For each of the sequenced libraries, we randomly down-sampled 4 million reads for analysis, using seqtk, to avoid saturation. Reads were trimmed from their adapter with cutadapt and aligned to the B. subtilis genome (subsp. subtilis str. NCIB 3610, NZ_CM000488.1) with Bowtie 2 version 2.3.4.1 (102). The number of uniquely mapped reads per gene was calculated with HTSeq (103). Normalization and testing for differential expression were performed with DESeq2 version 1.16. A gene was considered to be differentially expressed if its normalized mean read count ≥ 50, fold change ≥ 2, and adjusted P < 0.05. Using these criteria, 300 genes were found to be differentially expressed in the ∆cssRS transcriptome and 357 in the ∆tasA transcriptome. A significant number of genes, 72, were found to be differentially expressed in both transcriptomes (P < 1.8 × 1010, hypergeometric test). DAVID (Database for Annotation, Visualization and Integrated Discovery) Bioinformatics (104) was used to performed gene annotation enrichment analysis.

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