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1.
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
2.
RNA ; 26(10): 1431-1447, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32611709

RESUMO

RNA structure influences numerous processes in all organisms. In bacteria, these processes include transcription termination and attenuation, small RNA and protein binding, translation initiation, and mRNA stability, and can be regulated via metabolite availability and other stresses. Here we use Structure-seq2 to probe the in vivo RNA structurome of Bacillus subtilis grown in the presence and absence of amino acids. Our results reveal that amino acid starvation results in lower overall dimethyl sulfate (DMS) reactivity of the transcriptome, indicating enhanced protection owing to protein binding or RNA structure. Starvation-induced changes in DMS reactivity correlated inversely with transcript abundance changes. This correlation was particularly pronounced in genes associated with the stringent response and CodY regulons, which are involved in adaptation to nutritional stress, suggesting that RNA structure contributes to transcript abundance change in regulons involved in amino acid metabolism. Structure-seq2 accurately reported on four known amino acid-responsive riboswitches: T-box, SAM, glycine, and lysine riboswitches. Additionally, we discovered a transcription attenuation mechanism that reduces yfmG expression when amino acids are added to the growth medium. We also found that translation of a leader peptide (YfmH) encoded just upstream of yfmG regulates yfmG expression. Our results are consistent with a model in which a slow rate of yfmH translation caused by limitation of the amino acids encoded in YfmH prevents transcription termination in the yfmG leader region by favoring formation of an overlapping antiterminator structure. This novel RNA switch offers a way to simultaneously monitor the levels of multiple amino acids.


Assuntos
Aminoácidos/genética , Bacillus subtilis/genética , Proteínas de Bactérias/genética , RNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica/genética , Conformação de Ácido Nucleico , Estabilidade de RNA/genética , Transcrição Genética/genética , Transcriptoma/genética
3.
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
4.
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
5.
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
6.
PLoS One ; 15(4): e0231426, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32271848

RESUMO

Demand for agricultural crop continues to escalate in response to increasing population and damage of prime cropland for cultivation. Research interest is diverted to utilize soils with marginal plant production. Moisture stress has negative impact on crop growth and productivity. The plant growth promoting rhizobacteria (PGPR) and plant growth regulators (PGR) are vital for plant developmental process under moisture stress. The current study was carried out to investigate the effect of PGPR and PGRs (Salicylic acid and Putrescine) on the physiological activities of chickpea grown in sandy soil. The bacterial isolates were characterized based on biochemical characters including Gram-staining, P-solubilisation, antibacterial and antifungal activities and catalases and oxidases activities and were also screened for the production of indole-3-acetic acid (IAA), hydrogen cyanide (HCN) and ammonia (NH3). The bacterial strains were identified as Bacillus subtilis, Bacillus thuringiensis and Bacillus megaterium based on the results of 16S-rRNA gene sequencing. Chickpea seeds of two varieties (Punjab Noor-2009 and 93127) differing in sensitivity to drought were soaked for 3 h before sowing in fresh grown cultures of isolates. Both the PGRs were applied (150 mg/L), as foliar spray on 20 days old seedlings of chickpea. Moisture stress significantly reduced the physiological parameters but the inoculation of PGPR and PGR treatment effectively ameliorated the adverse effects of moisture stress. The result showed that chickpea plants treated with PGPR and PGR significantly enhanced the chlorophyll, protein and sugar contents. Shoot and root fresh (81%) and dry weights (77%) were also enhanced significantly in the treated plants. Leaf proline content, lipid peroxidation and antioxidant enzymes (CAT, APOX, POD and SOD) were increased in reaction to drought stress but decreased due to PGPR. The plant height (61%), grain weight (41%), number of nodules (78%) and pod (88%), plant yield (76%), pod weight (53%) and total biomass (54%) were higher in PGPR and PGR treated chickpea plants grown in sandy soil. It is concluded from the present study that the integrative use of PGPR and PGRs is a promising method and eco-friendly strategy for increasing drought tolerance in crop plants.


