Extended Data Fig. 10: FliC_P is expressed and incorporated into Enterobacter flagella, concomitantly with host FliC repression.

a, RNA-seq read coverage across the tldR-encoding prophage of Enterobacter sp. BIDMC93, demonstrating strong expression of fliC_P, tldR, and the gRNA, alongside other genes involved in lysogeny maintenance (e.g. CI). b, Motility assays (left) with wild-type (WT) and Enterobacter deletion strains reveal similar motility phenotypes, as visualized with LB-agar plate images (middle) and a bar graph quantifying motility via halo size (right). Plate images and bar graphs represent three biological replicates; bars indicate mean ± s.d. c, Schematic representation of FliC/FliC_P homologs encoded by Enterobacter sp. BIDMC93, with relative genomic positions indicated. FliC₂ is a second host flagellin gene copy encoded at an alternate flagellar assembly locus within this strain, which is not targeted by TldR and not commonly present in other Enterobacter strains. d, Results from liquid chromatography with tandem mass spectrometry (LC–MS/MS) analyses performed on digested peptides from purified flagellar filaments, isolated from the three indicated Enterobacter sp. BIDMC93 strains. The WT ( + CmR) strain encodes the cmR gene downstream of the tldR-gRNA locus (as in Fig. 5e). Data represent the label free quantification (LFQ) intensities reflecting the variable D2-3 regions of FliC, FliC_P, or FliC₂. Although the FliC₂ appears to be the most dominant flagellin component, the relevant amounts of host FliC and FliC_P demonstrate that prophage-encoded FliC_P readily assembles into extracellular flagellar filaments, and that host FliC production is de-repressed upon prophage deletion. e, Quantification of changes in the expression profiles of Enterobacter FliC homologs, measured from RNA-seq data of three biological replicates depicted in Fig. 5f,g. TPM, transcripts per million. f, Alignment of fliC/fliC_P/fliC₂ promoters indicates that guide RNA-target DNA mismatches prevent TldR-targeting of fliC₂ and fliC_P in Enterobacter sp. BIDMC93. g, RNA-seq read coverage in the host fliC promoter/5′-UTR region overlayed for three biological replicates of four Enterobacter strains, with labeled TAM and target sequences highlighted upstream of the TSS. Strain AR136 (top) does not encode a fliC_P-tldR locus; note the distinct expression levels, measured via relative counts per million (CPM). h, Alignment of host fliC promoter regions for the strains shown in g compared to E. coli K12, with percent sequence identities indicated on the right. Reported FliA/σ²⁸ promoter elements from E. coli K12 are shown below the alignment. i, RNA-seq read coverage in the prophage-encoded fliC_P promoter/5′-UTR region overlayed for three biological replicates of two representative Enterobacter strains, confirming the predicted TSS. j, Schematic of multiple sequence alignment of the promoter region driving fliC_P gene expression, across six verified prophages described in Extended Data Fig. 2, highlighting the region that was queried for MEME motif detection.