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1.
Nat Commun ; 11(1): 4963, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009406

RESUMO

Bacterial nanotubes are membranous structures that have been reported to function as conduits between cells to exchange DNA, proteins, and nutrients. Here, we investigate the morphology and formation of bacterial nanotubes using Bacillus subtilis. We show that nanotube formation is associated with stress conditions, and is highly sensitive to the cells' genetic background, growth phase, and sample preparation methods. Remarkably, nanotubes appear to be extruded exclusively from dying cells, likely as a result of biophysical forces. Their emergence is extremely fast, occurring within seconds by cannibalizing the cell membrane. Subsequent experiments reveal that cell-to-cell transfer of non-conjugative plasmids depends strictly on the competence system of the cell, and not on nanotube formation. Our study thus supports the notion that bacterial nanotubes are a post mortem phenomenon involved in cell disintegration, and are unlikely to be involved in cytoplasmic content exchange between live cells.


Assuntos
Bacillus subtilis/citologia , Bacillus subtilis/metabolismo , Viabilidade Microbiana , Nanotubos/química , Bacillus subtilis/genética , Bacillus subtilis/ultraestrutura , Conjugação Genética , DNA Bacteriano/genética , Plasmídeos/genética
2.
Nat Commun ; 11(1): 5078, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033266

RESUMO

Metabolic engineering facilitates chemical biosynthesis by rewiring cellular resources to produce target compounds. However, an imbalance between cell growth and bioproduction often reduces production efficiency. Genetic code expansion (GCE)-based orthogonal translation systems incorporating non-canonical amino acids (ncAAs) into proteins by reassigning non-canonical codons to ncAAs qualify for balancing cellular metabolism. Here, GCE-based cell growth and biosynthesis balance engineering (GCE-CGBBE) is developed, which is based on titrating expression of cell growth and metabolic flux determinant genes by constructing ncAA-dependent expression patterns. We demonstrate GCE-CGBBE in genome-recoded Escherichia coli Δ321AM by precisely balancing glycolysis and N-acetylglucosamine production, resulting in a 4.54-fold increase in titer. GCE-CGBBE is further expanded to non-genome-recoded Bacillus subtilis to balance growth and N-acetylneuraminic acid bioproduction by titrating essential gene expression, yielding a 2.34-fold increase in titer. Moreover, the development of ncAA-dependent essential gene expression regulation shows efficient biocontainment of engineered B. subtilis to avoid unintended proliferation in nature.


Assuntos
Acetilglucosamina/metabolismo , Bacillus subtilis/crescimento & desenvolvimento , Vias Biossintéticas , Escherichia coli/crescimento & desenvolvimento , Ácido N-Acetilneuramínico/metabolismo , Bacillus subtilis/metabolismo , Proliferação de Células , Escherichia coli/metabolismo , Código Genético , Proteínas de Fluorescência Verde/metabolismo , Engenharia Metabólica , Análise do Fluxo Metabólico , Regiões Promotoras Genéticas/genética , RNA de Transferência/genética , Tirosina/metabolismo
3.
Nucleic Acids Res ; 48(19): 10785-10801, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33045732

RESUMO

Quorum sensing plays crucial roles in bacterial communication including in the process of conjugation, which has large economical and health-related impacts by spreading antibiotic resistance. The conjugative Bacillus subtilis plasmid pLS20 uses quorum sensing to determine when to activate the conjugation genes. The main conjugation promoter, Pc, is by default repressed by a regulator RcopLS20 involving DNA looping. A plasmid-encoded signalling peptide, Phr*pLS20, inactivates the anti-repressor of RcopLS20, named RappLS20, which belongs to the large group of RRNPP family of regulatory proteins. Here we show that DNA looping occurs through interactions between two RcopLS20 tetramers, each bound to an operator site. We determined the relative promoter strengths for all the promoters involved in synthesizing the regulatory proteins of the conjugation genes, and constructed an in vivo system uncoupling these regulatory genes to show that RappLS20 is sufficient for activating conjugation in vivo. We also show that RappLS20 actively detaches RcopLS20 from DNA by preferentially acting on the RcopLS20 molecules involved in DNA looping, resulting in sequestration but not inactivation of RcopLS20. Finally, results presented here in combination with our previous results show that activation of conjugation inhibits competence and competence development inhibits conjugation, indicating that both processes are mutually exclusive.


Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Conjugação Genética , Regulação Bacteriana da Expressão Gênica , Percepção de Quorum , Bacillus subtilis/metabolismo , Bacillus subtilis/fisiologia , Proteínas de Bactérias/metabolismo , Plasmídeos/genética , Regiões Promotoras Genéticas
4.
Nat Commun ; 11(1): 3803, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732991

RESUMO

Microbial communities comprised of phototrophs and heterotrophs hold great promise for sustainable biotechnology. Successful application of these communities relies on the selection of appropriate partners. Here we construct four community metabolic models to guide strain selection, pairing phototrophic, sucrose-secreting Synechococcus elongatus with heterotrophic Escherichia coli K-12, Escherichia coli W, Yarrowia lipolytica, or Bacillus subtilis. Model simulations reveae metabolic exchanges that sustain the heterotrophs in minimal media devoid of any organic carbon source, pointing to S. elongatus-E. coli K-12 as the most active community. Experimental validation of flux predictions for this pair confirms metabolic interactions and potential production capabilities. Synthetic communities bypass member-specific metabolic bottlenecks (e.g. histidine- and transport-related reactions) and compensate for lethal genetic traits, achieving up to 27% recovery from lethal knockouts. The study provides a robust modelling framework for the rational design of synthetic communities with optimized growth sustainability using phototrophic partners.


Assuntos
Bacillus subtilis/metabolismo , Escherichia coli/metabolismo , Processos Heterotróficos/fisiologia , Processos Fototróficos/fisiologia , Synechococcus/metabolismo , Yarrowia/metabolismo , Aldeídos/metabolismo , Bacillus subtilis/genética , Reatores Biológicos/microbiologia , Escherichia coli/genética , Etanol/metabolismo , Formaldeído/metabolismo , Metanol/metabolismo , Microbiota/fisiologia , Modelos Biológicos , Ácido Succínico/metabolismo , Synechococcus/genética , Yarrowia/genética
5.
Nature ; 585(7823): 124-128, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32848247

RESUMO

Tight coupling of transcription and translation is considered a defining feature of bacterial gene expression1,2. The pioneering ribosome can both physically associate and kinetically coordinate with RNA polymerase (RNAP)3-11, forming a signal-integration hub for co-transcriptional regulation that includes translation-based attenuation12,13 and RNA quality control2. However, it remains unclear whether transcription-translation coupling-together with its broad functional consequences-is indeed a fundamental characteristic of bacteria other than Escherichia coli. Here we show that RNAPs outpace pioneering ribosomes in the Gram-positive model bacterium Bacillus subtilis, and that this 'runaway transcription' creates alternative rules for both global RNA surveillance and translational control of nascent RNA. In particular, uncoupled RNAPs in B. subtilis explain the diminished role of Rho-dependent transcription termination, as well as the prevalence of mRNA leaders that use riboswitches and RNA-binding proteins. More broadly, we identified widespread genomic signatures of runaway transcription in distinct phyla across the bacterial domain. Our results show that coupled RNAP-ribosome movement is not a general hallmark of bacteria. Instead, translation-coupled transcription and runaway transcription constitute two principal modes of gene expression that determine genome-specific regulatory mechanisms in prokaryotes.


Assuntos
Bacillus subtilis/genética , Regulação Bacteriana da Expressão Gênica , Biossíntese de Proteínas , Transcrição Genética , Regiões 5' não Traduzidas/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Filogenia , RNA Bacteriano/biossíntese , RNA Bacteriano/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fator Rho/metabolismo , Ribossomos/metabolismo , Riboswitch/genética
6.
Food Chem ; 332: 127438, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32645671

RESUMO

ß-N-acetylhexosaminidases have attracted much attention in recent years due to their potential application in oligosaccharide production, in particular lacto-N-triose II (LNT2) and lacto-N-neotetraose (LNnT) synthesis, which can be further used as backbone precursors for human milk oligosaccharides. A novel ß-N-acetylhexosaminidase gene from Tyzzerella nexilis (TnHex189) was heterologously expressed in Bacillus subtilis. The highest ß-N-acetylhexosaminidase activity of 14.5 U mL-1 was obtained in a 5-L fermentor by fed-batch fermentation for 27 h. TnHex189 was optimally active at pH 5.0 and 45 °C. It efficiently synthesized LNT2 with a conversion ratio of 57.2% (4.7 g L-1). The synthesized LNT2 was further converted to LNnT by a reported ß-galactosidase (BgaD-D) in 8 h, with a conversion ratio of 17.3% (6.1 g L-1). These unique synthesis activities may make this enzyme a good candidate for the food industry.


