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1.
Food Chem ; 399: 133993, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36029678

RESUMO

At present, uncovering how to preventandcontrol hyperuricemia has become an important public health issue. Fermented traditionalChinesemedicine has exhibited promising applications in the clinical management of hyperuricemia. In this study, we generated a hyperuricemic mouse model to explore the potent therapeutic ability of Bacillus subtilis-fermented Astragalus membranaceus (BFA) on this condition by multi-omics analysis. We found that the serum uric acid level was decreased in hyperuricemic mice after BFA treatment. BFA effectively attenuated renal inflammation and regulated the expression of urate transporters. Additionally, we found that BFA could increase the abundances of butyrate-producing bacteria, including Butyricimonas synergistica, Odoribacter splanchnicus, and Collinsella tanakaei, and probiotics, including Lactobacillus intestinalis and Bacillus mycoides, in hyperuricemic mice. Therefore, we believe that BFA has the potential to become a novel safe and valid functional food for addressing hyperuricemia.


Assuntos
Microbioma Gastrointestinal , Hiperuricemia , Animais , Astragalus propinquus/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Hiperuricemia/tratamento farmacológico , Hiperuricemia/genética , Rim , Camundongos , Ácido Úrico/metabolismo
2.
Braz. j. biol ; 83: e250550, 2023. tab, graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1345536

RESUMO

Abstract Vanillin is the major component which is responsible for flavor and aroma of vanilla extract and is produced by 3 ways: natural extraction from vanilla plant, chemical synthesis and from microbial transformation. Current research was aimed to study bacterial production of vanillin from native natural sources including sewage and soil from industrial areas. The main objective was vanillin bio-production by isolating bacteria from these native sources. Also to adapt methodologies to improve vanillin production by optimized fermentation media and growth conditions. 47 soil and 13 sewage samples were collected from different industrial regions of Lahore, Gujranwala, Faisalabad and Kasur. 67.7% bacterial isolates produced vanillin and 32.3% were non-producers. From these 279 producers, 4 bacterial isolates selected as significant producers were; A3, A4, A7 and A10. These isolates were identified by ribotyping as A3 Pseudomonas fluorescence (KF408302), A4 Enterococcus faecium (KT356807), A7 Alcaligenes faecalis (MW422815) and A10 Bacillus subtilis (KT962919). Vanillin producers were further tested for improved production of vanillin and were grown in different fermentation media under optimized growth conditions for enhanced production of vanillin. The fermentation media (FM) were; clove oil based, rice bran waste (residues oil) based, wheat bran based and modified isoeugenol based. In FM5, FM21, FM22, FM23, FM24, FM30, FM31, FM32, FM34, FM35, FM36, and FM37, the selected 4 bacterial strains produced significant amounts of vanillin. A10 B. subtilis produced maximum amount of vanillin. This strain produced 17.3 g/L vanillin in FM36. Cost of this fermentation medium 36 was 131.5 rupees/L. This fermentation medium was modified isoeugenol based medium with 1% of isoeugenol and 2.5 g/L soybean meal. ech gene was amplified in A3 P. fluorescence using ech specific primers. As vanillin use as flavor has increased tremendously, the bioproduction of vanillin must be focused.


Resumo A vanilina é o principal componente responsável pelo sabor e aroma do extrato de baunilha e é produzida de três formas: extração natural da planta da baunilha, síntese química e transformação microbiana. A pesquisa atual teve como objetivo estudar a produção bacteriana de vanilina a partir de fontes naturais nativas, incluindo esgoto e solo de áreas industriais. O objetivo principal era a bioprodução de vanilina por meio do isolamento de bactérias dessas fontes nativas. Também para adaptar metodologias para melhorar a produção de vanilina por meio de fermentação otimizada e condições de crescimento. Foram coletadas 47 amostras de solo e 13 de esgoto de diferentes regiões industriais de Lahore, Gujranwala, Faisalabad e Kasur; 67,7% dos isolados bacterianos produziram vanilina e 32,3% eram não produtores. Desses 279 produtores, 4 isolados bacterianos selecionados como produtores significativos foram: A3, A4, A7 e A10. Esses isolados foram identificados por ribotipagem como fluorescência A3 Pseudomonas (KF408302), A4 Enterococcus faecium (KT356807), A7 Alcaligenes faecalis (MW422815) e A10 Bacillus subtilis (KT962919). Os produtores de vanilina foram posteriormente testados para produção aprimorada de vanilina e foram cultivados em diferentes meios de fermentação sob condições de crescimento otimizadas para produção aprimorada de vanilina. Os meios de fermentação (FM) foram: à base de óleo de cravo, à base de resíduos de farelo de arroz (resíduos de óleo), à base de farelo de trigo e à base de isoeugenol modificado. Em FM5, FM21, FM22, FM23, FM24, FM30, FM31, FM32, FM34, FM35, FM36 e FM37, as 4 cepas bacterianas selecionadas produziram quantidades significativas de vanilina. A10 B. subtilis produziu quantidade máxima de vanilina. Essa cepa produziu 17,3 g / L de vanilina em FM36. O custo desse meio de fermentação 36 foi de 131,5 rúpias / L. Esse meio de fermentação foi um meio à base de isoeugenol modificado com 1% de isoeugenol e 2,5 g / L de farelo de soja. O gene ech foi amplificado em A3 P. fluorescence usando primers específicos para ech. Como o uso da vanilina como sabor aumentou tremendamente, a bioprodução da vanilina deve ser focada.


