Development of a molecular diagnostic test for the specific detection of Brettanomyces bruxellensis in red wine.

Brettanomyces bruxellensis is considered the main source of spoilage in red wine. This yeast, by producing volatile phenols, is responsible for the development of unpleasant aromas affecting the quality of final products and resulting in substantial economic losses for wine producers. This work therefore describes the development of an easy to-use colorimetric molecular diagnostic test for the rapid and specific detection of B. bruxellensis in wine. Detection was achieved using a sandwich hybridization format in which the target RNA was recognized by an immobilized DNA capture probe and a labelled DNA signal probe. The proposed device was highly specific to B. bruxellensis and showed a linear relationship between measured signal and target RNA concentration in the range 0.1-5 ng µL-1, with a limit of detection value of 0.1 ng µL-1 of total RNA. The colorimetric assay was validated on red wine samples, with a detection limit of 102 CFU mL-1. This study suggests that the reported method could be used for early detection of spoilage yeasts in wine and other alcoholic beverages.

Control of pellicle biogenesis involves the diguanylate cyclases PdgA and PdgB, the c-di-GMP binding protein MxdA and the chemotaxis response regulator CheY3 in Shewanella oneidensis.

Shewanella oneidensis is an aquatic proteobacterium with remarkable respiratory and chemotactic abilities. It is also capable of forming biofilms either associated to surfaces (SSA-biofilm) or at the air-liquid interface (pellicle). We have previously shown that pellicle biogenesis in S. oneidensis requires the flagellum and the chemotaxis regulatory system including CheA3 kinase and CheY3 response regulator. Here we searched for additional factors involved in pellicle development. Using a multicopy library of S. oneidensis chromosomal fragments, we identified two genes encoding putative diguanylate cyclases (pdgA and pdgB) and allowing pellicle formation in the non-pellicle-forming cheY3-deleted mutant. A mutant deleted of both pdgA and pdgB is affected during pellicle development. By overexpressing phosphodiesterase encoding genes, we confirmed the key role of c-di-GMP in pellicle biogenesis. The mxd operon, previously proposed to encode proteins involved in exopolysaccharide biosynthesis, is also essential for pellicle formation. In addition, we showed that the MxdA protein, containing a degenerate GGDEF motif, binds c-di-GMP and interacts with both CheY3 and PdgA. Therefore, we propose that pellicle biogenesis in S. oneidensis is controlled by a complex pathway that involves the chemotaxis response regulator CheY3, the two putative diguanylate cyclases PdgA and PdgB, and the c-di-GMP binding protein MxdA.

ChrASO, the chromate efflux pump of Shewanella oneidensis, improves chromate survival and reduction.

The chromate efflux pump encoding gene chrASO was identified on the chromosome of Shewanella oneidensis MR1. Although chrASO is expressed without chromate, its expression level increases when Cr(VI) is added. When deleted, the resulting mutant ΔchrASO exhibits a chromate sensitive phenotype compared to that of the wild-type strain. Interestingly, heterologous expression of chrASO in E. coli confers resistance to high chromate concentration. Moreover, expression of chrASO in S. oneidensis and E. coli significantly improves Cr(VI) reduction. This effect could result either from extracytoplasmic chromate reduction or from a better cell survival leading to enhanced Cr(VI) reduction.