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1.  Three-Component Lysine/Ornithine Decarboxylation System in Lactobacillus saerimneri 30a 
Journal of Bacteriology  2013;195(6):1249-1254.
Lactic acid bacteria play a pivotal role in many food fermentations and sometimes represent a health threat due to the ability of some strains to produce biogenic amines that accumulate in foods and cause trouble following ingestion. These strains carry specific enzymatic systems catalyzing the uptake of amino acid precursors (e.g., ornithine and lysine), the decarboxylation inside the cell, and the release of the resulting biogenic amines (e.g., putrescine and cadaverine). This study aimed to identify the system involved in production of cadaverine from lysine, which has not been described to date for lactic acid bacteria. Strain Lactobacillus saerimneri 30a (formerly called Lactobacillus sp. 30a) produces both putrescine and cadaverine. The sequencing of its genome showed that the previously described ornithine decarboxylase gene was not associated with the gene encoding an ornithine/putrescine exchanger as in other bacteria. A new hypothetical decarboxylation system was detected in the proximity of the ornithine decarboxylase gene. It consisted of two genes encoding a putative decarboxylase sharing sequence similarities with ornithine decarboxylases and a putative amino acid transporter resembling the ornithine/putrescine exchangers. The two decarboxylases were produced in Escherichia coli, purified, and characterized in vitro, whereas the transporter was heterologously expressed in Lactococcus lactis and functionally characterized in vivo. The overall data led to the conclusion that the two decarboxylases and the transporter form a three-component decarboxylation system, with the new decarboxylase being a specific lysine decarboxylase and the transporter catalyzing both lysine/cadaverine and ornithine/putrescine exchange. To our knowledge, this is an unprecedented observation of a bacterial three-component decarboxylation system.
doi:10.1128/JB.02070-12
PMCID: PMC3592000  PMID: 23316036
2.  Genome Sequence of Lactobacillus saerimneri 30a (Formerly Lactobacillus sp. Strain 30a), a Reference Lactic Acid Bacterium Strain Producing Biogenic Amines 
Genome Announcements  2013;1(1):e00097-12.
Lactobacillus sp. strain 30a (Lactobacillus saerimneri) produces the biogenic amines histamine, putrescine, and cadaverine by decarboxylating their amino acid precursors. We report its draft genome sequence (1,634,278 bases, 42.6% G+C content) and the principal findings from its annotation, which might shed light onto the enzymatic machineries that are involved in its production of biogenic amines.
doi:10.1128/genomeA.00097-12
PMCID: PMC3569274  PMID: 23405290
3.  Identification of pOENI-1 and Related Plasmids in Oenococcus oeni Strains Performing the Malolactic Fermentation in Wine 
PLoS ONE  2012;7(11):e49082.
Plasmids in lactic acid bacteria occasionally confer adaptive advantages improving the growth and behaviour of their host cells. They are often associated to starter cultures used in the food industry and could be a signature of their superiority. Oenococcus oeni is the main lactic acid bacteria species encountered in wine. It performs the malolactic fermentation that occurs in most wines after alcoholic fermentation and contributes to their quality and stability. Industrial O. oeni starters may be used to better control malolactic fermentation. Starters are selected empirically by virtue of their fermentation kinetics and capacity to survive in wine. This study was initiated with the aim to determine whether O. oeni contains plasmids of technological interest. Screening of 11 starters and 33 laboratory strains revealed two closely related plasmids, named pOENI-1 (18.3-kb) and pOENI-1v2 (21.9-kb). Sequence analyses indicate that they use the theta mode of replication, carry genes of maintenance and replication and two genes possibly involved in wine adaptation encoding a predicted sulphite exporter (tauE) and a NADH:flavin oxidoreductase of the old yellow enzyme family (oye). Interestingly, pOENI-1 and pOENI-1v2 were detected only in four strains, but this included three industrial starters. PCR screenings also revealed that tauE is present in six of the 11 starters, being probably inserted in the chromosome of some strains. Microvinification assays performed using strains with and without plasmids did not disclose significant differences of survival in wine or fermentation kinetics. However, analyses of 95 wines at different phases of winemaking showed that strains carrying the plasmids or the genes tauE and oye were predominant during spontaneous malolactic fermentation. Taken together, the results revealed a family of related plasmids associated with industrial starters and indigenous strains performing spontaneous malolactic fermentation that possibly contribute to the technological performance of strains in wine.
doi:10.1371/journal.pone.0049082
PMCID: PMC3489775  PMID: 23139835
5.  Biogenic amine production by the wine Lactobacillus brevis IOEB 9809 in systems that partially mimic the gastrointestinal tract stress 
BMC Microbiology  2012;12:247.
Background
Ingestion of fermented foods containing high levels of biogenic amines (BA) can be deleterious to human health. Less obvious is the threat posed by BA producing organisms contained within the food which, in principle, could form BA after ingestion even if the food product itself does not initially contain high BA levels. In this work we have investigated the production of tyramine and putrescine by Lactobacillus brevis IOEB 9809, of wine origin, under simulated gastrointestinal tract (GIT) conditions.