Assuntos
Benzaldeídos/metabolismo , Aromatizantes/metabolismo , Bacillus subtilis/metabolismo , Microbiologia Industrial , Pseudomonas fluorescens/metabolismo , Enterococcus faecium/metabolismo , Meios de Cultura , Alcaligenes faecalis/metabolismo , Fermentação
3.
Nat Commun ; 13(1): 7082, 2022 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-36400765

RESUMO

Many bacteria in nature exist in multicellular communities termed biofilms, where cells are embedded in an extracellular matrix that provides rigidity to the biofilm and protects cells from chemical and mechanical stresses. In the Gram-positive model bacterium Bacillus subtilis, TasA is the major protein component of the biofilm matrix, where it has been reported to form functional amyloid fibres contributing to biofilm structure and stability. Here, we present electron cryomicroscopy structures of TasA fibres, which show that, rather than forming amyloid fibrils, TasA monomers assemble into fibres through donor-strand exchange, with each subunit donating a ß-strand to complete the fold of the next subunit along the fibre. Combining electron cryotomography, atomic force microscopy, and mutational studies, we show how TasA fibres congregate in three dimensions to form abundant fibre bundles that are essential for B. subtilis biofilm formation. Our study explains the previously observed biochemical properties of TasA and shows how a bacterial extracellular globular protein can assemble from monomers into ß-sheet-rich fibres, and how such fibres assemble into bundles in biofilms.


Assuntos
Bacillus subtilis , Aranhas , Animais , Bacillus subtilis/metabolismo , Biofilmes , Proteínas de Bactérias/metabolismo , Amiloide/metabolismo , Aranhas/metabolismo
4.
Curr Microbiol ; 79(12): 398, 2022 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-36352269

RESUMO

A bacterium strain isolated from freshwater sediment of San Pablo river of Santiago de Cuba, Cuba was identified as a Bacillus sp. by Matrix-Assisted Laser Desorption/Ionization Time Of Flight Mass Spectrometry. A 16S rRNA gene analysis showed that the isolate A3 belongs to the operational group Bacillus amyloliquefaciens, while the phylogenetic analysis of the gyrA gene sequence grouped it within B. amyloliquefaciens subsp. plantarum cluster, referred now as Bacillus velezensis. In vitro antibacterial studies demonstrated the capacity of the isolate A3 to produce bioactive metabolites against Bacillus subtilis ATCC 11,778, Bacillus cereus ATCC 6633, and Staphylococcus aureus ATCC 25,923 by cross-streak, overlay, and microdilution methods. The strain also showed a high potential against the multidrug-resistant Staphylococcus aureus ATCC 700,699, ATCC 29,213, and ATCC 6538. At pH 8 and 96 h in the medium 2 of A3 culture conditions, the produced metabolites with antibacterial potential were enhanced. Some alterations in the morphology of the phytopathogens Aspergillus niger ATCC 9642, Alternaria alternata CECT 2662, and Fusarium solani CCEBI 3094 were induced by the cell-free supernatant of B. velezensis A3. A preliminary study of the nature of the bioactive compounds produced by the strain A3 showed the presence of both lipids and peptides in the culture. Those results highlight B. velezensis A3 as a promissory bacterium capable to produce bioactive metabolites with antibacterial and antifungal properties against pathogens.


