F. succinogenes: Difference between revisions

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==Classification (1)==
==Classification==


===Higher order taxa===
===Higher order taxa===
cellular organisms; Bacteria; Fibrobacteres/Acidobacteria group; Fibrobacteres; Fibrobacteres (class); Fibrobacterales; Fibrobacteraceae; Fibrobacter
cellular organisms; Bacteria; Fibrobacteres/Acidobacteria group; Fibrobacteres; Fibrobacteres (class); Fibrobacterales; Fibrobacteraceae; Fibrobacter [http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=833&lvl=3&lin=f&keep=1&srchmode=1&unlock (1)]


===Species===
===Species===
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==Description and significance==
==Description and significance==
Fibrobacter succinogenes has been considered as one of the most actively fibrolytic bacteria in the rumen (Forsberg et al. 1997), as this species possesses a varity of fibrolytic enzymes (Malburg & Forsberg 1993; Bera et al. 1999) and is detected in the rumen at high density (Michalet-Doreau et al. 2001; Koike et al. 2003). 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).
''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 [http://www.blackwell-synergy.com/doi/pdf/10.1111/j.1740-0929.2004.00207.x (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. ([http://microbewiki.kenyon.edu/index.php/User:Wccho#Application_to_Biotechnology see Application to Biotechnology]) [http://aem.asm.org/cgi/reprint/70/4/2172 (9)]
 
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).
 
B18 Fibrobacteres - Gram-negative anaerobes associated with the digestive tracts of herbivores. Representative species or genus: Fibrobacter


==Genome structure==
==Genome structure==
As of June 2007, the genome of L. jensenii 1153 has been fully sequenced using shotgun sequencing by the Lawrence Berkeley National Lab and Osel: The Bacterio-Therapeutics Company, but the data has to be yet released to public (4). For this reason, information on native plasmids and the genome is not available. The genome was sequenced for research conducted at Stanford University, Department of Medicine, that required a better understanding of gene regulation and promoter regions so to increase efficiency of HIV inhibitor cyanovirin-N expression in Lactobacillus jensenii. (See under Current Research for more details)
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. [http://www.springerlink.com/content/4vk3qgha397bbql5/ (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. [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T2W-3VYTM9R-R&_user=4429&_coverDate=05%2F15%2F1996&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000059602&_version=1&_urlVersion=0&_userid=4429&md5=541a45328b56cef22f6a2c6599761187 (11)]
Most of the sequencing methods include bacterial colony-based strain typing using PCR-fingerprinting and phylogenetic analysis of the partial 16S rRNA gene. Analysis shows that its genome contains over 1600 ORFs which include "novel cell wall anchor domains, unique signal sequences, powerful promoter elements, and possible sites for chromosomal integration of heterologous genes"(9). Its DNA has low G+C content (36.1 ± 2.3 moles % guanine + cytosine), similar to the DNA composition of L. acidophilus - L. jugurti group (36.1 ± 1.2 moles % guanine + cytosine); it produces D-lactic acid as its major metabolic product (10).
 
The genome of L. johnsonii strain NCC 533 was sequenced by the Nestle Research Center in Switzerland through the method of shotgun sequencing. The 1,992,676 base pair genome has a circular topology and is composed of 1,821 protein coding genes with 79 tRNAs (2, 4, 14). The Lactobacillus genus as a whole is characterized by its low Guanine+Cytosine content. L. johnsonii, in particular, contains a G+C content of 34.6% (2). Interestingly, L. johnsonii contains no genes which encode for the biosynthetic pathways necessary to generate amino acids and necessary cofactors. Rather, the genome contains of many amino acid proteases, peptidases, and phosphotransferase transporters and hence requires amino acids and peptides that come from its environment. In addition, genome sequencing has revealed that L. johnsonii contains all of the genes necessary for the synthesis of pyrimidines, but lacks genes necessary for the synthesis of purines. Thus, L. johnsonii also must depend on its environment in order to acquire purine nucleotides. Since this organism must obtain amino acids and purine nucleotides from exogenous sources, it is thought that it relies on its human host or other intestinal microorganisms in order to obtain such monomeric nutrients (2)
 
Describe the size and content of the genome.  How many chromosomes?  Circular or linear?  Other interesting features?  What is known about its sequence?
Does it have any plasmids?  Are they important to the organism's lifestyle?


==Cell structure and metabolism==
==Cell structure and metabolism==
This bacterium is one of the three most predominant cellulolytic organisms in the rumen, the other two being Ruminococcus sp.. 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. Increasing cellulose degradation is an important goal in industrial processes. This organism is highly specialized for cellulose degradation, and 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. Strain S85 if the type strain for this organism.
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. [http://www.blackwell-synergy.com/doi/abs/10.1111/j.1742-4658.2005.04662.x (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.[http://pubs.acs.org/cgi-bin/abstract.cgi/bipret/2006/22/i05/abs/bp060016e.html (7)]


http://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&orig_db=&term=fibrobacter%20succinogenes&cmd=search
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. [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&orig_db=&term=fibrobacter%20succinogenes&cmd=search (2)]


Describe any interesting features and/or cell structures; how it gains energy; what important molecules it produces.
==Ecology==
''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''. [http://www.blackwell-synergy.com/doi/abs/10.1111/j.1742-4658.2005.04662.x (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''. [http://www.blackwell-synergy.com/doi/abs/10.1046/j.1432-1327.2001.02300.x (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. [http://www.blackwell-synergy.com/doi/abs/10.1046/j.1432-1327.2001.02300.x (13)]


==Ecology==
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. [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&orig_db=&term=fibrobacter%20succinogenes&cmd=search (2)]
Describe any interactions with other organisms (included eukaryotes), contributions to the environment, effect on environment, etc.


