The human pathogen Listeria monocytogenes is able to transition from environmental saprophyte to facultative intracellular bacteria. In this process, virulence gene expression is controlled by the positive regulatory factor A (PrfA). Recent studies at the single cell level have shown that gene expression in response to stress exposure is stochastic in individual bacteria cells. Current studies applied those general findings to Listeria cells, revealing that PrfA as well is not regulated consistently, but that PrfA activity differs between individual cells. The aim of this study was to elucidate the mechanism by which Listeria regulates PrfA activation at the single cell level and whether this property is heritable or not. A reporter fusion, namely an eGFP sequence, integrated following the PrfA dependent promoter (Phly), was used to visualize the activation after heat stress exposure. Fluorescence activated cell sorting (FACS) was used to distinguish PrfA positive and negative cells. After passaging and sorting PrfA activating versus non-activating cells over several generations, two stable fluorescent phenotypes emerged. A comparison between the genome of the PrfA positive and its parent strain revealed a single-nucleotide polymorphism (SNP) in the CDS of LMRG_02823, as well as a mutation in the 5'UTR of LMRG_00195, both LPXTG family cell wall associated proteins regulated via small RNAs. The link between those mutations and PrfA activation is currently being investigated.