Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: Research was completed with a graduate doctoral student to test a computational model for optimizing translation of mRNA into higher protein yields. Specific genes (GST and pfu0132) were analyzed with the model, then synthesized to meet criteria for high-efficiency translation. The proteins GST and Pfu0132 were expressed in E. coli, where indeed improved (30% - 50% higher) synthesis was detected. The systems provides a generally useful approach to engineering genes for high-level translation and protein yield. Several newly sequenced phage genomes were analyzed to obtain new insights to phage genome evolution and novel anti-bacterial reagent development. Genomes were annotated and prepared for deposition in GenBank and for manuscript preparation. PARTICIPANTS: Tuttle, Anna. NCSU Department of Microbiology Ph.D. student. Vu, Scott. NCSU Biomedical Engineerng Ph.D. student. TARGET AUDIENCES: Biotechnology and pharmaceutical companies that seek improved gene expression and protein yields. Life Science researchers studying phage biology, phage-bacterial interactions, and phage-derived biotherapeutics and diagnostics. Biotechnology companies, including small business start-ups, can derive new knowledge or applications for the above and for new food safety applications. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Longterm scientific contributions are in the areas of improved gene expression; increased levels of diagnostic, therapeutic and industrial protein production; viral genome diversity; new viral gene products that can inhibit pathogenic bacteria; new diagnostics for food and agricultural biosafety; and new treatments for human, animal and plant infectious agents.
Publications
- Uzan, M. and E.S. Miller. 2010. Post-transcriptional control by bacteriophage T4: mRNA decay and inhibition of translation initiation. Virology Journal, 7:360
- Petrov V.M., S. Ratnayaka, J.M. Nolan, E.S. Miller, and J.D. Karam. 2010. Genomes of the T4-related bacteriophages as windows on microbial genome evolution. Virology Journal 7:292
- Karam, J.D and E.S Miller. 2010. Bacteriophage T4 and its relatives (Editorial). Virology Journal, 7:293
|
Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Research was completed to generate mutations in two important enzymes of NAD metabolism encoded by the Vibrio phage KVP40: nicotinamide phosphoribosyl transferase (NadV) and nicotinamide mononucleotide adenylyltransferase (NatV). The same genes were manipulated in phage RB43, a related virus that infects strains of Escherichia coli. Affects of mutational knock-outs on viral DNA replication and burst size are under investigation. To expand appreciation and understanding of genome diversity among the large T4-type myoviridae phages, several newly isolated phage and their genomes were sequenced. These include phages infecting Vibrio, Aeromonas (a pathogen of fish - trout and salmon in particular), Salmonella and Pseudomonas. The genomes were sequenced using current Roche FLX 454 technology. Sixteen new genomes range in size from approximately 100 kbp to 240 kbp. New phage products coded by the sequenced viral genomes can provide resources for bacterial pathogen control. PARTICIPANTS: Lee, Jae Lee. NCSU Department of Microbiology MS Thesis student. Degree conferred. Tuttle, Anna. NCSU Department of Microbiology Ph.D. student. Degree in progress. Edwards, Ginger. NCSU Department of Microbiology B.S. student. Degree conferred. TARGET AUDIENCES: Life Science researchers studying phage biology, phage-bacterial interactions, and phage-derived biotherapeutics and diagnostics. Biotechnology companies, including small business start-ups, can derive new knowledge or applications for the above and for new food safety applications. PROJECT MODIFICATIONS: - Increased focus on products and research initiatives deriving from whole genome sequencing of novel phages infecting pathogenic bacteria.
Impacts
Longterm scientific contributions are in the areas of viral metabolism and takeover of host metabolism; viral DNA replication efficiency; viral genome diversity; new viral gene products that can inhibit pathogenic bacterial propagation; new diagnostics for food and agricultural biosafety; new treatments for human, animal and plant infectious agents.
Publications
- Lee, J. Y. 2008. Enzyme Properties and mRNA Expression of an NAD+ Scavenging System: NatV and NadV of Vibrio parahaemolyticus Phage KVP40. Master of Science Thesis. North Carolina State University. Raleigh, NC.
|
Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Research was completed on two important enzymes of NAD metabolism encoded by the Vibrio phage KVP40: nicotinamide phosphoribosyl transferase (NadV) and nicotinamide mononucleotide adenylyltransferase (NatV). The reaction rates were determined using coupled reporter assays and the products formed were confirmed by mass spectrometry. The results yield new insights to NAD synthesis and present potentially new avenues for anti-infectives for food safety concerns (Vibrio parahaemolyticus). A project to address fungal disease of Tall Fescue was performed in collaboration with Dr. Ron Qu. In this approach, the phage T4 lysozyme gene was cloned and transfered into Festuca. Testing of fescue resistance to infection by Magnaporthe and Rhizoctonia fungi demonstrated expression of the transgenic lysozyme and improved fungal resistance. PARTICIPANTS: ES Miller - NCSU JY Lee - NCSU R Qu - NCSU L Treadway - NCSU S Dong - NCSU HD Shew - NCSU N Deighton - NCSU TARGET AUDIENCES: Researchers and programs in Food Safety; Human Health and Disease; Basic Research and Biology; Turfgrass management; Plant Biology and Pathology PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Tall fescue (Festuca arundinacea Schreb.) is an important turf and forage grass species worldwide. Fungal diseases present a major limitation in the maintenance of tall fescue lawns, landscapes, and forage fields. Two severe fungal diseases of tall fescue are brown patch, caused by Rhizoctonia solani, and gray leaf spot, caused by Magnaporthe grisea. We introduced the bacteriophage T4 lysozyme gene into tall fescue through Agrobacterium-mediated genetic transformation. In replicated experiments under controlled environments conducive to disease development, 6 of 13 transgenic events showed high resistance to inoculation of a mixture of two M. grisea isolates from tall fescue. Three of these six resistant plants also displayed significant resistance to an R. solani isolate from tall fescue. Thus, we have demonstrated that the bacteriophage T4 lysozyme gene confers resistance to both gray leaf spot and brown patch diseases in transgenic tall fescue plants. The gene may have wide applications in engineered fungal disease resistance in various crops.