Assuntos
Bacillus , Staphylococcus aureus Resistente à Meticilina , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Filogenia , Bacillus/genética , Bacillus/metabolismo , Fungos/genética , Bacillus subtilis/metabolismo , Antibacterianos/química , Água Doce
5.
World J Microbiol Biotechnol ; 39(1): 12, 2022 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-36372802

RESUMO

Transcriptional factors are well studied in bacteria for their global interactions and the effects they produce at the phenotypic level. Particularly, Bacillus subtilis has been widely employed as a model Gram-positive microorganism used to characterize these network interactions. Bacillus species are currently used as efficient commercial microbial platforms to produce diverse metabolites such as extracellular enzymes, antibiotics, surfactants, industrial chemicals, heterologous proteins, among others. However, the pleiotropic effects caused by the genetic modification of specific genes that codify for global regulators (transcription factors) have not been implicated commonly from a bioprocess point of view. Recently, these strategies have attracted the attention in Bacillus species because they can have an application to increase production efficiency of certain commercial interest metabolites. In this review, we update the recent advances that involve this trend in the use of genetic engineering (mutations, deletion, or overexpression) performed to global regulators such as Spo0A, CcpA, CodY and AbrB, which can provide an advantage for the development or improvement of bioprocesses that involve Bacillus species as production platforms. Genetic networks, regulation pathways and their relationship to the development of growth stages are also discussed to correlate the interactions that occur between these regulators, which are important to consider for application in the improvement of commercial-interest metabolites. Reported yields from these products currently produced mostly under laboratory conditions and, in a lesser extent at bioreactor level, are also discussed to give valuable perspectives about their potential use and developmental level directed to process optimization at large-scale.


Assuntos
Bacillus , Fatores de Transcrição , Fatores de Transcrição/genética , Bacillus/genética , Bacillus/metabolismo , Bacillus subtilis/metabolismo , Engenharia Genética , Redes Reguladoras de Genes , Proteínas de Bactérias/metabolismo , Transcrição Genética
6.
Int J Mol Sci ; 23(19)2022 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-36232729

RESUMO

ATP-dependent Lon proteases are key participants in the quality control system that supports the homeostasis of the cellular proteome. Based on their unique structural and biochemical properties, Lon proteases have been assigned in the MEROPS database to three subfamilies (A, B, and C). All Lons are single-chain, multidomain proteins containing an ATPase and protease domains, with different additional elements present in each subfamily. LonA and LonC proteases are soluble cytoplasmic enzymes, whereas LonBs are membrane-bound. Based on an analysis of the available sequences of Lon proteases, we identified a number of enzymes currently assigned to the LonB subfamily that, although presumably membrane-bound, include structural features more similar to their counterparts in the LonA subfamily. This observation was confirmed by the crystal structure of the proteolytic domain of the enzyme previously assigned as Bacillus subtilis LonB, combined with the modeled structure of its ATPase domain. Several structural features present in both domains differ from their counterparts in either LonA or LonB subfamilies. We thus postulate that this enzyme is the founding member of a newly identified LonBA subfamily, so far found only in the gene sequences of firmicutes.


Assuntos
Protease La , Proteases Dependentes de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Peptídeo Hidrolases/metabolismo , Protease La/genética , Protease La/metabolismo , Proteoma/metabolismo
7.
Biotechnol Adv ; 61: 108049, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36243207

RESUMO

Bio-derived materials have long been harnessed for their potential as backbones of biodegradable constructs. With increasing understanding of organismal biochemistry and molecular genetics, scientists are now able to obtain biomaterials with properties comparable to those achieved by the petroleum industry. Poly-γ-glutamic acid (γ-PGA) is an anionic pseudopolypeptide produced and secreted by several microorganisms, especially Bacillus species. γ-PGA is polymerised via the pgs intermembrane enzymatic complex expressed by many bacteria (including GRAS member - Bacillus subtilis). γ-PGA can exist as a homopolymer of L- glutamic acid or D- glutamic acid units or it can be a co-polymer comprised of D and L enantiomers. This non-toxic polymer is highly viscous, soluble, biodegradable and biocompatible. γ-PGA is also an example of versatile chiral-polymer, a characteristic that draws great attention from the industry. Increased understanding in the correlation between microbial genetics, substrate compositions, fermentation conditions and polymeric chemical characteristics have led to bioprocess optimisation to provide cost competitive, non-petroleum-based, biodegradable solutions. This review presents detailed insights into microbial synthesis of γ-PGA and summaries current understanding of the correlation between genetic makeup of γ-PGA-producing bacteria, range of culture cultivation conditions, and physicochemical properties of this incredibly versatile biopolymer. Additionally, we hope that review provides an updated overview of findings relevant to sustainable and cost-effective biosynthesis of γ-PGA, with application in medicine, pharmacy, cosmetics, food, agriculture and for bioremediation.


