Biomedical subjects

Angela Marcobal

Publications and source records attributed to Angela Marcobal.

10 recordsLinked to original sources

PubMed2006

Evidence for horizontal gene transfer as origin of putrescine production in Oenococcus oeni RM83.

The nucleotide sequence of a 17.2-kb chromosomal DNA fragment containing the odc gene encoding ornithine decarboxylase has been determined in the putrescine producer Oenococcus oeni RM83. This DNA fragment contains 13 open reading frames, including genes coding for five transposases and two phage proteins. This description might represent the first evidence of a horizontal gene transfer event as the origin of a biogenic amine biosynthetic locus.

Chromosomes, Bacterial↗

PubMed2006

A multifactorial design for studying factors influencing growth and tyramine production of the lactic acid bacteria Lactobacillus brevis CECT 4669 and Enterococcus faecium BIFI-58.

A central composite face design was used to study growth and tyramine production of two strains of lactic acid bacteria, Lactobacillus brevis CECT 4669 and Enterococcus faecium BIFI-58. The effects of five physicochemical factors (incubation temperature and time, environmental pH, added tyrosine concentration, and pyridoxal-5-phosphate (PLP) supplementation) on cell growth and tyramine production were analyzed under aerobic and anaerobic conditions. The parameters of the quadratic model for each response variable were estimated by multiple linear regression (MLR), and statistical analysis of the results led to the elucidation of mathematical models capable of predicting the behavior of the responses as a function of the main variables involved in the process. Incubation time was found to be the most important variable influencing growth in L. brevis, while pH showed the highest contribution in E. faecium. The production of tyramine was dependent on the added tyrosine concentration and incubation time. The proposed MLR model predicted the optimum conditions that gave maximum responses for L. brevis and E. faecium growth and tyramine production. In both strains, this model predicted that the anaerobic condition at acidic pH (4.4) in the presence of a high tyrosine concentration favors tyramine production.

Culture Media↗

PubMed2006

First genetic characterization of a bacterial beta-phenylethylamine biosynthetic enzyme in Enterococcus faecium RM58.

Enterococcus faecium RM58 produces beta-phenylethylamine and tyramine. A gene from Ent. faecium RM58 coding for a 625 amino-acid residues protein that shows 85% identity to Enterococcus faecalis tyrosine decarboxylase has been expressed in Escherichia coli, resulting in L-phenylalanine and L-tyrosine decarboxylase activities. Both activities were lost when a truncated protein lacking 84 amino acids at its C-terminus was expressed in E. coli. This study constitutes the first genetic characterization of a bacterial protein having L-phenylalanine decarboxylase activity and solves a long-standing question regarding the specificity of tyrosine decarboxylases in enterococci.

Carboxy-Lyases↗

PubMed2006

PCR detection of foodborne bacteria producing the biogenic amines histamine, tyramine, putrescine, and cadaverine.

This study describes an easy PCR method for the detection of foodborne bacteria that potentially produce histamine, tyramine, putrescine, and cadaverine. Synthetic oligonucleotide pairs for the specific detection of the gene coding for each group of bacterial histidine, tyrosine, ornithine, or lysine decarboxylases were designed. Under the conditions used in this study, the assay yielded fragments of 372 and 531 bp from histidine decarboxylase-encoding genes, a 825-bp fragment from tyrosine decarboxylases, fragments of 624 and 1,440 bp from ornithine decarboxylases, and 1,098- and 1,185-bp fragments from lysine decarboxylases. This is the first PCR method for detection of cadaverine-producing bacteria. The method was successfully applied to several biogenic amine-producing bacterial strains.

Bacteriological Techniques↗

PubMed2005

Improved multiplex-PCR method for the simultaneous detection of food bacteria producing biogenic amines.

This study describes a simple and rapid multiplex-PCR method to determine the ability to produce histamine, tyramine and putrescine by bacteria. The assay is an improved method based on an assay designed for lactic acid bacteria. This improved method includes a pair of primers based on sequences from histidine decarboxylases from Gram-negative bacteria. Under the optimised conditions, the assay yielded a 367-bp DNA fragment from histidine decarboxylases of Gram-positive bacteria, 534-bp fragment from histidine decarboxylases of Gram-negative bacteria, 924-bp from bacterial tyrosine decarboxylases, and 1446-bp fragment from bacterial ornithine decarboxylases. The method was successfully applied to several biogenic amine-producing bacterial strains, even when DNAs of several target organisms were included in the same reaction. This simple method could be easily incorporated in food control laboratories to detect potentially biogenic amine-producing bacteria in foods.

Bacteriological Techniques↗

PubMed2005

Multiplex PCR method for the simultaneous detection of histamine-, tyramine-, and putrescine-producing lactic acid bacteria in foods.

