The emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. Here we report the development of a series of CRISPR-Cas13a-based antibacterial nucleocapsids, termed Cap-sidCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus by recognizing corresponding antimicrobial resistance genes. CapsidCas13a constructs are generated by packaging programmed CRISPR-Cas13a into a bacteriophage capsid to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the CapsidCas13a(s) exhibit strong bacterial killing activities upon recognizing target genes regardless of their location. Moreover, we also demonstrate that the CapsidCas13a(s) can be applied to detect bacterial genes through gene-specific depletion of bacteria without employing nucleic acid manipulation and optical visualization devices. Our data underscore the potential of CapsidCas13a(s) as both therapeutic agents against antimicrobial-resistant bacteria and nonchemical agents for detection of bacterial genes.

The arms race between (bacterio)phages and their hosts are recognised hotspots for genome evolution. Indeed, phages and their components have historically paved the way for many molecular biology techniques and biotech applications. Further exploration into their complex lifestyles has revealed that phages are often parasitised by distinct types of hyperparasitic mobile genetic elements. These so-called phage satellites exploit phages to ensure their own propagation and horizontal transfer into new bacterial hosts, and their prevalence and peculiar lifestyle has caught the attention of many researchers. Here, we review the parasite-host dynamics of the known phage satellites, their genomic organisation, and their hijacking mechanisms. Finally, we discuss how these elements can be repurposed for diverse biotech applications, kindling a new catalogue of exciting tools for microbiology and synthetic biology.

It is commonly assumed that the horizontal transfer of most bacterial chromosomal genes is limited, in contrast to the frequent transfer observed for typical mobile genetic elements. However, this view has been recently challenged by the discovery of lateral transduction in Staphylococcus aureus, where temperate phages can drive the transfer of large chromosomal regions at extremely high frequencies. Here, we analyse previously published as well as new datasets to compare horizontal gene transfer rates mediated by different mechanisms in S. aureus and Salmonella enterica. We find that the horizontal transfer of core chromosomal genes via lateral transduction can be more efficient than the transfer of classical mobile genetic elements via conjugation or generalized transduction. These results raise questions about our definition of mobile genetic elements, and the potential roles played by lateral transduction in bacterial evolution.

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Phage-inducible chromosomal islands (PICIs) are a widespread family of highly mobile genetic elements that disseminate virulence and toxin genes among bacterial populations. Since their life cycle involves induction by helper phages, they are important players in phage evolution and ecology. PICIs can interfere with the lifecycle of their helper phages at different stages resulting frequently in reduced phage production after infection of a PICI-containing strain. Since phage defense systems have been recently shown to be beneficial for the acquisition of exogenous DNA via horizontal gene transfer, we hypothesized that PICIs could provide a similar benefit to their hosts and tested the impact of PICIs in recipient strains on host cell viability, phage propagation and transfer of genetic material. Here we report an important role for PICIs in bacterial evolution by promoting the survival of phage-mediated transductants of chromosomal or plasmid DNA. The presence of PICIs generates favorable conditions for population diversification and the inheritance of genetic material being transferred, such as antibiotic resistance and virulence genes. Our results show that by interfering with phage reproduction, PICIs can protect the bacterial population from phage attack, increasing the overall survival of the bacterial population as well as the transduced cells. Moreover, our results also demonstrate that PICIs reduce the frequency of lysogenization after temperate phage infection, creating a more genetically diverse bacterial population with increased bet-hedging opportunities to adapt to new niches. In summary, our results identify a new role for the PICIs and highlight them as important drivers of bacterial evolution.

Phage-inducible chromosomal islands (PICIs) are a widespread family of mobile genetic elements, which have an important role in bacterial pathogenesis. These elements mobilize among bacterial species at extremely high frequencies, representing an attractive tool for the delivery of synthetic genes. However, tools for their genetic manipulation are limited and timing consuming. Here, we have adapted a synthetic biology approach for rapidly editing of PICIs in Saccharomyces cerevisiae based on their ability to excise and integrate into the bacterial chromosome of their cognate host species. As proof of concept, we engineered several PICIs from Staphylococcus aureus and Escherichia coli and validated this methodology for the study of the biology of these elements by generating multiple and simultaneous mutations in different PICI genes. For biotechnological purposes, we also synthetically constructed PICIs as Trojan horses to deliver different CRISPR-Cas9 systems designed to either cure plasmids or eliminate cells carrying the targeted genes. Our results demonstrate that the strategy developed here can be employed universally to study PICIs and enable new approaches for diagnosis and treatment of bacterial diseases.

Emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. We established a series of CRISPR-Cas13a-based antimicrobials, termed PhagoCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus through promiscuous RNA cleavage after recognizing corresponding antimicrobial resistance genes. PhagoCas13a constructs were generated by packaging CRISPR-Cas13a into a bacteriophage to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the PhagoCas13a(s) exhibited strong bacterial killing activities upon recognizing target genes regardless of their location. The antimicrobials' treatment efficacy was confirmed using a Galleria mellonella larvae model. Further, we demonstrated that the PhagoCas13a(s) can assist in bacterial gene detection without employing nucleic acid amplification and optical devices.

Phage‐inducible chromosomal islands (PICIs) are a family of phage satellites that hijack phage components to facilitate their mobility and spread. Recently, these genetic constructs are repurposed as antibacterial drones, enabling a new toolbox for unorthodox applications in biotechnology. To illustrate a new suite of functions, the authors have developed a user‐friendly diagnostic system, based upon PICI transduction to selectively enrich bacteria, allowing the detection and sequential recovery of Escherichia coli and Staphylococcus aureus. The system enables high transfer rates and sensitivities in comparison with phages, with detection down to ≈50 CFU mL−1. In contrast to conventional detection strategies, which often rely on nucleic acid molecular assays, and cannot differentiate between dead and live organisms, this approach enables visual sensing of viable pathogens only, through the expression of a reporter gene encoded in the PICI. The approach extends diagnostic sensing mechanisms beyond cell‐free synthetic biology strategies, enabling new synthetic biology/biosensing toolkits.