RESUMEN
Clostridium cellulolyticum is a model mesophilic anaerobic bacterium that efficiently degrades plant cell walls. The recent genome release offers the opportunity to analyse its complete degradation system. A total of 148 putative carbohydrate-active enzymes were identified, and their modular structures and activities were predicted. Among them, 62 dockerin-containing proteins bear catalytic modules from numerous carbohydrate-active enzymes' families and whose diversity reflects the chemical and structural complexity of the plant carbohydrate. The composition of the cellulosomes produced by C. cellulolyticum upon growth on different substrates (cellulose, xylan, and wheat straw) was investigated by LC MS/MS. The majority of the proteins encoded by the cip-cel operon, essential for cellulose degradation, were detected in all cellulosome preparations. In the presence of wheat straw, the natural and most complex of the substrates studied, additional proteins predicted to be involved in hemicellulose degradation were produced. A 32-kb gene cluster encodes the majority of these proteins, all harbouring carbohydrate-binding module 6 or carbohydrate-binding module 22 xylan-binding modules along dockerins. This newly identified xyl-doc gene cluster, specialised in hemicellulose degradation, comes in addition of the cip-cel operon for plant cell wall degradation. Hydrolysis efficiencies determined on the different substrates corroborates the finding that cellulosome composition is adapted to the growth substrate.
Asunto(s)
Proteínas Bacterianas/metabolismo , Celulosomas/metabolismo , Clostridium cellulolyticum/metabolismo , Polisacáridos/metabolismo , Proteómica/métodos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Metabolismo de los Hidratos de Carbono/genética , Celulosomas/enzimología , Celulosomas/genética , Clostridium cellulolyticum/enzimología , Clostridium cellulolyticum/genética , Hidrólisis , Polisacáridos/genética , Especificidad por SustratoRESUMEN
Further understanding of the plant cell wall degradation system of Clostridium cellulolyticum and the possibility of metabolic engineering in this species highlight the need for a means of random mutagenesis. Here, we report the construction of a Tn1545-derived delivery tool which allows monocopy random insertion within the genome.
Asunto(s)
Clostridium cellulolyticum/genética , Elementos Transponibles de ADN/genética , Ingeniería Genética/métodos , Vectores Genéticos , Mutagénesis Insercional/métodos , ADN Bacteriano/genética , Vectores Genéticos/genética , Integrasas/genéticaRESUMEN
The composition of the cellulosomes (multi enzymatic complexes involved in the degradation of plant cell wall polysaccharides) produced by Clostridium cellulolyticum differs according to the growth substrate. In particular, the expression of a cluster of 14 hemicellulase-encoding genes (called xyl-doc) seems to be induced by the presence of straw and not of cellulose. Genes encoding a putative two-component regulation system (XydS/R) were found upstream of xyl-doc. First evidence for the involvement of the response regulator, XydR, part of this two-component system, in the expression of xyl-doc genes was given by the analysis of the cellulosomes produced by a regulator overproducing strain when grown on cellulose. Nano-LC MS/MS analysis allowed the detection of the products of all xyl-doc genes and of the product of the gene at locus Ccel_1656 predicted to bear a carbohydrate binding domain targeting hemicellulose. RT-PCR experiments further demonstrated that the regulation occurs at the transcriptional level and that all xyl-doc genes are transcriptionally linked. mRNA quantification in a regulator knock-out strain and in its complemented derivative confirmed the involvement of the regulator in the expression of xyl-doc genes and of the gene at locus Ccel_1656 in response to straw. Electrophoretic mobility shift assays using the purified regulator further demonstrated that the regulator binds to DNA regions located upstream of the first gene of the xyl-doc gene cluster and upstream of the gene at locus Ccel_1656.
Asunto(s)
Proteínas Bacterianas/genética , Celulosomas/genética , Clostridium cellulolyticum/genética , Regulación Bacteriana de la Expresión Génica , Polisacáridos/metabolismo , Proteínas Bacterianas/metabolismo , Western Blotting , Metabolismo de los Hidratos de Carbono/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Celulasa/genética , Celulasa/metabolismo , Celulosa/metabolismo , Celulosomas/metabolismo , Cromatografía Liquida , Clostridium cellulolyticum/metabolismo , Técnicas de Inactivación de Genes , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Espectrometría de Masas , Familia de Multigenes , Nanotecnología , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad por SustratoRESUMEN
The enzyme diversity of the cellulolytic system produced by Clostridium cellulolyticum grown on crystalline cellulose as a sole carbon and energy source was explored by two-dimensional electrophoresis. The cellulolytic system of C. cellulolyticum is composed of at least 30 dockerin-containing proteins (designated cellulosomal proteins) and 30 noncellulosomal components. Most of the known cellulosomal proteins, including CipC, Cel48F, Cel8C, Cel9G, Cel9E, Man5K, Cel9M, and Cel5A, were identified by using two-dimensional Western blot analysis with specific antibodies, whereas Cel5N, Cel9J, and Cel44O were identified by using N-terminal sequencing. Unknown enzymes having carboxymethyl cellulase or xylanase activities were detected by zymogram analysis of two-dimensional gels. Some of these enzymes were identified by N-terminal sequencing as homologs of proteins listed in the NCBI database. Using Trap-Dock PCR and DNA walking, seven genes encoding new dockerin-containing proteins were cloned and sequenced. Some of these genes are clustered. Enzymes encoded by these genes belong to glycoside hydrolase families GH2, GH9, GH10, GH26, GH27, and GH59. Except for members of family GH9, which contains only cellulases, the new modular glycoside hydrolases discovered in this work could be involved in the degradation of different hemicellulosic substrates, such as xylan or galactomannan.