Assuntos
Bacillus , Ácido Glutâmico , Ácido Poliglutâmico/genética , Bacillus subtilis/metabolismo , Bacillus/genética , Fermentação , Biopolímeros
8.
Proc Natl Acad Sci U S A ; 119(41): e2204042119, 2022 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-36206370

RESUMO

SMC complexes, loaded at ParB-parS sites, are key mediators of chromosome organization in bacteria. ParA/Soj proteins interact with ParB/Spo0J in a pathway involving adenosine triphosphate (ATP)-dependent dimerization and DNA binding, facilitating chromosome segregation in bacteria. In Bacillus subtilis, ParA/Soj also regulates DNA replication initiation and along with ParB/Spo0J is involved in cell cycle changes during endospore formation. The first morphological stage in sporulation is the formation of an elongated chromosome structure called an axial filament. Here, we show that a major redistribution of SMC complexes drives axial filament formation in a process regulated by ParA/Soj. Furthermore, and unexpectedly, this regulation is dependent on monomeric forms of ParA/Soj that cannot bind DNA or hydrolyze ATP. These results reveal additional roles for ParA/Soj proteins in the regulation of SMC dynamics in bacteria and yet further complexity in the web of interactions involving chromosome replication, segregation and organization, controlled by ParAB and SMC.


Assuntos
Bacillus subtilis , Cromossomos Bacterianos , Adenosina Trifosfatases , Trifosfato de Adenosina/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Segregação de Cromossomos , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos
9.
Microbiology (Reading) ; 168(10)2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36301085

RESUMO

Replication and segregation of the genetic information is necessary for a cell to proliferate. In Bacillus subtilis, the Par system (ParA/Soj, ParB/Spo0J and parS) is required for segregation of the chromosome origin (oriC) region and for proper control of DNA replication initiation. ParB binds parS sites clustered near the origin of replication and assembles into sliding clamps that interact with ParA to drive origin segregation through a diffusion-ratchet mechanism. As part of this dynamic process, ParB stimulates ParA ATPase activity to trigger its switch from an ATP-bound dimer to an ADP-bound monomer. In addition to its conserved role in DNA segregation, ParA is also a regulator of the master DNA replication initiation protein DnaA. We hypothesized that in B. subtilis the location of the Par system proximal to oriC would be necessary for ParA to properly regulate DnaA. To test this model, we constructed a range of genetically modified strains with altered numbers and locations of parS sites, many of which perturbed chromosome origin segregation as expected. Contrary to our hypothesis, the results show that regulation of DNA replication initiation by ParA is maintained when a parS site is separated from oriC. Because a single parS site is sufficient for proper control of ParA, the results are consistent with a model where ParA is efficiently regulated by ParB sliding clamps following loading at parS.


Assuntos
Bacillus subtilis , Cromossomos Bacterianos , Bacillus subtilis/metabolismo , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Replicação do DNA/genética , Segregação de Cromossomos , Origem de Replicação/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo
10.
Proc Natl Acad Sci U S A ; 119(42): e2211178119, 2022 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-36215496

RESUMO

Intrinsically disordered regions (IDRs) can function as autoregulators of folded enzymes to which they are tethered. One example is the bacterial cell division protein FtsZ. This includes a folded core and a C-terminal tail (CTT) that encompasses a poorly conserved, disordered C-terminal linker (CTL) and a well-conserved 17-residue C-terminal peptide (CT17). Sites for GTPase activity of FtsZs are formed at the interface between GTP binding sites and T7 loops on cores of adjacent subunits within dimers. Here, we explore the basis of autoregulatory functions of the CTT in Bacillus subtilis FtsZ (Bs-FtsZ). Molecular simulations show that the CT17 of Bs-FtsZ makes statistically significant CTL-mediated contacts with the T7 loop. Statistical coupling analysis of more than 1,000 sequences from FtsZ orthologs reveals clear covariation of the T7 loop and the CT17 with most of the core domain, whereas the CTL is under independent selection. Despite this, we discover the conservation of nonrandom sequence patterns within CTLs across orthologs. To test how the nonrandom patterns of CTLs mediate CTT-core interactions and modulate FtsZ functionalities, we designed Bs-FtsZ variants by altering the patterning of oppositely charged residues within the CTL. Such alterations disrupt the core-CTT interactions, lead to anomalous assembly and inefficient GTP hydrolysis in vitro and protein degradation, aberrant assembly, and disruption of cell division in vivo. Our findings suggest that viable CTLs in FtsZs are likely to be IDRs that encompass nonrandom, functionally relevant sequence patterns that also preserve three-way covariation of the CT17, the T7 loop, and core domain.


