Microbial defenses against mobile genetic elements and viruses: Who defends whom from what?

Prokaryotes have numerous mobile genetic elements (MGEs) that mediate horizontal gene transfer (HGT) between cells. These elements can be costly, even deadly, and cells use numerous defense systems to filter, control, or inactivate them. Recent studies have shown that prophages, conjugative elements, their parasites (phage satellites and mobilizable elements), and other poorly described MGEs encode defense systems homologous to those of bacteria. These constitute a significant fraction of the repertoire of cellular defense genes. As components of MGEs, these defense systems have presumably evolved to provide them, not the cell, adaptive functions. While the interests of the host and MGEs are aligned when they face a common threat such as an infection by a virulent phage, defensive functions carried by MGEs might also play more selfish roles to fend off other antagonistic MGEs or to ensure their maintenance in the cell. MGEs are eventually lost from the surviving host genomes by mutational processes and their defense systems can be co-opted when they provide an advantage to the cell. The abundance of defense systems in MGEs thus sheds new light on the role, effect, and fate of the so-called “cellular defense systems,” whereby they are not only merely microbial defensive weapons in a 2-partner arms race, but also tools of intragenomic conflict between multiple genetic elements with divergent interests that shape cell fate and gene flow at the population level.

Sir2-domain associated short prokaryotic Argonautes provide defence against invading mobile genetic elements through NAD+ depletion

Argonaute (Ago) proteins are found in all three domains of life. The so-called long Agos are composed of four major domains (N, PAZ, MID, and PIWI) and contribute to RNA silencing in eukaryotes (eAgos) or defence against invading mobile genetic elements in prokaryotes (pAgos). Intriguingly, the majority (~60%) of prokaryotic Agos (pAgos) identified bioinformatically are shorter (comprised of only MID and PIWI domains) and are typically associated with Sir2, Mrr or TIR domain-containing proteins. The cellular function and mechanism of short pAgos remain enigmatic. Here, we show that short pAgos from Geobacter sulfurreducens, Caballeronia cordobensis and Paraburkholderia graminis, together with the NAD+-bound Sir2-proteins form a stable heterodimeric Sir2/Ago complex that recognizes invading plasmid or phage DNA through the pAgos subunit and activates Sir2 subunit triggering the endogenous NAD+ depletion and cell death thus preventing the propagation of invading DNA. This is the first demonstration that short Sir2-associated pAgos provide defence against phages and plasmids and underscores the diversity of mechanisms of prokaryotic Agos.

Mobile Genetic Elements Drive Antimicrobial Resistance Gene Spread in Pasteurellaceae Species

Mobile genetic elements (MGEs) and antimicrobial resistance (AMR) drive important ecological relationships in microbial communities and pathogen-host interaction. In this study, we investigated the resistome-associated mobilome in 345 publicly available Pasteurellaceae genomes, a large family of Gram-negative bacteria including major human and animal pathogens. We generated a comprehensive dataset of the mobilome integrated into genomes, including 10,820 insertion sequences, 2,939 prophages, and 43 integrative and conjugative elements. Also, we assessed plasmid sequences of Pasteurellaceae. Our findings greatly expand the diversity of MGEs for the family, including a description of novel elements. We discovered that MGEs are comparable and dispersed across species and that they also co-occur in genomes, contributing to the family’s ecology via gene transfer. In addition, we investigated the impact of these elements in the dissemination and shaping of AMR genes. A total of 55 different AMR genes were mapped to 721 locations in the dataset. MGEs are linked with 77.6% of AMR genes discovered, indicating their important involvement in the acquisition and transmission of such genes. This study provides an uncharted view of the Pasteurellaceae by demonstrating the global distribution of resistance genes linked with MGEs.

The ESKAPE mobilome contributes to the spread of antimicrobial resistance and CRISPR-mediated conflict between mobile genetic elements

Mobile genetic elements (MGEs) mediate the shuffling of genes among organisms. They contribute to the spread of virulence and antibiotic resistance genes in human pathogens, including the particularly problematic group of ESKAPE pathogens, such as Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter sp. Here, we performed the first systematic analysis of MGEs, including plasmids, prophages, and integrative and conjugative/mobilizable elements (ICEs/IMEs), in the ESKAPE pathogens. We characterized over 1700 complete ESKAPE genomes and found that different MGE types are asymmetrically distributed across these pathogens. While some MGEs are capable of exchanging DNA beyond the genus (and phylum) barrier, horizontal gene transfer (HGT) is mainly restricted by phylum or genus. We further observed that most genes on MGEs have unknown functions and show intricate distribution patterns. Moreover, AMR genes and anti-CRISPRs are overrepresented in the ESKAPE mobilome. Our results also underscored species-specific trends shaping the number of MGEs, AMR, and virulence genes across pairs of conspecific ESKAPE genomes with and without CRISPR-Cas systems. Finally, we found that CRISPR targets vary according to MGE type: while plasmid CRISPRs almost exclusively target other plasmids, ICEs/IME CRISPRs preferentially target ICEs/IMEs and prophages. Overall, our study highlights the general importance of the ESKAPE mobilome in contributing to the spread of AMR and mediating conflict among MGEs.

