Paloma López

Identification and analysis of genes for tetracycline resistance and replication functions in the broad-host-range plasmid pLS1

The streptococcal plasmid pMV158 and its derivative pLS1 are able to replicate and confer tetracycline resistance in both Gram-positive and Gram-negative bacteria. Copy numbers of pLS1 were 24, 4 and 4 molecules per genome in Streptococcus pneumoniae, Bacillus subtilis and Escherichia coli, respectively. Replication of the streptococcal plasmids in E. coli required functional polA and recA genes. A copy-number mutation corresponding to a 332 base-pair deletion of pLS1 doubled the plasmid copy number in all three species. Determination of the complete DNA sequence of pLS1 revealed transcriptional and translational signals and four open reading frames. A putative inhibitory RNA was encoded in the region deleted by the copy-control mutation. Two putative mRNA transcripts encoded proteins for replication functions and tetracycline resistance, respectively. The repB gene encoded a trans-acting, 23,000 Mr protein necessary for replication, and the tet gene encoded a very hydrophobic, 50,000 Mr protein required for tetracycline resistance. The polypeptides corresponding to these proteins were identified by specific labeling of plasmid-encoded products. The tet gene of pLS1 was highly homologous to tet genes in two other plasmids of Gram-positive origin but different in both sequence and mode of regulation from tet genes of Gram-negative origin.

Biogenic amines in fermented foods.

Food-fermenting lactic acid bacteria (LAB) are generally considered to be non-toxic and non-pathogenic. Some species of LAB, however, can produce biogenic amines (BAs). BAs are organic, basic, nitrogenous compounds, mainly formed through decarboxylation of amino acids. BAs are present in a wide range of foods, including dairy products, and can occasionally accumulate in high concentrations. The consumption of food containing large amounts of these amines can have toxicological consequences. Although there is no specific legislation regarding BA content in many fermented products, it is generally assumed that they should not be allowed to accumulate. The ability of microorganisms to decarboxylate amino acids is highly variable, often being strain specific, and therefore the detection of bacteria possessing amino acid decarboxylase activity is important to estimate the likelihood that foods contain BA and to prevent their accumulation in food products. Moreover, improved knowledge of the factors involved in the synthesis and accumulation of BA should lead to a reduction in their incidence in foods.

Lactic acid bacteria producing B-group vitamins: a great potential for functional cereals products.

Wheat contains various essential nutrients including the B group of vitamins. However, B group vitamins, normally present in cereals-derived products, are easily removed or destroyed during milling, food processing or cooking. Lactic acid bacteria (LAB) are widely used as starter cultures for the fermentation of a large variety of foods and can improve the safety, shelf life, nutritional value, flavor and overall quality of the fermented products. In this regard, the identification and application of strains delivering health-promoting compounds is a fascinating field. Besides their key role in food fermentations, several LAB found in the gastrointestinal tract of humans and animals are commercially used as probiotics and possess generally recognized as safe status. LAB are usually auxotrophic for several vitamins although certain strains of LAB have the capability to synthesize water-soluble vitamins such as those included in the B group. In recent years, a number of biotechnological processes have been explored to perform a more economical and sustainable vitamin production than that obtained via chemical synthesis. This review article will briefly report the current knowledge on lactic acid bacteria synthesis of vitamins B2, B11 and B12 and the potential strategies to increase B-group vitamin content in cereals-based products, where vitamins-producing LAB have been leading to the elaboration of novel fermented functional foods. In addition, the use of genetic strategies to increase vitamin production or to create novel vitamin-producing strains will be also discussed.