Assuntos
Proteínas de Bactérias/metabolismo , Clostridiales/enzimologia , Trissacarídeos/biossíntese , beta-N-Acetil-Hexosaminidases/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Clostridiales/genética , Estabilidade Enzimática , Fermentação , Expressão Gênica , Concentração de Íons de Hidrogênio , Oligossacarídeos/metabolismo , beta-N-Acetil-Hexosaminidases/química , beta-N-Acetil-Hexosaminidases/genética
7.
Int J Food Microbiol ; 330: 108783, 2020 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-32659523

RESUMO

The contamination of Aspergillus carbonarius causes decreases and great decay of agricultural products, and threatens the human and animal health by producing mycotoxins, especially ochratoxin A. Bacillus subtilis has been proved to efficiently inhibit the growth of A. carbonarius. Revealing the major active compound and the mechanisms for the antifungal of B. subtilis are essential to enhance its antifungal activity and control the quality of antifungal products made of it. In this study, we determined that iturin A is the major compound that inhibits Aspergillus carbonarius, a widespread fungal pathogen of grape and other fruits. Iturin A significantly inhibited growth and ochratoxin A production of A. carbonarius with minimal inhibitory concentrations (MICs) of 10 µg/mL and 0.312 µg/mL, respectively. Morphological observations revealed that iturin A caused swelling of the fungal cells and thinning of the cell wall and membrane at 1/2 MIC, whereas it inhibited fungal spore germination and caused mitochondrial swelling at higher concentrations. A differential transcriptomic analysis indicated that the mechanisms used by iturin A to inhibit A. carbonarius were to downregulate the expression of genes related to cell membrane, transport, osmotic pressure, oxidation-reduction processes, and energy metabolism. Among the down-regulated genes, those related to the transport capacity were most significantly influenced, including the increase of energy-related transport pathways and decrease of other pathways. Notably, the genes related to taurine and hypotaurine metabolism were also decreased, indicating iturin A potentially cause the occurrence of osmotic imbalance in A. carbonarius, which may be the intrinsic cause for the swelling of fungal cells and mitochondria. Overall, iturin A produced by B. subtilis played important roles to inhibit A. carbonarius via changing the fungal cell structure and causing perturbations to energy, transport and osmotic pressure metabolisms in fungi. The results indicated a new direction for researches on the mechanisms for lipopeptides and provided useful information to develop more efficient antifungal agents, which are important to agriculture and biomedicine.


Assuntos
Antifúngicos/farmacologia , Aspergillus/efeitos dos fármacos , Aspergillus/metabolismo , Bacillus subtilis/metabolismo , Peptídeos Cíclicos/farmacologia , Antifúngicos/metabolismo , Aspergillus/genética , Aspergillus/crescimento & desenvolvimento , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Micotoxinas/metabolismo , Pressão Osmótica/efeitos dos fármacos , Peptídeos Cíclicos/metabolismo , Esporos Fúngicos/efeitos dos fármacos , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismo , Transcriptoma
8.
Nature ; 584(7821): 470-474, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32669712

RESUMO

The rate of cell growth is crucial for bacterial fitness and drives the allocation of bacterial resources, affecting, for example, the expression levels of proteins dedicated to metabolism and biosynthesis1,2. It is unclear, however, what ultimately determines growth rates in different environmental conditions. Moreover, increasing evidence suggests that other objectives are also important3-7, such as the rate of physiological adaptation to changing environments8,9. A common challenge for cells is that these objectives cannot be independently optimized, and maximizing one often reduces another. Many such trade-offs have indeed been hypothesized on the basis of qualitative correlative studies8-11. Here we report a trade-off between steady-state growth rate and physiological adaptability in Escherichia coli, observed when a growing culture is abruptly shifted from a preferred carbon source such as glucose to fermentation products such as acetate. These metabolic transitions, common for enteric bacteria, are often accompanied by multi-hour lags before growth resumes. Metabolomic analysis reveals that long lags result from the depletion of key metabolites that follows the sudden reversal in the central carbon flux owing to the imposed nutrient shifts. A model of sequential flux limitation not only explains the observed trade-off between growth and adaptability, but also allows quantitative predictions regarding the universal occurrence of such tradeoffs, based on the opposing enzyme requirements of glycolysis versus gluconeogenesis. We validate these predictions experimentally for many different nutrient shifts in E. coli, as well as for other respiro-fermentative microorganisms, including Bacillus subtilis and Saccharomyces cerevisiae.


