Characterization of a <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> Strain Able to Produce Tyramine and Partial Cloning of a Putative Tyrosine Decarboxylase Gene

The aim of this article was to analyze the ability of wine Lactobacillus plantarum strains to form tyramine. Preliminary identification of L. plantarum strains was performed by amplification of the recA gene. Primers pREV and PlanF, ParaF and PentF were used respectively as reverse and forward primers in the polymerase chain reaction tests as previously reported. Furthermore, the gene encoding for the tyrosine decarboxylase (TDC) was partially cloned from one strain identified as L. plantarum. The strain was further analyzed by 16S rDNA sequence and confirmed as belonging to L. plantarum species. The tyrosine decarboxylase activity was investigated and tyramine was determined by the high-performance liquid chromatography method. Moreover, a negative effect of sugars such as glucose and fructose and L-malic acid on tyrosine decarboxylase activity was observed. The results suggest that, occasionally, L. plantarum is able to produce tyramine in wine and this ability is apparently confined only to L. plantarum strains harboring the tdc gene.     

Expression of Lactobacillus brevis IOEB 9809 tyrosine decarboxylase and agmatine deiminase genes in wine correlates with substrate availability

Aims: Lactobacillus brevis IOEB 9809 is able to produce both tyramine and putrescine via tyrosine decarboxylase and agmatine deiminase enzymes, respectively, when cultured on synthetic media. The aims of this study were to assess the expression of L. brevis IOEB 9809 tdc and aguA1 genes, during wine fermentation and to evaluate the effect of substrate availability and pH on tdc and aguA1 expression, as well as on biogenic amine production and L. brevis viability. Methods and Results: The relative expression of L. brevis IOEB 9809 tdc and aguA1 genes was analysed in wine by quantitative real‐time RT‐PCR (qRT‐PCR) during a period of incubation of 30 days. Cell viability, pH values, putrescine and tyramine concentration were monitored throughout the experiments. Conclusions: The wine trials indicated that L. brevis IOEB 9809 is able to produce both tyramine and putrescine during wine fermentation. Increased cell viability was also observed in wine supplemented with tyrosine or agmatine. qRT‐PCR analysis suggests a strong influence of substrate availability on the expression of genes coding for tyrosine decarboxylase and agmatine deiminase in L. brevis IOEB 9809. Less evident is the relationship between putrescine and tyramine production and tolerance to wine pH. Significance and Impact of Study: To our knowledge, this study represents the first assessment of relative expression of L. brevis IOEB 9809 genes involved in biogenic amine production in wine. Furthermore, an effect of biogenic amine production on viability of L. brevis during wine fermentation was established.     

Characterization of rice tryptophan decarboxylases and their direct involvement in serotonin biosynthesis in transgenic rice

l-Tryptophan decarboxylase (TDC) and l-tyrosine decarboxylase (TYDC) belong to a family of aromatic l-amino acid decarboxylases and catalyze the conversion of tryptophan and tyrosine into tryptamine and tyramine, respectively. The rice genome has been shown to contain seven TDC or TYDC-like genes. Three of these genes for which cDNA clones were available were characterized to assign their functions using heterologous expression in Escherichia coli and rice (Oryza sativa cv. Dongjin). The purified products of two of the genes were expressed in E. coli and exhibited TDC activity, whereas the remaining gene could not be expressed in E. coli. The recombinant TDC protein with the greatest TDC activity showed a K _m of 0.69 mM for tryptophan, and its activity was not inhibited by phenylalanine or tyrosine, indicating a high level of substrate specificity toward tryptophan. The ectopic expression of the three cDNA clones in rice led to the abundant production of the products of the encoded enzymes, tyramine and tryptamine. The overproduction of TYDC resulted in stunted growth and a lack of seed production due to tyramine accumulation, which increased as the plant aged. In contrast, transgenic plants that produced TDC showed a normal phenotype and contained 25-fold and 11-fold higher serotonin in the leaves and seeds, respectively, than the wild-type plants. The overproduction of either tyramine or serotonin was not strongly related to the enhanced synthesis of tyramine or serotonin derivatives, such as feruloyltyramine and feruloylserotonin, which are secondary metabolites that act as phytoalexins in plants.     

