scholarly journals Study on the mechanism of production of γ-PGA and nattokinase in Bacillus subtilis natto based on RNA-seq analysis

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Min Li ◽  
Zilong Zhang ◽  
Shenwei Li ◽  
Zhengan Tian ◽  
Xia Ma
Keyword(s):  
Bacillus Subtilis ◽  
Solid State ◽  
Metabolic Pathways ◽  
Molecular Targets ◽  
Commercial Production ◽  
Rna Seq ◽  
Expression Of Genes ◽  
Bacillus Subtilis Natto ◽  
Genes Encoding ◽  
Application Prospects

AbstractPoly-γ-glutamic acid (γ-PGA) and nattokinase (NK) are the main substances produced by Bacillus subtilis natto in solid-state fermentation and have wide application prospects. We found that our strains had higher activity of nattokinase when soybeans were used as substrate to increase the yield of γ-PGA. Commercial production of γ-PGA and nattokinase requires an understanding of the mechanism of co-production. Here, we obtained the maximum γ-PGA yield (358.5 g/kg, w/w) and highest activity of NK during fermentation and analyzed the transcriptome of Bacillus subtilis natto during co-production of γ-PGA and NK. By comparing changes in expression of genes encoding key enzymes and the metabolic pathways associated with the products in genetic engineering, the mechanism of co-production of γ-PGA and nattokinase can be summarized based on RNA-seq analysis. This study firstly provides new insights into the mechanism of co-production of γ-PGA and nattokinase by Bacillus subtilis natto and reveals potential molecular targets to promote the co-production of γ-PGA and nattokinase.

scholarly journals Study on Mechanism of γ-PGA and Nattokinase Produced by Bacillus Subtilis Natto Based on RNA-Seq Analysis

2020 ◽  
Author(s):  
Min Li ◽  
Zilong Zhang ◽  
Shenwei Li ◽  
Zhengan Tian ◽  
Xia Ma
Keyword(s):  
Bacillus Subtilis ◽  
Solid State ◽  
Metabolic Pathways ◽  
Fermentation Process ◽  
Molecular Targets ◽  
Commercial Production ◽  
Production Mechanism ◽  
Rna Seq ◽  
Bacillus Subtilis Natto ◽  
Application Prospects

Abstract Poly-γ-glutamic acid (γ-PGA) and nattokinase (NK) are the main substances produced by Bacillus subtilis natto in solid-state fermentation and have wide application prospects. We found that our strains have higher nattokinase activity when soybean is used as a substrate to improve the yield of γ-PGA. Commercial production of γ-PGA and nattokinase requires an understanding of the co-production mechanism. Here, we monitored the metabolites of the fermentation process firstly, analyzed the transcriptome of Bacillus subtilis natto when co-producing γ-PGA and NK, and obtained the maximum γ-PGA yield (11.39 ± 0.38%, w/w) and highest activity of NK during fermentation. By comparing the up-regulated genes and down-regulated genes of key enzymes and product-related metabolic pathways for genetic engineering, the co-production mechanism of γ-PGA and nattokinase can be summarized. This study firstly provides new insights into the co-production mechanism of γ-PGA and nattokinase by Bacillus subtilis natto, and reveals potential molecular targets for promoting the co-production of γ-PGA and nattokinase.

scholarly journals A Transcriptomic Approach to the Metabolism of Tetrapyrrolic Photosensitizers in a Marine Annelid

Molecules ◽  
2021 ◽  
Vol 26 (13) ◽  
pp. 3924
Author(s):  
Maria Leonor Santos ◽  
Mariaelena D’Ambrosio ◽  
Ana P. Rodrigo ◽  
A. Jorge Parola ◽  
Pedro M. Costa
Keyword(s):  
Metabolic Pathways ◽  
Molecular Networks ◽  
Bile Pigments ◽  
Rna Seq ◽  
The Past ◽  
Wide Range ◽  
Genes Encoding ◽  
Organ Specific ◽  
Bright Green ◽  
Green Coloration

