Genomic Sequencing of High-Efficiency Transducing Streptococcal Bacteriophage A25: Consequences of Escape from Lysogeny

Abstract

Lytic bacteriophage A25, which infects and several related species, has been used to better understand phage-microbe interactions due to its ability to mediate high-efficiency transduction. Most of these studies, however, are decades old and were conducted prior to the advent of next-generation sequencing and bioinformatics. The aim of our study was to gain a better understanding of the mechanism of high-efficiency transduction through analysis of the A25 genome. We show here that phage A25 is related to a family of genome prophages and became a lytic phage following escape from lysogeny. A lambdoid-like residual lysogeny module consisting of an operator site with two promoters and a-like antirepressor gene was identified, but the genes for the cI-like repressor and integrase are missing. Additionally, the genetic organization of the A25 genome was found to be modular in nature and similar to that of many prophages of as well as from other streptococcal species. A study of A25 homology to all annotated prophages within revealed near identity within the remnant lysogeny module of the A25 phage genome to the corresponding regions in resident prophages of genome strains MGAS10270 (M2), MGAS315 (M3), MGAS10570 (M4), and STAB902 (M4). Host range studies of MGAS10270, MGAS315, and MGAS10750 demonstrated that these strains were resistant to A25 infection. The resistance mechanism of superinfection immunity was confirmed experimentally through complementation of the operator region and -like repressor from prophage MGAS10270.2 into susceptible strains SF370, CEM1Δ4 (SF370ΔSpyCIM1), and ATCC 12204, which rendered all three strains resistant to A25 infection. prediction of packaging through homology analysis of the terminase large subunit from bacteriophages within the known packaging mechanism of Gram-positive bacteria as well as the evidence of terminally redundant and/or circularly permuted sequences suggested that A25 grouped with phages employing the less stringent -type packaging mechanisms, which likely explains the characteristic A25 high-efficiency transduction capabilities. Only a few examples of lytic phages appearing following loss of part or all of the lysogeny module have been reported previously, and the genetic mosaicism of A25 suggests that this event may not have been a recent one. However, the discovery that this lytic bacteriophage shares some of the genetic pool of prophages emphasizes the importance of genetic and biological characterization of bacteriophages when selecting phages for therapeutics or disinfectants, as phage-phage and phage-microbe interactions can be complex, requiring more than just assessment of host range and carriage of toxoid or virulence genes. Bacteriophages (bacterial viruses) play an important role in the shaping of bacterial populations as well as the dissemination of bacterial genetic material to new strains, resulting in the spread of virulence factors and antibiotic resistance genes. This study identified the genetic origins of phage A25 and uncovered the molecular mechan...

Survival Strategies of Streptococcus pyogenes in Response to Phage Infection

“…Streptococcal phages typically feature a modular genome organization with a clear distinction between regions dedicated to phage structure and host cell modulation [ 49 ]. Recently, two S. pyogenes phage genomes were sequenced where this modular genetic arrangement was observed as well [ 30 , 50 ]. The Phage A1 genome was highly similar to temperate Phage Str01 and virulent Phage A25, with Phage Str01 being its closest relative.…”

“…Lytic S. pyogenes Phages A6, A12, A25 and A27 have a broad host range and are able to infect some strains of Group C Streptococci (GCS), and Phage A25 was shown to be adaptable to Group G Streptococci (GGS) [ 25 , 29 ]. Recently, the genome sequence of Phage A25 has been determined to be 33,900 bp in size with a GC content of 38.44% [ 30 ]. The phages of S. pyogenes comprise a modular genetic arrangement with regions dedicated to gene regulation, DNA replication, DNA cleavage, DNA packaging, capsid and tail morphogenesis and lysis.…”

“…The phages of S. pyogenes comprise a modular genetic arrangement with regions dedicated to gene regulation, DNA replication, DNA cleavage, DNA packaging, capsid and tail morphogenesis and lysis. In contrast to most prophages of S. pyogenes , however, no identifiable virulence genes were found in the genome of Phage A25 [ 30 ]. Genomic analysis of the Phage A25 and prophage genomes provided hints towards the broad host range of S. pyogenes phages.…”

“…Genomic analysis of the Phage A25 and prophage genomes provided hints towards the broad host range of S. pyogenes phages. A highly mosaic nature of these genomes was identified, and genetic modules were shown to be shared between phages of S. pyogenes , Streptococcus pneumoniae and Streptococcus suis , among others [ 30 ].…”

Integration of prophages into CRISPR loci remodels viral immunity inStreptococcus pyogenes

“…In S. pyogenes this process contributes to the spread of the virulence genes carried by the temperate phages (Fischetti, 2007). In addition, inaccurate prophage excision and/or DNA packaging during induction can lead to the transduction of bacterial sequences into new hosts (McCullor et al, 2018).…”