division of cell and molecular microbiology

Transcription

DIVISION OF CELL AND MOLECULAR
MICROBIOLOGY
Head
Jan Nešvera, PhD.
The Division consists of eight laboratories, each of which has its own scientific projects
and independent research budget. All research is financed from the scientific grants awarded
to the individual groups by domestic or foreign grant agencies.
Scientific work of the Division is focused on studies of molecular biology and genetics
in both prokaryotic and eukaryotic microorganisms. Many of the microorganism studied by
the individual groups of the Division (e.g. streptomycetes, yeasts, corynebacteria, rhodococci,
pathogenic bacteria Bordetella pertussis, Neisseria meningitidis, Streptococcus pneumoniae,
mycobacteria) have a practical importance in industry and medicine. Control of gene expression and cell differentiation, effects of stress factors on cellular functions and their role in cell
aging and population survival, arrangement of eukaryotic plasma membrane, mechanisms of
restriction and modification of DNA and molecular aspects of bacterial pathogenicity are the
main topics under study.
The research exploits complex approaches, utilizing dynamically growing data on genome sequences of the studied microorganisms. Proteomics, based on analysis of 2-D electrophoresis gels, is frequently used. DNA-arrays apparatus make possible transcriptome analysis
of the studied microorganisms. This device serves the whole Institute of Microbiology as well
as customers from other institutes of the Academy of Sciences or the universities. Other methods involve, e.g., bioluminiscence as a detection technique in reporter gene systems, fluorescence microscopy for cell biology studies and fluorescence methods for measuring membrane
potential and the performance of multidrug resistance transporters. These methods form the
basis for collaboration with groups from other Divisions of the Institute.
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SCIENTIFIC REPORT ’07–’09
Laboratory 121
MICROBIAL PROTEOMICS
Head
Jaroslav Weiser, PhD.
Scientific staff
Karolina Buriánková, PhD. (on maternity leave)
Part-time scientists
Jiří Janeček, PhD.
Technical staff
Silvia Bezoušková MSc.
Doctoral students
Šárka Nezbedová, MSc., Denisa Petráčková RNDr.
Undergraduate students
Barbora Sitařová, Eva Tesařová, Zuzana Sochorová
Research field and principal results
The microbiological research in the laboratory is oriented towards the complex studies
of gene expression on protein level in populations of bacteria growing under conditions resembling their natural living conditions. The methodology we use involves both classical
bacterial physiology practices and progressive proteomics techniques. Laboratory is also
involved in few collaborative projects involving use of “gel based” proteomics technology.
Potential of glass bead-based two-phase cultivation system for studies
of bacterial populations
The glass bead-based two-phase cultivation system, which was developed for the studies of morphological and biochemical differentiation, had shown a potential to be useful in
studies of other biological problems such as surface adhesion and formation of biofilms. We
have shown that Streptomycetes grown on different types of bead surfaces (glass or zirconium
silicate) in identical liquid media are influenced in both biochemical and morphological differentiation (Fig. 13). This indicates that degree of hydrofobicity of the surface supporting
formation of mycelial network plays an important role in the regulation of antibiotic biosyn-
CELL AND MOLECULAR MICROBIOLOGY
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thesis as well as cell development. We have also demonstrated formation of biofilms of Mycobacterium smegmatis on surface of glass and zirconia beads. It makes this two-phase cultivation system very useful tool for studies of biofilm formation.
Fig. 13. Mycobacterium smegmatis culture grown on the “zirkonia beads” was stained
with methylene blue, the close-up of the surface of the beads shows the formation of
biofilm covering individual beads.
Secondary metabolism and its regulation in Streptomyces ambofaciens
In order to identify new and seemingly silent antibiotic biosynthesis pathways and their
products we prepared triple mutant of Streptomyces ambofaciens with inactivated genes for
biosynthesis of three antibiotics, spiramycin, congocidin and alpomycin. We found cultivation
conditions under which the mutant produced compound with antibacterial activity. Identification of this possible new drug is under way.
In the second part of the project carried out in collaboration with the group in Orsay,
France, we identified analogs of two global regulators described in Streptomycetes: Rep and
DasR, participating on regulation of antibiotic biosynthesis.
Other collaborative projects
In collaboration with the group of Ondřej Hrušák at the 2nd Medical Faculty, Charles
University we looked for common alterations in bone marrow plasma proteome patterns of
children suffering from acute lymphoblastic leukaemia (ALL). Analysed patient proteomes
were compared to proteome patterns of children after a bone marrow transplantation. The
immuno-chromatography depletion was proved to be the most effective technique in sample
preparation and was then used throughout the study. The analysis revealed one new protein
(SSP3106) in the control sample, 13 proteins up- or down-regulated in the range of 2- to
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4-fold and 6 proteins up-regulated more than 4-fold in the control sample. The proteins were
quantified and most of them identified by mass spectrometry.
