Characterization and analysis of theptsH andptsI genes coding for HPr and enzyme I of the Listeria monocytogenes phosphotransferase system

The phosphoenolpyruvate-dependent phosphotransferase system (PEP-dependent PTS) is utilized for sugar uptake by a wide variety of bacteria. The PEP-dependent PTS generally consists of two non-specific energy coupling proteins, Enzyme I and HPr. An additional set of three activities distributed among one or more proteins, known as the Enzyme II complex, provides the substrate specificity for individual sugar transport. In addition to its role in sugar transport, the PEP-dependent PTS is known to play a vital role in catabolite repression in Gram-negative organisms and has recently been implicated in Gram-positive catabolite repression as well. This finding provides a possible mechanism by which the PEP-dependent PTS can play a role in a large variety of global activities and may contribute to the survival of intracellular pathogens such as Listeria monocytogenes.; The objective of this research was to physically demonstrate the presence of a PEP-dependent PTS in the deadly foodborne pathogen L. monocytogenes and determine the role of genes and resulting proteins in overall PTS activity and regulation. The results from these studies demonstrate that L. monocytogenes contains ptsH and ptsI genes coding for HPr and Enzyme I proteins of the PTS. These genes reside in a 2.1 Kb transcription unit that is expressed at pH levels as low as 4.4 in the presence of glucose. The ptsH gene, located directly upstream of ptsI, codes for a functional protein (HPr) that can be phosphorylated in a PEP-dependent manner (at histidine 15) or in an ATP-dependent manner (at serine 46). ATP-dependent phosphoryiation of serine 46 was catalyzed by a previously undefined L. monocytogenes membrane associated kinase resulting in down regulation of PTS activity. Serine 46 phosphorylated HPr is stably maintained for several hours in vitro under variable energy conditions suggesting that HPr does not possess auto-phosphatase activity but instead may be dephosphorylated by a specific phosphatase.