Investigating luxS gene expression in lactobacilli along lab-scale cocoa fermentations.

Previous metagenomic analyses have suggested that lactobacilli present potential for Quorum Sensing (QS) in cocoa fermentation, and in the present research, laboratory scale fermentations were carried out to monitor the expression of luxS, a universal marker of QS. For that, 96 h-fermentations were studied, as follows: F0 (non inoculated control), F1 (inoculated with yeasts, lactic acid bacteria, and acetic acid bacteria), F2 (inoculated with yeasts and acetic acid bacteria), F3 (inoculated with yeasts only). The parameters evaluated were: plate counting, quantification of key enzymes and analysis of volatile organic compounds associated with key sensory descriptors, using headspace gas chromatography-mass spectrometry (GC-MS). Furthermore, QS was estimated by the quantification of the expression of luxS genes by Reverse Transcriptase Real-Time PCR. The results demonstrated that microbial succession occurred in pilot scale fermentations, but no statistical differences for microbial enumeration and α-diversity index were observed among experiments and control. Moreover, it was not possible to make conclusive correlations of enzymatic profile and fermenting microbiota, likely due to the intrinsic activity of plant hydrolases. Regarding to the expression of luxS genes, in Lactiplantibacillus plantarum they were active along the fermentation, but for Limosilactobacillus fermentum, luxS was expressed only at early and middle phases. Correlation analysis of luxS expression and production of volatile metabolites evidenced a possible negative association of Lp. Plantarum with fermentation quality. In conclusion, these data corroborate former shotgun metagenomic analysis by demonstrating the expression of luxS by lactobacilli in pilot scale cocoa fermentation and evidence Lp. Plantarum is the main lactic acid bacteria related to its expression.

Design of ß-Keto Esters with Antibacterial Activity: Synthesis, In Vitro Evaluation, and Theoretical Assessment of Their Reactivity and Quorum-Sensing Inhibition Capacity.

This work proposes the design of ß-keto esters as antibacterial compounds. The design was based on the structure of the autoinducer of bacterial quorum sensing, N-(3-oxo-hexanoyl)-l-homoserine lactone (3-oxo-C6-HSL). Eight ß-keto ester analogues were synthesised with good yields and were spectroscopically characterised, showing that the compounds were only present in their ß-keto ester tautomer form. We carried out a computational analysis of the reactivity and ADME (absorption, distribution, metabolism, and excretion) properties of the compounds as well as molecular docking and molecular dynamics calculations with the LasR and LuxS quorum-sensing (QS) proteins, which are involved in bacterial resistance to antibiotics. The results show that all the compounds exhibit reliable ADME properties and that only compound 7 can present electrophile toxicity. The theoretical reactivity study shows that compounds 6 and 8 present a differential local reactivity regarding the rest of the series. Compound 8 presents the most promising potential in terms of its ability to interact with the LasR and LuxS QS proteins efficiently according to its molecular docking and molecular dynamics calculations. An initial in vitro antimicrobial screening was performed against the human pathogenic bacteria Pseudomonas aeruginosa and Staphylococcus aureus as well as the phytopathogenic bacteria Pseudomonas syringae and Agrobacterium tumefaciens. Compounds 6 and 8 exhibit the most promising results in the in vitro antimicrobial screening against the panel of bacteria studied.

Pangenome analyses of LuxS-coding genes and enzymatic repertoires in cocoa-related lactic acid bacteria.

Lactobacillaceae presents potential for interspecific Quorum Sensing (QS) in spontaneous cocoa fermentation, correlated with high abundance of luxS. Three Brazilian isolates from cocoa fermentation were characterized by Whole Genome Sequencing and luxS gene was surveyed in their genomes, in comparison with public databases. They were classified as Lactiplantibacillus plantarum, Limosilactobacillus fermentum and Pediococcus acidilactici. LuxS genes were conserved in core genomes of the novel isolates, but in some non-cocoa related Lactic Acid Bacteria (LAB) it was accessory and plasmid-borne. The conservation and horizontal acquisition of luxS reinforces that QS is determinant for bacterial adaptation in several environments, especially taking into account the luxS has been correlated with modulation of bacteriocin production, stress tolerance and biofilm formation. Therefore, in this paper, new clade and species-specific primers were designed for future application for screening of luxS gene in LAB to evaluate the adaptive potential to diverse food fermentations.

Modeling Quorum Sensing Dynamics and Interference on Escherichia coli.

Bacteria control the expression of specific genes by Quorum Sensing (QS). This works using small signaling molecules called Autoinducers (AIs), for example, the Autoinducer-2 (AI-2). In this work, we present a mathematical model that represents the AI-2 dynamics on Escherichia coli, which is linked to the cell growth and the lsr operon expression. The model is adjusted using experimental data. Our results suggest that the extracellular AI-2 activity level depends on the cell growth rate, and this activity depends on the cell exponential growth phase. The model was adapted to simulate the interference of QS mechanisms in a co-culture of two E. coli strains: a wild type strain and a knock out strain that detects AI-2 but does not produce it. Co-culture simulations unveiled two conditions to avoid the QS on the wild strain: when the knock out takes control of the growth medium and overcomes the wild strain, or when is pre-cultured to its mid-exponential phase and then added to the wild strain culture. Model simulations unveiled new insights about the interference of bacterial communication and offer new tools for QS control.