Publications
- Dong, S., H. D. Shew, L. P. Tredway, J. Lu, E. Sivamani, E. S. Miller and R. Qu. 2008. Expression of the bacteriophage T4 lysozyme gene in tall fescue confers resistance to gray leaf spot and brown patch diseases. Transgenic Res. 17(1):47 - 57.
|
Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: The outcomes of this work were presented in an invited symposium (title: "Phage directed reprogramming of cellular processes") held at the General Meeting of the American Society for Microbiology in Toronto, Canada on May 23, 2007. The data were presented in a talk titled "NAD metabolism in the phage infected cell". Other work in phage biology was presented in a publication that resulted from a collaboration with our colleagues in the Department of Crop Science at North Carolina State University. In addition, the PI consulted with a virus detection company that modeled new systems for field applications that used bacteriophage to optimize the performance of their equipment.
PARTICIPANTS: Miller, Eric S. (P.I.); Lee, Jae Y. (Ph.D. student); Belanger,K. (Ph.D. student); Gladstone, L. (Masters student); Department of Crop Science at NC State University; Proteomics and Metabolomics Research Lab at NC state University. Small business consulting representatives.
TARGET AUDIENCES: Audiences for new knowledge: Research scientists at universities and federal agencies (USDA, EPA, FDA, NSF); graduate and postdoctoral trainees at universities, including trainees at NC State University; corporations developing new agricultural, veterinarian and human medicine products; attendees to national meetings for continuing education. Efforts include: symposium participation, publications, trainees in the P.I.'s research laboratory, clients of the Department of Microbiology and College of Agriculture and Life Sciences at NC State University, seed companies who developed Fescue grass varieties and extension agents who wish to keep abreast of advances in Fescue gray leaf spot brown patch disease resistance.
Impacts
Our findings on NAD metabolism demonstrated, through experimental results, that virus-infected cells express viral genes for enzymes of NAD synthesis. These enzymes are active and catalyze the reactions leading to the salvage of nicotinamide and converting it to NAD. NAD is an important cofactor for catalytic reactions, cell energetics and reduction reactions during normal cell growth. Therefore, a critical metabolic sequence has been identified that can present new targets for developing antiviral compounds. Further, investigators and clinicians may consider targeting the NAD pathway for antimicrobial compounds that inhibit these reactions and would thus inhibit bacterial growth. Findings with Crop Science collaborators at NCSU demonstrated that a phage gene can be incorporated into the genome of Fescue grass, and provide added defense against infection by gray leaf spot and brown patch fungi. The outcome is that clients are made aware of new fungal resistance
mechanisms and will support new studies and applications using transgenic plants with phage genes. Consultation with a small business viral detection company has improved their technology for enumerating viruses, and their size, in environmental samples. Their product development efforts were enhanced.
Publications
- Dong, S., H. D. Shew, L. P. Tredway, J. Lu, E. Sivamani, E. S. Miller and R. Qu. 2007. Expression of the bacteriophage T4 lysozyme gene in tall fescue confers resistance to gray leaf spot and brown patch diseases. Transgenic Res. Feb;17(1):47-57.
|
Progress 10/01/04 to 09/30/05
Outputs
The long-term goals of these projects are to bring together basic and applied research with RNA aptamers, RNA biology and their relevance to phage and bacteria of food safety concern. In one approach, the RNA sequence elements for protein binding specificity in translational control have been defined. High affinity RNAs for RegA RNA-binding proteins were isolated. This work was recently published (2005) in a major peer-reviewed journal. In other projects, we have advanced the characterization of virus-encoded enzymes that synthesize the essential co-factor NAD. Finding these enzymes to be encoded by phage was unique, but they also have properties not observed in other NAD synthesizing enzymes. Because the phages of interest, KVP40 and T4, infect bacteria of food safety concern (Vibrio parahaemolyticus and Escherichia coli, respectively), there is the potential for developing new anti-microbials for these pathogens using the insights derived from the phage genomics and
biochemistry studies. For example, the NAD pathway may well be a good anti-microbial metabolic target for the pathogens as the viral enzymes are used to supplement a limited resource (NAD) in the host pathogen. The purified phage NAD enzymes provide a useful substrate for developing inhibitors against these and the essential pathway of the pathogen. Similarly, the infection specificity of bacteriophages provides an approach for pathogen detection and diagnostics. KVP40 infects V. parahaemolyticus cells that can be found in shellfish and other aquaculture environments. Manipulating the phage as a reporter for the presence of its Vibrio host in foods or aquatic environments is a possible application. For this purpose, we have been constructing KVP40 phage strains with reporter genes that will be expressed and then readily detected if its bacterial host is present. Using T4 and related phage, a similar approach could detect E. coli 0157:H7 strains in meats and other processed foods.
