Tagging Polyphosphate Kinase Alters Enzymatic Activity and Affect Stress Recovery in E. coli

Abstract

Many bacterial species, including Escherichia coli (E. coli) utilize the enzyme polyphosphate kinase (PPK) to synthesize polyphosphate (polyP) in response to biological stress. Multiple studies have shown that impairing PPK activity impairs bacterial pathogenicity and survival, which has made it a target for therapeutic development. Unfortunately, PPK regulation is poorly understood. To address this, we previously described a series of mutations in E. coli ppk, termed ppk*, that result in high polyP accumulation in vivo. However, the specific activity of PPK* enzyme in vitro, when purified using a C‐term 6X His‐tag (PPK*‐HT), was comparable to purified WT enzyme (PPK‐HT).PPK activity is tied to its oligomeric state, with the dimeric form synthesizing polyP. To determine if the ppk* mutation altered oligomerization, we tested PPK‐HT and PPK*‐HT enzymes in an analytical ultracentrifuge. We found that the PPK‐HT enzyme was mostly dimeric but had a small monomeric population. Conversely, the PPK*‐HT enzyme was exclusively dimeric. This suggested that the ppk* mutation may affect oligomerization, but the His‐tag masked any effect on activity.We next hypothesized that the C‐term His tag alters the in vitro activity. To test this, we compared the specific activities of PPK‐HT and PPK*‐HT to that of PPK purified using a smaller C‐term C tag (PPK‐CT) and PPK enzyme purified with no tags (PPK‐NT). PPK*‐HT and PPK‐HT had comparable activities in vitro but were significantly more active than the untagged enzyme. PPK‐CT and PPK‐NT had comparable activities. This suggests that the C‐tag does not alter PPK activity while the His‐tag does.We next tested the purified enzymes’ sensitivity to substrate by testing PPK activity in 6mM and 20mM ATP. We found that the His‐tag increased the enzyme’s activity in the higher ATP concentrations. Comparing the 20mM and 6mM samples, the His‐tag enzymes showed an increase in activity of ~3.5 fold compared to their 6mM conditions. Conversely, the PPK‐CT and PPK‐UT only showed a 1.4‐ and 1.9‐ fold increase in activity, respectively.PolyP synthesis impairs bacterial growth and replication. We hypothesized that a more active PPK would impair recovery following a prolonged stationary phase. We developed strains carrying one of four expression vectors: an empty vector control (EV), PPK‐HT, PPK‐CT, or native PPK. Using these four strains, we developed a bacterial growth recovery curve assay. We found that while the PPK‐CT and untagged PPK strains had comparable recovery and growth rates, the PPK‐HT strain had a slower recovery rate resulting in a later shift into log phase.Taken together, our results have significant implications for polyP biology. We have shown that the standard purification method produces an enzyme with altered substrate sensitivities, making it a poorer candidate for drug development studies. C‐tag enzyme is comparable to untagged enzyme, making it a reliable candidate for future studies. Finally, the in vitro differences produce an in vivo phenotype, making it easy to test the effect of the tags in living systems.