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1.
Artigo em Inglês | MEDLINE | ID: mdl-35100105

RESUMO

A yellow-coloured, Gram-stain-positive, motile, aerobic and rod-shaped bacteria, designated DKR-3T, was isolated from oil-contaminated experimental soil. Strain DKR-3T could grow at pH 5.0-10.5 (optimum, pH 7.0-8.5), at 10-40 °C (optimum, 25-32 °C) and tolerated 3.5 % of NaCl. Phylogenetic analyses based on its 16S rRNA gene sequence indicated that strain DKR-3T formed a lineage within the family Cellulomonadaceae and was clustered with members of the genus Cellulomonas. Strain DKR-3T had highest 16S rRNA gene sequence similarities to Cellulomonas gelida DSM 20111T (98.3 %), Cellulomonas persica JCM 18111T (98.2 %) and Cellulomonas uda DSM 20107T (97.8 %). The predominant respiratory quinone was tetrahydrogenated menaquinone with nine isoprene units [MK-9(H4)]. The principal cellular fatty acids were anteiso-C15 : 0, C16 : 0 and anteiso-C17 : 0. The major polar lipids were diphosphatidylglycerol and phosphatidylglycerol. The cell-wall diamino acid was l-ornithine whereas rhamnose and glucose were the cell-wall sugars. The DNA G+C content was 74.2mol %. The genome of strain DKR-3T was 3.74 Mb and contained three putative biosynthetic gene clusters. The average nucleotide identity and digital DNA-DNA hybridization relatedness values between strain DKR-3T and its phylogenetically related members were below the species threshold values. Based on a polyphasic study, strain DKR-3T represents a novel species belonging to the genus Cellulomonas, for which the name Cellulomonas fulva sp. nov. is proposed. The type strain is DKR-3T (=KACC 22071T=NBRC 114730T).


Assuntos
Cellulomonas , Poluição por Petróleo , Filogenia , Microbiologia do Solo , Técnicas de Tipagem Bacteriana , Composição de Bases , Cellulomonas/classificação , Cellulomonas/isolamento & purificação , DNA Bacteriano/genética , Ácidos Graxos/química , Hibridização de Ácido Nucleico , Fosfolipídeos/química , Pigmentação , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Poluentes do Solo
2.
Arch Microbiol ; 204(2): 139, 2022 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-35032191

RESUMO

In the present study, 27 bacterial strains were isolated from environmental samples and screened for higher lignocellulose-degrading efficiency. The best degrader was combined in pairs with 14 strains with high ß-glucosidase activity to formulate a consortium. Microbial consortium 625 showed high lignocellulose degradation efficiency. ZJW-6 with low ß-glucosidase activity and the best lignocellulose decomposer was identified as a member of Cellulomonas. Consortium 625 composed of ZJW-6 and DA-25, an Acinetobacter, showed the highest degradation rate (57.62%) under optimized conditions. The DA-25 filtrate promoted ZJW-6 growth, upregulating the activity of key lignocellulose-degrading enzymes, including ß-glucosidase, endoglucanase, xylanase, laccase, and lignin peroxidase. ZJW-6 and DA-25 worked in a subordination manner when co-cultivated. ZJW-6 acted as the major decomposer whose growth and enzymatic activities were promoted by DA-25. This study proposes a novel microbial consortium with improved lignocellulose degradation efficiency and reduce the C:N ratio of lignocellulose materials, which can enhance bioenergy production.


Assuntos
Acinetobacter , Cellulomonas , Lignina , Consórcios Microbianos
3.
Ying Yong Sheng Tai Xue Bao ; 32(8): 2982-2988, 2021 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-34664473

RESUMO

In this study, three dominant bacteria Cellulomonas flavigena (Ⅰ), Cellulomonas flavigena (Ⅱ), Sphingomonas paucimobilis (Ⅲ) from Fire Phoenix rhizosphere soil were used to develop a multi-microbial agent system. For oil-contaminated soil in the Dagang oilfield, the immobilized test bacteria were inoculated into the Fire Phoenix rhizosphere soil to examine the effects of bacterial agents on polycyclic aromatic hydrocarbons (PAHs)-contaminated soil. The results showed that PAHs degradation was promoted under the ⅠⅢ (with an effective number of viable bacteria of 109 cfu·mL-1) and ⅠⅡⅢ (with an effective number of viable bacteria of 107 cfu·mL-1) treatments. The PAHs degradation rates were 32.2% and 41.4%, respectively, being significantly higher than that in the control treatments. The ⅠⅡⅢ treatment significantly promoted the belowground biomass of Fire Phoenix, which was 31.2% higher than that of the control treatment. Our results suggested that the multi-microbial agent constructed by the three dominant bacteria ⅠⅡⅢ could be used as a strengthening method for the remediation of PAHs-contaminated soil by Fire Phoenix, which provided a novel method for microbial enhanced phytoremediation technology.