Evidence that the Essential Response Regulator YycF in Streptococcus pneumoniae Modulates Expression of Fatty Acid Biosynthesis Genes and Alters Membrane Composition

The YycFG two-component system, originally identified in Bacillus subtilis, is highly conserved among gram-positive bacteria with low G+C contents. In Streptococcus pneumoniae, the YycF response regulator has been reported to be essential for cell growth, but the signal to which it responds and the gene members of the regulon remain unclear. In order to investigate the role of YycFG in S. pneumoniae, we increased the expression of yycF by using a maltose-inducible vector and analyzed the genome-wide effects on transcription and protein expression during the course of yycF expression. The induction of yycF expression increased histidine kinase yycG transcript levels, suggesting an autoregulation of the yycFG operon. Evidence from both proteomic and microarray transcriptome studies as well as analyses of membrane fatty acid composition indicated that YycFG is involved in the regulation of fatty acid biosynthesis pathways and in determining fatty acid chain lengths in membrane lipids. In agreement with recent transcriptome data on pneumococcal cells depleted of YycFG, we also identified several other potential members of the YycFG regulon that are required for virulence and cell wall biosynthesis and metabolism.

Pediococcus parvulus gtf gene encoding the GTF glycosyltransferase and its application for specific PCR detection of beta-D-glucan-producing bacteria in foods and beverages.

Exopolysaccharide production by lactic acid bacteria is beneficial in the dairy and oat-based food industries and is used to improve the texture of the fermented products. However, beta-D-glucan-producing bacteria are considered spoilage microorganisms in alcoholic beverages because their secreted exopolysaccharides alter the viscosity of cider, wine, and beer, rendering them unpalatable. The plasmidic glycosyltransferase (gtf) gene of the Pediococcus parvulus 2.6 strain isolated from ropy cider has been cloned and sequenced, and its GTF product was functionally expressed in Streptococcus pneumoniae. The GTF protein, which has glycosyltransferase activity, belongs to the COG1215 membrane-bound glycosyltransferase family, and agglutination tests revealed that the enzyme enables S. pneumoniae to synthesize beta-D-glucan. PCR amplification and Southern blot hybridization showed that the gtf gene is also present at different genomic locations in the beta-D-glucan producers Lactobacillus diolivorans G77 and Oenococcus oeni I4 strains, also isolated from ropy cider. A PCR assay has been developed for the detection of exopolysaccharide-producing bacteria. Forward and reverse primers, included respectively in the coding sequences of the putative glycosyltransferase domain and the fifth trans-membrane segment of the GTF, were designed. Analysis of 76 ropy and nonropy lactic acid bacteria validated the method for specific detection of beta-D-glucan homopolysaccharide producer Pediococcus, Lactobacillus, and Oenococcus strains. The limit of the assay in cider was 3 X 10(2) CFU/ml. This molecular method can be useful for the detection of ropy bacteria in cider before spoilage occurs, as well as for isolation of new exopolysaccharide-producing strains of industrial interest.

Probiotic Properties of the 2-Substituted (1,3)-β-d-Glucan-Producing Bacterium Pediococcus parvulus 2.6

ABSTRACT Exopolysaccharides have prebiotic potential and contribute to the rheology and texture of fermented foods. Here we have analyzed the in vitro bioavailability and immunomodulatory properties of the 2-substituted (1,3)-β-d-glucan-producing bacterium Pediococcus parvulus 2.6. It resists gastrointestinal stress, adheres to Caco-2 cells, and induces the production of inflammation-related cytokines by polarized macrophages.

Beta-Glucans Improve Growth, Viability and Colonization of Probiotic Microorganisms

Probiotics, prebiotics and synbiotics are frequently-used components for the elaboration of functional food. Currently, most of the commercialized probiotics are limited to a few strains of the genera Bifidobacteria, Lactobacillus and Streptococcus, most of which produce exopolysaccharides (EPS). This suggests that the beneficial properties of these microorganisms may be related to the biological activities of these biopolymers. In this work we report that a 2-substituted-(1,3)-β-d-glucan of non-dairy bacterial origin has a prebiotic effect on three probiotic strains. Moreover, the presence of this β-d-glucan potentiates in vitro adhesion of the probiotic Lactobacillus plantarum WCFS1 to human intestinal epithelial cells.

Naturally occurring 2-substituted (1,3)-β-D-glucan producing Lactobacillus suebicus and Pediococcus parvulus strains with potential utility in the production of functional foods.