Assuntos
Agricultura , Bacillaceae/fisiologia , Cicer/crescimento & desenvolvimento , Reguladores de Crescimento de Planta/farmacologia , Amônia/metabolismo , Bacillaceae/genética , Bacillaceae/isolamento & purificação , Bacillus megaterium/genética , Bacillus megaterium/isolamento & purificação , Bacillus subtilis/genética , Bacillus subtilis/isolamento & purificação , Bacillus subtilis/fisiologia , Biomassa , Clorofila/análise , Cicer/efeitos dos fármacos , Cicer/metabolismo , Ácidos Indolacéticos/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Reguladores de Crescimento de Planta/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Putrescina/metabolismo , Putrescina/farmacologia , RNA Ribossômico 16S/química , RNA Ribossômico 16S/metabolismo , Chuva , Ácido Salicílico/metabolismo , Ácido Salicílico/farmacologia , Plântula/efeitos dos fármacos , Plântula/crescimento & desenvolvimento , Microbiologia do Solo
7.
PLoS One ; 15(4): e0231274, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32271828

RESUMO

We evaluated the minimum inhibitory concentrations of clindamycin and erythromycin toward 98 Bacillus licheniformis strains isolated from several types of fermented soybean foods manufactured in several districts of Korea. First, based on recent taxonomic standards for bacteria, the 98 strains were separated into 74 B. licheniformis strains and 24 B. paralicheniformis strains. Both species exhibited profiles of erythromycin resistance as an acquired characteristic. B. licheniformis strains exhibited acquired clindamycin resistance, while B. paralicheniformis strains showed unimodal clindamycin resistance, indicating an intrinsic characteristic. Comparative genomic analysis of five strains showing three different patterns of clindamycin and erythromycin resistance identified 23S rRNA (adenine 2058-N6)-dimethyltransferase gene ermC and spermidine acetyltransferase gene speG as candidates potentially involved in clindamycin resistance. Functional analysis of these genes using B. subtilis as a host showed that ermC contributes to cross-resistance to clindamycin and erythromycin, and speG confers resistance to clindamycin. ermC is located in the chromosomes of strains showing clindamycin and erythromycin resistance and no transposable element was identified in its flanking regions. The acquisition of ermC might be attributable to a homologous recombination. speG was identified in not only the five genome-analyzed strains but also eight strains randomly selected from the 98 test strains, and deletions in the structural gene or putative promoter region caused clindamycin sensitivity, which supports the finding that the clindamycin resistance of Bacillus species is an intrinsic property.


Assuntos
Bacillus licheniformis/genética , Bacillus/genética , Clindamicina/farmacologia , Farmacorresistência Bacteriana/genética , Genes Bacterianos , Genômica , Bacillus/efeitos dos fármacos , Bacillus/crescimento & desenvolvimento , Bacillus licheniformis/classificação , Bacillus licheniformis/efeitos dos fármacos , Bacillus licheniformis/crescimento & desenvolvimento , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Sequência de Bases , Farmacorresistência Bacteriana/efeitos dos fármacos , Eritromicina/farmacologia , Testes de Sensibilidade Microbiana
8.
Proc Natl Acad Sci U S A ; 117(19): 10313-10321, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32341169

RESUMO

The H+/Ca2+ (calcium ion) antiporter (CAX) plays an important role in maintaining cellular Ca2+ homeostasis in bacteria, yeast, and plants by promoting Ca2+ efflux across the cell membranes. However, how CAX facilitates Ca2+ balance in response to dynamic cytosolic Ca2+ perturbations is unknown. Here, we identified a type of Ca2+ "mini-sensor" in YfkE, a bacterial CAX homolog from Bacillus subtilis. The mini-sensor is formed by six tandem carboxylate residues within the transmembrane (TM)5-6 loop on the intracellular membrane surface. Ca2+ binding to the mini-sensor triggers the transition of the transport mode of YfkE from a high-affinity to a low-affinity state. Molecular dynamics simulation and fluorescence resonance energy transfer analysis suggest that Ca2+ binding to the mini-sensor causes an adjacent segment, namely, the exchanger inhibitory peptide (XIP), to move toward the Ca2+ translocation pathway to interact with TM2a in an inward-open cavity. The specific interaction was demonstrated with a synthetic peptide of the XIP, which inhibits YfkE transport and interrupts conformational changes mediated by the mini-sensor. By comparing the apo and Ca2+-bound CAX structures, we propose the following Ca2+ transport regulatory mechanism of YfkE: Ca2+ binding to the mini-sensor induces allosteric conformational changes in the Ca2+ translocation pathway via the XIP, resulting in a rearrangement of the Ca2+-binding transport site in the midmembrane. Since the Ca2+ mini-sensor and XIP sequences are also identified in other CAX homologs and/or Ca2+ transporters, including the mammalian Na+/Ca2+ exchanger (NCX), our study provides a regulatory mechanism for the Ca2+/cation transporter superfamily.


