A Microbial Biorealm page on the genus F. succinogenes
Higher order taxa
cellular organisms; Bacteria; Fibrobacteres/Acidobacteria group; Fibrobacteres; Fibrobacteres (class); Fibrobacterales; Fibrobacteraceae; Fibrobacter (1)
Description and significance
Fibrobacter succinogenes is a rod-shaped, Gram-negative, and strictly anaerobic ruminal bacterium that can be found in the rumen of bovine animals including cows and sheep (3). As a major ruminal cellulolytic bacterium in the rumen, F. succinogenes can efficiently adhere to and degrade plant cell walls. In terms of its properties related to plant cell wall degradation, the S85 F. succinogenes strain is representitive of all rumen Fibrobacter strains, and therefore, it has been extensively studied to elucidate the machanism involved in adhesion and digestion of plant cell walls ever since it was isolated about 50 years ago from the bovine rumen by Bryant et al. It is important to have its genome sequenced as this organism is also known to produce a varity of fibrolytic enzymes in the rumen. Therefore, many enzymes responsible for the degradation of cellulose were isolated from F. succinogenes S85 and characterized, and some genes encoding such enzymes have been cloned and sequenced. (see Application to Biotechnology) (9)
The genome of F. succinogenes was found to be represented by the single circular DNA molecule. The overall GC content of F. succinogenes is 47-49% and its genomic size is approximately 3.6 kb, which is about 76.6% of the E. coli genome. The genome of this bacterium contains at least three rRNA operons. (10) Although the whole genome sequence of the F. succinogenes S85 strain has not been completely sequenced (only random shotgun sequencing completed), the genes encoding some of its proteins and enzymes were determined. For example, F. succinogenes S85 has cellulose-binding protein 1 (CBP1), and its nucleotide sequence was determined to be 3727 bp with a GC contents of 51.33%. Analysis of the sequence also revealed the presence of two open-reading frames (ORFs), designated ORF1 and ORF2, each of which comprises 3162 bp and 3363 bp, respectively. (11)
Cell structure and metabolism
According to previous studies on resting cells of F. succinogenes, maltodextrins, but not cellodextrins, accumulate in the extracellular medium of cells metabolizing cellulose. Maltodextrins with DP ranging from 4 to 7 are found extracellularly, whereas maltotriose (DP =3) is only present in cell extracts, and such observations suggest that maltodextrin plays a key role in carbon metabolism of F. succinogenes. In turn, this organism is able to synthesize and release oligosaccharides as maltodextrins (MD) and maltodextrin-1-phosphate (MD1P), upon incubation with glucose. A recent study provides further details regarding the metabolism of MDs in F. succinogenes although the synthesis and release of MD1P is still puzzling. (8)Although it can account for as much as 70% of the dry mass of the bacteria, glycogen is not used as energy storage in F. succinogenes since it is not able to withstand nutrient starvation; glycogen is rather stored and degraded simultaneously. As probably in most bacteria, therefore, glycogen is unlikely to play the role of an energy source permitting long-term survival; glycogen biosynthesis would be involved in fine-tuning of carbon flow regulation when grown on cellulose. Studies also revealed that in F. succinogenes excess soluble carbohydrates, such as glucose or cellobiose, promote bacterial death but cellulose does not.(7)
Since F. succinogenes is highly specialized for cellulose degradation, it is only capable of utilizing cellulose and cellulolytic degradation products as carbon sources. Access to cellulose is a rate-liming step in degradation, and the cellulolytic organisms have devised a number of mechanisms for improving access to this insoluble substrate, one of which is the production of surface-localized cellulases. The active enzymes are cell wall associated, but the presence of cellulosomes, large multiprotein cellulase complexes, has not been detected in this organism. Adherence is another method used to promote cellulose degradation, and this organism produces an extracellular matrix of glycoprotein glycocalyx which allows attachment to insoluble cellulose. In addition, the glycocalyx protects against protozoan attack of the bacterium as well as protease attack of the cellulase enzymes. (2)
F. succinogenes plays a key role in the rumen by degrading cellulose into metabolic products that are available to noncellulolytic species such as Streptococus bovis, Selenomonas ruminantium or Treponema bryantii, which have been shown to grow on cellulose in the presence of F. succinogenes. (8) For example, Streptococus bovis can be cocultured with F. succinogenes S85 on cellulose as sole carbon substrate, which suggests S. bovis has been fed by the cellodextrins excreted by F. succinogenes. (13)Since it is usually accepted that cellulodextrins are responsible for such cross-feeding effects, this cellodextrin release could, therefore, have important ecological implications in the rumen ecosystem because cellodextrins can be used as substrate by other rumen microorganisms. (13)
Since cellulose is one of the most abundant carbohydrates on the planet, this organism is, therefore, an important part of the global carbon biogeochemical cycle, converting the mass of fixed carbon generated by photosynthetic organisms back to products that eventually end up as carbon dioxide. (2)
There are no known diseases that are caused by this organism. It has not been found to be pathogenic.