In the collaboration with the group of Marie Weiserova of this Institute we analysed
phosphorylation of Type I restriction-modification (R-M) enzymes in Escherichia coli.
We showed that in vitro phosphorylation of the HsdR subunit of purified EcoKI endonuclease occurs on Thr, and is strictly dependent on the addition of a catalytic amount of
cytoplasmic fraction isolated from E. coli. So far this is the first case of phosphorylation of
a Type I R-M enzyme reported.
Publications
Benada O., Kofroňová O., Weiser J.: Life cycle SEM monitoring of Streptomycetes cultured on ballotina. 8th
Multinat. Congr. Microscopy (8MCM). Prague June 17 -21, 2007, Czechoslovak Microscopy Society, eds.
Nebesářová J., Hozák P., pp. 443-444, ISBN 978-80-2399397-4 (2007).
Cajthamlová K., Šišáková E., Weiser J., Weiserová M.: Phosphorylation of Type IA restriction-modification
complex enzyme EcoKI on HsdR subunit. FEMS Microbiol. Letters 270, 171-177 (2007).
Holub M., Bezoušková S., Kalachová L., Weiser J.: Protein synthesis elongation factor Tu present in spores of
Streptomyces coelicolor can be phosphorylated in vitro by a spore protein kinase. Folia Microbiol. 52, 471478 (2007).
Juguet M., Lautru S., Francou F. X., Nezbedova S., Leblond P., Gondry M., Pernodet J. L.:. An iterative nonribosomal peptide synthetase assembles the pyrrole-amide antibiotic congocidine in Streptomyces ambofaciens. Chem. Biol. 16, 421-31 (2009).
Papa S., De Rasmo D., Scacco S., Signorile A., Technikova-Dobrova Z., Palmisano G., Sardanelli A.M., Papa
F., Panelli D., Scaringi R., Santeramo A.: Mammalian complex I: A regulable and vulnerable pacemaker in
mitochondrial respiratory function. Biochim. Biophys. Acta Bioenergetics 1777, 719-728 (2008).
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Laboratory 122
CELL REPRODUCTION
Head
Jiří Hašek, PhD.
Scientific staff
Miroslava Opekarová, PhD.
Pavla Vašicová, PhD.
Ivana Malcová-Janatová, PhD.
Jana Vojtová-Vodolánová, PhD.
Part-time senior scientist Eva Streiblová, PhD. DSc.
Technical staff
Dana Janošková, MSc.
Lenka Nováková, MSc.
Jarmila Serbousková
Doctoral students
Ivana Frýdlová, MSc., Tomáš Groušl, MSc.
Iveta Bartoňová, Eva Tarantová, Renata Slabá
The main topics of the laboratory concern the elucidation of mechanisms responsible
for trafficking of regulatory proteins in growing or stressed cells of budding yeast Saccharomyces cerevisiae. The life cell imaging microscopy and the specific molecular biology
techniques are frequently used to analyze distribution of GFP or RFP tagged proteins in wildtype or mutant cells of S. cerevisiae under variour condition of cultivation.
Stress-induced rearrangement of translation machinery
In higher eukaryotic cells exposed to various environmental stresses translation initiation factors accumulate in cytoplasmic aggregates, called stress granules. The stress granules
were identified in heat-stressed fission yeast but until now their formation has not been referred for budding yeast Saccharomyces cerevisiae. We constructed various S. cerevisiae strains
expressing fusion proteins tagged with GFP and/or mRFP from their chromosomal sites and
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analyzed redistribution of translational factors, ribosomal proteins, mRNA binding proteins
and mRNA under various stresses. Together, our data indicate that the protein accumulations
induced by robust heat shock represent dynamic structures of some translational components
resembling stress granules of higher eukaryotic cells
Plasma membrane compartmentalization
In cooperation with Dr. Tanner from the Institute of Cell Biology and Plant Physiology
at Regensburg University, we obtained important results with a key impact on understanding
of plasma membrane arrangement in eukaryotic cells. With the use of fluorescent markers
attached to various plasma membranes proteins (Fig. 14), we showed that the plasma
membrane of S. cerevisiae is subdivided into at least two stable lateral compartments. One
consists of 50 to 80 patches of about 300 nm in diameter is called MCC (Membrane
Compartment of Can1, the arginine permease). The other one- MCP (Membrane Compartment of Pma1, the H+/ATPase) fills the space in between the MCC patches. Some other
PM proteins, like Hxt1 or Gap1 are distributed homogeneously. The distribution of proton
symporters in MCC can be affected by, e.g., lipid composition or the plasma membrane
energization, the distribution of other proteins is resistant to these effects. Recently we have
documented that the dwelling in the MCC protects its residents against endocytosis. The physiological importance of the compartmentalization of the plasma membrane is under intensive
study in our group (Fig. 15).
