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1.
Environ Microbiol Rep ; 14(5): 755-765, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35940859

RESUMO

Several members of Cohnella genus have been reported as xylanolytic bacteria with significant capacity as carbohydrate-active enzyme producers (CAZymes), whose mechanisms involving xylan degradation are a key goal for suitable applications in bio-based industries. Using Cohnella sp. AR92 bacterium, we ensembled a genomic-proteomic approach to assess plant biomass conversion targeting its xylanolytic set of enzymes. Also, the genomic traits of the strain AR92 were compared to other Cohnella spp., showing a significant variability in terms of genome sizes and content of genes that code CAZymes. The AR92 strain genome harbours 209 CAZymes encoding sequences active on different polysaccharides, particularly directed towards xylans. Concurrent proteomic data recovered from cultures containing three kinds of lignocellulosic-derived substrates showed a broad set of xylan-degrading enzymes. The most abundant CAZymes expressed in the different conditions assayed were endo-ß-1,4-xylanases belonging to the GH11 and GH10 families, enzymes that were previously proved to be useful in the biotransformation of lignocellulosic biomass derived from sugarcane as well as onto xylan-enriched substrates. Therefore, considering the large reserve of CAZymes of Cohnella sp. AR92, a xylan processing model for AR92 strain is proposed.


Assuntos
Bacillales , Xilanos , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Humanos , Polissacarídeos , Proteoma , Proteômica , Xilanos/metabolismo
2.
Artigo em Inglês | MEDLINE | ID: mdl-35960646

RESUMO

A novel strictly aerobic, Gram-stain-positive, rod-shaped, motile, endospore-forming, white-coloured bacterium, designated strain MFER-1T, was isolated from a fermented liquor of wild grasses sampled in the Republic of Korea. The respiratory quinone of strain MFER-1T was menaquinone-7 and its major cellular fatty acids were anteiso-C15 : 0 (55.3 %), iso-C16 : 0 (17.5 %) and C16 : 0 (12.1 %). The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, four unidentified aminophospholipids and an unidentified phospholipid. The 16S rRNA gene sequence of strain MFER-1T showed similarity of 98.1 % to 'Cohnella cholangitidis' 1 605-214T and below 98.0 % sequence similarity to the other Cohnella species. The phylogenomic tree indicated that strain MFER-1T formed a reliable cluster with several Cohnella species. The estimated genome size of strain MFER-1T was 8.52 Mb. Genomic DNA G+C content was 50.7mol%. The orthologous average nucleotide identity, digital DNA-DNA hybridization and amino acid identity values of strain MFER-1T with the most closely related species 'Cohnella cholangitidis' 1 605-214T were 78.7, 23.0 and 79.6 %, respectively. Based on the phenotypic, chemotaxonomic and phylogenetic results, strain MFER-1T should represent a novel species of the genus Cohnella, for which the name Cohnella herbarum sp. nov. is proposed, with strain MFER-1T (=KACC 21 257T=NBRC 114 628T) as the type strain.


Assuntos
Bacillales , Poaceae , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Fermentação , Fosfolipídeos/química , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
3.
Appl Microbiol Biotechnol ; 106(12): 4669-4681, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35759037

RESUMO

Aneurinibacillus thermoaerophilus CCM 8960 is a thermophilic bacterium isolated from compost in Brno. The bacterium accumulates polyhydroxyalkanoates (PHAs), a biodegradable and renewable alternative to petrochemical polymers. The bacterium reveals several features that make it a very interesting candidate for the industrial production of PHA. At first, due to its thermophilic character, the bacterium can be utilized in agreement with the concept of next-generation industrial biotechnology (NGIB), which relies on extremophiles. Second, the bacterium is capable of producing PHA copolymers containing a very high portion of 4-hydroxybutyrate (4HB). Such materials possess unique properties and can be advantageously used in multiple applications, including but not limited to medicine and healthcare. Therefore, this work focuses on the in-depth characterization of A. thermoaerophilus CCM 8960. In particular, we sequenced and assembled the genome of the bacterium and identified its most important genetic features, such as the presence of plasmids, prophages, CRISPR arrays, antibiotic-resistant genes, and restriction-modification (R-M) systems, which might be crucial for the development of genome editing tools. Furthermore, we focused on genes directly involved in PHA metabolism. We also experimentally studied the kinetics of glycerol and 1,4-butanediol (1,4BD) utilization as well as biomass growth and PHA production during cultivation. Based on these data, we constructed a metabolic model to reveal metabolic fluxes and nodes of glycerol and 1,4BD concerning their incorporation into the poly(3-hydroxybutyrate-co-4-hydroxybutyrate (P(3HB-co-4HB)) structure. KEY POINTS: • Aneurinibacillus sp. H1 was identified as Aneurinibacillus thermoaerophilus. • PHA metabolism pathway with associated genes was presented. • Unique monomer composition of produced PHAs was reported.


