Agmatine Deiminase Research Articles

Identification and Characterization of Polyamine Metabolism in Citrus in Response to 'Candidatus Liberibacter asiaticus' Infection.

Citrus Huanglongbing, one of the most devastating citrus diseases, is caused by 'Candidatus Liberibacter asiaticus' (CLas). Polyamines are aliphatic nitrogen-containing compounds that play important roles in disease resistance and are synthesized primarily by two pathways: an arginine decarboxylation pathway and an ornithine decarboxylation pathway. However, it is unclear whether polyamines play a role in the tolerance of citrus to infection by CLas and, if so, whether one or both of the core polyamine metabolic pathways are important. We used high-performance liquid chromatography and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry to detect the contents of nine polyamine metabolism-related compounds in six citrus cultivars with varying levels of tolerance to CLas. We also systematically detected the changes in polyamine metabolism-related compounds and H2O2 contents and compared the gene expression levels and the activities of enzymes involved in the polyamine metabolic pathway among healthy, asymptomatic, and symptomatic leaves of Newhall navel oranges infected with CLas. The tolerant and moderately tolerant varieties showed higher polyamine metabolism-related compound levels than those of susceptible varieties. Compared with the healthy group, the symptomatic group showed significantly increased contents of arginine, ornithine, γ-aminobutyric acid, and putrescine by approximately 180, 19, 1.5, and 0.2 times, respectively, and upregulated expression of biosynthetic genes. Arginase and ornithine decarboxylase enzyme activities were the highest in the symptomatic group, whereas arginine decarboxylase and agmatine deiminase enzyme activities were the highest in the asymptomatic group. The two polyamine biosynthetic pathways showed different trends with the increase of the CLas titer, indicating that polyamines were mainly synthesized through the arginine decarboxylase pathway in the asymptomatic leaves and were synthesized via the ornithine decarboxylase pathway in symptomatic leaves. These findings provide new insight into the changes in polyamine metabolism in citrus infected with CLas.

Characterization of an Arginine Decarboxylase from Streptococcus pneumoniae by Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Polyamines are polycations derived from amino acids that play an important role in proliferation and growth in almost all living cells. In Streptococcus pneumoniae (the pneumococcus), modulation of polyamine metabolism not only plays an important regulatory role in central metabolism, but also impacts virulence factors such as the capsule and stress responses that affect survival in the host. However, functional annotation of enzymes from the polyamine biosynthesis pathways in the pneumococcus is based predominantly on computational prediction. In this study, we cloned SP_0166, predicted to be a pyridoxal-dependent decarboxylase, from the Orn/Lys/Arg family pathway in S. pneumoniae TIGR4 and expressed and purified the recombinant protein. We performed biochemical characterization of the recombinant SP_0166 and confirmed the substrate specificity. For polyamine analysis, we developed a simultaneous quantitative method using hydrophilic interaction liquid chromatography (HILIC)-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization. SP_0166 has apparent Km, kcat, and kcat/Km values of 11.3 mM, 715,053 min-1, and 63,218 min-1 mM-1, respectively, with arginine as a substrate at pH 7.5. We carried out inhibition studies of SP_0166 enzymatic activity with arginine as a substrate using chemical inhibitors DFMO and DFMA. DFMO is an irreversible inhibitor of ornithine decarboxylase activity, while DFMA inhibits arginine decarboxylase activity. Our findings confirm that SP_0166 is inhibited by DFMA and DFMO, impacting agmatine production. The use of arginine as a substrate revealed that the synthesis of putrescine by agmatinase and N-carbamoylputrescine by agmatine deiminase were both affected and inhibited by DFMA. This study provides experimental validation that SP_0166 is an arginine decarboxylase in pneumococci.