Assuntos
Hidrocarbonetos Policíclicos Aromáticos , Poluentes do Solo , Bactérias , Cellulomonas , Hidrocarbonetos Policíclicos Aromáticos/análise , Solo , Microbiologia do Solo , Poluentes do Solo/análise , Sphingomonas
4.
Artigo em Inglês | MEDLINE | ID: mdl-34569921

RESUMO

Cellulomonas algicola KZ-21T was compared with Cellulomonas aurantiaca THG-SMD2.3T to examine the taxonomic relationship between the two type strains. The 16S rRNA gene sequence of Cellulomonas algicola KZ-21T shared complete similarity (100.0 %) with that of Cellulomonas aurantiaca THG-SMD2.3T. The results of phylogenetic analyses based on 16S rRNA gene sequences indicated that the two strains formed a tight cluster within the genus Cellulomonas. Genome comparison between the two strains revealed an average nucleotide identity of 99.2 % and a digital DNA-DNA hybridization estimate of 93.7±1.8 %, strongly indicating that the two strains belong to a single species. In addition, neither strain displayed any striking differences in metabolic, physiological or chemotaxonomic features. Therefore, we propose Cellulomonas aurantiaca as a later heterotypic synonym of Cellulomonas algicola.


Assuntos
Cellulomonas , Técnicas de Tipagem Bacteriana , Composição de Bases , Cellulomonas/genética , DNA Bacteriano/genética , Ácidos Graxos/química , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
5.
J Microbiol Biotechnol ; 31(11): 1519-1525, 2021 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-34489371

RESUMO

Hexavalent chromium (Cr(VI)) is recognized to be carcinogenic and toxic and registered as a contaminant in many drinking water regulations. It occurs naturally and is also produced by industrial processes. The reduction of Cr(VI) to Cr(III) has been a central topic for chromium remediation since Cr(III) is less toxic and less mobile. In this study, fermentative Fe(III)-reducing bacterial strains (Cellu-2a, Cellu-5a, and Cellu-5b) were isolated from a groundwater sample and were phylogenetically related to species of Cellulomonas by 16S rRNA gene analysis. One selected strain, Cellu-2a showed its capacity of reduction of both soluble iron (ferric citrate) and solid iron (hydrous ferric oxide, HFO), as well as aqueous Cr(VI). The strain Cellu-2a was able to reduce 15 µM Cr(VI) directly with glucose or sucrose as a sole carbon source under the anaerobic condition and indirectly with one of the substrates and HFO in the same incubations. The heterogeneous reduction of Cr(VI) by the surface-associated reduced iron from HFO by Cellu-2a likely assisted the Cr(VI) reduction. Fermentative features such as large-scale cell growth may impose advantages on the application of bacterial Cr(VI) reduction over anaerobic respiratory reduction.


Assuntos
Biodegradação Ambiental , Cellulomonas/metabolismo , Cromo/metabolismo , Compostos Férricos/metabolismo , Fermentação , Água Subterrânea/microbiologia , Filogenia , RNA Ribossômico 16S , República da Coreia
6.
Microsc Res Tech ; 84(12): 2947-2959, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34196062

RESUMO

Drought is a persistent and complex natural vulnerability whose rate and extent of recurrence are expected to increase with climate change. Regardless of the progress made in responding and adapting to water scarcity, drought stress causes severe afflictions. Therefore, the present study has been accomplished in Department of Botany, University of Peshawar to investigate the effect of biochar and plant growth promoting rhizobacteria (PGPR) Cellulomonas pakistanensis (NCCP11) and Sphingobacterium pakistanensis (NCCP246) on Vicia faba under drought stress. Two varieties of seeds Desi (V1) and Pulista (V2) were obtained from Cereal Crop Research Institute (CCRI) Nowshera, sown in earthen pots in triplicate filled with 3 kg soil and sand (2:1) and biochar (0 and 5% w/w). Scanning electron microscopy of biochar showed porous nature and energy dispersive x-ray spectroscopy spectroscopy showed C, Ca, Mg, and Na elemental composition. Germination parameters including germination energy (GE), Timson germination index (TGI), germination index (GI), and water use efficiency (WUE) were amplified to 28.04, 19.17, 25.72, and 43.62% in V1, respectively, and 14.38, 16.66, 19.79, and 41.50% in V2 respectively, by the co-application of biochar and PGPR. Agronomical attributes including, fresh and dry weight of leaves, root, and shoot were significantly reduced, which were positively ameliorated by 28.57, 36.36, 16, 10.47, 14.28, and 10%, respectively, by the application of biochar and PGPR especially by NCCP246 in combination as well as individually. It has been concluded that, adversities of drought significantly condensed with the application of biochar and PGPR, which may be important in agricultural practices carried out in water-deficient regions.


