Progress 10/01/06 to 07/31/08
Outputs
OUTPUTS: A large amount of agricultural waste such as rice straw, corn stalks, and wheat straw is currently produced in this country. If these cellulosic materials could be degraded to sugars, and the sugars fermented to ethanol, it would solve many problems in terms of the environment and energy. Our research is continuing on the enzymatic degradation of lignocellulosic materials by investigating the structure, function, regulation, and assembly of the cellulosome from Clostridium cellulovorans. The cellulosome is an extracellular enzyme complex that is capable of degrading cellulose and hemicellulose, the major components of plant cell walls. Our recent studies have shown that the cellulosome is not a homogeneous enzyme complex, but consists of many subpopulations of cellulosomes that differ in terms of enzymatic composition. The subunit composition varies depending on the growth substrate. Thus the organism is capable of degrading a variety of plant cell walls that it may encounter. In recent studies we have been attempting to express the cellulosomal genes in Bacillus subtilis and convert this aerobic microorganism into an efficient cellulose degrader. This will allow the production of cellulases more rapidly and in a system that is easier to handle, since B. subtilis is a fast growing aerobic microorganism. In this regard we have developed a method to transfer cellulase genes from C. cellulovorans to B. subtilis by a lateral gene transfer techniqure. Preliminary evidence indicates the successful transfer of DNA from C. cellulovorans to B. subtilis which enhanced the Avicelase activity of B. subtilis. In a collaborative effort, we have shown that cellulosomal enzymes can act synergistically to degrade sugar cane bargasse. PARTICIPANTS: Participants Personnel Roy H. Doi - Principal Investigator Helen M. Chan - Research Associate Hyunju Cha - Postdoctoral Fellow Jaeho Cha - Postdoctoral Fellow Satoshi Matsuoka - Postdoctoral Fellow Collabortive Financial Support U.S. Department of Energy Training Postdoctoral fellows were trained in the molecular biology of cellulases from Clostridium cellulovorans and the transfer of genes from C. cellulovorans to B. subtilis. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The newly developed method of converting B. subtilis to a more efficient cellulase producer will reduce costs for enzymes required for the degradation of cellulosic materials such agricultural and forest wastes. The enzymatic digestion of plant cell wall materials to sugars and the conversion of the sugars to ethanol will provide biofuel. These methods will help to keep the environment cleaner and will lead to use of plant biomass for biofuels that could partially reduce our dependence on foreign petroleum fuels.
Publications
- Doi, R.H. Cellulases of mesophilic microorganisms: Cellulosome & non-cellulosome Producers, Ann. N.Y.Acad. Sci 1125:267-279 (2008).
- Doi, R.H. Cellulosomes from mesophilic bacteria. pp. 97-106. In J.D.Wall, C.S. Harwood, and A. Demain (Eds.), Bioenergy, American Society of Microbiology, Washington, DC. (2008).
- Beukes, N., Chan, H., Doi, R.H. and Pletschke, B.I. Synergistic associations between Clostridium cellulovorans enzymes XynA, ManA and EngE against sugarcane bagasse. Enzyme Microbial Technol. 42:492-498 (2008).
|
Progress 01/01/07 to 12/31/07
Outputs
A large amount of agricultural waste such as rice straw, corn stalks, and wheat straw is currently produced in this country. If these cellulosic materials could be degraded to sugars, and the sugars fermented to ethanol, it would solve many problems in terms of the environment and energy. Our research is continuing on the enzymatic degradation of lignocellulosic materials by investigating the structure, function, regulation, and assembly of the cellulosome from Clostridium cellulovorans. The cellulosome is an extracellular enzyme complex that is capable of degrading cellulose and hemicellulose, the major components of plant cell walls. Our recent studies have shown that the cellulosome is not a homogeneous enzyme complex, but consists of many subpopulations of cellulosomes that differ in terms of enzymatic composition. The subunit composition varies depending on the growth substrate. Thus the organism is capable of degrading a variety of plant cell walls that it may
encounter. In recent studies we have found that one of the components of the cellulase gene cluster, HbpA, is capable of binding to the cell surface as well as bind cellulosomal enzymes and this complex appears to act in a complementary fashion to the cellulosome. Furthermore we have shown that the number of cohesins in the cellulosomal scaffolding protein plays an important role in amplifying the activity of the cellulosomal enzymes. Finally we have been able to express cellulosomal genes in Bacillus subtilis and demonstrated intercellular complementation between B. subtilis strains which are capable of synthesizing minicellulosomes.
Impacts
These studies will lead to better methods for disposing and utilizing cellulosic agricultural and forest wastes. The enzymatic digestion of plant cell wall materials to sugars and the conversion of the sugars to ethanol will provide biofuel. These methods will help to keep the environment cleaner and will lead to use of plant biomass for biofuels that could partially reduce our dependence on foreign petroleum fuels.
Publications
- Norris, V., den Blaauwen, T., Cabin-Flaman, A., Doi, R.H., Harshey, R., Janniere, L., Jimenez-sanchez, A., Jin, D. J., Levin, P.A., Mileykovskaya, E., Minsky, A., Pugsley, T., Saier, M. Jr., and Skarstad, K. 2007. Functional taxonomy of bacterial hyperstructures. Microb. Mol. Biol. Rev. 71:230-253.
- Doi, R.H. and Matsuoka, S. 2007. Structure, function and application of cellulolytic complex, cellulosome, of Clostridium cellulovorans. Bioscience and Industry 65:121-125.
- Norris, V., den Blaauwen, T., Doi, R.H., Haarshey, R., Janniere, L., Jimenez-Sanchez, A., Jin, D.J., Levin, P.A., Mileykovskaya, E., Minsky, A., Misevic, G., Ripoli, C., Saier, M. Jr., Skarstad, K. and Thellier, M. 2007. Towards a hyperstructure taxonomy. Ann. Rev. Microbiol. 61:309-329.
- Matsuoka, S., Yukawa, H., Inui, M. and Doi, R.H. 2007. Synergistic interaction of Clostridium cellulovorans cellulosomal cellulases and HbpA. J. Bacteriol.189: 7190-7194.
- Cha, J., Matsuoka, S., Chan, H. and Doi, R.H. 2007. Effect of the multiple copies of cohesins on cellulase and hemicellulase activities of Clostridium cellulovorans minicellulosome. J. Microbiol. Biotech. 17:1782-1788.
- Doi, R.H. 2006. The Clostridium cellulovorans Cellulosome, pp. 153-168. In V. Uversky and I.A. Kataeva (Eds.), Cellulosome, Nova Science Publishers, Inc., Hauppauge, NY.
- Arai, T., Matsuoka, S., Cho, H.-Y., Yukawa, H., Inui, M., Wong, S.-L., and Doi, R.H. 2007. Synthesis of Clostridium cellulovorans minicellulosomes by intercellular complementation. Proc. Natl. Acad. Sci. USA 104:1456-1460.
|
Progress 01/01/06 to 12/31/06
Outputs
A large amount of agricultural waste such as rice straw, corn stalks, and wheat straw is currently produced in this country. If these cellulosic materials could be degraded to sugars, and the sugars fermented to ethanol, it would solve many problems in terms of the environment and energy, Our research is continuing on the enzymatic degradation of lignocellulosic materials by investigating the structure, function, regulation, and assembly of the cellulosome from Clostridium cellulovorans. The cellulosome is an extracellular enzyme complex that is capable of degrading cellulose and hemicellulose, the major components of plant cell walls. Our recent studies have shown that the cellulosome is not a homogeneous enzyme complex, but consists of many subpopulations of cellulosomes that differ in terms of enzymatic composition. The subunit composition varies depending on the growth substrate. Thus the organism is capable of degrading a variety of plant cell walls that it may
encounter. In another study we have found that an apparent redundant population of family 9 cellulosomal endoglucanases actually allows the cellulosome to attack substrates with differing structures. In addition the products that were produced by the family 9 endoglucanases also were varied indicating that the family 9 enzymes attacked substrates in a differing manner and increased the versatility of the cellulosome. Further studies on the interaction of cellulosome with non-cellulosomal enzymes indicate that there is synergistic activity between the cellulosome and non-cellulosomal enzymes. The non-cellulosomal hemicellulases apparently attack the plant cell wall and expose the cellulosic components and facilitates the activity of the cellulosome. These studies will allow us to construct more efficient designer cellulosomes with greater capacity for degrading lignocelluloses.
