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Folate is an essential nutrient for nearly all organisms. While the physiological function and mechanism aspects of folate have been extensively and deeply investigated in Eukarya, related researches in Bacteria remains poorly understood. In this study, we focus on physiological functions of folate in Lactiplantibacillus plantarum by employing a combination of genetics, biochemistry and microscopy approaches. Deletion of the genes folE, folP, or both folE and folK in the folate biosynthesis pathway generated the mutant strains ΔfolE, ΔfolP, and ΔfolKE, respectively. Folate production in ΔfolE, ΔfolKE, and ΔfolP decreased to 51 %, 32 %, and 74 % of the wild-type level, respectively. Simultaneous deletion folE and folK distinctly extended the glutamate tail of folate. These mutants exhibited severely impaired growth capacity under normal conditions. Notably, only ΔfolP cells precipitated in liquid culture. All mutant strains displayed increased cell length, with the extent of elongation correlating to intracellular folate levels. It is noticed that DNA content was increased along with the cell size in deletion mutants. Additionally, 12 % of ΔfolKE cells and 4 % of ΔfolP cells exhibited abnormal lysis, characterized by granular cytoplasm. These findings provide significant insights into the physiological roles of folate in Bacteria., (Copyright © 2024 Elsevier GmbH. All rights reserved.)