Identification of Non-dot/icmSuppressors of theLegionella pneumophilaΔdotLLethality Phenotype

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Abstract
Legionella pneumophila, a causative agent of bacterial pneumonia, survives inside phagocytic cells by avoiding rapid targeting to the lysosome. This bacterium utilizes a type IVB secretion system, encoded by thedot/icmgenes, to replicate inside host cells. DotL, a critical component of the Dot/Icm secretion apparatus, functions as the type IV coupling protein. In contrast to mostdot/icmgenes, which are dispensable for growth on bacteriological media,dotLis required for the viability of wild-typeL. pneumophila. Previously we reported that ΔdotLlethality could be suppressed by inactivation of the Dot/Icm complex via mutations in otherdot/icmgenes. Here we report the isolation of non-dot/icmsuppressors of this phenotype. These ΔdotLsuppressors include insertions that disrupt the function of theL. pneumophilahomologs ofcpxR,djlA,lysS, and two novel open reading frames, lpg0742 and lpg1594, that we have namedldsAandldsBforlethality of ΔdotL suppressor. In addition to suppressing ΔdotLlethality, inactivation of these genes in a wild-type strain background causes a range of defects inL. pneumophilavirulence traits, including intracellular growth, implicating these factors in the proper function of the Dot/Icm complex. Consistent with previous data showing a role for thecpxsystem in regulating expression of severaldot/icmgenes, thecpxRinsertion mutant produced decreased levels of three Dot/Icm proteins, DotA, IcmV, and IcmW. The remaining four suppressors did not affect the steady-state levels of any Dot/Icm protein and are likely to represent the first identified factors necessary for assembly and/or activation of the Dot/Icm secretion complex.