Unlike lytic phages, temperate phages that enter lysogeny maintain a long-term association with their bacterial host. In this context, mutually beneficial interactions can evolve that support efficient reproduction of both phages and bacteria. Temperate phages are integrated into the bacterial chromosome as large DNA insertions that can disrupt gene expression, and they may pose a fitness burden on the cell. However, they have also been shown to benefit their bacterial hosts by providing new functions in a bacterium-phage symbiotic interaction termed lysogenic conversion. In this Opinion article, we discuss another type of bacterium-phage interaction, active lysogeny, in which phages or phage-like elements are integrated into the bacterial chromosome within critical genes or operons and serve as switches that regulate bacterial genes via genome excision.

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Bacterial pathogens often carry multiple prophages and other phage-derived elements within their genome, some of which can produce viral particles in response to stress. Listeria monocytogenes 10403S harbors two phage elements in its chromosome, both of which can trigger bacterial lysis under stress: an active prophage (ϕ10403S) that promotes the virulence of its host and can produce infective virions, and a locus encoding phage tail-like bacteriocins. Here, we show that the two phage elements are co-regulated, with the bacteriocin locus controlling the induction of the prophage and thus its activity as a virulence-associated molecular switch. More specifically, a metalloprotease encoded in the bacteriocin locus is upregulated in response to stress and acts as an anti-repressor for CI-like repressors encoded in each phage element. Our results provide molecular insight into the phenomenon of polylysogeny and its intricate adaptation to complex environments.

Summary Some Listeria monocytogenes ( Lm ) strains harbor a prophage within the comK gene, which renders it inactive. During Lm infection of macrophage cells, the prophage turns into a molecular switch, promoting comK gene expression and therefore Lm intracellular growth. During this process, the prophage does not produce infective phages or cause bacterial lysis, suggesting it has acquired an adaptive behavior suited to the pathogenic lifestyle of its host. In this study, we demonstrate that this non-classical phage behavior, named active lysogeny, relies on a transcriptional response that is specific to the intracellular niche. While the prophage undergoes lytic induction, the process is arrested midway, preventing the transcription of the late genes. Further, we demonstrate key phage factors, such as LlgA transcription regulator and a DNA replicase, that support the phage adaptive behavior. This study provides molecular insights into the adaptation of phages to their pathogenic hosts, uncovering unusual cooperative interactions.

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. monocytogenes. IMPORTANCE L. monocytogenes is an opportunistic intracellular pathogen and a widely studied model organism. An efficient counterselection marker is a longstanding 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.