In a screening of primers, we have selected three pairs of primers for a multiplex PCR assay for the simultaneous detection of lactic acid bacteria (LAB) strains, which potentially produce histamine, tyramine, and putrescine on fermented foods. These primers were based on sequences from histidine, tyrosine, and ornithine decarboxylases from LAB. Under the optimized conditions, the assay yielded a 367-bp DNA fragment from histidine decarboxylases, a 924-bp fragment from tyrosine decarboxylases, and a 1,446-bp fragment from ornithine decarboxylases. When the DNAs of several target organisms were included in the same reaction, two or three corresponding amplicons of different sizes were observed. This assay was useful for the detection of amine-producing bacteria in control collection strains and in a LAB collection. No amplification was observed with DNA from nonproducing LAB strains. This article is the first describing a multiplex PCR approach for the simultaneous detection of potentially amine-producing LAB in foods. It can be easily incorporated into the routine screening for the accurate selection of starter LAB and in food control laboratories.

Amino Acid Sequence↗

PubMed2004

Tannase activity by lactic acid bacteria isolated from grape must and wine.

We examined a range of oenological lactic acid bacteria species and reference strains for their potential to degrade tannins. Bacterial tannase activity was checked by a spectrophotometric and a visual reading method. None of the strains belonging to the oenological species of the genus Lactobacillus, Leuconostoc, Oenococcus or Pediococcus were tannase producers, with the exception of Lactobacillus plantarum. All the L. plantarum strains analyzed were positive for tannase activity and their identities were reconfirmed by L. plantarum PCR-specific assay or by sequencing the 16S rDNA. Tannase activity could be considered an important criterion for the selection of malolactic starter cultures since it might confer advantages in the winemaking process by reducing astringency and haze in wine.

Base Sequence↗

PubMed2004

Identification of the ornithine decarboxylase gene in the putrescine-producer Oenococcus oeni BIFI-83.

We report here the identification of an ornithine decarboxylase (ODC) gene in the putrescine-producer Oenococcus oeni BIFI-83 strain. The gene contains a 2,235-nucleotide open reading frame encoding a 745-amino acid residues protein with a deduced molecular mass of 81 kDa. The primary structure of the ODC deduced from the nucleotide sequence has a consensus sequence containing the pyridoxal-5-phosphate (PLP) binding domain, and the critical amino acids residues involved in enzymatic activity are also conserved. As determined by BLAST analysis, the deduced amino acid sequence of the odc gene shares a 67% identity with the ODC protein from Lactobacillus 30a. The odc gene appears to be rarely present in the genome of O. oeni, since in a screening for the presence of this gene in 42 oenococcal strains none of the strains possessed an odc gene copy.

Amino Acid Sequence↗

PubMed2004

The tyrosine decarboxylation test does not differentiate Enterococcus faecalis from Enterococcus faecium.

According to the current edition of the Bergey's Manual of Systematic Bacteriology [11] the tyrosine decarboxylation test allows the differentiation of enterococci. Tyrosine is decarboxylated to the biogenic amine tyramine by E. faecalis and not by E. faecium strains. In the present study we sequenced the16S rDNA of two tyramine-producing strains, BIFI-56 and BIFI-58, presumptively classified as E. faecalis. Their 16S rDNA were identical to the same fragment from the E. faecium type strain. Several E. faecium strains were then checked for their ability to decarboxylate tyrosine and also a putative tyrosine decarboxylase-coding gene was PCR amplified from these strains. All the strains confirmed as E. faecium produced tyramine and possessed a DNA fragment coding for a putative tyrosine decarboxylase. The concordance of the two methods allows us to conclude that the tyrosine decarboxylase test cannot be used in the differentiation of E. faecalis from E. faecium since at least some E. faecium strains are tyramine producers.

Bacterial Typing Techniques↗

PubMed2004

Allelic diversity and population structure in Oenococcus oeni as determined from sequence analysis of housekeeping genes.

Oenococcus oeni is the organism of choice for promoting malolactic fermentation in wine. The population biology of O. oeni is poorly understood and remains unclear. For a better understanding of the mode of genetic variation within this species, we investigated by using multilocus sequence typing (MLST) with the gyrB, pgm, ddl, recP, and mleA genes the genetic diversity and genetic relationships among 18 O. oeni strains isolated in various years from wines of the United States, France, Germany, Spain, and Italy. These strains have also been characterized by ribotyping and restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S-23S rRNA gene intergenic spacer region (ISR). Ribotyping grouped the strains into two groups; however, the RFLP analysis of the ISRs showed no differences in the strains analyzed. In contrast, MLST in oenococci had a good discriminatory ability, and we have found a higher genetic diversity than indicated by ribotyping analysis. All sequence types were represented by a single strain, and all the strains could be distinguished from each other because they had unique combinations of alleles. Strains assumed to be identical showed the same sequence type. Phylogenetic analyses indicated a panmictic population structure in O. oeni. Sequences were analyzed for evidence of recombination by split decomposition analysis and analysis of clustered polymorphisms. All results indicated that recombination plays a major role in creating the genetic heterogeneity of O. oeni. A low standardized index of association value indicated that the O. oeni genes analyzed are close to linkage equilibrium. This study constitutes the first step in the development of an MLST method for O. oeni and the first example of the application of MLST to a nonpathogenic food production bacteria.

Alleles↗