L. paraplantarum CRL 1905 was originally isolated from Real Hornillos quinoa sourdough (Ruiz Rodríguez et al., 2016). This strain belongs to the public Culture Collection of the Centro de Referencia para Lactobacilos (CERELA-CONICET, Tucumán, Argentina). LAB cultures were propagated twice in MRS broth (Britania, Argentina) at 37°C for 16 h. Cells were harvested by centrifugation at 8,000 × g for 10 min and washed twice in sterile 0.85% (w/v) saline solution to eliminate residual nutrients. Washed cells were used to inoculate a chemically defined medium (CDM) (Hebert et al., 2004) to a final A_560nm of 0.1. CDM was modified in its original Pi content to achieve final concentrations of 2 or 60 mM potassium phosphate (named CDM-P or CDM + P, respectively). When indicated, media with different Pi concentrations were used. Cells were grown at 37°C under static conditions for different times.

Intracellular polyP levels were measured using a 4′, 6-diamidino-2-phenylindol (DAPI, Sigma, United States) based fluorescence approach (Aschar-Sobbi et al., 2008). Briefly, cells were washed twice with buffer T (100 mM Tris–HCl, pH 8) and resuspended in the same buffer. Measurements were performed at 37°C with constant agitation in cuvettes containing cell suspensions in buffer T at an A_560nm of 0.02 with 17 μM DAPI and 0.00075% SDS and chloroform for cell permeabilization. Then, the mixture was incubated for 10 min at 37°C with agitation. The DAPI fluorescence spectra (excitation, 415 nm; emission, 445–650 nm) were recorded using an ISS PCI spectrofluorometer (ISS Inc., Champaign, IL, United States). Fluorescence of DAPI-polyP complex at 550 nm was used as a measurement of intracellular polyP since emissions from free DAPI and DAPI-DNA are minimal at this wavelength.

The long-term survival of L. paraplantarum CRL 1905 was analyzed by culturing the cells statically at 37°C in CDM-P or CDM + P for 24, 48, and 120 h. Cell viability was quantitatively determined by plating the cells onto MRS-agar plates for 48 h at 37°C, and expressed as colony forming units per milliliter (CFU mL⁻¹).

The percentages of live, injured, and dead cells were determined by flow cytometry. 48 h- CRL 1905 cells grown in CDM-P or CDM + P were stained with a cell viability kit (BD™ Biosciences, United States). Cells were suspended in phosphate-buffered saline (PBS) to approximately a concentration of 10⁶ cells mL⁻¹ and incubated at room temperature for 5 min with 420 μM for thiazole orange and 48 μM for propidium iodine. Samples were analyzed using a FACS Calibur flow cytometer (BD™Biosciences, United States) using two different wavelengths, 488 nm and 635 nm. Assays were carried out in duplicate, and data analysis was performed using FCS Express 4Flow Cytometer (De Novo Software, Glendale, CA, United States).

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical assay was performed to evaluate the radical scavenging activity of CRL 1905 cells grown in different media. 7 and 48 h-cells were harvested by centrifugation at 8,000 × g for 10 min at 4°C. The obtained pellets were washed twice and resuspended in saline solution (A_560nm = 1). 1.0 mL of ethanolic DPPH radical solution (0.2 mM) was added to 1.0 mL of suspensions of cells grown in CDM-P or CDM + P. The mixtures were incubated in the dark at 25°C for 30 min. Then, suspensions were centrifuged at 6000 × g for 10 min, and the A_{517 nm} of the obtained supernatants were measured. The DPPH radical scavenging ability was calculated as follows: scavenging activity (%) = [1 − (A_sample − A_blank)/A_control] × 100%, where A_sample, A_blank and A_control represent the A_{517 nm} of the sample, blank and control, respectively. Controls used deionized water instead of intact cells, and blanks used anhydrous ethanol instead of DPPH.

