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

Biogenic amines degradation by Lactobacillus plantarum: toward a potential application in wine.

Biogenic amines in wine represent a toxicological risk for the health of the consumer, with several trade implications. In this study 26 strains of Lactobacillus plantarum were analysed for their ability to degrade biogenic amines commonly found during wine fermentation. Two strains of L. plantarum were selected in reason of their ability to degrade putrescine and tyramine. The degradation was assessed in vitro, both in presence of the biogenic amines and in presence of the specific chemical precursor and of producer bacteria. The two L. plantarum biotypes were found capable to work synergically. In addition, the survival in wine-like medium and the aptitude to degrade malic acid after alcoholic fermentation of the selected L. plantarum strains was analysed. Our results suggest the potential application of wine L. plantarum strains to design malolactic starter cultures able to degrade biogenic amines in wine

Production of Fumaric Acid by Rhizopus arrhizus NRRL 1526: A Simple Production Medium and the Kinetic Modelling of the Bioprocess

Fumaric acid is a promising monomer to obtain biomass-based polyesters and polyamides, and it is mainly produced by fungi of the Rhizopus genus in medium to high titters. The use of glucose, a main component of starchy and cellulosic food waste, as carbon source, together with a low-nitrogen source concentration, is a promising route to reduce process costs. In this work, the effects of nitrogen and carbonate sources on Rhizopus arrhizus NRRL 1526 morphology and fumaric acid productivity were analysed, simplifying the traditional production broth composition. Moreover, a non-structured, non-segregated kinetic model was proposed and fitted to concentration data of all relevant components obtained in batches performed in triplicate with the selected production broth at 34 °C and 200 rpm in an orbital shaker.Depto. de Ingeniería Química y de MaterialesFac. de Ciencias QuímicasTRUEMinisterio de Ciencia, InnovaciónGerman Bundestag, via the Agency of Renewable Resourcespu

Aminas biógenas en alimentos: métodos moleculares para la detección e identificación de bacterias productoras

Biogenic amines are low molecular weight nitrogenous compounds with biological activity that are formed by decarboxylation of certain amino acids. Biogenic amines are present in all living organisms, in which they have important physiological functions. However, due to the metabolism of certain microorganisms, these compounds can accumulate in food at high concentrations, constituting a health risk for consumers. The presence of biogenic amine-producing microorganisms is the indispensable condition for the presence and further accumulation of biogenic amines in food. Because of this, several methods for the detection and identification of these biogenic amine-producing microorganisms have been developed. Among them, those based on culture independent techniques, such as the PCR, have several advantages such as their specificity, they are methods that are fast and easy to perform, and in many cases there is no need for pre-processing of food material which facilitates their incorporation at production plants. In this work, some available methods for the detection of biogenic amine-producing microorganisms are described, as well as their potential applications.Las aminas biógenas son compuestos nitrogenados de pequeño tamaño con actividad biológica que se forman por la descarboxilación enzimática de ciertos aminoácidos. Las aminas biógenas se encuentran presentes en todos los seres vivos, en los que participan en procesos biológicas de gran importancia. Sin em­bargo, debido al metabolismo de algunos microorganismos, estos compuestos se pueden acumular en alimentos en concentraciones elevadas, constituyendo un riesgo para la salud de los consumido­res. Para que las aminas biógenas alcancen estas concentraciones elevadas en los alimentos se requiere, como condición indispensa­ble, la presencia de microrganismos productores, por lo que se han desarrollado diferentes métodos para detectar la presencia de los mismos. Entre estos métodos, aquellos basados en técnicas inde­pendientes de cultivo, como la PCR, presentan ventajas como su gran especificidad, el hecho de ser rápidos y de fácil realización, y que en muchos casos ni siquiera es necesario un tratamiento previo de la muestra, lo que facilita su incorporación a las plantas de elabo­ración. En este trabajo se describen algunos de los métodos dispo­nibles en la actualidad para la detección de microorganismos pro­ductores de aminas biógenas, así como sus posibles aplicaciones

Putrescine biosynthesis and export genes are essential for normal growth of avian pathogenic Escherichia coli

[Background] Avian pathogenic Escherichia coli (APEC) is the infectious agent of a wide variety of avian diseases, which causes substantial economic losses to the poultry industry worldwide. Polyamines contribute to the optimal synthesis of nucleic acids and proteins in bacteria. The objectives of this study were to investigate; i) whether APEC E. coli encodes the same systems for biosynthesis and uptake as described for E. coli K12 and ii) the role of polyamines during in vitro growth of an avian pathogenic E. coli strain (WT-ST117- O83:H4T).[Results] Following whole genome sequencing, polyamine biosynthesis and export genes present in E. coli MG1655 (K-12) were found to be identical in WT-ST117. Defined mutants were constructed in putrescine and spermidine biosynthesis pathways (ΔspeB, ΔspeC, ΔspeF, ΔspeB/C and ΔspeD/E), and in polyamines transport systems (ΔpotE, ΔyeeF, ΔpotABCD and ΔpotFGHI). Contrary to what was observed for MG1655, the ΔpotE-ST117 mutant was growth attenuated, regardless of putrescine supplementation. The addition of spermidine or orthinine restored the growth to the level of WT-ST117. Growth attenuation after induction of membrane stress by SDS suggested that PotE is involved in protection against this stress. The ΔspeB/C-ST117 mutant was also growth attenuated in minimal medium. The addition of putrescine or spermidine to the media restored growth rate to the wild type level. The remaining biosynthesis and transport mutants showed a growth similar to that of WT-ST117. Analysis by Ultra-High Performance Liquid Chromatography revealed that the ΔspeB/C mutant was putrescine-deficient, despite that the gene speF, which is also involved in the synthesis of putrescine, was expressed.[Conclusions] Deletion of the putrescine transport system, PotE, or the putrescine biosynthesis pathway genes speB/C affected in vitro growth of APEC (ST117- O83:H4) strain, but not E. coli MG1655, despite the high similarity of the genetic make-up of biosynthesis and transport genes. Therefore, blocking these metabolic reactions may be a suitable way to prevent APEC growth in the host without disturbing the commensal E. coli population.Priscila R. Guerra was supported by a scholarship from CAPES – Brazilian Federal Agency for Support and Evaluation of Graduate Education within the Ministry of Education of Brazil. This study received financial support from Spanish Ministry of Economy, Industry and Competitiveness (AGL2013–45431-R and AGL2016–78708-R), and from the Danish Council for Independent Research (Technology and Production), grant number DFF – 4184-00050. The sponsors had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.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