Application to Biotechnology
F. succinogenes degrades cellulose by a very efficient cellulolytic system. Cellulose is depolymerized at the bacterial surface by various cellulases, and the released cellodextrins are hydrolysed to give glucose and cellobiose by a cellodextrinase located in the periplasm. The two sugars are then taken up and metabolized by the cells to succinate, acetate and small amounts of formate. When the extracellular sugar concentration is high, part of the sugar is stored as glycogen or is polymerized as cellodextrins. (13) This organism has been considered as one of the most actively fibrolytic bacteria in the rumen because this organism possesses a varity of fibrolytic enzymes and is detected in the rumen at high density (3). It is known to produce at least seven different glucanases, a cellodextrinase, a cellobiosidase, and both a cellobiase and a cellobiose-phosphorylase. The F. succinogenes S85 strain produces also at least six xylanases, a lichenase, an a-glucuronidase, and ferrulic acid and acetylxylane esterases and an arabinofuranosidase. (9) Due to extensive studies on this bacterium to elucidate the mechanism involved in adhesion and digestion of plant cell walls, some of the genes coding for cellulolytic activities have been isolated and sequenced. As a result, five endoglucanases, one cellobiosidase, one cellodextrinase, four xylanases, two acetyl xylan esterases, and two cellulose-binding proteins have been purified, or cloned, and the catalytic properties of the enzymes determined (5).
Synergistic interactions of F. succinogenes glycoside hydrolases
The collective activity of F. succinogenes glycoside hydrolases is thought to be greater than the sum of the individual enzyme activities due to their synergistic interactions. Endoglucanase I (or EG1), which is encoded by a gene cel9B, is a major endoglucanase of F. succinogenes. Therefore, the degree of synergism of the predominant cellulase EG1 along with 4 other cellulases from different families, EG2, Cel5H, Cel8B, and Cel10A was determined. The two predominant endoglucanases produced by F. succinogenes, Cel9B and Cel51A, were shown to have a degree of synergism of up to 1.67, while Cel10A revealed little synergy in combination with Cel9B and Cel51A. Mixture containing all the enzymes resulted a higher synergism degree than those containing 2 or 3 enzymes, which reflected the complementarity in their modes of action as well as substrate specificities. Further experiments are still in progress to identify other cellulases that may be important for cellulose degradation by F. succinogenes. (4)
Outer membrane proteins of F. succinogenes with potential roles in adhesion
Similar to other anaerobic cellulolytic bacteria, adhesion of F. succinogenes cells to cellulose appears to be a prerequisite for rapid and efficient cellulose hydrolysis by this organism. Such adhesion to cellulose and cellulose hydrolysis are thought to be intimately associated with the outer membrane proteins of F. succinogenes. In order to identify proteins with potential roles in adhesion to cellulose and in cellulose hydrolysis, therefore, research was conducted to investigate whether normal F. succinogenes strain and adhesion defective mutants (AD1 and AD4, respectively) are able to bind to different forms of cellulose. Mass spectroscopy analysis of trypsin digests of the separated proteins in conjunction with access to the genome sequence of F. succinogenes S85 was further used to identify proteins with roles in adhesion to, and digestion of cellulose. Studies revealed 6 proteins in S85 bound to crystalline cellulose that were absent from the mutants and 5 proteins in Ad1 that bound to ASC that were absent from Ad4. Since the functions of ORFs of the S85 genome are not fully known, the functions of some proteins were remained unknown, and therefore, further research is necessary to fully understand the unique mechanism of cellulose digestion by F. succinogenes. (5)
