Characterization of wine Lactobacillus plantarum by PCR-DGGE and RAPD-PCR analysis and identification of Lactobacillus plantarum strains able to degrade arginine

Summary Screening of strains isolated from red wine undergoing malolactic fermentation allowed the identification of lactic acid bacteria able to degrade arginine. A denaturing gradient gel electrophoresis approach, using the rpoB gene as the molecular target, was developed in order to characterize the isolated strains. Several strains were identified as Lactobacillus plantarum and were typed by RAPD-PCR with several randomly designed primers. Almost all of the␣L. plantarum strains identified were able to produce citrulline and ammonia, suggesting that the ability of␣L.␣plantarum to degrade arginine is a common feature in wine. During the characterization of the newly identified L.␣plantarum strains, the presence of genes coding for the arginine deiminase (ADI) pathway was observed in the strains able to produce citrulline, while the lack of this genes was observed in strain unable to produce citrulline. These results suggest that the degradation of arginine in L. plantarum is probably strain-dependent.     

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%).     

Characterization of a new organization of the plantaricin locus in the inducible bacteriocin-producing<Emphasis Type="Italic"> Lactobacillus plantarum</Emphasis> J23 of grape must origin

Lactobacillus plantarum J23 was previously characterized as a bacteriocin-producer-strain when it was cocultured with other lactic acid bacteria. In this work, the genetic organization of the pln locus in the J23 strain was studied and compared with those of previously described L. plantarum C11, WCFS1 and NC8 strains. A new organization of the plantaricin locus was detected in the J23 strain. The sequenced fragment (20,266 bp) comprised plnJLR, plnMNOP, plnEFI, plnGHSTUVWXY, and plNC8IF-plNC8HK-plnD operons, as well as a new region that includes three new orfs (GenBank accession number DQ323671). When the J23 pln gene sequences were compared with those included in the GenBank database, the identity of the putative encoded proteins was in the range 67.1–100%. The regulatory system and the repertoire of putative bacteriocins of the J23 pln locus presented important differences with respect to the ones of C11, WCFS1 and NC8, such as the absence of plnK and the presence of a larger plnJ gene than the previously described for the other L. plantarum strains. The pln locus in L. plantarum strains seems to be a mosaic-like structure with different modules and reorganizations that presents highly conserved regions related to transport and bacteriocin maturation and variable regions related to regulation and bacteriocin production.     

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.     

不同来源植物乳杆菌基因组结构和功能差异的比较研究

[目的] 本试验研究不同来源植物乳杆菌（Lactobacillus plantarum）基因特点以及在不同环境下其基因多样性,探究2株L.plantarum A8和P9在肠道生境及植物表面适应性的异同,为优良菌株的开发提供理论基础。[方法] 本研究对从动物肠道和植物表面分离获得的L.plantarum A8和L.plantarum P9的基因组进行分析,利用第二代测序技术（NextGeneration Sequencing,NGS）,基于Illumina NovaSeq测序平台,同时利用第三代单分子测序技术,基于PacBio Sequel测序平台,对L.plantarum A8和L.plantarum P9进行测序。采用Carbohydrate-active enzymes（CAZy）、Koyto encyclopedia of genes and genomes（KEGG）和Clusters of orthologous genes（COG）数据库对基因组进行功能注释;采用CGView软件绘制菌株的基因组环形图谱。应用比较基因组学与已经公开发表的其他L.plantarum基因组进行比较分析。[结果] 由研究可知L.plantarum A8和L.plantarum P9基因组大小存在差异,通过构建系统发育树发现2株菌与其他来源的L.plantarum分在同一分支,并且L.plantarum P9与母乳来源的L.plantarum WLPL04菌株距离最近,而L.plantarum A8与L.paraplantarum DSM10667距离最近。通过基因家族分析可知,2株菌共有基因为2643个,其中包括一些抗应激蛋白如热休克蛋白、冷休克蛋白。L.plantarum A8和P9独特基因分别为321和336个,L.plantarum A8中独特基因主要参与DNA复制、ABC转运系统（ABC transfer system）、PTS系统（phosphotransferase system）、磺酸盐转运系统、氨基酸生物合成等代谢通路;L.plantarum P9的独特基因以参与碳水化合物的运输和代谢基因居多,例如rpiA基因、lacZ基因、FruA基因等。[结论] 通过比较基因组学方法解析L.plantarum的基因组信息,发现动物肠道来源的L.plantarum具有较好的氨基酸转运能力,植物表面附着的L.plantarum菌株具有较好碳水化合物利用能力,从而为益生菌的开发与利用提供理论依据。     

