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The ability to manipulate chromosomally encoded genes is a fundamental biological tool for the analysis of gene function. Here, we provide in greater depth a protocol for the creation of nonpolar unlabelled gene deletions in Listeria monocytogenes that are facilitated by the splicing overlap extension PCR technique. For mutagenesis in L. monocytogenes, we describe the pKSV7 plasmid-based approach, which facilitates the introduction of a spliced amplicon in place of the corresponding segment of chromosomal DNA.A plasmid preparation is a method used to extract and purify plasmid DNA. Methods developed to purify plasmid DNA from bacteria generally involve harvesting and alkaline lysis of the bacteria, precipitation of chromosomal DNA and protein, followed by purification of the plasmid DNA. Here, we describe the mini-preparation of plasmid DNA by a rapid small-scale method, adapted for Listeria monocytogenes. The quality of plasmid DNA isolated using this method is sufficient for analytical purposes but may be upscaled for further downstream analysis. Electrophoretic separation of the resultant lysate allows conclusions to be made on the presence, number, copy number, and size of the plasmids in the analyzed bacterial strains.Proteomics has become an essential tool to answer biologists’ questions. For bacteriologists, the proteome of bacteria is much less complex than that of eukaryotic organisms. However, not all the different cell “compartments” are easily accessible, and the analysis of cell envelope proteins is particularly challenging. For the Gram-positive bacterium Listeria monocytogenes, one of the main foodborne pathogen microorganisms, the study of surface proteins is crucial to better understand the mechanisms of pathogenicity, as well as adaptation/resistance to and persistence in hostile environments. The evolution of proteomic techniques, and particularly the possibility of separating and analyzing complex protein samples by off-gel (LC-MS/MS) versus in-gel (two-dimensional electrophoresis) approach, has opened the doors to new extraction and preparation methods to target the different subproteomes. Here, we describe three procedures to prepare and analyze intracellular, exocellular, and cell surface proteins (1) the cell fractionation, based on cell broken and separation of protein subfractions by differential centrifugation; (2) the biotinylation, based on the labeling of cell surface proteins and their selective extraction; and (3) the enzymatic shaving by the action of trypsin on intact cells. These complementary methods allow to encompass all L. monocytogenes subproteomes for general profiling or target studies and could be applicable to other Gram-positive bacteria.The behavior of Listeria monocytogenes communities in the food chain is closely associated with their spatial organization. Whether as biofilms on industrial surfaces or as microcolonies in food matrices, the resulting physiological diversification combined with the presence of extracellular polymeric substances (EPS) triggers emergent community functions involved in the pathogen survival and persistence (e.g., tolerance to dehydration, biocides, or preservatives). Tinlorafenib In this contribution, we present a noninvasive confocal laser microscopy (CLM) protocol allowing exploration of the spatial organization of L. monocytogenes communities on various inert or nutritive materials relevant for the food industry.Listeria monocytogenes is a foodborne pathogen capable of colonizing and persisting in the food production environment (FPE). While there are a variety of factors involved in L. monocytogenes’ ability to persist in FPE, the ability to form biofilms has the potential to increase their chance of survival and long-term colonization. Understanding the mechanisms involved in L. monocytogenes ability to form biofilms may potentially help food safety managers optimize control strategies targeting it in the FPE. In this chapter, a high-throughput method to determine L. monocytogenes ability to attach and form biofilms utilizing FPE-grade stainless steel is described. This method provides fast and efficient results, facilitating scaling up to large numbers of isolates to measure their ability to form biofilms, where lower-throughput approaches can then be utilized to further characterize isolates of interest.High-throughput biochemical screening techniques are an important tool in phenotypic analysis of bacteria. New methods, simultaneously measuring many phenotype responses, increase the output of such investigations and allow a more complete overview of the bacterial phenotype, facilitating large-scale correlation to related genotypes. This chapter describes the application of OmniLog phenotype microarray analysis, a high-throughput assay for the phenotypic characterization of bacterial strains across a variety of different traits such as nutrient utilization and antimicrobial sensitivity, to Listeria species.Nucleotide sequence-based methods focusing on the single-nucleotide polymorphisms (SNPs) of Listeria monocytogenes and L. innocua housekeeping genes (multilocus sequence typing) and in the core genome (core genome MLST) facilitate the rapid and interlaboratory comparison in open accessible databases as provided by Institute Pasteur ( https//bigsdb.web.pasteur.fr/listeria/listeria.html ). Strains can be compared on a global level and help to track forward and trace backward pathogen contamination events in food processing facilities and in outbreak scenarios.PFGE is a valuable tool for assessing L. monocytogenes strain interrelatedness. It is based on the study of total bacterial DNA restriction patterns. Cells are embedded in agarose plugs before being lysed. The released DNA is then digested into large fragments by restriction enzymes. As DNA fragments are too large to be separated by traditional electrophoresis in an agarose gel, changes in the direction of the electrical current are periodically applied in order to allow the proper migration of large DNA fragments. Strains are characterized by the obtained DNA fragment patterns or pulsotypes which vary depending on the number and size of bands.