LP02 is a streptomycin-resistant thymidine auxotroph derived from L. pneumophila LP01. An unmarked deletion of flaA (corresponding to nucleotides 1478105–1479574 of the LP01 genome [49 (link)]) was generated in LP02 by use of the allelic exchange vector pSR47S [22 (link)]. The ΔflaA strain was also complemented by inserting the flaA gene and its own promoter (corresponding to nucleotides 1478136–1479915 of the LP01 genome) on the chromosome just after the ahpC gene (at position 3354877 of the LP01 genome). The ahpC locus is highly expressed but is not essential for Legionella virulence [50 (link)]. The fliI null strain LP02 fliI::Cm [22 (link)] was the kind gift of R. Isberg (Tufts University School of Medicine). The broad-host-range plasmid pBBR1-MCS2 was used to express flagellin from Legionella, E. coli MG1655 (fliC, b1923), S. flexneri 2457T (fliC, S2062), and Salmonella typhimurium LT2 (fliC, STM1959). The various flagellin open reading frames were first cloned into pET28a (NcoI, XhoI) and then transferred to pBBR1-MCS2 (XbaI, PvuI), such that all flagellins were expressed from the lac promoter of pBBR and the ribosome-binding site of pET28a. Expression was induced with 1mM IPTG. The pBBR-flagellin constructs were also expressed in E. coli CM735ΔfliC [51 (link)], along with LLO (pACYC184-LLOc) [41 (link)]. Salmonella strains were on the LT2 background and were the kind gift of the Starnbach laboratory.
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