RESUMEN
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.
Asunto(s)
Bacillus subtilis , Biopelículas , Fermentación , Hidrolisados de Proteína , Proteínas de Soja , Vitamina K 2 , Bacillus subtilis/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/fisiología , Biopelículas/crecimiento & desarrollo , Vitamina K 2/análogos & derivados , Vitamina K 2/metabolismo , Hidrolisados de Proteína/metabolismo , Proteínas de Soja/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Percepción de QuorumRESUMEN
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.
Asunto(s)
Bacillus subtilis , Dominio Catalítico , Lacasa , Simulación del Acoplamiento Molecular , Lacasa/metabolismo , Lacasa/genética , Lacasa/química , Bacillus subtilis/enzimología , Bacillus subtilis/genética , Estabilidad de Enzimas , Cinética , Ácidos Sulfónicos/metabolismo , Catálisis , BenzotiazolesRESUMEN
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.
Asunto(s)
Bacillus subtilis , Proteínas Bacterianas , Hexosaminas , Ingeniería Metabólica , Bacillus subtilis/metabolismo , Bacillus subtilis/genética , Ingeniería Metabólica/métodos , Hexosaminas/biosíntesis , Hexosaminas/metabolismo , Hexosaminas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Vías Biosintéticas , Ingeniería de Proteínas/métodos , Biosíntesis de Proteínas , Trisacáridos/metabolismo , Trisacáridos/biosíntesis , Trisacáridos/química , Extracción Líquido-Líquido/métodosRESUMEN
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.
Asunto(s)
Bacillus subtilis , Bacterias , Cucumis sativus , Nitrógeno , Rizosfera , Microbiología del Suelo , Cucumis sativus/microbiología , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Nitrógeno/metabolismo , Bacterias/clasificación , Bacterias/genética , Bacterias/metabolismo , Bacterias/aislamiento & purificación , ARN Ribosómico 16S/genética , Fertilizantes/análisis , Suelo/química , Microbiota , FilogeniaRESUMEN
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.
Asunto(s)
Bacillus subtilis , Microbioma Gastrointestinal , Probióticos , Animales , Bacillus subtilis/genética , Porcinos , Alimentación Animal , Heces/microbiología , Aumento de Peso , ARN Ribosómico 16S/genéticaRESUMEN
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.
Asunto(s)
Bacillus subtilis , Raíces de Plantas , Raíces de Plantas/microbiología , Bacillus subtilis/genética , Bacillus subtilis/crecimiento & desarrollo , Bacillus subtilis/fisiología , Evolución Molecular Dirigida , Plantas/microbiología , Bacillus/genética , Bacillus/fisiología , Bacillus/clasificación , Bacillus/crecimiento & desarrolloRESUMEN
Resveratrol is a promising functional ingredient applied in food products. However, low bioavailability and poor water solubility, which can be improved by glycosylation, hinder its application. A uridine diphosphate-dependent glycosyltransferase (UGT) from Bacillus subtilis 168 (named UGTBS) presents potential application for resveratrol glycosylation; nonetheless, imprecise regioselectivity renders the synthesis of resveratrol-3-O-ß-D-glucoside (polydatin) difficult. Therefore, molecular evolution was applied to UGTBS. A triple mutant Y14I/I62G/M315W was developed for 3-OH glycosylation of resveratrol and polydatin accounted for 91% of the total product. Kinetic determination and molecular docking indicated that the enhancement of hydrogen bond interaction and altered conformation of the binding pocket increases the enzyme's affinity for the 3-OH group, stabilizing the enzyme-substrate intermediate and promoting polydatin formation. Furthermore, a fed-batch cascade reaction by periodic addition of resveratrol was conducted and nearly 20 mM polydatin was obtained. The mutant Y14I/I62G/M315W can be used for polydatin manufacture.
Asunto(s)
Bacillus subtilis , Glucósidos , Glicosiltransferasas , Simulación del Acoplamiento Molecular , Estilbenos , Glucósidos/química , Glucósidos/metabolismo , Estilbenos/química , Estilbenos/metabolismo , Glicosiltransferasas/genética , Glicosiltransferasas/química , Glicosiltransferasas/metabolismo , Bacillus subtilis/enzimología , Bacillus subtilis/genética , Bacillus subtilis/química , Cinética , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Glicosilación , Resveratrol/química , Resveratrol/metabolismo , Especificidad por Sustrato , Ingeniería de ProteínasRESUMEN
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.