A convenient method for multiple insertions of desired genes into target loci on the <Emphasis Type="Italic">Escherichia coli</Emphasis> chromosome

We developed a method to insert multiple desired genes into target loci on the Escherichia coli chromosome. The method was based on Red-mediated recombination, flippase and the flippase recognition target recombination, and P1 transduction. Using this method, six copies of the lacZ gene could be simultaneously inserted into different loci on the E. coli chromosome. The inserted lacZ genes were functionally expressed, and β-galactosidase activity increased in proportion to the number of inserted lacZ genes. This method was also used for metabolic engineering to generate overproducers of aromatic compounds. Important genes of the shikimate pathway (aroF ^fbr and tyrA ^fbr or aroF ^fbr and pheA ^fbr ) were introduced into the chromosome to generate a tyrosine or a phenylalanine overproducer. Moreover, a heterologous decarboxylase gene was introduced into the chromosome of the tyrosine or phenylalanine overproducer to generate a tyramine or a phenethylamine overproducer, respectively. The resultant strains selectively overproduced the target aromatic compounds. Thus, the developed method is a convenient tool for the metabolic engineering of E. coli for the production of valuable compounds.     

The tyrosine decarboxylation test does not differentiate Enterococcus faecalis from Enterococcus faecium

According to the current edition of the Bergey's Manual of Systematic Bacteriology [11] the tyrosine decarboxylation test allows the differentiation of enterococci. Tyrosine is decarboxylated to the biogenic amine tyramine by E. faecalis and not by E. faecium strains. In the present study we sequenced the16S rDNA of two tyramine-producing strains, BIFI-56 and BIFI-58, presumptively classified as E. faecalis. Their 16S rDNA were identical to the same fragment from the E. faecium type strain. Several E. faecium strains were then checked for their ability to decarboxylate tyrosine and also a putative tyrosine decarboxylase-coding gene was PCR amplified from these strains. All the strains confirmed as E. faecium produced tyramine and possessed a DNA fragment coding for a putative tyrosine decarboxylase. The concordance of the two methods allows us to conclude that the tyrosine decarboxylase test cannot be used in the differentiation of E. faecalis from E. faecium since at least some E. faecium strains are tyramine producers.     

Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 Alleviated Symptoms of Salmonella Infection, as Determined in Wistar Rats Challenged with Salmonella enterica Serovar Typhimurium

Wistar rats were administered daily with Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA through intragastric gavage (1 × 10⁸ cfu of each strain and a combination of the two strains). Sterile saline was used as placebo. After 7 days, the animals were challenged by infection with 2 × 10⁸ CFU Salmonella enterica serovar Typhimurium. After 1 day of treatment with L. plantarum 423 and E. mundtii ST4SA, the feed and water intake, and body weight of the rats increased. The faecal moisture content and β-glucuronidase activity remained more-or-less constant after 2 days of treatment with E. mundtii ST4SA, L. plantarum 423 and a combination of the two strains. Reduced levels of endotoxin were recorded in blood samples taken from rats that received L. plantarum 423 and E. mundtii ST4SA. Although both strains alleviated symptoms of S. enterica serovar Typhimurium infection, L. plantarum 423 administered as a single culture proved more effective than E. mundtii ST4SA. Less promising results were recorded when L. plantarum 423 was administered in combination with E. mundtii ST4SA. This suggests that L. plantarum 423 is more effective than E. mundtii and should be the preferred probiotic to alleviate symptoms of S. enterica serovar Typhimurium infection.     

First evidence of a membrane‐bound,tyramine and β‐phenylethylamine producing,tyrosine decarboxylase in Enterococcus faecalis: A two‐dimensional electrophoresis proteomic study

The soluble and membrane proteome of a tyramine producing Enterococcus faecalis, isolated from an Italian goat cheese, was investigated. A detailed analysis revealed that this strain also produces small amounts of β‐phenylethylamine. Kinetics of tyramine and β‐phenylethylamine accumulation, evaluated in tyrosine plus phenylalanine‐enriched cultures (stimulated condition), suggest that the same enzyme, the tyrosine decarboxylase (TDC), catalyzes both tyrosine and phenylalanine decarboxylation: tyrosine was recognized as the first substrate and completely converted into tyramine (100% yield) while phenylalanine was decarboxylated to β‐phenylethylamine (10% yield) only when tyrosine was completely depleted. The presence of an aspecific aromatic amino acid decarboxylase is a common feature in eukaryotes, but in bacteria only indirect evidences of a phenylalanine decarboxylating TDC have been presented so far. Comparative proteomic investigations, performed by 2‐DE and MALDI‐TOF/TOF MS, on bacteria grown in conditions stimulating tyramine and β‐phenylethylamine biosynthesis and in control conditions revealed 49 differentially expressed proteins. Except for aromatic amino acid biosynthetic enzymes, no significant down‐regulation of the central metabolic pathways was observed in stimulated conditions, suggesting that tyrosine decarboxylation does not compete with the other energy‐supplying routes. The most interesting finding is a membrane‐bound TDC highly over‐expressed during amine production. This is the first evidence of a true membrane‐bound TDC, longly suspected in bacteria on the basis of the gene sequence.     

