Survival and Spread of Engineered Mycobacterium smegmatis and Associated Mycobacteriophage in Soil Microcosms

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Abstract The inoculation of microbes into soil environments has numerous applications for improving soil quality and crop health; however, the ability of exogenous and engineered microbes to survive and spread in soil remains uncertain. To address this challenge, we assayed the survival and spread of Mycobacterium smegmatis , engineered with either plasmid transformation or genome integration, as well as its mycobacteriophage Kampy, in both sterilized and non-sterilized soil microcosms over a period of 49 days. While engineered M. smegmatis and Kampy persisted in all soil microcosms, there was minimal evidence of spread to 5 cm away from the inoculation site. There was a higher prevalence of Kampy observed in sterilized soil than non-sterilized soil, suggesting a detrimental effect of the native soil biotic and viral community on the ability of this phage to proliferate in the soil microcosm. Additionally, higher abundance of the genome-integrated bacteria relative to the plasmid-carrying bacteria as well as evidence for loss of plasmid over the duration of the experiment suggest a burden associated with bacteria harboring plasmids, although plasmids were still retained across 49 days. To our knowledge, this is the first study to simultaneously measure the persistence and spread of bacteria and their associated phage in both sterilized and non-sterilized soil microcosms, employing bacteria with plasmid-based and genome-integrated engineered circuits. As such, this study provides a novel understanding of challenges associated with the deployment of bioengineered microbes into soil environments. Importance Healthy soil is essential to sustain life, as it provides habitable land, enables food production, promotes biodiversity, sequesters and cycles nutrients, and filters water. Given the prevalence of soil degradation, treatment of soil with microbes that promote soil and crop health could improve global soil sustainability; furthermore, the application of bioengineering and synthetic biology to these microbes allows fine-tunable and robust control of gene-of-interest expression. These solutions require the successful deployment of bacteria into the soil, an environment in which abundant competition and often limited nutrients can result in bacterial death or dormancy. This study employs Mycobacterium smegmatis as a chassis alongside its bacteriophage Kampy in soil microcosms to assess the ability of non-native microbes to survive and spread in soil. Insights from this experiment highlight important challenges which must be overcome for successful deployment of engineered microbes in the field.

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