Virulence-associated factors as targets for phage infection.

Bacterial pathogens can infect a wide range of hosts and pose a threat to public and animal health as well as to agriculture. The emergence of antibiotic-resistant strains has increased this risk by making the treatment of bacterial infections even more challenging. Pathogenic bacteria thrive in various ecological niches, but they can also be specifically targeted and killed by bacteriophages (phages). Lytic phages are now investigated and even used, in some cases, as alternatives or complements to antibiotics for preventing or treating bacterial infections (phage therapy). As such, it is key to identify factors responsible for phage specificity and efficiency. Here, we review recent advances in virulence-associated factors that are targeted by phages. We highlight components of the bacterial cell surface, effector systems, and motility structures exploited by phages and the effects of phages on cell aggregation and communication. We also look at the fitness trade-off of phage resistance.

The impact of Brevibacterium aurantiacum virulent phages on the production of smear surface-ripened cheeses.

Phages are ubiquitous and are particularly abundant in environments where their bacterial hosts thrive, such as those in the cheese industry. Although it is well documented that phages infect lactic acid bacteria, their impact has been notably overlooked on cheese ripening strains, such as Brevibacterium aurantiacum. Here, we aimed to study the impact of B. aurantiacum phages on the production of smear-ripened cheeses. We used model cheeses in industrial settings to monitor the development of the color of the cheese rind as well as of its microbial composition in presence or absence of virulent B. aurantiacum phages. Our results showed that the presence of B. aurantiacum phages significantly slowed down the development of the orange rind color in the model cheeses. In the final days of cheese ripening, phages were also detected in the control curds. By analyzing a hypervariable region of B. aurantiacum phage genomes, we detected phages with tandem repeat patterns that were different from those used in the phage-inoculated cheeses. Our results highlight the risks of using a phage-sensitive strain in smear-ripened cheese production. This is the first study to report on the impact of B. aurantiacum phages on smear-ripened cheeses.

The never-ending battle between lactic acid bacteria and their phages.

Over the past few decades, the interest in lactic acid bacteria (LAB) has been steadily growing. This is mainly due to their industrial use, their health benefits as probiotic bacteria and their ecological importance in host-related microbiota. Phage infection represents a significant risk for the production and industrial use of LAB. This created the need to study the various means of defense put in place by LAB to resist their viral enemies, as well as the countermeasures evolved by phages to overcome these defenses. In this review, we discuss defense systems that LAB employ to resist phage infections. We also describe how phages counter these mechanisms through diverse and sophisticated strategies. Furthermore, we discuss the way phage-host interactions shape each other's evolution. The recent discovery of numerous novel defense systems in other bacteria promises a new dawn for phage research in LAB.

In through the Out Door: A Functional Virulence Factor Secretion System Is Necessary for Phage Infection in Ralstonia solanacearum.

Bacteriophages put intense selective pressure on microbes, which must evolve diverse resistance mechanisms to survive continuous phage attacks. We used a library of spontaneous Bacteriophage Insensitive Mutants (BIMs) to learn how the plant pathogen Ralstonia solanacearum resists the virulent lytic podophage phiAP1. Phenotypic and genetic characterization of many BIMs suggested that the R. solanacearum Type II Secretion System (T2SS) plays a key role in phiAP1 infection. Using precision engineered mutations that permit T2SS assembly but either inactivate the T2SS GspE ATPase or sterically block the secretion portal, we demonstrated that phiAP1 needs a functional T2SS to infect R. solanacearum. This distinction between the static presence of T2SS components, which is necessary but not sufficient for phage sensitivity, and the energized and functional T2SS, which is sufficient, implies that binding interactions alone cannot explain the role of the T2SS in phiAP1 infection. Rather, our results imply that some aspect of the resetting of the T2SS, such as disassembly of the pseudopilus, is required. Because R. solanacearum secretes multiple virulence factors via the T2SS, acquiring resistance to phiAP1 also dramatically reduced R. solanacearum virulence on tomato plants. This acute fitness trade-off suggests this group of phages may be a sustainable control strategy for an important crop disease. IMPORTANCE Ralstonia solanacearum is a destructive plant pathogen that causes lethal bacterial wilt disease in hundreds of diverse plant hosts, including many economically important crops. Phages that kill R. solanacearum could offer effective and environmentally friendly wilt disease control, but only if the bacterium cannot easily evolve resistance. Encouragingly, most R. solanacearum mutants resistant to the virulent lytic phage phiAP1 no longer secreted multiple virulence factors and had much reduced fitness and virulence on tomato plants. Further analysis revealed that phage phiAP1 needs a functional type II secretion system to infect R. solanacearum, suggesting this podophage uses a novel infection mechanism.

