Different Modes of Regulation of the Expression of Dextransucrase in Leuconostoc lactis AV1n and Lactobacillus sakei MN1

Leuconostoc lactis AV1 strain isolated from a Tunisian avocado was characterized as a dextran producer. The promoter PdsrLL and the dsrLL gene encoding the DsrLL dextransucrase responsible for the dextran synthesis were transcriptionally fused to the mCherry coding gene generating the pRCR20 plasmid. Upon plasmid transfer, both AV1n and the dextran non-producing Leuconostoc mesenteroides CM70 became red due to expression of the mCherry from the PdsrLL-dsr-mrfp transcriptional fusion. Characterization of the polymers present in cultures supernatants revealed that the DsrLL encoded from pRCR20 in the recombinant bacteria was able to synthesize dextran. The production of dextran by the DsrLL in AV1n increased in response to low temperature, reaching 10-fold higher levels at 20°C than at 37°C (4.15 g/L versus 0.41 g/L). To analyze if this stress response includes activation at the transcriptional level and if it was only restricted to Leuconostoc, AV1n was transformed with plasmids carrying either the PdsrLL-mrfp fusion or the PdsrLS of Lactobacillus sakei MN1 fused to the mrfp gene, and the influence of temperature and carbon source on expression from the Dsr promoters was monitored by measurement of the mCherry levels. The overall expression analysis confirmed an induction of expression from PdsrLL upon growth at low temperature (20°C versus 30°C and 37°C) in the presence of sugars tested (sucrose, glucose, maltose, and fructose). In addition, the presence of sucrose, the substrate of Dsr, also resulted in activation of expression from PdsrLL. A different behavior was detected, when expression from PdsrLS was evaluated. Similar levels of fluorescence were observed irrespectively of the carbon source or temperature, besides a sequential decrease at 30°C and 20°C, when sucrose was present in the growth medium. In conclusion, the two types of regulation of expression of Dsr presented here revealed two different mechanisms for environmental adaptation of Leuconostoc and Lactobacillus that could be exploited for industrial applications

Biotechnological applications of sourdough lactic acid bacteria. A source for vitamins fortification and exopolysaccharides improvement

Sourdough fermentation relies on complex microbial ecosystems composed of lactic acid bacteria (LAB) and yeasts. As in other matrices, the fermentative microbiota deeply influences sensory, nutritional, functional, and hygienic quality. In particular, lactic acid bacteria are also an important starting point for developing specific food biotechnological applications. This chapter focuses on the potential exploitation of selected LAB to bio-produce, in situ, B-group vitamins, exopolysaccharides such as dextrans, and prebiotics, with positive modulation of global sourdough features. Concerning these molecular classes, the presented overview encompasses the importance of LAB biodiversity, the study of the genetic basis for the production of LAB metabolites, and the design of biotechnological strategies, reporting selected case studies from the scientific literature. Bread making has been reported as a representative production, but the proposed applications are transferable to other sourdough-based and, more generally, plant-based fermented foods and beverages. The possibility to bio-produce the molecules of interest directly in the matrix is well in line with the needs of sustainable development in food systems.Peer reviewe

Proteomic and in silico analyses of dextran synthesis influence on Leuconostoc lactis AV1n adaptation to temperature change

21 p.-9 fig.-1 tab.Leuconostoc lactis is found in vegetables, fruits, and meat and is used by the food industry in the preparation of dairy products, wines, and sugars. We have previously demonstrated that the dextransucrase of Lc. lactis (DsrLL) AV1n produces a high-molecular-weight dextran from sucrose, indicating its potential use as a dextran-forming starter culture. We have also shown that this bacterium was able to produce 10-fold higher levels of dextran at 20°C than at 37°C, at the former temperature accompanied by an increase in dsrLL gene expression. However, the general physiological response of Lc. lactis AV1n to cold temperature in the presence of sucrose, leading to increased production of dextran, has not been yet investigated. Therefore, we have used a quantitative proteomics approach to investigate the cold temperature-induced changes in the proteomic profile of this strain in comparison to its proteomic response at 37°C. In total, 337 proteins were found to be differentially expressed at the applied significance criteria (adjusted p-value ≤ 0.05, FDR 5%, and with a fold-change ≥ 1.5 or ≤ 0.67) with 204 proteins overexpressed, among which 13% were involved in protein as well as cell wall, and envelope component biosynthesis including DsrLL. Proteins implicated in cold stress were expressed at a high level at 20°C and possibly play a role in the upregulation of DsrLL, allowing the efficient synthesis of the protein essential for its adaptation to cold. Post-transcriptional regulation of DsrLL expression also seems to take place through the interplay of exonucleases and endonucleases overexpressed at 20°C, which would influence the half-life of the dsrLL transcript. Furthermore, the mechanism of cold resistance of Lc. lactis AV1n seems to be also based on energy saving through a decrease in growth rate mediated by a decrease in carbohydrate metabolism and its orientation toward the production pathways for storage molecules. Thus, this better understanding of the responses to low temperature and mechanisms for environmental adaptation of Lc. lactis could be exploited for industrial use of strains belonging to this species.This study was supported by the Spanish Ministry of Science, Innovation and Universities (grant no. RTI2018-097114-B-I00) and the Tunisian Ministry of Higher Education and Scientific Research.Peer reviewe

Análisis comparativo de cepas tunecinas productoras de dextrano pertenecientes a los géneros Leuconostoc y Weisella

