RESUMO
BsCotA laccase is a promising candidate for industrial application due to its excellent thermal stability. In this research, our objective was to enhance the catalytic efficiency of BsCotA by modifying the active site pocket. We utilized a strategy combining the diversity design of the active site pocket with molecular docking screening, which resulted in selecting five variants for characterization. All five variants proved functional, with four demonstrating improved turnover rates. The most effective variants exhibited a remarkable 7.7-fold increase in catalytic efficiency, evolved from 1.54 × 105 M-1 s-1 to 1.18 × 106 M-1 s-1, without any stability loss. To investigate the underlying molecular mechanisms, we conducted a comprehensive structural analysis of our variants. The analysis suggested that substituting Leu386 with aromatic residues could enhance BsCotA's ability to accommodate the 2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonate (ABTS) substrate. However, the inclusion of charged residues, G323D and G417H, into the active site pocket reduced kcat. Ultimately, our research contributes to a deeper understanding of the role played by residues in the laccases' active site pocket, while successfully demonstrating a method to lift the catalytic efficiency of BsCotA. KEY POINTS: ⢠Active site pocket design that enhanced BsCotA laccase efficiency ⢠7.7-fold improved in catalytic rate ⢠All tested variants retain thermal stability.
Assuntos
Bacillus subtilis , Domínio Catalítico , Lacase , Simulação de Acoplamento Molecular , Lacase/metabolismo , Lacase/genética , Lacase/química , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Estabilidade Enzimática , Cinética , Ácidos Sulfônicos/metabolismo , Catálise , BenzotiazóisRESUMO
Transposon mutagenesis screening of Bacillus subtilis YB-1471, a novel rhizosphere biocontrol agent of Fusarium crown rot (FCR) of wheat, resulted in the identification of orf04391, linked to reduced biofilm formation. The gene encodes a protein possessing a putative tertiary structure of a "double-wing" DNA-binding domain. Expression of orf04391 increased during biofilm development in stationary cultures and during rapid growth in shaking cultures. An orf04391 deletion strain showed reduced biofilm production related to lower levels of the extracellular matrix, and the mutant also had reduced sporulation, adhesion, root colonization, and FCR biocontrol efficiency. Transcriptome analysis of YB-1471 and Δorf04391 in stationary culture showed that the loss of orf04391 resulted in altered expression of numerous genes, including sinI, an initiator of biofilm formation. DNA binding was shown with his-tagged Orf04391 binding to the sinIR operon in vivo and in vitro. Orf04391 appears to be a transcriptional regulator of biofilm formation in B. subtilis through the Spo0A-SinI/SinR pathway.
Assuntos
Bacillus subtilis , Proteínas de Bactérias , Fusarium , Doenças das Plantas , Triticum , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Biofilmes/crescimento & desenvolvimento , Fusarium/genética , Fusarium/metabolismo , Fusarium/fisiologia , Regulação Bacteriana da Expressão Gênica , Doenças das Plantas/microbiologia , Triticum/microbiologiaRESUMO
Antagonistic bacterial strains from Bacillus spp. have been widely studied and utilized in the biocontrol of phytopathogens and the promotion of plant growth, but their impacts on the rhizosphere microecology when applied to crop plants are unclear. Herein, the effects of applying the antagonistic bacterium Bacillus subtilis S1 as a biofertilizer on the rhizosphere microecology of cucumbers were investigated. In a pot experiment on cucumber seedlings inoculated with S1, 3124 bacterial operational taxonomic units (OTUs) were obtained from the rhizosphere soils using high-throughput sequencing of 16S rRNA gene amplicons, and the most abundant phylum was Proteobacteria that accounted for 49.48% in the bacterial community. S1 treatment significantly reduced the abundances of soil bacterial taxa during a period of approximately 30 days but did not affect bacterial diversity in the rhizosphere soils of cucumbers. The enzymatic activities of soil nitrite reductase (S-Nir) and dehydrogenase (S-DHA) were significantly increased after S1 fertilization. However, the activities of soil urease (S-UE), cellulase (S-CL), and sucrase (S-SC) were significantly reduced compared to the control group. Additionally, the ammonium- and nitrate-nitrogen contents of S1-treated soil samples were significantly lower than those of the control group. S1 fertilization reshaped the rhizosphere soil bacterial community of cucumber plants. The S-CL activity and nitrate-nitrogen content in rhizosphere soil affected by S1 inoculation play important roles in altering the abundance of rhizosphere soil microbiota.
