Professor and Chair
DNA-Protein Interactions; DNA Structure, Transcriptional Regulation
of Laboratory for Molecular Visualization and Assessment
1984 University at Buffalo
Postdoctoral work 1984-88 Harvard University
Department of Biological Sciences
Phone: (716) 645-4940 (Research Office) or 645-4904 (Chair’s Office)
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Find information on the Laboratory for Molecular Visualization and Analysis here
The research in the Koudelka lab is focused around two central themes:
Mechanisms of Indirect Readout:
The mechanisms whereby regulatory proteins recognize specific DNA sequences remains one of the most important areas of study in biology. This process requires that the protein be able to seek out and recognize its particular binding sequence, in the presence of an overwhelming number of potential non-specific binding sites. In our studies of direct readout of DNA sequence, we have uncovered the intimate details of how amino acids and base pairs can interact, and how these interactions can be regulated by both protein and DNA structure. In indirect readout, sequence-dependent differences in the structure and flexibility of noncontacted bases in a DNA binding site regulate the stability and sequence-specificity of a protein-DNA complex. Despite the high prevalence and functional importance of indirect readout it is unclear how DNA sequence differences lead to changes in DNA structure and flexibility. We are determining the structural basis for, and functional implications of the indirect readout mechanism used by DNA binding proteins.
Evolution of Bacteriophage-encoded Exotoxins
Bacterially-derived exotoxins are among the most deadly substances known. The genes that encode these exotoxins are usually carried by bacterial viruses (bacteriophages) integrated into the bacterial host chromosome. It is generally assumed that the targets of these toxins are mammals. However, these phage-encoded exotoxin genes are widespread in the environment and are found with unexpectedly high frequency in regions that lack the presumed mammalian targets. These observations suggest that humans and other susceptible mammals are not the primary “targets” of these toxins. We are exploring the hypothesis that exotoxins are part of an antipredator defense mechanism.
Readout: DNA Structure Effects on Protein-DNA Interactions
The binding of proteins to specific DNA sequences plays a central role in the regulation of gene expression in all organisms. These proteins regulate gene expression by binding DNA at specific sites and activating or repressing transcription. Structural and biochemical studies have provided a detailed insight into how the intimate contacts between proteins and DNA enable proteins to bind specifically and with high affinity only to their cognate DNA binding sites. One conclusion of these studies is that sequence specific DNA recognition involves both direct and indirect readout of the binding site sequence.
In indirect readout, the stability and specificity of a protein-DNA complex is regulated by the sequence of bases not in contact with the protein. These noncontacted bases can inhibit or prevent the contacted DNA from being properly juxtaposed with protein groups. DNA sequence-dependent differences in the structure and flexibility of noncontacted bases lead to alterations in the strength and/or ease of forming protein-DNA contacts. The DNA sequence-limited geometry changes thereby indirectly alter the affinity and/or specificity of a protein for its cognate binding site. Hence, indirect effects of DNA sequence on protein-DNA complex formation occur by a modulation of the structural complementarity between the interacting molecules.
While it is clear that indirect readout of DNA sequence is an important component of the DNA sequence recognition mechanisms of many proteins, until recently, it was unclear how DNA sequence directs changes in DNA structure and/or flexibility. Our current studies indicate that indirect readout by DNA binding proteins repressor is influenced by sequence-dependent interactions between the solvent and the unbound and/or protein-bound DNA. These data also show that manipulating the solvent environment inside cells leads to specific effects on gene regulation.
Hence, our ongoing studies are aimed at 1) providing a thermodynamic and structural framework to explain the mechanisms of solvent-dependent indirect readout of DNA sequence, and 2) understanding how these sequence- and solvent-dependent differences in DNA structure influence the stability and function of protein-DNA complexes.
of Bacteriophage-encoded Exotoxins
Phages encoding exotoxin genes are found ubiquitously as lysogens in environmental bacteria, but it is unclear what advantage there is to the bacteria to harbor phage that encode such toxic compounds. In the context of humans, these exotoxins cause diseases ranging from cholera to diphtheria to enterohemorrhagic diarrhea. However, the frequency of occurrence of the genes encoding any particular exotoxin gene in bacteriophage and/or lysogens far exceeds the number of potential animal hosts. Moreover, these phage-encoded exotoxin genes are found at high frequency in free phages and lysogenic bacteria isolated from environments where the corresponding human diseases are not prevalent. These observations suggest that mammals are neither the original nor primary “targets” of these toxins. The phage-encoded exotoxins like the well-studied Shiga toxin (Stx), kill eukaryotic cells by attacking features and pathways that are common to all eukaryotes both, uni- and multi-cellular. Thus the evolution of these toxins may have occurred before the appearance of multicellular organisms. Since predation by eukaryotic predators (e.g., ciliates and other protozoa), is a major source of bacterial mortality, these observations suggest that exotoxins may have arisen as part of an antipredator, (antiprotozoan) defense strategy. Hence humans may be innocent bystanders in the evolutionary battle between protozoans and their bacterial prey.