Impacts
These studies will lead to better methods for disposing and utilizing cellulosic agricultural and forest wastes. The enzymatic digestion of plant cell wall materials to sugars and the conversion of the sugars to ethanol will provide biofuel. These methods will help to keep the environment cleaner and will lead to use of plant biomass for biofuels that could partially reduce our dependence on foreign petroleum fuels.
Publications
- Arai, T., Kosugi, A., Chan, H., Koukiekolo, R., Yukawa, H., Inui, M. and Doi, R.H. 2006. Properties of cellulosomal family 9 cellulases from Clostridium cellulovorans. Appl. Microbiol Biotechnol. 71: 654-660.
- Kosugi, A., Arai, T. and Doi, R.H. 2006. Degradation of cellulosome-produced cellooligosaccharides by an extracellular non-cellulosomal -glucan glucohydrolase, BglA, from Clostridium cellulovorans. Biochem. Biophys. Res. Commun. 349:20-23.
|
Progress 01/01/05 to 12/31/05
Outputs
A large amount of agricultural biomass such as corn stalks, rice straw, and wheat straw is produced annually in the USA. If these agricultural waste products could be converted to utilizable energy and/or organic compounds, it would help to reduce air and water pollution and provide value-added products. Furthermore the amount of energy produced could be substantial and lead to less dependence on foreign energy sources. Our current research is concerned with studying the properties and functions of cellulases and hemicellulases produced by an anaerobic microorganism, Clostridium cellulovorans. This bacterium produces an enzyme complex called the cellulosome that is capable of degrading plant cell wall materials very efficiently. The cellulosome contains cellulases, hemicellulases and pectinase that work in a synergistic manner to degrade plant cell walls and produce cellobiose, xylobiose, and organic acids. In our current work we have isolated two new genes, xynB, and
engO. XynB encodes an active xylanase that is found consistently in cellulosomes. EngO is a family 9 endoglucanase that is non-cellulosomal. We have also characterized the cellulose binding site of the cellulose binding module of the scaffolding protein (CbpA) of the cellulosome. In addition we have shown that the cellulosome population is comprised of a number of different subpopulations that consist of a variety of cellulosomal enzymes associated with the scaffolding protein. The pattern of the subpopulations was shown to be affected by the carbon growth medium. We have found that different plant cell wall substrates induce different levels of expression of specific genes such as cellulosomal cellulases and xylanases.
Impacts
The conversion of agricultural cellulosic waste to sugars and to ethanol will have a significant impact on the energy requirements of our state and country. It will also lead to a better environment, since there will be less pollution of streams and the air. The use of enzymes for biomass conversion will benefit farmers as well as the general public.
Publications
- HAN, S-O., YUKAWA, H., INUI, M. AND DOI, R.H. 2004. Isolation and expression of the xynB gene and its product XynB, a consistent component of the Clostridium cellulovorans cellulosome. J. Bacteriol. 186:8347-8355.
- HAN, S-O., YUKAWA, H., INUI, M. AND DOI, R.H. 2005. Effect of carbon source on the cellulosomal subpopulations of Clostridium cellulovorans. Microbiology 151:1491-1497.
- KOUKIEKOLO, R., CHO, H-Y., KOSUGI, A., INUI, M., YUKAWA, H. AND DOI, R.H. 2005. Degradation of corn fiber by Clostridium cellulovorans cellulases and hemicellulases and contribution of scaffolding protein, CbpA. Appl. Environ. Microbiol. 71:3504-3511.
- HAN, S.-O., YUKAWA, H., INUI, M. AND DOI, R.H. 2005. Molecular cloning, transcriptional and expression analysis of engO, encoding a noncellulosomal family 9 enzyme from Clostridium cellulovorans. J. Bacteriol. 187:4884-4889.
- MURASHIMA, K., KOSUGI, A., AND DOI, R.H. 2005. Site-directed mutagenesis and expression of the soluble form of the family IIIa cellulose binding domain from the cellulosomal scaffolding protein of Clostridium cellulovorans. J. Bacteriol. 187:7146-7149.
|
Progress 01/01/04 to 12/31/04
Outputs
A large amount of agricultural biomass such as corn stalks, rice straw, and wheat straw is produced annually in the USA. If these agricultural waste products could be converted to utilizable energy and/or organic compounds, it would help to reduce air and water pollution and provide value-added products. Furthermore the amount of energy produced could be substantial and lead to less dependence on foreign energy sources. Our research is concerned with studying the properties and functions of cellulases and hemicellulases produced by an anaerobic microorganism, Clostridium cellulovorans. This bacterium produces an enzyme complex called the cellulosome that is capable of degrading plant cell wall materials very efficiently. The cellulosome contains cellulases, hemicellulases and pectinase that work in a synergistic manner to degrade plant cell walls and produce cellobiose, xylobiose, and organic acids. We are studying the genes that code for these enzymes and the manner
in which their expression is controlled. We have found that different plant cell wall substrates induce different levels of expression of specific genes such as cellulases and xylanases. In addition to characterizing the enzymes involved in plant cell wall degradation, we have succeeded in transferring the cellulosome genes to a non-cellulose utilizing bacterium, Bacillus subtilis. This strategy may be used to convert many valuable industrial non-cellulose utilizers that currently use expensive carbohydrate substrates into utilizers of inexpensive biomass. This will reduce the cost of producing valuable organic compounds.
Impacts
The conversion of agricultural cellulosic waste to ethanol will have a significant impact on the energy requirements of our state and country. It will also lead to a better environment, since there will be less pollution of streams and the air. The use of enzymes for biomass conversion will benefit farmers as well as the general public.
Publications
- HAN, S.-O., CHO, H.-Y., YUKAWA, H., INUI, M. AND DOI, R.H. 2004. Regulation of expression of cellulosomes and non-cellulosomal (hemi-)cellulolytic enzymes in Clostridium cellulovorans during growth on different carbon sources. J. Bacteriol. 186:4218-4227.
- DOI, R.H. AND KOSUGI, A. 2004. Cellulosomes: plant cell wall degrading enzyme complexes. Nature Reviews - Microbiology 2: 541-551.
- CHO, H.-Y., YUKAWA, H., INUI, M., DOI, R.H. AND WONG, S.-L. 2004. Production of minicellulosomes from Clostridium cellulovorans in Bacillus subtilis WB800. Appl. Environ. Microbiol. 70:5704-5707.
- KOSUGI, A., AMANO, Y. MURASHIMA, AND DOI, R.H. 2004. Hydrophjilic domains of scaffolding protein CbpA promote glycosyl hydrolase activity and localization of cellulosomes to the cell surface of Clostridium cellulovorans. J. Bacteriol. 186: 6351-6359.
- HAN, S.-O., CHO, H.-Y., AND DOI, R.H. 2004. Regulation of expression of cellulosomal genes and subunit composition of Clostridium cellulovorans cellulosomes, pp. 175-191. In K. Ohmiya, K. Hayashi, K. Sakka, Y. Kobayashi, T. Kimura, and S. Karita (eds), Biotechnology of Lignocellulose Degradation and Biomass Utilization, Uni Publishers, Co., Ltd., Tokyo, Japan.
