Tyrosine Decarboxylase Activity Research Articles

Comparative transcriptomic profiles of Paulownia catalpifolia under different degrees of chilling stress during the seedling stage

BackgroundPaulownia, an ecologically and economically valuable plant species native to China, is notable for its excellent timber quality and strong adaptability. Among them, Paulownia catalpifolia displays the ability to survive in cold climate, a trait associated with northern China. Yet, the molecular information for its cold-tolerance has not been explored. This study was to investigate the changes in physiological indices and transcript levels of P. catalpifolia following cold exposure, which could provide evidence for revealing whether there were differences in the genetic basis of inducing physiological perturbations between moderate low temperature (MLT) and extreme low temperature (ELT).ResultsThe detection of physiological indices under diverse degrees of chilling stress showed similar patterns of alteration. Enhanced accumulation of osmoregulatory substances, such as soluble sugar and soluble protein, were more conducive under ELT compared to MLT in P. catalpifolia. Moreover, we observed leaf wilting symptoms distinctly after exposure to ELT for 48 h, while this effect was not obvious after MLT exposure for 48 h. Comparative transcriptomic analysis between MLT and ELT demonstrated 13,688 differentially expressed genes (DEGs), most of them appeared after 12 h and 48 h of treatment. GO and KEGG analyses elucidated prominent enrichment in aromatic-L-amino-acid decarboxylase activity term and carbohydrate metabolism pathways. Therefore, it was speculated that the DEGs involved in the above processes might be related to the difference in the contents of soluble protein and soluble sugar between MLT and ELT. Time series clustering analyses further highlighted several key genes engaged in the ‘Glycosyltransferases’, ‘Galactose metabolism’ and ‘Starch and sucrose metabolism’ pathways as well as the ‘tyrosine decarboxylase activity’ term. For instance, cellulose synthase-like A (CLSA2/9), raffinose synthase (RafS2), β-amylase (BAM1) and tyrosine/DOPA decarboxylase (TYDC1/2/5) genes, diverging in their expression trends between MLT and ELT, might significantly affect the soluble sugar and soluble protein abundance within P. catalpifolia.ConclusionBetween MLT and ELT treatments, partial overlaps in response pathways of P. catalpifolia were identified, while several genes regulating the accumulation of osmotic adjustment substances had disparate expression patterns. These findings could provide a novel physiological and molecular perspective for P. catalpifolia to adapt to complex low temperature habitats.

Monitoring Enzymatic Reaction Kinetics and Activity Assays in Confined Nanospace.

Enzyme-mediating biotransformations commonly occur in micro- and nanospace, which is crucial to maintain the essential biochemical processes and physiological functions in living systems. Probing enzyme-catalytic reactions in a biomimetic fashion remains challenging due to the lack of competent tools and methodology. Here, we show that studying enzymatic reaction kinetics can be readily achieved by a well-designed solid-state nanopore. Using tyrosine as a classical substrate, we quantitatively characterize the catalytic activity of tyrosinase (TYR) and tyrosine decarboxylase (TDC) in a nanoconfined space. Tyrosine was first immobilized in the nanopipette, wherein the active sites of tyrosine were left unoccupied. When successively exposed to TYR and TDC, a two-step cascade reaction can spontaneously take place. In this process, the surface wettability and charge of the nanopipette stemming from the catalytic products can sensitively regulate ion transport and ionic current rectification behavior, which were monitored by ionic current signal. In this biomimetic scenario, we obtained the enzymatic reaction kinetics of monophenyl oxidase that were not previously actualized in the conventional macroenvironment. Significantly, TYR showed higher enzyme activity, with a Km value of 1.59 mM, which was lower than that measured in a free and open space (with a Km of 3.01 mM). This suggests that tyrosine should be the most appropriate substrate of TYR, thus improving our understanding of tyrosine-associated biochemical reactions. This work offers an applicable technical platform to mimic enzyme-mediated biotransformations and biometabolisms.

