The tyrosyl-tRNA synthetase like gene located in the tyramine biosynthesis cluster of Enterococcus durans is transcriptionally regulated by tyrosine concentration and extracellular pH

<p>Abstract</p> <p>Background</p> <p>The tyramine producer <it>Enterococcus durans </it>IPLA655 contains all the necessary genes for tyramine biosynthesis, grouped in the TDC cluster. This cluster includes <it>tyrS</it>, an aminoacyl-tRNA synthetase like gene.</p> <p>Results</p> <p>This work shows that <it>tyrS </it>was maximally transcribed in absence of tyrosine at acidic pH, showing a greater than 10-fold induction in mRNA levels over levels occurring in presence of tyrosine. Mapping of the <it>tyrS </it>transcriptional start site revealed an unusually long untranslated leader region of 322 bp, which displays the typical features of the T box transcriptional attenuation mechanism. The tyrosine concentration regulation of <it>tyrS </it>was found to be mediated by a transcription antitermination system, whereas the specific induction at acidic pH was regulated at transcription initiation level.</p> <p>Conclusions</p> <p>The expression of the <it>tyrS </it>gene present in the TDC cluster of <it>E. durans </it>is transcriptionally regulated by tyrosine concentration and extracelular pH. The regulation is mediated by both an antitermination system and the promoter itself.</p

Bacteriophages in Dairy Industry: PCR Methods as Valuable Tools

Under CC BY 3.0 license. © The Author(s).Microorganisms have been empirically used since ancestral times to produce fermented dairy products from milk. In the actual dair y industry, milk is subjected to large scale fermentation processes that involve microorgan isms mostly belonging to the Lactic Acid Bacteria (LAB) group. Bacteriophages that in fect LAB have been claimed as one of the principal sources of fermentation failure (spo ilage or delay) on the manufacture of many dairy products (Brüssow et al., 1998; Josephsen & Neve, 1998; Garneau & Moineau, 2011). Some estimates assume that virulent phages are the primary direct responsible of the largest-economic loss of dairy factories, since th ey affect negatively up to the 10% of all milk fermentations (Moineau & Levesque, 2005).This work was performed with financial support from the Ministry of Science and Innovation, Spain (AGL2010- 18430). B. del Río an d N. Martínez are beneficiary of a JAE DOC-CSIC contract (Spain). D. M. Linares is beneficiary of a FICYT contract (Asturias, Spain).Peer Reviewe

Lactic Acid Bacteria as a Live Delivery System for the in situ Production of Nanobodies in the Human Gastrointestinal Tract

Lactic acid bacteria (LAB) are among the most widely used microorganisms in food fermentation. However, some LAB species can also be used as live vehicles for the in situ delivery of therapeutic molecules to the mucosa of the human gastrointestinal tract (GIT). Many LAB species have ‘qualified presumption of safety’ status and survive passage through the GIT. Indeed, some are part of the usual GIT microbiota. These are appropriate candidates for the in situ production of recombinant prophylactic and therapeutic proteins. Live recombinant LAB that produce microbial antigens have been shown to elicit an immune response that confers protection against the corresponding pathogens; these LAB could therefore be used as oral vaccines. In addition, some LAB have been genetically engineered to produce therapeutic, neutralizing antibodies. The variable domain of heavy-chain-only antibodies from camelids – known as VHH antibodies or nanobodies – has peculiar properties (nanoscale size, robust structure, acid resistance, high affinity and specificity, easily produced in bacteria, etc.) that make them ideal choices as LAB-produced immunotherapeutic agents. The present review examines the advantages offered by LAB for the in situ production of therapeutic proteins in the human GIT, discusses the use of in situ produced VHH antibody fragments, and assesses the usefulness of this strategy in the treatment of infectious and non-infectious gastrointestinal diseases

Construction and characterization of a double mutant of Enterococcus faecalis that does not produce biogenic amines

Enterococcus faecalis is a lactic acid bacterium characterized by its tolerance of very diverse environmental conditions, a property that allows it to colonize many different habitats. This species can be found in food products, especially in fermented foods where it plays an important role as a biopreservative and influences the development of organoleptic characteristics. However, E. faecalis also produces the biogenic amines tyramine and putrescine. The consumption of food with high concentrations of these compounds can cause health problems. The present work reports the construction, via homologous recombination, of a double mutant of E. faecalis in which the clusters involved in tyramine and putrescine synthesis (which are located in different regions of the chromosome) are no longer present. Analyses showed the double mutant to grow and adhere to intestinal cells normally, and that the elimination of genes involved in the production of tyramine and putrescine has no effect on the expression of other genes

Immobilization-Stabilization of &beta;-Glucosidase for Implementation of Intensified Hydrolysis of Cellobiose in Continuous Flow Reactors

Cellulose saccharification to glucose is an operation of paramount importance in the bioenergy sector and the chemical and food industries, while glucose is a critical platform chemical in the integrated biorefinery. Among the cellulose degrading enzymes, &beta;-glucosidases are responsible for cellobiose hydrolysis, the final step in cellulose saccharification, which is usually the critical bottleneck for the whole cellulose saccharification process. The design of very active and stable &beta;-glucosidase-based biocatalysts is a key strategy to implement an efficient saccharification process. Enzyme immobilization and reaction engineering are two fundamental tools for its understanding and implementation. Here, we have designed an immobilized-stabilized solid-supported &beta;-glucosidase based on the glyoxyl immobilization chemistry applied in porous solid particles. The biocatalyst was stable at operational temperature and highly active, which allowed us to implement 25 &deg;C as working temperature with a catalyst productivity of 109 mmol/min/gsupport. Cellobiose degradation was implemented in discontinuous stirred tank reactors, following which a simplified kinetic model was applied to assess the process limitations due to substrate and product inhibition. Finally, the reactive process was driven in a continuous flow fixed-bed reactor, achieving reaction intensification under mild operation conditions, reaching full cellobiose conversion of 34 g/L in a reaction time span of 20 min

Screening sourdough samples for gliadin-degrading activity revealed Lactobacillus casei strains able to individually metabolize the coeliac disease-related 33-mer peptide

A selective culture medium containing acid-hydrolyzed gliadins as the sole nitrogen source was used in the search for sourdough-indigenous lactic acid bacteria (LAB) with gliadin-metabolizing activity. Twenty gliadin-degrading LAB strains were isolated from 10 sourdoughs made in different ways and from different geographical regions. Fifteen of the 20 isolated strains were identified as Lactobacillus casei, a species usually reported as subdominant in sourdough populations. The other five gliadin-degrading strains belonged to the more commonly encountered sourdough species Leuconostoc mesenteroides and Lactobacillus plantarum. All these strains were shown to be safe in terms of their resistance to antimicrobial agents. When individually incubated with the α2-gliadin-derived immunotoxic 33-mer peptide (97.5 ppm), half of the L. casei strains metabolized at least 50% of it within 24 h. One strain metabolized 82% of the 33-mer peptide within 8 h, and fully made it disappear within 12 h. These results reveal for the first time the presence in sourdough of proteolytic L. casei strains with the capacity to individually metabolize the coeliac disease-related 33-mer peptide.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author