ODC Gene Research Articles

Safety assessment of enterocin-producing Enterococcus strains isolated from sheep and goat colostrum

BackgroundThis study investigates the safety evaluation of enterocin-producing 11 E. mundtii and two E. faecium strains previously isolated from small livestock colostrums. Enterococcus species do not possess Generally Recognized as Safe (GRAS) status. Hence, it is critical to scrutinize enterococci’s antibiotic resistance, virulence characteristics, and biogenic amine production capabilities in order to assess their safety before using them as starter or adjunct cultures.ResultsEnterococcus strains showed susceptibility to medically significant antibiotics. Multiple-drug resistance (MDR) was found in only E. faecium HC121.4, and its multiple antibiotic resistance (MAR) index was detected to be 0.22. The tetL and aph(3')-IIIa were the most commonly found antibiotic resistance genes in the strains. However, E. mundtii strains HC56.3, HC73.1, HC147.1, and E. faecium strain HC121.4 were detected to lack any of the antibiotic resistance genes examined in this study. Only E. mundtii HC166.3 showed hemolytic activity, while none of the strains engage in gelatinase activity. The strains were identified to have virulence factor genes with a low rate. None of the virulence factor genes could be detected in E. mundtii HC26.1, HC56.3, HC73.1, HC165.3, HC166.8, and E. faecium HC121.4. The E. mundtii HC73.2 strain displayed the highest presence of virulence factor genes, namely gelE, efaAfs, cpd, and ccf. Similarly, the E. mundtii HC112.1 strain showed a significant presence of genes efaAfm, ccf, and acm. There was no decarboxylation of histidine, ornithine, or lysine seen in any of the strains. Nevertheless, E. faecium HC121.4 and HC161.1 strains could decarboxylate tyrosine, but E. mundtii HC26.1, HC56.3, HC73.1, HC73.2, HC112.1, HC147.1, HC155.2, HC165.3, HC166.3, HC166.5, and HC166.8 strains only showed a limited capacity for tyrosine decarboxylation. None of the strains possessed the hdc, odc, or ldc genes, but all of them had the tdc gene.ConclusionThe E. mundtii HC56.3 and HC73.1 strains were deemed appropriate for utilization in food production. Using the remaining 11 strains as live cultures in food production activities could pose a possible risk to consumer health.

Evaluating the impact of Torulaspora delbrueckii and amino acid concentration on the nitrogen metabolism of Oenococcus oeni

This study investigates the impact of Saccharomyces cerevisiae and Torulaspora delbrueckii inoculation strategies and amino acid supplementation on nitrogen metabolism and malolactic fermentation (MLF) performance in Oenococcus oeni. Oenococcus oeni has low demand of nitrogen and typically prefers peptides over amino acids. In this work we studied the nitrogen metabolism (proteins, peptides, amino acids and biogenic amines (BA)) of three O. oeni strains. Higher initial amino acid concentrations generally accelerated MLF, particularly with the PSU-1 strain, which exhibited stuck fermentation in some conditions with low initial amino acid concentration related with coinoculation of yeasts. Nitrogen metabolism in O. oeni showed a preference for peptide-bound amino acids, and gene expression analyses highlighted a general upregulation in response to increased amino acid concentrations. BA production, particularly cadaverine and putrescine, was strain-dependent and associated with the presence of the odc gene. Besides, the production of 2-phenylethylamine was related with alcoholic fermentation (AF) inoculation strategy. Overall, the study demonstrates the complexity of nitrogen metabolism in O. oeni, emphasizing that it is influenced by both the yeast species used in AF and the initial amino acid composition, highlighting the importance of peptide composition, offering a potential tool for optimizing MLF in winemaking.