Fig. 14. A single optical section of fermenting Saccharomyces cerevisiae cells showing
distribution of actin filaments (Abp140-GFP; green) and mitochondria (pMITO-RFP;
red) at the cortical domain.
Functional analysis of Isw2 protein
Strains of Saccharomyces cerevisiae lacking Isw2, the catalytic subunit of the Isw2
chromatin remodeling complex, show the mating type independent activation of the cell wall
integrity (CWI) signaling pathway. Since the CWI pathway activation usually reflects cell
wall defects, we searched for the cell wall-related genes changed in expression. The genes
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DSE1, CTS1 and CHS1 were upregulated as a result of the Isw2 absence, according to our
previously published gene expression profiles. Western blot analyses of double deletion mutants, however, did not indicate contribution of the chitin metabolism-related genes CTS1 and
CHS1 to the CWI pathway activation. Nevertheless, deletion of the DSE1 gene encoding
a daughter cell-specific protein with unknown function suppressed the CWI pathway activation in isw2Δ cells. In addition, deletion of DSE1 also abolished the budding-within-the-birthscar phenotype of isw2Δ cells. Plasmid-driven overexpression proved that deregulation of the
Dse1 synthesis was also responsible for the CWI pathway activation and manifestation of the
budding-within-the-birth-scar phenotype in wild-type cells. The overproduced Dse1-GFP
localized to both sides of the septum and persisted in unbudded cells. Although the exact
cellular role of this daughter cell-specific protein has to be elucidated, our data point to involvement of Dse1 in bud site selection in haploid cells.
Fig. 15. Nce102 is homogenously distributed in membranes of shmoos. Localization of
the proteins treated with 30 μg/mL α factr for 2 h; 3D reconstructions of confocal z stacks
are presented; bar = 2 μm.
Life cell imaging microscopy studies
The late endosome plays a key role in coordinating vesicular transport of proteins between Golgi, vacuole/lysosome, and plasma membrane. In cooperation with the laboratory of
Dr. Hinnebusch from NICHD NIH, Bethesda, USA we found that deleting multiple genes
involved in vesicle fusion at the multivesicle bodies (MVB) impairs transcriptional activation
by Gcn4, a global regulator of amino acid biosynthetic genes in S.cerevisiae.
Many aspects of plant development depend on distribution of the plant signalling molecule auxin. Auxin transport inhibitors (ATIs) interfere with directional auxin transport but the
molecular mechanism of ATIs action has remained largely elusive. In cooperation with the
laboratory of Dr. Friml from the University of Tübingen and others, we showed using live cell
imaging microscopy that ATIs inhibit multiple vesicle trafficking processes in yeast cells.
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Publications
Groušl T., Ivanov P., Frýdlová I., Vašicová P., Janda F., Vojtová J., Malínská K., Malcová I., Nováková L.,
Janošková D., Valášek L., Hašek J.: Robust heat shock induces eIF2α-phosphorylation- independent assembly of stress granules containing eIF3 and 40S ribosomal subunits in budding yeast S. cerevisiae.
J.Cell.Sci.122, 2078-2088 (2009).
Frýdlová I., Malcová I., Vašicová P., Hašek J.: Deregulation of DSE1 gene expression results in aberrant budding within the birth scar and CWI pathway activation in Saccharomyces cerevisiae. Eukaryot.Cell 8,
586-594 (2009).
Prevorovský M., Groušl T., Stanurová J., Rynes J., Nellen W., Půta F., Folk P.: Cbf11 and Cbf12, the fission
yeast CSL proteins, play opposing roles in cell adhesion and coordination of cell and nuclear division.
Exp Cell Res. 315, 1533-1547, (2009).
Jelínek F., Cifra M., Pokorný J., Vaniš J., Šimša J., Hašek J., Frýdlová I.: Measurement of electrical oscillations
and mechanical vibrations of yeast cells membrane around 1 kHz. Electromagn Biol Med. 28, 223-232
(2009).