Assuntos
Adaptação Fisiológica , Meio Ambiente , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Acetatos/metabolismo , Bacillus subtilis/citologia , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Divisão Celular , Escherichia coli/enzimologia , Escherichia coli/genética , Fermentação , Gluconeogênese , Glucose/metabolismo , Glicólise , Metabolômica , Modelos Biológicos , Mutação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo
9.
Medicine (Baltimore) ; 99(23): e20631, 2020 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-32502043

RESUMO

This retrospective study aimed to explore the benefits and safety of probiotics (live combined Bacillus subtilis and Enterococcus faecium granules with multivitamines) for the treatment of children with antibiotic-associated diarrhea (AAD).A total of 72 children with AAD were analyzed in this study. Of these, 36 children received routine treatment plus probiotics, and were assigned to a treatment group. The other 36 children underwent routine treatment alone, and were assigned to a control group. Patients in both groups were treated for a total of 7 days. The efficacy and safety were evaluated by duration of diarrhea (days), number of dressings needed daily, abdominal pain intensity, stool consistency (as assessed by Bristol Stool Scale (BSS)), and any adverse events.After treatment, probiotics showed encouraging benefits in decreasing duration of diarrhea (days) (P < .01), number of dressings needed every day (P < .01), abdominal pain intensity (P < .01), and stool consistency (BSS (3-5), P < .01; BSS (6-7), P < .01). In addition, no adverse events were documented in this study.The findings of this study demonstrated that probiotics may provide promising benefit for children with AAD. Further studies are still needed to warrant theses findings.


Assuntos
Diarreia/induzido quimicamente , Probióticos/efeitos adversos , Bacillus subtilis/metabolismo , Estudos de Casos e Controles , Criança , Pré-Escolar , Enterococcus faecium/metabolismo , Feminino , Humanos , Masculino , Probióticos/farmacologia , Estudos Retrospectivos
10.
Ecotoxicol Environ Saf ; 201: 110810, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32521369

RESUMO

Heavy metals pollution in river sediments is irreversible, and it is not easy to be found to be concealed. The pollution of heavy metals for river sediments is currently a serious environmental problem. In the paper, the Bacillus subtilis was selected to remove heavy metals by bio-mineralization method in river sediments. Optimal content of Bacillus subtilis powder and organophosphate monoester was 20 g and 0.2 mol in 1 L of bacterial solution, respectively. The optimum reaction time and temperature were 36 h and 30 °C, respectively. The optimal reaction conditions were applied to zinc ions in river sediments. After heavy metals treatment, there was little effect on the water content before and after flocculation and vacuum preloading. Treatment of heavy metals had no effect on the cross-plate shear strength of river sediments after vacuum preloading. After treatment of heavy metals, the effect of purifying water quality was the group B(Polyacrylamide + Polysilicate aluminium ferric) bigger than the group A (Polyacrylamide). The removal efficiency of zinc ions (Zn2+) in the group B was 89.59% and 74.99% before and after the vacuum preloading, respectively, which was better than that in the group A.


Assuntos
Bacillus subtilis/metabolismo , Floculação , Sedimentos Geológicos/química , Metais Pesados/análise , Rios/química , Vácuo , Poluentes Químicos da Água/análise , Biodegradação Ambiental , China , Sedimentos Geológicos/microbiologia , Metais Pesados/metabolismo , Medição de Risco , Rios/microbiologia , Poluentes Químicos da Água/metabolismo
11.
J Biosci Bioeng ; 130(3): 272-282, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32546403

RESUMO

The industrially relevant biopolymer poly-γ-glutamic acid (γ-PGA) is commonly synthesized using glycerol, citrate, and glutamic acid as carbon sources. In this study, two strains capable of utilizing glucose as sole carbon source for γ-PGA synthesis were constructed. Efficient γ-PGA production was achieved with derivatives of the well-investigated laboratory strain Bacillus subtilis 168, by replacing the native promoter of the PGA synthetase operon with the strong constitutive promoter Pveg or with the xylose-inducible promoter Pxyl. The carbon yield for γ-PGA increased by 129% to 0.131 C-mol C-mol-1 when using glucose as the sole substrate compared to the conventional carbon source mixture glycerol, citrate, and glutamic acid. The characterization of the produced γ-PGA demonstrated a time-dependent molecular weight of 1180-1850 kDa and a d-glutamic acid monomer content of 49-62%. To elucidate the consequences of γ-PGA production, we characterized the engineered strain by metabolomics. While the metabolite concentrations in the TCA cycle leading up to 2-oxoglutarate decreased in γ-PGA producer strains, the glutamic acid concentration was constant, despite the drastic increase in glutamic acid demand. The results are discussed in the context of metabolic regulation and future metabolic engineering strategies to enhance precursor supply for γ-PGA synthesis from glucose.


