Calcium determines Lactiplantibacillus plantarum intraspecies competitive fitness

The importance of individual nutrients for microbial strain robustness and coexistence in habitats containing different members of the same species is not well understood. To address this for Lactiplantibacillus plantarum in food fermentations, we performed comparative genomics and examined the nutritive requirements and competitive fitness for L. plantarum strains B1.1 and B1.3 isolated from a single sample of teff injera fermentation batter. Compared to B1.1 and other L. plantarum strains, B1.3 has a smaller genome, limited biosynthetic capacities, and large mobilome. Despite these differences, B1.3 was equally competitive with B1.1 in a suspension of teff flour. In commercially-sourced, nutrient-replete MRS (cMRS) medium, strain B1.3 reached three-fold higher numbers than B1.1 within two days of passage. Because B1.3 growth and competitive fitness was poor in mMRS, a modified MRS lacking beef extract, we used mMRS to identify nutrients needed for robust B1.3 growth. No improvement was observed when mMRS was supplemented with nucleotides, amino acids, vitamins, or monovalent metals. Remarkably, the addition of divalent metal salts increased the growth rate and cell yields of B1.3 in mMRS. Metal requirements were confirmed by Inductively Coupled Plasma Mass Spectrometry, showing that total B1.3 intracellular metal concentrations were significantly (up to 2.7-fold) reduced compared to B1.1. Supplemental CaCl 2 conferred the greatest effect, resulting in equal growth between B1.1 and B1.3 over successive five passages in mMRS. Moreover, calcium supplementation reversed a B1.3 strain-specific stationary phase, flocculation phenotype. These findings show how L. plantarum calcium requirements affect competitive fitness at the strain level. Importance Ecological theory states that the struggle for existence is stronger between closely related species. Contrary to this assertion, fermented foods frequently sustain conspecific individuals, despite their high levels of phylogenetic relatedness. Therefore, we investigated two isolates of Lactiplantibacillus plantarum B1.1 and B1.3 randomly selected from a single batch of teff injera batter. These strains spanned the known genomic and phenotypic range of the L. plantarum species, and in nutrient-replete, laboratory culture medium, strain B1.3 exhibited poor growth and was outcompeted by the more robust strain B1.1. Despite those differences, B1.1 and B1.3 were equally competitive in teff flour. This result shows how these bacteria have adapted for co-existence in that environment. The capacity for the single macronutrient calcium to restore B1.3 competitive fitness in laboratory culture medium suggests that L. plantarum intraspecies diversity found in food systems is fine-tuned to nutrient requirements at the strain level.