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

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.

Maintenance and thermal stabilization of NADH dehydrogenase-2 conformation upon elimination of its C-terminal region.

Development of an artificial enzyme with activity and structure comparable to that of natural enzymes is an important goal in biological chemistry. Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a peripheral membrane-bound flavoprotein, belonging to a group of enzymes with scarce structural information. By eliminating the C-terminal region of NDH-2, a water soluble version with significant enzymatic activity was previously obtained. Here, NDH-2 structural features were established, in comparison to those of the truncated version. Far-UV circular dichroism, Fourier transform infrared spectroscopy and limited proteolysis analysis showed that the overall structure of both proteins was similar at 30 °C. Experimental data agree with the predicted NDH-2 structure (PDB: 1OZK). The absence of C-terminal region stabilized in ∼5-10 °C the truncated protein conformation. However, truncation impaired enzymatic activity at low temperatures, probably due to the weak interaction of the mutant protein with FAD cofactor.

Inhibition of Penicillium expansum by an oxidative treatment.

Several oxidizing compounds such as sodium hypochlorite (NaClO) and hydrogen peroxide (H(2)O(2)) are used to control postharvest decay in fresh fruit due to their antimicrobial effects. Here, we applied these compounds in vitro, in the presence of CuSO(4), against Penicillium expansum, causal agent of apple blue mold. MICs were 50 mg L(-1) and 400 mmol L(-1) for NaClO and H(2)O(2), respectively, when these compounds were individually applied to conidia suspensions during 2 min. A combined oxidative treatment (OT) consisting on an incubation with 1 mg L(-1) NaClO and 200 mmol L(-1) H(2)O(2), in the presence of 6 mmol L(-1) CuSO(4), inhibited growth, conidial germination and fungal infectivity on apple. The fractional inhibitory concentration index for the interaction between NaClO and H(2)O(2) in the OT was 0.52 indicating a synergistic effect of the oxidizing compounds. These results suggest that the OT could be an interesting alternative for apple diseases postharvest control.

LuMPIS--a modified luminescence-based mammalian interactome mapping pull-down assay for the investigation of protein-protein interactions encoded by GC-low ORFs.

The GC content is highly variable among the genomes of different organisms. It has been shown that recombinant gene expression in mammalian cells is much more efficient when GC-rich coding sequences of a certain protein are used. In order to study protein-protein interactions in Varicella zoster virus, a GC-low herpesvirus, we have developed a novel luminescence-based maltose-binding protein pull-down interaction screening system (LuMPIS) that is able to overcome the impaired protein expression levels of GC-low ORFs in mammalian expression systems.