We have isolated three lactic acid bacteria (Lactobacillus suebicus CUPV221, Pediococcus parvulus CUPV1 and P. parvulus CUPV22) that produced high levels of 2-substituted (1,3)-beta-D-glucans which increased the viscosity of the growth media. The (1,3)-beta-D-glucan consisted of two main molecular species, with masses of approximately 10(7) and 10(4) Da, whose proportions varied among the strains. The three strains survived exposure to saliva and simulated gastric conditions at pH 5, with P. parvulus CUPV22 surviving at pH 3.1, and L. suebicus CUPV221 surviving at pH 1.8. All strains were resistant to pancreatin and bile salts. P. parvulus CUPV22 exhibited the highest adhesion (10.5%) to Caco-2 cells, which decreased to 1.2% after washing the cells. Finally, P. parvulus CUPV22 and L. suebicus CUPV221 induced the production of inflammation-related cytokines by polarized macrophages, and interestingly, L. suebicus stimulated the production of cytokine IL-10. These results indicate that the three strains have potential utility for the production of functional foods.

Selective advantage of deletions enhancing chloramphenicol acetyltransferase gene expression in Streptococcus pneumoniae plasmids.

A hybrid plasmid, pJS37, was made by combining pLS1, which confers tetracycline (Tc) resistance, and pC194, which confers chloramphenicol (Cm) resistance. Both pJS37 (7.3 kb) and its derivative pJS140 (6.0 kb), from which pC194 replication genes were removed, were structurally and segregationally stable when introduced into Streptococcus pneumoniae and grown either in the presence of Tc or in the absence of drug. However, both hybrid plasmids underwent systematic deletion when grown in the presence of Cm. One of the deleted forms, pJS4 (3.4 kb), could not be maintained in the absence of a helper plasmid; two others, pJS3 (4.1 kb) and pJS5 (3.8 kb), lost the tet gene but retained the replication functions of pLS1. They both expressed very high levels of Cm acetyltransferase (CAT), which, in the case of pJS5, were constitutive. Nucleotide sequence determination of the deletion junctions in pJS3 and pJS5 indicated that the deletions occurred, presumably by recombination, between short direct repeats of 6 and 9 bp, respectively. In both cases the tet promoter was juxtaposed to the cat gene. In the case of pJS5, the deletion removed a sequence that sequestered the ribosome-binding site (RBS) for cat, thereby rendering constitutive the production of CAT. The increased resistance to Cm afforded by the hyperexpression of the cat gene apparently provided a positive selective advantage for the accumulation of the deleted forms in the plasmid pool.

Activation of the diacetyl/acetoin pathway in Lactococcus lactis subsp. lactis bv. diacetylactis CRL264 by acidic growth.

ABSTRACT Lactococcus lactis subsp. lactis bv. diacetylactis strains are aroma-producing organisms used in starter cultures for the elaboration of dairy products. This species is essentially a fermentative microorganism, which cometabolizes glucose and citrate to yield aroma compounds through the diacetyl/acetoin biosynthetic pathway. Our previous results have shown that under acidic growth Lactococcus bv. diacetylactis CRL264 expresses coordinately the genes responsible for citrate transport and its conversion into pyruvate. In the present work the impact of acidic growth on glucose, citrate, and pyruvate metabolism of Lactococcus bv. diacetylactis CRL264 has been investigated by proteomic analysis. The results indicated that acid growth triggers the conversion of citrate, but not glucose, into α-acetolactate via pyruvate. Moreover, they showed that low pH has no influence on levels of lactate dehydrogenase and pyruvate dehydrogenase. Therefore, the influence of external pH on regulation of the diacetyl/acetoin biosynthetic pathway in Lactococcus bv. diacetylactis CRL264 has been analyzed at the transcriptional level. Expression of the als, aldB, aldC, and butBA genes encoding the enzymes involved in conversion of pyruvate into aroma compounds has been investigated by primer extension, reverse transcription-PCR analysis, and transcriptional fusions. The results support that this biosynthetic pathway is induced at the transcriptional level by acidic growth conditions, presumably contributing to lactococcal pH homeostasis by synthesis of neutral compounds and by decreasing levels of pyruvate.