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

AIMS: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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. SIGNIFICANCE AND IMPACT OF THE STUDY: This study gives deeper insight into the metabolic regulation of tyrDC activity of enterococci.

Tyramine biosynthesis is transcriptionally induced at low pH and improves the fitness of Enterococcus faecalis in acidic environments.

Enterococcus faecalis is a commensal bacterium of the human gut that requires the ability to pass through the stomach and therefore cope with low pH. E. faecalis has also been identified as one of the major tyramine producers in fermented food products, where they also encounter acidic environments. In the present work, we have constructed a non-tyramine-producing mutant to study the role of the tyramine biosynthetic pathway, which converts tyrosine to tyramine via amino acid decarboxylation. Wild-type strain showed higher survival in a system that mimics gastrointestinal stress, indicating that the tyramine biosynthetic pathway has a role in acid resistance. Transcriptional analyses of the E. faecalis V583 tyrosine decarboxylase cluster showed that an acidic pH, together with substrate availability, induces its expression and therefore the production of tyramine. The protective role of the tyramine pathway under acidic conditions appears to be exerted through the maintenance of the cytosolic pH. Tyramine production should be considered important in the adaptability of E. faecalis to acidic environments, such as fermented dairy foods, and to survive passage through the human gastrointestinal tract.

Tyrosine decarboxylase from Lactobacillus brevis: soluble expression and characterization.

Tyrosine decarboxylase (TDC, EC 4.1.1.25) is an enzyme that catalyzes the decarboxylation of l-tyrosine to produce tyramine and CO2. In this study, a 1881-bp tdc gene from Lactobacillus brevis was cloned and heterologously expressed in Escherichia coli BL21 (DE3). Glucose was discovered to play an important role in the soluble expression of rLbTDC. After optimization, recombinant TDC (rLbTDC) was achieved in excellent solubility and a yield of 224mg rLbTDC/L broth. The C-terminal His-Tagged rLbTDC was one-step purified with 90% recovery. Based on SDS-PAGE and gel filtration analysis, rLbTDC is a dimer composed of two identical subunits of approximately 70kDa. Using l-tyrosine as substrate, the specific activity of rLbTDC was determined to be 133.5U/mg in the presence of 0.2mM pyridoxal-5'-phosphate at 40°C and pH 5.0. The Km and Vmax values of rLbTDC were 0.59mM and 147.1µmolmin(-1)mg(-1), respectively. In addition to l-tyrosine, rLbTDC also exhibited decarboxylase activity towards l-DOPA. This study has demonstrated, for the first time, the soluble expression of tdc gene from L. brevis in heterologous host.

The sequential exposure to jasmonate, salicylic acid and yeast extract promotes sanguinarine accumulation in Argemone mexicana cell cultures.

The effects of the sequential application of methyl jasmonate (MeJa), salicylic acid (SA) and yeast extract (YE) to Argemone mexicana cell cultures were compared to either the sole application of each elicitor, or to the three-partite mixture. The highest sanguinarine accumulation occurred using the sequential treatment (ninefold over unexposed control cultures), followed by the single application of YE (fivefold). The elicitor mixture produced less sanguinarine than sole exposure to YE but higher than MeJa alone. SA did not produce any effect. Transcripts corresponding to tyrosine decarboxylase and berberine bridge enzyme accumulated in treated cells, but did not correlate with alkaloid accumulation. Discrete epifluorescence foci, surrounding the nucleus and scattered throughout the cytoplasm of elicited cells, suggested the presence of alkaloid-accumulating vesicles which could participate in a mechanism to avoid sanguinarine toxicity.

Endosperm-specific expression of tyramine N-hydroxycinnamoyltransferase and tyrosine decarboxylase from a single self-processing polypeptide produces high levels of tyramine derivatives in rice seeds.

The plant-specific tyramine derivatives, feruloyltyramine (FT) and 4-coumaroyltyramine (CT), represent bioactive compounds found at low levels in many plant species. We generated transgenic rice seeds that produce high levels of CT (14 microg g(-1) seeds) and FT (2.7 microg g(-1) seeds) through the dual expression of tyramine N-hydroxycinnamoyltransferase and tyrosine decarboxylase, using the self-processing foot-and-mouth disease virus 2A sequence and the endosperm-specific prolamin promoter.

Intrinsic neurons of Drosophila mushroom bodies express short neuropeptide F: relations to extrinsic neurons expressing different neurotransmitters.

