Biological Consequences of Atypical Phage Conversion in Gram-Positive Pathogens

Temperate bacteriophage have a complex, dynamic relationship with bacteria: parasitizing in the lytic cycle, but often increasing bacteria’s fitness as lysogens. The phage-bacteria relationship is vast and has evolved over more than an estimated three billion years, and there are likely many uncharacterized, intricate events between host and phage with important impacts on bacterial pathogenesis. This Thesis explores some of these lesser-studied phage-bacteria interactions, describing atypical mechanisms (“conversion events”) by which phage shape the populations of Bacillus anthracis and Staphylococcus aureus, driving their increased diversity and likely impacting their natural behaviors. In B. anthracis, phage contributions to virulence are largely unknown. The first part of this Thesis describes how an induced phage from a highly virulent, B. anthracis-like isolate affects the well-characterized strain Sterne and selects for a phage-resistant variant with a markedly altered phenotype, but with no apparent difference in virulence potential. In this work, we characterize this variant strain by a variety of techniques, including whole-genome DNA and RNA-sequencing. In addition, we connect the Sterne variant phenotype to that of the phage’s parent strain, B. cereus Biovar anthracis CA, uncovering lytic phage-bacteria interactions (i.e., selection by lysis) that may act to promote phenotypic diversity and shape populations of B. anthracis and B. anthracis-like pathogenic species in the wild. Unlike B. anthracis, S. aureus has well-characterized bacteriophage contributions to its virulence potential, with known lysogens carrying virulence factors stably integrated into the host chromosome. The second part of this Thesis describes an extra-chromosomal DNA sequencing screening that uncovers the presence of episomal prophages in a number of S. aureus clinical isolates. QPCR characterization of one of these strains, MSSA476, reveals that the episomal nature of one of its prophages, ɸSa4ms, would have been missed if sequencing whole genomic and not specifically extra-chromosomal DNA. In addition, we find that ɸSa4ms excision into the cytoplasm is a temporal event, and that the prophage does not appear to undergo lytic cycle replication after excision—suggesting that its excision is part of a lysogenic switch. Follow-up experiments show that ɸSa4ms excision can alter expression of htrA2 and promote increased heat-stress tolerance. This work suggests that for S. aureus, in addition to carrying important virulence determinants, phage may also play a rather widespread role as DNA-level switches to control virulence factor expression and/or generate distinct subpopulations. While this Thesis discusses atypical phage conversion events, it also illustrates perhaps the most important, universal role of phage in bacterial pathogens: tools to create diversity and allow for bacteria’s increased infection and success under different evolutionary selections and environmental conditions.