- ARAI, T., KOSUGI, A., CHAN, H., KOUKIEKOLO, R., TAMARU, Y. AND DOI, R.H. 2004. Properties and mutation analysis of the EngY, an enzymatic subunit of the Clostridium cellulovorans cellulosome, pp. 237-240. In K. Ohmiya, K. Hayashi, K. Sakka, Y. Kobayashi, T. Kimura, and S. Karita (eds), Biotechnology of Lignocellulose Degradation and Biomass Utilization, Uni Publishers, Co., Ltd., Tokyo, Japan.
|
Progress 01/01/03 to 12/31/03
Outputs
Agricultural biomass such as corn stalks, rice straw, and wheat straw represent a potential energy source if they can be converted efficiently to sugars and fermented to ethanol. It is estimated that the conversion of all the corn stalk produced in the USA to ethanol would result in substantial reduction in the importation of petroleum. Futhermore such utilization of agricultural wastes would lead to a cleaner environment. The rate limiting step in the utilization of biomass for energy purposes is the conversion of cellulose and hemicellulose to sugars. Our research is concerned with studying the properties of cellulases and hemicellulases in order to engineer more efficient enzymes. We are taking several complementary approaches to studying this problem. We have analyzed the enzymes produced by Clostridium cellulovorans, an anaerobic microorganism that produces copious amounts of cellulases and hemicellulases in an enzyme complex called the cellulosome. We
havestudied the structure and function of the cellulosomes and found that it is capable of degrading cellulose, xylan, and pectin. We have studied how the enzymes can work in a synergistic manner and have shown that cellulases can act synergistically and that cellulases and hemi-cellulases also are more effective in degrading corn fiber to sugars when they are both present. We are currently studying the regulation of cellulosome synthesis and have found that the composition and activity of the cellulosome is regulated by the growth substrate. Growth on xylan, for instance, induces a high level of xylanase and this has been demonstrated at both the transcript and protein levels. In addition by two dimensional chromatography, we have shown that the cellulosome population is heterogeneous and can be fractionated into several sub-populations that have discrete enzymatic properties. These results have shown that we may be able to construct 'designer' cellulosomes with specific functions
that can be used in synergy studies.
Impacts
The conversion of agricultural cellulosic waste to ethanol will have a significant impact on the energy requirements of our state and country. It will also lead to a better environment, since there will be less pollution of streams and the air. The use of enzymes for biomass conversion will benefit farmers as well as the general public.
Publications
- DOI, R.H. 2003. Conversion of corn stalks to riches. J. Bacteriol. 185:701-702.
- MURASHIMA, K., KOSUGI, A., AND DOI, R.H 2003. Synergistic effects of cellulosomal xylanase and cellulases from Clostridium cellulovorans on plant cell wall degradation. J. Bacteriol. 185:1518-1524.
- MURASHIMA, K., KOSUGI, A. AND DOI, R.H. 2003. Solubilization of cellulosomal cellulases by fusion with cellulose binding domain of non-cellulosomal cellulase EngD from Clostridium cellulovorans. Protein: Structure, Function, Genetics 50:620-628.
- HAN, S.O., YUKAWA, H., INUI, M., AND DOI, R.H. 2003. Transcription of Clostridium cellulovorans cellulosomal cellulase and hemicellulase genes. J. Bacteriol. 185:2520-2529.
- TAMARU, Y., KOSUGI, A., MURASHIMA, K. AND DOI, R.H. 2003. The Clostridium cellulovorans cellulosome and its application to plant biomass degradation. Recent Res. Devel. Microbiol. 7:1-18.
- MURASHIMA, K. AND DOI, R.H. 2003. Selection of heat stable Clostridium cellulovorans cellulases after in vitro recombination. Methods in Molecular Biology Series, Volume 230: 231-237 (Frances H. Arnold and George Georgiou, Eds), Humana Press, Totowa, NJ 07512, USA.
- DOI, R.H., A. KOSUGI, K. MURASHIMA, Y. TAMURA, AND S.O. HAN. 2003. Cellulosomes from mesophilic bacteria. J. Bacteriol. 185:5907-5914.
- HAN, S.O., YUKAWA, H., INUI, M. AND DOI, R.H. 2003. Regulation of expression of cellulosomal cellulase and hemicellulase genes in Clostridium cellulovorans. J. Bacteriol. 185:6067-6075.
|
Progress 01/01/02 to 12/31/02
Outputs
In order to convert agricultural biomass and waste to utilizable forms of energy, it is necessary to gain a better understanding of the enzymatic processes that can convert plant cell wall materials to sugars that can be fermented to alcohol. For this purpose we are investigating the structure, function, assembly, and regulation of the genes for the proteins subunits of the Clostridium cellulovorans cellulosome. During the past year we have concentrated on studying how the cellulosomal enzymes work in concert to degrade cellulose and hemicellulose, how the properties of cellulosomal enzymes might be improved by DNA shuffling techniques, and how cellulosomal enzyme genes are regulated. We have shown that there is active synergism between the cellulases in cellulosomes, between cellulases and hemi-cellulases in cellulosomes, and between cellulosomal and non-cellulosomal enzymes. By use of natural substrates we have shown that plant cell walls can be degraded by
combinations of designer mini-cellulosomes with specific functions. By a proper combination of cellulases, we obtained a degree of synergy of 3. By use of cellulases and xylanase, we have obtained a degree of synergy of 2.9 on a natural substrate such as corn stalk fiber. By DNA shuffling techniques we have recombined genes for two endoglucanases in vitro and obtained more heat resistant cellulase enzymes. This chimeric enzyme was stable at 55 C whereas the parent endoglucanases were completely inactivated at this temperature. This will serve as a model system for further directed evolution studies to obtain enzymes with properties that will make them suitable for biomass degradation. We have obtained evidence that the expression of the genes involved in cellulose and hemi-cellulose degradation are affected by the substrates that are available to C. cellulovorans during growth. For instance, xylanase activity is stimulated if the substrate is xylan. Pectin lyase is not produced at
all, unless pectin is present as the substrate. We are interested in learning how these genes are regulated I n order to construct mutants that can produce large quantities of these degradative enzymes. These studies are leading to new approaches that will allow us to construct novel cellulosomes that should be more efficient in their plant cell wall degrading ability.
Impacts
The cellulosomal and non-cellulosomal cellulases and hemi-cellulases of Clostridium cellulovorans can degrade plant cell walls. The conversion of agricultural biomass to sugars by these enzymes can yield sugars that can be converted to utilizable forms of energy such as alcohol. This will lead to an efficient use of agricultural wastes and lead to a cleaner environment as well. This will benefit the farmers as well as the general public.
Publications
- KOSUGI, A., MURASHIMA, K., TAMARU, Y. AND DOI, R.H. 2002. Cell surface anchoring role of N-terminal surface layer homology domains of Clostridium cellulovorans EngE. J. Bacteriol. 184:884-888.
- Murashima, K., Kosugi, A. and Doi, R.H. 2002. Determination of subunit composition of Clostridium cellulovorans cellulosomes that degrade plant cell walls. Appl. Environ. Microbiol. 68:1610-1615.
- TAMARU, Y., UI, S., MURASHIMA, K., KOSUGI, A., CHAN, H., DOI, R.H. AND LIU, B. 2002. Formation of protoplasts from cultured tobacco cells and Arabidopsis thaliana by the action of cellulosomes and pectate lyase from Clostridium cellulovorans. Appl. Environ. Microbiol. 68:2614-2618.
- MURASHIMA, K., KOSUGI, A. AND DOI, R.H. 2002. Thermostabilization of cellulosomal endoglucanase EngB from Clostridium cellulovorans by in vitro DNA recombination with non-cellulosomal endoglucanase EngD. Molec. Microbiol. 45:617-626.
- MURASHIMA, K., KOSUGI, A. AND DOI, R.H. 2002. Synergistic effects on crystalline cellulose degradation between cellulosomal cellulases from Clostridium cellulovorans. J. Bacteriol. 184: 5088-5095.
- KOSUGI, A., MURASHIMA, K. AND DOI, R.H. 2002. Xylanase and Acetyl Xylan Esterase Activities of XynA, a Key Subunit of the Clostridium cellulovorans Cellulosome for Xylan Degradation. Appl. Environ. Microbiol. 68:6399-6402.
- KOSUGI, A., MURASHIMA, K. AND DOI, R.H.. 2002. Characterization of non-cellulosomal subunits, ArfA and BgaA from Clostridium cellulovorans, that cooperate with the cellulosome in plant cell wall degradation. J. Bacteriol. 184:6859-6865.