Assuntos
Bacillus subtilis/metabolismo , Glucose/metabolismo , Metabolômica , Ácido Poliglutâmico/análogos & derivados , Bacillus subtilis/genética , Ciclo do Ácido Cítrico , Engenharia Metabólica , Peso Molecular , Óperon/genética , Ácido Poliglutâmico/biossíntese , Ácido Poliglutâmico/química
12.
Arch Microbiol ; 202(8): 2189-2196, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32524179

RESUMO

Three hydrocarbon degrading microorganisms were isolated from the soil samples collected from the storage sites of low viscosity spindle oil containers and identified on the basis of morphological and biochemical characteristics as Aeromonas hydrophila, Bacillus subtilis and Staphylococcus aureus. The study has revealed high ability of these microorganisms for oil biodegradation. The results have indicated that all isolates had the potential to breakdown the hydrocarbon. The most efficient bacteria among these examined was Aeromonas hydrophila which biodegraded almost all tested hydrocarbon giving a treatment percentage of 98% within 30 days which was considered as the perfect period for degradation. Also, a small scale was designed to treat the spindle oil with the using of oxidation process and all the tested organic materials were biodegraded in a treatment percentage of 100% within retention time of 20 days.


Assuntos
Bactérias/metabolismo , Biodegradação Ambiental , Petróleo/microbiologia , Microbiologia do Solo , Poluentes do Solo/metabolismo , Aeromonas hydrophila/metabolismo , Bacillus subtilis/metabolismo , Bactérias/isolamento & purificação , Hidrocarbonetos/metabolismo , Petróleo/metabolismo , Poluição por Petróleo , Staphylococcus aureus/metabolismo , Viscosidade
13.
J Food Sci ; 85(7): 2164-2170, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32572963

RESUMO

Sanguisorba officinalis L. (family Rosaceae, subfamily Rosoideae) is a plant found throughout Southern Europe, Northern Africa, and Eastern Asia. This study demonstrated the antibacterial activity of a purified polyphenolic extract (PPE) from S. officinalis L. against Bacillus subtilis using growth inhibitory and apoptosis assays, and investigated the antibacterial mechanism responsible for changes in cell membrane properties. Fourier transform infrared spectroscopy suggested that PPE altered the cell wall and membrane properties of B. subtilis. Further determination of cell membrane integrity and permeability revealed that B. subtilis membrane integrity was more severely damaged by PPE at the minimum inhibitory concentration (MIC) than at the minimum bactericidal concentrati on (MBC). Instead, PPE at the MBC reduced cell membrane fluidity by significantly decreasing the proportion of anteiso- and iso-branched phospholipid fatty acids (PLFAs) from 64.17 ± 0.28% and 27.23 ± 0.03% in the control to 5.57 ± 1.06% and 6.00 ± 1.40%, respectively (P < 0.001). Scanning electron microscopy revealed different effects of PPE on cell morphology, demonstrating that, at the MIC and MBC, PPE exerted antibacterial activity by disrupting the cell membrane and reducing cell membrane fluidity, respectively. Consequently, this study elucidated changes in the bacterial membrane due to exposure to PPE and its potential use as an antimicrobial agent. PRACTICAL APPLICATION: The abuse of synthetic chemical preservatives raises food safety concerns; however, plant-derived polyphenolic compounds may be a safe and effective alternative. This study demonstrated the strong antibacterial activity of a purified polyphenolic extract (PPE) of Sanguisorba officinalis L. and revealed its antibacterial mechanism against Bacillus subtilis, suggesting that it may provide a useful antimicrobial agent in food industry applications.


