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Rhs elements are some of the most positively selected genes known and were recently found to function as type II TA-systems in addition to their previous role as delivered toxins. Antitoxins of type II TA systems are often inherently unstable proteins. Here we discover that the RhsI immunity proteins in Salmonella enterica serovar Typhimurium are also unstable on their own, but stabilized in the presence of their cognate toxins. This raises interesting questions regarding how such unstable immunity proteins can protect against incoming toxins delivered by neighboring attackers. In this study, we observed that one Rhs immunity protein can protect against more than one toxin. Therefore, the internal expression of RhsCT-I could be important for protection against delivered Rhs toxins in addition to regulating Salmonellagrowth in macrophages. The internal transcripts of rhsCT-I are upregulated in InSPI-2 and within macrophages by the combined action of RpoS, PhoP/Q and H-NS. These are the same regulators that control the expression of the type 6 secretion system known to deliver Rhs effectors in Salmonella. This suggests that expression of rhsCT-I is elevated under conditions that favor delivery, which further supports a role for the internal expression of Rhs proteins in protection against toxin delivery. 

Diarrheal diseases still present a large problem for human kind, causing roughly half a million deaths annually. To cause infection, bacterial pathogens must first colonize the gut, a niche that is already occupied by the normal bacterial flora. The ability for a pathogenic strain to inhibit the growth of other bacteria can facilitate the colonization process. Although Enterotoxigenic E. coli (ETEC) is one of the major causative agents of diarrheal disease, very little is known about the competition systems found in and used by ETEC. Here we perform a comprehensive search for bacteriocins, type 6 secretion and contact-dependent growth inhibition systems in all ETEC strains found in the NCBI database. We discover a set of closely related strains, isolated from different geographical regions over several decades, all contain colicin Ia. Using at least one representative strain from each of the 8 ETEC families commonly associated with childhood diarrhea, we demonstrate that colicin Ia is expressed in these strains, allowing them to inhibit the growth of laboratory strains as well as commensal normal flora E. coli in vitro. In addition, we identify T6SS in 50% of all ETEC isolates found in the NCBI database. We observe that he most prevalent T6SS is active in the presence of bile salts, suggesting a role of this system within the host. We discovered that T6SS are under strong positive selection and we find evidence of frequent horizontal gene transfer of the T6SS loci. In summary, we demonstrate that bacterial toxin delivery systems and the ability to compete with other bacteria is important for ETEC during some parts of its lifecycle. 

Antimicrobial resistance (AMR) is an increasing problem worldwide, and new treatment options for bacterial infections are direly needed. Engineered probiotics show strong potential in treating or preventing bacterial infections. However, one concern with the use of live bacteria is the risk of the bacteria acquiring genes encoding for AMR or virulence factors through horizontal gene transfer (HGT), and the transformation of the probiotic into a superbug. Therefore, we developed an engineered CRISPR-Cas9 system that protects bacteria from horizontal gene transfer. We synthesized a CRISPR locus targeting eight AMR genes and cloned this with the Cas9 and transacting tracrRNA on a medium copy plasmid. We next evaluated the efficiency of the system to block HGT through transformation, transduction, and conjugation. Our results show that expression of the CRISPR-Cas9 system successfully protects E. coli MG1655 from acquiring the targeted resistance genes by transformation or transduction with 2–3 logs of protection depending on the system for transfer and the target gene. Furthermore, we show that the system blocks conjugation of a set of clinical plasmids, and that the system is also able to protect the probiotic bacterium E. coli Nissle 1917 from acquiring AMR genes.