Dysregulation of Porphyromonas gingivalis Agmatine Deiminase Expression in Alzheimer's Disease.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder, with a significant burden on global health. AD is characterized by a progressive cognitive decline and memory loss. Emerging research suggests a potential link between periodontitis, specifically the presence of oral bacteria such as Porphyromonas gingivalis (P. gingivalis), and AD progression. P. gingivalis produces an enzyme, Agmatine deiminase (AgD), which converts agmatine to N-carbamoyl putrescine (NCP), serving as a precursor to essential polyamines. Recent studies have confirmed the correlation between disruptions in polyamine metabolism and cognitive impairment. This study aims to investigate the dysregulation of P. gingivalis Agmatine deiminase (PgAgD) in the context of AD. Saliva samples were collected from a total of 54 individuals, including 27 AD patients and 27 healthy controls. The expression of the PgAgD gene was analyzed using quantitative Real-- Time PCR. The results showed a significant decrease in PgAgD gene expression in the saliva samples of AD patients compared to healthy controls. This downregulation was found in AD patients with advanced stages of periodontitis. Additionally, a correlation was observed between the decrease in PgAgD expression and the 30-item Mini-Mental State Examination (MMSE) score. These findings suggest that measuring PgAgD expression in saliva could be a noninvasive tool for monitoring AD progression and aid in the early diagnosis of patients with periodontitis. Further research is needed to validate our results and explore the underlying mechanisms linking periodontitis, PgAgD expression, and AD pathophysiology.

Transcriptomics in serum and culture medium reveal shared and differential gene regulation in pathogenic and commensal Streptococcus suis.

Streptococcus suis colonizes the upper respiratory tract of healthy pigs at high abundance but can also cause opportunistic respiratory and systemic disease. Disease-associated S. suis reference strains are well studied, but less is known about commensal lineages. It is not known what mechanisms enable some S. suis lineages to cause disease while others persist as commensal colonizers, or to what extent gene expression in disease-associated and commensal lineages diverge. In this study we compared the transcriptomes of 21 S. suis strains grown in active porcine serum and Todd-Hewitt yeast broth. These strains included both commensal and pathogenic strains, including several strains of sequence type (ST) 1, which is responsible for most cases of human disease and is considered to be the most pathogenic S. suis lineage. We sampled the strains during their exponential growth phase and mapped RNA sequencing reads to the corresponding strain genomes. We found that the transcriptomes of pathogenic and commensal strains with large genomic divergence were unexpectedly conserved when grown in active porcine serum, but that regulation and expression of key pathways varied. Notably, we observed strong variation of expression across media of genes involved in capsule production in pathogens, and of the agmatine deiminase system in commensals. ST1 strains displayed large differences in gene expression between the two media compared to strains from other clades. Their capacity to regulate gene expression across different environmental conditions may be key to their success as zoonotic pathogens.

Putrescine Biosynthesis from Agmatine by Arginase (TtARG) in Thermus thermophilus.

In the three domains of life, three biosynthetic pathways are known for putrescine. The first route is conversion of ornithine to putrescine by ornithine decarboxylase (ODC: SpeC), the second route is the conversion of arginine to agmatine by arginine decarboxylase (ADC: SpeA), followed by the conversion of agmatine to putrescine by agmatine ureohydrolase (AUH: SpeB), and the third route is the conversion of agmatine to N-carbamoylputrescine by agmatine deiminase (agmatine iminohydrolase, AIH), followed by the conversion of N-carbamoylputrescine to putrescine by N-carbamoylputrescine amidohydrolase (NCPAH). An extreme thermophile, Thermus thermophilus produces putrescine, although this bacterium lacks homologs for putrescine synthesizing pathways, such as ODC, AUH, AIH and NCPAH. To identify genes involved in putrescine biosynthesis in T. thermophilus, putrescine biosynthesis was examined by disruption of a predicted gene for agmatinase (agmatine ureohydrolase), or by using purified enzyme. It was found that arginase (TTHA1496) showed an agmatinase activity utilizing agmatine as a substrate. These results indicate that this bacterium can use arginase for putrescine biosynthesis. Arginase is a major contributor to putrescine biosynthesis under physiological conditions. The presence of an alternative pathway for converting agmatine into putrescine is functionally important for polyamine metabolism supporting survival at extreme environments.

Genomic Insights of Alnus-Infective Frankia Strains Reveal Unique Genetic Features and New Evidence on Their Host-Restricted Lifestyle.

The present study aimed to use comparative genomics to explore the relationships between Frankia and actinorhizal plants using a data set made of 33 Frankia genomes. The determinants of host specificity were first explored for "Alnus-infective strains" (i.e., Frankia strains belonging to Cluster Ia). Several genes were specifically found in these strains, including an agmatine deiminase which could possibly be involved in various functions as access to nitrogen sources, nodule organogenesis or plant defense. Within "Alnus-infective strains", Sp+ Frankia genomes were compared to Sp- genomes in order to elucidate the narrower host specificity of Sp+ strains (i.e., Sp+ strains being capable of in planta sporulation, unlike Sp- strains). A total of 88 protein families were lost in the Sp+ genomes. The lost genes were related to saprophytic life (transcriptional factors, transmembrane and secreted proteins), reinforcing the proposed status of Sp+ as obligatory symbiont. The Sp+ genomes were also characterized by a loss of genetic and functional paralogs, highlighting a reduction in functional redundancy (e.g., hup genes) or a possible loss of function related to a saprophytic lifestyle (e.g., genes involved in gas vesicle formation or recycling of nutrients).