Assuntos
Poli-Hidroxialcanoatos , Ácido 3-Hidroxibutírico , Bacillales , Butileno Glicóis , Glicerol , Poliésteres/metabolismo
4.
FEMS Microbiol Lett ; 369(1)2022 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-35687414

RESUMO

Biogenic coalbed methane is produced by biological processes mediated by synergistic interactions of microbial complexes in coal seams. However, the ecological role of functional bacteria in biogenic coalbed methane remains poorly understood. Here, we studied the metagenome assembled genomes (MAGs) of Bacillales and Clostridiales from coal seams, revealing further expansion of hydrogen and acetogen producers involved in organic matter decomposition. In this study, Bacillales and Clostridiales were dominant orders (91.85 ± 0.94%) in cultured coal seams, and a total of 16 MAGs from six families, including Bacillus, Paenibacillus, Staphylococcus, Anaerosalibacter, Hungatella and Paeniclostridium, were reconstructed. These microbial groups possessed multiple metabolic pathways (glycolysis/gluconeogenesis, pentose phosphate, ß-oxidation, TCA cycle, assimilatory sulfate reduction, nitrogen metabolism and encoding hydrogenase) that provided metabolic substrates (acetate and/or H2) for the methanogenic processes. Therein, the hydrogenase-encoding gene and hydrogenase maturation factors were merely found in all the Clostridiales MAGs. ß-oxidation was the main metabolic pathway involved in short-chain fatty acid degradation and acetate production, and most of these pathways were detected and exhibited different operon structures in Bacillales MAGs. In addition, assimilatory sulfate reduction and nitrogen metabolism processes were also detected in some MAGs, and these processes were also closely related to acetate production and/or organic matter degradation according to their operon structures and metabolic pathways. In summary, this study enabled a better understanding of the ecological roles of Bacillales and Clostridiales in biogenic methane in coal seams based on a combination of bioinformatic techniques.


Assuntos
Bacillales , Hidrogenase , Acetatos , Bacillales/metabolismo , Clostridiales/metabolismo , Carvão Mineral/microbiologia , Humanos , Metano/metabolismo , Nitrogênio , Sulfatos
5.
Sci Rep ; 12(1): 10301, 2022 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-35717508

RESUMO

Cellulases are hydrolytic enzymes with wide scientific and industrial applications. We described a novel cellulase, CelC307, from the thermophilic indigenous Cohnella sp. A01. The 3-D structure of the CelC307 was predicted by comparative modeling. Docking of CelC307 with specific inhibitors and molecular dynamic (MD) simulation revealed that these ligands bound in a non-competitive manner. The CelC307 protein was purified and characterized after recombinant expression in Escherichia coli (E. coli) BL21. Using CMC 1% as the substrate, the thermodynamic values were determined as Km 0.46 mM, kcat 104.30 × 10-3 (S-1), and kcat/Km 226.73 (M-1 S-1). The CelC307 was optimally active at 40 °C and pH 7.0. The culture condition was optimized for improved CelC307 expression using Plackett-Burman and Box-Behnken design as follows: temperature 20 °C, pH 7.5, and inoculation concentration with an OD600 = 1. The endoglucanase activity was positively modulated in the presence of Na+, Li+, Ca2+, 2-mercaptoethanol (2-ME), and glycerol. The thermodynamic parameters calculated for CelC307 confirmed its inherent thermostability. The characterized CelC307 may be a suitable candidate for various biotechnological applications.