Assuntos
Secas , Vicia faba , Cellulomonas , Carvão Vegetal , Sphingobacterium
7.
Biosci Biotechnol Biochem ; 85(9): 1971-1985, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34232281

RESUMO

Cellulomonas uda produces Xyn11A, moderately thermostable xylanase, with optimal activity at 50 °C and pH 6.5. An improvement in the biochemical properties of Xyn11A was achieved by site-directed mutagenesis approach. Wild-type xylanase, Xyn11A-WT, and its mutant Xyn11A-N9Y were expressed in Escherichia coli, and then both enzymes were purified and characterized. Xyn11A-N9Y displayed optimal activity at 60 °C and pH 7.5, an upward shift of 10 °C in the optimum temperature and an upward shift of 1 unit in optimum pH; also, it manifested an 11-fold increase in thermal stability at 60 °C, compared to that displayed by Xyn11A-WT. Molecular dynamics simulations of Xyn11A-WT and Xyn11A-N9Y suggest that the substitution N9Y leads to an array of secondary structure changes at the N-terminal end and an increase in the number of hydrogen bonds in Xyn11A-N9Y. Based on the significant improvements, Xyn11A-N9Y may be considered as a candidate for several biotechnological applications.


Assuntos
Cellulomonas/enzimologia , Endo-1,4-beta-Xilanases/genética , Mutação , Sequência de Aminoácidos , Catálise , Endo-1,4-beta-Xilanases/química , Escherichia coli/genética , Simulação de Dinâmica Molecular , Conformação Proteica
8.
BMC Microbiol ; 21(1): 177, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34116639

RESUMO

BACKGROUND: A bacterial consortium SCP comprising three bacterial members, viz. Stenotrophomonas acidaminiphila APG1, Pseudomonas stutzeri APG2 and Cellulomonas sp. APG4 was developed for degradation of the mono-azo dye, Reactive Blue 28. The genomic analysis of each member of the SCP consortium was done to elucidate the catabolic potential and role of the individual organism in dye degradation. RESULTS: The genes for glycerol utilization were detected in the genomes of APG2 and APG4, which corroborated with their ability to grow on a minimal medium containing glycerol as the sole co-substrate. The genes for azoreductase were identified in the genomes of APG2 and APG4, while no such trait could be determined in APG1. In addition to co-substrate oxidation and dye reduction, several other cellular functions like chemotaxis, signal transduction, stress-tolerance, repair mechanisms, aromatic degradation, and copper tolerance associated with dye degradation were also annotated. A model for azo dye degradation is postulated, representing the predominant role of APG4 and APG2 in dye metabolism while suggesting an accessory role of APG1. CONCLUSIONS: This exploratory study is the first-ever attempt to divulge the genetic basis of azo-dye co-metabolism by cross-genome comparisons and can be harnessed as an example for demonstrating microbial syntrophy.


Assuntos
Compostos Azo/metabolismo , Cellulomonas/metabolismo , Corantes/metabolismo , Pseudomonas stutzeri/metabolismo , Stenotrophomonas/metabolismo , Biodegradação Ambiental , Cellulomonas/genética , Cellulomonas/crescimento & desenvolvimento , Meios de Cultura/metabolismo , Genoma Bacteriano , Consórcios Microbianos , Pseudomonas stutzeri/genética , Pseudomonas stutzeri/crescimento & desenvolvimento , Stenotrophomonas/genética , Stenotrophomonas/crescimento & desenvolvimento
9.
Appl Microbiol Biotechnol ; 105(11): 4577-4588, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34019113

