Identification of the cellulose-binding domain of Fibrobacter succinogenes endoglucanase F

Mitsumori, Makoto

doi:10.1016/s0378-1097(99)00641-2

Cited by 12 publications

(18 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These genes were conserved between the two strains, whose DNA-DNA similarity is less than 5%. The EGF endoglucanase from F. succinogenes S85 is a complex modular enzyme displaying CMCase activity and comprising a cellulose binding module preceding a catalytic domain homologous to that of the family 51 glycoside hydrolase (28). The ubiquitous detection of the partial celF gene in all Fibrobacter strains studied was probably due to particularly strict conservation of the celF primer sequences, chosen in the N-terminal coding region of EGF, whose function is unknown and which has no homology with any sequence present at this time in the databases.…”

Section: Discussionmentioning

confidence: 99%

Fiber-Degrading Systems of Different Strains of the Genus Fibrobacter

Béra-Maillet

Ribot

Forano

2004

Appl Environ Microbiol

View full text Add to dashboard Cite

The S85 type strain of Fibrobacter succinogenes, a major ruminal fibrolytic species, was isolated 49 years ago from a bovine rumen and has been used since then as a model for extensive studies. To assess the validity of this model, we compared the cellulase-and xylanase-degrading activities of several other F. succinogenes strains originating from different ruminants, including recently isolated strains, and looked for the presence of 10 glycoside hydrolase genes previously identified in S85. The NR9 F. intestinalis type strain, representative of the second species of the genus, was also included in this study. DNA-DNA hybridization and 16S rRNA gene sequencing first classified the strains and provided the phylogenetic positions of isolates of both species. Cellulase and xylanase activity analyses revealed similar activity profiles for all F. succinogenes strains. However, the F E strain, phylogenetically close to S85, presented a poor xylanolytic system and weak specific activities. Furthermore, the HM2 strain, genetically distant from the other F. succinogenes isolates, displayed a larger cellulolytic profile on zymograms and higher cellulolytic specific activity. F. intestinalis NR9 presented a higher cellulolytic specific activity and a stronger extracellular xylanolytic activity. Almost all glycoside hydrolase genes studied were found in the F. succinogenes isolates by PCR, except in the HM2 strain, and few of them were detected in F. intestinalis NR9. As expected, the fibrolytic genes of strains of the genus Fibrobacter as well as the cellulase and xylanase activities are better conserved in closely related phylogenetic isolates.

show abstract

Section: Discussionmentioning

confidence: 99%

Fiber-Degrading Systems of Different Strains of the Genus Fibrobacter

Béra-Maillet

Ribot

Forano

2004

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…In the gastrointestinal tracts of herbivores and ruminants, Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes are widely believed to coordinate degradation of the cellulosic component of plant biomass. Our understanding of the adhesion mechanism(s) employed by these three bacteria has been limited by the lack of genetic tools available to dissect the process(es), but recent studies have identified a variety of structures including novel cellulosebinding modules present in cellulosomes (Ding et al, 2001;Rincon et al, 2001Rincon et al, , 2003, cell-surface-associated cellulases (Devillard et al, 2004;Mitsumori & Minato, 2000;Malburg et al, 1997), cell-surface exopolysaccharides (Mosoni & Gaillard-Martinie, 2001) and other uncharacterized cellulosebinding proteins (CBPs) (Gong et al, 1996;Miron & Forsberg, 1999). These different adhesion mechanisms are described in recent reviews (Morrison & Miron, 2000;Miron et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

The Ruminococcus albus pilA1-pilA2 locus: expression and putative role of two adjacent pil genes in pilus formation and bacterial adhesion to cellulose

et al. 2005

View full text Add to dashboard Cite

Ruminococcus albus produces fimbria-like structures that are involved with the bacterium's adhesion to cellulose. The subunit protein has been identified in strain 8 (CbpC) and strain 20 (GP25) and both are type IV fimbrial (Pil) proteins. The presence of a pil locus that is organized similarly in both strains is reported here together with the results of an initial examination of a second Pil protein. Downstream of the cbpC/gp25 gene (hereafter referred to as pilA1) is a second pilin gene (pilA2). Northern blot analysis of pilA1 and pilA2 transcripts showed that the pilA1 transcript is much more abundant in R. albus 8, and real-time PCR was used to measure pilA1 and pilA2 transcript abundance in R. albus 20 and its adhesion-defective mutant D5. Similar to the findings with R. albus 8, the relative expression of pilA1 in the wild-type strain was 73-fold higher than that of pilA2 following growth with cellobiose, and there were only slight differences between the wild-type and mutant strain in pilA1 and pilA2 transcript abundances, indicating that neither pilA1 nor pilA2 transcription is adversely affected in the mutant strain. Western immunoblots showed that the PilA2 protein is localized primarily to the membrane fraction, and the anti-PilA2 antiserum does not inhibit bacterial adhesion to cellulose. These results suggest that the PilA2 protein plays a role in the synthesis and assembly of type IV fimbriae-like structures by R. albus, but its role is restricted to cell-associated functions, rather than as part of the externalized fimbrial structure. INTRODUCTIONThe initial step in the degradation of cellulosic biomass by many anaerobic micro-organisms requires the adhesion of the organism to this substrate. Adhesion facilitates maximal enzyme-substrate interaction and gives the adherent cells an advantage for substrate uptake, and can be a ratelimiting step in polysaccharide hydrolysis. In the gastrointestinal tracts of herbivores and ruminants, Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes are widely believed to coordinate degradation of the cellulosic component of plant biomass. Our understanding of the adhesion mechanism(s) employed by these three bacteria has been limited by the lack of genetic tools available to dissect the process(es), but recent studies have identified a variety of structures including novel cellulosebinding modules present in cellulosomes (Ding et al., 2001;Rincon et al., 2001Rincon et al., , 2003, cell-surface-associated cellulases (Devillard et al., 2004;Mitsumori & Minato, 2000;Malburg et al., 1997), cell-surface exopolysaccharides (Mosoni & Gaillard-Martinie, 2001) and other uncharacterized cellulosebinding proteins (CBPs) (Gong et al., 1996;Miron & Forsberg, 1999). These different adhesion mechanisms are described in recent reviews (Morrison & Miron, 2000;Miron et al., 2001).Recent work in our laboratory has established that type IV Abbreviation: CBP, cellulose-binding protein.