- MURASHIMA, K., CHEN, C-L, KOSUGI, A., TAMARU, Y., DOI, R.H. AND WONG, S.L. 2002. Heterologous expression of Clostridium cellulovorans engB gene using protease deficient Bacillus subtilis , and preparation of active recombinant cellulosomes. J. Bacteriol. 184:76-81.
|
Progress 01/01/01 to 12/31/01
Outputs
The cellulosome (an enzyme complex for degradation of plant cell walls) from CLOSTRIDIUM CELLULOVORANS is being analyzed for its structure, function, assembly, and regulation, since microbial degradative enzymes play a major role in degrading plant cell wall materials. A knowledge of the enzymes involved is essential if the enzymes are to be improved so that the process can take place more efficiently in transforming biomass to more utilizable forms of energy such as ethanol. During the past year, we have cloned the pectate lyase A gene whose product PelA is also a subunit of the cellulosome, have demonstrated that the cohesins of the scaffolding protein (CbpA) of the cellulosome have different binding affinities for the cellulosomal enzymes, have characterized the xylanolytic enzymes that are induced by growth on xylan, have shown that the surface layer homology (SLH) domains of EngE can bind the enzyme to the cell surface, and have demonstrated the heterologous
expression of EngB in BACILLUS SUBTILIS and prepared a mini-cellulosome containing a mini-CbpA and EngB. The latter experiments are the first step in our plans to make "designer cellulosomes" with specific functions and to obtain efficient production of cellulosomes in a heterologous organism which may be easier to handle that C. CELLULOVORANS. Thus significant progress has been made in understanding the properties of the cellulosome as well as the regulation of expression of its subunits.
Impacts
The cellulosomal enzymes are capable of degrading plant cell wall materials. Thus agricultural biomass can be converted to more utilizable forms of energy such as ethanol and methane if this process can be improved. This research can lead to the development of more efficient enzymes to carry out this biomass conversion.
Publications
- DOI, R.H. AND TAMARU, Y. 2001. The Clostridium cellulovorans cellulosome: an enzyme complex with plant cell wall degrading activity. Chem. Rec.1: 24-32.
- TAMARU, Y. AND DOI, R.H. 2001. Pectate Lyase A, an Enzymatic Subunit of the Clostridium cellulovorans Cellulosome. Proc. Natl.Acad. Sci. USA 98: 4125- 4129.
- PARK, J.-S., MATANO, Y. AND DOI, R.H. 2001. Cohesin-Dockerin Interactions of Cellulosomal Subunits of Clostridium cellulovorans. J. Bacteriol. 183:5431-5435.
- KOSUGI, A., MURASHIMA, K. AND DOI, R.H. 2001. Characterization of Xylanolytic Enzymes in Clostridium cellulovorans: Expression of Xylanase Activity Dependent on Growth Substrates. J. Bacteriol. 183: 7037-7043.
|
Progress 01/01/00 to 12/31/00
Outputs
The CLOSTRIDIUM CELLULOVORANS cellulosome (cellulase) is being analyzed for its structure, function, assembly, and regulation, since microbial cellulases play a role in the degradation of cellulosic biomass and contribute to the CO2 cycle. Also in order to convert plant cell wall materials efficiently to cellobiose, pentose, and other carbohydrates, a knowledge of the enzymes that carry out the degradation of plant cell walls is necessary. We have cloned a gene cluster containing 9 genes involved in cellulase degradation, a gene for cellulosomal pectate lyase (pelA), and a mannanase gene, (manA). We have also cloned a gene cluster for xylanases that are specific for xylan and do not degrade cellulose. Thus the cellulosome has all the enzymes necessary for degrading plant cell walls. In fact we can convert plant tissue cells to protoplasts. Moreover, we also have recent evidence that C. cellulovorans expresses chitinase and alginase activities. Thus this microorganism
is a versatile degrader of various tpyes of biopolymers. We are currently studying the properties of the surface layer homology domain of CbpA and EngE to determine whether they are involved in binding the cellulosome to the cell surface. We are also making mutant enzymes that will have greater cellulase activity and studying the regulation of expression of the cellulosome genes.
Impacts
Enzymes that degrade plant cell walls are being studied for their genetic and biochemical characteristics. This research has potential for converting agricultural and forestry wastes to useful products such as ethanol, methane, and cellulase enzymes for industrial purposes.
Publications
- TAMARU, Y., KARITA, S., IBRAHIM, A., CHAN, H. AND DOI, R.H. 2000. A large gene cluster for the Clostridium cellulovorans cellulosome. J. Bacteriol. 182:5906-5910.
- MIYAGI, T., CHUANG, L.F., DOI, R.H., CARLOS, M.P., TORRES, J.V. AND CHUANG, R.Y. 2000. Morphine induces gene expression of CCR5 in human CEMx174 lymphocytes. J. Biol. Chem. 275: 31305-31310.
- SUZUKI, S., MIYAGI, T., CHUANG, T.K., CHUANG, L.F., DOI, R.H. AND CHUANG, R.Y. 2000. Mu opioid receptors of human lymphocytes; evidence of transcriptional activation by morphine. Biochem. Biophys. Res. Commun. In Press.
- NANAMIYA, H., FUGONO, N., ASAI, K., DOI, R.H. AND KAWAMURA, F. 2000. Suppression of temperature-sensitive sporulation mutation in the Bacillus subtilis sigA gene by rpoB mutation. FEMS Microbiol Lett. 192: 237-241.
- TAMARU, Y. AND DOI, R.H. 2000. engL gene cluster of Clostridium cellulovorans contains a gene for cellulosomal manA. J. Bacteriol. 182:244-247.
- PARK, J.-S., SHIN, H.-S., AND DOI, R.H. 2000. Fusion Proteins Containing Cellulose-Binding Domains. Meth.Enzymol. 326: 418-429.
|
Progress 01/01/99 to 12/31/99
Outputs
The CLOSTRIDIUM CELLULOVORANS cellulosome (cellulase) is being analyzed for its structure, function, assembly, and regulation, since microbial cellulases play a role in the degradation of cellulosic biomass and contributes to the CO2 cycle. We have been able to clone a gene cluster containing 9 genes involved in cellulase degradation. In addition we have cloned a cellulosomal pectate lyase gene, pelA, which codes for a pectinolytic enzyme. It is the first report of a pectinolytic enzyme that is a component of the cellulosome. A mannanase gene, manA, was also cloned, sequenced and expressed in E. coli. Thus the cellulosome has all the enzymes necessary for degrading plant cell walls. In fact we have been able to convert Arabidopsis and tobacco cells into protoplasts by treatment with cellulosomes. Cellulosome synthesis appears to be regulated by the carbohydrate substrate available to the organism, since the quantity and the enzymatic composition of the cellulosome is
altered by growth in different carbohydrate-containing media. The crystal structure of the cellulose binding domain (CBD) of the scaffolding protein CbpA was also determined. This provides information on how the cellulosome binds to its cellulose substrate.
Impacts
This research has potential for converting agricultural wastes to useful products such as ethanol, methane, and cellulase enzymes for industrial purposes. This will be good for the environment and for agriculture.
Publications
- Tamaru, Y., Liu, C.-C., Ichishi, A., Malburg, L., and Doi, R.H. 1999. The Clostridium cellulovorans cellulosome and non-cellulosomal cellulases, pp.488-494. In K. Ohmiya, K. Hayashi, K. Sakka, Y. Kobayashi, T. Kimura, and S. Karita (eds), Genetics, Biochemistry and Ecology of Cellulose Degradation, Uni Publishers, Co., Ltd., Tokyo, Japan.
- Wang, L.-F., Park, S.-S., and Doi, R.H. 1999. A novel Bacillus subtilis gene, antE, temporally regulated and convergent to and overlapping dnaE. J. Bacteriol. 181:353-356.