Results
An in vitro model that simulates the normal physiological conditions in the human digestive tract, as well as Caco-2 epithelial human cell lines, was used to challenge L. brevis IOEB 9809, which produced both tyramine and putrescine under all conditions tested. In the presence of BA precursors and under mild gastric stress, a correlation between enhancement of bacterial survival and a synchronous transcriptional activation of the tyramine and putrescine biosynthetic pathways was detected. High levels of both BA were observed after exposure of the bacterium to Caco-2 cells.
Conclusions
L. brevis IOEB 9809 can produce tyramine and putrescine under simulated human digestive tract conditions. The results indicate that BA production may be a mechanism that increases bacterial survival under gastric stress.
doi:10.1186/1471-2180-12-247
PMCID: PMC3499163  PMID: 23113922
Biogenic amines; Lactic acid bacteria; Putrescine; Tyramine; Food safety; Food toxicity
6.  Evidence of Two Functionally Distinct Ornithine Decarboxylation Systems in Lactic Acid Bacteria 
Biogenic amines are low-molecular-weight organic bases whose presence in food can result in health problems. The biosynthesis of biogenic amines in fermented foods mostly proceeds through amino acid decarboxylation carried out by lactic acid bacteria (LAB), but not all systems leading to biogenic amine production by LAB have been thoroughly characterized. Here, putative ornithine decarboxylation pathways consisting of a putative ornithine decarboxylase and an amino acid transporter were identified in LAB by strain collection screening and database searches. The decarboxylases were produced in heterologous hosts and purified and characterized in vitro, whereas transporters were heterologously expressed in Lactococcus lactis and functionally characterized in vivo. Amino acid decarboxylation by whole cells of the original hosts was determined as well. We concluded that two distinct types of ornithine decarboxylation systems exist in LAB. One is composed of an ornithine decarboxylase coupled to an ornithine/putrescine transmembrane exchanger. Their combined activities results in the extracellular release of putrescine. This typical amino acid decarboxylation system is present in only a few LAB strains and may contribute to metabolic energy production and/or pH homeostasis. The second system is widespread among LAB. It is composed of a decarboxylase active on ornithine and l-2,4-diaminobutyric acid (DABA) and a transporter that mediates unidirectional transport of ornithine into the cytoplasm. Diamines that result from this second system are retained within the cytosol.
doi:10.1128/AEM.07161-11
PMCID: PMC3298143  PMID: 22247134
7.  Multilocus Sequence Typing of Oenococcus oeni: Detection of Two Subpopulations Shaped by Intergenic Recombination▿ † 
Oenococcus oeni is the acidophilic lactic acid bacterial species most frequently associated with malolactic fermentation of wine. Since the description of the species (formerly Leuconostoc oenos), characterization of indigenous strains and industrially produced cultures by diverse typing methods has led to divergent conclusions concerning the genetic diversity of strains. In the present study, a multilocus sequence typing (MLST) scheme based on the analysis of eight housekeeping genes was developed and tested on a collection of 43 strains of diverse origins. The eight targeted loci were successfully amplified and sequenced for all isolates. Only three to 11 different alleles were detected for these genes. The average nucleotide diversity also was rather limited (0.0011 to 0.0370). Despite this limited allelic diversity, the combination of alleles of each strain disclosed 34 different sequence types, which denoted a significant genotypic diversity. A phylogenetic analysis of the concatenated sequences showed that all strains form two well distinct groups of 28 and 15 strains. Interestingly, the same groups were defined by pulsed-field gel electrophoresis, although this method targets different genetic variations. A minimum spanning tree analysis disclosed very few and small clonal complexes. In agreement, statistical analyses of MLST data suggest that recombination events were important during O. oeni evolution and contributed to the wide dissemination of alleles among strains. Taken together, our results showed that MLST is more efficient than pulsed-field gel electrophoresis for typing O. oeni strains, and they provided a picture of the O. oeni population that explains some conflicting results previously obtained.
doi:10.1128/AEM.02563-08
PMCID: PMC2648160  PMID: 19114515
8.  High Frequency of Histamine-Producing Bacteria in the Enological Environment and Instability of the Histidine Decarboxylase Production Phenotype▿  
Lactic acid bacteria contribute to wine transformation during malolactic fermentation. They generally improve the sensorial properties of wine, but some strains produce histamine, a toxic substance that causes health issues. Histamine-producing strains belong to species of the genera Oenococcus, Lactobacillus, and Pediococcus. All carry an hdcA gene coding for a histidine decarboxylase that converts histidine into histamine. For this study, a method based on quantitative PCR and targeting hdcA was developed to enumerate these bacteria in wine. This method was efficient for determining populations of 1 to 107 CFU per ml. An analysis of 264 samples collected from 116 wineries of the same region during malolactic fermentation revealed that these bacteria were present in almost all wines and at important levels, exceeding 103 CFU per ml in 70% of the samples. Histamine occurred at an often important level in wines containing populations of the above-mentioned bacteria. Fifty-four colonies of histamine producers isolated from four wines were characterized at the genetic level. All were strains of Oenococcus oeni that grouped into eight strain types by randomly amplified polymorphic DNA analysis. Some strains were isolated from wines collected in distant wineries. Moreover, hdcA was detected on a large and possibly unstable plasmid in these strains of O. oeni. Taken together, the results suggest that the risk of histamine production exists in almost all wines and is important when the population of histamine-producing bacteria exceeds 103 per ml. Strains of O. oeni producing histamine are frequent in wine during malolactic fermentation, but they may lose this capacity during subcultures in the laboratory.
doi:10.1128/AEM.01496-07
PMCID: PMC2227711  PMID: 18065614
9.  The Mechanism of the Tyrosine Transporter TyrP Supports a Proton Motive Tyrosine Decarboxylation Pathway in Lactobacillus brevis 
Journal of Bacteriology  2006;188(6):2198-2206.
