Method ArticleA novel suicide plasmid for efficient gene mutation in Listeria monocytogenes
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.
Section snippets
Bacterial strains, plasmids, and growth conditions
The bacterial strains and plasmids used in this study are described in Table 1. L. monocytogenes strain F2365 was originally isolated from Mexican-style soft cheese implicated in a 1985 outbreak of listeriosis in California (Linnan et al., 1988). L. monocytogenes strains were grown routinely overnight in brain heart infusion broth (BHI) (Difco Laboratories, Detroit, MI). Escherichia coli was grown in Luria–Bertani (LB) broth. Antibiotics (Sigma-Aldrich, St. Louis, MO) used as needed were:
Construction of the pHoss1 suicide plasmid
A novel 8995 bp pHoss1 suicide plasmid was constructed. It contains a heat-sensitive origin of replication, four unique restriction sites (SalI, EcoRI, SmaI and NcoI), erythromycin resistance gene, and a 1371 bp fragment encoding an antisense secY RNA expression cassette driven by an inducible Pxyl/tetO promoter (Fig. 1).
Construction of L. monocytogenes in-frame deletion mutants
Using the pHoss1 plasmid, we were able to construct two L. monocytogenes mutant strains, Lmf2365ΔispG and Lmf2365ΔispH (Fig. 2). Selection for the second allelic exchange was
Discussion
L. monocytogenes is an intracellular pathogen transmitted to humans and animals through the consumption of contaminated food. Clinical signs of listeriosis in healthy individuals include febrile gastroenteritis, whereas in immunocompromised individuals, listeriosis can be an invasive and systemic infection leading to sepsis, meningitis, and meningoencephalitis with a high mortality rate (25–30%) (Allerberger and Wagner, 2010). In addition, fetal infections cause spontaneous abortions,
Acknowledgments
We thank Drs. Dagmar Beier and Timothy Foster for providing us with pLSV2 and pIMAY plasmids. We also thank Dr. Robert Wills for his help on statistical analysis and Drs. John Harkness and Stephen Pruett for their review of our manuscript. This project was funded by USDA ARS Agreement #58-6402-2729, which is operated under USDA CRIS project MIS501170, “Mississippi Center for Food Safety and Post-Harvest Technology”.
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