Effects of pickle brine and glycine addition on biogenic amine production in pickle fermentation

This study investigated the effects of different pickle brines and glycine additions on biogenic amine formation in pickle fermentation. The results showed that the brines with higher biogenic amine content led to the production of more biogenic amines in the simulated pickle fermentation system. This was related to the abundance of biogenic amine-producing microorganisms in the microbial communities of the brines. Metagenome analysis of the brines and metatranscriptome analysis of the fermentation systems showed that putrescine was primarily from Lactobacillus, Oenococcus, and Pichia, while histamine and tyramine were primarily from Lactobacillus and Tetragenococcus. Addition of glycine significantly reduced the accumulation of biogenic amines in the simulated pickle fermentation system by as much as 70 %. The addition of glycine had no inhibitory effect on the amine-producing microorganisms, but it down-regulated the transcription levels of the genes for enzymes related to putrescine synthesis in Pichia, Lactobacillus, and Oenococcus, as well as the histidine decarboxylase genes in Lactobacillus and Tetragenococcus. Catalytic reaction assay using crude solutions of amino acid decarboxylase extracted from Lactobacillus brevis showed that the addition of glycine inhibited 45 %–55 % of ornithine decarboxylase and tyrosine decarboxylase activities. This study may provide a reference for the study and control of the mechanism of biogenic amine formation in pickle fermentation.

Description of Dellaglioa carnosa sp. nov., a novel species isolated from high-oxygen modified-atmosphere packaged meat

The study provides a taxonomic characterization of three bacterial strains isolated from high-oxygen modified-atmosphere packaged beef from Germany. The strains of the novel species shared identical 16S rRNA gene sequence to the closely related type strain of Dellaglioa algida. However, the in-silico DNA-DNA hybridization (DDH) values indicate that they belong to a different genomic species. The in silico DDH estimate value between TMW 2.2523T and the type strain of Dellaglioa algida DSM 15638T was only 63.2 %. The whole genome average nucleotide identity blast (ANIb) value of 95.1 % between TMW 2.2523T and the closely related type strain of D. algida was within the recommended threshold value of 95–96 % for bacterial species delineation. Additionally, the phylogenomic analyses based on multi locus sequence alignment (MLSA) showed that strain TMW 2.2523T and additional strains TMW 2.2444 and TMW 2.2533 formed a monophyletic group separate from D. algida strains. Furthermore, tyrosine decarboxylase activity could be attributed to strains of the new proposed species. The results of this polyphasic approach support the affiliation of these strains to a novel species within the genus Dellaglioa for which we propose the name Dellaglioa carnosa sp. nov. The designated respective type strain is TMW 2.2523T (DSM 114968T = LMG 32819T).

Antimicrobial activity and safety features assessment of Weissella spp. from environmental sources.

Weissella strains have been reported to be useful in biotechnological and probiotic determinations, and some of them are considered opportunistic pathogens. Given the widespread interest about antimicrobial susceptibilities, transmission of resistances, and virulence factors, there is little research available on such topics for Weissella. The aim of this study was to assess the safety aspects and antimicrobial potential of 54 Weissella spp. strains from different environmental sources. Antibiotic susceptibility, hemolytic activity, horizontal transfer, and antibacterial activity were studied, as well as the detection of biogenic amine BA production on decarboxylase medium and PCR was performed. All the strains were nonhemolytic and sensitive to chloramphenicol and ampicillin. Several strains were classified as resistant to fusidic acid, and very low resistance rates were detected to ciprofloxacin, tetracycline, streptomycin, lincomycin, erythromycin, and rifampicin, although all strains had intrinsic resistance to vancomycin, nalidixic acid, kanamycin, and teicoplanin. Two BA‐producing strains (W. halotolerans FAS30 and FAS29) exhibited tyrosine decarboxylase activity, and just one W. confusa FS077 produced both tyramine and histamine, and their genetic determinants were identified. Ornithine decarboxylase/odc gene was found in 16 of the Weissella strains, although 3 of them synthesize putrescine. Interestingly, eight strains with good properties displayed antibacterial activity. Conjugation frequencies of erythromycin from Bacillus to Weissella spp. varied in the average of 3 × 10−9 transconjugants/recipient. However, no tetracycline‐resistant transconjugant was obtained with Enterococcus faecalis JH2‐2 as recipient. The obtained results support the safe status of Weissella strains, derived from environmental sources, when used as probiotics in animal feed.

