Extended Data Fig. 2: Validation of IDTS mutant phenotypes predicated by Tn-seq.
a, Null mutations in IDTS genes do not impair L. pneumophila replication in nutrient rich bacteriological medium. The wild type (WT) and indicated null mutant strains were grown in CYE medium and the absorbance at 600 nm was monitored over time. Data are the mean ± standard deviation of 2 biological replicates, each generated from 2 technical replicates (Source Data 6). b, IDTS genes for which mutations were predicted to be neutral based on Tn-seq do not impair L. pneumophila growth in amoebae when compared to the WT strain. c, The phenotype predicted by Tn-seq for mavB (lgp1752) is due to polar effects of transposon insertion mutations on lpg1751. Top panel: The L. pneumophila genetic locus of lpg1751 and lpg1752 (mavB). Bottom panel: Deletion of lpg1751 but not mavB severely impairs L. pneumophila replication within A. castellanii and a ∆mavB∆lpg1751 double mutant phenocopies the ∆lpg1751 single deletion strain. d, The intracellular growth defects of IDTS null mutants can be rescued by reintroducing the corresponding gene on a self-replicating plasmid. Bacterial strains harboring the empty vector pJB908 or the respective complementation plasmid were used to challenge A. castellanii or H. vermiformis. In b,c,d, The WT and indicated IDTS null mutant strains were used to challenge amoebae and bacterial growth, based on recovered colony forming units (cfus) on solid media from lysed host cells, was assessed after 24 hours. Plotted is the total bacterial yield 24 hours post infection (hpi) normalized to the wild type strain by the number of intracellular bacteria 1–2 hpi. e, Uptake and/or survival phenotypes of IDTS mutants can be rescued by reintroducing the corresponding gene on a self-replicating plasmid. Bacterial strains harboring the empty vector pJB908 or the respective complementation plasmid were used to challenge A. castellanii or H. vermiformis for 1.5 hours. Cells were then treated with gentamycin to kill extracellular bacteria and internal bacteria were enumerated based on recovered cfus from host cell lysates plated on bacteriological medium. In b-e, Data are the mean ± standard deviation of 2–5 biological replicates, each generated from 3 technical replicates; *P < 0.02, 1-way ANOVA, relative to the WT strain (Source Data for Extended Data Fig. 2).