The past decade has seen growing interest in marine natural pigments for biotechnological applications. One of the most abundant classes of biological pigments is the tetrapyrroles, which are prized targets due their photodynamic properties; porphyrins are the best known examples of this group. Many animal porphyrinoids and other tetrapyrroles are produced through heme metabolic pathways, the best known of which are the bile pigments biliverdin and bilirubin. Eulalia is a marine Polychaeta characterized by its bright green coloration resulting from a remarkably wide range of greenish and yellowish tetrapyrroles, some of which have promising photodynamic properties. The present study combined metabolomics based on HPLC-DAD with RNA-seq transcriptomics to investigate the molecular pathways of porphyrinoid metabolism by comparing the worm’s proboscis and epidermis, which display distinct pigmentation patterns. The results showed that pigments are endogenous and seemingly heme-derived. The worm possesses homologs in both organs for genes encoding enzymes involved in heme metabolism such as ALAD, FECH, UROS, and PPOX. However, the findings also indicate that variants of the canonical enzymes of the heme biosynthesis pathway can be species- and organ-specific. These differences between molecular networks contribute to explain not only the differential pigmentation patterns between organs, but also the worm’s variety of novel endogenous tetrapyrrolic compounds.

scholarly journals The Purine Efflux Pump PbuE in Bacillus subtilis Modulates Expression of the PurR and G-Box (XptR) Regulons by Adjusting the Purine Base Pool Size

2005 ◽  
Vol 187 (2) ◽  
pp. 791-794 ◽  
Author(s):  
Per Nygaard ◽  
Hans H. Saxild
Keyword(s):  
Bacillus Subtilis ◽  
De Novo ◽  
Efflux Pump ◽  
Purine Base ◽  
Purine Bases ◽  
Purine Analogs ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Transcriptional Fusions

ABSTRACT In Bacillus subtilis, the expression of genes encoding enzymes and other proteins involved in purine de novo synthesis and salvage is affected by purine bases and phosphoribosylpyrophosphate (PRPP). The transcription of the genes belonging to the PurR regulon is negatively regulated by the PurR protein and PRPP. The expression of the genes belonging to the G-box (XptR) regulon, including the pbuE gene, is negatively regulated by a riboswitch-controlled transcription termination mechanism. The G-box regulon effector molecules are hypoxanthine and guanine. pbuE encodes a purine base efflux pump and is now recognized as belonging to a third purine regulon. The expression of the pbuE gene is positively regulated by a riboswitch that recognizes adenine. Here we show that the expression of pbuE′-lacZ transcriptional fusions are induced by adenine to the highest extent in mutants which do not express a functional PbuE pump. In a mutant defective in the metabolism of adenine, the ade apt mutant, we found a high intracellular level of adenine and constitutive high levels of PbuE. A growth test using a purine auxotroph provided further evidence for the role of PbuE in lowering the intracellular concentration of purine bases, including adenine. Purine analogs also affect the expression of pbuE, which might be of importance for the protection against toxic analogs. In a mutant that overexpresses PbuE, the expression of genes belonging to the PurR regulon was increased. Our findings provide further evidence for important functions of the PbuE protein, such as acting as a pump that lowers the purine base pool and affects the expression of the G-box and PurR regulons, including pbuE itself, and as a pump involved in protection against toxic purine base analogs.

scholarly journals The Metabolic Reprogramming of Frem2 Mutant Mice Embryos in Cryptophthalmos Development

2021 ◽  
Vol 8 ◽  
Author(s):  
Xiayin Zhang ◽  
Ruixin Wang ◽  
Ting Wang ◽  
Xulin Zhang ◽  
Meimei Dongye ◽  
...  
Keyword(s):  
Metabolic Reprogramming ◽  
Mutant Mice ◽  
Compound Heterozygous ◽  
Rna Seq ◽  
Targeted Metabolomics ◽  
Expression Of Genes ◽  
Fetal Mice ◽  
Genes Encoding ◽  
Go Terms ◽  
Increased Susceptibility