Approaches of directed recombination in the phage are used to construct the diagnostic reporter phages. Overall, progress has been made on the basic genetic, metabolic and macromolecular interactions of viral-host systems, with potential for new applications in treatments and detection strategies for food and animal safety concerns.
Impacts
Understanding the requirements of RNA-protein interactions, basic genetic and metabolic processes in phage-infected and non-infected Vibrio cells can potentially identify new targets for anti-bacterial treatments. Among the unique targets are RNA ligase, used in repair of tRNA, and the enzymes of nicotinamide metabolism, where NAD is an essential coenzyme of numerous cellular and viral reactions. V. parahaemolyticus and V. cholerae are of food safety (shellfish) and global human health concern, respectfully, and are the pathogens of focus. New RNA- and phage-based diagnostics are possible from these basic science research projects, that would address detection procedures for shellfish pathogens (Vibrio) and meat-borne pathogens (E. coli O157:H7) of broad prevelance.
Publications
- Dean, T.R., S.V. Allen and E.S. Miller. 2005. In vitro selection of phage RB69 RegA RNA binding sites yields UAA repeats. Virology 336:26-36.
- Pineda, M., Gregory, B. D., Szczypinski, B., Baxter, K., Hochschild, A., Miller, E. S. & Hinton, D. M. 2004. A family of anti-sigma 70 proteins in T-type phages and bacteria that are similar to AsiA, a transcription inhibitor and co-activator of bacteriophage T4. J. Mol. Biol. 344:1183-1197.
- Boyack, R. and E.S. Miller (mentor). 2005. Directed Gene Replacement Using Lambda Red in Escherichia coli and T4. NCSU undergraduate summer research symposium. Online at http://www.ncsu.edu/ugrs/summer/HHMIabs.htm
|
Progress 10/01/03 to 09/30/04
Outputs
In this project, we have brought together basic and applied research with RNA aptamers, RNA biology and their relevance to phage and bacteria of food safety concern. In one approach, the RNA sequence elements for protein binding specificity in translational control have been defined. High affinity RNAs for RegA RNA-binding proteins were isolated. In other projects, we have determined the complete genome sequence of phage KVP40 that infects important human and shellfish pathogens of the bacterium Vibrio. The genes of KVP40 include many that are of potential biotechnology application and provide a resource for future basic biology investigations. One published study addressed an RNA ligase whose properties are poorly understood yet the enzyme exists in all phylogenetic kingdoms. RNA ligases are important for RNA repair and have uses in molecular biollogy. Another project revealed that KVP40 and related phages encode an anti-sigma protein that inactivates gene
transcription of E. coli, Pseudomonas, Vibrio and other bacteria of human and agricultural concern. The program illustrates the advantages of basic biological research leading to several agricultural and biotechnology relevant applications.
Impacts
Understanding the requirements of RNA interactions, basic genetic and metabolic processes in phage-infected and noninfected Vibrio cells can potentially identify new targets for anti-bacterial treatments. V. parahaemolyticus and V. cholerae are of food safety (shellfish) and global human health concern, respectfully. New RNA-based diagnostics are also likely.
Publications
- Pineda, M., Gregory, B. D., Szczypinski, B., Baxter, K., Hochschild, A., Miller, E. S. & Hinton, D. M. 2004. A family of anti-s70 proteins in T-type phages and bacteria that are similar to AsiA, a transcription inhibitor and co-activator of bacteriophage T4. J. Mol. Biol. 344:1183-1197.
- Yin, S., C.K. Ho, E.S. Miller, and S. Shuman. 2004. Characterization of bacteriophage KVP40 and T4 RNA ligase 2. Virology 319:141-151.
|
Progress 10/01/02 to 09/30/03
Outputs
Vibriophage KVP40 is a Myoviridae phage that infects several Vibrio species, including V. cholerae, V. parahaemolyticus, V. anguillarum, V. natriegens, as well as Photobacterium leiognathi [1]. Coliphage T4 is it's closest, well-characterized relative. The large dsDNA genome has been sequenced and annotated at 244,835 bp [2], revealing that many essential T4-like genes for DNA replication and the virion structure are present, with the encoded proteins conserved at 25 - 55% identity. Only 26% of the KVP40 genes are T4-like, while the majority are of unknown function (ca. 65%) and another group (ca 8%) most closely resembles genes previously identified only in cellular genomes. Based on BLAST analyses, conserved domains, and pathway reconstruction, KVP40 appears to encode a complete pyridine nucleotide salvage pathway. A transport complex for nicotinamide mononucleotide (NMN) is encoded, resembling proteins of enteric bacteria. Nicotinamide in KVP40-infected cells can
be directly converted to NMN by the phage-encoded NadV enzyme that aligns with NadV of Deinococcus and Pasteurella. An NMN adenylyltransferase reaction that yields NAD+ can be catalyzed by either a bifunctional phage NadR protein, or by a second phage protein identified as a two-domain Nudix hydrolase. The KVP40 nudix hydrolase, which we designate as NatV, is related to a similar bifunctional enzyme from a marine Synechocystis that also adenylylates NMN. Turnover of NADH, to either NMN or nicotinamide, could occur via either the NatV Nudix or another enzyme identified as a Sir2/CobB homolog. Many of these enzymes are controlled by allosteric effectors (e.g., NAD+, ATP). An overview of the KVP40 genome will be presented, focusing on the reconstruction of the NAD+ salvage pathway. Preliminary results have advanced the cloning, expression and purification of the NadV enzyme. Scavenging and modulating the intracellular NAD+ pool under a diverse set of host and environmental growth
conditions would be a physiological advantage for the T4-like phage. New targets for anti-Vibrio therapies may also be suggested. 1. Matsuzaki, S., S. Tanaka et al. 1992. Microbiol Immunol. 36:93-97. 2. Miller, E., J. Heidelberg et al., 2003. J. Bacteriol. 185:5220-5233.