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.     

Predominance of malolactic fermentation overd-glucose utilization inLactobacillus plantarum andLactobacillus curvatus wine strains

Summary Two selected wine strains of the genusLactobacillus (L. plantarum 197 andL. curvatus 783) were tested for their ability to complete malolactic fermentation (MLF) in a synthetic medium (PBM-broth) supplemented withL-malic acid (7.5–74.6 mM) andD-glucose (5.5–55 mM). The 24 directed fermentation assays, 12 for each bacterial strain, were carried out at 20°C and pH 3.5. MLF was completed (residualL-malic acid 0.2 mM) in eight days in 19 of the 24 fermentation assays, even in the presence of 74.6 mML-malic acid or 55.5 mMD-Glucose utilization was generally simultaneous to MLF but was completed (residual concentrations 0.2 mM) only in 6 of the 24 fermentation assays. These results support the use of these strains in directed MLF assays at the very differentL-malic acid andD-glucose concentrations tested.     

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.     

Metabolic engineering of a Lactobacillus plantarum double ldh knockout strain for enhanced ethanol production

Lactobacillus plantarum ferments glucose through the Embden–Meyerhof–Parnas pathway: the central metabolite pyruvate is converted into lactate via lactate dehydrogenase (LDH). By substituting LDH with pyruvate decarboxylase (PDC) activity, pyruvate may be redirected toward ethanol production instead of lactic acid fermentation. A PDC gene from the Gram-positive bacterium Sarcina ventriculi (Spdc) was introduced into an LDH-deficient strain, L. plantarum TF103, in which both the ldhL and ldhD genes were inactivated. Four different fusion genes between Spdc and either the S. ventriculi promoter or three Lactococcus lactis promoters in pTRKH2 were introduced into TF103. PDC activity was detected in all four recombinant strains. The engineered strains were examined for production of ethanol and other metabolites in flask fermentations. The recombinant strains grew slightly faster than the parent TF103 and produced 90–130 mM ethanol. Although slightly more ethanol was observed, carbon flow was not significantly improved toward ethanol, suggesting that a further understanding of this organism’s metabolism is necessary.     

基于比较基因组学揭示不同植物乳杆菌的遗传特征及菌株差异——以Lactobacillus plantarum P9和Lp-6研究为例

[目的]植物乳杆菌(Lactbacillus plantarum,L.plantarum)在食品、医药和动物养殖等多个领域均有应用。本文以L.plantarum P9和Lp-6为例,解析L.plantarum遗传背景和基因组特征,为其鉴定和开发奠定基础。[方法]本研究采用PacBio SMRT测序技术完成了L.plantarum P9和Lp-6全基因组测序,结合已公开的110株L.plantarum全基因组数据和1株模式菌株ATCC 14917T数据,通过比较基因组学方法探究L.plantarum基因组的差异。[结果]L.plantarum P9和Lp-6基因组大小分别为3314.1和3482.5 kb,GC含量(%)分别为44.38%和44.32%,二者分别含有8个和9个质粒。113株L.plantarum系统发育树结果显示,L.plantarum P9与L.plantarum ATCC 14917T遗传距离更近,L.plantarum Lp-6更接近祖先群体分支。与L.plantarum WCSF1相比,含有xerS等基因的22.0 kb基因组片段在L.planarum Lp-6上发生了倒位,在L.plantarum P9基因组中缺失;L.plantarum Lp-6染色体插入含tagF等基因的13.0 kb片段;包含gpmA等基因的14.4 kb基因片段插入到L.plantarum P9染色体中。[结论]通过比较基因组学方法解析L.plantarum P9和Lp-6遗传信息,发现不同L.plan tarum菌株的遗传特征存在差异。     