Grossmann G., Malinsky J., Stahlschmidt W., Loibl M., Weig-Meckl I., Frommer W.B., Opekarová M., Tanner
W.: Plasma membrane microdomains regulate turnover of transport proteins in yeast. J. Cell. Biol. 183,
1075-1088 (2008).
Zhang F., Gaur N.A., Hasek J., Kim S-J., Qiu H., Swanson M.J., Hinnebusch A.G.: Disrupting vesicular
trafficking at the endosome attenuates transcriptional activation by Gcn4. Mol. Cell. Biol. 28, 6796-6818
(2008).
Pokorny J., Hašek J., Vanis J., Jelínek F.: Biophysical aspects of cancer-electromagnetic mechanism. Indian J.
Exp. Biol. 46, 310-321 (2008).
Dhonukshe P., Grigoriev I., Fischer R., Tominaga M., Robinson D.G., Hašek J. Paciorek T., Petrášek J.,
Seifertová D., Zažímalová E., Gadella Jr T.W.J., Stierhof Y-D., Ueda T., Oiwa K., Akhmanova A., Brock
R., Spang A., Friml J.: Auxin transport inhibitors impair vesicle motility and actin cytoskeleton dynamics
in diverse eukaryotes. PNAS 105, 4489-4494 (2008).
Frýdlová I., Basler M., Vašicová P., Malcová I., Hašek J.: Special type of pheromone-induced invasive growth
in Saccharomyces cerevisiae. Current Genetics 54, 87-96 (2007).
Grossmann G., Opekarová M., Malínský J., Weig-Meckl I., Tanner W.: Membrane potential governs lateral
segregation of plasma membrane proteins and lipids in yeast. EMBO J. 26, 1-8 (2007).
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Laboratory 123
MOLECULAR GENETICS OF BACTERIA
Head
Jan Nešvera, PhD.
Scientific staff
Maria Brennerová, PhD.
Libor Krásný, PhD.
Miroslav Pátek, PhD.
Lenka Rucká, PhD. (on maternity leave)
Marie Weiserová, PhD.
Vladimír Brenner, PhD.
Prof. Jiří Jonák, DSc.
Technical staff
Dana Lukavská
Věra Reimannová
Anna Stachová
Zuzana Štropová
Doctoral students
Andrea Benáková, MSc., Kamila Cajthamlová, MSc., Alena Guzanová, MSc.,
Jiří Holátko, MSc., Jiřina Josefiová, MSc., Pavla Kadeřábková, MSc.,
Monika Knoppová, MSc., Olesya Korotkevych, MSc., Mongkol Phensaijai, MSc.,
Martina Pravečková, MSc., Luděk Sojka, MSc., Juraj Szököl, MSc.,
Hana Šanderová, MSc., Radoslav Šilar, MSc., Eva Šišáková, MSc.,
Alžběta Švenková, MSc., Hana Tišerová, MSc., Martina Zemanová, MSc.
Vladimíra Fučíková, Tomáš Kouba
Research fields and principal results
Scientific program of the Laboratory covers four research fields: (1) Studies of function
of restriction-modification enzymes of Type I; (2) Studies of regulation of gene expression in
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Bacillus; (3) Studies of gene expression in biotechnologically important corynebacteria and
rhodococci; (4) Characterization of microbial communities in polluted localities.
Interrelationship between helicase and nuclease domains during DNA translocation
by the molecular motor EcoR124I
The Type I Restriction-Modification enzyme EcoR124I comprises three subunits with
the stoichiometry HsdR2:HsdM2:HsdS1. The HsdR subunit is an archetypical example of the
fusion between nuclease and helicase domains into a single polypeptide, a linkage that is
found in many other DNA processing enzymes. To explore the interrelationship between
these physically-linked domains, we examined the DNA translocation properties of EcoR124I
complexes in which the HsdR subunits had been mutated in the RecB-like nuclease motifs II
or III. Nuclease mutations had noteworthy effects on DNA translocation despite being discrete from the helicase domain. In addition to reductions in DNA cleavage activity, we also
observed decreased translocation and ATPase rates, different enzyme populations with different characteristic translocation rates, a tendency to stall during initiation and altered HsdR
turnover dynamics.
Regulation of gene expression in Bacillus
We use the Gram-positive bacterium Bacillus subtilis, a model organism for molecular
biologists, both in basic research and in the development of novel antibiotics. Our basic research is focused on analysis of regulation of gene expression at transcriptional as well as at
translational level.
We discovered the key importance of the identity of the first nucleoside triphosphate of
a mRNA molecule (most typically it is ATP or GTP) for changes in gene expression in response to amino acid starvation in B. subtilis. Upregulated promoters initiate mostly with
ATP, while downregulated promoters with GTP.