Assuntos
Antiporters/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Cálcio/metabolismo , Citoplasma/metabolismo , Escherichia coli/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Sequência de Aminoácidos , Antiporters/genética , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Escherichia coli/genética , Mutação , Conformação Proteica , Homologia de Sequência , Trocador de Sódio e Cálcio/genética
9.
Nucleic Acids Res ; 48(10): 5332-5348, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32324221

RESUMO

The genomes of organisms from all three domains of life harbor endogenous base modifications in the form of DNA methylation. In bacterial genomes, methylation occurs on adenosine and cytidine residues to include N6-methyladenine (m6A), 5-methylcytosine (m5C), and N4-methylcytosine (m4C). Bacterial DNA methylation has been well characterized in the context of restriction-modification (RM) systems, where methylation regulates DNA incision by the cognate restriction endonuclease. Relative to RM systems less is known about how m6A contributes to the epigenetic regulation of cellular functions in Gram-positive bacteria. Here, we characterize site-specific m6A modifications in the non-palindromic sequence GACGmAG within the genomes of Bacillus subtilis strains. We demonstrate that the yeeA gene is a methyltransferase responsible for the presence of m6A modifications. We show that methylation from YeeA does not function to limit DNA uptake during natural transformation. Instead, we identify a subset of promoters that contain the methylation consensus sequence and show that loss of methylation within promoter regions causes a decrease in reporter expression. Further, we identify a transcriptional repressor that preferentially binds an unmethylated promoter used in the reporter assays. With these results we suggest that m6A modifications in B. subtilis function to promote gene expression.


Assuntos
Adenosina/análogos & derivados , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Adenosina/análise , Adenosina/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos , Metilação de DNA , Enzimas de Restrição-Modificação do DNA , Epigênese Genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/fisiologia , Fatores de Transcrição/metabolismo
10.
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
11.
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
12.
Mol Biol (Mosk) ; 54(1): 137-145, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32163397

RESUMO

Bacillus subtilis bacteria play an important role in veterinary medicine, medicine, and biotechnology, and the permanently growing demand for biotechnological products fuels the improvement of the properties of biotechnological strains. B. subtilis strains with improved characteristics maybe obtained by rational design and the directed evolution technologies, or be found among newly described strains. In the course of the long-term microbiome composition studies in the Russian segment of the International Space Station, the B. subtilis 20 strain was isolated, this strain shows the capacity for rapid growth and considerable biomass accumulation, as well as increased resistance to acidification of the environment in comparison to the "terrestrial" B. subtilis 168 strain. What is more, B. subtilis 20 is hyperresistant to the DNA and protein damaging factors that are linked to the overexpression of the genes controlling DNA repair, hydrogen sulfide production, and reactive oxygen species neutralization. The described properties of B. subtilis 20 are indicative of its considerable potential as a promising producer of biologically active compounds.


Assuntos
Bacillus subtilis/classificação , Bacillus subtilis/fisiologia , Biotecnologia/tendências , Bacillus subtilis/genética , Bacillus subtilis/isolamento & purificação
15.
Microb Cell Fact ; 19(1): 52, 2020 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111210

