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Gene Expression Analysis Gets Gassy

Soil scientists use a gas-producing reporter system to assess gene activity in bacteria.

Jun 1, 2018
Ruth Williams
E. coli bacteria are genetically engineered to produce the EFE protein constitutively and MHT in response to the bacterial communication molecule AHL. The presence of the E. coli in the soil, and the levels of AHL, in this example produced by Rhizobium bacteria, can then be detected non-disruptively using headspace gas chromatography—with the ratio of MHT-produced CH3Br to EFE-produced ethylene reflecting the concentration of AHL.
See full infographic: WEB © GEORGE RETSECK

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 analysisReporter genes
 encode
How it works
 
Strengths
 
Weaknesses
 
Visualization with
a rhizotron
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
and processes
 
Soil samples must
be extremely thin,
so not easily scalable
 
Gas reportersMethyl 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.
 Easily scalable
No spatial information

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