Our observations show that when co-cultured with the ciliate predator, Tetrahymena thermophila, bacteria bearing a bacteriophage that codes for Stx kill this predator. We also showed that bacterial strains carrying Stx-encoding phage are more resistant to predation than strains that do not. Our data also indicate T. thermophila releases a factor that signals the bacteria to the presence of a predator and stimulates them to produce Stx. T. thermophila eat bacteria by capturing them with their oral apparatus. We showed that Stx can enter T. thermophila via this novel route. We also found that purified Stx can effectively kill T. thermophila after apparently entering the cell via a plasma membrane-mediated endocytotic process. These findings provide the possibility that Stx intoxicates T. thermophila through both these novel routes: a non-receptor mediated pathway through the oral apparatus and/or endocytosis through the plasma, a process that may or may not be receptor-mediated. Our central hypothesis is that exotoxins, such as Stx, may have evolved as part of an antipredator strategy to protect bacteria from protozoan predators..
Arnold, J.W, Koudelka, G.B. (2013)
The Trojan horse of the microbiological arms race: Phage encoded bacterial toxins as a weapon against eukaryotic predators,
Env. Micro. in press.
Mauro, S.A., Opalka, H., Lindsay, K., Colon, M.P., Koudelka, G.B. (2013)
The microcosm mediates the persistence of Shiga toxin producing E. coli (STEC) in freshwater ecosystems.
Applied and Env. Microbiology, 79, 4821-4842 (Full text).
Shkilnyj, P., Colon, M.P., and Koudelka, G.B., (2013)
Bacteriophage 434 Hex protein prevents RecA-mediated repressor autocleavage
Viruses 5, 111-126 (Full text)
Stolfa, G., and Koudelka, G.B., (2013)
Entry and Killing of Tetrahymena by Bacterially Produced Shiga toxin,
mBio, 4 e00416-12; doi:10.1128/mBio.00416-12. (Full text)
Harris, L.A., Watkins, D., Williams, L.D., and Koudelka G. B. (2013)
Indirect Readout of DNA Sequence by P22 Repressor: Roles of DNA and Protein Functional Groups in modulating DNA Conformation,
J. Mol. Biol 425, 133-143., http://dx.doi.org/10.1016/j.jmb.2012.10.008.
Bullwinkle, T.J., Samorodnitsky, D., Rosati, R.C. and Koudelka, G.B. (2012)
DNA binding specificity determinants of 933W repressor,
PLOS One, 7: e34563. doi:10.1371/journal.pone.0034563
Pawlowski , D.R., Raslawsky, A., Siebert, G., Metzger, D.J., Koudelka, G.B., Karalus, R.J. (2011)
Identification of Hylemonella gracilis as an antagonist of Yersinia pestis persistence.
Journal of Bioterrorism & Biodefense, S3:004. doi:10.4172/2157-2526.S3-004.
Mauro S.A. and Koudelka, G.B (2011)
Shiga Toxin: Expression, Distribution, and Its Role in the Environment
Toxins 3, 608-625; doi:10.3390/toxins3060608
Bullwinkle, T.J., and Koudelka (2011)
The Lysis-Lysogeny Decision of Bacteriophage 933W: A 933W Repressor-Mediated Long Distance Loop Has No Role in Regulating 933W PRM Activity,
J. Bacteriol, 193, 3313-3323 (Full text)
Watkins, D., Mohan, S., Koudelka, G.B., Williams, L.D., (2010)
Sequence Recognition of DNA by Protein-Induced Conformational Transitions.
J. Mol. Biol. 396, 1145-64. (Full text)
Lainhart, W, Stolfa, G. and Koudelka, G.B. (2009)
Shiga Toxin as a Bacterial Defense against a Eukaryotic Predator, Tetrahymena thermophila,
J. Bacteriol. 191 5116-5122 (Full text)
Watkins, D., Hsiao, C., Woods, K.K., Koudelka, G.B., Williams, L.D., (2008)
P22 c2 Repressor-Operator Complex: Mechanisms of Direct and Indirect Readout.
Biochemistry 47, 2325-2338 (Full text).
Shkilnyj, P. and Koudelka, G.B. (2007)
Effect of Salt Shock on the Stability of λimm434 Lysogens,
J. Bacteriol., 189:3115-3123 Full text.
McCabe, B.C., Pawlowski, D.R., Koudelka, G.B., (2005)
The bacteriophage 434 repressor dimer preferentially undergoes autoproteolysis by intramolecular mechanism.
