Phage selection drives resistance-virulence trade-offs in Ralstonia solanacearum plant pathogenic bacterium irrespective of the growth temperature

While temperature has been shown to affect the survival and growth of
bacteria and their phage parasites, it is unclear if trade-offs between
phage resistance and other bacterial traits depend on the temperature.
Here, we experimentally compared the evolution of phage
resistance-virulence trade-offs and underlying molecular mechanisms in
phytopathogenic Ralstonia solanacearum bacterium at 25 °C and 35 °C
temperature environments. We found that experimental growth conditions
selected for small colony variants (SCVs) with increased growth rate and
mutations in the quorum-sensing (QS) signalling receptor gene, phcS.
Interestingly, SCVs were also phage-resistant and reached higher
frequencies in the presence of phages in both temperature environments.
Evolving phage resistance was costly in terms of reduced carrying
capacity, biofilm formation and reduced virulence in planta possibly due
to loss of QS-mediated expression of key virulence genes. We also observed
mucoid phage-resistant colonies that showed loss of virulence and reduced
twitching motility likely due to parallel mutations in prepilin peptidase
gene pilD. Moreover, phage-resistant SCVs from 35 °C-phage treatment had
parallel mutations in genes encoding type II secretion system (T2SS) genes
(gspE and gspF), indicating that defects in pseudopilus made bacterium
resistant to the phage. Additional transcriptomic analysis revealed
upregulation of CBASS and type Ⅰ restriction-modification phage defence
systems in response to phage exposure, which coincided with reduced
expression of motility and virulence-associated genes, including pilD and
type II and III secretion systems. Together, these results suggest that
phage resistance-virulence trade-offs are not affected by the growth
temperature but can be mediated through both pre- and post-infection phage
resistance mechanisms.