BackgroundCryptophthalmos is characterized by congenital ocular dysplasia with eyelid malformation. The pathogenicity of mutations in genes encoding components of the FRAS1/FREM protein complex is well established, but the underlying pathomechanisms of this disease are still unclear. In the previous study, we generated mice carrying Frem2R725X/R2156W compound heterozygous mutations using CRISPR/Cas9 and showed that these mice recapitulated the human cryptophthalmos phenotype.MethodsIn this study, we tracked changes in the metabolic profile of embryos and expression of metabolism-related genes in Frem2 mutant mice on E13.5 compared with wild-type mice. RNA sequencing (RNA-seq) was utilized to decipher the differentiated expression of genes associated with metabolism. Untargeted metabolomics and targeted metabolomics analyses were performed to detect and verify the shifts in the composition of the embryonic metabolome.ResultsDifferentially expressed genes participating in amino acid metabolism and energy metabolism were observed by RNA-seq. Transcriptomic analysis suggests that 821 (39.89%) up-regulated genes and 320 (32.99%) down-regulated genes were involved in the metabolic process in the enriched GO terms. A total of 92 significantly different metabolites were identified including creatine, guanosine 5′-monophosphate, cytosine, cytidine 5′-monophosphate, adenine, and L-serine. Interestingly, major shifts related to ATP binding cassette transporters (ABC transporters) and the biosynthesis of amino acids in the composition of the embryonic metabolome were observed by KEGG metabolic analysis, indicating that these pathways could also be involved in the pathogenesis of cryptophthalmos.ConclusionWe demonstrate that Frem2 mutant fetal mice have increased susceptibility to the disruption of eye morphogenesis in association with distinct transcriptomic and metabolomic signatures. Our findings suggest that the metabolomic signature established before birth may play a role in mediating cryptophthalmos in Frem2 mutant mice, which may have important implications for the pathogenesis of cryptophthalmos.

scholarly journals Co-culturing Hyphomicrobium nitrativorans strain NL23 and Methylophaga nitratireducenticrescens strain JAM1 allows sustainable denitrifying activities under marine conditions

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e12424
Author(s):  
Alexandra Cucaita ◽  
Marianne Piochon ◽  
Richard Villemur
Keyword(s):  
Expression Profiles ◽  
Lag Phase ◽  
Rna Seq ◽  
Expression Of Genes ◽  
N Assimilation ◽  
Genes Encoding ◽  
Potential Synergy ◽  
Pure Cultures

Background Hyphomicrobium nitrativorans strain NL23 and Methylophaga nitratireducenticrescens strain JAM1 are the principal bacteria involved in the denitrifying activities of a methanol-fed, fluidized-bed marine denitrification system. Strain NL23 possesses the complete denitrification pathway, but cannot grow under marine conditions in pure cultures. Strain JAM1 is a marine bacterium that lacks genes encoding a dissimilatory nitrite (NO2−) reductase and therefore cannot reduce NO2−. Here, we report the characterization of some of their physiological traits that could influence their co-habitation. We also perform co-cultures to assess the potential synergy between the two strains under marine and denitrifying conditions. Methodology Anoxic planktonic pure cultures of both strains were grown with different concentrations of nitrate (NO3−). Anoxic planktonic co-cultures could only be cultured on low NaCl concentrations for strain NL23 to grow. Biofilm co-cultures were achieved in a 500-mL bioreactor, and operated under denitrifying conditions with increasing concentrations of NaCl. NO3− and NO2− concentrations and the protein content were measured to derive the denitrification rates. The concentrations of both strains in co-cultures were determined by quantitative PCR (qPCR). Ectoine concentration was measured by mass spectrometry in the biofilm co-culture. The biofilm was visualized by fluorescence in situ hybridization. Reverse-transcription-qPCR and RNA-seq approaches were used to assess changes in the expression profiles of genes involved in the nitrogen pathways in the biofilm cultures. Results Planktonic pure cultures of strain JAM1 had a readiness to reduce NO3− with no lag phase for growth in contrast to pure cultures of strain NL23, which had a 2-3 days lag phase before NO3− starts to be consumed and growth to occur. Compared to strain NL23, strain JAM1 has a higher µmax for growth and higher specific NO3− reduction rates. Denitrification rates were twice higher in the planktonic co-cultures than those measured in strain NL23 pure cultures. The biofilm co-cultures showed sustained denitrifying activities and surface colonization by both strains under marine conditions. Increase in ectoine concentrations was observed in the biofilm co-culture with the increase of NaCl concentrations. Changes in the relative transcript levels were observed in the biofilm culture with genes encoding NapA and NapGH in strain NL23. The type of medium had a great impact on the expression of genes involved in the N-assimilation pathways in both strains. Conclusions These results illustrate the capacity of both strains to act together in performing sustainable denitrifying activities under marine conditions. Although strain JAM1 did not contribute in better specific denitrifying activities in the biofilm co-cultures, its presence helped strain NL23 to acclimate to medium with NaCl concentrations >1.0%.