Assuntos
Bacillales , Celulase , Celulases , Bacillales/metabolismo , Celulase/metabolismo , Celulases/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Íons , Temperatura
6.
Microbiologyopen ; 11(3): e1299, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35765181

RESUMO

As a hallmark of Archaea, their cell membranes are comprised of ether lipids. However, Archaea-type ether lipids have recently been identified in Bacteria as well, with a somewhat different composition: In Bacillales, sn-glycerol 1-phosphate is etherified with one C35 isoprenoid chain, which is longer than the typical C20 chain in Archaea, and instead of a second isoprenoid chain, the product heptaprenylglyceryl phosphate becomes dephosphorylated and afterward diacetylated by the O-acetyltransferase YvoF. Interestingly, database searches have revealed YvoF homologs in Halobacteria (Archaea), too. Here, we demonstrate that YvoF from Haloferax volcanii can acetylate geranylgeranylglycerol in vitro. Additionally, we present the first-time identification of acetylated diether lipids in H. volcanii and Halobacterium salinarum by mass spectrometry. A variety of different acetylated lipids, namely acetylated archaeol, and acetylated archaetidylglycerol, were found, suggesting that halobacterial YvoF has a broad substrate range. We suppose that the acetyl group might serve to modify the polarity of the lipid headgroup, with still unknown biological effects.


Assuntos
Archaea , Bacillales , Archaea/metabolismo , Éteres/química , Éteres/metabolismo , Espectrometria de Massas , Terpenos/metabolismo
7.
J Org Chem ; 87(10): 6588-6600, 2022 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-35537215

RESUMO

Stereoselective synthesis of d-glycero- and l-glycero-ß-d-mannoheptosides has been achieved by cesium carbonate-mediated ß-selective anomeric O-alkylation of the corresponding d-mannoheptoses. In addition, this method has been utilized in the total synthesis of a tetrasaccharide repeat unit of Bacillus thermoaerophilus surface-layer glycoprotein.


Assuntos
Glicoproteínas de Membrana , Oligossacarídeos , Alquilação , Bacillales , Glicoproteínas
8.
Microb Cell Fact ; 21(1): 39, 2022 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-35292016

RESUMO

BACKGROUND: The microbial production of hemicellulasic cocktails is still a challenge for the biorefineries sector and agro-waste valorization. In this work, the production of hemicellulolytic enzymes by Thermobacillus xylanilyticus has been considered. This microorganism is of interest since it is able to produce an original set of thermostable hemicellulolytic enzymes, notably a xylanase GH11, Tx-xyn11. However, cell-to-cell heterogeneity impairs the production capability of the whole microbial population. RESULTS: Sequential cultivations of the strain on xylan as a carbon source has been considered in order to highlight and better understand this cell-to-cell heterogeneity. Successive cultivations pointed out a fast decrease of xylanase activity (loss of ~ 75%) and Tx-xyn11 gene expression after 23.5 generations. During serial cultivations on xylan, flow cytometry analyses pointed out that two subpopulations, differing at their light-scattering properties, were present. An increase of the recurrence of the subpopulation exhibiting low forward scatter (FSC) signal was correlated with a progressive loss of xylanase activity over several generations. Cell sorting and direct observation of the sorted subpopulations revealed that the low-FSC subpopulation was not sporulating, whereas the high-FSC subpopulation contained cells at the onset of the sporulation stage. The subpopulation differences (growth and xylanase activity) were assessed during independent growth. The low-FSC subpopulation exhibited a lag phase of 10 h of cultivation (and xylanase activities from 0.15 ± 0.21 to 3.89 ± 0.14 IU/mL along the cultivation) and the high-FSC subpopulation exhibited a lag phase of 5 h (and xylanase activities from 0.52 ± 0.00 to 4.43 ± 0.61 over subcultivations). Serial cultivations on glucose, followed by a switch to xylan led to a ~ 1.5-fold to ~ 15-fold improvement of xylanase activity, suggesting that alternating cultivation conditions could lead to an efficient population management strategy for the production of xylanase. CONCLUSIONS: Taken altogether, the data from this study point out that a cheating behavior is responsible for the progressive reduction in xylanase activity during serial cultivations of T. xylanilyticus. Alternating cultivation conditions between glucose and xylan could be used as an efficient strategy for promoting population stability and higher enzymatic productivity from this bacterium.