Functional analysis of Ornithine decarboxylase in manipulating the wing dimorphism in Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)

The brown planthopper (BPH, Nilaparvata lugens), a major insect pest of rice, can make a shift in wing dimorphism to adapt to complex external environments. Our previous study showed that NlODC (Ornithine decarboxylase in N. lugens) was involved in wing dimorphism of the brown planthopper. Here, further experiments were conducted to reveal possible molecular mechanism of NlODC in manipulating the wing dimorphism. We found that the long-winged rate (LWR) of BPH was significantly reduced after RNAi of NlODC or injection of DFMO (D, L-α-Difluoromethylornithine), and LWR of males and females significantly decreased by 21.7% and 34.6%, respectively. Meanwhile, we also examined the contents of three polyamines under DFMO treatment and found that the contents of putrescine and spermidine were significantly lower compared to the control. After 3rd instar nymphs were injected with putrescine and spermidine, LWR was increased significantly in both cases, and putrescine was a little bit more effective, with 5.6% increase in males and 11.4% in females. Three days after injection of dsNlODC, injection of putrescine and spermidine rescued LWR to the normal levels. In the regulation of wing differentiation in BPH, NlODC mutually antagonistic to NlAkt may act through other signaling pathways rather than the classical insulin signaling pathway. This study illuminated a physiological function of an ODC gene involved in wing differentiation in insects, which could be a potential target for pest control.

Antioxidant, antibacterial and antileishmanial potential of Micromeria nervosa extracts and molecular mechanism of action of the bioactive compound.

This study aimed to determine the antibacterial and antileishmanial potential of Micromeria nervosa extracts. The identification of the antileishmanial compound and the study of its molecular mechanism of action have also been undertaken. Ethanol extract showed high polyphenol content and diethyl ether extract exhibited high DPPH scavenging and low beta-carotene bleaching activity (IC50=13.04±0.99µg/mL and 200.18±3.32µg/mL, respectively). However, diethyl ether extract displayed high antibacterial activity against Gram-positive strains including methicillin-resistant Staphylococcus aureus (MIC=31.25µg mL-1), Staphylococcus aureus ATCC6538 (MIC=62.5µg mL-1) and Listeria monocytogenes ATCC 19115 (MIC=125µg mL-1) as well as high antileishmanial activity against the promastigote forms of L. infantum and L. major (IC50=11.45µg mL-1 and IC50=14.53µg mL-1, respectively). The active compound was purified using bioassay-guided fractionation and thin layer chromatography, and identified as ursolic acid using high performance liquid chromatography coupled with a photodiode array and mass spectrometry. The purified compound was strongly inhibitory against the promastigote and amastigote forms of L. infantum and L. major (IC50=5.87µg mL-1 and 6.95mL-1, versus 9.56µg mL-1 and 10. 68µg mL-1, respectively) without overt cytotoxicity against Raw 264.7 macrophage cells (SI=13.53 and 11.43, respectively). The commercial compound (ursolic acid) showed similar activity against amastigotes and promastigotes forms of L. infantum and L. major. Moreover, its molecular mode of action against leishmaniasis seems to involve the expression of the ODC and SPS genes involved in thiol pathway. Extracts of Micromeria nervosa can be considered as a potential alternative to antimicrobial and antileishmanial drugs.

Ornithine decarboxylase regulates putrescine-related metabolism and pollen development in Atropa belladonna

Polyamines, including putrescine, spermidine, and spermine, play critical roles in cell physiology by different forms. As a rate-limiting enzyme that converts ornithine to putrescine, ornithine decarboxylase (ODC, EC 1.1.1.37) has been studied in detail in animals and microorganisms, but its specific functions are poorly understood in plants. In this study, the metabolic and developmental roles of the ODC gene were studied through RNAi-mediated suppression of the ODC gene (AbODC) in A. belladonna. Suppression of AbODC reduced the production of precursors of medicinal tropane alkaloids, including putrescine and N-methylputrescine, as well as hyoscyamine and scopolamine. In AbODC-RNAi roots, the production of putrescine and spermidine in free form was reduced, but in the AbODC-RNAi leaves, the content of free polyamines was not altered. In the roots/leaves of AbODC-RNAi plants, the production of conjugated and bound polyamines was reduced. In addition, suppression of the ODC gene resulted in reduction of polyamines and pollen sterility in AbODC-RNAi flowers. In floral organs, GUS-staining results indicated that AbODC was domainantly expressed in pollen. In summary, ornithine decarboxylase not only plays a key role in regulating the biosynthesis of diverse forms of polyamines and medicinal tropane alkaloids, but also participates in pollen development.