Arbuscular mycorrhizal fungi enhanced cadmium uptake in Photinia frase through altering root transcriptomes and root-associated microbial communities

As a non-essential metal, cadmium (Cd) pollution poses severe threats to plant growth, environment, and human health. Phytoextraction using nursery stocks prior to their transplantation is a potential useful approach for bioremediation of Cd contaminated soil. A greenhouse pot experiment was performed to investigate the growth, Cd accumulation, profiles of transcriptome as well as root-associated microbiomes of Photinia frase in Cd-added soil, upon inoculation of two types of arbuscular mycorrhizal fungi (AMF) Sieverdingia tortuosa and Funneliformis mosseae. Compared with the control, inoculation of F. mosseae increased Cd concentrations in root, stem and leaf by 57.2%, 44.1% and 71.1%, respectively, contributing to a total Cd content of 182 μg/plant. KEGG pathway analysis revealed that hundreds of genes involved in 'Mitogen-activated protein kinase (MAPK) signaling pathway', 'plant hormone signal transduction', 'biosynthesis of secondary metabolites' and 'glycolysis/gluconeogenesis' were enriched upon inoculation of F. mosseae. The relative abundance of Acidobacteria was increased upon inoculation of S. tortuosa, while Chloroflexi and Patescibacteria were increased upon inoculation of F. mosseae, and the abundance of Glomerales increased from 23.0% to above 70%. Correlation analysis indicated that ethylene-responsive transcription factor, alpha-aminoadipic semialdehyde synthase, isoamylase and agmatine deiminase related genes were negatively associated with the relative abundance of Glomerales operational taxonomic units (OTUs) upon inoculation of F. mosseae. In addition, plant cysteine oxidase, heat shock protein, cinnamoyl-CoA reductase and abscisic acid receptor related genes were positively associated with the relative abundance of Patescibacteria OTUs upon inoculation of F. mosseae. These finding suggested that AMF can enhance P. frase Cd uptake by modulating plant gene expression and altering the structure of the soil microbial community. This study provides a theoretical basis for better understanding the relationship between root-associated microbiomes and root transcriptomes of P. frase, from which a cost-effective and environment-friendly strategy for phytoextraction of Cd in Cd-polluted soil might be developed.

The diversity among the species Tetragenococcus halophilus including new isolates from a lupine seed fermentation

BackgroundTetragenococcus (T.) halophilus can be isolated from a variety of fermented foods, such as soy sauce, different soy pastes, salted fish sauce and from cheese brine or degraded sugar beet thick juice. This species contributes by the formation of short chain acids to the flavor of the product. Recently, T. halophilus has been identified as a dominant species in a seasoning sauce fermentation based on koji made with lupine seeds.ResultsIn this study we characterized six strains of T. halophilus isolated from lupine moromi fermentations in terms of their adaptation towards this fermentation environment, salt tolerance and production of biogenic amines. Phylogenic and genomic analysis revealed three distinctive lineages within the species T. halophilus with no relation to their isolation source, besides the lineage of T. halophilus subsp. flandriensis. All isolated strains from lupine moromi belong to one lineage in that any of the type strains are absent. The strains form lupine moromi could not convincingly be assigned to one of the current subspecies. Taken together with strain specific differences in the carbohydrate metabolism (arabinose, mannitol, melibiose, gluconate, galactonate) and amino acid degradation pathways such as arginine deiminase pathway (ADI) and the agmatine deiminase pathway (AgDI) the biodiversity in the species of T. halophilus is greater than expected. Among the new strains, some strains have a favorable combination of traits wanted in a starter culture.ConclusionsOur study characterized T. halophilus strains that were isolated from lupine fermentation. The lupine moromi environment appears to select strains with specific traits as all of the strains are phylogenetically closely related, which potentially can be used as a starter culture for lupine moromi. We also found that the strains can be clearly distinguished phylogenetically and phenotypically from the type strains of both subspecies T. halophilus subsp. halophilus and T. halophilus subsp. flandriensis.

