Generation of Markerless Gene Deletion Mutants in Listeria monocytogenes Using a Mutated pheS for Counterselection.

Gene alteration/deletion by allelic exchange is the preferred strategy for gene manipulation in bacteria. Here we present the fundamentals for an efficient allelic exchange gene deletion method in the bacterial pathogen Listeria monocytogenes. Combining vector generation by Gibson assembly with a counterselection system based on the mutated phenylalanine synthetase (pheS*) makes the generation of gene deletion mutants straightforward and time efficient.

Bone Marrow-Derived Macrophage (BMDM ) Infection by Listeria monocytogenes.

Listeria monocytogenes is a gram-positive bacterium adapted to life as both an environmental saprophyte and a pathogenic parasite of mammalian hosts, with a transcriptomic program tailored for each niche. Study of the L. monocytogenes pathogenic lifestyle requires conditions that mimic the mammalian niche. Of the myriad experimental models used to achieve such conditions, the bone marrow-derived macrophage (BMDM) is a relatively simple and reliable primary immune cell model for L. monocytogenes infections. Here we describe the extraction, preparation, and storage of BMDMs and their use in L. monocytogenes infection experiments.

A dual-function phage regulator controls the response of cohabiting phage elements via regulation of the bacterial SOS response.

Listeria monocytogenes strain 10403S harbors two phage elements in its chromosome; one produces infective virions and the other tailocins. It was previously demonstrated that induction of the two elements is coordinated, as they are regulated by the same anti-repressor. In this study, we identified AriS as another phage regulator that controls the two elements, bearing the capacity to inhibit their lytic induction under SOS conditions. AriS is a two-domain protein that possesses two distinct activities, one regulating the genes of its encoding phage and the other downregulating the bacterial SOS response. While the first activity associates with the AriS N-terminal AntA/AntB domain, the second associates with its C-terminal ANT/KilAC domain. The ANT/KilAC domain is conserved in many AriS-like proteins of listerial and non-listerial prophages, suggesting that temperate phages acquired such dual-function regulators to align their response with the other phage elements that cohabit the genome.

Temperate bacteriophages as regulators of host behavior.

Bacteriophages are ubiquitous and affect most facets of life, from evolution of bacteria, through ecology and global biochemical cycling to human health. The interactions between phages and bacteria often lead to biological novelty and an important milestone in this process is the ability of phages to regulate their host's behavior. In this review article, we will focus on newly reported cases that demonstrate how temperate phages regulate bacterial gene expression and behavior in a variety of bacterial species, pathogenic and environmental. This regulation is mediated by diverse mechanisms such as transcription factors, sRNAs, DNA rearrangements, and even controlled bacterial lysis. The outcome is mutualistic relationships that enable adaptively enhanced communal phage-host fitness under specific conditions.