Mushroom bodies constitute prominent paired neuropils in the brain of insects, known to be involved in higher olfactory processing and learning and memory. In Drosophila there are about 2,500 intrinsic mushroom body neurons, Kenyon cells, and a large number of different extrinsic neurons connecting the calyx, peduncle, and lobes to other portions of the brain. The neurotransmitter of the Kenyon cells has not been identified in any insect. Here we show expression of the gene snpf and its neuropeptide products (short neuropeptide F; sNPFs) in larval and adult Drosophila Kenyon cells by means of in situ hybridization and antisera against sequences of the precursor and two of the encoded peptides. Immunocytochemistry displays peptide in intrinsic neuronal processes in most parts of the mushroom body structures, except for a small core in the center of the peduncle and lobes and in the alpha'- and beta'-lobes. Weaker immunolabeling is seen in Kenyon cell bodies and processes in the calyx and initial peduncle and is strongest in the more distal portions of the lobes. We used different antisera and Gal4-driven green fluorescent protein to identify Kenyon cells and different populations of extrinsic neurons defined by their signal substances. Thus, we display neurotransmitter systems converging on Kenyon cells: neurons likely to utilize dopamine, tyramine/octopamine, glutamate, and acetylcholine. Attempts to identify other neurotransmitter components (including vesicular glutamate transporter) in Kenyon cells failed. However, it is likely that the Kenyon cells utilize an additional neurotransmitter, yet to be identified, and that the neuropeptides described here may represent cotransmitters.

Two functional but noncomplementing Drosophila tyrosine decarboxylase genes: distinct roles for neural tyramine and octopamine in female fertility.

The trace biogenic amine tyramine is present in the nervous systems of animals ranging in complexity from nematodes to mammals. Tyramine is synthesized from tyrosine by the enzyme tyrosine decarboxylase (TDC), a member of the aromatic amino acid family, but this enzyme has not been identified in Drosophila or in higher animals. To further clarify the roles of tyramine and its metabolite octopamine, we have cloned two TDC genes from Drosophila melanogaster, dTdc1 and dTdc2. Although both gene products have TDC activity in vivo, dTdc1 is expressed nonneurally, whereas dTdc2 is expressed neurally. Flies with a mutation in dTdc2 lack neural tyramine and octopamine and are female sterile due to egg retention. Although other Drosophila mutants that lack octopamine retain eggs completely within the ovaries, dTdc2 mutants release eggs into the oviducts but are unable to deposit them. This specific sterility phenotype can be partially rescued by driving the expression of dTdc2 in a dTdc2-specific pattern, whereas driving the expression of dTdc1 in the same pattern results in a complete rescue. The disparity in rescue efficiencies between the ectopically expressed Tdc genes may reflect the differential activities of these gene products. The egg retention phenotype of the dTdc2 mutant and the phenotypes associated with ectopic dTdc expression contribute to a model in which octopamine and tyramine have distinct and separable neural activities.

Uncoupled defense gene expression and antimicrobial alkaloid accumulation in elicited opium poppy cell cultures.

Treatment of opium poppy (Papaver somniferum L.) cell cultures with autoclaved mycelial homogenates of Botrytis sp. resulted in the accumulation of sanguinarine. Elicitor treatment also caused a rapid and transient induction in the activity of tyrosine/dopa decarboxylase (TYDC, EC 4.1.1.25), which catalyzes the conversion of L-tyrosine and L-dopa to tyramine and dopamine, respectively, the first steps in sanguinarine biosynthesis. TYDC genes were differentially expressed in response to elicitor treatment. TYDC1-like mRNA levels were induced rapidly but declined to near baseline levels within 5 h. In contrast, TYDC2-like transcript levels increased more slowly but were sustained for an extended period. Induction of TYDC mRNAs preceded that of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) mRNAs. An elicitor preparation from Pythium aphanidermatum was less effective in the induction of TYDC mRNA levels and alkaloid accumulation; however, both elicitors equally induced accumulation of PAL transcripts. In contrast, treatment with methyl jasmonate resulted in an induction of TYDC but not PAL mRNAs. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and the protein kinase inhibitor staurosporine partially blocked the fungal elicitor-induced accumulation of sanguinarine. However, only staurosporine and okadaic acid, an inhibitor of protein phosphatases 1 and 2A, blocked the induction of TYDC1-like transcript levels, but they did not block the induction of TYDC2-like or PAL transcript levels. These data suggest that activation mechanisms for PAL, TYDC, and some later sanguinarine biosynthetic enzymes are uncoupled.

Elicitor-induced tyrosine decarboxylase in berberine-synthesizing suspension cultures of Thalictrum rugosum.

Tyrosine decarboxylase (EC 4.1.1.25) was induced in suspension cultures of Thalictrum rugosum by treatment with a yeast glucan elicitor. Maximum induction was observed at a carbohydrate concentration of 0.4 mg/g fresh weight of cells and maximum enzyme activity was reached 20 h after addition of elicitor. The enzyme was inducible in late exponential and early stationary growth phases. A good correlation between induced tyrosine decarboxylase activity and berberine biosynthesis has been established. It is suggested that tyrosine decarboxylase may be a key enzyme between primary and secondary metabolisms in the biosynthesis of norlaudanosoline-derived alkaloids.