Assuntos
Bacillales , Endo-1,4-beta-Xilanases , Bacillales/metabolismo , Carbono/metabolismo , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Xilanos/metabolismo
9.
Bioresour Technol ; 345: 126557, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34906701

RESUMO

To investigate the influences of sulfide oxidizing bacteria on H2S odor control in sewage sludge composting, a facultative chemolithotroph strain was isolated and identified as Cohnella thermotolerans LYH-2. Strain LYH-2 decreased the initially added sulfide by 94.6% when glucose and NH4Cl were used as the optimal energy substrates. The biotransformation of sulfide substrates followed first-order reaction kinetics, and the highest degradation rate constant (0.0537 h-1) and bacterial dry weight (0.745 g/L) were obtained at 300 mg/L of initial sulfide. The C. thermotolerans strain was inoculated as the bacterial agent into the sewage sludge and rice husk composting in forced ventilation composting reactors for 25 d; the bacterial inoculation prolonged the thermophilic period by 2 d, decreased 35.4% of H2S odor emission, and accelerated the composting process compared to the control group. The results demonstrated that C. thermotolerans inoculants effectively controlled H2S emission and promoted maturity in sewage sludge composting.


Assuntos
Compostagem , Sulfeto de Hidrogênio , Bacillales , Odorantes/análise , Oxirredução , Esgotos , Solo , Sulfetos
10.
Nat Commun ; 12(1): 6191, 2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34702830

RESUMO

Class 2 CRISPR systems are exceptionally diverse, nevertheless, all share a single effector protein that contains a conserved RuvC-like nuclease domain. Interestingly, the size of these CRISPR-associated (Cas) nucleases ranges from >1000 amino acids (aa) for Cas9/Cas12a to as small as 400-600 aa for Cas12f. For in vivo genome editing applications, compact RNA-guided nucleases are desirable and would streamline cellular delivery approaches. Although miniature Cas12f effectors have been shown to cleave double-stranded DNA, targeted DNA modification in eukaryotic cells has yet to be demonstrated. Here, we biochemically characterize two miniature type V-F Cas nucleases, SpCas12f1 (497 aa) and AsCas12f1 (422 aa), and show that SpCas12f1 functions in both plant and human cells to produce targeted modifications with outcomes in plants being enhanced with short heat pulses. Our findings pave the way for the development of miniature Cas12f1-based genome editing tools.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Edição de Genes , Bacillales/enzimologia , Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas , Clostridiales/enzimologia , Endodesoxirribonucleases/química , Células HEK293 , Humanos , Células Vegetais , Multimerização Proteica , RNA Guia/genética , RNA Guia/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Zea mays
11.
J Biotechnol ; 342: 64-71, 2021 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-34688788

RESUMO

The screening, identification, and study of the functional properties of cellulolytic xylanolytic bacteria are crucial for the construction of applicable bioprocesses. The thermophilic facultatively anaerobic, xylanolytic bacterial strain DA-C8 (=JCM34211=DSM111723) exhibiting efficient xylan degradation was newly isolated from compost. Strain DA-C8 completely degraded 1% beechwood xylan within 4 days under anaerobic conditions. By 16S rRNA gene sequence homology and phylogenetic tree analysis, strain DA-C8 was closely related to Paenibacillus cisolokensis and Xylanibacillus composti; however, the average nucleotide identity and digital DNA-DNA hybridization values based on genome information and the carbon source utilization properties indicated that strain DA-C8 belongs to Paenibacillus rather than Xylanibacillus. The gene numbers of xylanase and endoglucanase of strain DA-C8 and X. composti were not different; however, strain DA-C8 had higher abundance of α-L-arabinofuranosidase, ß-xylosidase, and ß-glucosidase than X. composti. Strain DA-C8 showed decreased xylan and corn hull degradation abilities and growth on xylan medium under aerobic conditions. Quantitative PCR showed high expression of xylan and cellulose degradation genes under anaerobic conditions, but the genes were repressed under aerobic conditions, indicating that strain DA-C8 controls polysaccharide degradation depending on the aeration conditions. Strain DA-C8 is a new species of Paenibacillus with a unique polysaccharide degradation system.