Purification and partial gene sequence of the tyrosine decarboxylase of Lactobacillus brevis IOEB 9809

Some lactic acid bacteria contain a tyrosine decarboxylase (TDC) which converts tyrosine to tyramine, a biogenic amine frequently encountered in fermented food and wine. Purification and microsequencing of the TDC of Lactobacillus brevis IOEB 9809 allowed us to determine a partial sequence of the TDC gene encoding 264 amino acids of the enzyme. Analysis of this protein sequence revealed typical features of pyridoxal phosphate-dependent amino acid decarboxylases while not any known decarboxylase was closely related to the TDC of L. brevis IOEB 9809. In addition, we could detect other L. brevis strains carrying a TDC gene in a rapid assay based on the polymerase chain reaction.     

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

Few 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.     

Identification and functional properties of dominant lactic acid bacteria isolated at different stages of solid state fermentation of cassava during traditional <Emphasis Type="Italic">gari</Emphasis> production

Culture-based technique was used to study the population dynamics of the bacteria and determine the dominant lactic acid bacteria (LAB) during cassava fermentation. LAB was consistently isolated from the fermented mash with an initial viable count of 6.00 log c.f.u. g⁻¹ observed at 12 h. The aerobic viable count of amylolytic lactic acid bacteria (ALAB) was higher than other group of LAB throughout the fermentation up to 96 h with the highest viable count of 8.08 log c.f.u. g⁻¹. Combination of phenotypic parameters and 16S rDNA gene sequencing identified the dominant group of LAB as Lactobacillus plantarum, L. fermentum and Leuconostoc mesenteroides while the pulse field gel electrophoresis determined that the strains were genotypically heterogeneous. The sugar fermentation profile of the isolates showed that indigestible sugars such as raffinose and stachyose can be fermented by the strains. Information was also generated about the functional properties of the strains. Only strain L. plantarum 9st0 isolate at 0 h of the fermentation produced bacteriocin with antagonism against closely related indicator strains. Quantitatively, the highest amylase activity was produced by strain L. plantarum 7st12, while appreciable amylase was also produced by L. fermentum 1st96. The result of this work showed that selection of mixed starter cultures of bacteriocin- and amylase-producing L. plantarum and L. fermentum will be highly relevant as starter cultures during the intermediate and large scale gari production.     

Characterization of lactic acid bacteria isolated from sourdoughs for Cornetto, a traditional bread produced in Basilicata (Southern Italy)

A total of 41 strains of lactic acid bacteria (LAB) isolated from durum wheat sourdoughs used to produce Cornetto di Matera bread, were identified by SDS-PAGE of whole cell proteins (WCP) and screened for acid production ability, antimicrobial activity and exopolysaccharide (EPS) production. The isolates were identified as Lactobacillus plantarum (49%), Leuconostoc mesenteroides (17%), Lactobacillus curvatus (15%), Lactobacillus paraplantarum (12%), Weissella cibaria (5%) and Lactobacillus pentosus (2%). Several strains of Lb. plantarum and Leuc. mesenteroides showed a high acid production ability. The antagonistic activity was tested using an agar-spot deferred antagonism assay against a set of five indicators. The species had different profiles of inhibition. Lb. plantarum had the largest spectrum of inhibition, while no isolates of W. cibaria and Leuc. mesenteroides showed antimicrobial activity. No strains had antimicrobial activity against Bacillus cereus. The inhibitory activity of five strains was confirmed to be sensitive to proteolytic enzymes and thus potentially due to bacteriocin production. All Leuc. mesenteroides and W. cibaria strains produced EPS from sucrose. Some Lb. plantarum and Lb. paraplantarum strains produced EPS from different sugars in solid media. EPS production in liquid media was different within the species, with the highest production in liquid media containing glucose and maltose. A defined strain starter culture (W. cibaria DBPZ1006, Lb. plantarum DBPZ1015 and S. cerevisiae MTG10) was selected on the basis of technological properties and tested in model sourdough fermentations.     