DNA tandem repeats contribute to the genetic diversity of Brevibacterium aurantiacum phages.

This report presents the characterization of the first virulent phages infecting Brevibacterium aurantiacum, a bacterial species used during the manufacture of surface-ripened cheeses. These phages were also responsible for flavour and colour defects in surface-ripened cheeses. Sixteen phages (out of 62 isolates) were selected for genome sequencing and comparative analyses. These cos-type phages with a long non-contractile tail currently belong to the Siphoviridae family (Caudovirales order). Their genome sizes vary from 35,637 to 36,825 bp and, similar to their host, have a high GC content (~61%). Genes encoding for an immunity repressor, an excisionase and a truncated integrase were found, suggesting that these virulent phages may be derived from a prophage. Their genomic organization is highly conserved, with most of the diversity coming from the presence of long (198 bp) DNA tandem repeats (TRs) within an open reading frame coding for a protein of unknown function. We categorized these phages into seven genomic groups according to their number of TR, which ranged from two to eight. Moreover, we showed that TRs are widespread in phage genomes, found in more than 85% of the genomes available in public databases.

Mobilome of Brevibacterium aurantiacum Sheds Light on Its Genetic Diversity and Its Adaptation to Smear-Ripened Cheeses.

Brevibacterium aurantiacum is an actinobacterium that confers key organoleptic properties to washed-rind cheeses during the ripening process. Although this industrially relevant species has been gaining an increasing attention in the past years, its genome plasticity is still understudied due to the unavailability of complete genomic sequences. To add insights on the mobilome of this group, we sequenced the complete genomes of five dairy Brevibacterium strains and one non-dairy strain using PacBio RSII. We performed phylogenetic and pan-genome analyses, including comparisons with other publicly available Brevibacterium genomic sequences. Our phylogenetic analysis revealed that these five dairy strains, previously identified as Brevibacterium linens, belong instead to the B. aurantiacum species. A high number of transposases and integrases were observed in the Brevibacterium spp. strains. In addition, we identified 14 and 12 new insertion sequences (IS) in B. aurantiacum and B. linens genomes, respectively. Several stretches of homologous DNA sequences were also found between B. aurantiacum and other cheese rind actinobacteria, suggesting horizontal gene transfer (HGT). A HGT region from an iRon Uptake/Siderophore Transport Island (RUSTI) and an iron uptake composite transposon were found in five B. aurantiacum genomes. These findings suggest that low iron availability in milk is a driving force in the adaptation of this bacterial species to this niche. Moreover, the exchange of iron uptake systems suggests cooperative evolution between cheese rind actinobacteria. We also demonstrated that the integrative and conjugative element BreLI (Brevibacterium Lanthipeptide Island) can excise from B. aurantiacum SMQ-1417 chromosome. Our comparative genomic analysis suggests that mobile genetic elements played an important role into the adaptation of B. aurantiacum to cheese ecosystems.

Complete Genome Sequence of Brevibacterium linens SMQ-1335.

Brevibacterium linens is one of the main bacteria found in the smear of surface-ripened cheeses. The genome of the industrial strain SMQ-1335 was sequenced using PacBio. It has 4,209,935 bp, a 62.6% G+C content, 3,848 open reading frames, and 61 structural RNAs. A new type I restriction-modification system was identified.