Assuntos
Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Membrana Celular/metabolismo , Ácidos Graxos/metabolismo , Fosfolipídeos/metabolismo , Extratos Vegetais/farmacologia , Polifenóis/farmacologia , Sanguisorba/química , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Membrana Celular/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Ácidos Graxos/química , Conservantes de Alimentos/farmacologia , Testes de Sensibilidade Microbiana , Fosfolipídeos/química
14.
Phys Rev Lett ; 124(15): 158102, 2020 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-32357050

RESUMO

We analyze the nonequilibrium shape fluctuations of giant unilamellar vesicles encapsulating motile bacteria. Owing to bacteria-membrane collisions, we experimentally observe a significant increase in the magnitude of membrane fluctuations at low wave numbers, compared to the well-known thermal fluctuation spectrum. We interrogate these results by numerically simulating membrane height fluctuations via a modified Langevin equation, which includes bacteria-membrane contact forces. Taking advantage of the lengthscale and timescale separation of these contact forces and thermal noise, we further corroborate our results with an approximate theoretical solution to the dynamical membrane equations. Our theory and simulations demonstrate excellent agreement with nonequilibrium fluctuations observed in experiments. Moreover, our theory reveals that the fluctuation-dissipation theorem is not broken by the bacteria; rather, membrane fluctuations can be decomposed into thermal and active components.


Assuntos
Vesículas Citoplasmáticas/química , Lipídeos de Membrana/química , Modelos Biológicos , Modelos Químicos , Bacillus subtilis/química , Bacillus subtilis/citologia , Bacillus subtilis/metabolismo , Vesículas Citoplasmáticas/metabolismo , Locomoção , Lipídeos de Membrana/metabolismo , Fosfatidiletanolaminas/química , Rodaminas/química , Termodinâmica , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo
15.
Mol Immunol ; 123: 88-96, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32447084

RESUMO

The anaerobic pathogen Clostridium perfringens is the most potent cause of intestinal diseases, such as enterotoxemia, hemorrhagic enteritis, and lamb dysentery, in sheep. Three toxinotypes (B, C, and D) are usually the cause of these diseases and are mainly mediated via three important exotoxins: alpha toxin (CPA), beta toxin (CPB), and epsilon toxin (ETX). We have designed a chimeric protein, rCpa-b-x, that contains the C-terminal binding region of CPA, partial sequence of CPB, and ETX (Cpa247-370, Cpb108-305, and EtxH118P, respectively) according to the principle of structural vaccinology. The rCpa-b-x protein was then expressed by pHT43 plasmid in vivo using Bacillus subtilis as a delivery vector (Bs-pHT43-Cpa-b-x). The immunological activity of the rCpa-b-x protein was verified by western blot and its immunological efficacy was evaluated in a murine model. Oral administration with a recombinant agent caused local mucosal and systemic immune responses, and serum lgG and intestinal mucosal secretory IgA (sIgA) antibody titers were significantly increased. Levels of IL-2, IL-4, and IFN-γ were significantly higher in lymphocytes isolated from the Bs-pHT43-Cpa-b-x group compared with levels from the control groups. The percentages of CD4+ and CD8+ T lymphocytes in the Bs-pHT43-Cpa-b-x and inactivated vaccine (IV) groups were in the normal range. Mice of vaccine groups and control groups were challenged with 1x LD100 unit filtrate containing alpha, beta, and epsilon toxins. Mice in the Bs-pHT43-Cpa-b-x group were found to have lower rates of morbidity. The active immunization of mice with Bs-pHT43-Cpa-b-x still maintained 85% to 90% survival at the end of the 10-day observation period, whereas mice of control groups died within two to five days. The results of this study demonstrate the effectiveness of Bs-pHT43-Cpa-b-x in preventing C. perfringens infection in mice, and that Bs-pHT43-Cpa-b-x could be considered a potential vaccine against C. perfringens.


Assuntos
Bacillus subtilis/metabolismo , Toxinas Bacterianas/imunologia , Vacinas Bacterianas/metabolismo , Vacinas Bacterianas/uso terapêutico , Infecções por Clostridium/prevenção & controle , Clostridium perfringens/imunologia , Animais , Bacillus subtilis/genética , Toxinas Bacterianas/metabolismo , Vacinas Bacterianas/química , Vacinas Bacterianas/genética , Infecções por Clostridium/imunologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Ligação Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/uso terapêutico , Vacinação/métodos , Vacinas Sintéticas/química , Vacinas Sintéticas/genética , Vacinas Sintéticas/metabolismo , Vacinas Sintéticas/uso terapêutico
16.
PLoS One ; 15(4): e0232096, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32339210

