TABLE 1.

Bacterial strains and plasmids

Strain or plasmidRelevant genotype/phenotypeaReference or source
Strains
    E. coli DH5αF endA1 hsdR17 (r m+) supE44 thi-l recA1 gyrA (Nal1) relA1 Δ(lacZYA-argF)U169 φ80dlacZΔM15 λpirRK6Laboratory collection
    L. pneumophila
        MB110Lp02 wild type; thyA hsdR rpsL10
        MB413Lp02 letA 22-3::kan mutant (lpg2646)36
        MB416Lp02 letS 36::kan mutant (lpg1912)36
        MB410Lp02 fliA 35::kan mutant (lpg1782)36
        MB510Lp02 fliA 35::kan mutant pMMBGent-FliAThis work
        MB550Lp02 fliA 35::kan mutant preg-FliAThis work
        MB462Lp02 fliA 35::kan mutant pMMBGent-Δmob vector controlThis work
        MB460Lp02 dotA::gent mutant (lpg2646)66
        MB379Lp02 rpoS::kan mutant (lpg1284)8
        MB473Lp02 pMMBGentΔmob; vector control35
        MB552Lp02 fliD::kan mutant (lpg1338)This work
        MB553Lp02 fliD::gent mutantThis work
        MB554Lp02 flhB::gent “A” mutant (lpg1786)This work
        MB555Lp02 flhB::gent “C” mutantThis work
        MB556Lp02 flhB::kan mutantThis work
        MB534Lp02 flaA::kan mutant (lpg1340)This work
        MB557Lp02 flaA::kan mutant pMMBGent-flaAThis work
        MB558Lp02 flaA::kan mutant pMMBGentΔmob, vector controlThis work
        MB532Lp02 flaA::gent mutantThis work
        MB560Lp02 motAB::gent “A” mutant (lpg1780-81)This work
        MB561Lp02 motAB::gent “C” mutantThis work
        MB562Lp02 motAB::gent “A” pMMB206-motABThis work
        MB563Lp02 motAB::gent “A” pMMB206; vector controlThis work
        MB564Lp02 fliI::cam mutant (lpg1757)61
        MB565Lp02 flI::cam fliD::gent “2A” double mutantThis work
        MB566Lp02 flI::cam fliD::gent “2B” double mutantThis work
        MB567Lp02 flaA::gent fliD::kan “A” double mutantThis work
        MB568Lp02 flaA::gent fliD::kan “B” double mutantThis work
        MB573Lp02 flhB::gent “A” fliA 35::kan double mutantThis work
Plasmids
    pMMBGent-ΔmobpMMB67EH derivative; Δmob lacIq Ptac Gentr; broad-host-range vector35
    pMMB206-ΔmobpMMB66EH derivative; Δmob lacIq PtaclacUV5 Camr66
    pUC4KSource of 1.3-kb kanamycin resistance GenBlockPharmacia
    pGEMT-EasyMCS within coding region of β-lactamase α fragment linearized with single-T overhangs, 3 kb; AmprPromega
    pGEMT-GentpGEMT-Easy with 1.7-kb Gentr cassette from BH038 cloned MCS, source of Gent cassette; Ampr GentrThis work
    pGEMTMCS within coding region of β-lactamase α fragment, lacking EcoRI sites of T-Easy; AmprPromega
    pGEM-flaApGEMT-Easy with 1.876-kb PCR-amplified flaA chromosomal region, ligated into T overhangs; AmprThis work
    pGEM-ΔflaA-gentpGem-flaA with 1.7-kb Gentr cassette disrupting internal EcoRI sites at 393-455 bases of 1,428-base FlaA ORFThis work
    pGEM-ΔflaA-kanpGem-flaA with 1.3-kb Kanr cassette disrupting internal EcoRI sites at 393-455 bases of 1,428-base FlaA ORFThis work
    pMMBGent-FlaApMMBGent-Δmob with ∼3.0 kb of FlaA genomic region with native promoter and ORF ligated into MCS Complementing plasmid, GentrThis work
    pGEM-fliApGEMT-Easy with 0.94-kb PCR-amplified flaA chromosomal region, ligated into T overhangs; AmprThis work
    pMMBGent-FliA940-pp FliA genomic region from pGEM-fliA cut with EcoRI, isolated, and ligated into EcoRI site of pMMBGent-Δmob, noninducible, complementing plasmid, GentrThis work
    pregFliApMMB206-Δmob with ∼750-bp FliA RBS and ORF PCR product cloned into PtaclacUV5 site, IPTG-inducible FliA expression; CamrThis work
    pGEM-fliDpGEMT (not T-Easy) with 3.457-kb PCR-amplified fliD chromosomal region, ligated into T overhangs; AmprThis work
    pGEM-ΔfliD-kanpGem-fliD with 1.3-kb Kanr cassette disrupting internal EcoRI site at base 641 of 1,626-base FliD ORF; Ampr KanrThis work
    pGEM-ΔfliD-gentpGem-fliD with 1.7-kb Gentr cassette disrupting internal EcoRI site at base 641 of 1,626-base FliD ORF; Ampr GentrThis work
    pGEM-motABpGEMT-Easy with 1,600-bp PCR-amplified motAB chromosomal region, ligated into T overhangs; AmprThis work
    pGEM-ΔmotAB-gent1 (A)pGEM-motAB with 1.9-kb Gentr cassette inserted between StuI and ClaI sites in opposite orientation to motAB, losing ∼130 bp of motAB region; Ampr GentrThis work
    pGEM-ΔmotAB-gent2 (C)pGEM-motAB with 1.9-kb Gentr cassette inserted between StuI and ClaI sites in same orientation as motAB, losing ∼130 bp of motAB region; Ampr GentrThis work
    pMMB206-motAB1.7-kb motAB ORF region amplified with BamH1-MotA5 and SalI-MotB3 primers, digested, and ligated into pMMB206-Δmob at the PtaclacUV5 site, IPTG-inducible MotAB expression; CamrThis work
    pGEM-flhBpGEMT-Easy with 3.4-kb PCR-amplified flhB chromosomal region ligated into T overhangs; AmprThis work
    pGEM-ΔflhB-EcoRVpGem-flhB with addition of internal N-terminal EcoRV site at base 137 of the 1,140-bp FlhB ORF and loss of ∼770 bp of the ORFThis work
    pGEM-ΔflhB-gentpGem-flhB-EcoR V with 1.7-kb Gentr cassette inserted at EcoRV site disrupting the truncated FlhB; Ampr GentrThis work
  • a MCS, multicloning site; RBS, ribosome binding site.