Assuntos
Paenibacillus , Xilanos , Anaerobiose , Bacillales , Composição de Bases , DNA Bacteriano , Paenibacillus/genética , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
12.
J Microbiol Methods ; 191: 106350, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34710512

RESUMO

Aerobic spore-forming Bacillales are a highly diverse and ubiquitous group that includes organisms that cause foodborne illnesses and food spoilage. Classical microbiological and biochemical identification of members of the order Bacillales represents a challenge due to the diversity of organisms in this group as well as the fact that the phenotypic-based taxonomic assignment of some named species in this group is not consistent with their phylogenomic characteristics. DNA-sequencing-based tools, on the other hand, can be fast and cost-effective, and can provide for a more reliable identification and characterization of Bacillales isolates. In comparison to 16S rDNA, rpoB was shown to better discriminate between Bacillales isolates and to allow for improved taxonomic assignment to the species level. However, the lack of a publicly accessible rpoB database, as well as the lack of standardized protocols for rpoB-based typing and strain identification, is a major challenge. Here, we report (i) the curation of a DNA sequence database for rpoB-based subtype classification of Bacillales isolates; (ii) the development of standardized protocols for generating rpoB sequence data, and a scheme for rpoB-based initial taxonomic identification of Bacillales isolates at the species level; and (iii) the integration of the database in a publicly accessible online platform that allows for the analysis of rpoB sequence data from uncharacterized Bacillales isolates. Specifically, we curated a database of DNA sequences for a 632-nt internal variable region within the rpoB gene from representative Bacillales reference type strains and a large number of isolates that we have previously isolated and characterized through multiple projects. As of May 21, 2021, the rpoB database contained more than 8350 rpoB sequences representing 1902 distinct rpoB allelic types that can be classified into 160 different genera. The database also includes 1129 rpoB sequences for representative Bacillales reference type strains as available on May 21, 2021 in the NCBI database. The rpoB database is integrated into the online Food Microbe Tracker platform (www.foodmicrobetracker.com) and can be queried using the integrated BLAST tool to initially subtype and taxonomically identify aerobic and facultative anaerobic spore-formers. While whole-genome sequencing is increasingly used in bacterial taxonomy, the rpoB sequence-based identification scheme described here provides a valuable tool as it allows for rapid and cost-effective initial isolate characterization, which can help to identify and characterize foodborne pathogens and food spoilage bacteria. In addition, the database and primers described here can also be adopted for metagenomics approaches that include rpoB as a target, improving discriminatory power and identification over what can be achieved using 16S rDNA as a target.


Assuntos
Bacillales/genética , Bacillales/isolamento & purificação , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana/métodos , RNA Polimerases Dirigidas por DNA/genética , Esporos/química , Alelos , Análise Custo-Benefício , Primers do DNA , DNA Ribossômico , Bases de Dados de Ácidos Nucleicos , Doenças Transmitidas por Alimentos , Metagenômica , Filogenia , Padrões de Referência , Sequenciamento Completo do Genoma
13.
World J Microbiol Biotechnol ; 37(10): 178, 2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34549358

RESUMO

Paludifilum halophilum DSM 102817T is the first member of the genus Paludifilum in the Thermoactinomycetaceae family. The thermohalophilic bacterium was isolated from the solar saltern of Sfax, Tunisia and was shown to be able to produce ectoines with a relatively high-yield and to cope with salt stress conditions. In this study, the whole genome of P. halophilum was sequenced and analysed. Analysis revealed 3,789,765 base pairs with an average GC% content of 51.5%. A total of 3775 genes were predicted of which 3616 were protein-coding genes and 73 were RNA genes. The genes encoding key enzymes for ectoines (ectoine and hydroxyectoine) synthesis (ectABCD) were identified from the bacterial genome next to a gene cluster (ehuABCD) encoding a binding-protein-dependent ABC transport system responsible for ectoines mobility through the cell membrane. With the aid of KEGG analysis, we found that the central catabolic network of P. halophilum comprises the pathways of glycolysis, tricarboxylic acid cycle, and pentose phosphate. In addition, anaplerotic pathways replenishing oxaloacetate and glutamate synthesis from central metabolism needed for high ectoines biosynthetic fluxes were identified through several key enzymes. Furthermore, a total of 18 antiSMASH-predicted putative biosynthetic gene clusters for secondary metabolites with high novelty and diversity were identified in P. halophilum genome, including biosynthesis of colabomycine-A, fusaricidin-E, zwittermycin A, streptomycin, mycosubtilin and meilingmycin. Based on these data, P. halophilum emerged as a promising source for ectoines and antimicrobials with the potential to be scaled up for industrial production, which could benefit the pharmaceutical and cosmetic industries.