- Rough, S.E., Yau, P.M., Chuang, L.F., Doi, R.H. and Chuang, R.Y. 1999. Effect of the chlorinated hydrocarbons heptachlor, chlordane, and toxaphene on retinoblastoma tumor suppressor in human lymphocytes. Toxicol. Lett. 104:127-135.
- Doi, R.H., Goldstein, M., Takagi, M., Hashida, S., Shoseyov, O. and Segel, I. 1999. Structure and function of Clostridium cellulovorans cellulase subunits, pp. 479-485. In Environment, Science and Technology: The challenge of the 21st century. Chulabhorn Research Institute, Bangkok, Thailand.
- Tamaru, Y. and Doi, R.H. 1999. Three surface layer homology domains at the N-terminus of the Clostridium cellulovorans major cellulosomal subunit, EngE. J. Bacteriol. 181: 3270-3276.
- Rought, S.E., Yau, P.M., Guo, X.-W., Chuang, L.F., Doi, R.H., and Chuang, R.Y. 1999. Modulation of CPP32 activity and induction of apoptosis by heptachlor, a chlorinated hydrocarbon pesticide. J. Biochem. Mol. Toxicol. 14: 42-50.
- Miyagi, T., Peng, C.Y.S., Chuang, R.Y., Mussen, E., Spivak, M.S., and Doi, R.H. 1999. Verification of oxytetracyline resistant American foulbrood pathogen Paenibacillus larvae in the United States. J. Invert. Pathol., in press.
|
Progress 01/01/98 to 12/01/98
Outputs
The structure, function, regulation, and assembly of the cellwallsome and non-cellwallsomal cellulases of CLOSTRIDIUM CELLULOVORANS are being investigated, since cellulases play a major role in the carbon cycle on earth by converting plant cell wall materials to cellobiose and glucose and finally to CO(subscript 2). We have cloned and sequenced two gene clusters during the past year which has given us an idea of the genes involved in plant cell wall degradation. The largest gene cluster called the engL cluster contains 9 genes including manA which is involved in mannan degradation. The engY cluster contains two genes engY and pelA, which codes for pectate lyase and is involved in pectin degradation. Therefore, the cellwallsome is capable of degrading cellulose, xylan, mannan and pectin. The engE gene coding for the most abundant endoglucanase has been cloned and it has the unusual property of containing three repeated surface layer homology (SLH) domains which suggests
that it can bind not only to the scaffolding protein CbpA of the cellwallsome, but is also capable of binding to the cell surface. The SLH domains of EngE and of CbpA are therefore capable of binding the cellwallsome to the cell surface.
Impacts
(N/A)
Publications
- ICHI-ISHI, A., SHEWEITA, S. and DOI, R.H. 1998. Characterization of EngF from Clostridium cellulovorans and identification of a novel cellulose binding domain. Appl. Environ. Microbiol. 64:1086-1090.
- LIU, C.-C. and DOI, R.H. 1998. Properties of exgS, a gene for a major subunit of the Clostridium cellulovorans cellulosome. Gene 211:39-47.
- DOI, R.H., PARK, J.-S., LIU, C.-C., MALBURG, L.M. JR., TAMARU, Y., ICHIISHI,A. and IBRAHIM, A. 1998. Cellulosome and non-cellulosomal cellulases of Clostridium cellulovorans. Extremophiles 2:53-60.
- WILLIAMS, J.R., PENG, C.Y.S., CHUANG, R.Y., DOI, R.H. and MUSSEN, E.C. 1998. The inhibitory effect of azadirachtin on American foulbrood pathogen Paernbacillus (formerly Bacillus) larvae of honey bee (Apis mellifera L.) and other gram pos.
|
Progress 01/01/97 to 12/01/97
Outputs
The cellulosome and non-cellulosomal cellulases of CLOSTRIDIUM CELLULOVORANS are being investigated for their structure, function, regulation, assembly, and synergism. We have isolated a novel non-cellulosomal cellulase EngF that has weak endoglucanase activity and is induced during growth on cellobiose. It has a novel cellulose binding sequence which has not been reported previously. In contrast to EngD, a previously characterized non-cellulosomal enzyme, EngF has about 1% of the specific activity of EngD on acid swollen cellulose. EngF may function as a cellodextrinase and may act synergistically with the cellulosome, since preliminary studies indicate that there is more than an additive activity when EngF and the cellulosome are present together. We have further isolated the gene for P100, the major, endoglucanase associated with the cellulosome, and another novel cellulosomal enzyme EngY. These genes are being sequenced and their products are also being
characterized. The regulation of these and previously isolated genes are being investigated by using the genes as probes for the expression of mRNA produced from these genes. The characterization of P100 will allow us to examine the assembly of the cellulosome with the other two major cellulosomal subunits, CbpA and ExgS. Mini-CbpA is being expressed in ESCHERICHIA COLI and will be used in assembly studies with P100 and ExgS.
Impacts
(N/A)
Publications
- PARK, J.-S. and DOI, R.H. 1997. Clostridium cellulovorans cellulose and its useful applications. Proc. Intl. Symp. Strain Development for Bioindustry, p. 71-78.
- LIAO, C.-T., WEN-Y.-D., WANG, W.-H., TSAI, S.-C., DOI, R.H., and CHANG, B.-Y. 1997. The importance of a proper helical structure in the promoter -10 binding region to Bacillus subtilis sigma-A structure and function. J. Biochem. (Tokyo).
|
Progress 01/01/96 to 12/30/96
Outputs
The structure, function, and assembly of the CLOSTRIDIUM CELLULOVORANS cellulosome and the regulation of synthesis of the subunits of the cellulosome are being investigated. We have found that a small segment of the nine conserved hydrophobic domains (HBD) of the major scaffolding protein, CbpA, of the cellulosome is capable of binding weakly to the hydrophobic domains of the enzymatic subunits that associate with the CbpA. On the other hand, the duplicated sequences (DS) of the enzymatic subunits bind strongly to the HBD; thus two types of binding forces allow the enzymatic subunits to bind to CbpA. The HBD1 and HBD6 bind the major enzymatic subunits, P70 and P100, with different affinity. Thus, the HBDs are not identical in their functions. We have sequenced the exgS gene of one of the major enzymatic subunits previously called P70. The exgS codes for an exoglucanase ExgS. Growth in different carbon sources indicated that the cellulose genes were highly regulated.
Impacts
(N/A)
Publications
- DOI, R.H. 1995. The future of food and agricultural biotechnology. Bulletin of Food Research 34:1-8.
|
Progress 01/01/95 to 12/30/95
Outputs
The structure, function and assembly of the CLOSTRIDIUM CELLULOVORANS cellulosome is being investigated. The major findings include the observation that the duplicated sequences (DS) of the enzymatic subunits (endoglucanases) are not necessary for their interaction with the scaffolding protein, cellulose binding protein A (CbpA), and that sequences upstream of the DS are involved in the binding of subunits to CbpA. This was demonstrated by deletion of the DS region and subsequent binding of the DS-less protein to the endoglucanase binding domain (EBD) of the CbpA. This was shown with EngB and EngD and the recently isolated EngF subunits. We have also cloned, sequenced and characterized the ExgS gene that codes for the major exoglucanase, ExgS, present in the cellulosome. ExgS is homologous to the previously reported CelS from C. THERMOCELLUM. We further observed that the major cellulosome subunit genes (cbpA, P100, and exgS) were repressed during growth of C.
CELLUVORANS on glucose or cellobiose, whereas engF, a minor component of the cellulosome, was highly derepressed.
Impacts
(N/A)
Publications
|
Progress 01/01/94 to 12/30/94
Outputs
The structure, function and assembly of the CLOSTRIDIUM CELLULOVORANS cellululosome are being characterized. Analysis of the cellulose binding domain (CBD) of the cellulose binding protein A (CbpA), the major scaffolding protein present in the cellulosome, revealed that mutations, whether small or large deletions or single site amino acid mutations, led to a significant decrease in the binding ability of CBD to crystalline cellulose. This suggested that the entire CBD structure may be critical for its binding to cellulose. Studies on the hydrophobic domain (HBD) of the CbpA indicated that about a 30 amino acid sequence (endoglucanase binding domain or EBD) is essential for binding of endoglucanases to CbpA. The studies on cellulosome assembly have revealed that crystalline cellulose promotes the assembly of the various subunits into the cellulosome. The key subunit appears to be CbpA which initially is present in a nascent form that is converted to a mature form by
binding to crystalline cellulose. After conversion to the mature form CbpA is able to bind the other major (P100 and P70) and minor subunits (various endoglucanases) that are present in the cellulosome.
