Identification of new loci involved in adhesion of Listeria monocytogenes to eukaryotic cells

Insertional mutagenesis was performed with Tn1545 in the genetic background of an inIAB deletion mutant to identify new adhesion determinants in Liste ria monocytogenes, Four insertion mutants defective in adhesion to eukaryot ic cells were identified, Insertion sites were cloned by inverse-PCR and se quenced, The genetic organization of insertion regions was further analysed by screening and sequencing DNA fragments from a HindIII library and by se arching databases, Three adhesion-defective mutants each had one copy of Tn 1545 inserted into their chromosome, The insertion sites were different in the three mutants: (i) upstream from two ORFs in tandem. similar to dfp and priA of Bacillus subtilis. respectively; (ii) within an ORF encoding a put ative 126 amino-acid-polypeptide with no significant similarity to any know n protein; (iii) within an ORF similar to a B. subtilis ORF with no known f unction. just upstream from an operon similar to an ABC (ATP-binding casset te) transporter operon from B, subtilis. The excisants obtained from these mutants using the excision reporter plasmid pTCR9 recovered full adhesion c apacity, A fourth mutant was the most severely defective in adhesion, It ha d five Tn1545 insertions, one of which was upstream from dfp and priA, and another of which was upstream from ami, a gene encoding a surface-exposed a utolysin with a C terminus similar to that of InIB, Ami was clearly involve d because an ami null mutant constructed in an ECD Delta inIA-F background was adhesion-defective. Thus new regions involved in the adhesion of L, mon ocytogenes to eukaryotic cells were identified, Further study is required t o define more accurately the roles of these regions in the adhesion process itself.