Effect of an Environment Friendly Heat and Relative Humidity Approach on γ-Aminobutyric Acid Accumulation in Different Highland Barley Cultivars.

In this study, heat and relative humidity (HRH) treatment was applied in highland barley for γ-aminobutyric acid (GABA) accumulation. Tibetan highland barley cultivars (25) were selected for comparison and analysis. HRH treatment could accumulate GABA in several hours with low moisture content and high temperature, and the grains were treated for 2.5 h at 65 °C in this study. The GABA content of processed grains under HRH optimal condition ranged from 26.91 to 76.28 mg·100 g−1, which was significantly higher than the initial content (12.78–43.00 mg·100 g−1). The highest GABA accumulation capacity was observed in two-row yellow cultivars (YT1), increasing from 36.52 to 76.28 mg·100 g−1. Correlation analysis showed that the accumulation of GABA after HRH treatment was positively and significantly (p < 0.05) correlated with the contents of protein (0.52), total free amino acids (0.68), threonine (0.53), serine (0.51), glutamate (0.69), glycine (0.49), alanine (0.46), cysteine (0.57), tyrosine (0.50), lysine (0.53), proline (0.40), and glutamate decarboxylase (GAD) activity (0.62), which were closely related to GABA-shunt pathway. The polyamines contents, diamine oxidase (DAO) and polyamine oxidase (PAO) activities, as the substrates and critical enzymes of polyamine degradation pathway, showed no significant correlation with GABA accumulation. The results suggested that the main pathway of GABA accumulation in highland barley under HRH treatment was GABA-shunt pathway.

Functional characterization of tyrosine decarboxylase genes that contribute to acteoside biosynthesis in Rehmannia glutinosa.

The RgTyDCs possess typical decarboxylase functional activity in vitro and in vivo and participate in acteoside biosynthesis in R. glutinosa, positively controlling its production via activated acteoside/tyrosine-derived pathways. Acteoside is an important ingredient in Rehmannia glutinosa and an active natural component that contributes to human health. Tyrosine decarboxylase (TyDC) is thought to play an important role in acteoside biosynthesis. Several plant TyDC family genes have been functionally characterized and shown to play roles in some bioactive metabolites' biosynthesis by mediating the decarboxylation of L-tyrosine and L-dihydroxyphenylalanine (L-DOPA); however, one TyDC (named RgTyDC1) in R. glutinosa has been identified to date, but the family genes that contribute to acteoside biosynthesis remain largely characterized. Here, by in silico and experimental analyses, we isolated and identified three RgTyDCs (RgTyDC2 to RgTyDC4) in this species; these genes' sequences showed 50.92-82.55% identity, included highly conserved domains with homologues in other plants, classified into two subsets, and encoded proteins that localized to the cytosol. Enzyme kinetic analyses of RgTyDC2 and RgTyDC4 indicated that they both efficiently catalysed L-tyrosine and L-dopa. The overexpression of RgTyDC2 and RgTyDC4 in R. glutinosa, which was associated with enhanced TyDC activity, significantly increased tyramine and dopamine contents, which was positively correlated with improved acteoside production; moreover, the overexpression of RgTyDCs led to upregulated expression of some other genes-related to acteoside biosynthesis. This result suggested that the overexpression of RgTyDCs can positively activate the molecular networks of acteoside pathways, enhancing the accumulation of tyramine and dopamine, and promoting end-product acteoside biosynthesis. Our findings provide an evidence that RgTyDCs play vital molecular roles in acteoside biosynthesis pathways, contributing to the increase in acteoside yield in R. glutinosa.

