High-throughput isotopic analysis of RNA microarrays to quantify microbial resource use

Area of Science:

• Microbial Ecology
• Environmental Microbiology
• Molecular Ecology

Background:

• Most microorganisms are uncultivated, limiting understanding of their ecological roles.
• Current methods often infer microbial functions from diversity and genomic data.
• Direct functional measurements are crucial for uncultivated microbial communities.

Purpose of the Study:

• To develop a high-sensitivity, high-throughput method for directly measuring the functional roles of uncultivated microbes.
• To enable the quantification of substrate use by specific microbial taxa in complex communities.
• To test genomics-generated hypotheses about microbial biogeochemical functions.

Main Methods:

• Developed Chip-stable isotope probing (Chip-SIP), integrating stable isotope probing (SIP) with phylogenetic microarrays.
• Incubated microbial communities with isotopically labeled substrates.
• Hybridized extracted rRNA to a microarray and measured isotope incorporation using NanoSIMS.

Main Results:

• Chip-SIP detected low levels of isotopic enrichment (0.5 atom % 13C, 0.1 atom % 15N), allowing short incubations and low substrate concentrations.
• Applied to an estuarine community, Chip-SIP quantified substrate incorporation by 81 distinct microbial taxa.
• Demonstrated resource partitioning among microbes utilizing simple organic substrates like amino acids, nucleic acids, and fatty acids.

Conclusions:

• Chip-SIP is a sensitive and high-throughput method for directly assessing microbial functions.
• The approach allows for testing genomics-based predictions in natural microbial communities.
• Expands the toolkit for microbial ecologists studying biogeochemical processes in diverse environments.