Characterization of a novel 4-guanidinobutyrase from Candida parapsilosis.

Enzymes of the ureohydrolase superfamily are specific in recognizing their substrates. While looking to broaden the substrate specificity of 4-guanidinobutyrase (GBase), we isolated a yeast, typed as Candida parapsilosis (NCIM 3689), that efficiently utilized both 4-guanidinobutyrate (GB) and 3-guanidinopropionate (GP) as a sole source of nitrogen. A putative GBase sequence was identified from its genome upon pBLAST query using the GBase sequence from Aspergillus niger (AnGBase). The C. parapsilosis GBase (CpGBase) ORF was PCR amplified, cloned, and sequenced. Further, the functional CpGBase protein expressed in Saccharomyces cerevisiae functioned as GBase and 3-guanidinopropionase (GPase). S. cerevisiae cannot grow on GB or GP. However, the transformants expressing CpGBase acquired the ability to utilize and grow on both GB and GP. The expressed CpGBase protein was enriched and analyzed for substrate saturation and product inhibition by γ-aminobutyric acid and ß-alanine. In contrast to the well-characterized AnGBase, CpGBase from C. parapsilosis is a novel ureohydrolase and showed hyperbolic saturation for GB and GP with comparable efficiency (Vmax/KM values of 3.4 and 2.0, respectively). With the paucity of structural information and limited active site data available on ureohydrolases, CpGBase offers an excellent paradigm to explore this class of enzymes.

miR­151a­5p promotes the proliferation and metastasis of colorectal carcinoma cells by targeting AGMAT.

Colorectal carcinoma (CRC) is one of the most common types of digestive cancer. It has been reported that the ectopic expression of microRNAs (miRs) plays a critical role in the occurrence and progression of CRC. In addition, it has also been suggested that miR­151a­5p may serve as a useful biomarker for the early detection and treatment of different types of cancer and particularly CRC. However, the specific effects and underlying mechanisms of miR­151a­5p in CRC remain elusive. The results of the current study demonstrated that miR­151a­5p was upregulated in CRC cell lines and clinical tissues derived from patients with CRC. Functionally, the results showed that miR­151a­5p significantly promoted CRC cell proliferation, migration and invasion. Additionally, dual luciferase reporter assays verified that agmatinase (AGMAT) was a direct target of miR­151a­5p and it was positively associated with miR­151a­5p expression. Mechanistically, miR­151a­5p could enhance the epithelial­mesenchymal transition of CRC cells. Taken together, the results of the current study revealed a novel molecular mechanism indicating that the miR­151a­5p/AGMAT axis could serve a crucial role in the regulation of CRC and could therefore be considered as a potential therapeutic strategy for CRC.

[Improving the activity of creatinase from Alcaligenes sp. KS-85 through semi-rational design].

Creatinine levels in biological fluids are important indicators for the clinical evaluation of renal function. Creatinase (CRE, EC3.5.3.3) is one of the key enzymes in the enzymatic measurement of creatinine concentration, and it is also the rate-limiting enzyme in the whole enzymatic cascade system. The poor catalytic activity of CRE severely limits its clinical and industrial applications. To address this issue, a semi-rational design is applied to increase the activity of a creatinase from Alcaligenes sp. KS-85 (Al-CRE). By high-throughput screen of saturation mutagenesis libraries on the selected hotspot mutations, multiple variant enzymes with increased activity are obtained. The five-point best variant enzyme (I304L/F395V/K351V/Y63S/Q88A) were further obtained by recombine the improved mutations sites that to showed a 2.18-fold increased specific activity. Additionally, structure analysis is conducted to understand the mechanism of the activity change. This study paves the way for a better practical application of creatinase and may help further understand its catalytic mechanism.

