Fig. 3: L. pneumophila genes lpg2370, lpg2369, and lpg2368 belong to the same operon and constitute a tripartite HipBST family TA system.

a The HipA-C-terminal domain-like proteins are found in bacteria, branches of the IQTree maximum likelihood phylogenetic tree of representative HipA-C-terminal sequences are colored by the taxonomic groupings as per the upper left panel. b Schematic of the lpg2368–lpg2369-lpg2370 operon in L. pneumophila and the HipBST operon from E. coli O127:H6 strain. In the genome of L. pneumophila, lpg2368, lpg2369, and lpg2370 (relative position of the conserved P-loop is highlighted in yellow) are located in the same operon and encode proteins that share homology with the HipBST TA system. c Comparison of elution profiles of Lpg2370 (11.3 mL), Lpg2370-Lpg2369 (10.9 mL), and Lpg2370-Lpg2369-Lpg2368 (9.6 mL) in size-exclusion chromatography (Superdex 75 increase column). The accompanying SDS-PAGE gel with peak fraction samples is provided on the right side. d Growth curve (upper panel) and the CFU count (bottom panel) of L. pneumophila ∆lpg2368-∆lpg2369-∆lpg2370 (∆3) expressing the kinase site active mutant H199A (H/A) and the wild-type Lpg2370, Lpg2370 causes cellular growth arrest upon induction, whereas the kinase site active mutant H199A (H/A) does not. e Growth curve (upper panel) and the CFU count (bottom panel) of overnight cultures L. pneumophila ∆lpg2368-∆lpg2369-∆lpg2370 (∆3) harboring pZL507 (pZL507:: lpg2370) or the empty pZL507 combined with pJL03 (pJL03:: lpg2368), pJL03 (pJL03:: lpg2369), pJL03 (pJL03:: lpg2368–lpg2369) or the empty low-copy-number pJL03 vector, as indicated, the tested bacterial strains were diluted in fresh AYE broth (supplemented with 10 μg/mL Gentamicin) to OD₆₀₀ = 0.1 and split into 2-mL subcultures. 200 μM IPTG was added to induce the expression of Lpg2370, and 1% arabinose was added to induce the expression of Lpg2368, Lpg2369, or Lpg2368 + Lpg2369. In panels d and e, data shown are mean values ± SD (n  =  3 independent experiments).