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.     

Effect of hematin on the activities of nitrite reductase and catalase in lactobacilli

The effect of the addition of hematin on the activities of nitrite reductase and catalase was studied with cell suspensions of strains of lactobacillus species. In cells of Lactobacillus plantarum grown under aerobic or anaerobic conditions nitrite reductase was present. This activity was not exhibited by cells of L. curvatus and only rarely by L. sake. In addition, catalase activity was detected in aerobically grown cells only, with all strains of L. plantarum and L. sake; again L. curvatus was devoid of this activity. Ammonia was formed as the main product of nitrite reduction by L. plantarum. With lactate as the electron donor, the end products of carbohydrate catabolism were carbon dioxide, acetoin and acetate. The activities of nitrite reductase and catalase in strains of lactobacillus species may be used for optimizing the quality of starter cultures applied for the production of raw sausages.     

短乳杆菌与植物乳杆菌的发酵特性

【背景】目前对于酸菜发酵的研究主要关注点是植物乳杆菌(Lactobacillus plantarum),有关短乳杆菌(Lactobacillus brevis)在酸菜方面的研究报道很少。【目的】为了挖掘短乳杆菌的发酵性能并开发酸菜发酵剂,将2株短乳杆菌分别与1株植物乳杆菌进行组合并发酵酸菜,分析短乳杆菌对酸菜发酵品质的影响。【方法】分别测定短乳杆菌与植物乳杆菌的单菌株生长产酸性能、耐酸性及亚硝酸盐降解力,并将两菌种组合后发酵酸菜,分析1-7d内酸度、乳酸菌活菌数、亚硝酸盐含量及酸菜质构特性的变化趋势。【结果】相较于短乳杆菌Lb-9-2,短乳杆菌Lb-5-3的生长和产酸速率较慢、酸耐受力较弱,但其亚硝酸盐降解力较强。两株短乳杆菌分别与植物乳杆菌Lp-9-1组合后产酸力显著增强,并在3 d时达到最低pH值(约3.10);植物乳杆菌Lp-9-1的添加使酸菜中总体乳酸菌生长延迟,在5 d时达到最高活菌数;组合菌种的样品中亚硝酸盐含量在1-7 d内变化较为平缓,前5天内两个组合之间差异不显著;接种乳酸菌会降低酸菜硬度和弹性,发酵3d时Lb-5-3/Lp-9-1组合的硬度最大,感官评价得分最高。【...     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

Preliminary characterization of wine lactobacilli able to degrade arginine

Lactobacillus strains able to degrade arginine were isolated and characterized from a typical red wine. All the strains were gram-positive, catalase-negative and produced both D- and L-lactate from glucose. Strains L2, L3, L4, and L6 were able to produce CO₂ from glucose; however, production of CO₂ from glucose was not observed in strains L1 and L5, suggesting that they belong to the homofermentative wine lactic acid bacteria (LAB) group. All of the lactobacilli were tested for their ability to ferment 49 carbohydrates. The sugar fermentation profile of strain L1 was unique, suggesting that this strain belonged to Lactococcus lactis ssp. cremoris, a non-typical wine LAB. Furthermore, a preliminary typing was performed by using a random amplified polymorphic DNA analysis (RAPD-PCR analysis).