Impacts
Understanding the requirements of NAD synthesis in phage-infected and noninfected Vibrio cells can potentially identify new targets for anti-bacterial treatments. V. parahaemolyticus and V. cholerae are of food safety (shellfish) and global human health concern, respectfully.
Publications
- Miller, E.S., E. Kutter, G. Mosig, T. Kunisawa, F. Arisaka and W. Ruger. 2003. The genome of bacteriophage T4. Microbiol. Molec. Biol. Rev. 67:86-156.
- Miller, E.S., J. Heidelberg, J.A. Eisen, W.C. Nelson, A.S. Durkin, A. Ciecko, T.V. Feldblyum, O. White, I.T. Paulsen, W.C. Neirman, J. Lee, B. Szczypinski and C.M. Fraser. 2003. Complete genome sequence of the broad-host-range vibriophage KVP40: comparative genomics of a T4-related bacteriophage. J. Bacteriol. 185:5220-5233.
- Li, Z. and E.S. Miller. 2003. Vibriophage KVP40 encodes a complete pyridine nucleotide (NAD+) salvage cycle. US-Japan cholera and other bacterial enteric infections, 38th joint panel meeting. p. 207.
|
Progress 10/01/01 to 09/30/02
Outputs
RNA-protein interactions directly modulate yields of protein expressed from mRNAs in all cells and are suitable targets for antimicrobial therapuetics, for molecular diagnostics, and provide reagents for commercial enzymes. In this project, the complete 245 kbp genomic sequence of the vibriophage KVP40 (which infects estuary Vibrio bacteria found in shelfish and oysters) was determined and evaluated for RNA-protein studies and applications. RNA binding proteins have been identified and targeted for further analysis. These include two species of RNA ligase and polynucleotide kinase. Complementing these studies, high affinity RNA aptamers are being isolated that can be used to bind and detect viruses and bacteria of food safety concern.
Impacts
Applications arising from bacteriophage genomics and their RNA-binding proteins, along with in vitro selection of high affinity RNA aptamers, should result in diagnostic and antimicrobial reagents for agriculture.
Publications
- Miller, E., J. Heidelberg, J. Eisen, J. Lee, B. Szczypinski, T. Dean and C. Fraser. 2002. Lessons from T4: the 245 kbp Genomic Sequence of Vibriophage KVP40. Abst. Gen. Meeting. Amer. Soc. for Microbiology, p. 300, ASM Press.
|
Progress 10/01/00 to 09/30/01
Outputs
RNA-protein interactions directly modulate yields of protein expressed from mRNAs in all cells. In this project, interactions of translational repressor proteins that control synthesis of DNA metabolism and replication enzymes have been studied at the molecular level. To expand this and related areas, the complete 245 kbp genomic sequence of the vibriophage KVP40 (which infects estuary Vibrio bacteria found in shelfish and oysters) has been determined. Inventions for molecular biology grade enzymes and their applications have been disclosed. Further, RNA binding proteins have been identified and targeted for further analysis. Complementing these studies, high affinity RNA aptamers are being isolated that can be used to bind and detect viruses and bacteria of food safety concern. In other oncgoing projects, bioprocessing of poultry feathers was addressed using proteases of natural bacterial isolates enriched and isolated from low-temperature environments. Three
bacterial isolates identified in the previous year were further characterized with respect to poultry feather degradation at temperatures between 15 - 30C. This is considerably lower than the optimum, 50C temperature previously recorded for feather degrading Bacillus licheniformis strains. The program has identified the psychrotolerant bacteria by chemotaxonomic properties and 16S rRNA sequence analysis. Gene sequences were determined for the principal protease likely to direct keratin degradation. The proteases are related to, but distinct from, enzymes identified in other bacteria. Subsequently, the induction and expression of these genes during growth of the bacteria on feather as sole nutrient source was monitored and quantified by the method of RT-PCR. The enzymes are a class of protease not previously recognized for keratin degradation.
Impacts
Fundamental studies of RNA interactions in gene regulation and for diagnostic development are underway that have applications in food safety and molecular diagnostics. Additional projects have identified and characterized bacillus enzymes for agricultural waste enzymes.