RESUMO

One of the main distinguishing features of bacteria belonging to the Cellulomonas genus is their ability to secrete multiple polysaccharide degrading enzymes. However, their application in biomass deconstruction still constitutes a challenge. We addressed the optimisation of the xylanolytic activities in extracellular enzymatic extracts of Cellulomonas sp. B6 and Cellulomonas fimi B-402 for their subsequent application in lignocellulosic biomass hydrolysis by culture in several substrates. As demonstrated by secretomic profiling, wheat bran and waste paper resulted to be suitable inducers for the secretion of xylanases of Cellulomonas sp. B6 and C. fimi B-402, respectively. Both strains showed high xylanolytic activity in culture supernatant although Cellulomonas sp. B6 was the most efficient xylanolytic strain. Upscaling from flasks to fermentation in a bench scale bioreactor resulted in equivalent production of extracellular xylanolytic enzymatic extracts and freeze drying was a successful method for concentration and conservation of the extracellular enzymes, retaining 80% activity. Moreover, enzymatic cocktails composed of combined extra and intracellular extracts effectively hydrolysed the hemicellulose fraction of extruded barley straw into xylose and xylooligosaccharides. KEY POINTS: • Secreted xylanase activity of Cellulomonas sp. B6 and C. fimi was maximised. • Biomass-induced extracellular enzymes were identified by proteomic profiling. • Combinations of extra and intracellular extracts were used for barley straw hydrolysis.


Assuntos
Cellulomonas , Biomassa , Endo-1,4-beta-Xilanases , Hidrólise , Proteômica
10.
Methods Mol Biol ; 2290: 187-201, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34009591

RESUMO

Polymerase chain reaction (PCR) is a popular molecular tool for detection of bacteria. PCR allows millions of copies of a target segment of DNA to be produced. The DNA is extracted from overnight grown cultures of pure bacterial isolates using either the organo-solvent method or a commercial DNA extraction kit. The quality and purity of the DNA is determined by performing gel electrophoresis on 0.8% agarose gel. The DNA is amplified by performing PCR assay. Bands of approximately 1.5 kb in size are obtained from the amplified products of DNA. The PCR products run on 1.5% agarose gel are visualized with UV light and imaged by gel documentation system. This chapter outlines the protocol for isolation and amplification of DNA from cellulolytic bacteria. Cellulolytic bacteria are considered a potential source of cellulases for pretreatment of crop residues during biogas production. PCR is considered a very powerful, sensitive, specific, fast, and reliable tool in molecular detection and diagnostics.


Assuntos
Biocombustíveis/microbiologia , DNA Bacteriano/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Bacillus/genética , Bactérias/classificação , Bactérias/genética , Cellulomonas/genética , Clostridium/genética , DNA Bacteriano/genética , Eletroforese/métodos , Pseudomonas/genética , Rhodothermus/genética
11.
Antonie Van Leeuwenhoek ; 114(5): 527-538, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33710455

RESUMO

A Gram-positive, smooth, sub-transparent, faint yellow,0.5-0.7 µm diameter, rod shaped aerobic or facultative aerobic strain P40-2Twas isolated from livestock farms in Northeast China. Strain P40-2T grew at 25-40 °C (optimum 30-38 °C), and in 0-4% (w/v) NaCl (optimum 0%) in LB medium. Based on 16S rRNA gene sequence analysis, strain P40-2T belongs to the class Cellulomonas and is most closely related to C. denverensis strain W6929, C. pakistanensis strain NCCP-11and C. hominis strain CE40.DNA-DNA hybridization rate of strain P40-2T was 29%, and the ANI with C.denverensisstrainW6929 was 85.33%. The genome is 3437431 bp long with a G + C content of 71.99%. Of the 3177 predicted genes, 3119 were protein-coding genes and 58 were RNA encoding genes. The chemotaxonomic data: menaquinone was MK-9(H4), anteiso-C15: 0, C16:0 and anteiso-C17: 0 were the major cellular fatty acids, and the main cell-wall amino acids were ornithine,alanine, glycine and glutamate. The cell wall peptidogly can sugars included glucose, rhamnose, galactose and mannose. The polar lipid present were DPG, PG, PE, and PIM. On the basis of DNA-DNA relatedness, phylogenetic position, complete genome sequence and physiological characteristics, strain P40-2T can be differentiated from other species of the genus Cellulomonas with validly published names and thus represents a novel species, for which the name Cellulomonas taurus is proposed. The type strain is Cellulomonas taurus P40-2T (= CGMCC No.1.17732T).The acute toxicity test in mice showed that LD50 of strain P40-2T was rather high with 1.5 × 1011 CFU/mouse, which indicated low pathogenicity. Drug susceptibility showed that strainP40-2T was resistant to most antibiotics and only sensitive to six antibiotics. Strain P40-2T contained a variety of hydrolytic enzymes including the ability to hydrolyze cellulose, ß-glucan, chitin, xylan, and casein. Microbial flocculant MBF-P40 for sewage was prepared with strain P40-2T, after strain P40-2T was confirmed that had good flocculation effect. MBF-P40 was used to prepare flocculation rate of 99.40%. MBF-P40 treatmented sewage from eight different sources. Flocculation rate for pig farm wastewater was 96.07%, COD removal rate is 71.05%, ammonia nitrogen removal rate is 18.22%. The result shows that MBF-P40 has a good flocculation effect, and good prospect of development and application for wastewater treatment.