Assuntos
Bacillus subtilis , Proteínas do Citoesqueleto , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Divisão Celular , Proteínas do Citoesqueleto/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Peptídeos/metabolismo
11.
J Agric Food Chem ; 70(43): 13935-13944, 2022 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-36278912

RESUMO

d-Allulose is an attractive rare sugar that can be used as a low-calorie sweetener with significant health benefits. To meet the increasing market demands, it is necessary to develop an efficient and extensive microbial fermentation platform for the synthesis of d-allulose. Here, we applied a comprehensive systematic engineering strategy in Bacillus subtilis WB600 by introducing d-allulose 3-epimerase (DAEase), combined with the deactivation of fruA, levDEFG, and gmuE, to balance the metabolic network for the efficient production of d-allulose. This resulting strain initially produced 3.24 g/L of d-allulose with a yield of 0.93 g of d-allulose/g d-fructose. We further screened and obtained a suitable dual promoter combination and performed fine-tuning of its spacer region. After 64 h of fed-batch fermentation, the optimized engineered B. subtilis produced d-allulose at titers of 74.2 g/L with a yield of 0.93 g/g and a conversion rate of 27.6%. This d-allulose production strain is a promising platform for the industrial production of rare sugar.


Assuntos
Bacillus subtilis , Frutose , Bacillus subtilis/metabolismo , Frutose/metabolismo , Racemases e Epimerases/metabolismo , Ciclo do Carbono
12.
Metab Eng ; 74: 108-120, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36257594

RESUMO

Lichenysin, producted by Bacillus licheniformis, is an important cyclic lipopeptide biosurfactant, which has potential applications in oil exploitation, drug development, biological control of agriculture and bioremediation. While studies are lacking on metabolism regulation of lichenysin biosynthesis, which limits metabolic engineering and large-scale production of lichenysin. In this study, the yield of lichenysin was improved obviously by 13.6 folds to 2.18 ± 0.03 g/L in degU deletion strain (WX02△degU) compared with the wild-type strain (WX02) and completely inhibited in degU overexpressed strain (WX02/pHY-degU). We further proved that DegU, a transcription factor plays a significant role in multicellular behavior, is a key negative regulator of lichenysin synthesis lchA operon. But interestingly, lichenysin yield was still inhibited by overexpressing DegU in the promoter-substituted strain (WX02-PP43lch), in which promoter of lchA operon cannot be controlled by DegU. Thus, through 13C-metabolic flux analysis, we found that deletion of degU also enhanced glucose uptake, branched chain amino acid synthesis, and fatty acid synthesis, while decrease acetoin synthesis, which is beneficial for the supply of lichenysin precursors. Further experiments demonstrate that DegU regulates these pathways by binding to the promoter regions of related genes. Overall, we systematically investigated the multi-pathway regulation network mediated by DegU on lichenysin biosynthesis, which not only contributes to the further metabolic engineering for lichenysin high-production, but sheds light on studies of transcription factor regulation.


Assuntos
Bacillus licheniformis , Bacillus licheniformis/genética , Bacillus licheniformis/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Lipoproteínas/química , Lipoproteínas/genética , Lipoproteínas/metabolismo , Anilidas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacillus subtilis/metabolismo
13.
ACS Infect Dis ; 8(11): 2253-2258, 2022 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-36268971

RESUMO

Metabolic profiling of the extracts from a library of actinobacteria led to the identification of a novel polyketide, demurilactone A, produced by Streptomyces strain DEM21308. The structure of the compound was assigned based on a detailed investigation of 1D/2D NMR spectra and HR-MS. Whole genome DNA sequencing, followed by bioinformatics analysis and insertional mutagenesis, identified type I polyketide synthases encoded by the dml gene cluster to direct the biosynthesis of this polyene macrolide. While the number of modules is consistent with the carbon backbone of the assigned structure, some discrepancies were identified in the domain organization of five modules. Close investigation of the amino acid sequences identified several mutations in the conserved motifs of nonfunctional domains. Furthermore, the absolute configuration of hydroxy-bearing stereocenters was proposed based on analyses of the ketoreductase domains. Remarkably, although demurilactone A has little detectable activity against normal-walled bacteria, it specifically inhibits the growth of cell wall-deficient "L-form" Bacillus subtilis at a minimum inhibitory concentration value of 16 µg/mL. Time-lapse microscopy analyses revealed that demurilactone affects membrane dynamics, probably by reducing membrane fluidity. This compound could be a powerful reagent for studying long-standing questions about the involvement of L-forms in recurrent infection.


