A novel suicide plasmid for efficient gene mutation in Listeria monocytogenes

Highlights

• A novel plasmid (pHoss1) for Listeria monocytogenes was constructed.

• pHoss1 is very efficient for the generation of in-frame deletion mutants.

• The L. monocytogenes ispG and ispH genes were in-frame mutated.

• The L. monocytogenes ispG and ispH genes are not involved in cell adhesion.

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.

Introduction

Deletion of bacterial genes by allelic exchange is a widely used method to study gene functions. The success of allelic exchange increases with the availability of selection markers that permit the effective isolation of transformants and recombinant strains resulting from single or double crossover events. Although several plasmids have been available for generating allelic exchange mutants in Gram-positive bacteria, construction of Listeria monocytogenes mutants has been very inefficient due to lack of a plasmid with effective selection markers for first and second allelic exchanges.

Two vectors constructed for allelic exchange in Gram-positive bacteria are pAUL-A and pLSV2 (Chakraborty et al., 1992, Wuenscher et al., 1991), which both have Ery marker for the first allelic exchange, but do not have a selection marker for the second allelic exchange, making the mutant identification extremely laborious due to the low frequency of the second recombination event (Reyrat et al., 1998).

A pMAD vector was developed and used for generating allelic replacements in several Gram-positive bacteria, including Staphylococcus aureus, L. monocytogenes, and Bacillus cereus (Arnaud et al., 2004). This vector features a temperature sensitive origin of replication, an erythromycin selection marker, and lacZ gene encoding β-galactosidase (bgaB) for blue-white screening. However, color screening does not permit positive selection of mutants for the second allelic exchange.

Recently, pKOR1 and pIMAY suicide plasmids were developed for allelic exchange in Staphylococcus (Bae and Schneewind, 2006, Monk et al., 2012). These two vectors have advantages of employing antisense secY RNA expression for positive selection of the second allelic exchange. The expression of secY antisense RNA in the presence of anhydrotetracycline prevents growth of cells that retain the integrated plasmid and provides selection for chromosomal excision and loss of plasmid (Bae and Schneewind, 2006). We attempted to use these two plasmids to construct in-frame deletions in L. monocytogenes, but the transformation attempts have been unsuccessful.

To expedite mutant construction in L. monocytogenes, we developed a new suicide plasmid named pHoss1, which combines the pMAD backbone and the secY antisense cassette from pIMAY. To assess the usefulness of pHoss1, ispG and ispH genes of L. monocytogenes (LMOf2365_1460 and LMOf2365_1470) were deleted in-frame. IspG and IspH are iron sulfate enzymes involved in isoprenoid biosynthesis via the mevalonate-independent 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway. Enzymes in this pathway are encoded by six genes (ispC, ispD, ispE, ispF, ispG, and ispH) and result in production of isopentenyl pyrophosphate (IPP) or its isomer dimethylallyl pyrophosphate (DMAPP) (Hunter, 2007, Rohmer, 1999). 1-hydroxy-2-methyl-2-(E)-butenol 4-diphosphate synthase (IspG) and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (IspH) are the last two enzymes of this pathway. The IspG protein catalyzes the conversion of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (ME-2,4cPP) into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (Hecht et al., 2001), whereas ispH converts 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate into IPP and DMAPP (Altincicek et al., 2002). Because the enzymes of the MEP pathway are not found in humans, this pathway has been used recently as an anti-infective drug target of various bacterial infections and malaria (Nakagawa et al., 2013, Obiol-Pardo et al., 2011). Interestingly, nonpathogenic strains of Listeria innocua and L. monocytogenes do not possess these two genes (Begley et al., 2008, Steele et al., 2011). Thus, we hypothesized that ispG and ispH genes could be essential for L. monocytogenes pathogenesis, and we tested this by developing mutants and determining their attachment properties in human Caco-2 cells, a transformed intestinal epithelial cell line.