ISCce
 1
 and ISCce
 2
 , Two Novel Insertion Sequences in
 Clostridium cellulolyticum

Maamar, Hédia; Philip, Pascale de; Bélaı̈ch, Jean-Pierre; Tardif, Chantal

doi:10.1128/jb.185.3.714-725.2003

Cited by 12 publications

(9 citation statements)

References 41 publications

(36 reference statements)

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“…Considering the phylogenetic distance between these three species, the presence of a common bacterial ancestor does not seem the most probable hypothesis for explaining the close taxonomic relatedness of cellulosomal genes. Recently, two novel insertion sequences (IS) suggested as transpositionally active, called ISCce1 and ISCce2, have been reported in C. cellulolyticum [40]. Southern blotting analysis with different Clostridium species of the ISCce2 region, revealed some DNA fragments homology with C. cellobioparum , C. papyrosolvens , C. termitidis and C. cellulovorans chromosomic DNA.…”

Section: The Cellulasic System Of C Cellulolyticummentioning

confidence: 99%

Clostridium cellulolyticum: model organism of mesophilic cellulolytic clostridia

Desvaux¹

2005

FEMS Microbiol Rev

182

102

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Clostridium cellulolyticum ATCC 35319 is a non-ruminal mesophilic cellulolytic bacterium originally isolated from decayed grass. As with most truly cellulolytic clostridia, C. cellulolyticum possesses an extracellular multi-enzymatic complex, the cellulosome. The catalytic components of the cellulosome release soluble cello-oligosaccharides from cellulose providing the primary carbon substrates to support bacterial growth. As most cellulolytic bacteria, C. cellulolyticum was initially characterised by limited carbon consumption and subsequent limited growth in comparison to other saccharolytic clostridia. The first metabolic studies performed in batch cultures suggested nutrient(s) limitation and/or by-product(s) inhibition as the reasons for this limited growth. In most recent investigations using chemostat cultures, metabolic flux analysis suggests a self-intoxication of bacterial metabolism resulting from an inefficiently regulated carbon flow. The investigation of C. cellulolyticum physiology with cellobiose, as a model of soluble cellodextrin, and with pure cellulose, as a carbon source more closely related to lignocellulosic compounds, strengthen the idea of a bacterium particularly well adapted, and even restricted, to a cellulolytic lifestyle. The metabolic flux analysis from continuous cultures revealed that (i) in comparison to cellobiose, the cellulose hydrolysis by the cellulosome introduces an extra regulation of entering carbon flow resulting in globally lower metabolic fluxes on cellulose than on cellobiose, (ii) the glucose 1-phosphate/glucose 6-phosphate branch point controls the carbon flow directed towards glycolysis and dissipates carbon excess towards the formation of cellodextrins, glycogen and exopolysaccharides, (iii) the pyruvate/acetyl-CoA metabolic node is essential to the regulation of electronic and energetic fluxes. This in-depth analysis of C. cellulolyticum metabolism has permitted the first attempt to engineer metabolically a cellulolytic microorganism.

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Section: The Cellulasic System Of C Cellulolyticummentioning

confidence: 99%

Clostridium cellulolyticum: model organism of mesophilic cellulolytic clostridia

Desvaux¹

2005

FEMS Microbiol Rev

182

102

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“…Here, the in vivo modification of the cellulosome composition was explored in order to obtain new information. For this purpose, we recently developed a reproducible gene transfer method for C. cellulolyticum (Jennert et al ., 2000; Tardif et al ., 2001) and isolated spontaneous cipC insertional mutants from a strain routinely subcultured in cellobiose‐containing liquid medium (Maamar et al ., 2003). By studying the molecular structure of their chromosomal cipC region, two types of clones have been characterized.…”

Section: Introductionmentioning

confidence: 99%

“…In the cipC Mut1 clones, the element was found to be composed of one insertion sequence (ISCce 1 ) interrupted by another one (ISCce 2 ). In the cipC Mut2 clones, only one copy of ISCce 1 was located at the same site (Maamar et al ., 2003). The present report describes the cipC Mut1 strain at the phenotypic, physiological, biochemical and gene expression levels compared to the wild‐type (WT) strain.…”