Assuntos
Diamino Aminoácidos/metabolismo , Bacillales , Metabolismo Secundário/genética , Bacillales/genética , Bacillales/metabolismo , Biologia Computacional , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , Salinidade , Estresse Salino
14.
Arch Microbiol ; 203(10): 6053-6060, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34562146

RESUMO

A Gram-positive, aerobic, rod-shaped bacterium, designated as strain 1605-214T, was isolated from the blood sample of a patient with cholangitis. Based on its 16S rRNA gene sequence, the strain 1605-214T belonged to the genus Cohnella and exhibited 97.9% sequence identity with Cohnella luojiensis DSM 24270T (GQ214052). DNA-DNA hybridization, digital DNA-DNA hybridization, and average nucleotide identity values between the two species were 23% ± 1.9, 21.1%, and 77.2%, respectively. The cellular fatty acids of strain 1605-214T were mainly comprised of anteiso-C15:0 (36.1%), iso-C16:0 (16.5%), and C16:0 (15.1%). The predominant quinone was menaquinone-7; predominant polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, and aminophospholipid-1. The cell wall peptidoglycan of strain 1605-214T contained meso-diaminopimelic acid. DNA G + C content of strain 1605-214T was 50.6 mol%. 5187 genes out of a total of 5413 (94.6%) were assigned putative functions using eggNOG v5.0. Based on genotypic characteristics and genomic sequence analysis results, strain 1605-214T was confirmed to represent a novel species of genus Cohnella, for which the name Cohnella cholangitidis sp. nov., was proposed.


Assuntos
Ácidos Graxos , Fosfolipídeos , Bacillales , Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , Ácidos Graxos/análise , Humanos , Fosfolipídeos/análise , Filogenia , RNA Ribossômico 16S/genética , República da Coreia , Análise de Sequência de DNA , Vitamina K 2
15.
Nucleic Acids Res ; 49(18): 10589-10603, 2021 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-34478554

RESUMO

SR1 is a dual-function sRNA from Bacillus subtilis. It inhibits translation initiation of ahrC mRNA encoding the transcription activator of the arginine catabolic operons. Base-pairing is promoted by the RNA chaperone CsrA, which induces a slight structural change in the ahrC mRNA to facilitate SR1 binding. Additionally, SR1 encodes the small protein SR1P that interacts with glyceraldehyde-3P dehydrogenase A to promote binding to RNase J1 and enhancing J1 activity. Here, we describe a new target of SR1, kinA mRNA encoding the major histidine kinase of the sporulation phosphorelay. SR1 and kinA mRNA share 7 complementary regions. Base-pairing between SR1 and kinA mRNA decreases kinA translation without affecting kinA mRNA stability and represses transcription of the KinA/Spo0A downstream targets spoIIE, spoIIGA and cotA. The initial interaction between SR1 and kinA mRNA occurs 10 nt downstream of the kinA start codon and is decisive for inhibition. The sr1 encoded peptide SR1P is dispensable for kinA regulation. Deletion of sr1 accelerates sporulation resulting in low quality spores with reduced stress resistance and altered coat protein composition which can be compensated by sr1 overexpression. Neither CsrA nor Hfq influence sporulation or spore properties.


Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Biossíntese de Proteínas , Proteínas Quinases/genética , Pequeno RNA não Traduzido/fisiologia , Bacillales/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/metabolismo , Pareamento de Bases , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Proteínas Quinases/biossíntese , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/metabolismo , Esporos Bacterianos/química , Esporos Bacterianos/genética , Esporos Bacterianos/fisiologia , Fatores de Transcrição/metabolismo
16.
Bioorg Chem ; 116: 105245, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34482168

RESUMO

The GH-51 α-l-arabinofuranosidase from Thermobacillus xylanilyticus (TxAbf) possesses versatile catalytic properties, displaying not only the ability to hydrolyze glycosidic linkages but also to synthesize furanobiosides in α-l-Araf and ß-d-Galf series. Herein, mutants are investigated to evaluate their ability to perform self-condensation, assessing both yield improvements and changes in regioselectivity. Overall yields of oligo-α-l-arabino- and oligo-ß-d-galactofuranosides were increased up to 4.8-fold compared to the wild-type enzyme. In depth characterization revealed that the mutants exhibit increased transfer rates and thus a hydrolysis/self-condensation ratio in favor of synthesis. The consequence of the substitution N216W is the creation of an additional binding subsite that provides the basis for an alternative acceptor substrate binding mode. As a result, mutants bearing N216W synthesize not only (1,2)-linked furanobiosides, but also (1,3)- and even (1,5)-linked furanobiosides. Since the self-condensation is under kinetic control, the yield of homo-disaccharides was maximized using higher substrate concentrations. In this way, the mutant R69H-N216W produced oligo-ß-d-galactofuranosides in > 70% yield. Overall, this study further demonstrates the potential usefulness of TxAbf mutants for glycosynthesis and shows how these might be used to synthesize biologically-relevant glycoconjugates.


Assuntos
Bacillales/enzimologia , Inibidores Enzimáticos/farmacologia , Furanos/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Configuração de Carboidratos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Furanos/síntese química , Furanos/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Modelos Moleculares , Relação Estrutura-Atividade
17.
Nat Chem Biol ; 17(11): 1132-1138, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34475565

RESUMO

The RNA-guided CRISPR-associated (Cas) nucleases are versatile tools for genome editing in various organisms. The large sizes of the commonly used Cas9 and Cas12a nucleases restrict their flexibility in therapeutic applications that use the cargo-size-limited adeno-associated virus delivery vehicle. More compact systems would thus offer more therapeutic options and functionality for this field. Here, we report a miniature class 2 type V-F CRISPR-Cas genome-editing system from Acidibacillus sulfuroxidans (AsCas12f1, 422 amino acids). AsCas12f1 is an RNA-guided endonuclease that recognizes 5' T-rich protospacer adjacent motifs and creates staggered double-stranded breaks to target DNA. We show that AsCas12f1 functions as an effective genome-editing tool in both bacteria and human cells using various delivery methods, including plasmid, ribonucleoprotein and adeno-associated virus. The small size of AsCas12f1 offers advantages for cellular delivery, and characterizations of AsCas12f1 may facilitate engineering more compact genome-manipulation technologies.


Assuntos
Bacillales/química , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Edição de Genes
18.
World J Microbiol Biotechnol ; 37(8): 144, 2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34351499

RESUMO

Spores of many species of the orders Bacillales and Clostridiales can be vectors for food spoilage, human diseases and intoxications, and biological warfare. Many agents are used for spore killing, including moist heat in an autoclave, dry heat at elevated temperatures, UV radiation at 254 and more recently 222 and 400 nm, ionizing radiation of various types, high hydrostatic pressures and a host of chemical decontaminants. An alternative strategy is to trigger spore germination, as germinated spores are much easier to kill than the highly resistant dormant spores-the so called "germinate to eradicate" strategy. Factors important to consider in choosing methods for spore killing include the: (1) cost; (2) killing efficacy and kinetics; (3) ability to decontaminate large areas in buildings or outside; and (4) compatibility of killing regimens with the: (i) presence of people; (ii) food quality; (iii) presence of significant amounts of organic matter; and (iv) minimal damage to equipment in the decontamination zone. This review will summarize research on spore killing and point out some common flaws which can make results from spore killing research questionable.