Assuntos
Bacillus subtilis , Streptomyces , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Inibidores do Crescimento/metabolismo , Policetídeo Sintases/genética , Streptomyces/genética , Streptomyces/química , Macrolídeos
14.
Nat Microbiol ; 7(11): 1918-1931, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36192538

RESUMO

The transcriptome-wide contributions of Rho-dependent and intrinsic (Rho-independent) transcription termination mechanisms in bacteria are unclear. By sequencing released transcripts in a wild-type strain and strains containing deficiencies in NusA, NusG and/or Rho (10 strains), we produced an atlas of terminators for the model Gram-positive bacterium Bacillus subtilis. We found that NusA and NusG stimulate 77% and 19% of all intrinsic terminators, respectively, and that both proteins participate in Rho-dependent termination. We also show that Rho stimulates termination at 10% of the intrinsic terminators in vivo. We recapitulated Rho-stimulated intrinsic termination at 5 terminators in vitro and found that Rho requires the KOW domain of NusG to stimulate this process at one of these terminators. Computational analyses of our atlas using RNAstructure, MEME suite and DiffLogo, combined with in vitro transcription experiments, revealed that Rho stimulates intrinsic terminators with weak hairpins and/or U-rich tracts by remodelling the RNA upstream of the intrinsic terminator to prevent the formation of RNA structures that could otherwise compete with the terminator hairpin. We also identified 56 putative examples of 'hybrid Rho-dependent termination', wherein classical Rho-dependent termination occurs after readthrough of a Rho-stimulated intrinsic terminator.


Assuntos
Bacillus subtilis , Transcrição Genética , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , RNA/metabolismo
15.
Genes Dev ; 36(17-18): 970-984, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36265902

RESUMO

Intrinsically disordered protein regions (IDRs) have been implicated in diverse nuclear and cytoplasmic functions in eukaryotes, but their roles in bacteria are less clear. Here, we report that extracytoplasmic IDRs in Bacillus subtilis are required for cell wall homeostasis. The B. subtilis σI transcription factor is activated in response to envelope stress through regulated intramembrane proteolysis (RIP) of its membrane-anchored anti-σ factor, RsgI. Unlike canonical RIP pathways, we show that ectodomain (site-1) cleavage of RsgI is constitutive, but the two cleavage products remain stably associated, preventing intramembrane (site-2) proteolysis. The regulated step in this pathway is their dissociation, which is triggered by impaired cell wall synthesis and requires RsgI's extracytoplasmic IDR. Intriguingly, the major peptidoglycan polymerase PBP1 also contains an extracytoplasmic IDR, and we show that this region is important for its function. Disparate IDRs can replace the native IDRs on both RsgI and PBP1, arguing that these unstructured regions function similarly. Our data support a model in which the RsgI-σI signaling system and PBP1 represent complementary pathways to repair gaps in the PG meshwork. The IDR on RsgI senses these gaps and activates σI, while the IDR on PBP1 directs the synthase to these sites to fortify them.


Assuntos
Bacillus subtilis , Proteínas Intrinsicamente Desordenadas , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Homeostase
16.
Int J Mol Sci ; 23(18)2022 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-36142543

RESUMO

The azo dye orange II is used extensively in the textile sector for coloring fabrics. High concentrations of it are released into aqueous environments through textile effluents. Therefore, its removal from textile wastewater and effluents is necessary. Herein, initially, we tested 11 bacterial strains for their capabilities in the degradation of orange II dye. It was revealed in the preliminary data that B. subtilis can more potently degrade the selected dye, which was thus used in the subsequent experiments. To achieve maximum decolorization, the experimental conditions were optimized whereby maximum degradation was achieved at: a 25 ppm dye concentration, pH 7, a temperature of 35 °C, a 1000 mg/L concentration of glucose, a 1000 mg/L urea concentration, a 666.66 mg/L NaCl concentration, an incubation period of 3 days, and with hydroquinone as a redox mediator at a concentration of 66.66 mg/L. The effects of the interaction of the operational factors were further confirmed using response surface methodology, which revealed that at optimum conditions of pH 6.45, a dye concentration of 17.07 mg/L, and an incubation time of 9.96 h at 45.38 °C, the maximum degradation of orange II can be obtained at a desirability coefficient of 1, estimated using the central composite design (CCD). To understand the underlying principles of degradation of the metabolites in the aliquot mixture at the optimized condition, the study steps were extracted and analyzed using GC-MS(Gas Chromatography Mass Spectrometry), FTIR(Fourier Transform Infrared Spectroscopy), 1H and carbon 13 NMR(Nuclear Magnetic Resonance Spectroscopy). The GC-MS pattern revealed that the original dye was degraded into o-xylene and naphthalene. Naphthalene was even obtained in a pure state through silica gel column isolation and confirmed using 1H and 13C NMR spectroscopic analysis. Phytotoxicity tests on Vigna radiata were also conducted and the results confirmed that the dye metabolites were less toxic than the parent dye. These results emphasize that B. subtilis should be used as a potential strain for the bioremediation of textile effluents containing orange II and other toxic azo dyes.


