Tools for functional post-genomic analysis of Listeria monocytogenes

Department of Microbiology, Alimentary Pharmabiotic Centre, School of Pharmacy, University College Cork, Cork, Ireland

^* To whom correspondence should be addressed. Email: c.gahan{at}ucc.ie.

	Abstract

We describe the development of genetic tools for regulated gene expression, the introduction of chromosomal mutations and improved plasmid transfer by electroporation in the foodborne pathogen Listeria monocytogenes. pIMK, a kanamycin resistant, site-specific integrative, listeriophage vector, was constructed and then modified for over-expression (pIMK2) or for IPTG regulated expression (pIMK3 and pIMK4). The dynamic range of the promoters was assessed by luciferase activity, P60 secretion and internalin A mediated invasion. These demonstrated that pIMK4 and pIMK3 achieve a stringently controlled dynamic range of 540-fold. Stable gene over-expression was achieved with pIMK2, giving a range of expression for the three vectors of 1350-fold. The lactococcal pORI280 system was optimised for the generation of chromosomal mutations and used to create five new prfA star mutants. The combination of pIMK4 and pORI280 allowed the streamlined creation of "IPTG dependant" mutants. This was exemplified by the creation a clean deletion mutant of the universally essential secA gene, with this mutant exhibiting a rapid loss in viability upon withdrawal of IPTG. We have also improved plasmid transfer by electroporation into three commonly used laboratory strains of L. monocytogenes. An increase in transformation efficiency of 125-fold for EGDe over the widely used protocol of Park and Stewart (Park, S. F., and G. S. Stewart. 1990. Gene 94:129-132) was observed. A maximal transformation efficiency of 5.7 x 10⁶ and 6.7 x 10⁶ cfu per µg for was achieved for EGDe and 10403S, respectively, with a replicating plasmid. An efficiency of 2 x 10⁷ cfu per µg represents the highest thus far reported for L. monocytogenes F2365.