Assuntos
Bacillus subtilis , Bactérias , Cucumis sativus , Nitrogênio , Rizosfera , Microbiologia do Solo , Cucumis sativus/microbiologia , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Nitrogênio/metabolismo , Bactérias/classificação , Bactérias/genética , Bactérias/metabolismo , Bactérias/isolamento & purificação , RNA Ribossômico 16S/genética , Fertilizantes/análise , Solo/química , Microbiota , FilogeniaRESUMO
Guanidinoacetic acid, as an energetic substance, has a wide range of applications in the food, pharmaceutical, and feed industries. However, the biosynthesis of guanidinoacetic acid has not been applied in industrial production. In this study, we designed the synthetic route of guanidinoacetic acid in a food-grade strain of Bacillus subtilis. By regulating the expression of key enzymes, lifting feedback inhibition, and increasing membrane permeability, we achieved the efficient synthesis of guanidinoacetic acid by whole-cell catalysis. Firstly, the optimal L-arginine:glycine amidinotransferase was screened based on the phylogenetic tree, and the expression of the key enzyme was enhanced by a strategy combining strong promoter and genome integration. Secondly, the ornithine cycle for L-arginine synthesis in Corynebacterium glutamicum was introduced to alleviate the feedback inhibition of the enzyme by the byproduct L-ornithine, and the L-arginine degradation pathway was knocked down to enhance substrate regeneration. Thirdly, the expression of N-acetylmuramoyl-L-alanine amidase (LytC) was up-regulated to increase the cell membrane permeability. Finally, after optimization of whole-cell production conditions, strain Bs-13 achieved guanidinoacetic acid production at a titer of 13.1 g/L after 24 h, with a proudction rate of 0.54 g/(L·h) and a glycine conversion rate of 92.7%. The above strategy improved the production of guanidinoacetic acid and provided a reference for the biosynthesis of guanidinoacetic acid.
Assuntos
Arginina , Bacillus subtilis , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Arginina/biossíntese , Arginina/metabolismo , Glicina/análogos & derivados , Glicina/metabolismo , Glicina/biossíntese , Amidinotransferases/genética , Amidinotransferases/metabolismo , Corynebacterium glutamicum/metabolismo , Corynebacterium glutamicum/genética , N-Acetil-Muramil-L-Alanina Amidase/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Engenharia Metabólica , Ornitina/biossíntese , Ornitina/metabolismoRESUMO
D-mannose is a natural hexose with great economic and application values in the food, medicine, and cosmetic fields. However, most biosynthesis methods of D-mannose rely on Escherichia coli as the host, which poses safety issues during the production process and imposes limitations on subsequent applications. This study compared the enzyme properties of mannose isomerases from multiple sources to select the most suitable source. B. subtilis 168/pMA5-EcMIaseA was constructed with "generally recognized as safe" (GRAS) Bacillus subtilis as the host and used as a whole-cell catalyst to synthesize D-mannose from d-fructose. Optimizing the conversion conditions such as culture temperature, pH, and substrate concentration increased the yield of D-mannose. The results showed that the conversion rates reached 27.75% and 27.22% and the yields of D-mannose were 138.74 g/L and 163.30 g/L after 6 h whole-cell transformation with d-fructose at the concentrations of 500 g/L and 600 g/L, respectively, in a 5 L fermentor. This study achieves the highest yield of D-mannose produced under the catalysis by recombinant B. subtilis that has ever been reported and provides a basis for the industrial production and application of D-mannose.
