Microbial species present on the surface of grape berries at harvest play an important role in winemaking, thus counting and identifying them is of great importance. The use of conventional microbial techniques and molecular methods allowed a quantitative and qualitative inventory of the different microbial species present on the grape berries. These experiments were carried out in several areas of the Bordeaux region on the red grape varieties Merlot, Cabernet Sauvignon and Cabernet Franc. Populations and species clearly varied according to berry development stage. The most widespread yeast species at berry set, Aureobasidium pullulans was never detected at harvest. Fermentative yeasts were detected at harvest and not in the first stage of grape growth. Oenoccocus oeni was detected on immature as well as on mature berries. Gluconobacter oxydans was detected mainly at harvest. Detection of Pediococcus parvulus, was dependent on the vineyard. Veraison appeared to be a key stage for yeast colonisation and the increase in population involved a change in the proportion of each species. The number of A. pullulans fell significantly at veraison as it was superseded by fermentative yeasts. Microbial populations peaked at harvest when the berry surface available for adhesion was largest and no agrochemical treatments had been applied for some weeks. Soil, grape variety and grapegrowing practices may also influence this microbial ecosystem. Based on these and published data, we formulated hypotheses to describe this microbial ecosystem, thus enabling us to develop the concept of a microbial biofilm.

Histamine production from histidine in fermented food products by lactic acid bacteria results in food spoilage and is harmful to consumers. We have isolated a histamine-producing lactic acid bacterium, Lactobacillus hilgardii strain IOEB 0006, which could retain or lose the ability to produce histamine depending on culture conditions. The hdcA gene, coding for the histidine decarboxylase of L. hilgardii IOEB 0006, was located on an 80-kb plasmid that proved to be unstable. Sequencing of the hdcA locus disclosed a four-gene cluster encoding the histidine decarboxylase, a protein of unknown function, a histidyl-tRNA synthetase, and a protein, which we named HdcP, showing similarities to integral membrane transporters driving substrate/ product exchange. The gene coding for HdcP was cloned downstream of a sequence specifying a histidine tag and expressed in Lactococcus lactis. The recombinant HdcP could drive the uptake of histidine into the cell and the exchange of histidine and histamine. The combination of HdcP and the histidine decarboxylase forms a typical bacterial decarboxylation pathway that may generate metabolic energy or be involved in the acid stress response. Analyses of sequences present in databases suggest that the other two proteins have dispensable functions. These results describe for the first time the genes encoding a histamine-producing pathway and provide clues to the parsimonious distribution and the instability of histamine-producing lactic acid bacteria.

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The evolution of the wine microbial ecosystem is generally restricted to Saccharomyces cerevisiae and Oenococcus oeni, which are the two main agents in the transformation of grape must into wine by acting during alcoholic and malolactic fermentation, respectively. But others species like the yeast Brettanomyces bruxellensis and certain ropy strains of Pediococcus parvulus can spoil the wine. The aim of this study was to address the composition of the system more precisely, identifying other components. The advantages of the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) approach to wine microbial ecology studies are illustrated by bacteria and yeast species identification and their monitoring at each stage of wine production. After direct DNA extraction, PCR-DGGE was used to make the most exhaustive possible inventory of bacteria and yeast species found in a wine environment. Phylogenetic neighbor-joining trees were built to illustrate microbial diversity. PCR-DGGE was also combined with population enumeration in selective media to monitor microbial changes at all stages of production. Moreover, enrichment media helped to detect the appearance of spoilage species. The genetic diversity of the wine microbial community and its dynamics during winemaking were also described. Most importantly, our study provides a better understanding of the complexity and diversity of the wine microbial consortium at all stages of the winemaking process: on grape berries, in must during fermentation, and in wine during aging. On grapes, 52 different yeast species and 40 bacteria could be identified. The diversity was dramatically reduced during winemaking then during aging. Yeast and lactic acid bacteria were also isolated from very old vintages. B. bruxellensis and O. oeni were the most frequent.

Agmatine deiminase pathway genes in Lactobacillus brevis are linked to the tyrosine decarboxylation operon in a putative acid resistance locus In lactic acid bacteria (LAB), amino acids and their derivatives may be converted into aminecontaining compounds designated biogenic amines, in pathways providing metabolic energy and/ or acid resistance to the bacteria. In a previous study, a pathway converting tyrosine to tyramine was detected in Lactobacillus brevis and a fragment of a gene possibly involved in the production of another biogenic amine, putrescine, from agmatine, was detected in the same locus. The present study was carried out to determine if Lb. brevis actually harbours two biogenic amineproducing pathways in the same locus and to investigate the occurrence of the two gene clusters in other bacteria. Sequencing of the DNA locus in Lb. brevis revealed a cluster of six genes that are related to previously reported genes of agmatine deiminase pathways but with marked differences such as two genes encoding putative agmatine deiminases rather than one. Heterologous expression of encoded enzymes confirmed the presence of at least one active agmatine deiminase and one amino acid transporter that efficiently exchanged agmatine and putrescine. It was concluded that the Lb. brevis gene cluster encodes a functional and highly specific agmatine deiminase pathway. Screening of a collection of 197 LAB disclosed the same genes in 36 strains from six different species, and almost all the positive bacteria also contained the tyrosine catabolic pathway genes in the same locus. These results support the hypothesis that the agmatine deiminase and tyrosine catabolic pathways belong to a genomic region that provides acid resistance and that is exchanged horizontally as a whole between LAB. INTRODUCTIONLactic acid bacteria (LAB) can produce metabolic energy or increase their acid resistance by using catabolic pathways that convert amino acids or their derivatives into aminecontaining compounds designated biogenic amines (Molenaar et al., 1993; Fernández & Zú ñiga, 2006). In bacteria, biogenic amines may serve physiological functions such as cell proliferation control, but they are more generally released into the medium as the end products of the catabolic pathways (Tabor & Tabor, 1985;Griswold et al., 2006). In fact, excretion of the amine is an essential step in the pathways (see below). LAB carrying amino acid catabolic pathways are an important source of biogenic amines in foods (Ten Brink et al., 1990;Stratton et al., 1991;Silla Santos, 1996). There is interest in avoiding the proliferation of these bacteria because ingestion of foods containing large quantities of biogenic amines may cause human health issues such as headache, palpitations, flushing or vomiting (Silla Santos, 1996). Recently the genes of diverse pathways producing biogenic amines were identified in LAB (for a review, see Fernández & Zú ñiga, 2006). Interestingly, the pathways are strain specific rather than species specific, suggesting that horizonta...