Ornithine decarboxylase deficiency critically impairs nitrogen metabolism and survival in Aedes aegypti mosquitoes.

Ornithine decarboxylase (ODC; EC 4.1.1.17) catalyzes the conversion of ornithine to putrescine, the rate-limiting first step for de novo polyamine biosynthesis. Previously, we reported that genetic knockdown of xanthine dehydrogenase 1 (XDH1)-a gene encoding the enzyme involved in the last two steps of uric acid synthesis-causes an increase in ODC transcript levels in fat body of blood-fed Aedes aegypti mosquitoes, suggesting a crosstalk at molecular level between XDH1 and ODC during nitrogen disposal. To further investigate the role of ODC in nitrogen metabolism, we conducted several biochemical and genetic analyses in sugar- and blood-fed A. aegypti females. Distinct ODC gene and protein expression patterns were observed in mosquito tissues dissected during the first gonotrophic cycle. Both pharmacological and RNA interference-mediated knockdown of ODC negatively impacted mosquito survival, disrupted nitrogen waste disposal, delayed oviposition onset, and decreased fecundity in vitellogenic blood-fed females. A lag in the expression of two major digestive serine proteases, a reduction of blood meal digestion in the midgut, and a decrease in vitellogenin yolk protein uptake in ovarian follicles were observed by western blots in ODC-deficient females. Moreover, genetic silencing of ODC showed a broad transcriptional modulation of genes encoding proteins involved in multiple metabolic pathways in mosquito fat body, midgut, and Malpighian tubules prior to and after blood feeding. All together, these data demonstrate that ODC plays an essential role in mosquito metabolism, and that ODC crosstalks with multiple genes and proteins to prevent deadly nitrogen perturbations in A. aegypti females.

Production of Putrescine and Cadaverine by Paucilactobacillus wasatchensis.

Lactic acid bacteria (LAB) play a key role in many food fermentations. However, some LAB species can also cause food spoilage, e.g., through the formation of biogenic amines. Paucilactobacillus wasatchensis is a LAB that causes late gas production in Cheddar cheese, the molecular causes of which are not fully understood. This study reports on the ability of P. wasatchensis WDC04 to produce cadaverine and putrescine in broth supplemented with lysine and ornithine, as well as in a model cheese. The raclette-type semi-hard cheese produced with P. wasatchensis as an adjunct culture contained 1,085 mg kg−1 of cadaverine and 304 mg kg−1 of putrescine after 120 days of ripening. We identified two ornithine decarboxylase genes (odc) and a putrescine-ornithine antiporter gene (potE) in the genome sequence of P. wasatchensis. We could show that the two odc genes, which are located on two contigs, are contiguous and form the genetic cluster odc2-odc1-potE. Alignment searches showed that similar gene clusters exist in the genomes of Levilactobacillus paucivorans DSMZ22467, Lentilactobacillus kribbianus YH-lac9, Levilactobacillus hunanensis 151-2B, and Levilactobacillus lindianensis 220-4. More amino acid sequence comparisons showed that Odc1 and Odc2 shared 72 and 69% identity with a lysine and ornithine decarboxylase from Ligilactobacillus saerimneri 30a, respectively. To clarify the catalytic activities of both enzymes, the odc-coding genes were cloned and heterologously expressed as His-tagged fusion protein. The purified Odc1 protein decarboxylated lysine into cadaverine, while the recombinant Odc2 protein preferentially produced putrescine from ornithine but also exhibited low lysine decarboxylating activity. Both enzymes were active at pH of 5.5, a value often found in cheese. To our knowledge, this is only the second lysine decarboxylase in LAB whose function has been verified. The tandem arrangement of the genes in a single cluster suggests a gene duplication, evolving the ability to metabolize more amino. Divergent substrate preferences highlight the necessity of verifying the functions of genes, in addition to automatic annotation based on sequence similarity. Acquiring new biochemical data allows better predictive models and, in this case, more accurate biogenic amine production potential for LAB strains and microbiomes.