Lipid peroxidation, normally used to assess oxidative damage, was determined by thiobarbituric acid reactive substances (TBARS) assay, as reported by Castro et al. (2021) with minor modifications. Briefly, suspensions of cells grown in CDM-P or CDM + P were centrifuged, washed twice with sterile saline solution, and lysed by adding glass beads to be vortex-agitated. Then, cells were centrifuged at 8,000 × g for 10 min at 4°C, and the supernatants were collected. 12.5 μL of 20% trichloroacetic acid (TCA) was added to 500 μL of supernatant to determine malondialdehyde (MDA). The reaction mixture contains 150 μL of cell extract, 150 μL of H₂O, 100 μL 0.1 M EDTA, and 600 μL of 1% thiobarbituric acid in 0.05 M NaOH. A reaction blank containing H₂O without cell extract was also prepared. The reaction mixture was incubated at 100°C for 15 min. The tubes were cooled and MDA was measured spectrophotometrically at 532 nm. TBARS content (expressed in pmol mg protein⁻¹) was determined using a molar extinction coefficient 156 mM⁻¹ cm⁻¹. Protein concentration was determined by the Bradford method (Bio-Rad, United States).

Microbial survival under oxidative stress was assessed by exposing CRL 1905 strain to hydrogen peroxide. Cells grown in CDM + P or CDM-P for 48 h were centrifuged and washed twice, resuspended in physiological solution and exposed to 10 mM hydrogen peroxide at 37°C for 15 min. The survival rate was calculated as follows: Survival rate (%) = (log_CFU N₁/log_CFU N₀) x 100, where N₁ is the total viable count after treatment with H₂O₂, and N₀ is the total viable count before treatment (Guo et al., 2009).

For SEM analysis, L. paraplantarum CRL 1905 cells grown in CDM-P or CDM + P during 48 h were fixed with 2.5% glutaraldehyde, 2.5% paraformaldehyde, acetone, and ethanol dehydrated, and gold coated with anion sputter JFC-1100 (JEOL). Samples under study were then attached to aluminum holders and analyzed using a Carl Zeiss SUPRA-55 scanning electron microscope from CIME (Centro Integral de Microscopía Electrónica, CONICET, Tucumán, Argentina) with a resolution of 1.0 nm at 15 kV and 1.7 nm at 1 kV in high-vacuum (HV) mode and 2 nm at 30 kV in variable-pressure mode.

Biofilm formation was assayed based on the cells ability to adhere and grow on polystyrene microtiter plates and stained with crystal violet. Cells were grown in microtiter plates containing CDM-P or CDM + P under static conditions at 37°C for 48 h. Unattached cells were removed by washing the plates with deionized water. 0.1% crystal violet solution was added to each well. Plates were incubated at room temperature for 20 min and rinsed 3 times with water. The absorbed crystal violet was extracted with 95% ethanol and measured at A_595nm (Spectra MaxPlus384 Absorbance Microplate Reader, United States). Six replicates were performed for each experimental condition.

Biofilm formation of L. paraplantarum CRL 1905 cells was carried out in CDM-P or CDM + P medium, using 6-well polystyrene plates with a glass coverslip inside. After incubation for 48 h, unattached cells were removed, and coverslips were washed with PBS and dried at 37°C for 10 min. The adhered cells were immersed in PBS containing 1.5 μL of dye Syto9 and incubated for 20 min in the dark. The coverslips were then rinsed with sterile water and removed from the wells. Images were captured using a Zeiss Confocal Microscope LSM800 at the IMMCA (Instituto de Investigación en Medicina Molecular y Celular Aplicada, CONICET-UNT-SIPROSA, Tucumán, Argentina).

