Microgravity effects on secondary metabolism of plant-affecting bacteria

Despite their miniscule mass, microgravity has been shown to impose physiological stresses on bacteria, leading to altered gene regulation and metabolic output in unpredictable ways. Changes in metabolic output can ultimately impact the production of bacterial secondary metabolites. While a few studies have explored the changes in bacterial secondary metabolites under simulated or spaceflight microgravity, the specific area of plant-beneficial compound production by bacteria remains largely unexplored in these contexts. In this study, the production of metabolites pyoluteorin, 2,4-diacetylphloroglucinol (DAPG), orfamide A produced by the plant-promoting bacterium <i>Pseudomonas protegens</i> Pf-5, and pyrroloquinoline quinone (PQQ) produced by <i>Burkholderia ambifaria</i> AMMD under simulated microgravity was analyzed. Transcriptomic analysis of the biosynthetic clusters, coupled with targeted metabolomics, revealed significant downregulation of <i>ofaABC</i> and <i>pqqBCDE</i> operons on Day 3 of incubation under simulated microgravity. This downregulation corresponded to reduced levels of orfamide A and pyrroloquinoline quinone, indicating a functional link between transcriptional suppression and decreased metabolite yield. Two complementary genetic engineering approaches were used to increase the production of these compounds: native chromosomal promoter replacement with either endogenous or inducible promoters, or plasmid-based operon overexpression. While the chromosomal strategy yielded modest expression improvements in <i>P. protegens</i>, the plasmid-based system enabled robust expression with a significant increase in pyrroloquinoline production in <i>B. ambifaria</i>. This study provides a first step for engineering these microbes to provide plant-promoting metabolites in microgravity as a mechanism of aiding the cultivation of plants aboard space-faring vehicles.<h4>Importance</h4>Bacteria play an essential role in supporting plant health by producing antifungal compounds that protect against fungal diseases. In space, these bacterial functions may be disrupted. This study examined how microgravity affects the production of such compounds in plant beneficial bacteria, <i>Pseudomonas protegens</i> and <i>Burkholderia ambifaria</i>. Under simulated microgravity, both bacteria showed reduced production of some antifungal compounds due to reduced gene expression. To address this, we used genetic strategies to boost the bacteria's ability to produce these compounds despite the stress of microgravity. Our results showed that engineered bacteria could either partially recover gene expression or even enhance production. These findings are important first steps for engineering microbes to not only enhance plant growth for space agriculture but also provide important information for engineering microbes to boost crop production on Earth as well.