Impacts
(N/A)
Publications
|
Progress 01/01/93 to 12/30/93
Outputs
The functional domains of the cellulose binding protein A (CbpA) of the CLOSTRIDIUM CELLULOVORANS cellulose are being identified and characterized. A single cellulose binding domain (CBD) was found at the N-terminus of CbpA and resides in a peptide sequence containing about 160 amino acids. The CBD was able to bind to crystalline forms of cellulose and to chitin with a Kd of about 1 (mu)M but did not bind to soluble forms of cellulose such as carboxymethylcellulose or to cellobiose. Thus the CBD appears to bind to a three dimensional feature of crystalline cellulose. Currently mutations are being made in CBD to determine which amino acid residues are responsible for cellulose binding. A 30 amino acid sequence found in each of the nine 120-amino acid-long hydrophobic domains (HBD) binds to endoglucanases EngB and EngD when tested by an Interaction Western blot system or a sandwich enzyme immunoassay technique. The binding of these domains to their substrates is
dependent only on the isolated sequences that are synthesized from mini-genes in ESCHERICHIA COLI and not on any part of the CbpA molecule.
Impacts
(N/A)
Publications
|
Progress 01/01/92 to 12/30/92
Outputs
Analysis of the functional domains of the cellulose binding protein A (CbpA) from CLOSTRIDIUM CELLULOVORANS has revealed the presence of a cellulose binding domain (CDB) at the N-terminus of CbpA. This domain was subcloned and expressed in ESCHERICHIA COLI and purified by its affinity to cellulose. The purified CBD bound to several crystalline forms of cellulose and the binding was not inhibited by soluble forms of cellulose such as carboxymethyl-cellulose (CMC) or by cellobiose. One of the 8 hydrophobic domains (HBD) was subcloned and expressed and found to bind endoglucanases B (EngB) and D (EngD). Thus two major functional domains of CbpA have been identified. Further studies on their properties are being pursued.
Impacts
(N/A)
Publications
|
Progress 01/01/91 to 12/30/91
Outputs
Analysis of the CLOSTRIDIUM CELLULOVORANS cellulase subunits has revealed the presence of more than 10 different subunits. Two general types of subunits are present, those with and those without any apparent enzymatic activity. Three genes, ENGB, ENGC and ENGD, coding for enzymes subunits have been cloned and sequenced. Chimeric proteins have been cnstructed between EngB and EngD which showed that specificity of the enzyme resided in the N-terminal portion of the molecules and not in the conserved C-termini. One gene (CBPA) coding for a cellulose binding protein (CboA) that has no apparent enzymatic activity has been cloned and sequenced. The 190 kDa CbpA binds tightly to cellulose and is essential for the degradation of crystalline cellulose. CbpA has a signal peptide sequence, 5 putative cellulose binding domains, and 8 repeated highly conserved hydrophobic domains. Further CbpA domain studies are underway.
Impacts
(N/A)
Publications
|
Progress 01/01/90 to 12/30/90
Outputs
Progress has been made in understanding how the expression of genes that code for extracellular proteins are regulated by signals from the external medium. In our BACILLUS SUBTILIS system the extracellular protein genes are regulated by a redundant mechanism involving several positive and negative regulators. We have characterized one of the positive regulatory genes, SENS, by cloning and sequencing the gene and by studying the effects of this gene on the expression of the APRE gene which codes for subtilisin, a protease that is secreted into the medium. The SENS gene codes for a small 65 amino acid protein that is highly basic, contains a helix-turn-helix motif found in DNA binding proteins, and is partially homologous to RNA polymerase sigma factors. The SENS gene itself is regulated by a complex system of negative regulators and an attenuator mechanism. By deletion studies of the APRE gene we have found that the SENS positive regulation requires the presence of a
sequence upstream of the APRE promoter as well as a sequence which is part of the promoter region.
Impacts
(N/A)
Publications
|
Progress 01/01/89 to 12/30/89
Outputs
We have made substantial gains in our efforts to understand the regulation of gene expression in Bacillus subtilis by the analysis of two model systems: (a) the transcriptional regulation of the sigA operon and (b) the regulation of expression of the aprE gene. The analysis of the sigA operon has been carried out by determining the location of promoters that are used to express this operon during growth and during sporulation. By use of the primer extension method we have shown that 6 promoters (P1 through P6) are utilized to express the operon. Promoters P1 and P2 express the operon strongly during growth; promoters P5 and P6 very weakly during growth, but very strongly during early sporulation; promoters P3 and P4 only during late sporulation. P1, P2 and P3 are SigA or SigA-like promoters, P5 and P6 are SigH promoters, while the P4 promoter is still unidentified. The expression of the aprE gene is regulated in a very complex way by the products of regulatory
genes. We have cloned one of the regulatory genes, senS, which in high copy numbers stimulates the expression of aprE by a 3-4 fold. The expression of senS itself is regulated probably by an anti-termination mechanism which affects an attenuation site between the promoter and the open reading frame of senS. Preliminary analyses suggest that a NusA-like Box A sequence exists in the attenuation region indicating that a NusA-like function exists in B. subtilis.
Impacts
(N/A)
Publications
|
Progress 01/01/88 to 12/30/88
Outputs
We are continuing our research on the mechanism of gene regulation in Bacillus subtilis by studying the regulation of (1) the expression of the RNA polymerase sigma-A operon and (2) the expression of the extracellular serine protease (subtilisin) gene, aprE. We have shown that the expression of the sigma-A operon depends on both promoter and RNA polymerase switching from promoters P1P2 to promoter P3 during sporulation. We have made mutations in the -35 region of promoter P3 by site directed mutagenesis and somewhat surprisingly these mutations have had little or no effect on promoter P3 activity as determined by use of promoter probe plasmids. Further studies are being directed towards analysis of the -10 region of promoter P3 to see what effects the deletion of P3 activity has on the expression of the sigma-A operon during sporulation. Our studies on the expression of the subtilisin gene, aprE, have resulted in the isolation and characterization of two regulator
genes, senN and senS. The overproduction of the products of these two regulator genes results in the hyperexpression of several extracellular protein genes. Both of these genes code for a small protein of about 7,000 daltons. The senS gene itself appears to be highly regulated since an attenuator site exists between its promoter and structural gene sequence. The effect of these regulator genes on the expression of aprE indicates that the regulation is at the transcription level.
Impacts
(N/A)
Publications
|
Progress 01/01/87 to 12/30/87
Outputs
Our studies on the mechanism of gene expression in Bacillus subtilis are currently focused on the regulation of the RNA polymerase sigma-43 operon and the extracellular alkaline protease (subtilisin) gene. In our attempt to identify the role and function of P23, the first gene of the sigma-43 operon, we have made insertional and deletion mutations in P23. The insertional mutation (the cat gene was inserted in place of the deleted P23 gene) caused no apparent deleterious effect on growth or sporulation. In fact somewhat surprisingly it caused an acceleration of the sporulation phase by about one hour. The deletion of P23 resulted in two phenotypes, a spo and spo suggesting the possibility that this region of the operon is critical in regulating the expression of the operon during sporulation. Since the promoter P3 is located within the open reading frame of P23 and is expressed only during sporulation, we identified the RNA polymerase holoenzyme which is responsible
for its expression. By use of null mutants of minor sigma genes we observed that sigH was required for expression of promoter P3 suggesting that SigH itself may be required for expression from P3. In collaboration with Charles Moran, we were able to prove in vitro that RNA polymerase with sigma-30 (sigH) was the enzyme involved in transcribing P3 during the sporulation phase. Thus the possibility exists that the removal of P23 can affect the level of SigA during sporulation.
