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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.

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As human exploration missions to Mars are on the horizon, microbial cross-contamination remains a key issue to address. These issues can be approached today using advances in molecular metagenomics methods, which include rapid and sensitive sequencing platforms for characterizing microbial populations. Combined with analog missions, these methods provide powerful tools for assessing the challenges associated with planetary exploration. Here, we designed a protocol to monitor forward and backward contamination events and progression in an 11-days Mars analog mission in the Ramon crater in Israel. Forward contamination soil samples were collected daily from three sites–two sites in close proximity to the habitat and one isolated site. Backward contamination was determined in samples from nitrile gloves of six analog astronauts before and after extravehicular activities Temperature, relative humidity and soil composition data were also collected for all sites. Environmental DNA samples were extracted in the main habitat and 16S (bacterial) and 18S (eukaryotic, fungal) rRNA gene amplicons were sequenced and analyzed to study microbial population diversity and composition. Shannon Diversity index analysis and Principal Coordinates analysis (PCoA) of rRNA genes indicated that differences in the diversity and population composition were significant in sites closer to the habitat when compared to a reference site. These samples also demonstrated the introduction of human-associated taxa to the environment. Backward contamination consisted of bacterial taxa found on gloves upon return from EVA and also detected in soil, altogether 44 genera, indicating backward contamination events. To our knowledge, this is the first protocol to utilize advanced molecular technologies to investigate forward and backward contamination in a Mars analog mission.