RESUMO

BACKGROUND: Bacillus subtilis is an important industrial workhorse applied in the production of many different commercially relevant proteins, especially enzymes. Virtually all of these proteins are secreted via the general secretion (Sec) pathway. Studies from different laboratories have demonstrated essential or non-essential contributions of various Sec machinery components to protein secretion in B. subtilis. However, a systematic comparison of the impact of each individual Sec machinery component under conditions of high-level protein secretion was so far missing. RESULTS: In the present study, we have compared the contributions of non-essential Sec pathway components and cell envelope-associated proteases on the secretion efficiency of three proteins expressed at high level. This concerned the α-amylases AmyE from B. subtilis and AmyL from Bacillus licheniformis, and the serine protease BPN' from Bacillus amyloliquefaciens. We compared the secretion capacity of mutant strains in shake flask cultures, and the respective secretion kinetics by pulse-chase labeling experiments. The results show that secDF, secG or rasP mutations severely affect AmyE, AmyL and BPN' secretion, but the actual effect size depends on the investigated protein. Additionally, the chaperone DnaK is important for BPN' secretion, while AmyE or AmyL secretion are not affected by a dnaK deletion. Further, we assessed the induction of secretion stress responses in mutant strains by examining AmyE- and AmyL-dependent induction of the quality control proteases HtrA and HtrB. Interestingly, the deletion of certain sip genes revealed a strong differential impact of particular signal peptidases on the magnitude of the secretion stress response. CONCLUSIONS: The results of the present study highlight the importance of SecDF, SecG and RasP for protein secretion and reveal unexpected differences in the induction of the secretion stress response in different mutant strains.


Assuntos
Bacillus subtilis/enzimologia , Membrana Celular/enzimologia , Peptídeo Hidrolases/biossíntese , Via Secretória , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Peptídeo Hidrolases/genética , Transporte Proteico , Canais de Translocação SEC/genética , Canais de Translocação SEC/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , alfa-Amilases/genética
16.
Can J Microbiol ; 66(6): 401-412, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32160477

RESUMO

Fusarium wilt is a devastating soil-borne disease mainly caused by highly host-specific formae speciales of Fusarium spp. Antagonistic microorganisms play a very important role in Fusarium wilt control. Isolation of potential biocontrol strains has become increasingly important. Bacterial strain SEM-2 was isolated from the high-temperature stage of silkworm excrement composting. SEM-2 exhibited a considerable antagonistic effect against Fusarium graminearum mycelial growth and spore germination. The results of pot experiments suggested that SEM-2 has a better inhibitory effect on the early stage of disease occurrence. The green fluorescent protein labelled SEM-2 coated on the surface of tomato seeds colonised the roots of tomato plants in 15 days. Genome sequencing identified SEM-2 as a new strain of Bacillus subtilis, and genome annotation and analysis determined gene clusters related to the biosynthesis of antimicrobials, such as bacillaene, fengycin, bacillibactin, subtilosin A, surfactin, and bacilysin. Interestingly, liquid chromatography - quadrupole time-of-flight mass spectrometry revealed that metabolites in pathways associated with the synthesis of secondary metabolites and antibiotics were highly differentially expressed. These findings may help to explain the mode of action of B. subtilis SEM-2 against Fusarium spp.


Assuntos
Antibiose , Bacillus subtilis/fisiologia , Bombyx/microbiologia , Fusarium/crescimento & desenvolvimento , Genoma Bacteriano/genética , Lycopersicon esculentum/microbiologia , Doenças das Plantas/prevenção & controle , Animais , Anti-Infecciosos/metabolismo , Bacillus subtilis/química , Bacillus subtilis/genética , Bacillus subtilis/isolamento & purificação , Agentes de Controle Biológico , Cromatografia Líquida , Fezes/microbiologia , Espectrometria de Massas , Família Multigênica/genética , Doenças das Plantas/microbiologia , Sementes/microbiologia
17.
PLoS Genet ; 16(3): e1008275, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32176689

RESUMO

Bacillus subtilis cells are well suited to study how bacteria sense and adapt to proteotoxic stress such as heat, since temperature fluctuations are a major challenge to soil-dwelling bacteria. Here, we show that the alarmones (p)ppGpp, well known second messengers of nutrient starvation, are also involved in the heat stress response as well as the development of thermo-resistance. Upon heat-shock, intracellular levels of (p)ppGpp rise in a rapid but transient manner. The heat-induced (p)ppGpp is primarily produced by the ribosome-associated alarmone synthetase Rel, while the small alarmone synthetases RelP and RelQ seem not to be involved. Furthermore, our study shows that the generated (p)ppGpp pulse primarily acts at the level of translation, and only specific genes are regulated at the transcriptional level. These include the down-regulation of some translation-related genes and the up-regulation of hpf, encoding the ribosome-protecting hibernation-promoting factor. In addition, the alarmones appear to interact with the activity of the stress transcription factor Spx during heat stress. Taken together, our study suggests that (p)ppGpp modulates the translational capacity at elevated temperatures and thereby allows B. subtilis cells to respond to proteotoxic stress, not only by raising the cellular repair capacity, but also by decreasing translation to concurrently reduce the protein load on the cellular protein quality control system.


Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Resposta ao Choque Térmico/genética , Ligases/genética , Regulação Bacteriana da Expressão Gênica/genética
18.
Nat Commun ; 11(1): 950, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32075967

RESUMO

Stochastic pulsing of gene expression can generate phenotypic diversity in a genetically identical population of cells, but it is unclear whether it has a role in the development of multicellular systems. Here, we show how stochastic pulsing of gene expression enables spatial patterns to form in a model multicellular system, Bacillus subtilis bacterial biofilms. We use quantitative microscopy and time-lapse imaging to observe pulses in the activity of the general stress response sigma factor σB in individual cells during biofilm development. Both σB and sporulation activity increase in a gradient, peaking at the top of the biofilm, even though σB represses sporulation. As predicted by a simple mathematical model, increasing σB expression shifts the peak of sporulation to the middle of the biofilm. Our results demonstrate how stochastic pulsing of gene expression can play a key role in pattern formation during biofilm development.


Assuntos
Bacillus subtilis/genética , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Bacillus subtilis/metabolismo , Bacillus subtilis/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Heterogeneidade Genética , Microscopia de Fluorescência , Modelos Biológicos , Fator sigma/genética , Fator sigma/metabolismo , Esporos Bacterianos/genética , Esporos Bacterianos/metabolismo , Esporos Bacterianos/fisiologia , Processos Estocásticos , Estresse Fisiológico , Imagem com Lapso de Tempo
19.
J Agric Food Chem ; 68(8): 2477-2484, 2020 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-32013418

RESUMO

Lacto-N-neotetraose (LNnT), one of the oligosaccharides in human milk, has many beneficial effects on infant health. In a recent work, we have constructed a recombinant Bacillus subtilis strain for the production of LNnT. Here, we further improved LNnT production with a xylose-induced clustered regularly interspaced short palindromic repeats interference system. In particular, the expressions of pfkA and pyk genes in the Embden-Meyerhof-Parnas pathway module, zwf gene in the pentose phosphate pathway module, and mnaA gene in the teichoic acid synthesis module were downregulated. The LNnT titer was increased from 1.32 to 1.55 g/L. Furthermore, to improve the conversion efficiency of lacto-N-triose II to LNnT, we knocked out tuaD gene in branch pathway and improved the expression of lgtB gene, resulting in the further increase of LNnT titer to 2.01 g/L. Finally, the addition time and amount of inducer xylose were optimized, and LNnT titer reached 2.30 g/L in shake flask and 5.41 g/L in 3 L bioreactor.


Assuntos
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Oligossacarídeos/biossíntese , Engenharia Metabólica , Xilose/metabolismo
20.
Nat Commun ; 11(1): 626, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-32005818

RESUMO

Potassium homeostasis is vital for all organisms, but is challenging in single-celled organisms like bacteria and yeast and immobile organisms like plants that constantly need to adapt to changing external conditions. KUP transporters facilitate potassium uptake by the co-transport of protons. Here, we uncover the molecular basis for transport in this widely distributed family. We identify the potassium importer KimA from Bacillus subtilis as a member of the KUP family, demonstrate that it functions as a K+/H+ symporter and report a 3.7 Å cryo-EM structure of the KimA homodimer in an inward-occluded, trans-inhibited conformation. By introducing point mutations, we identify key residues for potassium and proton binding, which are conserved among other KUP proteins.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Transporte de Cátions/química , Potássio/metabolismo , Bacillus subtilis/química , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Transporte Biológico , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Dimerização , Transporte de Íons , Modelos Moleculares , Família Multigênica , Domínios Proteicos
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