We demonstrated that the B. subtilis YbxF protein binds to the ribosome with dependence on
growth phase. We also identified amino acids important for this interaction.
During studies on the thermostability determinants of the translation elongation factor
Tu (EF-Tu) from Bacillus stearothermophilus we identified its N-terminal region (12 amino
acids) as important for the thermostability of both the isolated catalytic G-domain and the
full-length EF-Tu. Comparison with other G-proteins suggested that this region may play a stabilizing role in other proteins as well.
We are developing, in collaboration with the Institute of Organic Chemistry and Biochemistry in Prague, novel antibiotics targeting bacterial RNA polymerase.
Gene expression in rhodococci and corynebacteria
The strain Rhodococcus erythropolis A4 shows high nitrile hydratase and amidase activities that can be used for biotransformation of various nitriles and amides to produce pharmaceutically important compounds. The cluster containing genes oxd (coding for aldoxime
dehydratase), ami (amidase) and nha1-nha2 genes (α- and β-subunits of nitrile hydratase) as
well as nhr1, nhr2, nhr3 and nhr4 genes coding for regulatory proteins was isolated from this
strain and its nucleotide sequence was determined. Promoter of the ami gene was localized
and transcription of this gene into three different RNA transcripts was detected. The ami gene
was cloned in expression plasmid vectors and heterologous production of amidase in Escherichia coli was proved.
The strain R. erythropolis CCM2595 is capable of degrading various aromatic pollutants (e.g. phenol, benzoate, p-hydroxybenzoate). Four promoters were localized within the
gene cluster pheA2A1R-catRABC involved in phenol degradation and their induction by
phenol was proved. Binding of CRP protein within the regulatory region of phe and cat genes
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was observed. The CRP regulator is supposed to mediate catabolic repression and substrate
utilization hierarchy. Recombinant strains carrying pheA2A1R and catRABC genes on multicopy plasmids degraded phenol more efficiently than the wild-type strain.
Promoter regions of amino-acid producing Corynebacterium glutamicum genes involved in stress response (mainly heat shock) were analyzed. Using non-radioactive primer
extension technique (Fig. 16), multiple transcriptional start points were detected in the up-
Fig. 16. Mapping of transcriptional start points (TSPs) of the sigH gene from Corynebacterium glutamicum by non-radioactive primer extension (PEX) technique. The peaks in the lower part of the
figure represent cDNA synthesized in reverse transcription (primer extension) using total RNA from
C. glutamicum wild-type (WT) strain and ΔsigH mutant isolated after growth at 30 °C and after heat
shock (40 °C, 1 h), respectively. The peaks (A, C, G, T) generated by the automatic sequencer represent the products of sequencing reactions performed with the same fluorescein-labeled primer as that
used for primer extension. The peak area in PEX reactions is proportional to the amount of mRNA
whose synthesis initiated at the respective promoters.
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stream regions of all tested genes (clpB, clpC, dnaJ2, dnaK, sigH, arnA). Among the multiple
(2–4) promoters of these genes, both promoters recognized by primary sigma factor SigA and
those recognized by extracytoplasmic function stress-responsive sigma factors (mainly by
SigH) were detected.
Metagenomic analysis of highly polluted localities
Diversity of extradiol dioxygenase (enzyme involved in the biodegradation of various
pollutants) in a site highly polluted with aliphatic and aromatic hydrocarbons was assessed by
functional screening of a fosmid library in E. coli with catechol as substrate. The 235 positive
clones from inserts of DNA extracted from contaminated soil were equivalent to one extradiol
dioxygenase coding gene per 3.6 Mb of DNA screened, indicating a strong selection for genes
encoding this function. Three subfamilies were identified as predominant. Functional analysis
of representative proteins revealed a subcluster of extradiol dioxygenases showing very high
affinity towards different catecholic substrates and a task-sharing between different extradiol
dioxygenases in the community of the contaminated site may ensure effective degradation of
mixtures of aromatics.
Microbial diversity and activities after extensive uranium leaching were analyzed and
numerical ecology tools were used for correlating phylogenetic profiles with environmental
variables. Sulfate reduction and denitrification capacity was found in the extremely acidified
groundwater ecosystem.
Publications
Blombach B., Schreiner M.E., Holátko J., Bartek T., Oldiges M., Eikmanns B.J.: L-valine production with
pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum. Appl. Environ. Microbiol. 73,
2079–2084 (2007).
Brennerová M.V., Josefiová J., Brenner V., Pieper D.H., Junca H.: Metagenomics reveals diversity and abundance of meta-cleavage pathways in microbial communities from soil highly contaminated with jet fuel
under air-sparging bioremediation. Environ. Microbiol. 11, 2216-2227 (2009).