Detection of ruminal cellulolytic bacteria by the FISH detection protocol
Fluorescence in situ hybridization (FISH) is very useful for species- and group-specific detection of bacteria in complex communities such as that in rumen. Although FISH has not been effectively used for the detection of fiber-attaching bacteria due to the autofluorescence emitted by plant fibrous materials, if FISH were to be used for ruminal cellulolytic bacteria such as F. succinogenes associated with plant fragments, it would be useful for characterization of the niches of such bacteria and also for assessment of their physiological significance. Upon establishment of the FISH protocol, researchers successfully minimized the autofluorescence of orchard grass hay and detected cells of phylogenetic group 1 of F. succinogenes attached to many stem and leaf sheath fragments of the hay under a fluorescence microscope. Since the phylogenetic group 1 of F. succinogenes was clearly detectable by FISH and was the bacterium with the largest population size in the less easily degradable hay stem, researchers suggested that that particular group of this organism played an significant role in fiber digestion. (6)
3. Koike S, Pan J, Suzuki T, Takano T, Oshima C, Kobayashi Y, Tanaka K. (2004) Ruminal distribution of the cellulolytic bacterium Fibrobacter succinogenes in relation to its phylogenetic grouping. Animal Science Journal 75, 417-422.
4. Qi M, Jun HS, Forsberg CW. (2007) Characterization and Synergistic Interactions of Fibrobacter succinogenes Glycoside Hydrolases. Appl Environ Microbiol, published online ahead of print on 27 July. 1-36.
5. Jun HS, Qi M, Gong J, Egbosimba EE, Forsberg CW. (2007) Outer Membrane Proteins of Fibrobacter succinogenes With Potential Roles in Adhesion to Cellulose and in Cellulose Digestion. J Bacteriol, published online ahead of print on 20 July. 1-37.
6. Shinkai T, Kobayashi Y. (2007) Localization of ruminal cellulolytic bacteria on plant fibrous materials as determined by fluorescence in situ hybridization and real-time PCR. Appl Environ Microbiol 73, 1646-1652.
10. Ogata K, Aminov RI, Nagamine T, Sugiura M, Tajima K, Mitsumori M, Sekizaki T, Kudo H, Minato H, Benno Y. (1997) Construction of a Fibrobacter succinogenes genomic map and demonstration of diversity at the genomic level. Curr Microbiol 35, 22-27.
11. Mitsumori M, Minato H, Sekizaki T, Uchida I, Ito H. (1996) Cloning, nucleotide sequence and expression of the gene encoding the cellulose-binding protein 1 (CBP1) of Fibrobacter succinogenes S85. FEMS Microbiol Lett 139, 43-50.
12. Gong J, Forsberg CW. (1993) Separation of outer and cytoplasmic membranes of Fibrobacter succinogenes and membrane and glycogen granule locations of glycanases and cellobiase. J Bacteriol 175, 6810-6821.
13. Matulova M, Delort AM, Nouaille R, Gaudet G, Forano E. (2001) Concurrent maltodextrin and cellodextrin synthesis by Fibrobacter succinogenes S85 as identified by 2D NMR spectroscopy. Eur J Biochem 268, 3907-3915.