Histidine and tyrosine decarboxylase activities of lactic acid bacteria in sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax)

Biogenic amines (BAs) are formed by decarboxylation of amino acids, amination and transamination of aldehyde and ketone groups. The excess of BAs is harmful to human health. BAs play a significant role in determining the shelf life and quality of foods. Released type and amount of BAs depend on factors such as the quality of the raw material, the diversity of natural microbiota, processing and storage conditions. In fish, the release of BAs is affected primarily from microbial growth as well as other reasons and may cause poisoning. It was aimed to determine the possibility of histidine decarboxylase activity gene (hdc) and tyrosine decarbocylase activity gene (tyrdc) in lactic acid bacteria (LAB) which were isolated from sea bream and sea bass. A total of 18 Gram positive-catalase negative LAB was isolated from 84 fish samples from 14 different fish markets. It was found that 12 out of 18 LAB (67%) isolates showed negative histidine and tyrosine decarboxylase activities. While 2 out of 6 (11%) LAB isolates were determined positive only tyrosine decarboxylase and 4 of them (22%) were positive for histidine and tyrosine decarboxylase. As a result of the Polymerase Chain Reaction (PCR), 9 out of 12 LAB isolates (75%) were found to have histidine decarboxylase activity gene. As a result, the prevalence of histidine decarboxylase activity gene in the LAB has detected more extensive than tyrosine decarboxylase activity gene. Increasing the studies examining the presence of aminobiogenic microorganisms in fish is important for the protection of public health.

Crystal structures clarify cofactor binding of plant tyrosine decarboxylase

Plant tyrosine decarboxylase (TyrDC) is a group II pyridoxal 5′-phosphate (PLP)-dependent decarboxylase that mainly catalyzes the decarboxylation of tyrosine to tyramine. This is biologically important for diverting essential primary metabolites into secondary metabolic pathways. Intensive studies have characterized the effective of PLP-binding and the substrate specificity of mammalian 3,4-dihydroxyphenyl-l-alanine (Dopa) decarboxylases, a member of group II PLP-dependent decarboxylase. However, the characteristics of PLP binding and substrate specificity of plant TyrDCs remain unknown. In this study, we focus on the PLP binding manner, and determined the crystal structures of the apo and PLP binding form of type II TyrDC from Papaver somniferum (PsTyrDCII and PsTyrDCII-PLP). The structures showed that, unlike mammalian Dopa decarboxylase, the binding of PLP does not induce distinct conformational changes of PsTyrDCII regarding the overall structure, but the PLP binding pocket displays conformational changes at Phe124, His203 and Thr262. Combining structural comparation and the obtained biochemical findings, it is demonstrated that PsTyrDCII does not binds PLP tightly. Such characteristics of PLP binding may be required by its catalytic reaction and substrate binding. The activity of TyrDC probably regulated by the concentration of PLP in cells.

Genotype of Wolbachia pipientis Endosymbiont Affects Octopamine Metabolism in Drosophila melanogaster Females

The effect of the genotype of the endosymbiont Wolbachia pipientis on the metabolism of octopamine (one of the main biogenic amines in insects) was studied in young Drosophila melanogaster females. The activity of tyrosine decarboxylase (key octopamine synthesis enzyme) and the activity of octopamine-dependent N-acetyltransferase (the enzyme of its degradation) were measured. It was demonstrated that the activity of both studied enzymes is increased under normal conditions in females infected with bacteria of the pathogenic wMelPop strain and decreased in those infected with bacteria of the wMelCS genotype, while it does not differ from those in uninfected flies in females infected with the wMel genotype. It was also found that the intensity of tyrosine decarboxylase response to heat stress is decreased in all females infected with Wolbachia. Thus, the effect of Wolbachia on octopamine metabolism was for the first time demonstrated in the Drosophila females, and it was shown that the nature of this effect is determined by the symbiont genotype.