TRPC1‐mediated Ca2+signaling enhances intestinal epithelial restitution by increasing α4 association with PP2Ac after wounding

Gut epithelial restitution after superficial wounding is an important repair modality regulated by numerous factors including Ca2+ signaling and cellular polyamines. Transient receptor potential canonical‐1 (TRPC1) functions as a store‐operated Ca2+ channel in intestinal epithelial cells (IECs) and its activation increases epithelial restitution by inducing Ca2+ influx after acute injury. α4 is a multiple functional protein and implicated in many aspects of cell functions by modulating protein phosphatase 2A (PP2A) stability and activity. Here we show that the clonal populations of IECs stably expressing TRPC1 (IEC‐TRPC1) exhibited increased levels of α4 and PP2A catalytic subunit (PP2Ac) and that TRPC1 promoted intestinal epithelial restitution by increasing α4/PP2Ac association. The levels of α4 and PP2Ac proteins increased significantly in stable IEC‐TRPC1 cells and this induction in α4/PP2Ac complexes was accompanied by an increase in IEC migration after wounding. α4 silencing by transfection with siRNA targeting α4 (siα4) or PP2Ac silencing destabilized α4/PP2Ac complexes in stable IEC‐TRPC1 cells and repressed cell migration over the wounded area. Increasing the levels of cellular polyamines by stable transfection with the Odc gene stimulated α4 and PP2Ac expression and enhanced their association, thus also promoting epithelial restitution after wounding. In contrast, depletion of cellular polyamines by treatment with α‐difluoromethylornithine reduced α4/PP2Ac complexes and repressed cell migration. Ectopic overexpression of α4 partially rescued rapid epithelial repair in polyamine‐deficient cells. These results indicate that activation of TRPC1‐mediated Ca2+ signaling enhances cell migration primarily by increasing α4/PP2Ac associations after wounding and this pathway is tightly regulated by cellular polyamines.

Induced expression of rolC for study of its effect on the expression of genes associated with nicotine synthesis in tobacco

Background. Agrobacterium rhizogenes rol genes cause not only hairy root syndrome in plants, but also affect their secondary metabolism. There are cases of increasing of nicotine content in transgenic tobacco roots expressing rolC alone or in combination with other rol genes. In this work, we evaluated the change in the expression of nicotine synthesis genes and their regulators in response to the induction of expression of rolC.
 Materials and methods. Plant material was represented by three Nicotiana tabacum genotypes: cv. Samsun and two transgenic lines, derived from this cultivar and containing rolC under dexamethasone inducible promoter: A. rhizogenes rolC (Pdex-A4rolC) and N. tabacum rolC (Pdex-trolC) correspondingly. Fluidigm Biomark RT-PCR was used for evaluation of expression of QPT1, QPT2, A622, ODC, ADC, PMT1, PMT2, PMT3, PMT4, MPO1, MPO2, BBL, MATE1, MATE2, ARF6, ERF168, ERF189, A4rolC, NtrolC, and reference gene gapdh. HPLC-MS / MS analysis was used to determine content of nicotine and its derivatives in plant tissues.
 Results. Expression of PMT genes for the synthesis of the pyrrolidine ring, as well as the genes, controlling enzyme for final stages of nicotine synthesis, was higher in transgenic lines without induction of rolC expression. Regulatory genes were activated by dexamethasone in both transgenic and control lines, indicating the inapplicability of rolC dexamethasone induction for their study. The level of expression of PMT and MPO genes increased over time in transgenic dexamethasone-induced lines. Nicotine content decreased in transgenic dexamethasone-induced plants.
 Conclusions. The rolC gene does not play a primary role in the regulation of nicotine synthesis genes. The mechanism of regulation of different nicotine biosynthesis genes and TFs varies.