Assuntos
Bacillus subtilis , Água Carbonatada , Compostos Azo/química , Compostos Azo/toxicidade , Bacillus subtilis/metabolismo , Benzenossulfonatos , Biodegradação Ambiental , Carbono/análise , Água Carbonatada/análise , Corantes/química , Glucose , Hidroquinonas , Naftalenos/análise , Sílica Gel , Cloreto de Sódio , Vapor/análise , Têxteis , Ureia , Águas Residuárias/química , Água/análise
17.
Front Cell Infect Microbiol ; 12: 983757, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36159636

RESUMO

Magnaporthe oryzae is one of the most destructive crop pathogens in the world, causing huge losses in rice harvest every year. Bacillus subtilis is a potential biocontrol agent that has been explored in many crop systems because it is a potent producer of bioactive compounds. However, the mechanisms by which these agents control rice blasts are not fully understood. We show that B. subtilis KLBMPGC81 (KC81) and its supernatant (SUP) have high antimicrobial activity against M. oryzae strain Guy11. To better exploit KC81 as a biocontrol agent, the mechanism by which KC81 suppresses rice blast pathogens was investigated. This study shows that KC81 SUP is effective in controlling rice blast disease. The SUP has a significant effect on suppressing the growth of M. oryzae and appressorium-mediated plant infection. KC81 SUP compromises cell wall integrity, microtubules and actin cytoskeleton, mitosis, and autophagy, all of which are required for M. oryzae growth, appressorium development, and host infection. We further show that the SUP reduces the activity of the cyclin-dependent kinase Cdc2 by enhancing the phosphorylation of Cdc2 Tyr 15, thereby impairing mitosis in M. oryzae cells. SUP induces the cell wall sensor MoWsc1 to activate the cell wall integrity pathway and Mps1 and Pmk1 mitogen-activated protein kinases. Taken together, our findings reveal that KC81 is an effective fungicide that suppresses M. oryzae growth, appressorium formation, and host infection by abnormally activating the cell wall integrity pathway, disrupting the cytoskeleton, mitosis, and autophagy.


Assuntos
Fenômenos Biológicos , Fungicidas Industriais , Magnaporthe , Oryza , Ascomicetos , Bacillus subtilis/metabolismo , Parede Celular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Doenças das Plantas/prevenção & controle , Transdução de Sinais
18.
J Agric Food Chem ; 70(38): 12128-12134, 2022 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-36099523

RESUMO

As a natural sweetener with low calories and various physiological activities, d-allulose has drawn worldwide attention. Currently, d-allulose 3-epimerase (DAEase) is mainly used to catalyze the epimerization of d-fructose to d-allulose. Therefore, it is quite necessary to enhance the food-grade expression of DAEase to meet the surging market demand for d-allulose. In this study, initially, the promising variant H207L/D281G/C289R of Clostridium cellulolyticum H10 DAEase (CcDAEase) was generated by protein engineering, the specific activity and the T1/2 of which were 2.24-fold and 13.45-fold those of the CcDAEase wild type at 60 °C, respectively. After that, PamyE was determined as the optimal promoter for the recombinant expression of CcDAEase in Bacillus subtilis, and catabolite-responsive element (CRE) box engineering was further performed to eliminate the carbon catabolite repression (CCR) effect. Lastly, high-density fermentation was carried out and the final activity peaked at 4971.5 U mL-1, which is the highest expression level and could effectively promote the industrial production of DAEase. This research provides a theoretical basis and technical support for the molecular modification of DAEase and its efficient fermentation preparation.