The effect of leaf galls of Cynipidae on accumulation and biosynthesis of plant amines in oak trees

Polyamines (PAs) are involved in plant response to abiotic and biotic stresses, however, their role in biochemical insect-plant interactions is not clear. Therefore, we compared the involvement of polyamines and key enzymes of their biosynthesis in gall formation process. The present study had used galls on oak leaves caused by asexual generation (♀♀) of three Cynipidae species, namely Cynips quercusfolii L., Neuroterus numismalis (Fourc.) and N. quercusbaccarum L., as a model. The obtained results indicate that gall formation on oak leaves affected amine content, but intensity of the changes in their levels were strongly dependent on the insect species. Nevertheless the downward trend was dominant among those changes. Changes in the activity of lysine decarboxylase (LDC), tyrosine decarboxylase (TyDC) and ornithine decarboxylase (ODC) usually corresponded with the direction of changes in polyamine contents. Several cases of divergence between changes in amine levels and the rate of their biosynthesis may suggest the involvement of other regulation mechanisms such as: arginine decarboxylase (ADC) and S-adenosylmethionine decarboxylase (SAMDC) as well as amine oxidases involved in its catabolic pathways. Thus, the future studies on biochemical mechanism of regulation of PAs accumulation during galls formation should be focused on importance of these enzymes.

Improving Soluble Expression of Tyrosine Decarboxylase from Lactobacillus brevis for Tyramine Synthesis with High Total Turnover Number.

The soluble expression of tyrosine decarboxylase (TDC) in heterologous host is often challenging. Here, acidic condition was found to be favorable for improving the soluble expression of TDC from Lactobacillus brevis in Escherichia coli, while addition of carbohydrates (such as glucose, arabinose, and fructose) was vital for decreasing the insoluble fraction. By simple pH control and addition of glucose, the specific activity of TDC in crude extract was enhanced to 46.3Umg-1, 3.67-fold of that produced from LB medium. Optimization of the reaction conditions revealed that Tween-80 was effective in improving the tyramine production catalyzed by TDC, especially at high tyrosine loadings. As much as 400mM tyrosine could be completely converted into tyramine with a substrate to catalyst ratio of 29.0gg-1 and total turnover number of 23,300. This study provides efficient strategies for the highly soluble expression of TDC and biocatalytic production of tyramine.

Tyramine reduction by tyrosine decarboxylase inhibitor in Enterococcus faecium for tyramine controlled cheonggukjang.

This study was carried out to find a method to control tyrosine decarboxylase activity (TDC) of a strain of Enterococcus faecium capable of producing high levels of tyramine. To select a TDC inhibitor, enzyme assay was first performed using purified TDC enzyme and 0.1% of TDC inhibiting chemicals. When 0.23% of nicotinic acid was added, tyramine content (363ug/mL) was lower than that of the control group (873ug/mL). At the same time, bacterial growth was decreased 1 log cycle from 8.62 to 7.56log CFU/mL. TDC expression level in E. faecium was measured by using RT-qPCR. Lower expression level (below 0.7) was observed after the addition of 0.23% nicotinic acid (in vitro). When cheonggukjang was manufactured with addition of nicotinic acid, tyramine contents were decreased from 698.67 to 117.27mg/kg when the concentration of nicotinic acid added was increased from 0.10 to 0.30%. These results suggest that nicotinic acid could be used as an agent (TDC inhibitor) to reduce tyramine content in cheonggukjang.

Growth, biogenic amine production and tyrDC transcription of Enterococcus faecalis in synthetic medium containing defined amino acid concentrations.

The tyraminogenic potential of the strains Enterococcus faecalis EF37 and ATCC 29212 was investigated in a synthetic medium containing defined amounts of tyrosine and phenylalanine at different temperatures. Enterococci growth and the production of biogenic amines (BA) were evaluated in relation to their pre-growth in medium containing tyrosine. Significant differences between the two strains were evidenced at metabolic level. Both the pre-adapted strains grew faster in all the tested conditions, independently of the presence of the precursor. Temperatures of 30 and 40°C positively affected the growth parameters. The tyrosine decarboxylase (tyrDC) activity of the strain EF37 was positively affected by pre-adaptation, while ATCC 29212 showed a faster and higher tyramine accumulation with not-adapted cells. The expression analysis of the gene tyrDC confirmed the influence of the growth conditions on gene transcription. The small differences found between the two strains in the maximum transcript level reached rapidly after the inoculum and the different behaviour in the tyramine accumulation suggested the possible involvement of complex regulation mechanisms on the tyrDC or on the membrane transport systems, which could affect the different BA accumulation trend. This study gives deeper insight into the metabolic regulation of tyrDC activity of enterococci.