Cajthamlová K., Šišáková E., Weiser J., Weiserová M.: Phosphorylation of Type IA restriction-modification
complex enzyme EcoKI on the HsdRsubunit. FEMS Microbiol. Lett. 270, 171–177 (2007).
Hänssler E., Müller T., Palumbo K., Pátek M., Brocker M., Krämer R., Burkovski A.: A game with many
players: control of gdh transcription in Corynebacterium glutamicum. J. Biotechnol. 142, 114-122
(2009).
Holátko J., Elišáková V., Prouza M., Nešvera J., Pátek M.: Promoter-activity modulation for branched-chain
amino acid production. European Patent EP 1860193, Publication date: 11/28/2007.
Holátko J., Elišáková V., Prouza M., Sobotka M., Nešvera J., Pátek M.: Metabolic engineering of the L-valine
biosynthesis pathway in Corynebacterium glutamicum using promoter activity modulation. J. Biotechnol.
139, 203-210 (2009).
Jelínek M., Weiserová M., Kocourek T., Jurek K., Strnad J.: Doped biocompatible layers prepared by laser.
Laser Physics, in press.
Kabelitz N., Macháčková J., Imfeld G., Brennerová M., Pieper D.H., Heipieper H.J., Junca H.: Enhancement of
the microbial community biomass and diversity during air sparging bioremediation of a soil highly contaminated with kerosene and BTEX. Appl. Microbiol. Biotechnol. 82, 565-577 (2009).
Knoppová M., Phensaijai M., Veselý M., Zemanová M., Nešvera J., Pátek M.: Plasmid vectors for testing in
vivo promoter activities in Corynebacterium glutamicum and Rhodococcus erythropolis. Curr. Microbiol. 55, 234-239 (2007).
Kolberg J., Hammerschmidt S., Frank R., Jonák J., Šanderová H., Aase A.: The surface-associated elongation
factor Tu is concealed for antibody binding on viable pneumococci and meningococci. FEMS Immunol.
Med. Microbiol. 53, 222–230 (2008).
Krásný L., Tišerová H., Jonák J., Rejman D. Šanderová H.: The identity of the transcription +1 position is
crucial for changes in gene expression in response to amino acid starvation in Bacillus subtilis. Mol. Microbiol; 69, 42–54 (2008).
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Kubáč D., Kaplan O., Elišáková V., Pátek M., Vejvoda V., Slámová K., Tóthová A., Lemaire M., Gallienne E.,
Lutz-Wahl S., Fischer L., Kuzma M., Pelantová H., van Pelt S., Bolte J., Křen V., Martínková L.: Biotransformation of nitriles to amides using soluble and immobilized nitrile hydratase from Rhodococcus
erythropolis A4. J. Mol. Catal. B: Enzym. 50, 107–113 (2008).
Martínková L., Uhnáková B., Pátek M., Nešvera J., Křen V.: Biodegradation potential of the genus Rhodococcus. Environ. Int. 35,162-177 (2009).
Nešvera J., Pátek M.: Plasmids and promoters in corynebacteria and their applications, pp. 113 – 154, in A. Burkovski (Ed): Corynebacteria: Genomics and Molecular Biology. Caister Academic Press, Wymondham
(UK) 2008.
Pátek M.: Branched-chain amino acids, pp. 129-162 in V.F. Wendisch (Ed): Amino Acids – Pathways, Regulation and Metabolic Engineering. Springer, Berlin 2007.
Rejman D., Pohl R., Kovačková S., Kočalka P., Švenková A., Šanderová H., Krásný L., Rosenberg I.: Pyrrolidine analogues of nucleosides and nucleotides, Nucl. Acids Symp. Ser. 52, 577-578 (2008).
Šanderová H., Tišerová H., Barvík I., Sojka L., Jonák J., Krásný L.: The N-terminal region is crucial for the
thermostability of the G-domain of Bacillus stearothermophilus EF-Tu. Biochim. Biophys. Acta 1804,
147-155 (2010).
Šišáková E., Stanley L.K., Weiserová M., Szczelkun M.D.: A RecB-family nuclease motif in the Type I restriction endonuclease EcoR124I. Nucl. Acids Res. 36, 3939–3949 (2008).
Šišáková E., Weiserová M., Dekker C., Seidel R., Szczelkun M.D.: The interrelationship of helicase and nuclease domains during DNA translocation by the molecular motor EcoR124I. J. Mol. Biol. 384, 1273–1286
(2008).