Quorum sensing in Enterococcus faecium, Enterococcus faecalis and Bacillus cereus strains isolated from ricotta processing / Quorum sensing em cepas de Enterococcus faecium, Enterococcus faecalis e Bacillus cereus isoladas do processamento de ricota

The quorum sensing phenomenon is a process of intra- and inter-species microbial communication involving the production and detection of extracellular signaling molecules. The autoinducer AI-2 has been proposed to serve as a universal signal for interspecies communication. This study aimed to evaluate the capability of Enterococcus faecium, Enterococcus faecalis, and Bacillus cereus strains isolated from ricotta processing to produce quorum sensing signalling molecules (AI-2). The strains were evaluated for the presence of the luxS gene using the polymerase chain reaction. AI-2 quorum sensing signalling molecules were measured in relative light units (RLUs) using a luminometer. A total of 74% of E. faecium, 91% of E. faecalis, and 95% of B. cereus isolates were positive for luxS gene. In addition, the induced bioluminescence in Vibrio harveyi BB170 was observed in all strains, indicating the presence of the AI-2 autoinducer.(AU)

O fenômeno quorum sensing corresponde a um processo de comunicação intra e interespécies microbianas e é mediado por sinais químicos extracelulares, denominados moléculas sinalizadoras ou auto indutoras (AI). A molécula AI2 está envolvida na comunicação interespécies, denominada sistema universal de comunicação. Este estudo teve como objetivo avaliar a capacidade de Enterococcus faecium, Enterococcus faecalis e Bacillus cereus isolados do processamento de ricota em produzir moléculas sinalizadoras de Quorum sensing (AI-2). Os isolados foram avaliados quanto à presença do gene luxS utilizando a reação em cadeia da polimerase (PCR). As moléculas sinalizadoras (AI-2) foram medidas em unidades relativas de luz (RLU) através de um luminômetro. Um total de 74% dos isolados de E. faecium, 91% de E. faecalis e 95% de B. cereus foram positivos para o gene luxS. Além disso, todos os isolados apresentaram capacidade de induzir o fenômeno de bioluminescência em Vibrio harveyi BB170, indicando a presença de auto indutores AI-2.(AU)

Quorum sensing in Enterococcus faecium, Enterococcus faecalis and Bacillus cereus strains isolated from ricotta processing / Quorum sensing em cepas de Enterococcus faecium, Enterococcus faecalis e Bacillus cereus isoladas do processamento de ricota

ABSTRACT: The quorum sensing phenomenon is a process of intra- and inter-species microbial communication involving the production and detection of extracellular signaling molecules. The autoinducer AI-2 has been proposed to serve as a 'universal signal' for interspecies communication. This study aimed to evaluate the capability of Enterococcus faecium, Enterococcus faecalis, and Bacillus cereus strains isolated from ricotta processing to produce quorum sensing signalling molecules (AI-2). The strains were evaluated for the presence of the luxS gene using the polymerase chain reaction. AI-2 quorum sensing signalling molecules were measured in relative light units (RLUs) using a luminometer. A total of 74% of E. faecium, 91% of E. faecalis, and 95% of B. cereus isolates were positive for luxS gene. In addition, the induced bioluminescence in Vibrio harveyi BB170 was observed in all strains, indicating the presence of the AI-2 autoinducer.

RESUMO: O fenômeno quorum sensing corresponde a um processo de comunicação intra e interespécies microbianas e é mediado por sinais químicos extracelulares, denominados moléculas sinalizadoras ou auto indutoras (AI). A molécula AI2 está envolvida na comunicação interespécies, denominada sistema "universal" de comunicação. Este estudo teve como objetivo avaliar a capacidade de Enterococcus faecium, Enterococcus faecalis e Bacillus cereus isolados do processamento de ricota em produzir moléculas sinalizadoras de Quorum sensing (AI-2). Os isolados foram avaliados quanto à presença do gene luxS utilizando a reação em cadeia da polimerase (PCR). As moléculas sinalizadoras (AI-2) foram medidas em unidades relativas de luz (RLU) através de um luminômetro. Um total de 74% dos isolados de E. faecium, 91% de E. faecalis e 95% de B. cereus foram positivos para o gene luxS. Além disso, todos os isolados apresentaram capacidade de induzir o fenômeno de bioluminescência em Vibrio harveyi BB170, indicando a presença de auto indutores AI-2.

Production of AI-2 is mediated by the S-ribosylhomocystein lyase gene luxS in Bacteroides fragilis and Bacteroides vulgatus.

Quorum sensing is a cell-cell signaling mechanism based on cell density and that involves the production of hormone-like molecules called autoinducers (AI). One of the most studied AIs has been termed AI-2, and its biosynthesis requires the enzyme encoded by luxS. We have previously described for the first time that Bacteroides species can produce molecules with AI-2 activity. In this study, we focus on the detection of luxS and its activity as the AI-2 synthase in Bacteroides species. The strains Bacteroides fragilis B3b and Bacteroides vulgatus ATCC 8482 were selected based on a positive phenotype for AI-2 production and the presence of a putative luxS in the genome, respectively. In order to identify the luxS gene, cloning and heterologous expression strategies were utilized. We demonstrate that both strains contain functional luxS orthologs that can complement AI-2 production in Escherichia coli.