Eric Miller

4510A Thomas Hall

Campus Box 7615
North Carolina State University
Raleigh, NC 27695
919.515.2391 (main office)
919.515.7230 (lab)
919.515.7867 (fax)
eric_miller@ncsu.edu


Courses

MB 758
Prokaryotic Molecular Genetics

MB 680/880
Microbiology Research Presentations


Publications
Aeromicrobium paper


Phage Page
Biography

 

Eric S. Miller
Professor

Bacteriophage Biology: Pyridine scavenging, Translational control Bioinformatics


Research Brief

Bacteriophage Biology: T4 and Vibrio Phage KVP40
The Myoviridae (Myophage) are dsDNA phages with contractile tails. T4 is the type phage of its myophage subgroup and is one of the better studied genetic and biochemical systems, having provided numerous advances in DNA replication, viral morphogenesis, gene expression and almost every process in molecular biology. Until recently, the 170 kbp genome of T4 was the only genome sequence available for this group of widely distributed phages. We helped to ameliorate this problem by sequencing the 245 kbp genome of phage KVP40, a broad host range phage that infects Vibrio parahaemolyticus, V. cholerae and other species of Vibrio. Among the 386 protein coding genes of KVP40, only 99 (25%) are also found in T4, primarily representing DNA replication and metabolism enzymes, and phage capsid, tail and other morphogenic proteins. Fully 65% of the KVP40 proteins have no homologs in the database, and the remainder are found in other genomes but not previously in viral genomes. KVP40 encodes 25 tRNAs and 5 pseudo-tRNAs in one gene cluster. The dirth of T4-type phage genome sequences in GenBank is being rectified to some extent by the sequencing of other related phage genomes (see: phage.bioc.tulane.edu), but this is still only a small sampling of the sequence diversity presented by these interesting phages. T4 genetic systems faciliate analysis of novel and extraordinary biochemical processes presented by the KVP40 genome and the other T4-type genomes.

Pyridine Nucleotide Metabolism. KVP40 has five genes that encode proteins that are involved in pyridine nucleotide (NAD+) metabolism. An important enzyme for scavenging nicotinamide and incorporating it into the pyridine nucleotide pool is nicotinamide phosphoribosyltransferase that KVP40 nadV gene encodes. To study this novel phage-directed salvage pathway, we have cloned, expressed, purified and characterized some of the genes and enzymes. It will be interesting to determine the importance of NAD synthesis by phages, which can lead to new insights into phage-host interactions ("metabolomics") and potentially to new anti-viral, anti-microbial compounds.

T4 Reg ATranslational Control and RNA repair. An interest of our laboratory is understanding how proteins recognize and bind messenger RNA to control the translational yield of protein. Recognition of RNA by protein is essential for cellular processes including gene regulation, macromolecular assembly, nucleic acid replication, viral infection, and catalysis. We study the molecular biology of RNA recognition by RegA translational repressor proteins from T4-type bacteriophages. RegA binds RNA near the initiator AUG but, unlike many other RNA-binding proteins, it does not appear to use RNA structure to recognize the binding site. RegA proteins have conserved sequence, and apparent structure, in the T4-type phages. We immobilized a histidine-tagged phage RB69 RegA protein and used in vitro selection methods to isolate a high-affinity RNA ligand. Interpretation of the selected sequences suggest that RegA proteins are not simple translational repressors, but perform other functions through their RNA binding function.
T4-type phages have an interesting set of tRNA repair enzymes that "put back together" a cleaved tRNA-Lys that cells (and/or phage) use to prevent phage infection. During studies of tRNA repair, a second RNA ligase (Rnl2) was discovered in T4, then in KVP40; these enzymes are phylogenetically related to RNA editing enzymes found in all domains of life. We have collaborated with Dr. Stewart Shuman (www.mskcc.org/mskcc/html/10421.cfm) to study the KVP40 Rnl2 and are conducting both genetic and biochemical experiments.

Bioinformatics and T4-Type Phage Gene Expression Signals. Using sequence analysis programs based on Information Theory (see: www.lecb.ncifcrf.gov/~toms/index.html), we have aligned the T4 early, middle and late promoters, and all of the ribosome binding sites, to obtain sequence logos and the information content of the respective transcription and translaiton initiation signals. A sequence logo of T4 late promoters recognized by the phage sigma 55 protein is shown below. We are analyzing other T4-type phage genomes using similar methods and have made interesting observations that suggest experimental strategies to test our hypotheses. Working with Dr. Debbie Hinton at NIH (www.niddk.nih.gov/intram/faculty.asp?People_ID=1619), one approach is examining the properties of the KVP40 AsiA protein, an anti-sigma protein encoded by several of the T4-type phages.