The Mysterious Case of the Microbe in the Soil



Microbial biosensors sense and environmental signal and,
in response, produce a measurable reporter such as a gas.
Image by E. Fulk


By Emm Fulk, graduate student, Rice University, Houston, TX, USA

 

Flagella? Check. Cell wall? Check. Cheesy music? Yes, that's right. What's a microbial detective without a melodramatic theme song?

Studies of soil microbiology have traditionally - and necessarily - been conducted from the outside in. Net fluxes of nutrients and gases from the soil environment can be chemically measured. High-throughput sequencing techniques can give metagenomic data, which provides a snapshot of the general composition of a soil microbiome. This is, essentially, a stakeout - we can get a general sense of a soil  community by observing the surrounding environment and may be able to infer some activities by measuring who and what comes in and out. These strategies give us an overall picture of soil communities and their net interactions within the ecosystem but lack the spatial, temporal and chemical sensitivity to fully understand the internal  dynamics of soil microbiomes.

We need a microbe on the inside.

The idea of using living microbes as biosensors is not especially new. To survive and adapt to new environmental stresses, such as nutrient or water deprivation, microbes have evolved networks to sense these changes and adapt their metabolism accordingly. Tying these naturally-evolved systems to a measurable reporter (for example, a fluorescent protein) is a logical step for understanding how microbes interact with their environment. Think of a light bulb and a light switch. The light switch senses whether it is on or off. The light bulb reports on the ON/OFF state of the light switch. Even if you can't see the switch, you can infer whether it is on or off by looking at the light bulb. Likewise, we can detect when a particular environmental condition elicits a microbial response by monitoring the production of the reporter.

I know just the microbe for the job… he's sensitive, discreet and reports only to me.

For microbial biosensors to be useful in soil, their sensors and reporters must both be suitable for monitoring interesting  
environmental conditions - for example, drought conditions, concentrations of nitrogen or carbon species, or cell-cell  
communication signals. Sensors for -osmotic stress, nitrate, quorum sensing molecules and various heavy metals have been or are being  
developed. These systems must also be sensitive to an environmentally relevant level of the signal. If too sensitive or not sensitive  
enough, reporter production is not triggered at the right times to give useful information. Ideally, reporters need to be measurable in  
situ. Traditional fluorescent or pigmented reports can't be used because, well, you can't see them in soil. Expanding our toolbox of  
reporters would allow us to monitor cell growth as well as environmental response and to measure multiple signals.

The name's coli. Escherichia coli.

Even after engineering a useful biosensor, it remains to find suitable host microbe. Most synthetic biology is initially done in E. coli, because it is relatively well understood and easy to engineer. E. coli can also a good first organism to test biosensor function. Expanding our ability to engineer other microorganisms is a key challenge both for biosensors in soil ecology and for synthetic biology as a whole. Numerous other organisms - for example, several soil-dwelling Pseudomonas species - have successfully been engineered as biosensors. However expanding our repertoire of bacterial hosts will enable biosensors to be used in more microbial communities.

Where were the suspects on the night of the…high nitrate concentration?

Recent developments in synthetic biology have greatly expanded our ability to manipulate microbes and perform increasingly difficult computations. For example, microbes can now be programmed to produce a reporter only if both signal 1 AND signal 2 are present. Other logic functions (1 OR 2, 1 AND NOT 2, etc.) could allow for studying specific combinations of environmental conditions, such as in hot spots or during hot moments.




In standard biosensors without memory, microbes produce a reporter directly
in proportion to the level of signal. In biosensors with memory,
the reporter is induced by the first instance of the environmental cue and remains on thereafter.
The top cartoon shows how these two scenarios compare at the level of individual microbes.
The bottom graph illustrates how reporter production differs at the whole-sample level.
Image by E. Fulk



Additionally, microbes can now be programmed to "remember" certain events. By using a class of enzymes called DNA recombinases, permanent changes can be made in the DNA of a microbial biosensor in response to the environmental signal. This change catalyzed by the DNA recombinase - specifically, a reversal of a short section of DNA - can be induced by the environmental signal and results in permanent, stable, and heritable expression of the reporter. Thus, the microbes have memory of the signal. Think again about the light switch - once the light switch is turned on for the first time, the light bulb is permanently on whether the switch turns off again. This could be useful for studying long-term behavior of microbes where consistent measuring of the signal is not always possible, or for situations where suitable reporters are limited (since the recombinase-mediated reversal of the DNA sequence is a sort of reporter in itself).

It was the Rhizobia! Case closed.

Well, not quite. This is just a small slice of the synthetic biology tools that may be of interest, and there are still engineering challenges to be addressed. Most crucially, however, we need to find the right soil ecology mystery to solve.

 

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