Extended Data Table 1. Exogenous dipeptide analogs rescue growth of cells that have been depleted of DA-DA.

Growth rescue of a D-alanine-D-alanine ligase double mutant (ΔddlA ΔddlB) of E. coli was tested with varying concentrations of natural and modified dipeptides. Similarly, E. coli and B. subtilis cells were rendered auxotrophic for DA-DA by inhibiting their respective D-alanine-D-alanine ligases with D-cycloserine (DCS).. Data represent the average of at least two biological replicates conducted with technical duplicates.

Condition	E. coli ΔddlA, ΔddlB	Condition	E. coli wt	B. subtilis wt
MM1	−	MM	+++++	+++++
MM+ DA-DA (25 µM)2	++	MM + DCS2 (20 µM for E. coli and 150 µM for B. subtilis)	−	−
MM+ DA-DA (200 µM)2	+++++	MM + DCS + DA-DA2(50 µM for E. coli and 50 µM for B.susbtilis)	++	++++
MM + DA-EDA (100 µM)2	+++++	MM + DCS + DA-EDA2(50 µM for E. coli and 800 µM for B. subtilis)	+++	+++
MM + DA-ADA (50 µM)2	++++	MM + DCS + DA-ADA2(50 µM for E. coli and 400 µM for B. subtilis)	+++	++++
MM + ELA-LA (0.8 mM)2	−
MM + EDA–DA (0.8 mM)2	−
MM + ADA–DA (0.8 mM)2	−
MM+ DA-DA (25 µM)3	+
MM + DA-DA (25 µM) + ELA–LA (0.8 mM)3	+
MM + DA-DA (25 µM) + EDA–DA (0.8 mM)3	++++
MM + DA-DA (25 µM) + ADA–DA (0.8 mM)3	+++

¹MM = minimal medium

²Initial inoculum was OD₆₀₀ = 0.025

³Initial inoculum was OD₆₀₀ = 0.0025

+++++ indicates the highest culture density achieved within a set and − indicates no bacterial growth, with intermediate values representing the relative fractions of the highest culture density within a set.