Gerald B. Koudelka
Professor and Chair
DNA-Protein Interactions; DNA
Structure, Transcriptional Regulation
Bacterial Pathogenesis
co-Director
of Laboratory for Molecular Visualization and Assessment
Ph.D.
1984 University at Buffalo
Postdoctoral work 1984-88 Harvard University
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Gerald B.
Koudelka
Department of Biological Sciences
607
Phone: (716) 645-2363 Ext.
158 (C607) or Ext 101 (C109)
To send e-mail: koudelka@buffalo.edu
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RESEARCH SUMMARY
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.
SELECTED PROJECTS
Indirect
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.
Evolution
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 preliminary 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..
PUBLICATIONS
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 (link to 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 (link to 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
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
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)
Recognition 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.
(1997)
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,
G.B. (1994)
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,
C.-Y. (1993)
Differential recognition of OR1 and OR3 by bacteriophage 434 repressor and
Cro
J. Biol. Chem. 268:23812-23817 Abstract
Bell, A.C., and Koudelka,
G.B. (1993)
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.
(1993)
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,
P.A. (1992)
DNA twisting and the effects of non-contacted bases on the affinity of 434
operator for 434 repressor
Nature 355:89-91 Abstract
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last modified: 08/03/2009 by G. Koudelka
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