Sojka L., Fučík V., Krásný L, Barvík I., Jonák J.: YbxF – a protein associated with exponential phase ribosomes
in Bacillus subtilis. J. Bacteriol 189, 4809–4814 (2007).
Vejvoda V., Šveda O., Kaplan O., Přikrylová V., Elišáková V., Himl M., Kubáč D., Pelantová H., Kuzma M.,
Křen V., Martínková L.: Biotransformation of heterocyclic dinitriles by Rhodococcus erythropolis and
fungal nitrilases. Biotechnol. Lett. 29, 1119–1124 (2007).
Veselý M., Knoppová M., Nešvera J., Pátek M.: Analysis of catRABC operon for catechol degradation from
phenol-degrading Rhodococcus erythropolis. Appl. Microbiol. Biotechnol. 76, 159–168 (2007).
Weiserová M., Ryu J.: Characterization of a restriction modification system from the commensal Escherichia
coli strain A0 34/86 (O83:K24:H31). BMC Microbiol. 8,106–112 (2008).
Zemanová M., Kadeřábková P., Pátek M., Knoppová M., Šilar R., Nešvera J.: Chromosomally encoded small
antisense RNA in Corynebacterium glutamicum. FEMS Microbiol. Lett. 279, 195-201 (2008).
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Laboratory 124
CELL BIOLOGY
Head
Libuše Váchová, PhD.
Scientific staff
Karel Sigler, PhD. DSc.
Alena Pichová, Assoc.Prof., PhD.
Otakar. Hlaváček, PhD.
Dana Gášková, Assoc.Prof., PhD.
Zdena Palková, Prof., PhD.
Technical staff
Marcela Kittlerová, MSc.
Helena Kučerová, MSc.
Alexandra Pokorná
Anna Machová
Doctoral students
Karel Harant, MSc., Dita Strachotová, MSc., Barbora Škaloudová, MSc.
Jan Bártl, Lenka Belicová, Jana Hlousková, Lenka Marešová, Daniela Paňková,
Aneta Sajdová, Milada Smetková, Ilona Urbářová, Andrea Volejníková
The fundamental research in our laboratory is focused mainly on physiology and molecular mechanisms of stress response, development, ageing and apoptosis and on membrane
processes in individual yeast cells as well as in yeast multicellular populations. Applied research concerns brewery biotechnology.
Caspases in yeast apoptosis-like death
It is still not clear whether yeast can die by apoptosis, i.e. by the dying process involving the function of caspases, or by other kinds of programmed cell death (PCD). Saccharomy-
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ces cerevisiae is able to produce one caspase homologue, metacaspase Mca1p/Yca1p, but its
substrate specificity is questionable. In addition, other caspase-like activities have been identified in yeast cells, e.g. by in situ staining of cells from colonies by fluorescent substrate
D2R. On the contrary, detection of caspase-like activities in yeast by fluorochrome-labelled
cell-permeable inhibitors of caspases (FLICA) was shown to provide false results.
Impact of mitochondrial status on aging and apoptosis in yeast
We are interested in the role of mitochondria during cellular aging and the cross-talk of
mitochondrial physiology with the physiology of the aging cell in the order to elucidate the
signals and molecular mechanisms influencing the role of mitochondria in, and events leading
to aging and apoptosis in yeast mutants affecting aging. We investigate the morphology,
quantity and respiratory activity of mitochondria and their disruption during aging and release
of free radicals, oxidative damage of proteins and lipids on the background of apoptotic
markers and replicative age.
Expression and localisation of ammonium exporters Ato1p, Ato2p and Ato3p
Colonies of S. cerevisiae pass through three main developmental phases, which are defined by changes in pH of the medium. Two acidic phases are separated by alkali phase characterised by ammonia production. We showed that the expression of ATO genes coding for
putative ammonium exporters is controlled by ammonia. All three Ato-GFP proteins localise
to the detergent-resistant compartments of plasma membrane and their appearance correlates
with the beginning of ammonia release. Ato1p-GFP and Ato3p-GFP form patches in the
membrane visible under the fluorescence microscope (Fig. 17). Ato3p-GFP patches are quite
stable; the formation of those of Ato1p-GFP is pH dependent. Ato1p-GFP patches form at pH
above 6 and they disappear at pH 5 or lower. Both the Ato1p-GFP clustering and patches
spreading are reversible. This suggests that besides the ammonia induction of Ato protein
synthesis, pH may rapidly regulate Ato1p function.