Publications
- Evans, K. L, J. Crowder, and E. S. Miller. 2000. Subtilisins of Bacillus sp. hydrolyze keratin and allow growth on feathers. Can J. Microbiol. 46: 1004-1011.
|
Progress 10/01/99 to 09/30/00
Outputs
The RNA-protein interface, characterized by specific RNA sequences or structures, and specific protein amino acids, is a potential therapeutic or diagnostic target. RNA sequences that bind macromolecules are candidates for target-specific reagents and diagnostics. The investigations in this program addressed 1) basic principles of RNA-protein interactions, and 2) strategies for developing therapeutics, diagnostics or environmental sensors from RNAs and proteins. Bacteriophage RegA proteins have been used with randomized RNAs in successive rounds of selection for increased affinity to these proteins. This in vitro selection method is now yielding high-affinity nucleic acids for environmental detection of viruses present in swine waste components, and to detect bacteria of food safety concern. The laboratory also studies microbial enzymes produced by bacteriophage and bacteria. Newly isolated bacteria and their proteases have been evaluated for feather degradation and
low temperature, feather-degrading enzymes identified. Overall, molecular diversity systems are in use for developing high affinity RNAs and novel microbial products of fundamental and applied interest.
Impacts
RNA molecules can be used as diagnostics to monitor viruses and bacteria present in water samples and foods. Enzymes have applications for converting by-products to usable resources. These projects will benefit agricultural waste management and food safety concerns.
Publications
- Evans, K.L., J. Crowder and E.S. Miller. 2000. Subtilisins of Bacillus spp. hydrolyze keratin and allow growth on feather. Can. J. Microbiol. 46:1004 - 1011.
- Dean, T.R. and E.S. Miller. 2000. In vitro selection of RNA aptamers that bind intact bacteriophage. Phage Biology Meeting, Montreal, Canada. p. 16 (Abstract)
- Evans,K.L., Crowder,J. and Miller,E.S. 2000. Bacillus licheniformis strain ATCC12759 subtilisin precursor (subC) gene, partial CDs, gi 11127683 gb AF205191.1 AF205191[11127683]
- Evans,K.L., Crowder,J. and Miller,E.S. 2000. Bacillus licheniformis strain NCIMB 10689 subtilisin precursor (subC) gene, partial CDs, gi 11127681 gb AF205190.1 AF205190[11127681]
- Evans,K.L., Crowder,J. and Miller,E.S. 2000. Bacillus licheniformis strain NCIMB 6816 subtilisin precursor (subC) gene, partial CDs, gi 11127679 gb AF205189.1 AF205189[11127679]
- Evans,K.L., Crowder,J. and Miller,E.S. 2000. Bacillus licheniformis Carlsberg strain NCIMB 6816 16S ribosomal RNA gene, partial sequence gi 2661091 gb AF035430.1 [2661091]
- Evans,K.L., Crowder,J. and Miller,E.S. 2000. Bacillus licheniformis strain NCIMB 10689 16S small subunit ribosomal RNA gene, partial sequence, gi 6644146 gb AF204165.1 AF204165[6644146]
- Evans,K.L., Crowder,J. and Miller,E.S. 2000. Bacillus licheniformis strain ATCC 12759 16S small subunit ribosomal RNA gene, partial sequence, gi 6644145 gb AF204164.1 AF204164[6644145]
- Evans,K.L., Crowder,J. and Miller,E.S. 2000. Bacillus licheniformis strain ATCC 53757 16S small subunit ribosomal RNA gene, partial sequence, gi 6644144 gb AF204163.1 AF204163[6644144]
- Evans,K.L., Crowder,J. and Miller,E.S. 2000. Bacillus licheniformis North Carolina isolate PWD-1 16S ribosomal RNA gene, partial sequence, gi 2661092 gb AF035431.1 [2661092]
|
Progress 01/01/99 to 12/31/99
Outputs
The RNA-protein interface, characterized by specific RNA sequences or structures, and specific protein amino acids, is a potential therapeutic or diagnostic target. RNA sequences that bind macromolecules are candidates for target-specific reagents and diagnotics. The investigations in this laboratory addressed 1) basic principles of RNA-protein interactions in translational repression by a viral protein, and 2) strategies for developing therapeutics, diagnostics or environmental sensors for RNAs. The bacteriophage RB69 RegA protein has been configured to have six histidine residues at the carboxyl terminus for chelation to immobilized nickel. Randomized RNAs were subjected to successive rounds of selection for increased affinity to immobilized phage RB69 RegA protein. This system has been used to select high-affinity RNA ligands that bind the His-tagged protein. Extrapolation of this in vitro selection method is now yielding high-affinity nucleic acids for
environmental detection of viruses present in swine waste components. Diagnostic RNA aptamers for bacteria are also being developed. The laboratory also studies microbial proteases that hydrolyze poultry feathers. Protease-encoding genes have been expressed in protease-deficient strains of Bacillus subtilis. This expression system is in use for mutagenesis and directed evolution of the protease for enhancing feather degradation under broader conditions of pH and temperature. Naturally occurring proteases have also been evaluated for feather degradation and new ones have been identified. Overall, molecular diversity systems are in use for developing high-affinity RNAs and novel proteases of fundamental and applied interest.
Impacts
(N/A)
Publications
- Allen, S.V. and Miller, E.S. 1999. RNA binding properties of in vitro expressed histidine-tagged RB69 RegA translational repressor protein. Analytical Biochemistry 26:32-37.