RESUMO

Endophytic bacteria, which are common in plant tissues, may help to control plant pathogens and enhance plant growth. Camellia oleifera, an oil-producing plant, is widely grown in warm, subtropical, hilly regions in China. However, C. oleifera is strongly negatively affected by C. oleifera anthracnose, which is caused by Colletetrichum fructicola. To find a suitable biocontrol agent for C. oleifera anthracnose, 41 endophytes were isolated from the stems, leaves, and roots of C. oleifera. Bacterial cultures were identified based on analyses of 16S rDNA sequences; most strains belonged to the genus Bacillus. The antagonistic effects of these strains on C. fructicola were tested in vitro. In total, 16 strains inhibited C. fructicola growth, with B. subtilis strain 1-L-29 being the most efficient. Strain 1-L-29 demonstrated antagonistic activity against C. siamense, C. asianum, Fusarium proliferatum, Agaricodochium camellia, and Pseudomonas syringae. In addition, this strain produced indole acetic acid, solubilized phosphate, grew on N-free media, and produced siderophores. To facilitate further microecological studies of this strain, a rifampicin-resistant, green fluorescent protein (GFP)-labeled strain, 1-L-29gfpr, was created using protoplast transformation. This plasmid had good segregational stability. Strain 1-L-29gfpr was re-introduced into C. oleifera and successfully colonized root, stem, and leaf tissues. This strain remained at a stable concentration in the root more than 20 d after inoculation. Fluorescence microscopic analysis showed that strain 1-L-29gfpr thoroughly colonized the root surfaces of C. fructicola as well as the root vascular tissues of Arabidopsis thaliana.


Assuntos
Antibacterianos/metabolismo , Bacillus subtilis/metabolismo , Camellia/crescimento & desenvolvimento , Endófitos/metabolismo , Doenças das Plantas/prevenção & controle , Raízes de Plantas/crescimento & desenvolvimento , Bacillus subtilis/classificação , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/isolamento & purificação , Camellia/metabolismo , Camellia/microbiologia , Endófitos/crescimento & desenvolvimento , Endófitos/isolamento & purificação , Controle Biológico de Vetores , Desenvolvimento Vegetal , Doenças das Plantas/microbiologia , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia
17.
Int J Food Microbiol ; 323: 108592, 2020 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-32315871

RESUMO

Microbial surface contamination of equipment or of food contact material is a recurring problem in the food industry. Spore-forming bacteria are far more resistant to a wide variety of treatments than their vegetative forms. Understanding the mechanisms underlying decontamination processes is needed to improve surface decontamination strategies against endospores potentially at the source of foodborne diseases or food-spoilage. Pulsed light (PL) with xenon lamps delivers high-energy short-time pulses of light with wavelengths in the range 200 nm-1100 nm and a high UV-C fraction. Bacillus subtilis spores were exposed to either PL or to continuous UV-C. Gel electrophoresis and western blotting revealed elimination of various proteins of the spore coat, an essential outer structure that protects spores from a wide variety of environmental conditions and inactivation treatments. Proteomic analysis confirmed the elimination of some spore coat proteins after PL treatment. Transmission electron microscopy of PL treated spores revealed a gap between the lamellar inner spore coat and the outer spore coat. Overall, spores of mutant strains with defects in genes coding for spore coat proteins were more sensitive to PL than to continuous UV-C. This study demonstrates that radiations delivered by PL contribute to specific damage to the spore coat, and overall to spore inactivation.


Assuntos
Bacillus subtilis/metabolismo , Bacillus subtilis/efeitos da radiação , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/efeitos da radiação , Descontaminação/métodos , Luz , Bacillus subtilis/genética , Parede Celular/metabolismo , Parede Celular/efeitos da radiação , Descontaminação/normas , Proteômica , Esporos Bacterianos/fisiologia , Esporos Bacterianos/efeitos da radiação
18.
PLoS One ; 15(4): e0231348, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32298338

