THE LEGIONELLA PNEUMOPHILA INCOMPLETE PHOSPHOTRANSFERASE SYSTEM (PTS) IS REQUIRED FOR OPTIMAL INTRACELLULAR GROWTH AND MAXIMAL EXPRESSION OF PMRA REGULATED EFFECTORS

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: 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 mutants 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 level 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 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.