The tyrosine decarboxylase operon of Lactobacillus brevis IOEB9809 contains, adjacent to the tyrosine decarboxylase gene, a gene for TyrP, a putative tyrosine transporter. The two genes potentially form a proton motive tyrosine decarboxylation pathway. The putative tyrosine transporter gene of L. brevis was expressed in Lactococcus lactis and functionally characterized using right-side-out membranes. The transporter very efficiently catalyzes homologous tyrosine-tyrosine exchange and heterologous exchange between tyrosine and its decarboxylation product tyramine. Tyrosine-tyramine exchange was shown to be electrogenic. In addition to the exchange mode, the transporter catalyzes tyrosine uniport but at a much lower rate. Analysis of the substrate specificity of the transporter by use of a set of 19 different tyrosine substrate analogues showed that the main interactions between the protein and the substrates involve the amino group and the phenyl ring with the para hydroxyl group. The carboxylate group that is removed in the decarboxylation reaction does not seem to contribute to the affinity of the protein for the substrates significantly. The properties of the TyrP protein are those typical for precursor-product exchangers that operate in proton motive decarboxylation pathways. It is proposed that tyrosine decarboxylation in L. brevis results in proton motive force generation by an indirect proton pumping mechanism.
doi:10.1128/JB.188.6.2198-2206.2006
PMCID: PMC1428153  PMID: 16513749
10.  Histamine-Producing Pathway Encoded on an Unstable Plasmid in Lactobacillus hilgardii 0006 
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.
doi:10.1128/AEM.71.3.1417-1424.2005
PMCID: PMC1065165  PMID: 15746344
11.  Rapid evolution of the DNA-binding site in LAGLIDADG homing endonucleases 
Nucleic Acids Research  2001;29(4):960-969.
Sequence analysis of chloroplast and mitochondrial large subunit rRNA genes from over 75 green algae disclosed 28 new group I intron-encoded proteins carrying a single LAGLIDADG motif. These putative homing endonucleases form four subfamilies of homologous enzymes, with the members of each subfamily being encoded by introns sharing the same insertion site. We showed that four divergent endonucleases from the I-CreI subfamily cleave the same DNA substrates. Mapping of the 66 amino acids that are conserved among the members of this subfamily on the 3-dimensional structure of I-CreI bound to its recognition sequence revealed that these residues participate in protein folding, homodimerization, DNA recognition and catalysis. Surprisingly, only seven of the 21 I-CreI amino acids interacting with DNA are conserved, suggesting that I-CreI and its homologs use different subsets of residues to recognize the same DNA sequence. Our sequence comparison of all 45 single-LAGLIDADG proteins identified so far suggests that these proteins share related structures and that there is a weak pressure in each subfamily to maintain identical protein–DNA contacts. The high sequence variability we observed in the DNA-binding site of homologous LAGLIDADG endonucleases provides insight into how these proteins evolve new DNA specificity.
PMCID: PMC29605  PMID: 11160929
12.  Biochemical characterization of I-CmoeI reveals that this H-N-H homing endonuclease shares functional similarities with H-N-H colicins 
Nucleic Acids Research  2000;28(22):4566-4572.
Endonuclease assays of the H-N-H proteins encoded by two group I introns in the Chlamydomonas moewusii chloroplast psbA gene revealed that the CmpsbA·1 intron specifies a site-specific DNA endonuclease, designated I-CmoeI. Like most previously reported intron-encoded endonucleases, I-CmoeI generates a double-strand break near the insertion site of its encoding intron, leaving 3′ extensions of 4 nt. This enzyme was purified from Escherichia coli as a fusion protein with a His tag at its N-terminus. The recombinant protein (rI-CmoeI) requires a divalent alkaline earth cation for DNA cleavage (Mg2+ > Ca2+ > Sr2+ > Ba2+). It also requires a metal cofactor for DNA binding, a property shared with H-N-H colicins but not with the homing endonucleases characterized to date. rI-CmoeI binds its recognition sequence as a monomer, as revealed by gel retardation assays. Km and kcat values of 100 ± 40 pM and 0.26 ± 0.04 min–1, respectively, were determined. Replacement of the first histidine of the H-N-H motif by an alanine residue abolishes both rI-CmoeI activity and binding to its substrate. We propose that this conserved histidine residue plays a role in binding the metal cofactor and that such binding induces a structural modification of the enzyme which is required for DNA recognition.
PMCID: PMC113871  PMID: 11071947

Results 1-12 (12)