Monitoring the proton gradient in yeast membrane vesicles by fluorescent probes
The basics of yeast bioenergetics were explored by using pH-sensitive fluorescent probe
pyranine for quantitative measurements of pH inside proteoliposomes carrying reconstituted
yeast plasma membrane H+-ATPase in their lipid membrane. This model system is suitable
for simultaneous monitoring of both membrane potential and ΔpH components of the proton
electrochemical gradient and elucidating their interconnection in biomembranes.
Oxidative stress in yeast cells and the action of new antioxidants
Superoxide dismutases serve S. cerevisiae cells for defence against superoxide radical
but the phenotypes of sod1Δ and sod2Δ mutants are different. Unlike the Sod1p protein,
whose major role is oxidative stress defence, Sod2p also plays a role in protecting cells
against other stresses – high osmolarity, heat and metalloid stress. Amphiphilic mono- and di(alkanoylamino) ethyldimethylamine-N-oxides protect sodΔ strains of S. cerevisiae against
the effect of peroxyl and superoxide radicals and also protect membrane lipids isolated from
these strains against peroxidation. The antioxidative actions of phenolic and N-oxide antioxidants differ. The former undergo reactivation, whereas the latter cannot be reactivated. Both
groups of antioxidants can mimic the role of superoxide dismutases.
Assessing the brewer’s yeast quality and brewery fermentation outcome
As a practical spin-off of the study of yeast bioenergetics, we optimized the acidification power (AP) test of brewer’s yeast quality and then used it in a brewery for assessing the
effect of modern technological steps on yeast quality in a set of fermentations performed in
cylindro-conical tanks. The effect of the yeast vitality can be masked by variations in pitching
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rate, wort composition, ambient conditions in the cylindro-conical tank, and other technological factors. A method was developed for a contact-free optical pH measurement of AP.
Fig. 17. Localisation of Ato-GFP ammonium exporters in Saccharomyces cerevisiae cells. A. AtoGFP proteins localise to plasma membrane, to its detergent-resistant compartments. Ato1p-GFP and
Ato2p-GFP form there visible patches (red arrow). Some molecules of Ato-GFP proteins are delivered
to vacuoles (white arrow) for degradation. B. Ato1p-GFP patches gradually disappear at pH 5 and
they reconstitute at pH 8. C. Three-dimensional reconstruction of S. cerevisiae cell with plasma membrane localised Ato1p-GFP in patches (processed from confocal microscopy sections). TV, transversal
and TG tangential optical sections. Blue bar, 5µm.
Constituents of cell membrane lipids and other biologically relevant compounds
In a broad-base study, we identified and characterized a number of very long chain unsaturated fatty acids and triacylglycerols in plants and medium chain polyunsaturated fatty
acids in algae. Other biologically important compounds included, e.g., astaxanthin diglucoside diesters and polysaccharides in algae, antibiotics produced by various microorganisms
and other compounds.
Yeast colony architecture and cell differentiation
Yeasts, when growing on solid surfaces, form organised multicellular structures, colonies, which build specific 3-D architecture. Cells within colonies differentiate into specialised
variants dependently on their position within the structure. Ammonium exporter Ato1p thus
appears synchronously in cells of surface layer of uniform thickness over the whole colony of
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laboratory S. cerevisiae strain (Fig. 18A). Another example is the uppermost one-cell layer of
cells tightly joined by thick cell walls and surface proteins protecting the colony against
harmful attack from environment like a “skin”. Contrary to smooth colonies of laboratory
strains, wild S. cerevisiae strains form markedly structured colonies (Fig. 18B). The building
of their wrinkled 3-D structure depends on dimorphic transition from yeast-cells to pseudohyphae and on production of adhesin Flo11p (Fig. 18C).
Fig. 18. Architecture of yeast colonies. A: Reconstruction of Ato1p-GFP pattern in 4-day-old microcolony on the basis of bottom and side-view pictures of the whole colony obtained by 2-photon confocal microscopy (from Env. Microbiol. 11, 1866, 2009). B: Wrinkled colonies formed by wild S. cerevisiae strain Σ1278. C: Localization of Flo11p-GFP to bud-neck and tip of the cells at the time when
colony starts to form wrinkles.
Publications
Cajthaml T., Křesinová Z., Svobodová K., Sigler K., Řezanka T.: Microbial transformation of synthetic estrogen
17alpha-ethynilestradiol. Environment. Pollution 153(12), 3325-3335 (2009).
Čáp M., Váchová L., Palková Z.: Yeast colony survival depends on metabolic adaptation and cell differentiation
rather than on stress defense. J. Biol. Chem., in press, doi:10.1074/jbc.M109.022871 (2009).
Čížková M., Pichová A., Vítová M., