线性聚合物多磷酸盐(poly-P)存在于生命的所有三个领域中,并具有不同的生理功能。聚磷酸激酶(Ppk)负责poly-P合成,而poly-P降解是通过酶外聚磷酸酶(Ppx)进行的。在许多乳杆菌科中,发现Ppk编码基因(ppk)与两个编码推定的外聚磷酸酶(ppx1和ppx2)的基因聚集在一起,每个基因具有不同的结构域组成,与基因顺序ppx1-ppk-ppx2。然而,这些ppx基因的特定功能仍未被探索。副干酪乳杆菌BL23中ppx1的框内缺失导致细菌无法积累poly-P,而ppx2的破坏不影响poly-P的合成。在Δppx1菌株中ppk的表达没有改变,只有通过反式表达ppx1才能恢复该菌株中的poly-P合成。此外,当从Δppx1菌株中的质粒表达ppk时,未观察到poly-P合成。纯化的Ppx2表现出体外外聚磷酸酶活性,而Ppx1没有体外酶活性。此观察结果与Ppx1中不存在在表征的胞外磷酸酶中发现的催化所必需的保守基序相对应。此外,用纯化的Ppk和Ppx1进行的测定证明Ppx1增强了Ppk活性。这些结果表明,Ppx1对于Lc中的poly-P合成至关重要。paracasei并公布了,第一次,Ppx1外聚磷酸酶在poly-P合成中的意想不到的作用。IMPORTANCEPoly-P是细菌中的关键分子,参与从应激恢复能力到发病机制的各种过程,同时也作为益生菌的功能成分。poly-P的合成受到严格的调控,但是潜在的机制仍未完全阐明。我们的研究揭示了在乳杆菌科细菌群中发现的两种外聚磷酸酶(Ppx)所起的独特作用,与食品和健康相关。这个特殊的群体值得注意的是拥有两种Ppx酶,据说参与poly-P降解。值得注意的是,我们的调查揭示了Ppx1在副干酪乳杆菌中前所未有的功能,它的缺失导致poly-P合成的完全停止,平行于消除poly-P形成酶时观察到的影响,poly-P激酶。与作为常规外聚磷酸酶的预期作用不同,Ppx1演示了一个意外的函数。我们的结果为我们对细菌中poly-P动力学的理解增加了一层复杂性。
The 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 many Lactobacillaceae, the Ppk-encoding gene (ppk) is found clustered together with two genes encoding putative exopolyphosphatases (ppx1 and ppx2) each having different domain compositions, with the gene order ppx1-ppk-ppx2. However, the specific function of these ppx genes remains unexplored. An in-frame deletion of ppx1 in Lacticaseibacillus paracasei BL23 resulted in bacteria unable to accumulate poly-P, whereas the disruption of ppx2 did not affect poly-P synthesis. The expression of ppk was not altered in the Δppx1 strain, and poly-P synthesis in this strain was only restored by expressing ppx1 in trans. Moreover, no poly-P synthesis was observed when ppk was expressed from a plasmid in the Δppx1 strain. Purified Ppx2 exhibited in vitro exopolyphosphatase activity, whereas no in vitro enzymatic 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 in Lc. paracasei and 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 the Lactobacillaceae bacterial 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 in Lacticaseibacillus 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.