Inactivation of Lgt Allows Systematic Characterization of Lipoproteins fromListeria monocytogenes

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Abstract
Lipoprotein anchoring in bacteria is mediated by the prolipoprotein diacylglyceryl transferase (Lgt), which catalyzes the transfer of a diacylglyceryl moiety to the prospective N-terminal cysteine of the mature lipoprotein. Deletion of thelgtgene in the gram-positive pathogenListeria monocytogenes(i) impairs intracellular growth of the bacterium in different eukaryotic cell lines and (ii) leads to increased release of lipoproteins into the culture supernatant. Comparative extracellular proteome analyses of the EGDe wild-type strain and the Δlgtmutant provided systematic insight into the relative expression of lipoproteins. Twenty-six of the 68 predicted lipoproteins were specifically released into the extracellular proteome of the Δlgtstrain, and this proved that deletion oflgtis an excellent approach for experimental verification of listerial lipoproteins. Consequently, we generated ΔlgtΔprfAdouble mutants to detect lipoproteins belonging to the main virulence regulon that is controlled by PrfA. Overall, we identified three lipoproteins whose extracellular levels are regulated and one lipoprotein that is posttranslationally modified depending on PrfA. It is noteworthy that in contrast to previous studies ofEscherichia coli, we unambiguously demonstrated that lipidation by Lgt is not a prerequisite for activity of the lipoprotein-specific signal peptidase II (Lsp) inListeria.