Ppx1 putative exopolyphosphatase is essential for polyphosphate accumulation inLacticaseibacillus paracasei

Abstract

ABSTRACTThe linear polymer polyphosphate (poly-P) is present across all three domains of life and serves diverse physiological functions. The enzyme polyphosphate kinase (Ppk) is responsible for poly-P synthesis, whereas poly-P degradation is carried out by the enzyme exopolyphosphatase (Ppx). In manyLactobacillaceae, the Ppk-encoding gene (ppk) is found clustered together with two genes encoding putative exopolyphosphatases (ppx1andppx2) each having different domain compositions, with the gene orderppx1-ppk-ppx2. However, the specific function of theseppxgenes remains unexplored. An in-frame deletion ofppx1inLacticaseibacillus paracaseiBL23 resulted in bacteria unable to accumulate poly-P, whereas disruption ofppx2had no effect on poly-P synthesis. Expression ofppkwas not altered in the Δppx1strain, and poly-P synthesis in this strain was only restored by expressingppx1intrans. Moreover, no poly-P synthesis was observed whenppkwas expressed from a plasmid in the Δppx1strain. Purified Ppx2 exhibitedin vitroexopolyphosphatase activity, whereas noin vitroenzymatic activity could be demonstrated for Ppx1. This observation corresponds with the absence in Ppx1 of conserved motifs essential for catalysis found in characterized exopolyphosphatases. Furthermore, assays with purified Ppk and Ppx1 evidenced that Ppx1 enhanced Ppk activity. These results demonstrate that Ppx1 is essential for poly-P synthesis inLc. paracaseiand have unveiled, for the first time, an unexpected role of Ppx1 exopolyphosphatase in poly-P synthesis.ImportancePoly-P is a pivotal molecular player in bacteria, participating in a diverse array of processes ranging from stress resilience to pathogenesis, while also serving as a functional component in probiotic bacteria. The synthesis of poly-P is tightly regulated, but the underlying mechanisms remain incompletely elucidated. Our study sheds light on the distinctive role played by the two exopolyphosphatases (Ppx) found in theLactobacillaceaebacterial group, of relevance in food and health. This particular group is noteworthy for possessing two Ppx enzymes, supposedly involved in poly-P degradation. Remarkably, our investigation uncovers an unprecedented function of Ppx1 inLacticaseibacillus paracasei, where its absence leads to the total cessation of poly-P synthesis, paralleling the impact observed upon eliminating the poly-P forming enzyme, poly-P kinase. Unlike the anticipated role as a conventional exopolyphosphatase, Ppx1 demonstrates an unexpected function. Our results added a layer of complexity to our understanding of poly-P dynamics in bacteria.