Assuntos
Bacillus subtilis , Frutose , Manose , Bacillus subtilis/metabolismo , Bacillus subtilis/genética , Manose/metabolismo , Manose/biossíntese , Frutose/metabolismo , Frutose/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Soil microbial communities host a large number of microbial species that support important ecological functions such as biogeochemical cycling and plant nutrition. The extent and stability of these functions are affected by inter-species interactions among soil microorganisms, yet the different mechanisms underpinning microbial interactions in the soil are not fully understood. Here, we study the extent of nutrient-based interactions among two model, plant-supporting soil microorganisms, the fungi Serendipita indica, and the bacteria Bacillus subtilis. We found that S. indica is unable to grow with nitrate - a common nitrogen source in the soil - but this inability could be rescued, and growth restored in the presence of B. subtilis. We demonstrate that this effect is due to B. subtilis utilising nitrate and releasing ammonia, which can be used by S. indica. We refer to this type of mechanism as ammonia mediated nitrogen sharing (N-sharing). Using a mathematical model, we demonstrated that the pH dependent equilibrium between ammonia (NH3) and ammonium (NH+4) results in an inherent cellular leakiness, and that reduced amonnium uptake or assimilation rates could result in higher levels of leaked ammonia. In line with this model, a mutant B. subtilis - devoid of ammonia uptake - showed higher S. indica growth support in nitrate media. These findings highlight that ammonia based N-sharing can be a previously under-appreciated mechanism underpinning interaction among soil microorganisms and could be influenced by microbial or abiotic alteration of pH in microenvironments.
Assuntos
Amônia , Bacillus subtilis , Nitratos , Nitrogênio , Microbiologia do Solo , Amônia/metabolismo , Nitrogênio/metabolismo , Nitratos/metabolismo , Bacillus subtilis/metabolismo , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/genética , Solo/química , Interações Microbianas , Concentração de Íons de Hidrogênio , Compostos de Amônio/metabolismoRESUMO
Tobacco Etch virus (TEV) protease is one of the most common tools for removing fusion tags, but no study has shown that TEV can be expressed at high levels in the GRAS host strain Bacillus subtilis and purified for further application. In this study, the fusion protein BsLysSN-TEV C/S-His-TEV consisting of a fusion tag, N-terminal domain of a lysyl-tRNA synthetase (BsLysSN) coded by B. subtilis lysS gene, placed at the N-terminus followed by an endoprotease TEV cleavage site and then the expression of this fusion protein in the cytoplasm of B. subtilis was investigated. The SDS-PAGE and Western-blot analysis demonstrated that His-TEV was overexpressed under the induction of IPTG. This result infers that His-TEV protease showed promising activity in the B. subtilis cytoplasm by the cleavage of the fusion protein. These cleavage products could be purified using the Ni-NTA column, which effectively cleaved the purified recombinant protein substrate, which can be applied in the protein purification process to remove the fusion tag. Significantly, since both His-TEV protease and the fusion recombinant protein substrate are expressed in the endotoxin-free host strain, the tag removal and purified product should be theoretically endotoxin-free, which could be a promising approach for producing therapeutic proteins and also for other relevant biomedical applications.