J. Bacteriol., 187 5624-30. Full text
Koudelka, G.B., Hufnagel, LA, Koudelka, A.P. (2004)
Isolation, Purification and Characterization of a Repressor from the Toxin-encoding Bacteriophage 933W
J. Bacteriol., 186 7659-7669. Full text
Mauro, S.A., Koudelka, G.B. (2004)
Monovalent Cations Direct Sequence Recognition by 434 Repressor,
J. Mol. Biol 340 445-457. Abstract
Pawlowski, D.R., Koudelka, G.B. (2004)
The Preferred Substrate for RecA-Mediated Cleavage of Bacteriophage 434 Repressor is the DNA-Bound Dimer
J. Bacteriol., 185 1-6. Full text
Ciubotaru, M., Koudelka, G.B. (2003)
DNA Allosterically Modulates the Cooperative DNA Binding Interactions of 434 Bacteriophage Repressor.
Biochemistry 42,4253-4264. Abstract
Mauro, S.A., Koudelka, G.B. (2003)
The Role of the Minor Groove Substituents in Indirect Readout of DNA Sequence by 434 Repressor
J. Biol. Chem, 278, 12955-12960. Abstract
Xu, J., Koudelka, G.B. (2001)
Repression of Transcription Initiation at 434 PR by 434 Repressor: Effects on Transition of a Closed to an Open Promoter Complex.
J. Mol. Biol . 309, 583-597. Abstract
Xu, J., McCabe, B.C., Koudelka, G.B. (2001)
Function-Based Selection and Characterization of Base pair Polymorphisms in a Promoter of E. coli RNA polymerase-s 70,
J. Bacteriol., 183, 2866-2873. Abstract
Xu, J. Koudelka, G.B. (2000)
DNA Sequence Requirements for the Activation of 434 PRM Transcription by 434 Repressor.
DNA and Cell Biol. 19, 621-630. Abstract
Xu, J., Koudelka, G.B. (2000)
Mutually Exclusive Utilization of PR and PRM Promoters in Bacteriophage 434 OR.
J. Bacteriol. 182, 3165-3174 Abstract
Ciubotaru, M., Bright, F.V., Ingersoll, C.M., Koudelka, G.B. (1999)
DNA-Induced Conformational Changes in Bacteriophage 434 Repressor.
J. Mol. Biol. 294, 859-873 Abstract .
Donner, A.L., Paa, K. & Koudelka, G.B. (1998)
Carboxyl-Terminal Domain Dimer Interface Mutant 434 Repressors Have Altered Dimerization and DNA Binding Specificities.
J. Mol. Biol. 283, 931-946. Abstract
Xu, J., Koudelka, G.B. (1998)
DNA-based Positive Control Mutants in the Binding Site of 434 Repressor.
J. Biol. Chem. 273, 24165-24172. Abstract
Koudelka, G.B. (1998)
Recognition of DNA Structure by 434 Repressor
Nucleic Acids Res. 26, 669-675. Abstract
Hilchey, S.P., Wu, L. & Koudelka, G.B. (1997)
of Nonconserved Bases in the P22 Operator by P22 Repressor Requires Specific
Interactions between Repressor and Conserved Bases.
J. Biol. Chem. 272, 19898-19906. Abstract
Donner, A.L., Carlson, P.A.
& Koudelka, G.B. (1997)
Dimerization Specificity of P22 and 434 Repressors Is Determined by Multiple Polypeptide Segments.
J. Bacteriol. 179, 1253-1261 Abstract
Hilchey, S.P., Koudelka G.B.
DNA-Based Loss of Specificity Mutations: Effects of DNA Sequence on the Contacted and Noncontacted Base Preferences of Bacteriophage P22 Repressor.
J. Biol. Chem. 272, 1646-1653. Abstract
How 434 repressor discriminates between OR1 and OR3: the influence of contacted and non-contacted base pairs
J. Biol. Chem. 270:1205-1212 Abstract
Carlson, P.A., and Koudelka,
Expression, purification, and functional characterization of the carboxyl terminal domain fragment of 434 repressor
J. Bacteriol. 176:6907-6914 Abstract
Koudelka, G.B., and Lam,
Differential recognition of OR1 and OR3 by bacteriophage 434 repressor and Cro
J. Biol. Chem. 268:23812-23817 Abstract
Bell, A.C., and Koudelka,
Operator sequence context influences amino acid-base pair interactions in 434 repressor-operator complexes
J. Mol. Biol. 234:542-553 Abstract
Wu, L., and Koudelka, G.B.
Sequence-dependent differences in DNA structure affect the affinity of P22 operators for P22 repressor
J. Biol. Chem. 268:18975-18981 Abstract
Wu, L., Vertino, A., and
Koudelka, G.B. (1992)
Non-contacted bases in the P22 operator affect its affinity for P22 repressor
J. Biol. Chem. 267:9134-9139 Abstract
Koudelka, G.B., and Carlson,
DNA twisting and the effects of non-contacted bases on the affinity of 434 operator for 434 repressor
Nature 355:89-91 Abstract
Page last modified: 12/06/2012 by G. Koudelka