Technological Parameters, Anti-Listeria Activity, Biogenic Amines Formation and Degradation Ability of L. plantarum Strains Isolated from Sheep-Fermented Sausage.

The aim of this work was to identify and characterize, from a technological and safety point of view, the lactic acid bacteria (LAB) isolated from traditional sheep-fermented sausage. First, LABs were identified then were screened for some technological parameters such as acidifying and growth ability, proteolytic and lipolytic activity and for antimicrobial activity. Finally, biogenic amine production and degradation abilities were also evaluated. This research reveals the predominance of Lactiplantibacillus (L.) plantarum on LAB community. Almost all L. plantarum strains were active against Listeria monocytogenes strains (inhibition zone diameters > 1 cm). None of the tested strains were positive in histidine (hdcA), lysine (ldc) and tyrosine (tyrdc) decarboxylase genes and only one (L. plantarum PT9-2) was positive to the agmatine deiminase (agdi) gene. Furthermore, given the positive results of the sufl (multi-copper oxidase) gene detection, all strains showed a potential degradation ability of biogenic amines.

Metagenomic insights into the bacteria responsible for producing biogenic amines in sufu

Harmful levels of biogenic amines (BAs) are frequently identified in sufu. The microorganisms and mechanisms responsible for BA production in sufu, however, are not well documented. In this study, sufu samples were randomly obtained from various regions of China. Putrescine, tyramine, and histamine were quantitated as the most abundant BAs. According to the metagenome sequencing, the abundances and diversities of genes encoding the critical enzymes in BA production were acquired. The results showed that genes encoding arginine-, ornithine-, tryptophan-, and histidine decarboxylases were the predominant amino acid decarboxylase genes. Furthermore, 34 metagenome-assembled genomes (MAGs) were generated, of which 23 encoded at least one gene involved in BA production. Genetic analysis of MAGs indicated genera affiliated with Enterococcus, Lactobacillus-related, and Lactococcus were the major histamine-synthesizing bacteria, and tyrosine may be utilized by Bacillus, Chryseobacterium, Kurthia, Lysinibacillus, Macrococcus, and Streptococcus to product tyramine. The critical species involved in two putrescine-producing pathways were also explored. In the ornithine decarboxylase pathway, Lactobacillus-related and Veillonella were predicted to be the main performers, whereas Sphingobacterium and unclassified Flavobacteriaceae were the dominant executors in the agmatine deiminase pathway. The present study not only explained the BAs formation mechanism in sufu but also identified specific bacteria used to control BAs in fermented soybean products.

Effects of NaCl on gene expression of agmatine deiminase pathway genes of putrescine inLactobacillus delbrueckiiandEscherichia coli

To investigated the mechanism of NaCl concentration on the production of putrescine in lactobacilli mf661779 and Escherichia coli mf661780. We explored the effect of NaCl concentration on the putrescine production mechanism in Lactobacillus delbrueckii MF661779 and Escherichia coli MF661780, both of which produce putrescine via the agmatine deiminase (AGDI) pathway. Reverse-transcription quantitative PCR (RT-qPCR) was used to analyse expression of AGDI pathway genes, and ultra-performance liquid chromatography (UPLC) was used to monitor putrescine production over 48 hr. The results showed that NaCl had a lager inhibitory effect on the growth of E. coli MF661780, while NaCl affected the pH of L. delbrueckii MF661779 cultures to a greater extent. NaCl had minimal influence on expression of the transmembrane transcription activator aguR, but it markedly inhibited the transcription of the aguBDAC operon (p < .01). Differences in augB gene expression in L. delbrueckii at different NaCl concentrations were greater than in E. coli. Practical applications In this research paper, we explored the effect of NaCl concentration on the putrescine production mechanism in Lactobacillus delbrueckii MF661779 and Escherichia coli MF661780, both of which produce putrescine via the agmatine deiminase (AGDI) pathway. NaCl had a lager inhibitory effect on the growth of E. coli MF661780, while NaCl affected the pH of L. delbrueckii MF661779 cultures to a greater extent. NaCl had minimal influence on expression of the transmembrane transcription activator aguR, but it markedly inhibited the transcription of the aguBDAC operon. This study was to determine the effects of NaCl on the putrescine production mechanism in L. delbrueckii and E. coli. According to the experiment, the research work is beneficial for evaluated the safety of fermented foods.