Assuntos
Bacillus subtilis , Endopeptidases , Proteínas Recombinantes de Fusão , Bacillus subtilis/genética , Bacillus subtilis/enzimologia , Endopeptidases/genética , Endopeptidases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/química , Endotoxinas/genética , Endotoxinas/metabolismo , Lisina-tRNA Ligase/genética , Lisina-tRNA Ligase/metabolismo , Lisina-tRNA Ligase/química , Expressão GênicaRESUMO
Bacillus subtilis is ubiquitously and broadly distributed in various environments but is mostly isolated from soil. Given that B. subtilis is known as a plant growth-promoting rhizobacterium in agriculture, we aimed to describe the natural distribution of this species and uncover how biotic and abiotic factors affect its distribution. When comparing different soils, we discovered that B. subtilis group species are most abundant in grasslands but can rarely be isolated from forest soil, even if the soil sample sites are situated in proximity. Differential analysis revealed that spore-forming bacteria exhibited enrichments in the grassland, suggesting niche overlap or synergistic interactions leading to the proliferation of certain Bacillus species in grassland environments. Network analysis further revealed that Bacillus and other Bacillota established a densely interconnected hub module in the grassland, characterised by positive associations indicating co-occurrence, a pattern not observed in the forest soil. Speculating that this difference was driven by abiotic factors, we combined amplicon sequencing with physico-chemical analysis of soil samples and found multiple chemical variables, mainly pH, to affect microbial composition. Our study pinpoints the factors that influence B. subtilis abundance in natural soils and, therefore, offers insights for designing B. subtilis-based biocontrol products in agricultural settings.
Assuntos
Bacillus subtilis , Florestas , Microbiologia do Solo , Solo , Bacillus subtilis/genética , Bacillus subtilis/classificação , Solo/química , PradariaRESUMO
There is a growing interest in the creation of engineered condensates formed via liquid-liquid phase separation (LLPS) to exert precise cellular control in prokaryotes. However, de novo design of cellular condensates to control metabolic flux or protein translation remains a challenge. Here, we present a synthetic condensate platform, generated through the incorporation of artificial, disordered proteins to realize specific functions in Bacillus subtilis. To achieve this, the "stacking blocks" strategy is developed to rationally design a series of LLPS-promoting proteins for programming condensates. Through the targeted recruitment of biomolecules, our investigation demonstrates that cellular condensates effectively sequester biosynthetic pathways. We successfully harness this capability to enhance the biosynthesis of 2'-fucosyllactose by 123.3%. Furthermore, we find that condensates can enhance the translation specificity of tailored enzyme fourfold, and can increase N-acetylmannosamine titer by 75.0%. Collectively, these results lay the foundation for the design of engineered condensates endowed with multifunctional capacities.
Assuntos
Bacillus subtilis , Proteínas de Bactérias , Hexosaminas , Engenharia Metabólica , Bacillus subtilis/metabolismo , Bacillus subtilis/genética , Engenharia Metabólica/métodos , Hexosaminas/biossíntese , Hexosaminas/metabolismo , Hexosaminas/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Vias Biossintéticas , Engenharia de Proteínas/métodos , Biossíntese de Proteínas , Trissacarídeos/metabolismo , Trissacarídeos/biossíntese , Trissacarídeos/química , Extração Líquido-Líquido/métodosRESUMO
N-terminal coding sequence (NCS) influences gene expression by impacting the translation initiation rate. The NCS optimization problem is to find an NCS that maximizes gene expression. The problem is important in genetic engineering. However, current methods for NCS optimization such as rational design and statistics-guided approaches are labor-intensive yield only relatively small improvements. This paper introduces a deep learning/synthetic biology codesigned few-shot training workflow for NCS optimization. Our method utilizes k-nearest encoding followed by word2vec to encode the NCS, then performs feature extraction using attention mechanisms, before constructing a time-series network for predicting gene expression intensity, and finally a direct search algorithm identifies the optimal NCS with limited training data. We took green fluorescent protein (GFP) expressed by Bacillus subtilis as a reporting protein of NCSs, and employed the fluorescence enhancement factor as the metric of NCS optimization. Within just six iterative experiments, our model generated an NCS (MLD62) that increased average GFP expression by 5.41-fold, outperforming the state-of-the-art NCS designs. Extending our findings beyond GFP, we showed that our engineered NCS (MLD62) can effectively boost the production of N-acetylneuraminic acid by enhancing the expression of the crucial rate-limiting GNA1 gene, demonstrating its practical utility. We have open-sourced our NCS expression database and experimental procedures for public use.
