Parallel engineering of environmental bacteria and performance over years under jungle-simulated conditions

Abstract

Engineered bacteria could perform many functions in the environment, for example, to remediate pollutants, deliver nutrients to crops or act as in-field biosensors. Model organisms can be unreliable in the field, but selecting an isolate from the thousands that naturally live there and genetically manipulating them to carry the desired function is a slow and uninformed process. Here, we demonstrate the parallel engineering of isolates from environmental samples by using the broad-host-range XPORT conjugation system (Bacillus subtilis mini-ICEBs1) to transfer a genetic payload to many isolates in parallel. Bacillus and Lysinibacillus species were obtained from seven soil and water samples from different locations in Israel. XPORT successfully transferred a genetic function (reporter expression) into 25 of these isolates. They were then screened to identify the best-performing chassis based on the expression level, doubling time, functional stability in soil, and environmentally-relevant traits of its closest annotated reference species, such as the ability to sporulate and temperature tolerance. From this library, we selected Bacillus frigoritolerans A3E1, re-introduced it to soil, and measured function and genetic stability in a contained environment that replicates jungle conditions. After 21 months of storage, the engineered bacteria were viable, could perform their function, and did not accumulate disruptive mutations.

Engineering the soil bacteriumPseudomonas synxantha2-79 into a ratiometric bioreporter for phosphorus limitation

“…All cultures were grown for 24 hours at 30°C and 220 rpm shaking. The soil in the tubes was then separated from the bacteria following the protocol in Chemla et al [9]. All cultures were then diluted 1:100 and filtered through a 10 µ m filter before proceeding to flow cytometry measurements.…”

“…Not only are they well-adjusted to the soil environment, they are in many cases known to promote plant-growth and protect against plant pathogens [7, 8]. Although it is not certain that engineered isolates can persist long-term in soil, it has been reporter previously that can persist and retain their engineered function for months in soil [9].…”

Engineering the Soil Bacterium Pseudomonas synxantha 2–79 into a Ratiometric Bioreporter for Phosphorus Limitation

“…All cultures were grown for 24 h at 30 °C and 220 rpm shaking. The soil in the tubes was then separated from the bacteria following the protocol in Chemla et al 9 The soil slurries were centrifuged at 1000g for 2 min at room temperature. The supernatant containing bacteria was then removed from the tubes.…”

“…7,8 Although it is not certain that engineered isolates can persist long-term in soil, it has been reported previously that they can persist and retain their engineered function for months in soil. 9 The first engineered bioreporter for phosphorus limitation in a pseudomonad was developed by de Weger et al: 10 they inserted lacZ in random places in the Pseudomonas putida WCS358 genome and found colonies that responded to phosphorus limitation with β-galactosidase activity. A similar approach was taken by Kragelund et al 11 who instead integrated the lux operon onto the Pseudomonas fluorescens DF57 genome.…”

“…Pseudomonads, a class of bacteria that are ubiquitous in soil, have been a common target chassis for engineering soil biosensors. Not only are they well-adjusted to the soil environment, but they also are in many cases known to promote plant growth and protect against plant pathogens. , Although it is not certain that engineered isolates can persist long-term in soil, it has been reported previously that they can persist and retain their engineered function for months in soil …”