Improving the activity of creatinase from Alcaligenes sp. KS-85 through semi-rational design / 生物工程学报

Creatinine levels in biological fluids are important indicators for the clinical evaluation of renal function. Creatinase (CRE, EC3.5.3.3) is one of the key enzymes in the enzymatic measurement of creatinine concentration, and it is also the rate-limiting enzyme in the whole enzymatic cascade system. The poor catalytic activity of CRE severely limits its clinical and industrial applications. To address this issue, a semi-rational design is applied to increase the activity of a creatinase from Alcaligenes sp. KS-85 (Al-CRE). By high-throughput screen of saturation mutagenesis libraries on the selected hotspot mutations, multiple variant enzymes with increased activity are obtained. The five-point best variant enzyme (I304L/F395V/K351V/Y63S/Q88A) were further obtained by recombine the improved mutations sites that to showed a 2.18-fold increased specific activity. Additionally, structure analysis is conducted to understand the mechanism of the activity change. This study paves the way for a better practical application of creatinase and may help further understand its catalytic mechanism.

Carbon catabolite repressor MoCreA is required for the asexual development and pathogenicity of the rice blast fungus.

During the infection and colonization process, the rice blast fungus Magnaporthe oryzae faces various challenges from hostile environment, such as nutrient limitation and carbon stress, while carbon catabolite repression (CCR) mechanism would facilitate the fungus to shrewdly and efficiently utilize carbon nutrients under fickle nutritional conditions since it ensures the preferential utilization of most preferred carbon sources through repressing the expression of enzymes required for the utilization of less preferred carbon sources. Researches on M. oryzae CCR have made some progress, however the involved regulation mechanism is still largely obscured, especially, little is known about the key carbon catabolite repressor CreA. Here we identified and characterized the biological functions of the CreA homolog MoCreA in M. oryzae. MoCreA is constitutively expressed throughout all the life stages of the fungus, and it can shuttle between nucleus and cytoplasm which is induced by glucose. Following functional analyses of MoCreA suggested that it was required for the vegetative growth, conidiation, appressorium formation and pathogenicity of M. oryzae. Moreover, comparative transcriptomic analysis revealed that disruption of MoCreA resulted in the extensive gene expression variations, including a large number of carbon metabolism enzymes, transcription factors and pathogenicity-related genes. Taken together, our results demonstrated that, as a key regulator of CCR, MoCreA plays a vital role in precise regulation of the asexual development and pathogenicity of the rice blast fungus.

Improved thermostability of creatinase from Alcaligenes Faecalis through non-biased phylogenetic consensus-guided mutagenesis.

BACKGROUND: Enzymatic quantification of creatinine has become an essential method for clinical evaluation of renal function. Although creatinase (CR) is frequently used for this purpose, its poor thermostability severely limits industrial applications. Herein, we report a novel creatinase from Alcaligenes faecalis (afCR) with higher catalytic activity and lower KM value, than currently used creatinases. Furthermore, we developed a non-biased phylogenetic consensus method to improve the thermostability of afCR. RESULTS: We applied a non-biased phylogenetic consensus method to identify 59 candidate consensus residues from 24 creatinase family homologs for screening afCR mutants with improved thermostability. Twenty-one amino acids of afCR were selected to mutagenesis and 11 of them exhibited improved thermostability compared to the parent enzyme (afCR-M0). Combination of single-site mutations in sequential screens resulted in a quadruple mutant D17V/T199S/L6P/T251C (M4-2) which showed ~ 1700-fold enhanced half-life at 57 °C and a 4.2 °C higher T5015 than that of afCR-M0. The mutant retained catalytic activity equivalent to afCR-M0, and thus showed strong promise for application in creatinine detection. Structural homology modeling revealed a wide range of potential molecular interactions associated with individual mutations that contributed to improving afCR thermostability. CONCLUSIONS: Results of this study clearly demonstrated that the non-biased-phylogenetic consensus design for improvement of thermostability in afCR is effective and promising in improving the thermostability of more enzymes.

Three Related Enzymes in Candida albicans Achieve Arginine- and Agmatine-Dependent Metabolism That Is Essential for Growth and Fungal Virulence.

