Accumulation of polyphosphate in Lactobacillus spp. and its involvement in stress resistance

Abstract

Polyphosphate (poly-P) is a polymer of phosphate residues synthesized and in some cases accumulated by microorganisms, where it plays crucial physiological roles such as the participation in the response to nutritional stringencies and environmental stresses. Poly-P metabolism has received little attention in Lactobacillus, a genus of lactic acid bacteria of relevance for food production and health of humans and animals. We show that among 34 strains of Lactobacillus, 18 of them accumulated intracellular poly-P granules, as revealed by specific staining and electron microscopy. Poly-P accumulation was generally dependent on the presence of elevated phosphate concentrations in the culture medium, and it correlated with the presence of polyphosphate kinase (ppk) genes in the genomes. The ppk gene from Lactobacillus displayed a genetic arrangement in which it was flanked by two genes encoding exopolyphosphatases of the Ppx-GppA family. The ppk functionality was corroborated by its disruption (LCABL_27820 gene) in Lactobacillus casei BL23 strain. The constructed ppk mutant showed a lack of intracellular poly-P granules and a drastic reduction in poly-P synthesis. Resistance to several stresses was tested in the ppk-disrupted strain, showing that it presented a diminished growth under high-salt or low-pH conditions and an increased sensitivity to oxidative stress. These results show that poly-P accumulation is a characteristic of some strains of lactobacilli and may thus play important roles in the physiology of these microorganisms.

FIG 1

Poly-P granules in several Lactobacillus strains. Bacterial smears were stained with Neisser staining and observed at ×100 magnification. (A) Examples of poly-P⁻ and poly-P⁺ strains are presented. (B) Poly-P granules in L. plantarum WCFS1 and L. casei BL23 strains grown under high- or low-phosphate conditions (MEI or LP-MEI, respectively).

FIG 2

Electron micrographs of selected Lactobacillus strains. Different strains were grown in MEI medium, fixed, and stained, and ultrathin preparations were observed using transmission electron microscopy. Under these conditions, the positions that were occupied by poly-P granules appeared as holes in the bacterial cytoplasm. The horizontal scale bars represent 0.5 μm.

FIG 3

Electron micrographs of L. casei BL23 cells grown in MEI and LP-MEI media. The characteristic holes caused by the removal of the poly-P inclusions are not observed in bacteria grown in LP-MEI. The horizontal scale bars represent 5 μm.

FIG 4

Genomic context of ppk genes identified in sequenced genomes of bacterial species belonging to Lactobacillales. The presence of ppk, ppx1, and ppx2 homologues and the occurrence of similar flanking genes in different strains and species is depicted. An asterisk above the strain name indicates the presence of truncated genes.

FIG 5

Effect of a mutation in ppk on poly-P synthesis in L. casei BL23. (A) Neisser staining of wild-type L. casei BL23 and strain BL379, disrupted in ppk, grown in different media. Staining of the BL379 strain complemented by the expression of ppk from a plasmid (pT1ppk) is also shown; (B) separation of poly-P isolated from BL23 and BL379 strains grown on MEI medium on a denaturing polyacrylamide gel. Poly-P was stained with toluidine blue.

FIG 6

Effect of a mutation in ppk from L. casei on growth under different stress conditions. Growth curves of L. casei strains (BL23, wild type; BL379, ppk mutant, and BL379[pT1ppk]) are shown. (A) Growth in MRS medium; (B) growth in MRS medium adjusted to pH 4; (C) growth in MEI medium with or without 0.6 M NaCl or with pH adjusted to 4; (D) growth in LP-MEI medium with or without 0.6 M NaCl or with pH adjusted to 4;. The results are the means of at least three experiments. Standard deviations were always lower than 15%.

FIG 7

Effect of a mutation in ppk from L. casei BL23 on growth under oxidative stress conditions. The diameters of the inhibition halos of plumbagin on agar plates are shown. The results are from at least 17 measurements. ***, P < 0.0003; **, P < 0.003.