Combined evolutionary engineering and genetic manipulation improve low pH tolerance and butanol production in a synthetic microbial Clostridium community.

Synthetic microbial communities have become a focus of biotechnological research since they can overcome several of the limitations of single-specie cultures. A paradigmatic example is Clostridium cellulovorans DSM 743B, which can decompose lignocellulose but cannot produce butanol. Clostridium beijerinckii NCIMB 8052 however, is unable to use lignocellulose but can produce high amounts of butanol from simple sugars. In our previous studies, both organisms were cocultured to produce butanol by consolidated bioprocessing. However, such consolidated bioprocessing implementation strongly depends on pH regulation. Since low pH (pH 4.5-5.5) is required for butanol fermentation, C. cellulovorans cannot grow well and saccharify sufficient lignocellulose to feed both strains at a pH below 6.4. To overcome this bottleneck, this study engineered C. cellulovorans by adaptive laboratory evolution, inactivating cell wall lyases genes (Clocel_0798 and Clocel_2169), and overexpressing agmatine deiminase genes (augA, encoded by Cbei_1922) from C. beijerinckii NCIMB 8052. The generated strain WZQ36: 743B*6.0*3△lyt0798△lyt2169-(pXY1-Pthl -augA) can tolerate a pH of 5.5. Finally, the alcohol aldehyde dehydrogenase gene adhE1 from Clostridium acetobutylicum ATCC 824 was introduced into the strain to enable butanol production at low pH, in coordination with solvent fermentation of C. beijerinckii in consortium. The engineered consortium produced 3.94 g/L butanol without pH control within 83 hr, which is more than5-fold of the level achieved by wild consortia under the same conditions. This exploration represents a proof of concept on how to combine metabolic and evolutionary engineering to coordinate coculture of a synthetic microbial community.

Genetic attenuation of alkaloids and nicotine content in tobacco (Nicotiana tabacum).

The role of six alkaloid biosynthesis genes in the process of nicotine accumulation in tobacco was investigated. Downregulation of ornithine decarboxylase, arginine decarboxylase, and aspartate oxidase resulted in viable plants with a significantly lower nicotine content. Attenuation of nicotine accumulation in Nicotiana tabacum was addressed upon the application of RNAi technologies. The approach entailed a downregulation in the expression of six different alkaloid biosynthesis genes encoding upstream enzymes that are thought to function in the pathway of alkaloid and nicotinebiosynthesis. Nine different RNAi constructs were designed to lower the expression level of the genes that encode the enzymes arginine decarboxylase, agmatine deiminase, aspartate oxidase, arginase, ornithine decarboxylase, and SAM synthase. Agrobacterium-based transformation of tobacco leaves was applied, and upon kanamycin selection, T0 and subsequently T1 generation seeds were produced. Mature T1 plants in the greenhouse were topped to prevent flowering and leaf nos. 3 and 4 below the topping point were tested for transcript levels and product accumulation. Down-regulation in arginine decarboxylase, aspartate oxidase, and ornithine decarboxylase consistently resulted in lower levels of nicotine in the leaves of the corresponding plants. Transformants with the aspartate oxidase RNAi construct showed the lowest nicotine level in the leaves, which varied from below the limit of quantification (20μg per g dry leaf weight) to 1.3mg per g dry leaf weight. The amount of putrescine, the main polyamine related to nicotine biosynthesis, showed a qualitative correlation with the nicotine content in the arginine decarboxylase and ornithine decarboxylase RNAi-expressing transformants. A putative early senescence phenotype and lower viability of the older leaves was observed in some of the transformant lines. The results are discussed in terms of the role of the above-mentioned genes in the alkaloid biosynthetic pathway and may serve to guide efforts to attenuate nicotine content in tobacco leaves.

Theaflavin-3,3'-Digallate Suppresses Biofilm Formation, Acid Production, and Acid Tolerance in Streptococcus mutans by Targeting Virulence Factors.