Effects of Amino Acid Decarboxylase Genes and pH on the Amine Formation of Enteric Bacteria From Chinese Traditional Fermented Fish (Suan Yu).

The formation of biogenic amines (BAs) is an important potential risk in Suan yu. This study investigated the amine production abilities of 97 strains of enteric bacteria screened from Suan yu. The genotypic diversity of amino acid decarboxylase and the effect of pH were explored on 27 strains of high-yield BAs. Results showed that high levels of putrescine, histamine, and cadaverine were produced by the 97 strains. In addition, 27 strains carried odc, speA, speB, adiA, and ldc genes. Thirteen carried hdc gene. Morganella morganii 42C2 produced the highest putrescine content of 880 mg/L via the ornithine decarboxylase pathway. The highest histamine content was produced by Klebsiella aerogenes 13C2 (1,869 mg/L). The highest cadaverine content was shown by Klebsiella pneumoniae 47C2 (1,821 mg/L). odc, adiA, speB, ldc, and hdc play important roles in the cellular acid stress response. Acid stress caused the growth delay but can increase the contents of putrescine, histamine, and cadaverine. Decarboxylase was strain specific rather than species specific. This study provides a reference for the genotypic diversity of decarboxylase and effect of pH on the types and abilities of BAs produced by enteric bacteria in Suan yu.

Temporal changes in gene expression and genotype frequency of the ornithine decarboxylase gene in native silverside Basilichthys microlepidotus: Impact of wastewater reduction due to implementation of public policies.

Human activity has caused a deterioration in the health and population size of riverine species; thus, public policies have been implemented to mitigate the anthropogenic impacts of water use, watercourse transformation, and pollution. We studied the Maipo River Basin, one of the most polluted with untreated wastewater in Chile, for a period of 12 years (2007‐2019). Since the implementation of new public policies, including the operation of a wastewater collector (2012), the Maipo River Basin is currently much less polluted by untreated water than before. To analyze the impact of wastewater reduction in this river basin, we studied the native silverside (Basilichthys microlepidotus), which inhabits both polluted and unpolluted areas of the river basin. Previous studies reported the overexpression of the ornithine decarboxylase (odc) gene, heterozygote deficit, and high frequency of a homozygote odc genotype in silverside populations that inhabit wastewater‐polluted sites, suggesting a phenotypic change and genotypic selection in response to pollution. Here, a population affected and another population unaffected by wastewater were studied before and after implementing the wastewater collector. The physicochemical data of water samples, changes in odc expression and microsatellite variability, and odc genotype frequencies were analyzed. The results showed physicochemical changes in the affected site before and after the operation of the wastewater collector. The microsatellite loci showed no changes in either population. The odc expression in the affected site was higher before the operation of the wastewater collector. Significant changes in the genotype frequencies of the odc gene before and after the wastewater collector operation were detected only at the affected site, wherein the homozygous dominant genotype decreased from >59% to <25%. Our results suggest that public policies aimed at mitigating aquatic pollution can indirectly affect both gene expression and genotype frequencies of important functional genes.

Characterization of natural Oenococcus oeni strains for Montepulciano d’Abruzzo organic wine production

Montepulciano d'Abruzzo is a red wine grape variety of Vitis vinifera L., grown in Central Italy. It is mainly identified with Abruzzo region, where it currently accounts for around 50% of the regional vineyard. Malolactic fermentation (MLF) has a crucial role in red wines giving microbial stabilization, biological deacidification through the decarboxylation of l-malic acid to l( +)-lactic acid and carbon dioxide, and increasing complexity of wine aroma. Studies are focusing on the selection of yeast starter cultures for this wine, while few studies are available on malolactic bacteria. Therefore, a technological (ability to grow up at different pH, concentration of SO2, ethanol, presence of hdc, tdc and odc genes, conversion of malic acid into lactic acid) and genetic characterization of autochthonous Oenococcus oeni strains was performed. Moreover, O. oeni strain with the best traits was selected and produced by a local starter industry and used in cellar to produce Montepulciano d’Abruzzo organic wine without added SO2. Obtained wines not only maintained the typical traits of Montepulciano d’Abruzzo wines but also showed healthy characteristics since wines were histamine free. Selected starter is actually produced and dispensed on demand and in a frozen concentrate culture for wineries.