Amino acid metabolism is crucial for fungal growth and development. Ureohydrolases produce amines when acting on l-arginine, agmatine, and guanidinobutyrate (GB), and these enzymes generate ornithine (by arginase), putrescine (by agmatinase), or GABA (by 4-guanidinobutyrase or GBase). Candida albicans can metabolize and grow on arginine, agmatine, or guanidinobutyrate as the sole nitrogen source. Three related C. albicans genes whose sequences suggested that they were putative arginase or arginase-like genes were examined for their role in these metabolic pathways. Of these, Car1 encoded the only bona fide arginase, whereas we provide evidence that the other two open reading frames, orf19.5862 and orf19.3418, encode agmatinase and guanidinobutyrase (Gbase), respectively. Analysis of strains with single and multiple mutations suggested the presence of arginase-dependent and arginase-independent routes for polyamine production. CAR1 played a role in hyphal morphogenesis in response to arginine, and the virulence of a triple mutant was reduced in both Galleria mellonella and Mus musculus infection models. In the bloodstream, arginine is an essential amino acid that is required by phagocytes to synthesize nitric oxide (NO). However, none of the single or multiple mutants affected host NO production, suggesting that they did not influence the oxidative burst of phagocytes.IMPORTANCE We show that the C. albicans ureohydrolases arginase (Car1), agmatinase (Agt1), and guanidinobutyrase (Gbu1) can orchestrate an arginase-independent route for polyamine production and that this is important for C. albicans growth and survival in microenvironments of the mammalian host.

Constructing a novel expression system by specific activation of amylase expression pathway in Penicillium.

BACKGROUND: Filamentous fungi have long been used as hosts for the production of proteins, enzymes and valuable products in various biotechnological applications. However, recombinant proteins are expressed with highly secreted host proteins when stronger promoters are used under inducing conditions. In addition, the efficiency of target protein expression can be limited by the application of constitutive promoters in recently developed filamentous fungal expression systems. RESULTS: In this study, a novel expression system was constructed by using a Penicillium oxalium strain that has powerful protein secretion capability. The secretory background of the host was reduced by knocking out the Amy13A protein and utilizing the starch as a carbon source. The strong promoter amy15A(p) was further improved by overexpressing the transcription activator AmyR and deleting of putative repressor CreA. By using the native amylase Amy15A as a reporter, the efficiency of expression from the amy15A promoter was dramatically and specifically enhanced after redesigning the regulatory network of amylase expression. CONCLUSIONS: Our researches clearly indicated that the triple-gene recombinant strain Δ13A-OamyR-ΔCreA, with the amy15A(p) promoter could be used as a suitable expression system especially for high-level and high-purity protein production.

Insights into the Mn2+ Binding Site in the Agmatinase-Like Protein (ALP): A Critical Enzyme for the Regulation of Agmatine Levels in Mammals.

Agmatine is a neurotransmitter with anticonvulsant, anti-neurotoxic and antidepressant-like effects, in addition it has hypoglycemic actions. Agmatine is converted to putrescine and urea by agmatinase (AGM) and by an agmatinase-like protein (ALP), a new type of enzyme which is present in human and rodent brain tissues. Recombinant rat brain ALP is the only mammalian protein that exhibits significant agmatinase activity in vitro and generates putrescine under in vivo conditions. ALP, despite differing in amino acid sequence from all members of the ureohydrolase family, is strictly dependent on Mn2+ for catalytic activity. However, the Mn2+ ligands have not yet been identified due to the lack of structural information coupled with the low sequence identity that ALPs display with known ureohydrolases. In this work, we generated a structural model of the Mn2+ binding site of the ALP and we propose new putative Mn2+ ligands. Then, we cloned and expressed a sequence of 210 amino acids, here called the "central-ALP", which include the putative ligands of Mn2+. The results suggest that the central-ALP is catalytically active, as agmatinase, with an unaltered Km for agmatine and a decreased kcat. Similar to wild-type ALP, central-ALP is activated by Mn2+ with a similar affinity. Besides, a simple mutant D217A, a double mutant E288A/K290A, and a triple mutant N213A/Q215A/D217A of these putative Mn2+ ligands result on the loss of ALP agmatinase activity. Our results indicate that the central-ALP contains the active site for agmatine hydrolysis, as well as that the residues identified are relevant for the ALP catalysis.