Cells grown during 48 h in CDM-P or CDM + P media were harvested by centrifugation (8,000 × g, 10 min, 4°C), washed with 50 mM Tris–HCl buffer, and resuspended in 50 mM Tris–HCl buffer (pH 7.5) containing 1 mM PMSF and 10 mM EDTA. Cell extracts were obtained by homogenizing the bacterial suspensions with glass beads (0.15- to 0.25-mm diameter; Sigma, United States) in a MiniBeadbeater-16 Cell Disrupter (Biospec, United States) for 10 min. Glass beads, unbroken cells, and cell debris were removed by centrifugation (14,000 × g, 10 min, 4°C). Protein concentration was determined by the bicinchoninic acid assay (BCA assay). The label-free proteomics was conducted at the Proteomics Core Facility CEQUIBIEM (University of Buenos Aires-CONICET). 30 μg protein extract of each sample was reduced with 20 mM dithiothreitol (DTT), alkylated with 50 mM iodoacetamide for 45 min in the dark, and then digested with trypsin overnight. Peptides extraction was performed with acetonitrile. Samples were dried with a SpeedVac device, resuspended with 30 μL of 0.1% trifluoroacetic acid, and desalted with a ZipTip C18 column (Merck). Then, peptides were analyzed by nanoHPLC (EASY-nLC 1000) coupled to an Orbitrap technology mass spectrometer (Q-Exactive with High Collision Dissociation cell and Orbitrap analyzer). Sample ionization was carried out by nanoelectrospray (Thermo Scientific brand, model EASY-SPRAY. Spray voltage: 1.8 kV). The instrument was equipped with an HCD (High Collision Dissociation) cell and an Orbitrap analyzer yielding the identification of peptides simultaneously to their separation by chromatography. The analysis of the raw files delivered by the mass spectrometer was performed by using the Proteome Discover software (version 2.1.1.21 Thermo Scientific) using the peptide peak area for identification. Searches were performed throughout the L. plantarum (strain ATCC BAA-793/NCIMB8826/WCFS1) protein sequence database (https://www.uniprot.org/proteomes/UP000000432). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2019) partner repository with the dataset identifier PXD018988. The statistical data analysis and visualization were performed using Perseus software (version 1.5.6.0, Max Planck Institute, Germany) (Tyanova et al., 2016). Proteins displaying at least 1.5-fold changes, with a value of p ≤0.05, were considered as differentially expressed. In some exceptional cases, proteins with value of p of <0.1 and fold change values of ≥2.5 were also considered as regulated due to their biological significance.

When indicated, data were subjected to analysis of variance (ANOVA) followed by Tukey’s test with Infostat for Windows, 2016 version (Di Rienzo et al., 2016).

The effect of Pi concentration on stationary phase polyP levels was investigated in L. paraplantarum CRL 1905. Cells grown in CDM supplemented with different Pi concentrations presented a Pi dependent-polyP accumulation at 48 h (Figure 1A). Those grown in media containing Pi concentrations below 60 mM presented low polyP levels in stationary phase. In contrast, high polymer levels were observed in cells grown in media supplemented with 60 mM Pi. Based on this critical concentration, media containing sufficient (2 mM, CDM-P) and high Pi (60 mM, CDM + P) were selected for subsequent studies. The analysis of polymer content through the growth curve revealed that cells cultivated in both media exhibited high polyP levels during exponential phase (7 h, see in Figure 1B). The achieved elevated polyP levels were maintained in CDM + P cells for up to 120 h, whereas significantly declined in those grown in CDM-P during the stationary phase (Figure 1B). It is worth mentioning that there was no Pi deficiency in any of the tested conditions and that media Pi concentration did not affect cell growth (Figure 1C) nor the final pH, starting in values of 6 and reaching ~4 after 24 h (data not shown).

Considering that environmental Pi regulates polyP levels in L. paraplantarum CRL 1905, further physiological responses to differential Pi concentrations were studied in the stationary phase. Viability assay showed that cell population in both media was approximately 10⁷ CFU mL⁻¹ at 24 h. Subsequently, viable cells in CDM-P started to decrease, reaching to 3 × 10⁴ CFU mL⁻¹ at 120 h, whereas cells cultured in CDM + P presented values of ~10⁶ CFU mL⁻¹. In addition, a flow cytometry analysis was performed to characterize the cell population in both media (Figure 2). The percentage of living cells at 120 h was higher when grown in CDM + P (~20%) compared to those grown in CDM-P (less than 3%), whereas most of the cells were alive at 24 h in both media (~ 80%). It should be noted that the effects of modifying the Pi concentration were also evident in a complex medium. Indeed, L. paraplantarum CRL1905 cells grown in MRS with the addition of Pi to achieve 60 mM maintained high polyP levels and enhanced viability in stationary phase when compared to those grown in MRS (20 mM Pi) (data not shown).

