Phage proofing Pseudomonas putida uncovers novel broad-spectrum phage resistance protein Psh

Broad-spectrum phage resistance offers an important layer of protection against bacterial fermenter crashes during biomanufacturing processes, yet the underlying mechanisms are often poorly defined or come at a fitness cost. Using experimental evolution, we generated two Pseudomonas putida strains that were resistant to at least six phage genera. Genome sequencing revealed a single frameshift deletion in each strain that restored functionality of a Type I secretion system (T1SS) ATPase. We also found strong transcriptional upregulation of a nearby protein, PP_1794, which we coin P hage sh ielding h elix rich protein (Psh). Overexpression experiments show that Psh protein is secreted by this restored T1SS to confer complete phage resistance. When compared to wild-type P. putida KT2440, no growth or expression defects were evident, but pyoverdine production was reduced. The Psh gene and T1SS are widely distributed across Gram-negative bacteria. These findings uncover a previously uncharacterized phage defense system in P. putida based on secretion-mediated receptor masking.