Mycolicibacterium smegmatis possesses operational agmatinase but contains no detectable polyamines.

Background: Polyamines are widespread intracellular molecules able to influence antibiotic susceptibility, but almost nothing is known on their occurrence and physiological role in mycobacteria. Methods: here, we analyzed transcriptomic, proteomic and biochemical data and obtained the first evidence for the post-transcriptional expression of some genes attributed to polyamine metabolism and polyamine transport in Mycolicibacterium smegmatis (basionym Mycobacterium smegmatis). Results: in our experiments, exponentially growing cells demonstrated transcription of 21 polyamine-associated genes and possessed 7 enzymes of polyamine metabolism and 2 polyamine transport proteins. Conclusion: Mycolicibacterium smegmatis putrescine synthesizing enzyme agmatinase SpeB was originally shown to catalyze agmatine conversion to putrescine in vitro. Nevertheless, we have not found any polyamines in mycobacterial cells.

In Ganoderma lucidum, Glsnf1 regulates cellulose degradation by inhibiting GlCreA during the utilization of cellulose.

Cellulose is a by-product of agricultural production and an abundant waste. As a carbon source, cellulose can be degraded and utilized by fungi. Carbon sources, which act as nutrients, not only provide energy but also serve as regulators of gene expression, metabolism and growth, through various signalling networks that enable cells to sense and adapt to varying environmental conditions. Nutrient-sensing pathways prioritize the use of preferred carbon sources and regulate the production of cellulose-degrading enzymes when necessary. Understanding the regulation of the fungal cellulolytic response will become increasingly important because we strive to increase the efficiency of the utilization of these renewable energy sources. Here, we show that Glsnf1, a sucrose-nonfermenting serine-threonine-protein kinase 1 (Snf1)/AMP-activated protein kinase homologue in medicinal macro basidiomycete Ganoderma lucidum, actively responds to carbon alterations and positively regulates cellulase activity and cellulase-related gene transcription. The carbon catabolite repressor CreA, a zinc binuclear cluster transcription factor that mediates the sensing of nutrients and suppression of the transcription of a number of genes necessary for the consumption of a less preferred carbon source, participates in the Glsnf1-mediated regulation of cellulases. Glsnf1 not only negatively regulates the transcription level of the CreA gene but also hinders its localization in the nucleus. Overall, our findings reveal a key nutrient-sensing mechanism that is critical for the modulation of carbon source adaptation in G. lucidum.

Agmatinase promotes the lung adenocarcinoma tumorigenesis by activating the NO-MAPKs-PI3K/Akt pathway.

Lung adenocarcinoma (LUAD) is one of the leading causes of cancer-related death worldwide. There is an urgent need to uncover the pathogenic mechanism to develop new treatments. Agmatinase (AGMAT) expression and its association with clinicopathological characteristics were analyzed via GEO, Oncomine, and TCGA databases, and IHC staining in human LUAD specimens. An EdU cell proliferation kit, propidiumiodide staining, colony formation, cell migration, and invasion assays, and a xenograft tumor model were used to detect the biological function of AGMAT in LUAD. Furthermore, the expression level of nitric oxide (NO) was detected using a DAF-FMDA fluorescent probe or nitrite assay kit, and further validated with Carboxy-PTIO (a NO scavenger). The roles of three isoforms of nitric oxide synthases (nNOS, eNOS, and iNOS) were validated using L-NAME (eNOS inhibitor), SMT (iNOS inhibitor), and spermidine (nNOS inhibitor). AGMAT expression was up-regulated in LUAD tissues. LUAD patients with high AGMAT levels were associated with poorer prognoses. AGMAT promoted LUAD tumorigenesis in NO released by iNOS both in vitro and in vivo. Importantly, NO signaling up-regulated the expression of cyclin D1 via activating the MAPK and PI3K/Akt-dependent c-myc activity, ultimately promoting the malignant proliferation of tumor cells. On the whole, AGMAT promoted NO release via up-regulating the expression of iNOS. High levels of NO drove LUAD tumorigenesis via activating MAPK and PI3K/Akt cascades. AGMAT might be a potential diagnostic and therapeutic target for LUAD patients.

