Role of the Pho regulon and genetic reconstruction of a phosphite-dependent Escherichia coli.

A phosphite (Pt)-dependent biological containment strategy, achieved by introducing a Pt-metabolic pathway and disrupting endogenous phosphate transporters, renders Escherichia coli growth strictly dependent on Pt, a compound rarely detected in natural environments, thereby preventing unintended environmental spread. In this study, we demonstrated that expression of phosphate regulon (Pho regulon) genes was markedly upregulated in a Pt-dependent E. coli strain due to the elimination of phoU, a negative regulator of the Pho regulon, along with the high-affinity phosphate transporter pstSCAB. However, further genetic modification of this strain for detailed analysis was hindered by the presence of multiple antibiotic resistance markers. To overcome this limitation, we reconstructed a Pt-dependent E. coli strain using CRISPR-Cas12a-mediated genome editing, enabling the removal of the antibiotic resistance markers and facilitating subsequent genetic manipulation. Using this strain, we disrupted the PhoBR two-component regulatory genes and found that deletion of phoBR alleviated the constitutive overexpression of Pho regulon genes and partially restored growth of the Pt-dependent strain. These findings provide mechanistic insights and technical advances for the refinement and practical application of Pt-dependent biocontainment strategy.