A combined approach was performed to compare antioxidant ability of cells grown in each condition, including the determination of the cellular scavenging activity on DPPH radicals, the malondialdehyde content (MDA, as a measure of lipid peroxidation), and the tolerance to exogenous hydrogen peroxide. According to results in Table 1, there was a significant decrease in the scavenging capacity of CDM-P cells after 48 h. In contrast, the scavenging ability of CDM + P cells was maintained through time, with values similar to those observed during the exponential growth phase. In addition, CDM-P cells exhibited an enhanced MDA production compared to cells grown in high Pi, indicating stronger damage at the lipid level in the first condition. Furthermore, after 15 min-treatment with H₂O₂, cells grown in sufficient Pi medium were more affected than that of CDM + P cells, producing a drop in viability of approximately one order of magnitude (Table 1). It is noteworthy that the scavenging activity, MDA content and exogenous peroxide tolerance during the exponential growth phase revealed no significant differences between both conditions (Table 1). Together, results suggest that CRL 1905 cells grown in high Pi condition exhibited a higher ability to neutralize reactive oxygen species (ROS) during stationary phase, exerting a better antioxidant response.

Taking into account the observed differences between stationary phase cells, SEM analysis was carried out to observe possible morphological changes in bacteria grown in both conditions. Figure 3 shows that the 48 h-cells cultured in CDM + P exhibited a typical rod shape and smooth surfaces. However, CDM-P cells tended to clamp and presented rough surfaces due to the appearance of many membrane vesicles (MVs) (Figure 3). It is worth mentioning that the length of cells grown in sufficient Pi medium increased ~25% compared to those grown in high Pi condition.

The ability of CRL 1905 strain to form biofilm was studied in both conditions at 48 h (Figure 4). Biofilm formation was enhanced when cells were grown in sufficient Pi medium, being approximately 3-fold higher than that produced in high Pi medium (Figure 4, left). As visualized by CLSM, cells grown in CDM-P produced a thicker biofilm than those grown in CDM + P (Figure 4, right), in correlation with the quantitative results obtained through the cristal violet technique.

To evaluate possible effects of extracellular Pi concentration on L. paraplantarum CRL 1905 protein profile during stationary phase, a comparative proteomic analysis was carried out with cells grown in the differential conditions during 48 h. CDM + P versus CDM-P expression profile showed 46 differentially expressed proteins (> 1.5 folds, value of p < 0.05), from which 28 were up-regulated and 18 were down-regulated. As shown in Table 2, proteins involved in glycolysis, pyruvate metabolism, and pentose phosphate pathway (PdhA, PdhB, PdhC, Pox3, Ack2, PflB, GlpK1, Xfp, RpiA, GalM3, RbsK, and Pgm1), and in nucleotide metabolism (Upp, PyrB, and PyrR) were upregulated under high Pi conditions. On the contrary, chaperones implicated in stress response (Hsp1 and Hsp3) and enzymes involved in the modification of the cell surface (Lp_3421, Acm2, Alr, MurF, and FlmA) were down-regulated in cells grown in CDM + P when compared to those grown in CDM-P (Table 3). According to COG classifications, up-regulated proteins were mainly involved in “carbohydrate transport and metabolism,” “energy production and conversion,” and “nucleotide transport and metabolism,” while most of the down-regulated proteins belonged to the “amino acid transport and metabolism,” “cell wall/membrane/envelope biogenesis,” and “posttranslational modification, protein turnover, chaperones” categories (Tables 2, 3, respectively).