Stx-phages are responsible for the horizontal transmission of Shiga toxin-encoding genes between bacteria, and can enter the lysogenic cycle in their host cells before being induced into lytic replication cycle. The size of most Stx-phage genomes is ~50% > λ phage genome. Due to the ability of prophages to carry and introduce additional genetic material to the host cell, Stx-phages can confer traits on their bacterial lysogen. This research focuses on identifying the function of a hypothetical gene, vb_24B_13c, from Stx-phages Φ24B [GenBank: HM 208303] towards the fitness of its bacterial host. In order to ascertain the function of it, multiple approaches including RNA-seq, NanoString datasets, bioinformatics analyses, and motility tests were used to compare phenotypes of various strains: Naïve (Escherichia coli MC1061), lysogen (MC1061/Φ24B::Cat), and naïve carrying an arabinose inducible plasmid (MC1061 pBAD_13c).

Motility tests were conducted on semi-solid agar plates at different temperatures. With one drop of bacterial culture on the centre, plates were incubated for 12 hours at 37°C or over 24 hours at 25°C or 30°C. Results showed that motility of MC1061/Φ24B::Cat (m = 6.85 cm) was enhanced compared to MC1061 naïve cell (5.0 cm), at both 37℃ and 30℃, suggesting prophage Φ24B contributes to the motility of its host. Similarly, the motility of MC1061 pBAD_13c induced by 0.5%, 1.0% or 2.0% arabinose (m > 9.0 cm) was enhanced at 37℃ compared to the naïve cell, indicating prophage gene vb_24B_13c can contribute to the motility of naïve cells even without the presence of other prophage genes. However, there was no enhanced motility of MC1061 pBAD_13c at 30℃ with arabinose, suggesting that temperature is a factor that affects the host’s motility.

RNA-seq data, as validated by an amylase assay using DNS Reducing Sugar Reagent, showed that α-amylase activity is upregulated in the lysogen. Moreover, NanoString data revealed that overexpression of prophage repressor gene, cI, could potentially inhibit the expression of other prophage genes, including vb_24B_13c, res and stk. This indicates that the lysogen’s expression of cI is not high enough to repress these prophage genes.

In conclusion, the expression of prophage vb_24B_13c can enhance the motility of its host cell by upregulating genes involved in flagellar synthesis and rotation, and it is able to inhibit the expression of some its downstream genes, such as res and stk. Moreover, these results confirm that the prophage plays a significant role in reprogramming the metabolic function of the host, indicating a profound influence on the host's phenotype.