Safety Evaluation of Enterocin Producer Enterococcus sp. Strains Isolated from Traditional Turkish Cheeses

The purpose of this study was to determine the antimicrobial activity and occurrence of bacteriocin structural genes in Enterococcus spp. isolated from different cheeses and also investigate some of their virulence factors. Enterococcus strains were isolated from 33 different cheeses. Enterococcus faecium (6 strains) and Enterococcus faecalis (5 strains) enterocin-producing strains were identified by 16S rDNA analyses. Structural genes entA, entB, entP and entX were detected in some isolates. Multiple enterocin structural genes were found in 7 strains. None of the tested enterococci demonstrated anyβ-haemolytic activity and only one strain had gelatinase activity. Six strains showed multiple antibiotic resistance patterns and in addition, vanA and several virulence genes were detected in many strains. Only E. faecalis MBE1-9 showed tyrosine decarboxylase activity and tdc gene was detected only in this strain.

The impact of FOXO on dopamine and octopamine metabolism in Drosophila under normal and heat stress conditions.

ABSTRACTThe forkhead boxO transcription factor (FOXO) is a component of the insulin signalling pathway and plays a role in responding to adverse conditions, such as oxidative stress and starvation. In stressful conditions, FOXO moves from the cytosol to the nucleus where it activates gene expression programmes. Here, we show that FOXO in Drosophila melanogaster responds to heat stress as it does to other stressors. The catecholamine signalling pathway is another component of the stress response. In Drosophila, dopamine and octopamine levels rise steeply under heat, nutrition and mechanical stresses, which are followed by a decrease in the activity of synthesis enzymes. We demonstrate that the nearly twofold decline of FOXO expression in foxoBG01018 mutants results in dramatic changes in the metabolism of dopamine and octopamine and the overall response to stress. The absence of FOXO increases tyrosine decarboxylase activity, the first enzyme in octopamine synthesis, and decreases the enzymatic activity of enzymes in dopamine synthesis, alkaline phosphatase and tyrosine hydroxylase, in young Drosophila females. We identified the juvenile hormone as a mediator of FOXO regulation of catecholamine metabolism. Our findings suggest that FOXO is a possible trigger for endocrinological stress reactions.

Insulin‐like receptor substrate gene chico regulates octopamine metabolism in Drosophila melanogaster

AbstractOctopamine, one of the main insect biogenic amines, plays an important role in the control of fitness in Drosophila melanogaster Meigen. The present study examines the effects of a null mutation of the gene of the insulin‐like receptor substrate (chico), in the heterozygous state, on octopamine metabolism, heat stress resistance and fecundity of D. melanogaster. A rise in the activity of one of the key enzymes of octopamine synthesis, tyrosine decarboxylase, as well as that of an enzyme of its degradation, octopamine‐dependent N‐acetyl transferase, is observed in chico1/+ females. It is also found that the resistance to heat stress is decreased and fecundity is reduced dramatically in chico1/+ flies. Such changes in these parameters in D. melanogaster females result from a rise in octopamine titre, which suggests that chico affects the octopamine level by regulating the activity of tyrosine decarboxylase.

Tyrosine decarboxylase activity of Enterococcus mundtii: new insights into phenotypic and genetic aspects.