scholarly journals Characterization of Spores of Bacillus subtilis That Lack Most Coat Layers

2008 ◽  
Vol 190 (20) ◽  
pp. 6741-6748 ◽  
Author(s):  
Sonali Ghosh ◽  
Barbara Setlow ◽  
Paul G. Wahome ◽  
Ann E. Cowan ◽  
Marco Plomp ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Dipicolinic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Great Majority ◽  
Coat Proteins ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Wet Heat ◽  
Coat Material

ABSTRACT Spores of Bacillus subtilis have a thick outer layer of relatively insoluble protein called the coat, which protects spores against a number of treatments and may also play roles in spore germination. However, elucidation of precise roles of the coat in spore properties has been hampered by the inability to prepare spores lacking all or most coat material. In this work, we show that spores of a strain with mutations in both the cotE and gerE genes, which encode proteins involved in coat assembly and expression of genes encoding coat proteins, respectively, lack most extractable coat protein as seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as well as the great majority of the coat as seen by atomic force microscopy. However, the cotE gerE spores did retain a thin layer of insoluble coat material that was most easily seen by microscopy following digestion of these spores with lysozyme. These severely coat-deficient spores germinated relatively normally with nutrients and even better with dodecylamine but not with a 1:1 chelate of Ca2+ and dipicolinic acid. These spores were also quite resistant to wet heat, to mechanical disruption, and to treatment with detergents at an elevated temperature and pH but were exquisitely sensitive to killing by sodium hypochlorite. These results provide new insight into the role of the coat layer in spore properties.

scholarly journals Global Transcriptional Response of Bacillus subtilis to Treatment with Subinhibitory Concentrations of Antibiotics That Inhibit Protein Synthesis

2005 ◽  
Vol 49 (5) ◽  
pp. 1915-1926 ◽  
Author(s):  
Janine T. Lin ◽  
Mariah Bindel Connelly ◽  
Chris Amolo ◽  
Suzie Otani ◽  
Debbie S. Yaver
Keyword(s):  
Protein Synthesis ◽  
Bacillus Subtilis ◽  
Time Course ◽  
Expression Patterns ◽  
Transcriptional Response ◽  
Global Gene Expression ◽  
Protein Synthesis Inhibitors ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Subinhibitory Concentrations

ABSTRACT Global gene expression patterns of Bacillus subtilis in response to subinhibitory concentrations of protein synthesis inhibitors (chloramphenicol, erythromycin, and gentamicin) were studied by DNA microarray analysis. B. subtilis cultures were treated with subinhibitory concentrations of protein synthesis inhibitors for 5, 15, 30, and 60 min, and transcriptional patterns were measured throughout the time course. Three major classes of genes were affected by the protein synthesis inhibitors: genes encoding transport/binding proteins, genes involved in protein synthesis, and genes involved in the metabolism of carbohydrates and related molecules. Similar expression patterns for a few classes of genes were observed due to treatment with chloramphenicol (0.4× MIC) or erythromycin (0.5× MIC), whereas expression patterns of gentamicin-treated cells were distinct. Expression of genes involved in metabolism of amino acids was altered by treatment with chloramphenicol and erythromycin but not by treatment with gentamicin. Heat shock genes were induced by gentamicin but repressed by chloramphenicol. Other genes induced by the protein synthesis inhibitors included the yheIH operon encoding ABC transporter-like proteins, with similarity to multidrug efflux proteins, and the ysbAB operon encoding homologs of LrgAB that function to inhibit cell wall cleavage (murein hydrolase activity) and convey penicillin tolerance in Staphylococcus aureus.