Asunto(s)
Bacillus subtilis , Proteínas Bacterianas , Polisacárido Liasas , Regiones Promotoras Genéticas , Señales de Clasificación de Proteína , Bacillus subtilis/genética , Bacillus subtilis/enzimología , Bacillus subtilis/metabolismo , Polisacárido Liasas/genética , Polisacárido Liasas/metabolismo , Polisacárido Liasas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Señales de Clasificación de Proteína/genética , Vibrio/genética , Vibrio/enzimología , Vectores Genéticos/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/química , Iniciación de la Cadena Peptídica TraduccionalRESUMEN
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.
Asunto(s)
Procesamiento Postranscripcional del ARN , ARN de Transferencia , Temperatura , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Seudouridina/metabolismoRESUMEN
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.
Asunto(s)
Bacillus subtilis , Replicación del ADN , Bacillus subtilis/genética , Bacillus subtilis/enzimología , Ribonucleasas/metabolismo , Ribonucleasas/genética , Ribonucleasa H/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Rayos UltravioletaRESUMEN
RNase Y is a key endoribonuclease that regulates global mRNA turnover and processing in Bacillus subtilis and likely many other bacteria. This enzyme is anchored to the cell membrane, creating a pseudo-compartmentalization that aligns with its role in initiating the decay of mRNAs primarily translated at the cell periphery. However, the reasons behind and the consequences of RNase Y's membrane attachment remain largely unknown. In our study, we examined a strain expressing wild-type levels of a cytoplasmic form of RNase Y from its chromosomal locus. This strain exhibits a slow-growth phenotype, similar to that of an RNase Y null mutant. Genome-wide data reveal a significant impact on the expression of hundreds of genes. While certain RNA substrates clearly depend on RNase Y's membrane attachment, others do not. We observed no correlation between mRNA stabilization in the mutant strains and the cellular location or function of the encoded proteins. Interestingly, the Y-complex, a specificity factor for RNase Y, also appears also recognize the cytoplasmic form of the enzyme, restoring wild-type levels of the corresponding transcripts. We propose that membrane attachment of RNase Y is crucial for its functional interaction with many coding and non-coding RNAs, limiting the cleavage of specific substrates, and potentially avoiding unfavorable competition with other ribonucleases like RNase J, which shares a similar evolutionarily conserved cleavage specificity.
Asunto(s)
Bacillus subtilis , Proteínas Bacterianas , Membrana Celular , Regulación Bacteriana de la Expresión Génica , Bacillus subtilis/genética , Bacillus subtilis/enzimología , Bacillus subtilis/metabolismo , Membrana Celular/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Estabilidad del ARN , Endorribonucleasas/metabolismo , Endorribonucleasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
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.
Asunto(s)
Bacillus subtilis , Genoma Bacteriano , Bacillus subtilis/genética , Filogenia , Variación Genética , Composición de Base , Genómica , Cromosomas Bacterianos/genética , Análisis de Secuencia de ADNRESUMEN
Cellular processes require precise and specific gene regulation, in which continuous mRNA degradation is a major element. The mRNA degradation mechanisms should be able to degrade a wide range of different RNA substrates with high efficiency, but should at the same time be limited, to avoid killing the cell by elimination of all cellular RNA. RNase Y is a major endoribonuclease found in most Firmicutes, including Bacillus subtilis and Staphylococcus aureus. However, the molecular interactions that direct RNase Y to cleave the correct RNA molecules at the correct position remain unknown. In this work we have identified transcripts that are homologs in S. aureus and B. subtilis, and are RNase Y targets in both bacteria. Two such transcript pairs were used as models to show a functional overlap between the S. aureus and the B. subtilis RNase Y, which highlighted the importance of the nucleotide sequence of the RNA molecule itself in the RNase Y targeting process. Cleavage efficiency is driven by the primary nucleotide sequence immediately downstream of the cleavage site and base-pairing in a secondary structure a few nucleotides downstream. Cleavage positioning is roughly localised by the downstream secondary structure and fine-tuned by the nucleotide immediately upstream of the cleavage. The identified elements were sufficient for RNase Y-dependent cleavage, since the sequence elements from one of the model transcripts were able to convert an exogenous non-target transcript into a target for RNase Y.
