Sinorhizobium meliloti uses proline betaine (PB) as an osmoprotectant when osmotically stressed and as an energy source in low-osmolarity environments. To fulfill this dual function, two separate PB transporters, BetS and Hut, that contribute to PB uptake at high and low osmolarity, respectively, have been previously identified. Here, we characterized a novel transport system that mediates the uptake of PB at both high and low osmolarities. Sequence analysis of Tn5-luxAB chromosomal insertions from several PB-inducible mutants has revealed the presence of a four-gene locus encoding the components of an ABC transporter, Prb, which belongs to the oligopeptide permease (Opp) family. Surprisingly, prb mutants were impaired in their ability to transport PB, and oligopeptides were not shown to be competitors for PB uptake. Further analysis of Prb specificity has shown its ability to take up other quaternary ammonium compounds such as choline and, to a lesser extent, glycine betaine. Interestingly, salt stress and PB were found to control prb expression in a positive and synergistic way and to increase Prb transport activity. At low osmolarity, Prb is largely implicated in PB uptake by stationary-phase cells, likely to provide PB as a source of carbon and nitrogen. Furthermore, at high osmolarity, the analysis of prb and betS single and double mutants demonstrated that Prb, together with BetS, is a key system for protection by PB.

Monitoring the hydrolysis or disappearance of starch by the starch iodine test is a simple procedure for determining the maturity of cider apples, but it does not indicate the exact amount of starch present.

Lactic acid bacteria and yeasts were numerated, isolated and identified from ciders prepared with the same variety of apple from apple trees grown on three different lands: silt, siliceous clay and green clay. The land has an incidence on the time (earliness) of ripeness of the apples and on their sugar content and, therefore, on the development of the microbial flora in the ciders obtained from these fruits. The same genera of yeasts and lactic acid bacteria were isolated from the three ciders, except the yeast Candida famata exclusively found in the must obtained from apples grozvn on silt. At the quantitative level, the highest development of yeasts was observed in the must from the land green clay whereas lactic acid bacteria growth was enhanced in the must from the land siliceous clay.Key Words: Lactic acid bacteria, yeasts, apple, lands, cider making. INTRODUCTIONThe fermentation of apple juice to produce an alcoholic beverage is believed to have been practised for over 2000 years10. The production of cider is carried out by sequential apple juice extraction using mechanical presses followed by two microbiological processes of great significance : alcoholic fermentation and malolactic conversion316. In traditional cider making no external source of yeast is added to the must and alcoholic fermentation is effected by wild yeasts from apples and utensils. Spontaneous fermentation will begin within a few hours if the temperature of the must is above 10°C14. press. The juices were poured into barrels and stored at 10°C under inert gas. Microbial samples from ciderSamples for analysis of the musts were collected aseptically via a sampling port and cider was allowed to run out for few seconds before the samples were collected. Media and culture conditionsA series of 10-fold dilutions in physiological saline (NaCl 0.9%) was made for each sample. Aliquots (0.1 ml) of appropriate dilution were plated onto duplicate plates of raka ray agar (obtained from Oxoid Unipath, UK) and yeast extract glucose (yeast extract 5g/litre, agar 15 g/litre, glucose 20 g/litre YEG). Lactic acid bacteria (LAB) were detected on raka ray agar. The YEG supplemented with chloramphenicol at a 100 ppm concentration was used to enumerate yeasts. Plates were incubated microaerophilically at 30°C After incubation for 5 days no increase in colony number was found and total colony counts were subsequently made. Identification of microorganismsYeasts were identified using API20 galleries (APISystem-Biomerieux, France) complemented by other tests (sporulation and pseudomycelium formation). These tests although not always sufficient to identify species, were suitable for genera determination. The identification of lactic acid bacteria was achieved using API50-CH galleries. The profile drawn for each isolated microorganism allowed determination between species.Analytical methods Sugars (glucose, fructose and saccharose) were analysed by HPLC according to the procedure described by Morvan-Bertrand et al.u using mannitol as internal standard or by refr...

Lactic acid bacteria and yeasts were numerated, isolated and identified from ciders prepared with the same variety of apple from apple trees grown on three different lands: silt, siliceous clay and green clay. The land has an incidence on the time (earliness) of ripeness of the apples and on their sugar content and, therefore, on the development of the microbial flora in the ciders obtained from these fruits. The same genera of yeasts and lactic acid bacteria were isolated from the three ciders, except the yeast Candida famata exclusively found in the must obtained from apples grown on silt. At the quantitative level, the highest development of yeasts was observed in the must from the land green clay whereas lactic acid bacteria growth was enhanced in the must from the land siliceous clay.