Fig 2. Initial characterisation of kfiB and kfiC deletion mutants.

Isogenic mutants deleted for the K5 capsule biosynthesis genes kfiB (EcNΔkfiB) or kfiC (EcNΔkfiC) were assessed to confirm attenuation of capsule production, ensure gene deletions did not impact expression of downstream genes, and assess impact on biofilm formation and Caco-2 adherence. A) Loss of capsule production was confirmed using the ΦK5 bacteriophage sensitivity assay, in which cells lacking a K5 capsule are resistant. Images show results from soft ager overlay plates for strains exposed to 10⁶ to 10⁴ pfu/ml of bacteriophage, in which phage replication is manifest as plaques or clearing of the confluent bacterial growth. EcN WT—E. coli Nissle 1917 wild-type; MG1655—E. coli K12 control strain naturally lacking a K5 capsule; EcNΔkfiB—EcN kfiB deletion mutant; EcNΔkfiC—EcN kfiC deletion mutant. B) Expression of associated genes in the kfi gene cluster were assessed in deletion mutants using RT-PCR, and figures indicate gene found to be active in each mutant. C) The impact of kfiB or kfiC gene deletion on the ability of EcN to form biofilms on abiotic surfaces was assessed using the CV biofilm assay, as originally used to screen mini-Tn5 mutants, and compared to the levels of WT biofilm formation. Data shows absorbance readings obtained following elution of CV stain from biofilms. D) The impact of kfiB or kfiC gene deletion on the ability of EcN to adhere to cultured Caco-2 cells was assessed using a co-culture system as for Fig. 1. EcN WT—E. coli Nissle 1917 wild-type; EcNΔkfiB—EcN kfiB deletion mutant; EcNΔkfiC—EcN kfiC deletion mutant. All figures show the mean of three replicate experiments, and error bars show SE of the mean. Significant differences between WT EcN and mutants is indicated by ** (P ≤ 0.01) or **** (P <0.0001).