Assuntos
Bacillales/crescimento & desenvolvimento , Clostridiales/crescimento & desenvolvimento , Desinfecção/métodos , Esporos Bacterianos/crescimento & desenvolvimento , Bacillales/efeitos dos fármacos , Clostridiales/efeitos da radiação , Desinfecção/instrumentação , Temperatura Alta , Humanos , Esporos Bacterianos/efeitos da radiação , Raios Ultravioleta
19.
J Econ Entomol ; 114(5): 2043-2050, 2021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34463330

RESUMO

The grain aphid Sitobion avenae (Fabricius) is one of the most important cereal pests, damaging crops through sap sucking and virus transmission. Sitobion avenae harbors the secondary endosymbiont Regiella insecticola, which is highly prevalent in populations in south-central Chile and other regions of the world. In order to develop ecological alternatives for biological control, we studied the effect of applying the spores of a strain of the bacterium Bacillus subtilis on the survival and fecundity of the most prevalent genotype of S. avenae in central Chile. The strain selected was one that in previous studies had shown the ability to outcompete other bacteria. Using clones of this aphid genotype infected and uninfected with R. insecticola, we found that applying B. subtilis spores through artificial diets and spraying on leaves decreased both adult survival and nymph production. The detection of spores within the aphid body was negatively correlated with nymph production and was lower in the presence of R. insecticola when applied in diets. B. subtilis spores applied on leaves reduced the number of aphids, an effect that was stronger on aphids harboring R. insecticola. A possible interaction between endosymbiotic bacteria and bacterial antagonists within the aphid body is discussed.


Assuntos
Afídeos , Bacillaceae , Bacillales , Animais , Bacillus subtilis , Enterobacteriaceae , Crescimento Demográfico , Esporos Bacterianos , Simbiose
20.
Appl Microbiol Biotechnol ; 105(18): 6759-6778, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34458936

RESUMO

The genus Cohnella belongs to a group of Gram-positive endospore-forming bacteria within the Paenibacillaceae family. Although most species were described as xylanolytic bacteria, the literature still lacks some key information regarding their repertoire of xylan-degrading enzymes. The whole genome sequence of an isolated xylan-degrading bacterium Cohnella sp. strain AR92 was found to contain five genes encoding putative endo-1,4-ß-xylanases, of which four were cloned, expressed, and characterized to better understand the contribution of the individual endo-xylanases to the overall xylanolytic properties of strain AR92. Three of the enzymes, CoXyn10A, CoXyn10C, and CoXyn11A, were shown to be effective at hydrolyzing xylans-derived from agro-industrial, producing oligosaccharides with substrate conversion values of 32.5%, 24.7%, and 10.6%, respectively, using sugarcane bagasse glucuronoarabinoxylan and of 29.9%, 19.1%, and 8.0%, respectively, using wheat bran-derived arabinoxylan. The main reaction products from GH10 enzymes were xylobiose and xylotriose, whereas CoXyn11A produced mostly xylooligosaccharides (XOS) with 2 to 5 units of xylose, often substituted, resulting in potentially prebiotic arabinoxylooligosaccharides (AXOS). The endo-xylanases assay displayed operational features (temperature optima from 49.9 to 50.4 °C and pH optima from 6.01 to 6.31) fitting simultaneous xylan utilization. Homology modeling confirmed the typical folds of the GH10 and GH11 enzymes, substrate docking studies allowed the prediction of subsites (- 2 to + 1 in GH10 and - 3 to + 1 in GH11) and identification of residues involved in ligand interactions, supporting the experimental data. Overall, the Cohnella sp. AR92 endo-xylanases presented significant potential for enzymatic conversion of agro-industrial by-products into high-value products.Key points• Cohnella sp. AR92 genome encoded five potential endo-xylanases.• Cohnella sp. AR92 enzymes produced xylooligosaccharides from xylan, with high yields.• GH10 enzymes from Cohnella sp. AR92 are responsible for the production of X2 and X3 oligosaccharides.• GH11 from Cohnella sp. AR92 contributes to the overall xylan degradation by producing substituted oligosaccharides.


Assuntos
Bacillales , Saccharum , Endo-1,4-beta-Xilanases/genética , Hidrólise , Oligossacarídeos , Xilanos
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