show abstract

“…Further analysis of the binding specificity and structure-function relationships of this module will clearly be of interest. There have been several instances of novel bacterial CBMs that show unique sequences, suggesting that there is considerable sequence diversity among CBMs (6,25,44).…”

Section: Discussionmentioning

confidence: 99%

EndB, a Multidomain Family 44 Cellulase from Ruminococcus flavefaciens 17, Binds to Cellulose via a Novel Cellulose-Binding Module and to Another R. flavefaciens Protein via a Dockerin Domain

Rincón¹,

McCrae²,

Kirby³

et al. 2001

Appl Environ Microbiol

View full text Add to dashboard Cite

The mechanisms by which cellulolytic enzymes and enzyme complexes in Ruminococcus spp. bind to cellulose are not fully understood. The product of the newly isolated cellulase gene endB from Ruminococcus flavefaciens 17 was purified as a His-tagged product after expression in Escherichia coli and found to be able to bind directly to crystalline cellulose. The ability to bind cellulose is shown to be associated with a novel cellulose-binding module (CBM) located within a region of 200 amino acids that is unrelated to known protein sequences. EndB (808 amino acids) also contains a catalytic domain belonging to glycoside hydrolase family 44 and a C-terminal dockerin-like domain. Purified EndB is also shown to bind specifically via its dockerin domain to a polypeptide of ca. 130 kDa present among supernatant proteins from Avicel-grown R. flavefaciens that attach to cellulose. Cellulolytic Ruminococcus spp. play an important role in the degradation of plant cell wall polysaccharides in the rumen and hindgut of mammals (5,15,20). Early biochemical and microscopic evidence indicated that their plant cell wall-degrading enzymes are organized into high-molecular-weight complexes on the cell surface (22,24,43). Analysis of cloned polysaccharidase genes has, however, produced somewhat conflicting evidence concerning the molecular organization of these enzymes, and the mechanisms by which they might attach to their substrate and to the cell surface have thus remained unclear. Many of the cellulase genes first isolated from R. flavefaciens and R. albus (7,18,30,33,40,41) were reported to encode single domain enzymes smaller than 50 kDa that carry no obvious substrate binding domains, and no regions that might be responsible for the types of protein-protein interactions that are found, for example, in the assembly of cellulosome complexes from Clostridium spp. (3, 4, 9). On the other hand, it has been known for some time that xylanases from R. flavefaciens display complex multidomain organization (11, 45). Furthermore, the endoglucanase EndA from R. flavefaciens 17 was shown to be a multidomain enzyme carrying an 80-amino-acid dockerin-like region that is also present in three xylanases and an esterase from the same strain (2, 19). Dockerin-like regions have also been reported recently in multidomain endoglucanases from R. albus F40 (29, 30). Since dockerins are responsible for the assembly of cellulosome complexes via specific dockerin-cohesin interactions in Clostridium species (3,4,17,31,34), this provides a strong indication that complexes resembling cellulosomes may be involved in the organization of many plant cell wall-degrading enzymes in ruminococci. In support of this, two linked genes that encode structural proteins containing cohesin domains have recently been identified in R. flavefaciens 17 (8). Cellulosome organization provides one potential mechanism for binding of cellulolytic enzymes to their substrate since cellulosomal scaffolding proteins from clostridia carry cellulose-binding modules (CBMs) (4, 39). On t...

show abstract

Identification of the cellulose-binding domain of Fibrobacter succinogenes endoglucanase F

Cited by 12 publications

References 8 publications

Fiber-Degrading Systems of Different Strains of the Genus Fibrobacter

Fiber-Degrading Systems of Different Strains of the Genus Fibrobacter

The Ruminococcus albus pilA1-pilA2 locus: expression and putative role of two adjacent pil genes in pilus formation and bacterial adhesion to cellulose

EndB, a Multidomain Family 44 Cellulase from Ruminococcus flavefaciens 17, Binds to Cellulose via a Novel Cellulose-Binding Module and to Another R. flavefaciens Protein via a Dockerin Domain

Contact Info

Product

Resources

About