==Pathology==
==Pathology==
How does this organism cause disease?  Human, animal, plant hosts?  Virulence factors, as well as patient symptoms.
There are no known diseases that are caused by this organism. It has not been found to be pathogenic.


==Application to Biotechnology==
==Application to Biotechnology==
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.
''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. [http://www.blackwell-synergy.com/doi/abs/10.1046/j.1432-1327.2001.02300.x (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 [http://www.blackwell-synergy.com/doi/pdf/10.1111/j.1740-0929.2004.00207.x (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. [http://aem.asm.org/cgi/reprint/70/4/2172 (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 [http://jb.asm.org/cgi/reprint/JB.00560-07v1?view=long&pmid=17644604 (5)].
 
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==
==Current Research==
===Synergistic interactions of ''F. succinogenes'' glycoside hydrolases===
====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''. [http://aem.asm.org/cgi/reprint/AEM.01037-07v1?view=long&pmid=17660301 (4)]
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''. [http://aem.asm.org/cgi/reprint/AEM.01037-07v1?view=long&pmid=17660301 (4)]


===Outer membrane proteins of ''F. succinogenes'' with potential roles in adhesion===
====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''. [http://jb.asm.org/cgi/reprint/JB.00560-07v1?view=long&pmid=17644604 (5)]
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''. [http://jb.asm.org/cgi/reprint/JB.00560-07v1?view=long&pmid=17644604 (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. [http://aem.asm.org/cgi/content/full/73/5/1646?view=long&pmid=17209077 (6)]


==References==
==References==
(1) NCBI: Fibrobacter succinogenes, Accessed Aug 22, 2007, <http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=833&lvl=3&lin=f&keep=1&srchmode=1&unlock>
1. [http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=833&lvl=3&lin=f&keep=1&srchmode=1&unlock NCBI: ''Fibrobacter succinogenes'', Accessed Aug 22, 2007.]
 
2. [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&orig_db=&term=fibrobacter%20succinogenes&cmd=search NCBI: ''Fibrobacter succiongenes subsp.'' S85, Accessed Aug 25, 2007.]
 
3. [http://www.blackwell-synergy.com/doi/pdf/10.1111/j.1740-0929.2004.00207.x 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. [http://aem.asm.org/cgi/reprint/AEM.01037-07v1?view=long&pmid=17660301 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. [http://jb.asm.org/cgi/reprint/JB.00560-07v1?view=long&pmid=17644604 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. [http://aem.asm.org/cgi/content/full/73/5/1646?view=long&pmid=17209077 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. [http://pubs.acs.org/cgi-bin/abstract.cgi/bipret/2006/22/i05/abs/bp060016e.html Desvaux M. (2006) Unravelling Carbon Metabolism in Anaerobic Cellulolytic Bacteria. Biotechnol Prog 22, 1229-1238.]
 
8. [http://www.blackwell-synergy.com/doi/abs/10.1111/j.1742-4658.2005.04662.x 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. [http://aem.asm.org/cgi/reprint/70/4/2172 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. [http://www.springerlink.com/content/4vk3qgha397bbql5/ 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.]


(2) Forsberg CW, Cheng KJ, White BA. 1997. Polysaccharide degradation in the rumen and large intestine. In: Mackie RI. White BA (eds), Gastrointestinal Microbiology, pp. 319-379. Chapman and Hall, New York.
11. [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T2W-3VYTM9R-R&_user=4429&_coverDate=05%2F15%2F1996&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000059602&_version=1&_urlVersion=0&_userid=4429&md5=541a45328b56cef22f6a2c6599761187 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.]


(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, pp. 417-422.  
12. [http://jb.asm.org/cgi/reprint/175/21/6810?view=long&pmid=8226622 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.]


4. [http://aem.asm.org/cgi/reprint/AEM.01037-07v1?view=long&pmid=17660301 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. pp. 1-36.]
13. [http://www.blackwell-synergy.com/doi/abs/10.1046/j.1432-1327.2001.02300.x 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.]


5. [http://jb.asm.org/cgi/reprint/JB.00560-07v1?view=long&pmid=17644604 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. pp. 1-37.]
14. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17292641&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Seon Park J, Russell JB, Wilson DB. (2007) Characterization of a family 45 glycosyl hydrolase from ''Fibrobacter succinogenes'' S85. Anaerobe 13, 83-85.]


Edited by [mailto:wccho@ucsd.edu Woo Cheal Cho], student of [mailto:ralarsen@ucsd.edu Rachel Larsen]
Edited by [mailto:wccho@ucsd.edu Woo Cheal Cho], student of [mailto:ralarsen@ucsd.edu Rachel Larsen]

Latest revision as of 17:55, 15 April 2011

This is a curated page. Report corrections to Microbewiki.

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)

Ecology

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)

Pathology

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).

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. NCBI: Fibrobacter succiongenes subsp. S85, Accessed Aug 25, 2007.

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.

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.

14. Seon Park J, Russell JB, Wilson DB. (2007) Characterization of a family 45 glycosyl hydrolase from Fibrobacter succinogenes S85. Anaerobe 13, 83-85.

Edited by Woo Cheal Cho, student of Rachel Larsen