- Allen S.V. 1999. Analysis of RNA binding by the RB69 RegA translational repressor protein using affinity chromatography and in vitro selection. Ph.D. Dissertation, North Carolina State University.
|
Progress 01/01/98 to 12/31/98
Outputs
The RNA-protein interface, characterized by specific RNA sequences or structures, and specific protein amino acids, is a potential therapeutic or diagnostic target. RNA sequences that bind macromolecules are candidates for target-specific reagents and diagnostics. The investigations in this laboratory addressed 1) basic principles of RNA-protein interactions in translational repression by a viral protein, and 2) strategies for developing therapeutics, diagnostics or environmental sensors from RNAs and proteins. The bacteriophage RB69 RegA protein has been configured to have six histidine residues at the carboxyl terminus for chelation to immobilized nickel. Randomized RNAs are then subjected to successive rounds of selection for increased affinity to the immobilized RegA protein. This system has been used to select high-affinity RNA ligands that bind the His-tagged protein. Extrapolation of this in vitro selection method is now yielding high-affinity nucleic acids for
environmental detection of viruses present in swine waste components. The laboratory also studies microbial proteases that hydrolyze poultry feathers. Protease-encoding genes have been expressed in protease-deficient strains of Bacillus subtilis. This expression system is in use for mutagenesis and directed evolution of the protease for enhancing feather degradation under broader conditions of pH and temperature. Naturally occurring proteases have also been evaluated for feather degradation and new ones have been identified. Overall, molecular diversity systems are in use for developing high-affinity RNAs and novel proteases of fundamental and applied interest.
Impacts
(N/A)
Publications
- Allen, S.V. and Miller, E.S. 1998. RNA-binding properties of in vitro his-tagged RB69 RegA translational repressor protein. Proc. Evergreen Intl. Phage Biology Meeting. p. 39. (Abstract)
|
Progress 01/01/97 to 12/31/97
Outputs
The RNA-protein interface, characterized by specific RNA sequences or structures, and specific protein amino acids, is a potential therapeutic or diagnostic target. RNA sequences that bind macromolecules are candidates for target-specific reagents and diagnostics. The investigations in this laboratory addressed 1)basic principles of RNA- protein interactions in translational repression by a viral protein, and 2)strategies for developing therapeutics, diagnostics or environmental sensors from RNAs and proteins. The bacteriophage RB69 RegA protein has been configured to have six histidine residues at the carboxyl terminus for chelation to immobilized nickel. Randomized RNAs are then subjected to successive rounds of selection for increased affinity to the immobilized RegA protein. This system has been optimized and is in progress. Extrapolation of the high-affinity RNAs for environmental detection of swine waste component is in progress. The laboratory also studies
microbial proteases that hydrolyze poultry feathers. Protease- encoding genes have been expressed in protease-deficient strains of Bacillus subtilis. This expression system is in use for mutagenesis and directed evolution of the protease for enhancing feather degradation under broader conditions of pH and temperature. Naturally occurring proteases have also been evaluated for feather degradation.
Impacts
(N/A)
Publications
- LIN, X., WONG, S.-L., MILLER, E.S. and SHIH, J.C.H. 1997. Expression of the Bacillus licheniformis PWD-1 keratinase gene in B. subtilis. J. Indust. Microbiol. 19:134-138.
- MILLER, E.S., SHIH, G.C., CHUNG, S.K. and BALLARD, D.N. 1997. An E. coli B mutation, rpoB5081, that prevents growth of phage T4 strains defective in DNA degradation. FEMS Microbiol. Let. 157:109-116.---
- ALLEN, S.V. and MILLER, E.S. 1997. His-tagged phage RegA translational repressor proteins. 97th Gen. Mtg. Amer. Soc. Microbiol. May 4-8, 1997, M12, p. 381 (Abstract).---
- MILLER, E., FALK, K., SHIH, J. and LIN, X. 1997. Hydrolysis of feather keratin by bacterial serine proteases. NCSU Animal and Poultry Waste Management Center Inauguration Program, pg. 5
|
Progress 01/01/96 to 12/30/96
Outputs
The RNA-protein interface, characterized by specific RNA sequences or structuresand specific protein amino acids, is a potential therapeutic or diagnostic target. Derivatives of RNA-binding proteins, or RNA sequences that bind macromolecules, are candidates for target-specific reagents and diagnostics. Investigations addressed 1)basic principles of RNA-protein interactions in translational repression by a viral protein, 2)strategies for developing therapeutics, diagnostics or environmental sensors from RNAs and proteins. A system was implemented to randomize amino acids in the RNA-binding protein RegA and to select from the randomized pool those proteins that bind RNA with higher affinity. The RB69 phage RegA protein has been configured to have 6 histidine residues at the carboxyl terminus for chelation to immobilized nickel. Randomized RNAs are subjected to successive rounds of selection for increased affinity to the immobilized RegA protein. Use of the high affinity
RNAs to therapeutics and environmental diagnostics is possible. The lab also studies a microbial protease that hydrolyses poultry feathers. The protease gene has been expressed in a protease-deficient strain of Bacillus subtilis. Studies on the substrate specificity of feather-degrading enzymes show two related enzymes have similar catalytic properties. A system has been constructed for directed evolution of the protease for enhancing feather degradation under broader conditions of pH and temperature.
Impacts
(N/A)
Publications
- LIN, X., WONG, S.L., MILLER, E. S., and SHIH, J.C.H. 1996. Cloning and expressionof Bacillus licheniformis PWD 1 kerA in Bacillus subtilis. Ann.Mtg. Amer. Soc. Microbiol., 115:079, p. 368. (Abstract).
