Complementation ofListeria monocytogenesNull Mutants with SelectedListeria seeligeriVirulence Genes Suggests Functional Adaptation of Hly and PrfA and Considerable Diversification ofprfARegulation inL. seeligeri

WhileListeria seeligeri andL. monocytogenes contain the mainListeria virulence gene cluster, onlyL. monocytogenes is considered an intracellular pathogen. Initial evolutionary analyses showed that the virulence genesprfA ,hly , andplcA are conserved inL. seeligeri , with specific Hly and PrfA amino acid residues showing evidence for positive selection inL. seeligeri . Our data also show that temperature-dependent transcript patterns forprfA , which encodes a transcriptional regulator of virulence genes, differed betweenL. monocytogenes andL. seeligeri . To further investigate the divergence of virulence gene function and regulation,L. seeligeri prfA (prfA LS ),hly (hly LS ), andplcA (plcA LS ), as well asprfA LS constructs with differentprfA promoter regions, were introduced into appropriateL. monocytogenes null mutants. Only whenprfA LS was under the control of theL. monocytogenes prfA promoters (P1- and P2prfA ) (P1P2LM prfA LS ) wasprfA LS able to fully complement the ΔprfA LM deletion.hly LS introduced into anL. monocytogenes background under its native promoter showed transcript levels similar to those ofhly LM and was able to partially restoreL. monocytogenes wild-type-level hemolysis and intracellular growth, even though HlyLM and HlyLS showed distinct patterns of cell- and supernatant-associated hemolytic activities. Our data indicate that (i) regulation ofprfA expression differs betweenL. monocytogenes andL. seeligeri , althoughhly transcription is temperature dependent in both species, and (ii) PrfA and Hly functions are largely, but not fully, conserved betweenL. seeligeri andL. monocytogenes . Virulence gene homologues and their expression thus appear to have adapted to distinct but possibly related functions in these two species.