R. parkeri strain generation and validation

WT R. parkeri strain Portsmouth [National Center for Biotechnology Information (NCBI) accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_017044.1","term_id":"383483341","term_text":"NC_017044.1"}}NC_017044.1; originally a gift from C. Paddock, Center for Disease Control and Prevention] and bacterial stocks of ompB^STOP::tn [the genome sequences of these bacterial strains are available at the Sequence Read Archive as accession no. SRP154218 (WT, SRX4401164; ompB^STOP::tn, SRX4401167)], pkmt1::tn, pkmt2::tn, wecA::tn, and rlmD::tn mutants were propagated and purified every ~6 to 10 months as described below. Side-by-side experimental comparisons were made between stocks prepared at similar times.

R. parkeri pkmt1::tn, pkmt2::tn, wecA::tn, and 114 other transposon insertion mutant strains, screened for pUb, were previously isolated in a screen for small-plaque (Sp) mutants (21, 32). The ompB^STOP::tn was previously isolated in a suppressor screen and lacked expression of OmpB (11). The rmlD::tn and 132 other transposon insertion mutant strains, screened for pUb, were isolated in an independent screen in which mutants were isolated without regard for plaque size (table S1). The genomic locations of transposon insertion sites for all mutants were determined by semirandom nested PCR. To verify the insertions and clonality, we used PCRs that amplified the transposon insertion site using primers for flanking chromosomal regions: 5′-GCTCACTAGATAGCACTCG-′3 and 5′-GCTCGATTTATCTCACTTTATG-′3 for rlmD::tn, 5′-CGTTTAATAGTCCAGTTAATTTGT-′3 and 5′-CCGTCTATACCGTCCATAAAAT-′3 for wecA::tn, 5′-GCATCGAAATAACCCTGAG-′3 and 5′-GCAAACTTCTCAAAGAAATTAACG-′3 for pkmt1::tn, 5′-GCTAAGAAATCTTCTAATTTGATATTTTAC-′3 and 5′-CGAAAATTTACCTGAGCCTT-′3 for pkmt2::tn, and 5′-CGACACATAATAGCACAAACTAC-′3 and 5′-GCGGAGGCGGTAGTAAAG-′3 for mrdA::tn (fig. S10).