Assuntos
Bacillus subtilis , Aprendizado Profundo , Proteínas de Fluorescência Verde , Biologia Sintética , Biologia Sintética/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Expressão Gênica/genética , Algoritmos , Engenharia Genética/métodosRESUMO
A 50:50 blend of two Bacillus subtilis strains positively impacted the productivity of finishing pigs. Given this observed effect, we hypothesized that each strain has distinct effects on weight gain and their influence on gut microbiota. In a 16-week test, 160 pigs were divided into four groups: basal diet, B. subtilis ps4100, B. subtilis ps4060, and 50:50 mixture supplemented. Subsequently, we compared body weight and fecal microbiota. Among the supplements, ps4100, ps4060, and the 50:50 mix yielded respective average daily weight gains (ADG) of 3.6%, 4.6%, and 3.9% by the 6th week. The weight difference was maintained through the 16th week. At the 11th week, the difference in α-diversity among the fecal microbiota was marginal, and 17 of 229 genera showed differential abundance between the control and either of the treatment groups. A total of 12 of the 17 genera, including Lactobacillus, showed differential abundance between the ps4100 and ps4060-fed groups, and only Eubacterium consistently decreased in abundance in both the ps4100 and ps4060 groups. In comparison, microbial diversity was significantly different at the 16th week (p < 0.05), with 96 out of 229 genera exhibiting differential abundance. A total of 42 of the 96 genera exhibited similar patterns in both the ps4100 and ps4060 groups compared to the control group. Additionally, 236 of 687 microbial enzymes with differential abundance deduced from 16S rRNA reads showed similar differential abundance in both groups compared to the control group. We concluded that the overall microbial balance, rather than the dominance or significant decrease of a few specific genera, likely caused the enhanced ADG until the 11th week. Substantial changes in microbiota manifested at the 16th week did not cause dramatically increased ADG but were a consequence of weight gain and could positively affect animal physiology and health afterward.
Assuntos
Bacillus subtilis , Microbioma Gastrointestinal , Probióticos , Animais , Bacillus subtilis/genética , Suínos , Ração Animal , Fezes/microbiologia , Aumento de Peso , RNA Ribossômico 16S/genéticaRESUMO
Menaquinone-7 (MK-7) is a form of vitamin K2 with health-beneficial effects. A novel fermentation strategy based on combining soy protein hydrolysates (SPHs) with biofilm-based fermentation was investigated to enhance menaquinone-7 (MK-7) biosynthesis by Bacillus subtilis natto. Results showed the SPHs increased MK-7 yield by 199.4% in two-stage aeration fermentation as compared to the SP-based medium in submerged fermentation, which was related to the formation of robust biofilm with wrinkles and the enhancement of cell viability. Moreover, there was a significant correlation between key genes related to MK-7 and biofilm synthesis, and the quorum sensing (QS) related genes, Spo0A and SinR, were downregulated by 0.64-fold and 0.39-fold respectively, which promoted biofilm matrix synthesis. Meanwhile, SPHs also enhanced the MK-7 precursor, isoprene side chain, supply, and MK-7 assembly efficiency. Improved fermentation performances of bacterial cells during fermentation were attributed to abundant oligopeptides (Mw < 1 kDa) and moderate amino acids, particularly Arg, Asp, and Phe in SPHs. All these results revealed that SPHs were a potential and superior nitrogen source for MK-7 production by Bacillus subtilis natto.
Assuntos
Bacillus subtilis , Biofilmes , Fermentação , Hidrolisados de Proteína , Proteínas de Soja , Vitamina K 2 , Bacillus subtilis/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/fisiologia , Biofilmes/crescimento & desenvolvimento , Vitamina K 2/análogos & derivados , Vitamina K 2/metabolismo , Hidrolisados de Proteína/metabolismo , Proteínas de Soja/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Percepção de QuorumRESUMO
Bacteria thrive in diverse environments and must withstand various stresses. A key stress response mechanism is the reprogramming of macromolecular biosynthesis and metabolic processes through alarmones - signaling nucleotides that accumulate intracellularly in response to metabolic stress. Diadenosine tetraphosphate (Ap4A), a putative alarmone, is produced in a noncanonical reaction by universally conserved aminoacyl-tRNA synthetases. Ap4A is ubiquitous across all domains of life and accumulates during heat and oxidative stress. Despite its early discovery in 1966, Ap4A's alarmone status remained inconclusive. Recent discoveries identified Ap4A as a precursor to RNA 5' caps in Escherichia coli. Additionally, Ap4A was found to directly bind to and allosterically inhibit the purine biosynthesis enzyme inosine 5'-monophosphate dehydrogenase, regulating guanosine triphosphate levels and enabling heat resistance in Bacillus subtilis. These findings, along with previous research, strongly suggest that Ap4A plays a crucial role as an alarmone, warranting further investigation to fully elucidate its functions.
Assuntos
Fosfatos de Dinucleosídeos , Proteostase , Estabilidade de RNA , Fosfatos de Dinucleosídeos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Bactérias/metabolismo , Bactérias/genética , Estresse FisiológicoRESUMO
The bacterium Bacillus subtilis is a widely used study model and industrial workhorse organism that belongs to the group of gram-positive bacteria. In this study, we report the analysis of a newly sequenced complete genome of B. subtilis strain SRCM117797 along with a comparative genomics of a large collection of B. subtilis strain genomes. B. subtilis strain SRCM117797 has 4,255,638 bp long chromosome with 43.4% GC content and high coding sequence association with macromolecules, metabolism, and phage genes. Genomic diversity analysis of 232 B. subtilis strains resulted in the identification of eight clusters and three singletons. Of 147 B. subtilis strains included, 89.12% had strain-specific genes, of which 6.75% encoded strain-specific insertion sequence family transposases. Our analysis showed a potential role of strain-specific insertion sequence family transposases in intra-cellular accumulation of strain-specific genes. Furthermore, the chromosomal layout of the core genes was biased: overrepresented on the upper half (closer to the origin of replication) of the chromosome, which may explain the fast-growing characteristics of B. subtilis. Overall, the study provides a complete genome sequence of B. subtilis strain SRCM117797, show an extensive genomic diversity of B. subtilis strains and insights into strain diversification mechanism and non-random chromosomal layout of core genes.
Assuntos
Bacillus subtilis , Genoma Bacteriano , Bacillus subtilis/genética , Filogenia , Variação Genética , Composição de Bases , Genômica , Cromossomos Bacterianos/genética , Análise de Sequência de DNARESUMO
Removal of RNA/DNA hybrids for the maturation of Okazaki fragments on the lagging strand, or due to misincorporation of ribonucleotides by DNA polymerases, is essential for all types of cells. In prokaryotic cells such as Escherichia coli, DNA polymerase 1 and RNase HI are supposed to remove RNA from Okazaki fragments, but many bacteria lack HI-type RNases, such as Bacillus subtilis. Previous work has demonstrated in vitro that four proteins are able to remove RNA from RNA/DNA hybrids, but their actual contribution to DNA replication is unclear. We have studied the dynamics of DNA polymerase A (similar to Pol 1), 5'->3' exonuclease ExoR, and the two endoribonucleases RNase HII and HIII in B. subtilis using single-molecule tracking. We found that all four enzymes show a localization pattern similar to that of replicative DNA helicase. By scoring the distance of tracks to replication forks, we found that all four enzymes are enriched at DNA replication centers. After inducing UV damage, RNase HIII was even more strongly recruited to the replication forks, and PolA showed a more static behavior, indicative of longer binding events, whereas RNase HII and ExoR showed no response. Inhibition of replication by 6(p hydroxyphenylazo)-uracil (HPUra) demonstrated that both RNase HII and RNase HIII are directly involved in the replication. We found that the absence of ExoR increases the likelihood of RNase HIII at the forks, indicating that substrate availability rather than direct protein interactions may be a major driver for the recruitment of RNases to the lagging strands. Thus, B. subtilis replication forks appear to be an intermediate between E. coli type and eukaryotic replication forks and employ a multitude of RNases, rather than any dedicated enzyme for RNA/DNA hybrid removal.
