Ecological Functions of Agricultural Soil Bacteria and Microeukaryotes in Chitin Degradation: A Case Study.

Chitin provides a valuable carbon and nitrogen source for soil microorganisms and is a major component of particulate organic matter in agricultural soils. To date, there is no information on interaction and interdependence in chitin-degrading soil microbiomes. Since microbial chitin degradation occurs under both oxic and anoxic conditions and both conditions occur simultaneously in soil, the comparison of the active microbiome members under both conditions can reveal key players for the overall degradation in aerated soil. A time-resolved 16S rRNA stable isotope probing experiment was conducted with soil material from the top soil layer of a wheat-covered field. [13CU]-chitin was largely mineralized within 20 days under oxic conditions. Cellvibrio, Massilia, and several Bacteroidetes families were identified as initially active chitin degraders. Subsequently, Planctomycetes and Verrucomicrobia were labeled by assimilation of 13C carbon either from [13CU]-chitin or from 13C-enriched components of primary chitin degraders. Bacterial predators (e.g., Bdellovibrio and Bacteriovorax) were labeled, too, and non-labeled microeukaryotic predators (Alveolata) increased their relative abundance toward the end of the experiment (70 days), indicating that chitin degraders were subject to predation. Trophic interactions differed substantially under anoxic and oxic conditions. Various fermentation types occurred along with iron respiration. While Acidobacteria and Chloroflexi were the first taxa to be labeled, although at a low 13C level, Firmicutes and uncultured Bacteroidetes were predominantly labeled at a much higher 13C level during the later stages, suggesting that the latter two bacterial taxa were mainly responsible for the degradation of chitin and also provided substrates for iron reducers. Eventually, our study revealed that (1) hitherto unrecognized Bacteria were involved in a chitin-degrading microbial food web of an agricultural soil, (2) trophic interactions were substantially shaped by the oxygen availability, and (3) detectable predation was restricted to oxic conditions. The gained insights into trophic interactions foster our understanding of microbial chitin degradation, which is in turn crucial for an understanding of soil carbon dynamics.

Metabolic Regulation of "Ca. Methylacidiphilum Fumariolicum" SolV Cells Grown Under Different Nitrogen and Oxygen Limitations.

Aerobic methanotrophic bacteria can use methane as their sole energy source. The discovery of "Ca. Methylacidiphilum fumariolicum" strain SolV and other verrucomicrobial methanotrophs has revealed that the ability of bacteria to oxidize CH(4) is much more diverse than has previously been assumed in terms of ecology, phylogeny, and physiology. A remarkable characteristic of the methane-oxidizing Verrucomicrobia is their extremely acidophilic phenotype, growing even below pH 1. In this study we used RNA-Seq to analyze the metabolic regulation of "Ca. M. fumariolicum" SolV cells growing at µ(max) in batch culture or under nitrogen fixing or oxygen limited conditions in chemostats, all at pH 2. The analysis showed that two of the three pmoCAB operons each encoding particulate methane monoxygenases were differentially expressed, probably regulated by the available oxygen. The hydrogen produced during N(2) fixation is apparently recycled as demonstrated by the upregulation of the genes encoding a Ni/Fe-dependent hydrogenase. These hydrogenase genes were also upregulated under low oxygen conditions. Handling of nitrosative stress was shown by the expression of the nitric oxide reductase encoding genes norB and norC under all conditions tested, the upregulation of nitrite reductase nirK under oxygen limitation and of hydroxylamine oxidoreductase hao in the presence of ammonium. Unraveling the gene regulation of carbon and nitrogen metabolism helps to understand the underlying physiological adaptations of strain SolV in view of the harsh conditions of its natural ecosystem.

Draft genome sequence of the volcano-inhabiting thermoacidophilic methanotroph Methylacidiphilum fumariolicum strain SolV.

The draft genome of Methylacidiphilum fumariolicum SolV, a thermoacidophilic methanotroph of the phylum Verrucomicrobia, is presented. Annotation revealed pathways for one-carbon, nitrogen, and hydrogen catabolism and respiration together with central metabolic pathways. The genome encodes three orthologues of particulate methane monooxygenases. Sequencing of this genome will help in the understanding of methane cycling in volcanic environments.

Autotrophic methanotrophy in verrucomicrobia: Methylacidiphilum fumariolicum SolV uses the Calvin-Benson-Bassham cycle for carbon dioxide fixation.

Genome data of the extreme acidophilic verrucomicrobial methanotroph Methylacidiphilum fumariolicumstrain SolV indicated the ability of autotrophic growth. This was further validated by transcriptome analysis, which showed that all genes required for a functional Calvin-Benson-Bassham (CBB) cycle were transcribed. Experiments with (13)CH(4) or (13)CO(2) in batch and chemostat cultures demonstrated that CO(2) is the sole carbon source for growth of strain SolV. In the presence of CH(4), CO(2) concentrations in the headspace below 1% (vol/vol) were growth limiting, and no growth was observed when CO(2)concentrations were below 0.3% (vol/vol). The activity of the key enzyme of the CBB cycle, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), measured with a (13)C stable-isotope method was about 70 nmol CO(2) fixed · min(-1)· mg of protein(-1). An immune reaction with antibody against the large subunit of RuBisCO on Western blots was found only in the supernatant fractions of cell extracts. The apparent native mass of the RuBisCO complex in strain SolV was about 482 kDa, probably consisting of 8 large (53-kDa) and 8 small (16-kDa) subunits. Based on phylogenetic analysis of the corresponding RuBisCO gene, we postulate that RuBisCO of the verrucomicrobial methanotrophs represents a new type of form I RuBisCO.

Organic acids and ethanol inhibit the oxidation of methane by mire methanotrophs.

Aerobic methane (CH(4) ) oxidation reduces the emission of CH(4) from mires and is regulated by various environmental factors. Organic acids and alcohols are intermediates of the anaerobic degradation of organic matter or are released by plant roots. Methanotrophs isolated from mires utilize these compounds preferentially to CH(4) . Thus, the effect of organic acids and ethanol on CH(4) oxidation by methanotrophs of a mire was evaluated. Slurries of mire soil oxidized supplemental CH(4) down to subatmospheric concentrations. The dominant pmoA and mmoX genotypes were affiliated with sequences from Methylocystis species capable of utilization of acetate and atmospheric CH(4) . Soil slurries supplemented with acetate, propionate or ethanol had reduced CH(4) oxidation rates compared with unsupplemented or glucose-supplemented controls. Expression of Methylocystis-affiliated pmoA decreased when CH(4) consumption decreased in response to acetate and was enhanced after acetate was consumed, at which time the consumption of CH(4) reached control levels. The inhibition of methanotroph activity might have been due to either toxicity of organic compounds or their preferred utilization. CH(4) oxidation was reduced at 5 and 0.5 mM of supplemental organic compounds. Acetate concentrations may exceed 3 mM in the investigated mire. Thus, the oxidation of CH(4) might decrease in microzones where organic acids occur.