show Abstracthide AbstractEffect on gene transcript levels upon up- or down-regulation of two component system WalRK (Wal-ON and Wal-OFF) Overall design: Wal-ON: overexpress the regulator walR from a single copy integrated on the chromosome under xylose inducible control; produce either the wildtype WalR, or a WalR-D54E mutant that is expected to mimic phosphorylated WalR and thus be constitutively active. Wal-OFF: CRISPR interference (CRISPRi) machinery integrated into the chromosome under xylose induction. sgRNA is either a negative control that does not target anywhere on the C. difficile chromosome, or targets the first gene in the wal operon, cd630_17810. Growth under no xylose conditions relies on leaky expression, growth with low xylose induces the CRISPRi machinery further.