptake rates in the three study sites varied seasonally between 0 and 70 μg CH4 m-2 h-1. Greatest uptake rates coincided with lowest soil moisture in spring and summer. Over all sites and seasons, the methanotrophic communities were dominated by uncultivated methanotrophs belonging to the upland soil cluster alphaproteobacteria (USCα)-like clades RA14, MHP and JR1. The copy numbers of pmoA genes ranged from 3.8 x 105 to 1.9 x 106 copies g-1 of soil. The soil moisture was shown to have a significant but opposed effect on the CH4 uptake rates and the changes in USCα-like diversity and pmoA gene abundance. These changes were greatest at low net CH4 uptake rates during winter times and coincided with an overall increase in bacterial 16S rRNA gene abundances. The methanotrophic population dynamics were increasingly stimulated by soil moisture contents >50 vol% and were primarily related to members of the MHP clade (Shrestha et al., 2011).

In a collaborative study, we examined the activity and diversity of methanotrophic bacteria in glacier forefields on siliceous and calcareous bedrock in the Swiss Alps. All the siliceous sites were dominated by upland soil cluster gammaproteobacteria (USCγ), while the calcareous sites were colonized by either USCγ or Methylocystis. The presence of Methylocystis may be related to the fact that profiles from all calcareous sites revealed a substantial deep-soil CH4 source with soil-CH4 concentrations greater than 1000 ppmv. Members of USCγ are putative atmospheric methane oxidizers that are widely distributed across permafrost and arctic soils.

(iii) Some Methylocystis spp., such as strain H2s, are capable of slow growth on acetate in the absence of CH4. Acetate-grown cells of strain H2s respond faster to CH4 availability than those kept for the same period of time without any substrate. Presumably, this is related to the fact that pMMO transcripts are detectable in strain H2s even after several transfers on acetate. Strain H2s represents a numerically abundant methanotroph population in northern Sphagnum-dominated wetlands. The ability to utilize acetate may be an important part of its survival strategy, allowing strain SC2 and relatives to maintain the methane oxidation machinery in the absence of CH4 (Belova et al., 2011).

Soil metatranscriptomics

We have begun to explore the global transcriptome, or metatranscriptome, of soil microbial communities using oxic versus anoxic paddy soil as the model system. Using conventional Sanger sequencing, our first attempt to examine global gene expression allowed us to detect the active components of the microbial communities but provided limited insight into their ecosystem functions (Shrestha et al. 2009). More recently, we developed an efficient method for extracting high quality mRNA from soil. Key steps in the isolation of total RNA are low-pH extraction (pH 5.0) and Q-Sepharose chromatography. The removal efficiency of humic acids was 94 to 98% for all soils tested. To enrich mRNA, subtractive hybridization of rRNA is most efficient. The rRNA-depleted RNA is of sufficient purity and integrity (size range of 0.3 to 4 kb) for further applications (Mettel et al., 2010). Using this extraction protocol, we established a reliable procedure for generating soil metatranscriptomic data sets by 454 Titanium pyrosequencing.

Bacterial communities in Sphagnum-dominated peatlands

This research is carried out in collaboration with Dr. Svetlana N. Dedysh (Winogradsky Institute, RAS, Moscow) and her group. The research has been continuously funded for more than 10 years by the Deutsche Forschungsgemeinschaft and the Russian Fund for Basic Research.

Over the last two years, we investigated temperature and nitrogen induced changes in the activity and composition of the cellulolytic bacterial community in Sphagnum peat, in order to assess the potential impact of global change on microbial processes. Peatlands in the Northern Hemisphere harbor 200-450 Pg of carbon, which is about one-third of the global soil carbon pool. A large proportion of these peatlands consists of Sphagnum-dominated ombrotrophic bogs, which are characterized by low pH values of 3.5 to 5 and extremely low rates of plant debris decomposition. The degradation of cellulose, the major component of Sphagnum-derived litter, was almost undetectable at 10°C and occurred at low rates at 20°C, while it was significantly accelerated at both temperature regimes by the addition of available nitrogen. Acidobacteria were shown to represent cellulolytic members of the indigenous bacterial community in acidic peat, but were easily out-competed by Cytophaga-like bacteria under conditions of increasing temperature and nitrogen availability. Members of the phylum Firmicutes, known to be key players in cellulose degradation in neutral habitats, were not detected in the cellulolytic community enriched at low pH (Pankratov et al., 2011). In our future research, we will continue to explore the metabolic diversity and functional roles of members of the Acidobacteria in northern wetlands, linking microbial community composition and environmental transcriptomics with cultivation studies.

We also characterized new moderately acidophilic planctomycete isolates for which we propose the name Telmatocola sphagniphila gen. nov., sp. nov. In contrast to other taxonomically described members of the family Planctomycetaceae, cells of Telmatocola sphagniphila have a weak cellulolytic potential (Kulichevskaya et al., in press).