Characterization of the tyraminergic system in the central nervous system of the locust,Locusta migratoria migratoides

Tyramine occurs in the central nervous system (CNS) of the migratory locust,Locusta migratoria migratoides. The distribution of tyramine within the CNS does not parallel that of octopamine. Tyramine is synthesised from tyrosine in the presence of tyrosine decarboxylase. A second decarboxylase in the CNS is active against 5HTP and DOPA. The locust ganglia incorporate tyramine by high- and low-affinity uptake processes that appear to be independent of dopamine and octopamine. Depolarisation of the locust ganglia by high potassium concentration results in calcium-dependent release of incorporated [³H]tyramine.     

Tyramine accumulation in rice cells caused a dwarf phenotype via reduced cell division

Transgenic rice plants overexpressing a rice tyrosine decarboxylase (TyDC) exhibited a dwarf phenotype with a high level of tyramine accumulation. The height of transgenic rice was reduced on average to 35% of the wild-type height, whereas the number of tillers increased to 190% that of wild type. When judged by cellular distribution of tyramine and tyramine derivatives, the level of tyramine in soluble and insoluble fractions was higher than that of tyramine derivatives such as 4-coumaroyltyramine (CT) in the transgenic rice plants, suggesting that tyramine rather than its derivatives was a causative compound triggering the dwarf phenotype. Microscopic observation revealed that cell size in the transgenic lines was maintained, with a slightly irregular arrangement in the leaf mesophyll cells. When wild-type rice seeds were grown in the presence of tyramine, rice seedlings also showed stunted phenotypes in a dose-dependent manner. When these stunted seedlings were employed to measure the degree of cellular proliferation by bromodeoxyuridine incorporation, only small numbers of cells were found to retain labeled nuclei in shoot tips compared with the untreated control. These results show that the dwarf phenotype associated with tyramine accumulation in transgenic rice plants is attributable to a reduction in cell number rather than cell size. In addition, our dwarf phenotype caused by tyramine was not closely associated with known dwarf genes such as D88.     

Genetic delineation between and within the widespread coccolithophore morpho‐species Emiliania huxleyi and Gephyrocapsa oceanica (Haptophyta)

Emiliania huxleyi and Gephyrocapsa oceanica are abundant coccolithophore morpho‐species that play key roles in ocean carbon cycling due to their importance as both primary producers and cal‐cifiers. Global change processes such as ocean acidification impact these key calcifying species. The physiology of E. huxleyi, a developing model species, has been widely studied, but its genetic delineation from G. oceanica remains unclear due to a lack of resolution in classical genetic markers. Using nuclear (18S rDNA and 28S rDNA), mitochondrial (cox1, cox2, cox3, rpl16, and dam), and plastidial (16S rDNA, rbcL, tufA, and petA) DNA markers from 99 E. huxleyi and 44 G. oceanica strains, we conducted a multigene/multistrain survey to compare the suitability of different markers for resolving phylogenetic patterns within and between these two morpho‐species. The nuclear genes tested did not provide sufficient resolution to discriminate between the two morpho‐species that diverged only 291Kya. Typical patterns of incomplete lineage sorting were generated in phylogenetic analyses using plastidial genes. In contrast, full morpho‐species delineation was achieved with mitochondrial markers and common intra‐morpho‐species phylogenetic patterns were observed despite differing rates of DNA substitution. Mitochondrial genes are thus promising barcodes for distinguishing these coccolithophore morpho‐species, in particular in the context of environmental monitoring.     

Genomic comparative analysis of the environmental Enterococcus mundtii against enterococcal representative species

Background

Enterococcus mundtii is a yellow-pigmented microorganism rarely found in human infections. The draft genome sequence of E. mundtii was recently announced. Its genome encodes at least 2,589 genes and 57 RNAs, and 4 putative genomic islands have been detected. The objective of this study was to compare the genetic content of E. mundtii with respect to other enterococcal species and, more specifically, to identify genes coding for putative virulence traits present in enterococcal opportunistic pathogens.