- FALK, K. L., SHIH, J.C.H. and MILLER, E.
|
Progress 01/01/95 to 12/30/95
Outputs
RegA proteins of bacterial viruses are translational repressors that selectivelybind mRNAs at sequences that are coincident with the translation start site and inhibit ribosome binding; protein synthesis is abruptly stopped and the mRNAs rapidly decay. During the current period, the macromolecular interactions of phage RegA proteins with biologically relevant mRNAs was quantified by fluorescence quenching methods. Binding constants and cooperative protein-protein interactions were characterized. In addition, a system has been developed to randomize amino acids of the RNA-binding domains and to select from the randomized pool those proteins that bind to RNA with higher affinity. The first objective of these studies is to identify amino acids of RegA that confer translational repression activity to the protein, and thus define the amino acids needed to bind mRNA. A phage has been configured by molecular cloning that places RegA, as part of a RegA-gpVIII hybrid capsid
protein, on the surface of the filamentous phage fd. By the surface display of RegA, these phage now bind to RNA. Current efforts focus on randomizing the RegA RNA-binding domains to select for alternative and high- affinity RNA-binding proteins. Future objectives include the isolation of display phages that bind to ligands for diagnostic or therapeutic applications, particularly applications in environmental sensoring.
Impacts
(N/A)
Publications
- JOZWIK, C.E. and MILLER, E.S. 1995. RNA-protein interactions of the bacteriophage RB69 RegA translational repressor protein. Nucleic Acids Symp. Series 33:256 - 257.
- LIN, X., KELEMEN, D.W., MILLER, E.S., and SHIH, J.C. 1995. Sequence and expression of kerA, the gene encoding a keratinolytic protease of Bacillus licheniformis PWD-1. Appl. Environ. Microbiol. 61:1469 - 1474.
- MILLER, E.S., SHIH, J.C., and CHANG, S.K. 1995. An amino acid substitution in the Beta subunit of RNA polymerase affects growth of T4 strains defective in DNA and mRNA turnover. Gen. Mtg. Amer. Soc. Microbiol., p. 573 (Abstract).
- JOZWIK, C.E. and MILLER, E.S. 1995. Macromolecular interactions of the RegA translational repressor proteins from T-even and RB bacteriophages. Gen. Mtg. Amer. Soc. Microbiol., p. 573 (Abstract).
- KELEMEN, D.W., LIN, X., MILLER, E.S., and SHIH, J. 1995. Cloning and expression of the keratinase gene isolated from B. licheniformis PWD-1. Gen. Mtg. Amer. Soc. Microbiol., p. 528 (Abstract).
|
Progress 01/01/94 to 12/30/94
Outputs
RegA proteins of bacterial viruses are translational repressors of mRNAs that encode enzymes for viral DNA replication. RegA selectively binds these mRNAs at a sequence that is coincident with the translation start site and inhibits ribosome binding; protein synthesis is thereby inhibited and the mRNAs rapidly decay. T4 RegA is the best characterized of this class of protein, and more recently, this laboratory has cloned, sequenced and characterized a similar protein from the enteric bacteriophage designated RB69. During the current period, purified T4 and RB69 RegA proteins have been used in quantitative fluorescence quenching analyses to determine the site size (6 - 9 nucleotides) of RegA when the protein binds nucleic acids. The fluorescence data are also used to determine the equilibrium binding parameter, Kd, and the apparent cooperativity constant. In the course of these studies, RegA was shown to behave as a probable dimeric protein, rather than as a monomer as
previously suggested. Studies of virus-host interactions have shown that a mutation (P806S) in the Beta subunit of host RNA polymerase, causing increased transcription termination, prevents phage development when phage regA or denA (Endonuclease II) genes are defective. Mechanisms of aborting viral infection can be derived from these studies. Additional studies have addressed proteolytic degradation of poultry waste products using a secreted bacterial enzyme, keratinase.
Impacts
(N/A)
Publications
- JOZWIK, C.E. and MILLER, E.S. 1994. RNA-protein interactions of the RegA translational repressor protein from the T4 and RB69 bacteriophage. Reg. Mtg. Amer. Soc. Microbiol., Charlotte, NC., p. 469 (abstract).
- LIN, X., KELEMEN, D.W., MILLER, E.S., and SHIH, J. 1994. Isolation and sequencing of a keratinase gene by PCR walking. Ann. Mtg. Amer. Soc. Microbiol., p. 5 (abstract).
- MILLER, E.S., LIN, X., and SHIH, J.C.H. 1994. Characterization of a keratinolytic protease from a strain of Bacillus etc. J. Cell. Biochem. supp. 18D, p.170. (abstract).
- MILLER, E.S. et al. 1994. Control of translation initiation: mRNA structures and translational repressors. In: Molecular Biology of Bacteriophage T4, Karam et al. Ed, ASM Press, Washington, D.C., p. 193-205.
- CARLSON, K. and MILLER, E.S. 1994. Experiments in T4 genetics. In: Molecular Biology of Bacteriophage T4, Karam et al. Ed, ASM Press, Washington, D.C., p. 421-483.
|
Progress 01/01/93 to 12/30/93
Outputs
RegA proteins of bacterial viruses are translational repressors of mRNAs that encode enzymes for viral DNA replication. RegA selectively binds these mRNAs at a sequence that is coincident with the translation start site and inhibits ribosome binding; protein synthesis is thereby inhibited and the mRNAs rapidly decay. T4 RegA is the best characterized of this class of protein, and more recently, this laboratory has cloned, sequenced and characterized a similar protein from the enteric bacteriophage designated RB69. During the current period, RB69 RegA protein has been purified and its RNA-binding activities examined. When purified RB69 RegA is added to in vitro translation systems, translation of specific target mRNAs is repressed. RNAse protection experiments with purified protein and RNA demonstrate binding of RB69 RegA to sites similar, but not identical, to those recognized by bacteriophage T4 RegA protein. In addition, properties of mutant RB69 RegA proteins and
the RNase protection data suggest that RB69 RegA binds mRNAs with an apparent cooperativity parameter. Related studies in the laboratory have resulted in the identification and sequence determination of mutations in the catalytic Beta subunit of the bacterial host RNA polymerase that affect phage development when phage regA or denA (Endonuclease II) genes are mutationally altered. Therefore, physiological relations between mRNA (RegA), DNA (DenA) turnover, and RNA polymerase affect viral growth.