Assuntos
Bacillus subtilis , Replicação do DNA , Bacillus subtilis/genética , Bacillus subtilis/enzimologia , Ribonucleases/metabolismo , Ribonucleases/genética , Ribonuclease H/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Raios UltravioletaRESUMO
L-arabinose isomerase (L-AI) is a functional enzyme for the isomerizing of D-galactose to produce D-tagatose. In this study, L-AI-C6-encoding gene from the probiotic Lactobacillus fermentum C6 was cloned and expressed in Bacillus subtilis WB600 for investigating enzymatic characteristics and bioconverting D-tagatose by means of whole-cell catalysis. Results showed that the engineered B. subtilis WB600-pMA5-LAI achieved a maximum specific activity of L-AI-C6 (232.65 ± 15.54 U/mg protein) under cultivation in LB medium at 28 °C for 40 h. The recombinant L-AI-C6 was purified, and enzymatic characteristics test showed its optimum reaction temperature and pH at 60 °C and 8.0, respectively. In addition, L-AI-C6 exhibited good stability within the pH range of 5.5-9.0. By using B. subtilis WB600-pMA5-LAI cells as whole-cell catalyst, the highest D-tagatose yield reached 42.91 ± 0.28 % with D-galactose as substrate, which was 2.41 times that of L. fermentum C6 (17.79 ± 0.11 %). This suggested that the cloning and heterologous expression of L-AI-C6 was an effective strategy for improving D-tagatose conversion by whole-cell catalysis. In brief, the present study demonstrated that the reaction temperature, pH, and stability of L-AI-C6 from L. fermentum C6 meet the demands of industrial application, and the constructed B. subtilis WB600-pMA5-LAI shows promising potential for the whole-cell biotransformation of D-tagatose.
Assuntos
Aldose-Cetose Isomerases , Bacillus subtilis , Hexoses , Limosilactobacillus fermentum , Proteínas Recombinantes , Bacillus subtilis/genética , Bacillus subtilis/enzimologia , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , Hexoses/metabolismo , Hexoses/biossíntese , Limosilactobacillus fermentum/enzimologia , Limosilactobacillus fermentum/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Concentração de Íons de Hidrogênio , Temperatura , Clonagem Molecular/métodos , Estabilidade Enzimática , Galactose/metabolismo , CinéticaRESUMO
Alginate lyases (ALys) whose degrading products, alginate oligosaccharides, exhibit various outstanding biochemical activities have aroused increasing interest of researchers in the marine bioresource field. However, their predominant sourcing from marine bacteria, with limited yields and unclear genetic backgrounds, presents a challenge for industrial production. In this study, ALys (Aly01) from Vibrio natriegens SK 42.001 was expressed in Bacillus subtilis (B. subtilis), a nonpathogenic microorganism recognized as generally safe (GRAS). This accomplishment was realized through a comprehensive strategy involving vector and host selection, promoter and signal peptide screening, and engineering of the ribosome binding site (RBS) and the N-terminal coding sequence (NCS). The optimal combination was identified as the pP43NMK and B. subtilis WB600. Among the 19 reported strong promoters, PnprE exhibited the best performance, showing intracellular enzyme activities of 4.47 U/mL. Despite expectations, dual promoter construction did not yield a significant increase. Further, SPydhT demonstrated the highest extracellular activity (1.33 U/mL), which was further improved by RBS/NCS engineering, reaching 4.58 U/mL. Finally, after fed-batch fermentation, the extracellular activity reached 18.01 U/mL, which was the highest of ALys with a high molecular weight expressed in B. subtilis. These findings are expected to offer valuable insights into the heterologous expression of ALys in B. subtilis.