Role and dynamics of an agmatinase-like protein (AGM-1) in Neurospora crassa.

Agmatinase is known as a metalloenzyme which hydrolyzes agmatine to produce urea and putrescine, being crucial in the alternative pathway to produce polyamines. In this study, an agmatinase-like protein (AGM-1) (NCU 01348) in the filamentous fungus Neurospora crassa is reported. Purified AGM-1 from N. crassa displays enzymatic activity hydrolyzing agmatine; therefore, it can be considered as an agmatinase-like protein. However, its role in the alternative pathway to produce polyamines apparently is not its main function since only a slight reduction of polyamines concentration was detected in the Δagm-1 het strain. Moreover, the null mutant Δagm-1 (homokaryon strain) was unable to grow and the deficiency of agm-1 in the heterokaryon strain provoked a decrease in elongation rate, conidia and biomass production, despite of having de constitutive pathway via the ornithine decarboxylase (ODC). Additionally, mature hyphae of the Δagm-1 het strain presented unusual apical branching and a disorganized Spitzenkörper (Spk). Trying to reveal the role of AGM-1in N. crassa, the protein was tagged with GFP and interestingly the dynamics and intracellular localization of AGM-1 closely resembles the F-actin population. This finding was further examined in order to elucidate if AGM-1is in a close association with F-actin. Since polyamines, among them agmatine, have been reported to act as stabilizers of actin filaments, we evaluated in vitro G-actin polymerization in the presence of agmatine and the effect of purified AGM-1 from N. crassa on these polymerized actin filaments. It was found that polymerization of actin filaments increases in the presence of agmatine and the addition of purified AGM-1 from N. crassa depolymerizes these actin filaments. Also, it was determined that an intact substrate binding site of the enzyme is necessary for the localization pattern of the native AGM-1. These results suggest that in N. crassa AGM-1 has a close association with the F-actin population via its substrate agmatine, playing an essential role during cell development.

A Novel CreA-Mediated Regulation Mechanism of Cellulase Expression in the Thermophilic Fungus Humicola insolens.

The thermophilic fungus Humicola insolens produces cellulolytic enzymes that are of great scientific and commercial interest; however, few reports have focused on its cellulase expression regulation mechanism. In this study, we constructed a creA gene (carbon catabolite repressor gene) disruption mutant strain of H. insolens that exhibited a reduced radial growth rate and stouter hyphae compared to the wild-type (WT) strain. The creA disruption mutant also expressed elevated pNPCase (cellobiohydrolase activities), pNPGase (ß-glucosidase activities), and xylanase levels in non-inducing fermentation with glucose. Unlike other fungi, the H. insolens creA disruption mutant displayed lower FPase (filter paper activity), CMCase (carboxymethyl cellulose activity), pNPCase, and pNPGase activity than observed in the WT strain when fermentation was induced using Avicel, whereas its xylanase activity was higher than that of the parental strain. These results indicate that CreA acts as a crucial regulator of hyphal growth and is part of a unique cellulase expression regulation mechanism in H. insolens. These findings provide a new perspective to improve the understanding of carbon catabolite repression regulation mechanisms in cellulase expression, and enrich the knowledge of metabolism diversity and molecular regulation of carbon metabolism in thermophilic fungi.

Characterization of 4-guanidinobutyrase from Aspergillus niger.

Arginase is the only fungal ureohydrolase that is well documented in the literature. More recently, a novel route for agmatine catabolism in Aspergillus niger involving another ureohydrolase, 4-guanidinobutyrase (GBase), was reported. We present here a detailed characterization of A. niger GBase - the first fungal (and eukaryotic) enzyme to be studied in detail. A. niger GBase is a homohexamer with a native molecular weight of 336 kDa and an optimal pH of 7.5. Unlike arginase, the Mn2+ enzyme from the same fungus, purified GBase protein is associated with Zn2+ ions. A sensitive fluorescence assay was used to determine its kinetic parameters. GBase acted 25 times more efficiently on 4-guanidinobutyrate (GB) than 3-guanidinopropionic acid (GP). The Km for GB was 2.7±0.4 mM, whereas for GP it was 53.7±0.8 mM. While GB was an efficient nitrogen source, A. niger grew very poorly on GP. Constitutive expression of GBase favoured fungal growth on GP, indicating that GP catabolism is limited by intracellular GBase levels in A. niger. The absence of a specific GPase and the inability of GP to induce GBase expression confine the fungal growth on GP. That GP is a poor substrate for GBase and a very poor nitrogen source for A. niger offers an opportunity to select GBase specificity mutations. Further, it is now possible to compare two distinct ureohydrolases, namely arginase and GBase, from the same organism.

