Transposon library anomaly reveals importance of cell wall teichoic acids for kin discrimination

Genome-wide transposon insertion sequencing (Tn-seq) is a powerful tool to measure the importance of genes for growth. In this study, we applied Tn-seq to the Gram-positive model system Bacillus subtilis, and found that after growth in liquid medium the transposon library lacked transposon insertions in several genes related to lipoteichoic acid biosynthesis and cell wall teichoic acid modification. This was unexpected since these genes are not essential for normal growth. By growing the transposon library as a confluent layer of cells, and as discrete colonies, we found that these genes are only important when the transposon library is grown as a confluent layer. Apparently, growing the transposon library as a mixed population reduces the fitness of teichoic acid mutants, which was confirmed by coculturing experiments. This phenomenon can be explained when lipoteichoic acid and teichoic acid D-alanylation mutants become sensitive to secreted autologous antimicrobials and/or toxins. Extensive mutant analyses suggested that multiple autologous antimicrobials are involved. Finally, we show that the reduced fitness of teichoic acid mutants can be countered by the addition of divalent cations. These data raise several questions concerning the evolution of kin discrimination, and show that growing genome-wide mutant libraries as mixed cultures can influence library composition.