Resumen del trabajo presentado en el XI Workshop de la Sociedad Española de Microbiota, Probióticos y Prebióticos, celebrado en Granada (España), del 12 al 14 de febrero de 2020Introducción/Objetivos. Los exopolisacáridos (EPS) producidos por las bacterias ácido lácticas (BAL) poseen una amplia aplicación industrial. Así, los dextranos se emplean en la industria alimentaria para mejorar las propiedades reológicas de alimentos fermentados y poseen capacidad inmunomoduladora y antiviral. El objetivo de este trabajo fue identificar weisellas productoras de dextrano y caracterizar su naturaleza y el efecto de sus polímeros comparándolo con los de Leuconostoc lactis AV1n. Metodología. Los EPS se caracterizaron por análisis de la composición de azúcares y de metilación. Su tamaño se determinó por SEC-MALS. La producción de dextrano y el flujo metabólico se cuantificaron por el método del fenol sulfúrico y por cromatografía de gases. Se analizó la interacción bacteria-células Caco-2, y la formación de biopelícula por tinción con cristal violeta. Resultados. Se demostró que los EPS eran dextranos con elevadas masas moleculares (5,84x107-2,61x108 Da). Su producción se detectó durante la fase exponencial de crecimiento, con un consumo de sacarosa de > 95% por AV1n y de 57% por las weisellas. El EPS producido por W. confusa 11.3b disminuyó en la fase estacionaria. Este resultado indicativo de una actividad dextranasa, fue confirmado por la detección de halos de degradación de azul dextrano. W. confusa V30 mostró menor adherencia a los enterocitos en presencia de glucosa (38,1%) versus sacarosa (14,3%). El tipo de azúcar no afectó la adhesión de W. cibaria AV2ou (10,5±1,4%) y 11.3b (11,2±3,0%). AV1n mostró mayor adherencia en presencia de sacarosa (48,8%) versus glucosa (27,8%) y se categorizó como fuerte formador de biopelícula sobre poliestireno en presencia de sacarosa (3,38±0,38) versus glucosa (0,78±0,21). Dichos azúcares no influyeron diferencialmente en la formación de biopelícula por las weisellas. Conclusiones. Por primera vez, L. lactis AV1n ha evidenciado al dextrano como efector positivo de la adhesión y agregación de BAL, y se ha detectado una actividad dextranasa en una weisella

Different modes of regulation of the expression of dextransucrase in Leuconostoc lactis AV1n and Lactobacillus sakei MN1

Leuconostoc lactis AV1 strain isolated from a Tunisian avocado was characterized as a dextran producer. The promoter PdsrLL and the dsrLL gene encoding the DsrLL dextransucrase responsible for the dextran synthesis were transcriptionally fused to the mCherry coding gene generating the pRCR20 plasmid. Upon plasmid transfer, both AV1n and the dextran non-producing Leuconostoc mesenteroides CM70 became red due to expression of the mCherry from the PdsrLL-dsr-mrfp transcriptional fusion. Characterization of the polymers present in cultures supernatants revealed that the DsrLL encoded from pRCR20 in the recombinant bacteria was able to synthesize dextran. The production of dextran by the DsrLL in AV1n increased in response to low temperature, reaching 10-fold higher levels at 20∘C than at 37∘C (4.15 g/L versus 0.41 g/L). To analyze if this stress response includes activation at the transcriptional level and if it was only restricted to Leuconostoc, AV1n was transformed with plasmids carrying either the PdsrLL-mrfp fusion or the PdsrLS of Lactobacillus sakei MN1 fused to the mrfp gene, and the influence of temperature and carbon source on expression from the Dsr promoters was monitored by measurement of the mCherry levels. The overall expression analysis confirmed an induction of expression from PdsrLL upon growth at low temperature (20∘C versus 30∘C and 37∘C) in the presence of sugars tested (sucrose, glucose, maltose, and fructose). In addition, the presence of sucrose, the substrate of Dsr, also resulted in activation of expression from PdsrLL. A different behavior was detected, when expression from PdsrLS was evaluated. Similar levels of fluorescence were observed irrespectively of the carbon source or temperature, besides a sequential decrease at 30∘C and 20∘C, when sucrose was present in the growth medium. In conclusion, the two types of regulation of expression of Dsr presented here revealed two different mechanisms for environmental adaptation of Leuconostoc and Lactobacillus that could be exploited for industrial applications.This work was supported by the Spanish Ministry of Economy and Competitiveness (Grants AGL2015-65010-C3-1-R and RTI2018-097114-B-100) and the Tunisian Ministry of Higher Education and Scientific Research.Peer Reviewe

The role of dextran production in the metabolic context of Leuconostoc and Weissella Tunisian strains

High molecular weight dextrans improve the rheological properties of fermented products and have immunomodulatory and antiviral activity. We report on 5.84 × 10–2.61 × 10 Da dextrans produced by Leuconostoc lactis AV1n, Weissella cibaria AV2ou and Weissella confusa V30 and FS54 strains. Dextransucrases catalyze dextran synthesis by sucrose hydrolysis concomitant with fructose generation. The four bacteria have dextransucrases with molecular weight of about 160 kDa detected by zymograms. Each bacterium showed different interplay of dextran production and metabolic fluxes. All bacteria produced lactate, and AV2ou apart, synthesized mannitol from fructose. FS54 hydrolyzed dextran blue and the concentration of dextran produced by this bacterium decreased during the stationary phase. The AV1n binding to Caco-2 cells and polystyrene plates was higher under conditions for dextran synthesis. Thus, this is the first instance of a Weissella dextranase, associated with a dextransucrase ability, and of a positive influence of dextran on adhesion and aggregation properties of a bacterium.This work was supported by the Spanish Ministry of Science, Innovation and Universities (grant RTI2018-097114-B-I00) and the TunisianMinistry of Higher Education and Scientific research