Polyphasic Characterisation of Non-Starter Lactic Acid Bacteria from Algerian Raw Camel’s Milk and Their Technological Aptitudes

Pozadina istraživanja. Spontano fermentirano devino mlijeko često se konzumira u Alžiru, što ga čini održivim izvorom različitih bakterija mliječno-kiselog vrenja, koje se mogu dodati starter kulturi radi poboljšanja kakvoće i sigurnosti fermentiranih mliječnih proizvoda. Eksperimentalni pristup. Dvanaest uzoraka sirovog devinog mlijeka upotrijebljeno je kao izvor autohtonih bakterija mliječno-kiselog vrenja, koje su zatim okarakterizirane pomoću 39 fenotipskih osobina što imaju tehnološki značaj. Rezultati i zaključci. Trideset i devet nestarterskih bakterija mliječno-kiselog vrenja izolirano je iz 12 uzoraka alžirskog sirovog devinog mlijeka, te je mikrobiološki, biokemijski i genetički okarakterizirano. Neki su izolati imali proteolitičku aktivnost, svojstvo zakiseljavanja, sposobnost korištenja citrata i proizvodnje dekstrana i acetoina. Etanol, acetaldehid, metil-acetat, acetoin i octena kiselina glavni su hlapljivi spojevi pronađeni u uzorcima. Klasterskom analizom pomoću metode UPGMA (engl. unweighted pair group method with arithmetic mean) određena je prosječna udaljenost klastera na osnovu trideset i devet ispitanih fenotipskih značajki, te je potvrđena mikrobiološka raznolikost sirovog devinog mlijeka. Novina i znanstveni doprinos. Sojevi izolirani iz netipičnog izvora imali su zanimljive tehnološke značajke koje ih čine prikladnim za dodavanje starter kulturi. Klasterska analiza na osnovu ispitanih fenotipskih osobina pokazala se kao koristan alat za tipiziranje izolata u slučaju kad ne postoje genetske informacije o sojevima. Ove spoznaje mogu se primijeniti u industrijskoj proizvodnji prerađevina devinog mlijeka.Research background. Consumption of spontaneously fermented camel’s milk is common in Algeria, making it a feasible source of diverse lactic acid bacteria (LAB) with the potential to be used as adjunct cultures to improve quality and safety of fermented dairy products. Experimental approach. Twelve raw camel´s milk samples were used as a source of indigenous LAB, which were further characterised by examining 39 phenotypic traits with technological relevance. Results and conclusions. Thirty-five non-starter LAB (NSLAB) were isolated from 12 Algerian raw camel\u27s milk samples and they were microbiologically, biochemically and genetically characterised. Some isolates showed proteolytic activity, acidifying capacity, the ability to use citrate, and to produce dextran and acetoin. Ethanol, acetaldehyde, methyl acetate, acetoin and acetic acid were the major volatile compounds detected. Cluster analysis performed using the unweighted group with arithmetic average (UPGMA) method, and based on the thirty-nine phenotypic characteristics investigated, reflected the microbial diversity that can be found in raw camel´s milk. Novelty and scientific contribution. The isolated strains, from a non-typical source, showed interesting technological traits to be considered as potential adjunct cultures. Cluster analysis based on the examined phenotypic characteristics proved to be a useful tool for the typification of isolates when no genetic information is available. These findings may be of use towards an industrialised production of camel\u27s milk dairy products

Enterococcus faecalis Bacteriophage 156 Is an Effective Biotechnological Tool for Reducing the Presence of Tyramine and Putrescine in an Experimental Cheese Model

Biogenic amines (BA) – nitrogenous compounds of low molecular weight – are the result of metabolism of certain amino acids. They are biologically present in all living organisms and play essential physiological roles. However, their accumulation in foodstuffs due to the metabolic activity of certain microorganisms represents a toxicological risk. Containing such microorganisms, and with an abundance of precursor substrate amino acids, fermented foods in general, and cheeses in particular, provide an ideal matrix for the accumulation of these toxic compounds. Unfortunately, the main microorganisms responsible for BA accumulation are members of the lactic acid bacteria (LAB) group, which are also essential for the development of the organoleptic characteristics of the final product. The methods used to reduce the BA content of cheese, such as milk pasteurization, commonly fail to do so, and affect desirable non-BA-producing LAB as well. Bacteriophages have been proposed as biotechnological tools for diminishing the presence of undesirable microorganisms in dairy products. Given their specificity, they could be used to target the population of BA-producing bacteria. In this work, we aimed to explore the use of Enterococcus faecalis infecting phages as a tool to reduce the content of BA in dairy products. For this, we proceeded to the isolation and characterization of E. faecalis bacteriophage 156, a member of the family Myoviridae. Its genome was sequenced and compared with that of E. faecalis family Myoviridae phages available in public databases. Its capacity to decrease the accumulation of the BA tyramine and putrescine in an experimental laboratory-scale cheese model was proven