Asunto(s)
Bacillus subtilis , Regulación Bacteriana de la Expresión Génica , División del ARN , Estabilidad del ARN , ARN Bacteriano , Staphylococcus aureus , Staphylococcus aureus/genética , Staphylococcus aureus/enzimología , Bacillus subtilis/genética , Bacillus subtilis/enzimología , Bacillus subtilis/metabolismo , ARN Bacteriano/metabolismo , ARN Bacteriano/genética , Estabilidad del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Endorribonucleasas/metabolismo , Endorribonucleasas/genética , Conformación de Ácido Nucleico , Secuencia de BasesRESUMEN
BACKGROUND: To effectively introduce plasmids into Bacillus species and conduct genetic manipulations in Bacillus chassis strains, it is essential to optimize transformation methods. These methods aim to extend the period of competence and enhance the permeability of the cell membrane to facilitate the entry of exogenous DNA. Although various strategies have been explored, few studies have delved into identifying metabolites and pathways associated with enhanced competence. Additionally, derivative Bacillus strains with non-functional restriction-modification systems have demonstrated superior efficiency in transforming exogenous DNA, lacking more explorations in the regulation conducted by the restriction-modification system to transformation process. RESULTS: Transcriptomic comparisons were performed to discover the competence forming mechanism and the regulation pathway conducted by the BsuMI methylation modification group in Bacillus. subtilis 168 under the Spizizen transformation condition, which were speculated to be the preferential selection of carbon sources by the cells and the preference for specific metabolic pathway when utilizing the carbon source. The cells were found to utilize the glycolysis pathway to exploit environmental glucose while reducing the demand for other phosphorylated precursors in this pathway. The weakening of these ATP-substrate competitive metabolic pathways allowed more ATP substrates to be distributed into the auto-phosphorylation of the signal transduction factor ComP during competence formation, thereby increasing the expression level of the key regulatory protein ComK. The expression of ComK upregulated the expression of the negative regulator SacX of starch and sucrose in host cells, reinforcing the preference for glucose as the primary carbon source. The methylation modification group of the primary protein BsuMI in the restriction-modification system was associated with the functional modification of key enzymes in the oxidative phosphorylation pathway. The absence of the BsuMI methylation modification group resulted in a decrease in the expression of subunits of cytochrome oxidase, leading to a weakening of the oxidative phosphorylation pathway, which promoted the glycolytic rate of cells and subsequently improved the distribution of ATP molecules into competence formation. A genetic transformation platform for wild-type Bacillus strains was successfully established based on the constructed strain B. subtilis 168-R-M- without its native restriction-modification system. With this platform, high plasmids transformation efficiencies were achieved with a remarkable 63-fold improvement compared to the control group and an increased universality in Bacillus species was also obtained. CONCLUSIONS: The enhanced competence formation mechanism and the regulation pathway conducted by the functional protein BsuMI of the restriction-modification system were concluded, providing a reference for further investigation. An effective transformation platform was established to overcome the obstacles in DNA transformations in wild-type Bacillus strains.
Asunto(s)
Bacillus subtilis , Proteínas Bacterianas , Transformación Bacteriana , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Plásmidos/genética , Plásmidos/metabolismo , Competencia de la Transformación por ADNRESUMEN
Bacterial interactions are vital for adapting to changing environments, with quorum sensing (QS) systems playing a central role in coordinating behaviors through small signaling molecules. The RRNPPA family is the prevalent QS systems in Bacillota and mediating communication through secreted oligopeptides, which are processed into active pheromones by extracellular proteases. Notably, in several cases the propeptides show the presence of multiple putative pheromones within their sequences, which has been proposed as a mechanism to diversify peptide-receptor specificity and potentially facilitate new functions. However, neither the processes governing the maturation of propeptides containing multiple pheromones, nor their functional significance has been evaluated. Here, using 2 Rap systems from bacteriophages infecting Bacillus subtilis that exhibit different types of pheromone duplication in their propeptides, we investigate the maturation process and the molecular and functional activities of the produced pheromones. Our results reveal that distinct maturation processes generate multiple mature pheromones, which bind to receptors with varying affinities but produce identical structural and biological responses. These findings add additional layers in the complexity of QS communication and regulation, opening new possibilities for microbial social behaviors, highlighting the intricate nature of bacterial interactions and adaptation.
Asunto(s)
Bacillus subtilis , Feromonas , Proteolisis , Percepción de Quorum , Feromonas/metabolismo , Bacillus subtilis/metabolismo , Bacillus subtilis/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Bacteriófagos/metabolismo , Bacteriófagos/genética , Secuencia de AminoácidosRESUMEN
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.