Assuntos
Cellulomonas , Purificação da Água , Animais , Técnicas de Tipagem Bacteriana , Composição de Bases , Cellulomonas/genética , DNA Bacteriano/genética , Ácidos Graxos/análise , Hidrolases , Gado , Camundongos , Fosfolipídeos/análise , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Suínos , Vitamina K 2
12.
Curr Microbiol ; 78(4): 1135-1141, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33683416

RESUMO

Cellulomonas sp. JZ18 is a gram-positive, rod shaped bacterium that was previously isolated from the root endosphere of the perennial desert tussock-grass Panicum turgidum. Genome coverage of PacBio sequencing was approximately 199X. Genome assembly generated a single chromosome of 7,421,843 base pairs with a guanine-cytosine (GC) content of 75.60% with 3240 protein coding sequences, 361 pseudo genes, three ribosomal RNA operons, three non-coding RNAs and 45 transfer RNAs. Comparison of JZ18's genome with type strains from the same genus, using digital DNA-DNA hybridization and average nucleotide identity calculations, revealed that JZ18 might potentially belong to a new species. Functional analysis revealed the presence of genes that may complement previously observed biochemical and plant phenotypes. Furthermore, the presence of a number of enzymes could be of potential use in industrial processes as biocatalysts. Genome sequencing and analysis, coupled with comparative genomics, of endophytic bacteria for their potential plant growth promoting activities under different soil conditions will accelerate the knowledge and applications of biostimulants in sustainable agriculture.


Assuntos
Cellulomonas , Panicum , Bactérias , Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , Ácidos Graxos , Hibridização de Ácido Nucleico , Filogenia , RNA Ribossômico 16S , Análise de Sequência de DNA
13.
J Am Chem Soc ; 143(6): 2500-2508, 2021 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-33529004

RESUMO

Electrostatic forces are important for protein folding and are favored targets of protein engineering. However, interactions between charged residues are difficult to study because of the complex network of interactions found in most proteins. We have designed a purposely simple system to investigate this problem by systematically introducing individual and pairs of charged and titratable residues in a protein otherwise free of such residues. We used constant pH molecular dynamics simulations, NMR spectroscopy, and thermodynamic double mutant cycles to probe the structure and energetics of the interaction between the charged residues. We found that the partial burial of surface charges contributes to a shift in pKa value, causing an aspartate to titrate in the neutral pH range. Additionally, the interaction between pairs of residues was found to be highly context dependent, with some pairs having no apparent preferential interaction, while other pairs would engage in coupled titration forming a highly stabilized salt bridge. We find good agreement between experiments and simulations and use the simulations to rationalize our observations and to provide a detailed mechanistic understanding of the electrostatic interactions.


Assuntos
Celulase/química , Eletricidade Estática , Ácido Aspártico/química , Celulase/genética , Cellulomonas/enzimologia , Histidina/química , Simulação de Dinâmica Molecular , Mutação , Conformação Proteica , Domínios Proteicos/genética , Desdobramento de Proteína , Termodinâmica
14.
Int J Syst Evol Microbiol ; 70(10): 5304-5311, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32877326