Results

An in-depth mining of the annotated genome was performed in order to uncover the unique properties of this microorganism, which allowed us to detect a gene encoding the antimicrobial peptide mundticin among other relevant features. Moreover, in this study a comparative genomic analysis against commensal and pathogenic enterococcal species, for which genomic sequences have been released, was conducted for the first time. Furthermore, our study reveals significant similarities in gene content between this environmental isolate and the selected enterococci strains (sharing an “enterococcal gene core” of 805 CDS), which contributes to understand the persistence of this genus in different niches and also improves our knowledge about the genetics of this diverse group of microorganisms that includes environmental, commensal and opportunistic pathogens.

Conclusion

Although E. mundtii CRL1656 is phylogenetically closer to E. faecium, frequently responsible of nosocomial infections, this strain does not encode the most relevant relevant virulence factors found in the enterococcal clinical isolates and bioinformatic predictions indicate that it possesses the lowest number of putative pathogenic genes among the most representative enterococcal species. Accordingly, infection assays using the Galleria mellonella model confirmed its low virulence.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-489) contains supplementary material, which is available to authorized users.     

Isolation and characterisation of lactic acid bacterium for effective fermentation of cellobiose into optically pure homo <Emphasis Type="SmallCaps">l</Emphasis>-(+)-lactic acid

Effective utilisation of cellulosic biomasses for economical lactic acid production requires a microorganism with potential ability to utilise efficiently its major components, glucose and cellobiose. Amongst 631 strains isolated from different environmental samples, strain QU 25 produced high yields of l-(+)-lactic acid of high optical purity from cellobiose. The QU 25 strain was identified as Enterococcus mundtii based on its sugar fermentation pattern and 16S rDNA sequence. The production of lactate by fermentation was optimised for the E. mundtii QU25 strain. The optimal pH and temperature for batch culturing were found to be 7.0°C and 43°C, respectively. E. mundtii QU 25 was able to metabolise a mixture of glucose and cellobiose simultaneously without apparent carbon catabolite repression. Moreover, under the optimised culture conditions, production of optically pure l-lactic acid (99.9%) increased with increasing cellobiose concentrations. This indicates that E. mundtii QU 25 is a potential candidate for effective lactic acid production from cellulosic hydrolysate materials.     

Tyramine degradation and tyramine/histamine production by lactic acid bacteria and Kocuria strains

Of 53 strains of lactic acid bacteria and Kocuria, screened for production or degradation of biogenic amines, 29 Kocuria varians and four strains of Enterococcus faecalisproduced tyramine and, at lower concentrations, histamine. In contrast, Lactobacillus strains that did not possess amino acid decarboxylase activity degraded tyramine. The greatest tyramine oxidase activity was present in the strains L. casei CRL705 (98% degradation) and CRL678 (93%) as well as in L. plantarum CRL681 (69%) and CRL682 (60%).     

PCR detection of enzyme-encoding genes in <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> strains of wine origin

Fifteen isolates of lactic acid bacteria originating from South African grape and wine samples were identified as Leuconostoc mesenteroides subsp. mesenteroides through the taxonomic analysis of their 16S rDNA gene sequences. These isolates were further tested for the presence of genes coding for enzymes of oenological relevance using PCR detection technique. A type strain of Leuc. mesenteroides (NCDO 529^T) was also incorporated for comparative analysis. From the PCR detection results, the estA, prtP, alsD, alsS, metK, metC and metB genes were present in all the strains tested. The bgl and gshR genes encoding β-glucosidase and glutathione reductase, respectively, were not detected in some strains. On the other hand, none of the tested strains possessed the genes encoding phenolic acid decarboxylase (padA), citrate permease (citP), citrate lyase (citD, citE and citF) and arginine deiminase pathway enzymes (arcA, arcB and arcC). The verification of PCR-generated fragments was performed by sequencing. GenBank database was used to search for homologous DNA sequences. Neighbour-joining trees based on nucleotide sequences of alsS, estA, metK and mleA genes were also constructed in order to study the phylogenetic relationship between Leuc. mesenteroides strains and closely related species. The phylogenetic analyses revealed that there are genetic heterogeneities between strains of Leuc. mesenteroides species. In conclusion, this study has improved our knowledge on the genetics of oenological strains of Leuc. mesenteroides and their genetic potential to contribute to certain wine aroma compounds.     

