F. succinogenes

A Microbial Biorealm page on the genus F. succinogenes

Classification

Higher order taxa

cellular organisms; Bacteria; Fibrobacteres/Acidobacteria group; Fibrobacteres; Fibrobacteres (class); Fibrobacterales; Fibrobacteraceae; Fibrobacter (1)

Species

NCBI: Taxonomy

Fibrobacter succinogenes

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)

Genome structure

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)

Since this bacterium is Gram-negative, it has the outer membrane. The OM from both glucose- and cellulose-grown cells and the extracellular membrane vesicles from cellulose-grown culture exhibited higher endoglucanase, xylanase, and acetlyesterase activities than the CM and other cell fractions. Endoglucanase 2 was absent from the isolated OM fractions of glucose- and cellulose-grown cells and from the extracellular membrane vesicles of cellulose-grown cells but was present in the CM and intracellular glycogen granule fractions, while endoglucanase 3 was enriched in the OM. Cellobiosidase was located primarily in the periplasm as previously reported, while cellobiase was mainly present in the glycogen granule fraction of glucose-grown cells and in a nongranular glycogen and CM complex in cellulose-grown cells.

Ecology

Cellulolytic bacteria including F. succinogenes play an important role in nature, born out by the fact that microbial cellulose utilization is responsible for one of the largest material flows in the biosphere. Cellulolytic bacteria have thus been studied in detail, but, because of methodological difficulties in using solid cellulosic substrates, the majority of the studies were carried out on bacteria utilizing soluble substrates. (8)

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. It is usually accepted that cellulodextrins are responsible for such cross-feeding effects. (8) Streptococus bovis, a noncellulolytic rumen bacterium, could be cocultured with F. succinogenes S85 on cellulose as sole carbon substrate and suggested that S. bovis was fed by the cellodextrins excreted by F. succinogenes. (13)

Cellodextrins were also produced by growing cells and excreted into the culture medium. This cellodextrin release could have important ecological implications in the rumen ecosystem because cellodextrins can be used as substrate by other rumen microorganisms. (13)

Pathology

There are no known diseases that are caused by this organism. It has not been found to be pathogenic.

Application to Biotechnology

Fibrobacter 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)

Fibrobacter succinogenes is a rod-shaped, Gram-negative, and strictly anaerobic ruminal bacterium. F. succinogenes 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). Due to the ability of this bacterium to efficiently adhere to and degrade plant cell walls, it has been extensively studied to elucidate the mechanism involved in adhesion and digestion of plant cell walls. 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).

All previous isolates of this species, comprising 10 strains, use limited soluble substrates such as glucose or short glucose polymer and produce similar fermentation metabolites. Meanwhile, these 10 isolates of F. succinegenes are divided into four different phylogenetic groups (Amann et al. 1992), based on comparative sequence analysis of 16S rRNA gene (16S rDNA). A few differences in phenotypic characters among these four groups have been reported: (i) group 1 is differentiated from the other groups by its pleomorphic coccoid morphology and poor ability to digest cellulose in agar medium (Montgomery et al. 1988); (ii) group 3 is distinguishable from group 2 by production of yellow pigment and requirement for vitamin B12; (iii) groups 2 and 4 cannot be distinguished by any phenotypic characters (Amann et al. 1992).

Because F. succinogenes is better characterized in phenotypic variation depending on its phylogenetic grouping (Shinkai et al. 2004), attention should now be paid to the contribution of each group to plant fiber digestion in the rumen. The contribution might be indirectly evaluated by ecological information of each group of E. succinogenes, for example, bacterial mass and localization in the rumen (3).

Fibrobacter succinogenes is one of the most active cellulolytic bacteria ever isolated from the rumen, but enzymes from F. succinogenes capable of hydrolyzing native (insoluble) cellulose at a rapid rate have not been identified. However, the genome sequence of F. succinogenes is now available, and it was hoped that this information would yield new insights into the mechanism of cellulose digestion. The genome has a single family 45 beta-glucanase gene, and some of the enzymes in this family have good activity against native cellulose. The gene encoding the family 45 glycosyl hydrolase from F. succinogenes S85 was cloned into Escherichia coli JM109(DE3) using pMAL-c2 as a vector. Recombinant E. coli cells produced a soluble fusion protein (MAL-F45) that was purified on a maltose affinity column and characterized. MAL-F45 was most active on carboxymethylcellulose between pH 6 and 7 and it hydrolyzed cellopentaose and cellohexaose but not cellotetraose. It also cleaved p-nitrophenyl-cellopentose into cellotriose and p-nitrophenyl-cellobiose. MAL-F45 produced cellobiose, cellotriose and cellotetraose from acid swollen cellulose and bacterial cellulose, but the rate of this hydrolysis was much too low to explain the rate of cellulose digestion by growing cultures. Because the F. succinogenes S85 genome lacks dockerin and cohesin sequences, does not encode any known processive cellulases, and most of its endoglucanase genes do not encode carbohydrate binding modules, it appears that F. succinogenes has a novel mechanism of cellulose degradation.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17292641&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

Does this organism produce any useful compounds or enzymes? What are they and how are they used?

Current Research

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)

References

1. NCBI: Fibrobacter succinogenes, Accessed Aug 22, 2007.

(2) Forsberg CW, Cheng KJ, White BA. 1997. Polysaccharide degradation in the rumen and large intestine. In: Mackie RI. White BA (eds), Gastrointestinal Microbiology 319-379. Chapman and Hall, New York.

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.

7. Desvaux M. 2006. Unravelling Carbon Metabolism in Anaerobic Cellulolytic Bacteria. Biotechnol Prog 22, 1229-1238.

8. Nouaille R, Matulova M, Delort AM, Forano E. 2005. Oligosaccharide synthesis in Fibrobacter succinogenes S85 and its modulation by the substrate. FEBS J 272, 2416-27.

9. Béra-Maillet C, Ribot Y, Forano E. (2004) Fiber-degrading system of different strains of the genus Fibrobacter. Appl Environ Microbiol 70, 2172-2179.

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.

Edited by Woo Cheal Cho, student of Rachel Larsen