An Effective Counterselection System for Listeria monocytogenes and Its Use To Characterize the Monocin Genomic Region of Strain 10403S

Abstract

Construction of Listeria monocytogenes mutants by allelic exchange has been laborious and time-consuming due to lack of proficient selection markers for the final recombination event, that is, a marker conveying substance sensitivity to the bacteria bearing it, enabling the exclusion of merodiploids and selection for plasmid loss. In order to address this issue, we engineered a counterselection marker based on a mutated phenylalanyl-tRNA synthetase gene (pheS*). This mutation renders the phenylalanine-binding site of the enzyme more promiscuous and allows the binding of the toxic p-chloro-phenylalanine analog (p-Cl-phe) as a substrate. When pheS* is introduced into L. monocytogenes and highly expressed under control of a constitutively active promoter, the bacteria become sensitive to p-Cl-phe supplemented in the medium. This enabled us to utilize pheS* as a negative selection marker and generate a novel, efficient suicide vector for allelic exchange in L. monocytogenes We used this vector to investigate the monocin genomic region in L. monocytogenes strain 10403S by constructing deletion mutants of the region. We have found this region to be active and to cause bacterial lysis upon mitomycin C treatment. The future applications of such an effective counterselection system, which does not require any background genomic alterations, are vast, as it can be modularly used in various selection systems (e.g., genetic screens). We expect this counterselection marker to be a valuable genetic tool in research on L. monocytogenesIMPORTANCEL. monocytogenes is an opportunistic intracellular pathogen and a widely studied model organism. An efficient counterselection marker is a long-standing need in Listeria research for improving the ability to design and perform various genetic manipulations and screening systems for different purposes. We report the construction and utilization of an efficient suicide vector for allelic exchange which can be conjugated, leaves no marker in the bacterial chromosome, and does not require the use of sometimes leaky inducible promoters. This highly efficient genome editing tool for L. monocytogenes will allow for rapid sequential mutagenesis, introduction of point mutations, and design of screening systems. We anticipate that it will be extensively used by the research community and yield novel insights into the diverse fields studied using this model organism.

Keywords: Listeria monocytogenes; allelic exchange; bacteriocins; counterselection; monocin; mutagenesis; pheS.

FIG 1

Construction and calibration of the pheS* counterselection marker. (A) Amino acid multiple-sequence alignment of PheS protein C termini, achieved with Clustal Omega. The sequences of the indicated species were aligned, and the conserved alanine corresponding to A294 of E. coli was identified (arrow); on the right is the number of amino acids in the protein. (B) Growth of the indicated strains on BHI agar plates with or without 18 mM p-Cl-phe.

FIG 2

pLR16-pheS* annotated plasmid map. The plasmid layout of pLR16-pheS*, including the key functions annotated, is shown. Ori⁺ is a temperature-sensitive Gram-positive origin of replication first published as a part of pKSV7, and the origin of transfer (OriT), chloramphenicol resistance genes, and multiple cloning site (MCS) were published as a part of pPL2 (18, 35). The figure was generated using SnapGene software (GSL Biotech).

FIG 3

Construction and validation of the Δmonocin and ΔftbQR mutants using pLR16-pheS*. (A) Schematic representation of the monocin gene region. The region is ~10.7 kbp long and consists of 17 genes (LMRG_2362 to LMRG_2378, here presented as ftbB to -R). Deletion of the whole region is designated Δmonocin, and deletion of the 1,132-bp long LMRG_2377 and LMRG_2378, encoding the holin and endolysin (marked in gray), is designated ΔftbQR. Annotation of gene groups is marked above the diagram; above the arrows, representing genes, are the gene names as designated by Lee et al. (21). (B) PCR verification of the deletion mutants. For the Δmonocin mutant, primers 15 and 18 (Table 1) were used, expecting ∼12,700-bp- and ∼2,000-bp-long products from A118 phage-cured L. monocytogenes and the Δmonocin mutant, respectively. For ΔftbQR, primers 19 and 22 (Table 1) were used, expecting ∼2,700-bp- and ∼1,600-bp-long products from A118-phage cured L. monocytogenes and ΔftbQR mutant, respectively. A 1-kb DNA ladder (GeneDireX) is shown on the right.

FIG 4

Growth of L. monocytogenes with and without mitomycin C treatment. A118-cured L. monocytogenes (Lm) and Δmonocin mutant and ΔftbQR mutant bacteria were grown in BHI rich medium at 30°C without (A) and with (B) mitomycin C (MC) at 1.5 μg/ml. The experiment was performed in a 96-well format in a Synergy HT BioTek plate reader. Growth curves from one representative experiment are shown. Some error bars, representing the standard deviation for a triplicate sample, are hidden by the symbols.