Fluorescent reporter proteins have revolutionized gene expression analysis, but their use is limited to more-or-less transparent systems, such as single cells or zebrafish larvae. For researchers studying organisms that live in soil, using these visual reporters is infeasible in any but the thinnest of samples, says environmental and synthetic biologist Jonathan Silberg of Rice University.
“We can analyze cultured microbes in exquisite detail,” adds biogeochemist Caroline Masiello, also at Rice. “But the question is—does it matter at the scale of an ecosystem?” Silberg, Masiello, and colleagues have devised a new gas reporter system that can be used to detect the presence and activity of microbes in opaque samples of any size.
Ecologists often use headspace gas analysis to measure common bacterially produced gases, such as carbon dioxide and methane, without disrupting the soil or sediment sample. If bacteria could be genetically engineered to produce unusual gases, the team reasoned, they could be readily detected and analyzed by this technique, which involves quantifying mixtures of gases in closed containers.
The team identified two potential reporter genes—one from a plant, Batis maritima, and one from the bacterium Pseudomonas syringae—encoding enzymes, MHT and EFE, respectively, that synthesize methyl bromide and ethylene. The researchers then cloned the genes into a plasmid, placing efe under the control of a constitutive promoter and mht under a promoter responsive to acylhomoserine lactone (AHL), a bacterial signaling molecule key in quorum sensing. The plasmid was transferred into Escherichia coli.
In gas from soil samples containing the engineered E. coli, the team could detect both the bacteria’s presence (via ethylene) and the production, or destruction, of AHL (via methyl bromide). The system, which also worked in the sediment-dwelling bacterium Shewanella oneidensis, could be used as-is for studying bacterial communication, or could be engineered to respond to other environmental signals, such as pollutants.
“This is a fascinating new technique,” says University of North Carolina at Chapel Hill microbial biogeochemist Carol Arnosti, who was not involved with the project. “[It] promises to revolutionize the manner in which we investigate the activities and interactions of bacterial communities in soils.” (ACS Synth Biol, 7:903-11, 2018)
|In-soil bacterial analysis||Reporter genes|
|How it works||Strengths||Weaknesses|
|Fluorescent proteins||Soil is placed in thin glass or plastic|
cassettes. Reporter-expressing bacteria, typically in association with reporter-expressing plant roots, can be visualized via fluorescence microscopy.
|Yields information about|
spatial and temporal
distribution of bacteria
|Soil samples must|
be extremely thin,
so not easily scalable
|Gas reporters||Methyl halide transferase (MHT) and ethylene|
forming enzyme (EFE)
|Methyl bromide and ethylene gases|
produced by reporter-expressing, soil-
dwelling bacteria are analyzed using headspace gas chromatography.
|Provides temporal information|
about bacterial processes.
|No spatial information|