Assuntos
Bacillus subtilis , Racemases e Epimerases , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Frutose/metabolismo , Concentração de Íons de Hidrogênio , Engenharia de Proteínas , Racemases e Epimerases/metabolismo , Edulcorantes/metabolismo
19.
Appl Environ Microbiol ; 88(18): e0087122, 2022 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-36094206

RESUMO

Microorganisms in nature form multicellular groups called biofilms. In biofilms, bacteria embedded in the extracellular matrix (ECM) interact intensely due to their proximity. Most studies have investigated genetically homogeneous biofilms, leaving a gap in knowledge on genetically heterogeneous biofilms. Recent insights show that a Gram-positive model bacterium, Bacillus subtilis, discriminates between strains of high (kin) and low (nonkin) genetic similarity, reflected in merging (kin) and boundaries (nonkin) between swarms. However, it is unclear how kinship between interacting strains affects their fitness, the genotype assortment, and incorporation of the mutant lacking the main structural ECM polysaccharide (EpsA-O) into floating biofilms (pellicles). We cultivated Bacillus subtilis strains as mixtures of isogenic, kin, and nonkin strain combinations in the biofilm-promoting minimal medium under static conditions, allowing them to form pellicles. We show that in nonkin pellicles, the dominant strain strongly reduced the frequency of the other strain. Segregation of nonkin mixtures in pellicles increased and invasion of nonkin EpsA-O-deficient mutants into pellicles decreased compared to kin and isogenic floating biofilms. Kin and isogenic strains had comparable relative frequencies in pellicles and showed more homogenous cell mixing. Overall, our results emphasize kin discrimination as a social behavior that shapes strain distribution, spatial segregation, and ECM mutant ability to incorporate into genetically heterogenous biofilms of B. subtilis. IMPORTANCE Biofilm communities have beneficial and harmful effects on human societies in natural, medical, and industrial environments. Bacillus subtilis is a biotechnologically important bacterium that serves as a model for studying biofilms. Recent studies have shown that this species engages in kin discriminatory behavior during swarming, which may have implications for community assembly, thus being of fundamental importance. Effects of kin discrimination on fitness, genotype segregation, and success of extracellular matrix (ECM) polysaccharide (EpsA-O) mutant invasion into biofilms are not well understood. We provide evidence that kin discrimination depends on the antagonism of the dominant strain against nonkin by using environmental strains with determined kin types and integrated fluorescent reporters. Moreover, this antagonism has important implications for genotype segregation and for when the bacteria are mixed with ECM producers. The work advances the understanding of kin-discrimination-dependent bacterial sociality in biofilms and its role in the assembly of multicellular groups.


Assuntos
Bacillus subtilis , Biofilmes , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Matriz Extracelular/metabolismo , Humanos , Polissacarídeos
20.
Elife ; 112022 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-36125244

RESUMO

Oxidative stress-mediated formation of protein hydroperoxides can induce irreversible fragmentation of the peptide backbone and accumulation of cross-linked protein aggregates, leading to cellular toxicity, dysfunction, and death. However, how bacteria protect themselves from damages caused by protein hydroperoxidation is unknown. Here, we show that YjbI, a group II truncated haemoglobin from Bacillus subtilis, prevents oxidative aggregation of cell-surface proteins by its protein hydroperoxide peroxidase-like activity, which removes hydroperoxide groups from oxidised proteins. Disruption of the yjbI gene in B. subtilis lowered biofilm water repellence, which associated with the cross-linked aggregation of the biofilm matrix protein TasA. YjbI was localised to the cell surface or the biofilm matrix, and the sensitivity of planktonically grown cells to generators of reactive oxygen species was significantly increased upon yjbI disruption, suggesting that YjbI pleiotropically protects labile cell-surface proteins from oxidative damage. YjbI removed hydroperoxide residues from the model oxidised protein substrate bovine serum albumin and biofilm component TasA, preventing oxidative aggregation in vitro. Furthermore, the replacement of Tyr63 near the haem of YjbI with phenylalanine resulted in the loss of its protein peroxidase-like activity, and the mutant gene failed to rescue biofilm water repellency and resistance to oxidative stress induced by hypochlorous acid in the yjbI-deficient strain. These findings provide new insights into the role of truncated haemoglobin and the importance of hydroperoxide removal from proteins in the survival of aerobic bacteria.


Assuntos
Bacillus subtilis , Hemoglobinas Truncadas , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Biofilmes , Heme/metabolismo , Peróxido de Hidrogênio/metabolismo , Ácido Hipocloroso/metabolismo , Proteínas de Membrana/metabolismo , Oxirredutases/metabolismo , Peroxidases/metabolismo , Fenilalanina/metabolismo , Agregados Proteicos , Soroalbumina Bovina/metabolismo , Hemoglobinas Truncadas/metabolismo , Água/metabolismo
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