As one of the most important cariogenic pathogens, Streptococcus mutans has strong abilities to form biofilms, produce acid and tolerate acid. In present study, we found that theaflavin-3,3′-digallate (TF3) had an inhibitory effect on S. mutans UA159 in vitro. Visualized by field emission-scanning electron microscopy, the suppressed formation of S. mutans biofilms grown with TF3 at sub-inhibitory concentrations could be attributed to the reduced biofilm matrix, which was proven to contain glucans and extracellular DNA (eDNA). Glucan-reduced effect of TF3 was achieved by down-regulating expression levels of gtfB, gtfC, and gtfD encoding glucosyltransferases. Besides, TF3 reduced eDNA formation of S. mutans by negatively regulating lrgA, lrgB, and srtA, which govern cell autolysis and membrane vesicle components. Furthermore, TF3 also played vital roles in antagonizing preformed biofilms of S. mutans. Bactericidal effects of TF3 became significant when its concentrations increased more than twofold of minimum inhibitory concentration (MIC). Moreover, the capacities of S. mutans biofilms to produce acid and tolerate acid were significantly weakened by TF3 at MIC. Based on real-time PCR (RT-PCR) analysis, the mechanistic effects of TF3 were speculated to comprise the inhibition of enolase, lactate dehydrogenase, F-type ATPase and the agmatine deiminase system. Moreover, TF3 has been found to downregulate LytST, VicRK, and ComDE two component systems in S. mutans, which play critical roles in the regulatory network of virulence factors. Our present study found that TF3 could suppress the formation and cariogenic capacities of S. mutans biofilms, which will provide new strategies for anti-caries in the future.

Genome analysis of lactic acid bacterial strains selected as potential starters for traditional Slovakian bryndza cheese.

Genomes of 21 strains of lactic acid bacteria isolated from Slovakian traditional cheeses were sequenced on an Illumina MiSeq platform. Subsequently, they were analysed regarding taxonomic classification, presence of genes encoding defence systems, antibiotic resistance and production of biogenic amines. Thirteen strains were found to carry genes encoding at least one bacteriocin, 18 carried genes encoding at least one restriction-modification system, all strains carried 1-6 prophages and 9 strains had CRISPR-Cas systems. CRISPR-Cas type II-A was the most common, containing 0-24 spacers. Only 10% spacers were found to be homological to known bacteriophage or plasmid sequences in databases. Two Enterococcus faecium strains and a Lactococcus lactis strain carried antibiotic resistance genes. Genes encoding for ornithine decarboxylase were detected in four strains and genes encoding for agmatine deiminase were detected in four strains. Lactobacillus paraplantarum 251 L appeared to be the most interesting strain, as it contained genes encoding for two bacteriocins, a restriction-modification system, two CRISPR-Cas systems, four prophages and no genes connected with antibiotic resistance or production of biogenic amines.

Genome analysis of lactic acid bacterial strains selected as potential starters for traditional Slovakian bryndza cheese

ABSTRACT Genomes of 21 strains of lactic acid bacteria isolated from Slovakian traditional cheeses were sequenced on an Illumina MiSeq platform. Subsequently, they were analysed regarding taxonomic classification, presence of genes encoding defence systems, antibiotic resistance and production of biogenic amines. Thirteen strains were found to carry genes encoding at least one bacteriocin, 18 carried genes encoding at least one restriction–modification system, all strains carried 1–6 prophages and 9 strains had CRISPR-Cas systems. CRISPR-Cas type II-A was the most common, containing 0–24 spacers. Only 10% spacers were found to be homological to known bacteriophage or plasmid sequences in databases. Two Enterococcus faecium strains and a Lactococcus lactis strain carried antibiotic resistance genes. Genes encoding for ornithine decarboxylase were detected in four strains and genes encoding for agmatine deiminase were detected in four strains. Lactobacillus paraplantarum 251 L appeared to be the most interesting strain, as it contained genes encoding for two bacteriocins, a restriction–modification system, two CRISPR-Cas systems, four prophages and no genes connected with antibiotic resistance or production of biogenic amines.

Bioactive polyamine production by a novel hybrid system comprising multiple indigenous gut bacterial strategies.

Metabolites of the intestinal microbiota are thought to be generated through metabolic pathways spanning multiple taxa of intestinal bacteria. We have previously shown that the level of putrescine, a polyamine found abundantly in the human intestinal lumen, is increased in the colonic lumen following administration of arginine and the probiotic Bifidobacterium sp.; however, the underlying mechanism remained poorly understood. We report a novel pathway for putrescine production from arginine through agmatine involving the collaboration of two bacterial groups, and triggered by environmental acidification (drop in pH to below 6.5 from neutral). This pathway comprises the acid tolerance system of Escherichia coli, representing bacteria that have an arginine-dependent acid resistance system; the energy production system of Enterococcus faecalis, representing bacteria that have an agmatine deiminase system; and the acid production system of the acid-producing bacteria, represented by Bifidobacterium spp. This pathway is unique in that it represents a relationship between the independent survival strategies of multiple bacteria.