Disruption of ureide degradation affects plant growth and development during and after transition from vegetative to reproductive stages.

BACKGROUND: The ureides allantoin and allantoate are major metabolic intermediates of purine catabolism with high nitrogen-to-carbon ratios. Ureides play a key role in nitrogen utilization in ureide-type legumes, but their effects on growth and development in non-legume plants are poorly understood. Here, we examined the effects of knocking out genes encoding ureide-degrading enzymes, allantoinase (ALN) and allantoate amidohydrolase (AAH), on the vegetative-to-reproductive transition and subsequent growth of Arabidopsis plants. RESULTS: The ureide-degradation mutants (aln and aah) showed symptoms similar to those of nitrogen deficiency: early flowering, reduced size at maturity, and decreased fertility. Consistent with these phenotypes, carbon-to-nitrogen ratios and nitrogen-use efficiencies were significantly decreased in ureide-degradation mutants; however, adding nitrogen to irrigation water did not alleviate the reduced growth of these mutants. In addition to nitrogen status, levels of indole-3-acetic acid and gibberellin in five-week-old plants were also affected by the aln mutations. To test the possibility that ureides are remobilized from source to sink organs, we measured ureide levels in various organs. In wild-type plants, allantoate accumulated predominantly in inflorescence stems and siliques; this accumulation was augmented by disruption of its catabolism. Mutants lacking ureide transporters, ureide permeases 1 and 2 (UPS1 and UPS2), exhibited phenotypes similar to those of the ureide-degradation mutants, but had decreased allantoate levels in the reproductive organs. Transcript analysis in wild-type plants suggested that genes involved in allantoate synthesis and ureide transport were coordinately upregulated in senescing leaves. CONCLUSIONS: This study demonstrates that ureide degradation plays an important role in supporting healthy growth and development in non-legume Arabidopsis during and after transition from vegetative to reproductive stages.

Early Senescence in Older Leaves of Low Nitrate-Grown Atxdh1 Uncovers a Role for Purine Catabolism in N Supply.

The nitrogen (N)-rich ureides allantoin and allantoate, which are products of purine catabolism, play a role in N delivery in Leguminosae. Here, we examined their role as an N source in nonlegume plants using Arabidopsis (Arabidopsis thaliana) plants mutated in XANTHINE DEHYDROGENASE1 (AtXDH1), a catalytic bottleneck in purine catabolism. Older leaves of the Atxdh1 mutant exhibited early senescence, lower soluble protein, and lower organic N levels as compared with wild-type older leaves when grown with 1 mm nitrate but were comparable to the wild type under 5 mm nitrate. Similar nitrate-dependent senescence phenotypes were evident in the older leaves of allantoinase (Ataln) and allantoate amidohydrolase (Ataah) mutants, which also are impaired in purine catabolism. Under low-nitrate conditions, xanthine accumulated in older leaves of Atxdh1, whereas allantoin accumulated in both older and younger leaves of Ataln but not in wild-type leaves, indicating the remobilization of xanthine-degraded products from older to younger leaves. Supporting this notion, ureide transporter expression was enhanced in older leaves of the wild type in low-nitrate as compared with high-nitrate conditions. Elevated transcripts and proteins of AtXDH and AtAAH were detected in low-nitrate-grown wild-type plants, indicating regulation at protein and transcript levels. The higher nitrate reductase activity in Atxdh1 leaves compared with wild-type leaves indicated a need for nitrate assimilation products. Together, these results indicate that the absence of remobilized purine-degraded N from older leaves of Atxdh1 caused senescence symptoms, a result of higher chloroplastic protein degradation in older leaves of low-nitrate-grown plants.

