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. 2023 Sep 28;89(9):e0062323.
doi: 10.1128/aem.00623-23. Epub 2023 Sep 5.

Diverse Durham collection phages demonstrate complex BREX defense responses

Affiliations

Diverse Durham collection phages demonstrate complex BREX defense responses

Abigail Kelly et al. Appl Environ Microbiol. .

Abstract

Bacteriophages (phages) outnumber bacteria ten-to-one and cause infections at a rate of 1025 per second. The ability of phages to reduce bacterial populations makes them attractive alternative antibacterials for use in combating the rise in antimicrobial resistance. This effort may be hindered due to bacterial defenses such as Bacteriophage Exclusion (BREX) that have arisen from the constant evolutionary battle between bacteria and phages. For phages to be widely accepted as therapeutics in Western medicine, more must be understood about bacteria-phage interactions and the outcomes of bacterial phage defense. Here, we present the annotated genomes of 12 novel bacteriophage species isolated from water sources in Durham, UK, during undergraduate practical classes. The collection includes diverse species from across known phylogenetic groups. Comparative analyses of two novel phages from the collection suggest they may be founding members of a new genus. Using this Durham phage collection, we determined that particular BREX defense systems were likely to confer a varied degree of resistance against an invading phage. We concluded that the number of BREX target motifs encoded in the phage genome was not proportional to the degree of susceptibility. IMPORTANCE Bacteriophages have long been the source of tools for biotechnology that are in everyday use in molecular biology research laboratories worldwide. Phages make attractive new targets for the development of novel antimicrobials. While the number of phage genome depositions has increased in recent years, the expected bacteriophage diversity remains underrepresented. Here we demonstrate how undergraduates can contribute to the identification of novel phages and that a single City in England can provide ample phage diversity and the opportunity to find novel technologies. Moreover, we demonstrate that the interactions and intricacies of the interplay between bacterial phage defense systems such as Bacteriophage Exclusion (BREX) and phages are more complex than originally thought. Further work will be required in the field before the dynamic interactions between phages and bacterial defense systems are fully understood and integrated with novel phage therapies.

Keywords: BREX; autographiviridae; bacteriophage; phage defense; phylogenetics.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig 1
Fig 1
The Durham collection phages represent diverse species. Phylogenetic analysis of the relatedness of the selected Durham phages shows the phages belong to multiple independent species. Mak and Bam could not be classified at this point. Scale represents the genomic distance scores (SG) calculated by tBLASTx.
Fig 2
Fig 2
Electron microscopy of representative Durham phages. Transmission electron micrographs of representative phages from each group. TB34, Baz, Alma, and Pau represent families with myoviral morphologies, having contractile tails. Baseplate and tail fibers are visible for TB34. BB1 samples showed great variation in phage particles, with either filled heads and short tails of commonly expected length or empty phage heads connected to vastly extended tails (Fig. S2). Jura and Mak have short tails and represent podoviral families, with tail fibers clearly visible for Jura. BB1, Mav, and Sip represent groups with siphoviral morphology, having flexible tails. All Mav and Sip particles visualized had empty capsids. Scale bars represent 100 nm.
Fig 3
Fig 3
Durham phages are spread throughout class Caudoviricetes. The Durham phages were compared to representative Caudoviricetes phages, the T phages and Lambda. The Durham phages are spread across the diversity of Caudoviricetes, with Trib and Baz, CS16 and Mav, and Mak and Bam clustering together. Again, Mak and Bam were unlike any of the sampled representative genomes. The tree scale reported refers to the branch length metadata of the tree. Similarly, an internal scale of the tree, based on branch length values, is also shown. Colored strips indicate the taxonomy of each branch at the family level.
Fig 4
Fig 4
Circular representation of the Mak genome (A), with predicted CDS annotations (B). The genome of Mak is approximately 40 kb, with 47% GC content, and with no predicted tRNA genes. Predicted CDS were assigned a function based on similarity to homologs in the reference database. NSPP, No Significant Phage Protein.
Fig 5
Fig 5
Mak and Bam are distinct from other Autographviridae. VIPTree was used to build a phylogenetic tree including Mak and Bam, based on protein homology with selected members of Autographiviridae. Mak and Bam cluster together but away from other sub-families within Autographiviridae. An internal scale of the tree, based on branch length values, is also shown. Colored strips indicate the taxonomy of each branch to sub-family level.
Fig 6
Fig 6
Mak and Bam are distinct within their closest subfamily and Genus. VIPTree was used again to try and determine the relationship of Mak and Bam to other members of their expected (A) subfamily, Studiervirinae and (B) genus, Chatterjeevirus. An internal scale of the tree, based on branch length values, is also shown.
Fig 7
Fig 7
Durham collection phages show diverse responses to BREX systems. (A) BREX loci were used in this study. (B) Locations of the respective target BREX sites for BREXEferg (GCTA A T), BREXEcoli (GGTA A G), and BREXStyariAΔariB (GATC A G) in the phage genomes, combining locations on the forward and reverse strands. For a plot showing separate strands, see Fig. S4. Numbers give the total number of hits for the respective motif in each phage genome. (C) EOPs of the 12 Durham phages in this study against the three BREX systems. For full data see Table S7.

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