Genome-based characterization of a plasmid-associated micrococcin P1 biosynthetic gene cluster and virulence factors in Mammaliicoccus sciuri IMDO-S72

Analysis of the de novo assembled genome of Mammaliicoccus sciuri IMDO-S72 revealed the genetically encoded machinery behind its earlier reported antibacterial phenotype and gave further insight into the repertoire of putative virulence factors of this recently reclassified species. A plasmid-encoded biosynthetic gene cluster was held responsible for the antimicrobial activity of M. sciuri IMDO-S72, comprising genes involved in thiopeptide production. The compound encoded by this gene cluster was structurally identified as micrococcin P1. Further examination of its genome highlighted the ubiquitous presence of innate virulence factors mainly involved in surface colonization. Determinants contributing to aggressive virulence were generally absent, with exception of a plasmid-associated ica cluster. The native antibiotic resistance genes sal (A) and mecA were detected within the genome, amongst others, but were not consistently linked with a resistant phenotype. While mobile genetic elements were identified within the genome, such as an untypeable SCC element, they proved to be generally free of virulence- and antibiotic-related genes. These results further suggest a commensal lifestyle of M. sciuri and indicate the association of antibiotic resistance determinants with mobile genetic elements, as an important factor in conferring antibiotic resistance, in addition to their unilateral annotation. Importance Mammaliicoccus sciuri has been put forward as an important carrier of virulence and antibiotic resistance genes, which can be transmitted to clinically important staphylococcal species such as Staphylococcus aureus . As a common inhabitant of mammal skin, this species is believed to have a predominant commensal lifestyle although it has been reported as an opportunistic pathogen in some cases. This study provides an extensive genome-wide description of its putative virulence potential taking into consideration the genomic context in which these genes appear, an aspect that is often overlooked during virulence analysis. Additional genome and biochemical analysis linked M. sciuri with the production of micrococcin P1, gaining further insight to which extent these biosynthetic gene cluster are distributed amongst different related species. The frequent plasmid-associated character hints that these traits can be horizontally transferred and might confer a competitive advantage to its recipient within its ecological niche.

Prediction of prophages and their host ranges in pathogenic and commensal Neisseria species

The genus Neisseria includes two pathogenic species, N. gonorrhoeae and N. meningitidis, and numerous commensal species. Neisseria species frequently exchange DNA with one other, primarily via transformation and homologous recombination, and via multiple types of mobile genetic elements (MGEs). Few Neisseria bacteriophages (phages) have been identified and their impact on bacterial physiology is poorly understood. Furthermore, little is known about the range of species that Neisseria phages can infect. In this study, we used three virus prediction tools to scan 248 genomes of 21 different Neisseria species and identified 1302 unique predicted prophages. Using comparative genomics, we found that many predictions are dissimilar from other prophages and MGEs previously described to infect Neisseria species. We also identified similar predicted prophages in genomes of different Neisseria species. Additionally, we examined CRISPR-Cas targeting of each Neisseria genome and predicted prophage. While CRISPR targeting of chromosomal DNA appears to be common among several Neisseria species, we found that 20% of the prophages we predicted are targeted significantly more than the rest of the bacterial genome in which they were identified (i.e., backbone). Furthermore, many predicted prophages are targeted by CRISPR spacers encoded by other species. We then used these results to infer additional host species of known Neisseria prophages and predictions that are highly targeted relative to the backbone. Together, our results suggest that we have identified novel Neisseria prophages, several of which may infect multiple Neisseria species. These findings have important implications for understanding horizontal gene transfer between members of this genus. IMPORTANCE: Drug-resistant N. gonorrhoeae is a major threat to human health. Commensal Neisseria species are thought to serve as reservoirs of antibiotic resistance and virulence genes for the pathogenic species N. gonorrhoeae and N. meningitidis. Therefore, it is important to understand both the diversity of mobile genetic elements (MGEs) that can mediate horizontal gene transfer within this genus, and the breadth of species these MGEs can infect. In particular, few bacteriophages (phages) have been identified and characterized in Neisseria species. In this study, we identified a large number of candidate phages integrated within the genomes of commensal and pathogenic Neisseria species, many of which appear to be novel phages. Importantly, we discovered extensive interspecies targeting of predicted phages by Neisseria CRISPR-Cas systems, which may reflect their movement between different species. Uncovering the diversity and host range of phages is essential for understanding how they influence the evolution of their microbial hosts.

