Comparative transcriptional analysis of flavour-biosynthetic genes of a native Saccharomyces cerevisiae strain fermenting in its natural must environment, vs. a commercial strain and correlation of the genes' activities with the produced flavour compounds.

BACKGROUND: During alcoholic fermentation, Saccharomyces cerevisiae synthesizes more than 400 different compounds with higher alcohols, acetate esters of higher alcohols and ethyl esters of medium-chain fatty acids being the most important products of its metabolism, determining the particular flavour profile of each wine. The concentration of the metabolites produced depends to a large extent on the strain used. The use of indigenous strains as starting cultures can lead to the production of wines with excellent organoleptic characteristics and distinct local character, superior in quality when compared to their commercial counterparts. However, the relationship of these wild-type genotypes, linked to specific terroirs, with the biosynthetic profiles of flavour metabolites is not completely clarified and understood. To this end, qRT-PCR was employed to examine, for the first time on the transcriptional level, the performance of an indigenous Saccharomyces cerevisiae strain (Z622) in its natural environment (Debina grape must). The expression of genes implicated in higher alcohols and esters formation was correlated with the concentrations of these compounds in the produced wine. Furthermore, by applying the same fermentation conditions, we examined the same parameters in a commercial strain (VL1) and compared its performance with the one of strain Z622. RESULTS: Strain Z622, exhibited lower concentrations of 2-methylbutanol, 3-methylbutanol and 2-phenyl ethanol, than VL1 correlating with the elevated expression levels of transaminase and decarboxylase genes. Furthermore, the significantly high induction of ADH3 throughout fermentation of Z622 probably explains the larger population numbers reached by Z622 and reflects the better adaptation of the strain to its natural environment. Regarding acetate ester biosynthesis, Z622 produced higher concentrations of total acetate esters, compared with VL1, a fact that is in full agreement with the elevated expression levels of both ATF1 and ATF2 in strain Z622. CONCLUSIONS: This study provides evidence on the transcriptional level that indigenous yeast Z622 is better adapted to its natural environment able to produce wines with desirable characteristics, i.e. lower concentrations of higher alcohol and higher ester, verifying its potential as a valuable starter for the local wine-industry.

Release of Cell-Free Ice Nucleators from Three Recombinant Ice Zymomonas mobilis Strains.

BACKGROUND/AIMS: This work is a study of the ability of three recombinant Zymomonas mobilis strains to release ice nucleators into their growth medium. METHODS: The recombinant ice(+)Z. mobilis cells were tested for their ability to produce cell-free ice nucleators, under three different growth temperatures and three different glucose concentrations. RESULTS: Cell-free ice nucleators were obtained from all the recombinant ice(+)Z. mobilis cells tested. The cell-free ice nucleation activity was not affected by the glucose concentration in the growth medium or the growth temperature. The freezing temperature threshold was below -7.6°C, demonstrating a class C nucleating structure of the ice nucleation protein. The size of the ice nucleators was less than 0.22 µm and their density was estimated as 1.024 ± 0.004 g/ml by Percoll density centrifugation. The properties of the detected ice nucleators, in addition to the absence of pyruvate decarboxylase activity in the spent medium (a cytosolic marker), support that the cell-free ice nucleation activity was due to the extracellular release of ice nucleators. CONCLUSION: These findings indicate that the recombinant ice(+)Z. mobilis cells could be valuable for future use as a source of active cell-free ice nucleation protein.

Complete genome sequence of Mycobacterium sp. strain (Spyr1) and reclassification to Mycobacterium gilvum Spyr1.

Mycobacterium sp.Spyr1 is a newly isolated strain that occurs in a creosote contaminated site in Greece. It was isolated by an enrichment method using pyrene as sole carbon and energy source and is capable of degrading a wide range of PAH substrates including pyrene, fluoranthene, fluorene, anthracene and acenapthene. Here we describe the genomic features of this organism, together with the complete sequence and annotation. The genome consists of a 5,547,747 bp chromosome and two plasmids, a larger and a smaller one with sizes of 211,864 and 23,681 bp, respectively. In total, 5,588 genes were predicted and annotated.

Complete genome sequence of Arthrobacter phenanthrenivorans type strain (Sphe3).

Arthrobacter phenanthrenivorans is the type species of the genus, and is able to metabolize phenanthrene as a sole source of carbon and energy. A. phenanthrenivorans is an aerobic, non-motile, and Gram-positive bacterium, exhibiting a rod-coccus growth cycle which was originally isolated from a creosote polluted site in Epirus, Greece. Here we describe the features of this organism, together with the complete genome sequence, and annotation.

The effect of Debina grapevine indigenous yeast strains of Metschnikowia and Saccharomyces on wine flavour.

The spontaneous alcoholic fermentation of grape must is a complex microbiological process involving a large number of various yeast species, to which the flavour of every traditional wine is largely attributed. Whilst Saccharomyces cerevisiae is primarily responsible for the conversion of sugar to alcohol, the activities of various non-Saccharomyces species enhance wine flavour. In this study, indigenous yeast strains belonging to Metschnikowia pulcherrima var. zitsae as well as Saccharomyces cerevisiae were isolated and characterized from Debina must (Zitsa, Epirus, Greece). In addition, these strains were examined for their effect on the outcome of the wine fermentation process when used sequentially as starter cultures. The resulting wine, as analyzed over three consecutive years, was observed to possess a richer, more aromatic bouquet than wine from a commercial starter culture. These results emphasize the potential of employing indigenous yeast strains for the production of traditional wines with improved flavour.

Proton inventories constitute reliable tools in investigating enzymatic mechanisms: application on a novel thermo-stable extracellular protease from a halo-alkalophilic Bacillus sp.

A novel protease designated protease-A-17N-1, was purified from the halo-alkalophilic Bacillus sp. 17N-1, and found active in media containing dithiothreitol and EDTAK(2). This enzyme maintained significant activity from pH 6.00 to 9.00, showed optimum k(cat)/K(m) value at pH 7.50 and 33 degrees C. It was observed that only specific inhibitors of cysteine proteinases inhibited its activity. The pH-(k(cat)/K(m)) profile of protease-A-17N-1 was described by three pK(a)s in the acid limb, and one in the alkaline limb. Both are more likely due t3o the protonic dissociation of an acidic residue, and the development and subsequent deprotonation of an ion-pair, respectively, in its catalytic site, characteristic for cysteine proteinases. Moreover, both the obtained estimates of rate constant k(1) and the ratio k(2)/k(-1) at 25 degrees C, from the temperature-(k(cat)/K(m)) profile of protease-A-17N-1, were found similar to those estimated from the proton inventories of the same parameter, verifying the reliability of the latter methodology. Besides, the bowed-downward proton inventories of k(cat)/K(m), as well as the large inverse SIE observed for this parameter, in combination with its dependence versus temperature, were showed unambiguously that k(cat)/K(m) = k(1). Such results suggest that the novel enzyme is more likely to be a cysteine proteinase functioning via a general acid-base mechanism.