RESUMO

Two Gram-stain-positive, facultatively anaerobic, motile, aerobic, rod-shaped and non-spore-forming actinobacteria, strains AO-9T and AO-18, were isolated from paddy soil collected from Daejeon, Republic of Korea. Colonies were smooth, lemon-yellow and circular and 0.5-0.8×2.0-2.4 µm in diameter after 3 days of incubation at 28 °C on tryptic soy agar. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strains AO-9T and AO-18 belonged to the genus Cellulomonas, showing the highest sequence similarities to Cellulomonas marina FXJ8.089T (96.6 %), Cellulomonas endophytica SYSUP0004T (96.5 %), Cellulomonas gelida DSM 20111T (96.2 %), Cellulomonas uda DSM 20107T (96.1 %), Cellulomonas rhizosphaerae NEAU-TCZ24T (96.1 %), Cellulomonas composti TR7-06T (96.0 %), Cellulomonas persica JCM 18111T (96.0 %) and less than 96 % to other closely related species. The DNA-DNA hybridization values between strains AO-9T and AO-18 were 87 %. The average nucleotide identity and digital DNA-DNA hybridization values between strain AO-9T and type strains of related species of the genus Cellulomonas were 84.0-85.8 % and 20.3-20.9 %, respectively. The major cellular fatty acids are anteiso-C15:0 (49.9 %), C14:0 (12.9 %) and iso-C14:0 (12.1 %). The predominant isoprenoid quinone was MK-9 (H4). The polar lipid profile consists of diphosphatidylglycerol, phosphatidylglycerol and one unidentified lipid. The DNA G+C content was 72.9 mol%. Based on its distinctive phenotypic, phylogenetic and chemotaxonomic characteristics, the two strains are considered to represent novel species of the genus Cellulomonas, for which the name Cellulomonas citrea sp. nov. is proposed. The type strain is AO-9T (=KACC 19069T=NBRC 112523T).


Assuntos
Cellulomonas/classificação , Oryza , Filogenia , Microbiologia do Solo , Técnicas de Tipagem Bacteriana , Composição de Bases , Cellulomonas/isolamento & purificação , DNA Bacteriano/genética , Ácidos Graxos/química , Hibridização de Ácido Nucleico , Pigmentação , RNA Ribossômico 16S/genética , República da Coreia , Análise de Sequência de DNA , Vitamina K 2/análogos & derivados , Vitamina K 2/química
15.
Proc Natl Acad Sci U S A ; 117(33): 19896-19903, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747547

RESUMO

Cellulose is the most abundant biomass on Earth, and many microorganisms depend on it as a source of energy. It consists mainly of crystalline and amorphous regions, and natural degradation of the crystalline part is highly dependent on the degree of processivity of the degrading enzymes (i.e., the extent of continuous hydrolysis without detachment from the substrate cellulose). Here, we report high-speed atomic force microscopic (HS-AFM) observations of the movement of four types of cellulases derived from the cellulolytic bacteria Cellulomonas fimi on various insoluble cellulose substrates. The HS-AFM images clearly demonstrated that two of them (CfCel6B and CfCel48A) slide on crystalline cellulose. The direction of processive movement of CfCel6B is from the nonreducing to the reducing end of the substrate, which is opposite that of processive cellulase Cel7A of the fungus Trichoderma reesei (TrCel7A), whose movement was first observed by this technique, while CfCel48A moves in the same direction as TrCel7A. When CfCel6B and TrCel7A were mixed on the same substrate, "traffic accidents" were observed, in which the two cellulases blocked each other's progress. The processivity of CfCel6B was similar to those of fungal family 7 cellulases but considerably higher than those of fungal family 6 cellulases. The results indicate that bacteria utilize family 6 cellulases as high-processivity enzymes for efficient degradation of crystalline cellulose, whereas family 7 enzymes have the same function in fungi. This is consistent with the idea of convergent evolution of processive cellulases in fungi and bacteria to achieve similar functionality using different protein foldings.


Assuntos
Proteínas de Bactérias/química , Celulases/química , Cellulomonas/enzimologia , Proteínas Fúngicas/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Evolução Biológica , Celulases/genética , Celulases/metabolismo , Cellulomonas/química , Cellulomonas/genética , Cellulomonas/metabolismo , Celulose/química , Celulose/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Cinética , Microscopia de Força Atômica
16.
J Biol Chem ; 295(43): 14606-14617, 2020 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-32816991

