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Applied and Environmental Microbiology, July 2008, p. 3921-3934, Vol. 74, No. 13
0099-2240/08/$08.00+0     doi:10.1128/AEM.00314-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Tools for Functional Postgenomic Analysis of Listeria monocytogenes{triangledown}

Ian R. Monk,1,2 Cormac G. M. Gahan,1,2,3* and Colin Hill1,2

Department of Microbiology,1 Alimentary Pharmabiotic Centre,2 School of Pharmacy, University College Cork, Cork, Ireland3

Received 6 February 2008/ Accepted 16 April 2008

We describe the development of genetic tools for regulated gene expression, the introduction of chromosomal mutations, and improved plasmid transfer by electroporation in the food-borne pathogen Listeria monocytogenes. pIMK, a kanamycin-resistant, site-specific, integrative listeriophage vector was constructed and then modified for overexpression (pIMK2) or for isopropyl-β-D-thiogalactopyranoside (IPTG)-regulated expression (pIMK3 and pIMK4). The dynamic range of promoters was assessed by determining luciferase activity, P60 secretion, and internalin A-mediated invasion. These analyses demonstrated that pIMK4 and pIMK3 have a stringently controlled dynamic range of 540-fold. Stable gene overexpression was achieved with pIMK2, giving a range of expression for the three vectors of 1,350-fold. The lactococcal pORI280 system was optimized for the generation of chromosomal mutations and used to create five new prfA star mutants. The combination of pIMK4 and pORI280 allowed streamlined creation of "IPTG-dependent" mutants. This was exemplified by creation of a clean deletion mutant with deletion of the universally essential secA gene, and this mutant exhibited a rapid loss of viability upon withdrawal of IPTG. We also improved plasmid transfer by electroporation into three commonly used laboratory strains of L. monocytogenes. A 125-fold increase in transformation efficiency for EGDe compared with the widely used protocol of Park and Stewart (S. F. Park and G. S. Stewart, Gene 94:129-132, 1990) was observed. Maximal transformation efficiencies of 5.7 x 106 and 6.7 x 106 CFU per µg were achieved for EGDe and 10403S, respectively, with a replicating plasmid. An efficiency of 2 x 107 CFU per µg is the highest efficiency reported thus far for L. monocytogenes F2365.

* Corresponding author. Mailing address: Department of Microbiology, University College Cork, Cork, Ireland. Phone: 353-21-4901363. Fax. 353-21-4903101. E-mail: c.gahan{at}ucc.ie

{triangledown} Published ahead of print on 25 April 2008.

Applied and Environmental Microbiology, July 2008, p. 3921-3934, Vol. 74, No. 13
0099-2240/08/$08.00+0     doi:10.1128/AEM.00314-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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