RESUMO

Salt stress is one of the devastating factors that hampers growth and productivity of soybean. Use of Pseudomonas pseudoalcaligenes to improve salt tolerance in soybean has not been thoroughly explored yet. Therefore, we observed the response of hydroponically grown soybean plants, inoculated with halotolerant P. pseudoalcaligenes (SRM-16) and Bacillus subtilis (SRM-3) under salt stress. In vitro testing of 44 bacterial isolates revealed that four isolates showed high salt tolerance. Among them, B. subtilis and P. pseudoalcaligenes showed ACC deaminase activity, siderophore and indole acetic acid (IAA) production and were selected for the current study. We determined that 106 cells/mL of B. subtilis and P. pseudoalcaligenes was sufficient to induce tolerance in soybean against salinity stress (100 mM NaCl) in hydroponics by enhancing plant biomass, relative water content and osmolytes. Upon exposure of salinity stress, P. pseudoalcaligenes inoculated soybean plants showed tolerance by the increased activities of defense related system such as ion transport, antioxidant enzymes, proline and MDA content in shoots and roots. The Na+ concentration in the soybean plants was increased in the salt stress; while, bacterial priming significantly reduced the Na+ concentration in the salt stressed soybean plants. However, the antagonistic results were observed for K+ concentration. Additionally, soybean primed with P. pseudoalcaligenes and exposed to 100 mM NaCl showed a new protein band of 28 kDa suggesting that P. pseudoalcaligenes effectively reduced salt stress. Our results showed that salinity tolerance was more pronounced in P. pseudoalcaligenes as compared to B. subtilis. However, a detailed study at molecular level to interpret the mechanism by which P. pseudoalcaligenes alleviates salt stress in soybean plants need to be explored.


Assuntos
Bacillus subtilis/patogenicidade , Pseudomonas pseudoalcaligenes/patogenicidade , Tolerância ao Sal , Soja/metabolismo , Bacillus subtilis/metabolismo , Transporte de Íons , Malondialdeído/metabolismo , Peroxidase/genética , Peroxidase/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Prolina/metabolismo , Pseudomonas pseudoalcaligenes/metabolismo , Espécies Reativas de Oxigênio , Soja/microbiologia
19.
Nat Commun ; 11(1): 1859, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313019

RESUMO

Bacteria can form biofilms that consist of multicellular communities embedded in an extracellular matrix (ECM). In Bacillus subtilis, the main protein component of the ECM is the functional amyloid TasA. Here, we study further the roles played by TasA in B. subtilis physiology and biofilm formation on plant leaves and in vitro. We show that ΔtasA cells exhibit a range of cytological symptoms indicative of excessive cellular stress leading to increased cell death. TasA associates to the detergent-resistant fraction of the cell membrane, and the distribution of the flotillin-like protein FloT is altered in ΔtasA cells. We propose that, in addition to a structural function during ECM assembly and interactions with plants, TasA contributes to the stabilization of membrane dynamics as cells enter stationary phase.


Assuntos
Proteínas Amiloidogênicas/metabolismo , Bacillus/fisiologia , Proteínas de Bactérias/metabolismo , Proteínas Amiloidogênicas/genética , Bacillus subtilis/metabolismo , Aderência Bacteriana , Proteínas de Bactérias/genética , Biofilmes , Morte Celular , Membrana Celular/metabolismo , Cucurbitaceae/microbiologia , Ecologia , Lipopeptídeos , Mutação , Transcriptoma
20.
Nat Commun ; 11(1): 1938, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32321911

RESUMO

Bacteria can produce membranous nanotubes that mediate contact-dependent exchange of molecules among bacterial cells. However, it is unclear how nanotubes cross the cell wall to emerge from the donor or to penetrate into the recipient cell. Here, we report that Bacillus subtilis utilizes cell wall remodeling enzymes, the LytC amidase and its enhancer LytB, for efficient nanotube extrusion and penetration. Nanotube production is reduced in a lytBC mutant, and the few nanotubes formed appear deficient in penetrating into target cells. Donor-derived LytB molecules localize along nanotubes and on the surface of nanotube-connected neighbouring cells, primarily at sites of nanotube penetration. Furthermore, LytB from donor B. subtilis can activate LytC of recipient bacteria from diverse species, facilitating cell wall hydrolysis to establish nanotube connection. Our data provide a mechanistic view of how intercellular connecting devices can be formed among neighbouring bacteria.


Assuntos
Amidoidrolases/metabolismo , Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Extensões da Superfície Celular/metabolismo , Parede Celular/enzimologia , Conjugação Genética , Amidoidrolases/genética , Bacillus subtilis/química , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Extensões da Superfície Celular/genética , Parede Celular/química , Parede Celular/genética , Parede Celular/metabolismo , Transporte Proteico
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