RESUMO

Cellobiohydrolases directly convert crystalline cellulose into cellobiose and are of biotechnological interest to achieve efficient biomass utilization. As a result, much research in the field has focused on identifying cellobiohydrolases that are very fast. Cellobiohydrolase A from the bacterium Cellulomonas fimi (CfCel6B) and cellobiohydrolase II from the fungus Trichoderma reesei (TrCel6A) have similar catalytic domains (CDs) and show similar hydrolytic activity. However, TrCel6A and CfCel6B have different cellulose-binding domains (CBDs) and linkers: TrCel6A has a glycosylated peptide linker, whereas CfCel6B's linker consists of three fibronectin type 3 domains. We previously found that TrCel6A's linker plays an important role in increasing the binding rate constant to crystalline cellulose. However, it was not clear whether CfCel6B's linker has similar function. Here we analyze kinetic parameters of CfCel6B using single-molecule fluorescence imaging to compare CfCel6B and TrCel6A. We find that CBD is important for initial binding of CfCel6B, but the contribution of the linker to the binding rate constant or to the dissociation rate constant is minor. The crystal structure of the CfCel6B CD showed longer loops at the entrance and exit of the substrate-binding tunnel compared with TrCel6A CD, which results in higher processivity. Furthermore, CfCel6B CD showed not only fast surface diffusion but also slow processive movement, which is not observed in TrCel6A CD. Combined with the results of a phylogenetic tree analysis, we propose that bacterial cellobiohydrolases are designed to degrade crystalline cellulose using high-affinity CBD and high-processivity CD.


Assuntos
Proteínas de Bactérias/química , Cellulomonas/enzimologia , Celulose 1,4-beta-Celobiosidase/química , Proteínas Fúngicas/química , Hypocreales/enzimologia , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Domínio Catalítico , Cellulomonas/química , Cellulomonas/metabolismo , Celulose/metabolismo , Celulose 1,4-beta-Celobiosidase/metabolismo , Cristalografia por Raios X , Proteínas Fúngicas/metabolismo , Hypocreales/química , Hypocreales/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Especificidade por Substrato
17.
Appl Microbiol Biotechnol ; 104(19): 8327-8337, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32803296

RESUMO

Cellodextrins are non-digestible oligosaccharides that have attracted interest from the food industry as potential prebiotics. They are typically produced through the partial hydrolysis of cellulose, resulting in a complex mixture of oligosaccharides with a varying degree of polymerisation (DP). Here, we explore the defined synthesis of cellotriose as product since this oligosaccharide is believed to be the most potent prebiotic in the mixture. To that end, the cellobiose phosphorylase (CBP) from Cellulomonas uda and the cellodextrin phosphorylase (CDP) from Clostridium cellulosi were evaluated as biocatalysts, starting from cellobiose and α-D-glucose 1-phosphate as acceptor and donor substrate, respectively. The CDP enzyme was shown to rapidly elongate the chains towards higher DPs, even after extensive mutagenesis. In contrast, an optimised variant of CBP was found to convert cellobiose to cellotriose with a molar yield of 73%. The share of cellotriose within the final soluble cellodextrin mixture (DP2-5) was 82%, resulting in a cellotriose product with the highest purity reported to date. Interestingly, the reaction could even be initiated from glucose as acceptor substrate, which should further decrease the production costs.Key points• Cellobiose phosphorylase is engineered for the production of cellotriose.• Cellotriose is synthesised with the highest purity and yield to date.• Both cellobiose and glucose can be used as acceptor for cellotriose production.


Assuntos
Cellulomonas , Glucosiltransferases , Celobiose , Celulose , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Especificidade por Substrato , Trioses
18.
J Appl Microbiol ; 129(3): 590-600, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32259336

RESUMO

AIMS: This study was done to obtain denitrifiers that could be used for bioaugmentation in woodchip bioreactors to remove nitrate from agricultural subsurface drainage water. METHODS AND RESULTS: We isolated denitrifiers from four different bioreactors in Minnesota, and characterized the strains by measuring their denitrification rates and analysing their whole genomes. A total of 206 bacteria were isolated from woodchips and thick biofilms (bioslimes) that formed in the bioreactors, 76 of which were able to reduce nitrate at 15°C. Among those, nine potential denitrifying strains were identified, all of which were isolated from the woodchip samples. Although many nitrate-reducing strains were isolated from the bioslime samples, none were categorized as denitrifiers but instead as carrying out dissimilatory nitrate reduction to ammonium. CONCLUSIONS: Among the denitrifiers confirmed by 15 N stable isotope analysis and genome analysis, Cellulomonas cellasea strain WB94 and Microvirgula aerodenitrificans strain BE2.4 appear to be promising for bioreactor bioaugmentation due to their potential for both aerobic and anaerobic denitrification, and the ability of strain WB94 to degrade cellulose. SIGNIFICANCE AND IMPACT OF THE STUDY: Denitrifiers isolated in this study could be useful for bioaugmentation application to enhance nitrate removal in woodchip bioreactors.