Homospermidine biosynthesis in the cyanobacterium Anabaena requires a deoxyhypusine synthase homologue and is essential for normal diazotrophic growth.

Polyamines are primordial, small organic polycations present in almost all cells, but their roles in bacteria are poorly understood. sym-Homospermidine is the dominant polyamine in the filamentous, N2 -fixing, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Synthesis of homospermidine was dependent on speA (encoding arginine decarboxylase), speB (agmatinase) and speY (deoxyhypusine synthase homologue), which in bacteria is an unprecedented pathway. Inactivation of any of these genes impaired diazotrophic growth. Heterocyst differentiation in the speA mutant was blocked at an early step, after induction of the regulatory gene hetR but before production of heterocyst-specific glycolipids (HGL). In contrast, the speY mutant produced HGL and showed slow diazotrophic growth. Analysis of fusions to green fluorescent protein revealed that SpeA (like SpeB previously described) accumulates at higher levels in vegetative cells than in heterocysts, and that SpeY accumulates in vegetative cells but also at significant levels in heterocysts. The homospermidine biosynthetic pathway is therefore active primarily in vegetative cells but the last step can be completed in heterocysts. Our findings indicate an important role for polyamines in the diazotrophic biology of Anabaena. Furthermore, inactivation of a gene cluster (potADB) encoding a polyamine ABC transporter disrupted diazotrophic growth, corroborating the importance of polyamine homeostasis in Anabaena.

Effects of Bisphenol-A on proliferation and expression of genes related to synthesis of polyamines, interferon tau and insulin-like growth factor 2 by ovine trophectoderm cells.

This study evaluated the effects of bisphenol A (BPA) on proliferation of ovine trophectoderm (oTr1) cells, as well as expression of genes for transport of arginine and synthesis of polyamines. BPA reduced proliferation of oTr1 cells at concentrations of 1 × 10-6, 1 × 10-5, 1 × 10-4 M compared to concentrations of 0, 1 × 10-9, and 1 × 10-8 M at 24 and 96 h of culture. Lower concentrations of BPA significantly increased expression of mRNAs for agmatinase (AGMAT), arginine decarboxylase (ADC), ornithine decarboxylase (ODC1) and solute carrier family 7 member 1 (SLC7A1). Similarly, synthesis of polyamines by oTr1 cells was greatest at lower concentrations of BPA and decreased as the dose of BPA increased. Expression of mRNAs for interferon tau (IFNT) and insulin-like growth factor 2 (IGF2) by oTr1 cells was greater than for controls at 1 × 10-9 M BPA. Overall, the effects of BPA on proliferation and gene expression by oTr1 cells were highly dose-dependent.

Carbon catabolite repression gene creA regulates morphology, aflatoxin biosynthesis and virulence in Aspergillus flavus.

Carbon catabolite repression (CCR) is a very important mechanism employed in the utilization of carbon as an energy source, required for the regulation of growth, development and secondary metabolite production in fungi. Despite the wide study of this mechanism in fungi, little is known about the major CCR gene creA in A. flavus. Hence, we report identification of A. flavus carbon catabolite repression gene creA, which is responsible for the repression of secondary carbon sources. Gene deletion and over-expression was employed to explicate the role of creA in the morphology, pathogenicity, and secondary metabolite production in A. flavus. We investigated these factors using three carbon sources including glucose, sucrose and maltose. Gene deletion mutant (ΔcreA) had a significant growth defect on complete medium and minimal medium containing maltose. Conidia production in ΔcreA was significantly impaired irrespective of the carbon source available, while sclerotia production was significantly increased, compared to wild type (WT) and over-expression strain (OE::creA). Importantly, ΔcreA produced insignificant amount of aflatoxin in complete medium, and its ability to colonize hosts was also impaired. Concisely, we showed that creA played an important role in the morphology, pathogenicity and secondary metabolite production of A. flavus.