Intracellular water structure dynamics in transformed Lactobacillus Plantarum

OLUWADAMILOLA FADIYA; PEDRO D. CLOP; MARIA A. PERILLO

Rosario - Santa Fé

Congreso; L Reunión Anual de la Sociedad Argentina de Biofísica; 2022

Sociedad Argentina de Biofísica

The aim of this study is to detect possible effects of protein overexpression on thedynamics of intracellular water during lactic acid production in transformed Lactobacillusplantarum, in order to provide elements to understand the coupling between chemicaland mechanical levels of cellular metabolism.L. plantarum was transformed (through electroporation) by inserting a DNA plasmidcontaining a β-Galactosidase (β-Gal) coding gen. Isopropyl β-D-1-thiogalactopyranoside(IPTG) was the inducer of β-Gal-gene expression. Experiments were performed with thefollowing samples: wild type (wt) and transformed (treated with (trIPTG) or without (tr)inducer) L.plantarum cells. PAGE-SDS confirmed β-Gal expression. For the fermentation,cells were incubated in lactose (Lac) broth at 37°C for 24hr, under continuous agitation,at different temperatures and pH. Lactic acid (LA), residual Lac, and glucose (Glu)concentrations were measured spectophotometrically to evaluate Lac hydrolysis. Thecolony-forming units (CFU) were counted by dilution method in MRS agar plates.Cytoplasmic water polarization dynamics was inferred from the fluorescent behavior of thehydrophilic ACDAN probe. So, tr, trIPTG, and wt cells were suspended in 100 mM, pH 6.8potassium phosphate buffer in the presence of 5 μM of ACDAN probe, incubated for 60min at room temperature, and washed twice by centrifugation at 10,000 g for 2 min. toeliminate the probe not internalized. Then, a suspension of labelled cells was prepared at10% w/v density in fresh 100 mM pH 6.8 potassium phosphate buffer and transferred toquartz cuvettes for immediate fluorescence measurement (lex=366 nm). Emission spectra(lem= from 400nm to 600nm) and generalized polarization value (GP = IB-IR/ IB + IR;B=440 nm and R=490 nm) were determined.Improved production of LA was observed in L.plantarum (trIPTG) 18g/L in comparison toL.plantarum (wt) (8g/L) and L.plantarum (tr) (16g/L). The fluorescent spectrum of ACDANtrapped inside cells was significantly polarized in contrast to the relaxed characteristics ofACDAN spectrum in isotropic aqueous solutions and in detergent lysed cells.