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.

Mucin induces CRISPR-Cas defence in an opportunistic pathogen

Parasitism by bacteriophages has led to the evolution of a variety of defense mechanisms in their host bacteria. However, it is unclear what factors lead to specific defenses being deployed upon phage infection. To explore this question, we exposed the bacterial fish pathogen Flavobacterium columnare to its virulent phage V156 in the presence of a eukaryotic host signal (mucin). All tested conditions led to some level of innate immunity, but the presence of mucin led to a dramatic increase in CRISPR spacer acquisition, especially in low nutrient conditions where over 60% of colonies had obtained at least one new spacer. Additionally, we show that the presence of a competitor bacterium further increases CRISPR spacer acquisition in F. columnare. These results suggest that ecological factors are important in determining defense strategies against phages, and that the concentration of phages on metazoan surfaces may select for the diversification of bacterial immune systems.

Hundreds of viral families in the healthy infant gut

The gut microbiome (GM) is shaped through infancy and plays a major role in determining susceptibility to chronic inflammatory diseases later in life. Bacteriophages (phages) are known to modulate bacterial populations in numerous ecosystems, including the gut. However, virome data is difficult to analyse because it mostly consists of unknown viruses, i.e. viral dark matter. Here, we manually resolved the viral dark matter in the largest human virome study published to date. Fecal viromes from a cohort of 647 infants at 1 year of age were deeply sequenced and analysed through successive rounds of clustering and curation. We uncovered more than ten thousand viral species distributed over 248 viral families falling within 17 viral order-level clades. Most of the defined viral families and orders were novel and belonged to the Caudoviricetes viral class. Bacterial hosts were predicted for 79% of the viral species using CRISPR spacers, including those in metagenomes from the same fecal samples. While Bacteroides-infecting Crassphages were present, novel viral families were more predominant, including phages infecting Clostridiales and Bifidobacterium. Phage lifestyles were determined for more than three thousand caudoviral species. Lifestyles were homogeneous at the family level for 149 Caudoviricetes families, including 32 families that were found to be virulent, while 117 were temperate. Virulent phage families were more abundant but temperate ones were more diverse and widespread. Together, the viral families found in this study represent a major expansion of existing bacteriophage taxonomy.

Manual resolution of virome dark matter uncovers hundreds of viral families in the infant gut

The gut microbiome (GM) is shaped through infancy and plays a major role in determining susceptibility to chronic diseases later in life. Bacteriophages (phage) are known to modulate bacterial populations in numerous ecosystems, including the gut. However, virome data is difficult to analyse because it mostly consists of unknown viruses, i.e. viral dark matter. Here, we manually resolved the viral dark matter in the largest human virome study published to date. Fecal viromes from a cohort of 647 infants at 1 year of age were deeply sequenced and analysed through successive rounds of clustering and curation. This uncovered more than ten thousand viral species distributed over 248 viral families falling within 17 viral order-level clusters. Most of the defined viral families and orders were novel and belonged to the Caudoviricetes viral class. Bacterial hosts were predicted for 79% of the viral species using CRISPR spacers in metagenomes from the same fecal samples. While Bacteroides-infecting Crassphages were present, novel viral families were more predominant, including phages infecting Clostridiales and Bifidobacterium. Phage lifestyles were determined for more than three thousand caudoviral species. Lifestyles were homogeneous at the family level for 149 caudiviral families. 32 families were found to be virulent, while 117 families were temperate. Virulent phage families were more abundant but temperate phage families were more diverse and widespread. Together, the viral families found in this study represent a major expansion of current bacteriophage taxonomy, and the sequences have been put online for use and validation by the community.

Equine intestinal O-seroconverting temperate coliphage Hf4s: genomic and biological characterization

Tailed bacteriophages constitute the bulk of the intestinal viromes of the vertebrate animals. However, the relationships between lytic and lysogenic lifestyles of the phages in these ecosystems are not always clear and may vary between the species or even between the individuals. The human intestinal (fecal) viromes are believed to be dominated by temperate phages, while in the horse feces the virulent phages are more prevalent. Almost all the isolates of horse fecal coliphages are virulent. Phage Hf4s is the first temperate equine intestinal coliphage characterized. It was isolated from the horse feces on the indigenous equine E. coli 4s strain. It is a podovirus, related to Lederbergvirus genus (including the well-characterized Salmonella phage P22). Hf4s recognizes the host O antigen as its primary receptor and possesses a functional O-antigen seroconversion cluster that renders the lysogens protected from the superinfection by the same phage and also abolishes the adsorption of some indigenous equine virulent coliphages, such as DT57C, while the other phages, such as G7C or phiKT retain the ability to infect E. coli 4s (Hf4s) lysogens.

Primed CRISPR-Cas Adaptation and Impaired Phage Adsorption in Streptococcus mutans

ABSTRACT Streptococcus mutans strain P42S possesses a type II-A CRISPR-Cas system that protects against phage infection and plasmid transformation. The analysis of 293 bacteriophage-insensitive mutants (BIMs) obtained upon exposure to the virulent phage M102AD revealed the acquisition of 399 unique spacers, including several ectopic spacer acquisitions and a few cases of native spacer deletions. The acquisition of multiple spacers was also observed and appears to be mostly due to priming, which has been rarely reported for type II-A systems. Analyses of the acquired spacers indicated that 88% of them are identical to a region of the phage M102AD genome. The remaining 12% of spacers had mismatches with the phage genome, primarily at the 5′ end of the spacer, leaving the seed sequence at the 3′ end largely intact. When a high multiplicity of infection (MOI) was used in the phage challenge assays, we also observed the emergence of CRISPR BIMs that, in addition to the acquisition of new spacers, displayed a reduced phage adsorption phenotype. While CRISPR-Cas and adsorption resistance work in tandem to protect S. mutans P42S against phage M102AD, nonidentified antiviral mechanisms are also likely at play in this strain. IMPORTANCE Bacteria are under the constant threat of viral predation and have therefore developed several defense mechanisms, including CRISPR-Cas systems. While studies on the mode of action of CRISPR-Cas systems have already provided great insights into phage-bacterium interactions, still more information is needed on the biology of these systems. The additional characterization of the type II-A CRISPR-Cas system of Streptococcus mutans P42S in this study provides novel information on the spacer acquisition step, especially regarding protospacer-adjacent motif (PAM) recognition, multiple-spacer acquisition, and priming.

Characterization of the First Virulent Phage Infecting Oenococcus oeni, the Queen of the Cellars

There has been little exploration of how phages contribute to the diversity of the bacterial community associated with winemaking and may impact fermentations and product quality. Prophages of Oenococcus oeni, the most common species of lactic acid bacteria (LAB) associated with malolactic fermentation of wine, have been described, but no data is available regarding phages of O. oeni with true virulent lifestyles. The current study reports on the incidence and characterization of the first group of virulent oenophages named Vinitor, isolated from the enological environment. Vinitor phages are morphologically very similar to siphoviruses infecting other LAB. Although widespread during winemaking, they are more abundant in musts than temperate oenophages. We obtained the complete genomic sequences of phages Vinitor162 and Vinitor27, isolated from white and red wines, respectively. The assembled genomes shared 97.6% nucleotide identity and belong to the same species. Coupled with phylogenetic analysis, our study revealed that the genomes of Vinitor phages are architecturally mosaics and represent unique combinations of modules amongst LAB infecting-phages. Our data also provide some clues to possible evolutionary connections between Vinitor and (pro)phages associated to epiphytic and insect-related bacteria.