P4-NR∆galUand P4-NR∆galEare rough mutants of P4-NR WT strain. Phage transduction was used to transfer the mutation from E. coli BW2511 mutants in the same genes. The rough mutants form clumps upon mixing with acriflavine (A), as opposed to P4-NR that has smooth colonies and does not aggregate. The rough phenotype is attributed to loss of O-antigen chain of bacterial LPS while wild type strain P4-NR with full length O-antigen is smooth (A). All strains show similar growth in LB broth including those expressing the fluorescent protein mCherry (pKB4985 in B). The rough mutants lack resistance to bovine serum, as indicated by no growth after 60 min of incubation with 80% normal bovine serum (N in C) and growth in heat-inactivated bovine serum (HI in C). Bacterial growth in heat-inactivated (D; HI) or normal (D; N) serum was also quantified and presented in D. Figures are of representative results from 3 or more similar experiments. P4-NR is serum resistant growing well in normal (C; N in D) and heat-inactivated (HI in D) bovine serum.