Tools for Functional Postgenomic Analysis ofListeria monocytogenes

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 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-␤-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

A Listeria monocytogenes RNA Helicase Essential for Growth and Ribosomal Maturation at Low Temperatures Uses Its C Terminus for Appropriate Interaction with the Ribosome

“…Why the complemented strain displayed low levels of Lmo1722 protein is unclear, but it could be due to transcript or plasmid instability. To analyze complementation phenotypes at wild-type Lmo1722 levels, lmo1722 alleles were placed under the control of an IPTGinducible promoter in the chromosomally integrative pIMK3 vector (47). The resulting constructs were streaked on agar plates with or without 1 mM IPTG and incubated at 16 or 37°C (Fig.…”

Listeria monocytogenes aguA1, but Not aguA2, Encodes a Functional Agmatine Deiminase

“…A homologous recombination strategy with SOE-PCR procedure was used for in-frame deletion to construct aguA1 and aguA2 single and double deletion mutants as described previously (15,27). The DNA fragments containing homologous arms upstream and downstream of aguA1 or aguA2 were obtained by PCR amplification of 10403S DNA templates using the SOE primers (supplemental Table S1).…”

“…The recombinant vectors containing the target gene deletion cassettes were confirmed by sequencing. A previous protocol was followed for deletion of the targeted genes via allelic exchange (27). Briefly, the competent L. monocytogenes 10403S cells were electroporated with one of the vector constructs.…”

The SOS response of Listeria monocytogenes is involved in stress resistance and mutagenesis

“…This resulted in 915 bp and 306 bp internal deletions for recA and yneA, respectively. Vector pIMK2 (Monk et al, 2008), containing the PSA phage integrase system, was used for construction of the yneA complementation mutant. Primers yneA-E and yneA-F (Table 2) were used for amplification of yneA and its promoter region, and the amplified fragment was cloned into pIMK2 as a SacIPstI fragment resulting in vector pIMK-yneA.…”