SAMPLE SOLUTION

The purpose of this essay is to summarize on how stable isotope probing has been applied in Li et al.’s study titled Genome-Resolved Proteomic Stable Isotope Probing of Soil Microbial Communities Using ¹³CO2 and ¹³C-Methanol”. Stable isotope probing is method that enables tracking of the flow of nutrients in an ecosystem through the use of isotopically labelled substrates. Plants release significant quantities of their photosynthetically-fixed carbon from their roots into the rhizosphere in form of organic acids, amino acids, carbohydrates, and other compounds. The carbon-containing substrates are then consumed by rhizosphere microbial communities. The rhizosphere microbial communities also help plants in return by defending plants against soil-borne pathogens, extraction of nutrients from the soil, and modulating plant growth with phytohormones (Li et al. 2). Studies of the nature and composition of rhizosphere microbial communities have been helpful in development of an understanding on the cycle of nutrients and carbon between soil and plants in various natural terrestrial ecosystems (Li et al. 2). Staple isotope probing is a novel approach of tracking nutrient flows in an ecosystem.

In Li et al. study, proteomic stable isotope probing was coupled with the targeted metagenomic binning with the goal of identifying ¹³C-labelled proteins and to rebuild metagenome-assembled genomes of the ¹³C-labeled microbial communities in a ¹³carbon (IV) oxide rhizosphere stable isotope probing experiment (2).  Metagenomics and proteomic stable isotope probing provided a cultivation-independent approach for validating the enzyme substrates utilized by microbial communities for carbon uptake. The approach was applied in a ¹³C-methanol stable isotope experiment with the aim of supporting methanol as a putative substrate of the abundant XoxF-type-methanol dehydrogenase in the rhizosphere (Li et al. 2). Many methylotrophic taxa consist of two different methanol dehydrogenase systems whose purpose is to oxidize methanol into formaldehyde. The two systems include the calcium-dependent MxaFI-type, which is well-studied and lanthanide-containing XoxF-type, which was discovered recently (Chu & Lidstrom 1317).  Aerobic methanotrophs oxidize methane to energy and carbon, are need methanol dehydrogenase enzyme so as to transform methanol into formaldehyde (Chu & Lidstrom 1317).

Cultivation-independent stable isotope probing has been applied in tracking of carbon flows from plant to certain microorganisms of the rhizosphere communities. By growing in the ¹³Carbon (IV) oxide atmosphere, plants possess the capability of fixing ¹³C and in consequence release ¹³C-labeled compounds into the rhizosphere. The guiding principle is that microorganisms that can directly assimilate or utilize the ¹³C-labeled compounds released by plants are expected to produce ¹³C-labeled biomass. The ¹³C-labeled biomass, in this case, include RNA, DNA, and proteins (Li et al., 2).

Li and colleagues used three model plants, including Arabidopsis thaliana, Triticum

aestivum and Zea mays (1,2). These plants were cultivated in the same baseline soil collected from the field in four pots for each species of plants for a total of 12 pots. After growing for 29 days in the normal atmospheric conditions, these plants were then transferred to grow in a chamber with a ¹³carbon (IV) oxide atmosphere. Identical pots from each plant species were collected after three days of growth in a ¹³carbon (IV) oxide chamber to generate baseline rhizosphere samples. The other remaining identical pots for each plant species were collected after 8 days of growth in a ¹³carbon (IV) atmosphere to generate time point 2 rhizosphere samples (Li et al. 2). The 12 rhizosphere soil samples as well as the two baseline soil samples as the controls were then analyzed using meta-proteomics and meta-genomics (Li et al. 2). Li and colleagues found that the average percentage of the ¹³C atom of the ¹³C-labeled proteins was greater in time point 2 rhizosphere samples than from on baseline samples using Mann-Whitney U test (p value = 0.00262) (Li et al., 2,3).  Most of the identified ¹³C-labelled proteins in Li et al. (3) study, were predominantly house-keeping proteins, such as glycolysis enzymes, ribosomal proteins, and chaperones.

The predominant series of scaffolds in the metagenome assembly across the 14-soil samples were utilized to bin scaffolds into the metagenome-assembled genomes. The untargeted binning yielded 41-medium-quality bacterial metagenome-assembled genomes and one medium-quality archaeal-metagenome-assembled genomes (Li et al., 3). All these metagenome-assembled genomes comprised of less than 10% genome contamination and more than 70% genome completeness. Thirty-two (32) of the 41 identified bacterial metagenome-assembled genomes were found to have belonged to Bacteroidetes, Proteobacteria, and Actinobacteria (Li t al. 3). Metagenome-assembled genomes derived from rhizosphere microorganisms, which yielded the ¹³C-labelled proteins were recovered through application of a targeted binning approach (Li et al. 4). Fourteen (14) out of 28 identified ¹³C-labelled proteins, 14 were binned into four metagenome-assembled genomes, which comprised of the medium-quality metagenome-assembled genome-45 (MAG45) with 54% genome completeness and three low-quality MAGs, which were MAG46, MAG44, and MAGG43 with genome completeness between 42% and 21%. The percentage of ¹³C-labelled atoms ranged from 15-46%. MAG43 belonged to unclassified taxon associated to the Chloroflexi-related group, MAG46 to Archrobacter genus, MAG44 to Pseudomonas genus, and MAG45 to oxalobacteraceae family (Li et al. 4).

In conclusion, rhizosphere microorganisms have the capability of controlling the plant physiology through secretion of phytohormones, such as auxin and cytokinin (Li et al. 4). Li and colleagues hypothesized that methanol can act as an important source of carbon for soil microorganism through the XoxF-based methylotrophic pathways (6). The authors identified four soil-based microorganisms (MAG45, MAG46, MAG44, and MAGG43) that could synthesize ¹³C-labeled proteins in the plant rhizosphere for plants growing under ¹³Carbon (IV) oxide atmosphere. The ¹³C-labelled microbial proteins demonstrated transfer of photosynthetically-fixed ¹³C from ¹³Carbon (IV) oxide in plant hosts and then transferred to rhizosphere microorganisms (Li et al. 7).