Ppx1 putative exopolyphosphatase is essential for polyphosphate accumulation in Lacticaseibacillus paracasei

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Polyphosphate (poly-P) is a linear polymer vital for bacterial physiology, synthesized by polyphosphate kinase (Ppk) and degraded by exopolyphosphatases (Ppx).
• Lactobacillaceae often possess two putative exopolyphosphatase genes (ppx1 and ppx2) clustered with the ppk gene, but their specific roles are unclear.

Purpose of the Study:

• To investigate the specific functions of the two putative exopolyphosphatase genes, ppx1 and ppx2, in polyphosphate metabolism in Lacticaseibacillus paracasei.
• To elucidate the role of Ppx1 in polyphosphate synthesis and its relationship with Ppk.

Main Methods:

• Construction and analysis of in-frame deletion mutants (Δppx1, Δppx2) in L. paracasei BL23.
• Analysis of poly-P accumulation in wild-type and mutant strains.
• Gene expression analysis of ppk.
• Complementation studies by expressing ppx1 in trans.
• In vitro enzymatic assays of purified Ppx1 and Ppx2.
• Assays to determine the effect of Ppx1 on Ppk activity.

Main Results:

• Deletion of ppx1, but not ppx2, abolished poly-P accumulation in L. paracasei, despite unaltered ppk expression.
• Poly-P synthesis was restored in Δppx1 strains only by expressing ppx1 in trans, and not by expressing ppk from a plasmid.
• Purified Ppx2 showed exopolyphosphatase activity, while Ppx1 lacked detectable catalytic activity but enhanced Ppk activity in vitro.

Conclusions:

• Ppx1 is essential for poly-P synthesis in L. paracasei, contrary to its expected role in degradation.
• Ppx1 plays an unexpected regulatory role in poly-P synthesis, likely by modulating Ppk activity, adding complexity to bacterial poly-P dynamics.