Inorganic phosphate modifies stationary phase fitness and metabolic pathways in Lactiplantibacillus paraplantarum CRL 1905

Abstract

Inorganic phosphate (Pi) concentration modulates polyphosphate (polyP) levels in diverse bacteria, affecting their physiology and survival. Lactiplantibacillus paraplantarum CRL 1905 is a lactic acid bacterium isolated from quinoa sourdough with biotechnological potential as starter, for initiating fermentation processes in food, and as antimicrobial-producing organism. The aim of this work was to evaluate the influence of the environmental Pi concentration on different physiological and molecular aspects of the CRL 1905 strain. Cells grown in a chemically defined medium containing high Pi (CDM + P) maintained elevated polyP levels up to late stationary phase and showed an enhanced bacterial survival and tolerance to oxidative stress. In Pi sufficiency condition (CDM-P), cells were ~ 25% longer than those grown in CDM + P, presented membrane vesicles and a ~ 3-fold higher capacity to form biofilm. Proteomic analysis indicated that proteins involved in the "carbohydrate transport and metabolism" and "energy production and conversion" categories were up-regulated in high Pi stationary phase cells, implying an active metabolism in this condition. On the other hand, stress-related chaperones and enzymes involved in cell surface modification were up-regulated in the CDM-P medium. Our results provide new insights to understand the CRL 1905 adaptations in response to differential Pi conditions. The adjustment of environmental Pi concentration constitutes a simple strategy to improve the cellular fitness of L. paraplantarum CRL 1905, which would benefit its potential as a microbial cell factory.

Keywords: biofilm; lactic acid bacteria; polyphosphate; proteomics; stationary phase.

Figure 1

Influence of medium Pi concentration on intracellular polyP levels and bacterial growth. (A) Fluorescence emission at 550 nm of the DAPI- polyP complex measured in 48 h- CRL 1905 cells grown in CDM supplemented with the indicated Pi concentrations. (B) Fluorescence of the DAPI- polyP complex determined in Lactiplantibacillus paraplantarum CRL 1905 grown in CDM supplemented with 2 (CDM-P) or 60 (CDM + P) mM Pi at 7, 48 and 120 h. (C) Representative growth curves of CRL 1905 strain in the indicated media measured by absorbance at 560 nm during 120 h. Fluorescence is expressed in arbitrary units (AU) and the values are the average of three independent experiments. In panel A, different letters indicate significant differences among tested conditions according to Tukey’s test with a value of p of 0.05.

Figure 2

Survival of Lactiplantibacillus paraplantarum CRL 1905 grown in high and sufficient Pi conditions analyzed by flow cytometry at 24 and 120 h of culture. According to the cell state, results were grouped into three quadrants in: live, injured or dead. Dot plots are representative of three independent experiments and values are the average of the obtained percentages.

Figure 3

Morphology of Lactiplantibacillus paraplantarum CRL 1905 grown in high and sufficient Pi condition. Cells were grown in CDM-P or CDM + P during 48 h and analyzed by SEM. Magnification: 30000x (upper panels) and 50,000x (bottom panels). Data are representative from three independent experiments.

Figure 4

Effect of Pi concentration on the biofilm formation in Lactiplantibacillus paraplantarum CRL 1905. Cells were grown in CDM-P and CDM + P during 48 h. Cultures were diluted in fresh medium, containing the respective Pi concentrations, and biofilm formation was quantified after 48 h of incubation by crystal violet (bars graphic) or visualized and analyzed by CLSM (3D graphic constructed with ImageJ software). Data are representative of at least three independent experiments.