Assuntos
Bacillus subtilis , Proteínas de Bactérias , Polissacarídeo-Liases , Regiões Promotoras Genéticas , Sinais Direcionadores de Proteínas , Bacillus subtilis/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Polissacarídeo-Liases/genética , Polissacarídeo-Liases/metabolismo , Polissacarídeo-Liases/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Sinais Direcionadores de Proteínas/genética , Vibrio/genética , Vibrio/enzimologia , Vetores Genéticos/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/química , Iniciação Traducional da Cadeia PeptídicaRESUMO
Many strains from the Bacillus subtilis species complex exert strong plant growth-promoting activities. However, their efficacy in relevant conditions is variable, due in part to their inability to establish a strong interaction with roots in stressful environmental conditions. Adaptative laboratory evolution (ALE) is a powerful tool to generate novel strains with traits of interest. Many Bacillus evolved isolates, stemming from ALE performed with plants, possess a stronger root colonization capacity. An in-depth analysis of these isolates also allowed the identification of key features influencing the interaction with plant roots. However, many variables can influence the outcome of these assays, and thus, caution should be taken when designing ALE destined to generate better root colonizers.
Assuntos
Bacillus subtilis , Raízes de Plantas , Raízes de Plantas/microbiologia , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/fisiologia , Evolução Molecular Direcionada , Plantas/microbiologia , Bacillus/genética , Bacillus/fisiologia , Bacillus/classificação , Bacillus/crescimento & desenvolvimentoRESUMO
Transfer RNA (tRNA) modifications are essential for the temperature adaptation of thermophilic and psychrophilic organisms as they control the rigidity and flexibility of transcripts. To further understand how specific tRNA modifications are adjusted to maintain functionality in response to temperature fluctuations, we investigated whether tRNA modifications represent an adaptation of bacteria to different growth temperatures (minimal, optimal, and maximal), focusing on closely related psychrophilic (P. halocryophilus and E. sibiricum), mesophilic (B. subtilis), and thermophilic (G. stearothermophilus) Bacillales. Utilizing an RNA sequencing approach combined with chemical pre-treatment of tRNA samples, we systematically profiled dihydrouridine (D), 4-thiouridine (s4U), 7-methyl-guanosine (m7G), and pseudouridine (Ψ) modifications at single-nucleotide resolution. Despite their close relationship, each bacterium exhibited a unique tRNA modification profile. Our findings revealed increased tRNA modifications in the thermophilic bacterium at its optimal growth temperature, particularly showing elevated levels of s4U8 and Ψ55 modifications compared to non-thermophilic bacteria, indicating a temperature-dependent regulation that may contribute to thermotolerance. Furthermore, we observed higher levels of D modifications in psychrophilic and mesophilic bacteria, indicating an adaptive strategy for cold environments by enhancing local flexibility in tRNAs. Our method demonstrated high effectiveness in identifying tRNA modifications compared to an established tool, highlighting its potential for precise tRNA profiling studies.
Assuntos
Processamento Pós-Transcricional do RNA , RNA de Transferência , Temperatura , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Pseudouridina/metabolismoRESUMO
The transcriptional control of sporulation in Bacillus subtilis is reasonably well understood, but its translational control is underexplored. Here, we use RNA-seq, ribosome profiling and fluorescence microscopy to study the translational dynamics of B. subtilis sporulation. We identify two events of translation silencing and describe spatiotemporal changes in subcellular localization of ribosomes during sporulation. We investigate the potential regulatory role of ribosomes during sporulation using a strain lacking zinc-independent paralogs of three zinc-dependent ribosomal proteins (L31, L33 and S14). The mutant strain exhibits delayed sporulation, reduced germination efficiency, dysregulated translation of metabolic and sporulation-related genes, and disruptions in translation silencing, particularly in late sporulation.