Impacts
(N/A)
Publications
|
Progress 01/01/86 to 12/30/86
Outputs
We are continuing our study of the regulation of gene expression in Bacillus subtilis by analyzing the sigma-43 operon and the subtilisin (aprA) gene. The sigma-43 operon is transcribed during growth from two tandem sigma-43 promoters and during stationary phase from a downstream sigma-37 promoter. S1 nuclease mapping experiments indicated that the sigma-37 promoter was located within the N-terminus of the first gene (P23) of the operon. The transcripts from the sigma-43 promoters encoded three putative proteins with MWs of 23,000, 19,000 and 9,000, whereas the transcript from the sigma-37 promoter encoded only the protein with a MW of 9,000. All three putative proteins are in the same reading frame and three potential translational initiation sites have been tentatively identified from the base sequence analysis of the first gene. The promoter switching that occurs at the end of the log phase of growth, in turn causes the translational switching. This is the
first demonstration of promoter switching for an operon that is expressed primarily during the log phase of growth. This type of regulatory mechanism allows this operon to be expressed during both good and poor nutritional conditions. The analysis of the expression of the subtilisin gene has revealed the precise signal peptidase cleavage site for preprosubtilisin. Further conditions were found for maximum expression of a foreign procaryotic gene in B. subtilis. These findings should allow us to construct both a recombinant vector and a B.
Impacts
(N/A)
Publications
|
Progress 01/01/85 to 12/30/85
Outputs
The regulation of gene expression in Bacillus subtilis is being investigated by two gene systems: the RNA polymerase sigma-43 operon and the extracellular alkaline serine protease (subtilisin) gene (aprA). The entire sigma-43 operon has been cloned and sequenced. The operon consists of a promoter proximal gene which codes for a protein of 23,000 daltons (P23), the dnaE gene which codes for the DNA primase, and the rpoD gene which codes for sigma-43, the major sigma factor of B. subtilis. The transcription of this operon is controlled by tandem sigma-43 and sigma-37 promoters which results in prometer switching during the transition from the growth phase to the sporulation phase. In addition a heat shock promoter has been identifed which lies in the C-terminus of the dnaE gene and which controls the expression of the sigma-43 gene (rpoD) under heat shock conditions. Thus the regulation of expression of this operson appears to be extremely complex and requires at
least three different forms of RNA polymerase holoenzyme. The subtilisin gene is being analyzed for its transcriptional regulation and for its use in the expression and secretion of foreign gene products from B. subtilis. Deletion analyses upstream of the structural part of the gene revealed the presence of three promoters. The promoter farthest upstream appars to be a sigma-43 promoter which is regulated by a positive regulatory factor.
Impacts
(N/A)
Publications
|
Progress 01/01/84 to 12/30/84
Outputs
The regulations of gene expression in Bacillus subtilis is being investigated byanalyzing the properties of the subtilisin gene (apr) which codes for an extracellular alkaline serine protease. This gene has been cloned and sequenced and shown to be transcribed by the sigma-37 form of RNA polymerase. This is the first demonstration of a gene controlled by a minor sigma enzyme whose product has been well characterized. A sequence analysis of the gene showed that it codes for a preproenzyme. The signal peptide has 29 amino acids and the propeptide contains 77 residues. The N-terminus of the mature subtilisin starts at residue 107. The promoter region of this gene appears to be quite complex and it appears to have more than one promoter, since S1 mapping has shown the presence of at least two transcription initiation points. This promoter and signal peptide may be useful for expression and secretion of foreign proteins from B. subtilis in cloning experiments.
Impacts
(N/A)
Publications
|
Progress 01/01/83 to 12/30/83
Outputs
The structure and function of Bacillus subtilis RNA polymerase is being analyzedby three approaches: enzymology of the RNA polymerase holoenzymes; a genetic analysis of the major sigma-55 gene; and an analysis of the specificity of the holoenzymes. Significant progress has been made in all three areas. By use of the previously reported promoter probe plasmid pGR71, we have isolated a temporally regulated promoter which is expressed during stationary phase, but not during growth. The promoter is transcribed by the sigma-37 enzyme and the gene controlled by this enzyme is the B. subtilis subtilisin gene. The gene codes for a signal peptide, propeptide and then the protease gene. The sequence of the promoter region revealed a typical sigma-37 promoter which was also overlapped by a region of dyad symmetry, suggesting some type of regulatory site is present. This promoter and its signal peptide may be very useful for cloning and expressing heterologous genes. The
second major aspect of our work is our analysis of the sigma-55 gene, which we were able to clone and identify. We have physically mapped the gene to a 1.6 kbp fragment of DNA; this gene resides next to the dnaE gene which appears to be the DNA primase gene, since it has a high amino acid sequence homology with the E. coli DNA primase. We are currently sequencing the dnaE and rpoD (sigma-55) genes.
Impacts
(N/A)
Publications
|
Progress 01/01/82 to 12/30/82
Outputs
The structure and function of the multiple RNA polymerase holoenzymes and the expression of heterologous genes in Bacillus subtilis are being studied. For this purpose a promoter-probe plasmid has been developed which is capable of replication in both E. coli and B. subtilis and has a chloramphenicol acetyl transferase (CAT) gene originally found in Tn9. This gene which was integrated into the co-integrate plasmid, pGR1, is missing its promoter but has a HindIII restriction endonuclease site in front of the structural CAT gene. By insertion of HindIII fragments of B. subtilis into this site, we have been able to express the CAT gene. Since E coli genes are ordinarily not expressed in B. subtilis, we analyzed the product which was being synthesized in B. subtilis and E. coli. We found that a fusion product was being synthesized in B. subtilis indicating that translation was starting from a ribosome binding site (rbs) from the B. subtilis part of the mRNA. On the
other hand in E. coli two CAT products were found, one a native size CAT and the other a fusion product with the same molecular weight as that found in B. subtilis. Thus our interpretation is that the E. coli translation mechanism can recognize and utilize the rbs of both E. coli and B. subtilis, wherease the B. subtilis translation mechanism is able to utilize only the B. subtilis rbs on the mRNA. As part of our long range analysis of the RNA polymerase holoenzymes in B.
Impacts
(N/A)
Publications
|
Progress 01/01/81 to 12/30/81
Outputs
The structure and function of the multiple RNA polymerase holoenzymes of Bacillus subtilis are being analyzed by two major approaches. In the first approach the various holoenzyme forms are being purified and characterized as to their subunit structure and function. We have found that the major sigma factor (sigma-55) can bind to super-coiled but not to linear DNA. These results support the notion that sigma factors can play a role in the binding of RNA polymerase to the promoter site. To demonstrate the generality of this phenomenon, we were also able to demonstrate that the E. coli sigma factor could also bind to super-coiled, but not to linear DNA. To our surprise we also found that the DNA-sigma complex could be assayed by the nitrocellulose filter technique which should facilitate the analysis of complex formation. Current work is in progress in which we are comparing the properties of sigma-37, sigma-29, delta factor, and other core associated polypeptides
which have been isolated from both vegetative and sporulating cells of B. subtilis. As a complement to these studies we have constructed useful plasmid cloning vectors to facilitate the isolation of promoter-containing DNA fragments which we will be able to use as specific templates for the various RNA polymerase holoenzymes. One of these vector plasmids, pGR71, has an E. coli gene for chloramphenicol resistance which codes for chloramphenicol acetyl transferase (CAT), but this gene is missing its natural promoter.
