A novel suicide plasmid for efficient gene mutation in Listeria monocytogenes

Hossam Abdelhamed; Mark L Lawrence; Attila Karsi

doi:10.1016/j.plasmid.2015.05.003

A novel suicide plasmid for efficient gene mutation in Listeria monocytogenes

Plasmid. 2015 Sep:81:1-8. doi: 10.1016/j.plasmid.2015.05.003. Epub 2015 May 31.

Authors

Hossam Abdelhamed¹, Mark L Lawrence², Attila Karsi³

Affiliations

¹ Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762, USA.
² Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762, USA. Electronic address: lawrence@cvm.msstate.edu.
³ Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762, USA. Electronic address: a.karsi@msstate.edu.

PMID: 26038185
DOI: 10.1016/j.plasmid.2015.05.003

Abstract

Although several plasmids have been used in Listeria monocytogenes for generating mutants by allelic exchange, construction of L. monocytogenes mutants has been inefficient due to lack of effective selection markers for first and second recombination events. To address this problem, we have developed a new suicide plasmid, pHoss1, by using the pMAD plasmid backbone and anhydrotetracycline selection marker (secY antisense RNA) driven by an inducible Pxyl/tetO promoter. Expression of the secY antisense RNA eliminates merodiploids and selects for the loss of plasmid via a second allelic exchange, which enriches the number of mutants with deleted genes. To assess the effectiveness of pHoss1 for the generation of stable in-frame deletion mutations, we deleted the ispG and ispH genes of L. monocytogenes serotype 4b strain F2365. Results showed that identification of the second allelic exchange mutants was very efficient with 80-100% of the colonies yielding desired deletion mutants. L. monocytogenes' intestinal cell attachment was not altered when ispG and ispH genes were deleted. We expect that this new plasmid will be very useful for construction of marker-free deletion mutants in L. monocytogenes and in other Gram-positive bacteria, including Staphylococcus aureus and Bacillus cereus.

Keywords: In-frame deletion mutants; Listeria; Suicide plasmid; ispG; ispH.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Bacterial Adhesion / genetics
Gene Order
Genes, Bacterial*
Genetic Complementation Test
Listeria monocytogenes / genetics*
Mutation*
Plasmids / genetics*
Reading Frames
Sequence Deletion