Section: Introductionmentioning

confidence: 99%

Cellulolysis is severely affected in Clostridium cellulolyticum strain cipCMut1

Maamar

Valette

Fierobe

et al. 2004

Molecular Microbiology

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SummaryProgress towards understanding the molecular basis of cellulolysis by Clostridium cellulolyticm was obtained through the study of the first cellulolysis defective mutant strain, namely cipC Mut1. In this mutant, a 2 659 bp insertion element, disrupts the cipC gene at the sequence encoding the seventh cohesin of the scaffoldin CipC. cipC is the first gene in a large ' cel ' gene cluster, encoding several enzymatic subunits of the cellulosomes, including the processive cellulase Cel48F, which is the major component. Physiological and biochemical studies showed that the mutant strain was affected in cellulosome synthesis and severely impaired in its ability to degrade crystalline cellulose. It produced small amounts of a truncated CipC protein (P120), which had functional cohesin domains and assembled complexes which did not contain any of the enzymes encoded by genes of the ' cel ' cluster. The mutant cellulolytic system was mainly composed of three proteins designated P98, P105 and P125. Their Ntermini did not match any of the known cellulase sequences from C. cellulolyticum . A large amount of entire CipC produced in the cipC Mut1 strain by transcomplementation with plasmid pSOS cipC did not restore the cellulolytic phenotype, in spite of the assembly of a larger amount of complexes. The complexes produced in the mutant and complemented strains contained at least 12 different dockerincontaining proteins encoded by genes located outside of the ' cel ' cluster. The disturbances observed in the mutant and trans -complemented strains were the result of a strong polar effect resulting from the cipC gene disruption. In conclusion, this study provided genetic evidence that the cellulases encoded by the genes located in the ' cel ' cluster are essential for the building of cellulosomes efficient in crystalline cellulose degradation.

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“…Two mutants defective in cellulose degradation have been isolated from the mesophilic cellulosome-forming bacterium Clostridium cellulolyticum (18,19). The two mutant strains did not degrade crystalline cellulose.…”

mentioning

confidence: 99%

Mutations in the Scaffoldin Gene, cipA , of Clostridium thermocellum with Impaired Cellulosome Formation and Cellulose Hydrolysis: Insertions of a New Transposable Element, IS 1447 , and Implications for Cellulase Synergism on Crystalline Cellulose

et al. 2008

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Mutants of Clostridium thermocellum that had lost the ability to adhere to microcrystalline cellulose were isolated. Six of them that showed diminished ability to depolymerize crystalline cellulose were selected. Size exclusion chromatography of the proteins from the culture supernatant revealed the loss of the supramolecular enzyme complex, the cellulosome. However, denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis resulted in extracellular protein patterns comparable to those of isolated cellulosomes, except for a missing CipA band. Sequencing of the six mutant cipA genes revealed a new insertion (IS) element, IS1447, belonging to the IS3 family. It was inserted into the cipA reading frame in four different locations: cohesin module 1, two different positions in the carbohydrate binding module, and cohesin module 3. The IS sequences were identical and consisted of a transposase gene and the inverted repeats IRR and IRS. The insertion resulted in an obviously nonspecific duplication of 3 base pairs within the target sequence. This lack of specificity allows transposition without the need of a defined target DNA sequence. Eighteen copies of IS1447 were identified in the genomic sequence of C. thermocellum ATCC 27405. At least one of them can be activated for transposition. Compared to the wild type, the mutant culture supernatant, with a completely defective CipA protein, showed equal specific hydrolytic activity against soluble ␤-glucan but a 15-fold reduction in specific activity with crystalline cellulose. These results identify a genetic basis for the synergistic effect of complex formation on crystalline-cellulose degradation.

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ISCce 1 and ISCce 2 , Two Novel Insertion Sequences in Clostridium cellulolyticum

Cited by 12 publications

References 41 publications

Clostridium cellulolyticum: model organism of mesophilic cellulolytic clostridia

Clostridium cellulolyticum: model organism of mesophilic cellulolytic clostridia

Cellulolysis is severely affected in Clostridium cellulolyticum strain cipCMut1

Mutations in the Scaffoldin Gene, cipA , of Clostridium thermocellum with Impaired Cellulosome Formation and Cellulose Hydrolysis: Insertions of a New Transposable Element, IS 1447 , and Implications for Cellulase Synergism on Crystalline Cellulose

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