Inhibition of Biogenic Amines in Shewanella baltica by Anthocyanins Involving a Quorum Sensing System

Put was the main biogenic amine produced by Shewanella baltica. PL and PP were the main QS autoinducers of S. baltica. PL and PP displayed significant positive correlation with Put. DKPs and Put production and odc gene expression were inhibited by anthocyanins. Anthocyanins were proposed as new QSIs and seafood preservative candidates.

Gene expression analysis of antimony resistance in Leishmania tropica using quantitative real-time PCR focused on genes involved in trypanothione metabolism and drug transport

Pentavalent antimonials remain the treatment of choice for all the clinical forms of leishmaniasis. The increasing rates of antimony resistance are becoming a serious health problem in treatment of anthroponotic cutaneous leishmaniasis (ACL). Accordingly, unraveling molecular markers is crucial for improving medication strategies and monitoring of drug-resistant parasites. Different studies have suggested the importance of genes involved in trypanothione metabolism and drug transport. In this regard, presentstudy was designed to investigate the RNA expression level of five genes including γ-GCS, ODC, TRYR (involved in trypanothione metabolism), AQP1 (acts in drug uptake) and MRPA (involved in sequestration of drug) in sensitive and resistant Leishmania tropica isolates. Seven antimony-resistant and seven antimony-sensitive L. tropica clinical isolates were collected from ACL patients. Drug sensitivity test was performed on the samples as well as reference strains; afterwards, gene expression analysis was performed on clinical isolates by quantitative real-time PCR. The results revealed that the average expression level of AQP1 gene was decreased (0.47-fold) in resistant isolates compared to sensitive ones whereas MRPA (2.45), γ-GCS (2.1) and TRYR (1.97) was upregulated in resistant isolates. The average expression of ODC (1.24-fold) gene was not different significantly between sensitive and resistant isolates. Our findings suggest that AQP1, MRPA, GSH1 and TRYR can be considered as potential molecular markers for screening of antimony resistance in some L. tropica clinical isolates.

Molecular Characterization of Histamine-Producing Psychrotrophic Bacteria Isolated from Red Octopus (Octopus maya) in Refrigerated Storage.

The present study aimed at determining the histamine production capacity of Gram (+) and Gram (−) bacteria isolated from Octopus maya, along with identifying the presence of amino acid decarboxylase genes. Of the total 80 psychrotrophic microorganisms, 32 strains were identified as histamine-forming bacteria. The recombinant DNA technique was used for genotypic identification of histidine (hdc), ornithine (odc), and lysine decarboxylases (ldc) genes. Thirty-two strains were able to produce 60–100 ppm in trypticase soy broth with 1.0% l-histidine after 6 h at 20 °C. NR6B showed 98% homology with Hafnia alvei. NR73 represented 18.8% of the total isolates and showed 98% homology with Enterobacter xianfengensis and Enterobacter cloacae. NR6A represented 6% of the total isolates, which were identified as Lactococcus sp. The hdc gen from NR6B showed 100% identity with hdc from Morganella morganii; ldc showed 97.7% identity with ldc from Citrobacter freundii. The Odc gene was detected only in NR73 and showed 100% identity with Enterobacter sp. All the isolated were identified as weak histamine–former. The ingestion of a food containing small amounts of histamine has little effect on humans; however, the formation of biogenic amines is often considered as an indicator of hygienic quality; this emphasizes the importance of improving good management practices and storage.