SummaryFew information is available about the tyraminogenic potential of the species Enterococcus mundtii. In this study, two plant‐derived strains of E. mundtii were selected and investigated to better understand the phenotypic behaviour and the genetic mechanisms involved in tyramine accumulation. Both the strains accumulated tyramine from the beginning of exponential phase of growth, independently on the addition of tyrosine to the medium. The strains accumulated also 2‐phenylethylamine, although with lower efficiency and in greater extent when tyrosine was not added. Accordingly, the tyrosine decarboxylase (tyrDC) gene expression level increased during the exponential phase with tyrosine added, while it remained constant and high without precursor. The genetic organization as well as sequence identity levels of tyrDC and tyrosine permease (tyrP) genes indicated a correlation with those of phylogenetically closer enterococcal species, such as E. faecium, E. hirae and E. durans; however, the gene Na+/H+ antiporter (nhaC) that usually follow tyrP is missing. In addition, BLAST analysis revealed the presence of additional genes encoding for decarboxylase and permease in the genome of several E. mundtii strains. It is speculated the occurrence of a duplication event and the acquisition of different specificity for these enzymes that deserves further investigations.

Gene dilp6 regulates octopamine metabolism in Drosophila melanogaster

The effect of strong hypomorphic mutation of the insulin-like protein gene (dilp6) on metabolism of octopamine (one of the main biogenic amines in insects) was studied in Drosophila melanogaster males and females. The activity of tyrosine decarboxylase (the key enzyme of octopamine synthesis) and the activity of octopamine-dependent N-acetyltransferase (the enzyme of its degradation) were measured. It was demonstrated that the activity of both studied enzymes is decreased under normal conditions in the dilp6 41 mutants (as we previously demonstrated, this is correlated with an increased level of octopamine). It was also found that hypomorphic mutation of the dilp6 gene decreases the intensity of tyrosine decarboxylase response to heat stress. Thus, it was demonstrated for the first time that insulin-like DILP6 protein in drosophila influences the level of octopamine (regulating the activity of the enzyme degrading octopamine).

The Capability of Tyramine Production and Correlation between Phenotypic and Genetic Characteristics of Enterococcus faecium and Enterococcus faecalis Strains.

The aim of this study was to investigate the diversity of tyramine production capability of four Enterococcus strains in buffered systems in relation to their genetic characteristics and environmental conditions. Cells of the strains Enterococcus faecalis EF37 and ATCC 29212, and E. faecium FC12 and FC643 were re-suspended in phosphate/citrate buffers with different pH, NaCl concentration and incubation temperature. At intervals, cell viability and tyramine production were assessed by plate counting and HPLC analysis, respectively. The activity of a purified tyrosine decarboxylase (TDC) was determined under the same conditions, as a reference. Reduced loss in cell viability was observed in all the tested conditions, except for pH 4 after 24 h. The TDC activity was greatly heterogeneous within the enterococci: EF37 and FC12 produced the higher tyramine concentrations, ATCC 29212 showed a reduced decarboxylase activity, while EF643 did not accumulate detectable amounts of tyramine in all the conditions assayed. Among the considerate variables, temperature was the most influencing factor on tyramine accumulation for enterococcal cells. To further correlate the phenotypic and genetic characteristics of the enterococci, the TDC operon region carrying the genes tyrosine decarboxylase (tyrDC), tyrosine/tyramine permease (tyrP), and Na+/H+ antiporter (nhaC-2) was amplified and sequenced. The genetic organization and nucleotide sequence of this operon region were highly conserved in the enterococcal strains of the same species. The heterogeneity in tyramine production found between the two E. faecalis strains could be ascribed to different regulation mechanisms not yet elucidated. On the contrary, a codon stop was identified in the translated tyrDC sequence of E. faecium FC643, supporting its inability to accumulate tyramine in the tested conditions. In addition, the presence of an additional putative tyrosine decarboxylase with different substrate specificity and genetic organization was noticed for the first time. Concluding, the high TDC activity heterogeneity within enterococci determined different accumulation of tyramine, depending on different genetic determinants, regulation mechanisms, and environmental factors. The present research contributes to elucidate the genetic characteristics of enterococcal strains and correlate specific mutations to their different strain-dependent tyraminogenic activity.