Adhesion of the probiotic strains <Emphasis Type="Italic">Enterococcus mundtii</Emphasis> ST4SA and <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> 423 to Caco-2 cells under conditions simulating the intestinal tract,and in the presence of antibiotics and anti-inflammatory medicaments

Adhesion of Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 to Caco-2 (human carcinoma epithelial) cells was visualized by fluorescent staining. Both strains showed good adhesion compared to L. casei MB1, L. casei Shirota, L. johnsonii La1 and L. rhamnosus GG. No correlation was found between hydrophobicity, aggregation and adhesion to Caco-2 cells. Presence of antibiotics and anti-inflammatory medicaments reduced adhesion of bacterial strains to Caco-2 cells. Proteins sensitive to pepsin, trypsin and pronase are involved in the adhesion of E. mundtii ST4SA and L. plantarum 423 to Caco-2 cells. Adhesion of Listeria monocytogenes ScottA to Caco-2 cells was not prevented by E. mundtii ST4SA and L. plantarum 423. Cell-free culture supernatants of strains ST4SA and 423, containing the antimicrobial peptides plantaricin 423 and peptide ST4SA, prevented the invasion of L. monocytogenes ScottA into Caco-2 cells.     

Endosperm-specific expression of tyramine <Emphasis Type="Italic">N</Emphasis>-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 μg g⁻¹ seeds) and FT (2.7 μg 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.     

Specific detection of Escherichia coli isolated from water samples using polymerase chain reaction targeting four genes: cytochrome bd complex, lactose permease, beta-D-glucuronidase, and beta-D-galactosidase

Aims: To develop a PCR-based method for reliable detection of Escherichia coli that enables its differentiation from biochemically and phylogenetically related bacteria. Methods and Results: Using multiplex PCR targeting four genes (cytochrome bd complex, lactose permease, β-d -glucuronidase, and β-d -galactosidase) the possibility of specific detection of various control E. coli strains was tested. It was found that four PCR fragments of the predicted size were observed only for E. coli strains, but not for relatives as close as Shigella sp. or other enterobacteria. Not surprisingly, this method enabled us to identify also E. coli strains which did not exhibit the β-d -glucuronidase activity. Our multiplex PCR was also successfully used for identification of 95 environmental isolates of E. coli. Conclusions: The developed PCR-based method, in which four genes coding for lactose permease, cytochrome bd complex, β-d -glucuronidase, and β-d -galactosidase, serve as target DNA sequences, allows precise and reliable detection of E. coli strains. Significance and Impact of the study: The suggested approach increases the specificity of detection of E. coli since it enables to distinguish E. coli from Shigella sp. and other relative enterobacteria.     

First genetic characterization of a bacterial beta-phenylethylamine biosynthetic enzyme in Enterococcus faecium RM58

Enterococcus faecium RM58 produces beta-phenylethylamine and tyramine. A gene from Ent. faecium RM58 coding for a 625 amino-acid residues protein that shows 85% identity to Enterococcus faecalis tyrosine decarboxylase has been expressed in Escherichia coli, resulting in L-phenylalanine and L-tyrosine decarboxylase activities. Both activities were lost when a truncated protein lacking 84 amino acids at its C-terminus was expressed in E. coli. This study constitutes the first genetic characterization of a bacterial protein having L-phenylalanine decarboxylase activity and solves a long-standing question regarding the specificity of tyrosine decarboxylases in enterococci.     

Sensitivity to Enterocins of Biogenic Amine-Producing Faecal Enterococci from Ostriches and Pheasants

Enterococci are widespread bacteria forming the third largest genus among lactic acid bacteria. Some possess probiotic properties or they can produce beneficial proteinaceous antimicrobial substances called enterocins. On the other hand, some enterococci produce biogenic amines (BAs), so this study is focused on the sensitivity to enterocins of biogenic amine-producing faecal enterococci from ostriches and pheasants. Altogether, 60 enterococci isolated from faeces of ostriches and pheasants were tested for production of BAs. This target of the identified enterococci involved 46 strains selected from 140 ostriches and 17 from 60 pheasants involving the species Enterococcus hirae, E. faecium, E. faecalis, and E. mundtii. Although BAs histamine, cadaverine, putrescine, and tryptamine were not detected in the enterococci tested, in general high BA production by the tested enterococci was noted. The species E. hirae formed the majority of the enterococcal strains from ostrichs faeces (34 strains). High production of tyramine (TYM) was measured with an average amount of 958.16 ± 28.18 mg/ml. Among the enterococci from pheasants, the highest was production of TYM compared to phenylethylamine, spermidine, and spermine. Enterococci featured high BA production; however, they were sensitive to seven enterocins with inhibition activity ranging from 100 up to 25,600 AU/ml.