Biogenic Amine Production by and Phylogenetic Analysis of 23 Photobacterium Species

Photobacterium species are members of the bacterial communities typically associated with scombrotoxin-forming fish. Reclassification and discovery of new Photobacterium species has caused confusion as to which species are capable of biogenic amine production. We analyzed histamine, cadaverine, and putrescine production by 104 Photobacterium strains representing 23 species. The presence of the genes for histidine decarboxylase ( hdc), lysine decarboxylase ( ldc), and ornithine decarboxylase ( odc) was determined by real-time or conventional PCR and whole genome sequencing. Significant histamine production (>200 ppm) was detected in five Photobacterium species: P. angustum, P. aquimaris, P. kishitanii, P. damselae, and P. phosphoreum. The hdc gene was detected in all of these histamine-producing species except P. phosphoreum. Cadaverine was produced by eight Photobacterium species: P. angustum, P. aquimaris, P. damselae, P. iliopiscarium, P. kishitanii, P. leiognathi, P. mandapamensis, and P. phosphoreum. Putrescine was produced by six Photobacterium species: P. angustum, P. aquimaris, P. kishitanii, P. leiognathi, P. mandapamensis, and Photobacterium sp. Cadaverine production correlated closely with the presence of the ldc gene, but putrescine production did not correlate closely with the presence of the odc gene. Characterization of the biogenic amine production by Photobacterium species will allow identification of these marine bacteria and help ensure that current guidelines account for mitigation of these bacteria.

Identification and in vitro assessment of potential probiotic characteristics and antibacterial effects of Lactobacillus plantarum subsp. plantarum SKI19, a bacteriocinogenic strain isolated from Thai fermented pork sausage.

A total of 2257 lactic acid bacteria were preliminarily screened for antagonistic activity against Lactobacillus sakei subsp. sakei JCM 1157. Strain SKI19 was selected and identified at the subspecies level as Lactobacillus plantarum subsp. plantarum SKI19, using 16S rRNA gene sequence analysis combined with recA and dnaK genes' amplification. Antibacterial activity of SKI19 was completely lost after treatment of neutralized cell free culture supernatant with proteolytic enzymes, suggesting that SKI19 produced a bacteriocin-like substance that inhibited not only closely related species, but was also effective against Listeria monocytogenes DMST 17303. Viewed under scanning electron microscope, cell membranes of the indicator strain appeared to collapse after exposure to the bacteriocin-like substance. In vitro tests concerning probiotic properties, SKI19 survived under simulated gastrointestinal tract conditions, and adhesion of its cell surface to xylene and chloroform was 90.14 and 89.85%, respectively. Complete inhibition by SKI19 against pathogenic bacteria (Escherichia coli DMST 4212, L. monocytogenes DMST 17303, and Staphylococcus aureus DMST 8840) was observed in co-cultivation under anaerobic conditions. A safety assessment showed that SKI19 was susceptible to several antibiotics and had no haemolytic activity. PCR amplification of virulence factors with the specific primers for ace, asa1, cylLS , efaAfs , hyl, and gelE genes were negative for SKI19. Also, SKI19 did not harbor any hdc, tdc, odc or ldc genes involved in biogenic amine production. The results reveal that SKI19 has probiotic potential and antibacterial activity, and is safe for further application in certain food products.

Lactobacillus rossiae strain isolated from sourdough produces putrescine from arginine

This work reports a Lactobacillus rossiae strain (L. rossiae D87) isolated from sourdough that synthesizes putrescine - a biogenic amine that raises food safety and spoilage concerns - from arginine via the ornithine decarboxylase (ODC) pathway. The odc and potE genes were identified and sequenced. These genes respectively encode ornithine decarboxylase (Odc), which participates in the decarboxylation of ornithine to putrescine, and the ornithine/putrescine exchanger (PotE), which exchanges ornithine for putrescine. Transcriptional analysis showed that odc and potE form an operon that is regulated transcriptionally by ornithine in a dose-dependent manner. To explore the possible role of the ODC pathway as an acid stress resistance mechanism for this bacterium, the effect of acidic pHs on its transcriptional regulation and on putrescine biosynthesis was analysed. Acidic pHs induced the transcription of the odc-potE genes and the production of putrescine over that seen at neutral pH. Further, putrescine production via the ODC system improved the survival of L. rossiae D87 by counteracting the acidification of the cytoplasm when the cells were subjected to acidic conditions. These results suggest the ODC pathway of L. rossiae D87 provides a biochemical defence mechanism against acidic environments.