Sir2-domain associated short prokaryotic Argonautes provide defence against invading mobile genetic elements through NAD+ depletion

Argonaute (Ago) proteins are found in all three domains of life. The so-called long Agos are composed of four major domains (N, PAZ, MID, and PIWI) and contribute to RNA silencing in eukaryotes (eAgos) or defence against invading mobile genetic elements in prokaryotes (pAgos). Intriguingly, the majority (~60%) of prokaryotic Agos (pAgos) identified bioinformatically are shorter (comprised of only MID and PIWI domains) and are typically associated with Sir2, Mrr or TIR domain-containing proteins. The cellular function and mechanism of short pAgos remain enigmatic. Here, we show that short pAgos from Geobacter sulfurreducens, Caballeronia cordobensis and Paraburkholderia graminis, together with the NAD+-bound Sir2-proteins form a stable heterodimeric Sir2/Ago complex that recognizes invading plasmid or phage DNA through the pAgos subunit and activates Sir2 subunit triggering the endogenous NAD+ depletion and cell death thus preventing the propagation of invading DNA. This is the first demonstration that short Sir2-associated pAgos provide defence against phages and plasmids and underscores the diversity of mechanisms of prokaryotic Agos.

Bacillus cytotoxicus Genomics: Chromosomal Diversity and Plasmidome Versatility

Bacillus cytotoxicus is the thermotolerant representative of the Bacillus cereus group. This group, also known as B. cereus sensu lato, comprises both beneficial and pathogenic members and includes psychrotolerant and thermotolerant species. Bacillus cytotoxicus was originally recovered from a fatal outbreak in France in 1998. This species forms a remote cluster from the B. cereus group members and reliably contains the cytk-1 gene, coding for a cytotoxic variant of cytotoxin K. Although this species was originally thought to be homogenous, intra-species diversity has been recently described with four clades, six random amplified polymorphic DNA (RAPD) patterns, and 11 plasmids profiles. This study aimed to get new insights into the genomic diversity of B. cytotoxicus and to decipher the underlying chromosomal and plasmidial variations among six representative isolates through whole genome sequencing (WGS). Among the six sequenced strains, four fitted the previously described genomic clades A and D, while the remaining two constituted new distinct branches. As for the plasmid content of these strains, three large plasmids were putatively conjugative and three small ones potentially mobilizable, harboring coding genes for putative leaderless bacteriocins. Mobile genetic elements, such as prophages, Insertion Sequences (IS), and Bacillus cereus repeats (bcr) greatly contributed to the B. cytotoxicus diversity. As for IS elements and bcr, IS3 and bcr1 were the most abundant elements and, along with the group II intron B.c.I8, were found in all analyzed B. cytotoxicus strains. When compared to other B. cytotoxicus strains, the type-strain NVH 391-98 displayed a relatively low number of IS. Our results shed new light on the contribution of mobile genetic elements to the genome plasticity of B. cytotoxicus and their potential role in horizontal gene transfer.

Hierarchical length and sequence preferences establish a single major piRNA 3'-end

PIWI-interacting RNAs (piRNAs) guard germline genomes against the deleterious action of retroviruses and other mobile genetic elements. How piRNAs faithfully discriminate between self and non-self to restrict all mobile elements while sparing essential genes remains a key outstanding question in genome biology. PiRNAs use extensive base-pairing to recognize their targets and variable 3'ends could change the specificity and efficacy of piRNA silencing. Here, we identify conserved rules that ensure the generation of a single major piRNA 3'end in flies and mice. Our data suggest that the PIWI proteins initially define a short interval on pre-piRNAs that grants access to the ZUC-processor complex. Within this Goldilocks zone, the preference of the ZUC-processor to cut in front of a Uridine determines the ultimate processing site. We observe a mouse-specific roadblock that relocates the Goldilocks zone and generates an opportunity for consecutive trimming by PNLDC1. Our data reveal a conserved hierarchy between length and sequence preferences that controls the piRNA sequence space. The unanticipated precision of 3'end formation bolsters the emerging understanding that the functional piRNA sequence space is tightly controlled to ensure effective defense.