The Legionella pneumophila Incomplete Phosphotransferase System Is Required for Optimal Intracellular Growth and Maximal Expression of PmrA-Regulated Effectors

Abstract

ABSTRACT The nitrogen phosphotransferase system (PTS Ntr ) is a regulatory cascade present in many bacteria, where it controls different functions. This system is usually composed of three basic components: enzyme I Ntr (EI Ntr ), NPr, and EIIA Ntr (encoded by the ptsP , ptsO , and ptsN genes, respectively). In Legionella pneumophila , as well as in many other Legionella species, the EIIA Ntr component is missing. However, we found that deletion mutations in both ptsP and ptsO are partially attenuated for intracellular growth. Furthermore, these two PTS Ntr components were found to be required for maximal expression of effector-encoding genes regulated by the transcriptional activator PmrA. Genetic analyses which include the construction of single and double deletion mutants and overexpression of wild-type and mutated forms of EI Ntr , NPr, and PmrA indicated that the PTS Ntr components affect the expression of PmrA-regulated genes via PmrA and independently from PmrB and that EI Ntr and NPr are part of the same cascade and require their conserved histidine residues in order to function. Furthermore, expression of the Legionella micdadei EII Ntr component in L. pneumophila resulted in a reduction in the levels of expression of PmrA-regulated genes which was completely dependent on the L. pneumophila PTS components and the L. micdadei EII Ntr conserved histidine residue. Moreover, reconstruction of the L. pneumophila PTS in vitro indicated that EI Ntr is phosphorylated by phosphoenolpyruvate (PEP) and transfers its phosphate to NPr. Our results demonstrate that the L. pneumophila incomplete PTS Ntr is functional and involved in the expression of effector-encoding genes regulated by PmrA.