Occurrence of biogenic amine-forming lactic acid bacteria during a craft brewing process

The aims of this study were to assess the lactic acid bacteria population present during a craft brewing process and to determine their potential to produce biogenic amines (BA). Samples at different stages were taken and the isolates grown on MRS agar plates were genotyped by randomly amplified polymorphic DNA-PCR and identified by MALDI-TOF as belonging to Leuconostoc mesenteroides (80%), Lactobacillus brevis (15%) and Enterococcus faecium (5%).To assay the aminobiogenic capacity of the strains, both a qualitative assay, using a synthetic medium, and a quantitative assay, using wort and beer, were carried out and contents of different BA and free amino acids (FAA) were measured by RP-HPLC. Presence in strains of tdc, hdc, odc and ldc decarboxylase genes was assayed by multiplex PCR.Tyramine was the only BA produced by all the strains in the qualitative assay while the 90% did it significantly in wort and beer. Concentrations of the remaining BA analyzed did not change significantly. In contrast, all the strains harboured the tdc, hdc, odc decarboxylases genes. Total BA content in the beers ranged between 30.3 and 48.3 mg/L. None of the strains produced any significant change in total FAA content in wort or beer.

Spermidine Inversely Influences Surface Interactions and Planktonic Growth in Agrobacterium tumefaciens.

In bacteria, the functions of polyamines, small linear polycations, are poorly defined, but these metabolites can influence biofilm formation in several systems. Transposon insertions in an ornithine decarboxylase (odc) gene in Agrobacterium tumefaciens, predicted to direct synthesis of the polyamine putrescine from ornithine, resulted in elevated cellulose. Null mutants for odc grew somewhat slowly in a polyamine-free medium but exhibited increased biofilm formation that was dependent on cellulose production. Spermidine is an essential metabolite in A. tumefaciens and is synthesized from putrescine in A. tumefaciens via the stepwise actions of carboxyspermidine dehydrogenase (CASDH) and carboxyspermidine decarboxylase (CASDC). Exogenous addition of either putrescine or spermidine to the odc mutant returned biofilm formation to wild-type levels. Low levels of exogenous spermidine restored growth to CASDH and CASDC mutants, facilitating weak biofilm formation, but this was dampened with increasing concentrations. Norspermidine rescued growth for the odc, CASDH, and CASDC mutants but did not significantly affect their biofilm phenotypes, whereas in the wild type, it stimulated biofilm formation and depressed spermidine levels. The odc mutant produced elevated levels of cyclic diguanylate monophosphate (c-di-GMP), exogenous polyamines modulated these levels, and expression of a c-di-GMP phosphodiesterase reversed the enhanced biofilm formation. Prior work revealed accumulation of the precursors putrescine and carboxyspermidine in the CASDH and CASDC mutants, respectively, but unexpectedly, both mutants accumulated homospermidine; here, we show that this requires a homospermidine synthase (hss) homologue. Polyamines are small, positively charged metabolites that are nearly ubiquitous in cellular life. They are often essential in eukaryotes and more variably in bacteria. Polyamines have been reported to influence the surface-attached biofilm formation of several bacteria. In Agrobacterium tumefaciens, mutants with diminished levels of the polyamine spermidine are stimulated for biofilm formation, and exogenous provision of spermidine decreases biofilm formation. Spermidine is also essential for A. tumefaciens growth, but the related polyamine norspermidine exogenously rescues growth and does not diminish biofilm formation, revealing that the growth requirement and biofilm control are separable. Polyamine control of biofilm formation appears to function via effects on the cellular second messenger cyclic diguanylate monophosphate, regulating the transition from a free-living to a surface-attached lifestyle.