Structural and Functional Basis for Targeting Campylobacter jejuni Agmatine Deiminase To Overcome Antibiotic Resistance.

Campylobacter jejuni is the most common bacterial cause of gastroenteritis and a major contributor to infant mortality in the developing world. The increasing incidence of antibiotic-resistant C. jejuni only adds to the urgency to develop effective therapies. Because of the essential role that polyamines play, particularly in protection from oxidative stress, enzymes involved in the biosynthesis of these metabolites are emerging as promising antibiotic targets. The recent description of an alternative pathway for polyamine synthesis, distinct from that in human cells, in C. jejuni suggests this pathway could be a target for novel therapies. To that end, we determined X-ray crystal structures of C. jejuni agmatine deiminase (CjADI) and demonstrated that loss of CjADI function contributes to antibiotic sensitivity, likely because of polyamine starvation. The structures provide details of key molecular features of the active site of this protein. Comparison of the unliganded structure (2.1 Å resolution) to that of the CjADI-agmatine complex (2.5 Å) reveals significant structural rearrangements that occur upon substrate binding. The shift of two helical regions of the protein and a large conformational change in a loop near the active site generate a narrow binding pocket around the bound substrate. This change optimally positions the substrate for catalysis. In addition, kinetic analysis of this enzyme demonstrates that CjADI is an iminohydrolase that effectively deiminates agmatine. Our data suggest that C. jejuni agmatine deiminase is a potentially important target for combatting antibiotic resistance, and these results provide a valuable framework for guiding future drug development.

The Relationship among Tyrosine Decarboxylase and Agmatine Deiminase Pathways in Enterococcus faecalis.

Enterococci are considered mainly responsible for the undesirable accumulation of the biogenic amines tyramine and putrescine in cheeses. The biosynthesis of tyramine and putrescine has been described as a species trait in Enterococcus faecalis. Tyramine is formed by the decarboxylation of the amino acid tyrosine, by the tyrosine decarboxylase (TDC) route encoded in the tdc cluster. Putrescine is formed from agmatine by the agmatine deiminase (AGDI) pathway encoded in the agdi cluster. These biosynthesis routes have been independently studied, tyrosine and agmatine transcriptionally regulate the tdc and agdi clusters. The objective of the present work is to study the possible co-regulation among TDC and AGDI pathways in E. faecalis. In the presence of agmatine, a positive correlation between putrescine biosynthesis and the tyrosine concentration was found. Transcriptome studies showed that tyrosine induces the transcription of putrescine biosynthesis genes and up-regulates pathways involved in cell growth. The tyrosine modulation over AGDI route was not observed in the mutant Δtdc strain. Fluorescence analyses using gfp as reporter protein revealed PaguB (the promoter of agdi catabolic genes) was induced by tyrosine in the wild-type but not in the mutant strain, confirming that tdc cluster was involved in the tyrosine induction of putrescine biosynthesis. This study also suggests that AguR (the transcriptional regulator of agdi) was implicated in interaction among the two clusters.

Putrescine biosynthesis in Lactococcus lactis is transcriptionally activated at acidic pH and counteracts acidification of the cytosol

Lactococcus lactis subsp. cremoris CECT 8666 is a lactic acid bacterium that synthesizes the biogenic amine putrescine from agmatine via the agmatine deiminase (AGDI) pathway. The AGDI genes cluster includes aguR. This encodes a transmembrane protein that functions as a one-component signal transduction system, the job of which is to sense the agmatine concentration of the medium and accordingly regulate the transcription of the catabolic operon aguBDAC. The latter encodes the proteins necessary for agmatine uptake and its conversion into putrescine. This work reports the effect of extracellular pH on putrescine biosynthesis and on the genetic regulation of the AGDI pathway. Increased putrescine biosynthesis was detected at acidic pH (pH5) compared to neutral pH. Acidic pH induced the transcription of the catabolic operon via the activation of the aguBDAC promoter PaguB. However, the external pH had no significant effect on the activity of the aguR promoter PaguR, or on the transcription of the aguR gene. The transcriptional activation of the AGDI pathway was also found to require a lower agmatine concentration at pH5 than at neutral pH. Finally, the following of the AGDI pathway counteracted the acidification of the cytoplasm under acidic external conditions, suggesting it to provide protection against acid stress.