Impacts
(N/A)
Publications
|
Progress 01/01/80 to 12/30/80
Outputs
The structure and function of RNA polymerase of sporulating Bacillus subtilis cells are being investigated by two major approaches in order to understand the role of this enzyme in gene selection during differentiation. By one enzymological approach we have discovered that the enzyme can actually exist in three forms during the vegetative phase of growth. These forms differ by having small molecular weight polypeptides associated with the core enzyme (E). These forms have been designated as ESigma 5 5, ESigma 5 5, ESigma 3 7, and EDelta. In sporulating cells three additional forms have been found with the composition ESigma. In sporulating cells three additional forms have been found with the composition ESigma 2 9, EDelta 2, and EDelta 3. In order to determine the promoters specificities of these form, we have been using recombinant DNA techniques to isolate DNA fragments containing one or few promoters to use as templates for these RNA polymerases. Data have
been obtained which show that the promoters recognized by ESigma 5 5 and ESigma 3 7 are different and occupy different promoter spectra. The promoter recognized by ESigma 3 7 can also be recognized to some degree by ESigma 2 9. These differences in promoter specificity are being determined by comparing RNA transcripts and showing their transcription from specific DNA fragments.
Impacts
(N/A)
Publications
|
Progress 01/01/79 to 12/30/79
Outputs
We are continuing our studies on the structure and function of the transcriptionapparatus of Bacillus subtilis in order to gain an understanding of the mechanism of interaction between RNA polymerase (RPase) and promoters, the factors which influence promoter selection during growth and sporulation of the organism and the properties of promoters which influence their efficiency. Two major approaches have been taken including the isolation of various forms of RPase and an analysis of subunit and functional properties of these forms and the isolation of promoter-containing DNA fragments in order to study RPase binding and their base sequences. The latter approach is quite new for my research program, but it has progessed very well to the point that we have identified and cloned many promoter-containing fragments of B. subtilis DNA. These fragments are currently being studied for their relative binding efficiencies to vegetative cell RPase, their initiation ability for
transcription and their base sequences. The relative paucity of information about the mechanism of action of B. subtilis RPase has forced us to do extensive basic analyses of the interactions of RPase with DNA, but this will be extremely beneficial for all of our future studies. The second major approach is a continuing effort to characterize the RPase found in vegetative and sporulating cells of B. subtilis.
Impacts
(N/A)
Publications
|
Progress 01/01/78 to 12/30/78
Outputs
Our analyses of the genetic transcription machinery of Cacillus subtilis have revealed significant differences between its RNA polymerase and that from E. coli. The subunits of the B. subtilis enzyme differ in size and function. In E. coli the Beta subunit is responsible for both rifampicin and streptolydigin resistance. In B. subtilis the Beta subunit is responsible for rifampicin resistance, but the Beta' subunit is responsible for streptolydigin resistance. Also the size of the B. subtilis Beta subunit is larger than that of the B' subunit. Thus our results indicate that reconstitution studies are required before definitive functions can be assigned to the subunits of prokaryotic RNA polymerases. A second significant finding indicates that the initiation mechanism of B. subtilis may require two polypeptides instead of one as in E. coli. We have found that a small polypeptide, called delta factor, is involved in DNA site recognition and that sigma factor is
involved in initiation of RNA synthesis. Thus two factors working sequentially in B. subtilis initiate RNA synthesis. This is in contrast to E. coli in which one large polypeptide appears to have both the recognition and initiation functions. A third study has indicated that the RNA polymerases from vegetative and sporulating cells may have different promoter specificities. The antibiotic netropsin which binds to A-T rich regions of DNA inhibits the sporulation RNA polymerase at a much loiwer concentration than the vegetative enzyme.
Impacts
(N/A)
Publications
|
Progress 01/01/77 to 12/30/77
Outputs
Our analyses of the RNA polymerase from the sporulating bacterium Bacillus subtilis have been continued in order to gain an understanding of its role in gene expression during differentiation. A major goal was accomplished during the past year when methods were perfected for the separation of the subunits of the RNA polymerase core. The use of Blue Dextran-Sepharose and phosphocellulose column chromatography has enabled us to separate the Beta, Beta', and Alpha subunits in good yield. This has allowed us to do reconstitution experiments between wild type and mutant subunits in order to study the function of the individual subunits. The data to date indicate that the largest polypeptide (Beta) is responsible for rifampicin resistance and the second largest polypeptide (Beta') is responsible for streptolydigin resistance. These properties of the subunit differ from that reported previously with E. coli. It was possible to make a double antibiotic resistance enzyme
in vitro by this technique. Another aspect of our work with significant results is the use of the DNA-cellulose column to obtain fine separation of RNA polymerase activity into several different forms of the enzyme. The elution pattern from the column shows the following sequence of enzyme forms: Alpha(2)Beta Beta'Delta, Alpha(2)Beta Beta'Conjunction', and Alpha(2) Beta Beta'Conjunction.
Impacts
(N/A)
Publications
|
Progress 01/01/76 to 12/30/76
Outputs
The analysis of the regulation of transcription during sporulation of Bacillus subtilis has been continued. Two major directions have been undertaken. One concerns the purification of large amounts of both log phase and sporulation RNApolymerase (RPase) in order to analyze its subunit structure and function. A new method has been developed to remove protease from crude extracts; it involves the use of a hemoglobin-Sepharose affinity column which effectively removes all peptidases and proteases from the crude extract. This has increasedyield of enzyme by 10-fold and assures us that proteolytic cleavage products arenot obtained. A new method has been developed to purify RPase by a Blue DextranSepharose column which allows rapid purification of the beta subunit when the enzyme is treated with urea. We have been able to show that the zinc atoms associated with the RPase is located on the beta subunit. This indicates that the beta subunit is probably involved in the
catalytic function of the enzyme. An extensive characterization of the sporulation RPase has been accomplished. This enzyme has a new subunit associated with the RPase core which we have called differentiation factor delta. When the delta factor is associated with the core, the enzyme has a greater affinity to DNA at high ionic conditions, hasa higher specific activity than holoenzyme from vegetative cells, and is inhibited at lower concentrations of netropsin, an antibiotic which binds A-T rich regions of DNA. We have also found that the delta
Impacts
(N/A)
Publications
|
Progress 01/01/75 to 12/30/75
Outputs
Previous studies with competition hybridization techniques demonstrated that sporulating cells of Bacillus subtilis were expressing both vegetative cell genes and sporulation cell specific genes. It was of interest to determine whether the expression of sporulation genes was dependent on a modification of RNA polymerase. The purification of RNA polymerase from both vegetative and sporulating cells was undertaken. Great difficulty was encountered initially because of the high protease activity of sporulating cells which degraded RNA polymerase in cell free extracts. The use of diisopropylfluorophosphate, a potent protease inhibitor, has allowed us to purify the enzyme. We have found the usual RNA polymerase core (aybb') and holoenzyme (aybb'o) in vegetative cells. At TPG.5 of sporulation the same pattern was found. At TDT.5, in additionto core and holoenzyme a new form of RNA polymerase was observed which had a newpolypeptide (8') with a molecular weight of 27,000.
The composition of this enzyme is aybb'8'. At T1.5 another modified enzyme was found with composition aybb'8(2). The 8(2) subunit had a molecular weight of 20,000. A mutant which stopped sporulating at T(1) did not form these modified enzymes. These results suggest the exciting possibility that RNA polymerase can be modified and that this may be a mechanism to regulate its template specificity.
Impacts
(N/A)
Publications
|
Progress 01/01/74 to 12/30/74
Outputs
The analysis of messenger RNA (mRNA) in sporulating cells and dormant spores of B. subtilis has revealed that sporulation specific mRNA is made in a sequential manner during the various stages of sporulation. However, it was interesting that some vegetative cell genes are continually expressed even late in sporulation resulting in the presence of both vegetative and sporulation specific mRNAs in sporulating cells. Even the dormant spore was found to have amixture of vegetative and sporulation specific mRNAs. This indicates that both types of genes are expressed during sporulation in the forespore. These resultsindicate that the RNA polymerase must be able to recognize and transcribe at least two different classes of genes. The pulse-labeling pattern of sporulatingcells was also analyzed by short pulses of uridine-HDT at half hour intervals during sporulation. A reproducible pattern of RNA synthesis was obtained. The results indicated that the rate of synthesis of
RNA varied quite dramatically depending on the stage of sporulation.
Impacts
(N/A)
Publications
|
|