Genome-Resolved Proteomic Stable Isotope Probing of Soil Microbial Communities Using 13CO2 and 13C-Methanol

Abstract

Stable isotope probing (SIP) enables tracking the nutrient flows from isotopically labeled substrates to specific microorganisms in microbial communities. In proteomic SIP, labeled proteins synthesized by the microbial consumers of labeled substrates are identified with a shotgun proteomics approach. Here, proteomic SIP was combined with targeted metagenomic binning to reconstruct metagenome-assembled genomes (MAGs) of the microorganisms producing labeled proteins. This approach was used to track carbon flows from 13CO2 to the rhizosphere communities of Zea mays, Triticum aestivum, and Arabidopsis thaliana. Rhizosphere microorganisms that assimilated plant-derived 13C were capable of metabolic and signaling interactions with their plant hosts, as shown by their MAGs containing genes for phytohormone modulation, quorum sensing, and transport and metabolism of nutrients typical of those found in root exudates. XoxF-type methanol dehydrogenases were among the most abundant proteins identified in the rhizosphere metaproteomes. 13C-methanol proteomic SIP was used to test the hypothesis that XoxF was used to metabolize and assimilate methanol in the rhizosphere. We detected 7 13C-labeled XoxF proteins and identified methylotrophic pathways in the MAGs of 8 13C-labeled microorganisms, which supported the hypothesis. These two studies demonstrated the capability of proteomic SIP for functional characterization of active microorganisms in complex microbial communities.

Impact of plants on the diversity and activity of methylotrophs in soil

“…Previous studies have indicated that methylotrophic bacteria are enriched in the rhizosphere of certain plant species, for example, Methylobacteraceae and Hyphomicrobiaceae in the rhizosphere of Arabidopsis thaliana [12], Methylophilaceae and Comamonadaceae in the pea rhizosphere and Methylophilaceae and Methylocaldum in the wheat rhizosphere [13]. Methanol dehydrogenase genes have been detected in the rhizosphere of rice, grasses, soybeans, cereals and pea plants [14][15][16], methanol dehydrogenase enzymes have been detected in the rhizosphere soils of oat, wheat and A. thaliana [17], and soils in association with A. thaliana had higher rates of methanol dissimilation than non-plantassociated soils [18]. However, the reasons for changes in the abundance of methylotrophs in the soil in response to plant growth are hard to identify, since many of these methylotrophs can also use multi-carbon compounds, which could be supplied either directly from the plant or from the exudate-induced accelerated breakdown of recalcitrant soil organic matter (SOM) [19].…”

Modes of Action of Microbial Biocontrol in the Phyllosphere

“…Members of the genus Methylobacterium enhance plant growth by producing auxins and cytokinins (Koenig et al, 2002;Kwak et al, 2014;Jorge et al, 2019;Li et al, 2019). Interaction with the host is beneficial for the symbiont's growth since they metabolize the methanol released as the plant grows (Kutschera, 2007).…”

“…The presence of these pathways can be detected by the presence of the following essential genes: ipd C and ald H for the indole-3-pyruvate pathway (encoding the enzymes necessary for the decarboxylation of indole-3-pyruvate and subsequent oxidation, respectively), dcc and ald H for the tryptamine pathway (encoding the enzymes necessary for the decarboxylation of tryptophan and subsequent oxidation, respectively), iaa M and iaa H for the indole-3-acetamide pathway (encoding for tryptophan-2-monooxygenase and indole-3-acetamide hydrolase, respectively), and nth A for the indole-3-acetonitrile pathway (encoding for nitrile hydratase α) ( de Souza et al, 2019 ). It is important to note that although the indole-3-acetamide pathway was considered as being exclusive for the excessive IAA production by gall forming bacteria like P. savastanoi , Erwinia spp., and Agrobacterium transformed plant tissue ( Jameson, 2000 ), these genes are also present in methylotrophic rhizosphere microorganisms ( Li et al, 2019 ). The ipd C gene is of special interest since it was demonstrated that the specific growth conditions in the phyllosphere trigger the expression of the ipd C gene in symbiotic P. agglomerans (syn.…”

Genome Sequences of 42 Bacteria Isolated from Sorghum bicolor Roots

“…Eight of the genomes contain nitrogenase gene clusters ( nifHDK ) for biological nitrogen fixation. These genomes will aid in investigations of plant-microbe interactions with sorghum ( 11 ).…”