Impacts
(N/A)
Publications
- JOZWIK, C.E. and MILLER, E.S. 1993. RNA-protein interactions of RegA translational repressor proteins from bacteriophage T4 and RB69. Gordon Research Conference on Nucleic Acids. p. 8. (abstract)
- JOSWIK, C.E. and MILLER, E.S. 1993. RNA-protein interactions of RegA translational repressor proteins from bacteriophage T4 and RB69. Ann. Meeting Amer. Soc. Biochem. Molec. Biol. p. A1217. (abstract).
|
Progress 01/01/92 to 12/30/92
Outputs
RegA proteins of bacterial viruses are translational repressors of several messenger RNAs required for viral DNA replication. RegA selectively binds these mRNAs, at a sequence that is coincident with the ribosome binding site, using nucleotide-amino acid interactions that are not yet understood. Because translational regulation and RNA recognition are essential to gene regulation in all cell types, understanding this process will provide new insights to cellular metabolism, development and pathogenesis. We have cloned and sequenced the regA gene from bacteriophage RB69 and compared it to the better characterized phage T4 RegA. While numerous amino acid differences occur in the carboxy-terminal half of the two proteins, the amino-terminal portions are highly conserved. The RB69 RegA protein was demonstrated to function essentially like the T4 protein since expression of the cloned gene in T4-infected Escherichia coli cells resulted in translational repression of the
RegA-sensitive T4 MRNAS. Several mutations were isolated in T4 and RB69 regA and their nucleotide changes were determined. Focusing on the missense alleles, single amino acid substitutions that affect RegA function cluster at common, conserved residues in both viral proteins. These residues, particularly Val-15 to Ala-25, are inferred to determine RNA-recognition specificity for RegA. The residues between Val-15 and Ala-25 are predicted to be in an helical conformation; protein database searches reveal etc.
Impacts
(N/A)
Publications
- JOZWIK, C.E. and MILLER, E.S. 1992. Regions of bacteriophage T4 and RB69 RegA translational repressor proteins that determine RNA-binding specificity. Proc. Natl. Acad. Sci. USA 89:5053-5057.
- SHIH, G. and MILLER, E.S. 1992. Characterization of an E. coli strain (TabRegA) that restricts growth of bacteriophage T4 regA mutants. Abst. Amer. Soc. Microbiol. p. 286. (abstract).
|
Progress 01/01/91 to 12/30/91
Outputs
The level of cellular proteins is highly regulated, usually by transcriptional or translational control mechanisms. Translational control in bacteria is mediated by RNA secondary structures or by RNA-binding proteins. Our laboratory investigates the molecular genetics and potential translational control mechanisms operative in an erythromycin-producing soil bacterium. Formerly designated Arthrobacter sp. NRRL B-3381, our taxonomic analysis of fatty acid content, 16S rRNA sequence and other phenotypic traits, resulted in reclassification to a new genus and species which we designated as Aeromicrobium erythreum. The bacterium is most closely associated with the Nocardioides coryneform bacteria. Genetic systems for study of A. erythreum were developed. These include mutagenesis procedures, plasmid cloning vectors and methods for directed gene disruption. ermR, the erythromycin-resistance gene, was disrupted and shown to be the sole determinant of macrolide antibiotic
resistance. The rRNA methylase appears to regulate its own translation in other bacteria; the ermR methylase deletions we have constructed may be useful for translational control studies in A. erythreum. Progress has also been made in our studies of translational regulation of DNA replication by bacteriophage T4.
Impacts
(N/A)
Publications
- MILLER, E.S. 1991. Cloning vectors, mutagenesis, and gene disruption (ermR) for the erythromycin-producing bacterium Aeromicrobium erythreum. Applied and Environmental Microbiology 57:2758-2761.
- MILLER, E.S., WOESE, C.R., and BRENNER, S. 1991. Description of the erythromycin-producing bacterium Arthrobacter sp. strain NRRL B-3381 as Aeromicrobium erythreum gen. nov., sp. nov. International Journal of.
- KREUZER, H.E., and MILLER, E.S. 1991. Development of a tandem mass spectrometric method for analysis of RNA-protein cross links. Intl. Bacteriophage T4 Meeting, p. 60.
- JOZWIK, C.E., and MILLER, E.S. 1991. Identification of conserved and mutationally sensitive amino acids in the bacteriophage T4 and RB69 RegA translational repressor proteins. Intl. Bacteriophage T4 Meeting, p. 59.
- MILLER, E.S. and JOZWIK, C.E. 1991. Identification of conserved and mutationally sensitive amino acids in RegA translational repressor proteins. Keystone Symposium on Translational Control, p. 220.
|
|