Asunto(s)
Isomerasas Aldosa-Cetosa , Bacillus subtilis , Hexosas , Limosilactobacillus fermentum , Proteínas Recombinantes , Bacillus subtilis/genética , Bacillus subtilis/enzimología , Isomerasas Aldosa-Cetosa/genética , Isomerasas Aldosa-Cetosa/metabolismo , Hexosas/metabolismo , Hexosas/biosíntesis , Limosilactobacillus fermentum/enzimología , Limosilactobacillus fermentum/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Concentración de Iones de Hidrógeno , Temperatura , Clonación Molecular/métodos , Estabilidad de Enzimas , Galactosa/metabolismo , CinéticaRESUMEN
Orotic acid is widely used in healthcare and cosmetic industries. However, orotic acid-producing microorganisms are auxotrophic, which results in inefficient microbial production. Herein, a plasmid-free, uninduced, non-auxotrophic orotic acid hyperproducer was constructed from Escherichia coli W3110. Initially, the orotic acid degradation pathway was blocked and the carbamoyl phosphate supply was enriched. Subsequently, pyr operon from Bacillus subtilis F126 was heterologously expressed and precursors' supply was optimized. Thereafter, pyrE was dynamically regulated to reconstruct the non-auxotrophic pathway. Employing fed-batch cultivation, orotic acid titer, yield, and productivity of strain Ora21 reached 182.5 g/L, 0.58 g/g, and 3.80 g/L/h, respectively, the highest levels reported so far. Finally, a novel "Chaos to Order Cycles (COC)" fermentation was developed, which effectively increased the yield to 0.63 g/g. This research is a remarkable achievement in orotic acid production by microbial fermentation and has vast potential for industrial applications.
Asunto(s)
Bacillus subtilis , Escherichia coli , Fermentación , Ácido Orótico , Ácido Orótico/análogos & derivados , Ácido Orótico/metabolismo , Escherichia coli/metabolismo , Escherichia coli/genética , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Ingeniería Metabólica/métodos , Técnicas de Cultivo Celular por LotesRESUMEN
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.
Asunto(s)
Fosfatos de Dinucleósidos , Proteostasis , Estabilidad del ARN , Fosfatos de Dinucleósidos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Bacterias/metabolismo , Bacterias/genética , Estrés FisiológicoRESUMEN
Nucleosides, nucleotides, and their analogues are an important class of molecules that are used as substrates in research of enzymes and nucleic acid, or as antiviral and antineoplastic agents. Nucleoside phosphorylation is usually achieved with chemical methods; however, enzymatic phosphorylation is a viable alternative. Here, we present a chemoenzymatic synthesis of modified cytidine monophosphates, where a chemical synthesis of novel N4-modified cytidines is followed by an enzymatic phosphorylation of the nucleosides by nucleoside kinases. To enlarge the substrate scope, multiple mutant variants of Drosophila melanogaster deoxynucleoside kinase (DmdNK) (EC:2.7.1.145) and Bacillus subtilis deoxycytidine kinase (BsdCK) (EC:2.7.1.74) have been created and tested. It has been determined that certain point mutations in the active sites of the kinases alter their substrate specificities noticeably and allow phosphorylation of compounds that had been otherwise not phosphorylated by the wild-type DmdNK or BsdCK.
Asunto(s)
Citidina Monofosfato , Drosophila melanogaster , Animales , Fosforilación , Especificidad por Sustrato , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Citidina Monofosfato/análogos & derivados , Citidina Monofosfato/metabolismo , Citidina Monofosfato/química , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Fosfotransferasas/química , Bacillus subtilis/enzimología , Bacillus subtilis/genética , Mutación , Desoxicitidina Quinasa/genética , Desoxicitidina Quinasa/metabolismo , Desoxicitidina Quinasa/químicaRESUMEN
BACKGROUND: D-Allulose is one of the most well-known rare sugars widely used in food, cosmetics, and pharmaceutical industries. The most popular method for D-allulose production is the conversion from D-fructose catalyzed by D-allulose 3-epimerase (DAEase). To address the general problem of low catalytic efficiency and poor thermostability of wild-type DAEase, D-allulose biosensor was adopted in this study to develop a convenient and efficient method for high-throughput screening of DAEase variants. RESULTS: The catalytic activity and thermostability of DAEase from Caballeronia insecticola were simultaneously improved by semi-rational molecular modification. Compared with the wild-type enzyme, DAEaseS37N/F157Y variant exhibited 14.7% improvement in the catalytic activity and the half-time value (t1/2) at 65°C increased from 1.60 to 27.56 h by 17.23-fold. To our delight, the conversion rate of D-allulose was 33.6% from 500-g L-1 D-fructose in 1 h by Bacillus subtilis WB800 whole cells expressing this DAEase variant. Furthermore, the practicability of cell immobilization was evaluated and more than 80% relative activity of the immobilized cells was maintained from the second to seventh cycle. CONCLUSION: All these results indicated that the DAEaseS37N/F157Y variant would be a potential candidate for the industrial production of D-allulose.