Assuntos
Agricultura/métodos , Reatores Biológicos/microbiologia , Desnitrificação , Purificação da Água/métodos , Madeira/microbiologia , Betaproteobacteria/isolamento & purificação , Betaproteobacteria/metabolismo , Biodegradação Ambiental , Cellulomonas/isolamento & purificação , Cellulomonas/metabolismo , Minnesota , Nitratos/isolamento & purificação , Nitratos/metabolismo , Madeira/metabolismo
19.
Int J Syst Evol Microbiol ; 70(5): 3091-3095, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32238231

RESUMO

A Gram-stain-positive, facultatively anaerobic and non-motile strain, designated SYSUP0004T, was isolated from the tubers of Gastrodia elata Blume collected from Yunnan Province, PR China. The 16S rRNA gene sequence result showed that the strain SYSUP0004T shared low similarity (97.7 %) with the type strain of Cellulomonas marina. SYSUP0004T grew at pH 6.0-9.0 (optimum, pH 8.0), temperature 4-30 °C (optimum, 28 °C) and could tolerate NaCl up to 4 % w/v (optimum in the absence of NaCl). The cell-wall peptidoglycan type was A4ß with an interpeptide bridge l-ornithine-d-glutamic acid. Cell-wall sugars were mannose, ribose, glucose, galactose and fucose. The menaquinone was MK-9(H4). The major fatty acids were anteiso-C15:0, anteiso-C15 : 1 A, C16 : 0 and anteiso-C17 : 0. The polar lipids of SYSUP0004T were diphosphatidylglycerol, unidentified phosphoglycolipid, phosphatidylinositol mannosides and unidentified glycolipid. The genomic DNA G+C content was 76.5 %. The average nucleotide identity values between SYSUP0004T and members of the genus Cellulomonas were below the cut-off level (95-96 %) recommended as the ANI criterion for interspecies identity. Thus, based on the above results strain SYSUP0004T represents a novel species of the genus Cellulomonas, for which the name Cellulomonas endophytica sp. nov. is proposed. The type strain, SYSUP0004T (=KCTC 49025T=CGMCC 1.16405T).


Assuntos
Cellulomonas/classificação , Gastrodia/microbiologia , Filogenia , Técnicas de Tipagem Bacteriana , Composição de Bases , Cellulomonas/isolamento & purificação , China , DNA Bacteriano/genética , Ácidos Graxos/química , Glicolipídeos/química , Peptidoglicano/química , Fosfolipídeos/química , Tubérculos/microbiologia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Vitamina K 2/análogos & derivados , Vitamina K 2/química
20.
J Hazard Mater ; 391: 122184, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32092674

RESUMO

A facultative exoelectrogen strain Lsc-8 belonging to the Cellulomonas genus with the ability to degrade carboxymethyl cellulose (CMC) coupled with the reduction of Cr(VI), was successfully isolated from rumen content. The maximum output power density of the microbial fuel cells (MFCs) inoculated strain Lsc-8 was 9.56 ±â€¯0.37 mW·m-2 with CMC as the sole carbon source. From the biomass analysis it can be seen that the electricity generation of the MFCs was primarily attributed to the planktonic cells of strain Lsc-8 rather than the biofilm attached on the electrode, which was different from Geobacter sulfurreducens. Especially, during electricity generation of the MFCs using CMC as carbon source in the anode chamber, the Cr(VI) reduction were simultaneously realized. And it is also found that the Cr(VI) reduction ratio by strain Lsc-8 is directly related to the initial Cr(VI) concentration, and it increased with the increase of initial Cr(VI) concentration at first, then started to decrease when the Cr(VI) concentration was above 21 mg ·L-1. Meanwhile, the highest output power density of 3.47 ±â€¯0.28 mW·m-2 was observed coupling with 95.22 ±â€¯2.72 % of Cr(VI) reduction. These data suggested that the strain Lsc-8 could reduce high toxicity Cr(VI) to low toxicity Cr(III) coupled with electricity generation in MFCs with CMC as the carbon source. Our results also suggested that this study will provide a possibility to simultaneously degrade Cr(VI) and generate electricity by using cellulose as the carbon source via MFCs.


Assuntos
Fontes de Energia Bioelétrica , Carboximetilcelulose Sódica/metabolismo , Cellulomonas/metabolismo , Cromo/metabolismo , Eletricidade , Oxirredutases/metabolismo , Riboflavina/metabolismo
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