scholarly journals Differential transcription of expanded gene families in central carbon metabolism of Streptomyces coelicolor A3(2)

2019 ◽  
Author(s):  
Jana K Schniete ◽  
Richard Reumerman ◽  
Leena Kerr ◽  
Nicholas P Tucker ◽  
Iain S Hunter ◽  
...  
Keyword(s):  
Carbon Source ◽  
Streptomyces Coelicolor ◽  
Carbon Sources ◽  
Gene Families ◽  
Gene Clusters ◽  
Central Carbon Metabolism ◽  
Rna Seq ◽  
Enzymatic Function ◽  
Expression Of Genes ◽  
Genes Encoding

AbstractBackgroundStreptomycete bacteria are prolific producers of specialised metabolites, many of which have clinically relevant bioactivity. A striking feature of their genomes is the expansion of gene families that encode the same enzymatic function. Genes that undergo expansion events, either by horizontal gene transfer or duplication, can have a range of fates: genes can be lost, or they can undergo neo-functionalisation or sub-functionalisation. To test whether expanded gene families in Streptomyces exhibit differential expression, an RNA-Seq approach was used to examine cultures of wild-type Streptomyces coelicolor grown with either glucose or tween as the sole carbon source.ResultsRNA-Seq analysis showed that two-thirds of genes within expanded gene families show transcriptional differences when strains were grown on tween compared to glucose. In addition, expression of specialised metabolite gene clusters (actinorhodin, isorenieratane, coelichelin and a cryptic NRPS) was also influenced by carbon source.ConclusionsExpression of genes encoding the same enzymatic function had transcriptional differences when grown on different carbon sources. This transcriptional divergence enables partitioning to function under different physiological conditions. These approaches can inform metabolic engineering of industrial Streptomyces strains and may help develop cultivation conditions to activate the so-called silent biosynthetic gene clusters.

scholarly journals Overexpression and Activation of αvβ3 Integrin Differentially Affects TGFβ2 Signaling in Human Trabecular Meshwork Cells

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1923
Author(s):  
Mark S. Filla ◽  
Kristy K. Meyer ◽  
Jennifer A. Faralli ◽  
Donna M. Peters
Keyword(s):  
Trabecular Meshwork ◽  
Open Angle Glaucoma ◽  
Integrin Signaling ◽  
Αvβ3 Integrin ◽  
Rna Seq ◽  
Significant Differential Expression ◽  
Protein Protein Interaction ◽  
Trabecular Meshwork Cells ◽  
Expression Of Genes ◽  
Genes Encoding

Studies from our laboratory have suggested that activation of αvβ3 integrin-mediated signaling could contribute to the fibrotic-like changes observed in primary open angle glaucoma (POAG) and glucocorticoid-induced glaucoma. To determine how αvβ3 integrin signaling could be involved in this process, RNA-Seq analysis was used to analyze the transcriptomes of immortalized trabecular meshwork (TM) cell lines overexpressing either a control vector or a wild type (WT) or a constitutively active (CA) αvβ3 integrin. Compared to control cells, hierarchical clustering, PANTHER pathway and protein-protein interaction (PPI) analysis of cells overexpressing WT-αvβ3 integrin or CA-αvβ3 integrin resulted in a significant differential expression of genes encoding for transcription factors, adhesion and cytoskeleton proteins, extracellular matrix (ECM) proteins, cytokines and GTPases. Cells overexpressing a CA-αvβ3 integrin also demonstrated an enrichment for genes encoding proteins found in TGFβ2, Wnt and cadherin signaling pathways all of which have been implicated in POAG pathogenesis. These changes were not observed in cells overexpressing WT-αvβ3 integrin. Our results suggest that activation of αvβ3 integrin signaling in TM cells could have significant impacts on TM function and POAG pathogenesis.

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