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) 645-4940
(Research Office) or 645-4904 (Chair’s Office)
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 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
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)
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: 08/09/2010 by G. Koudelka
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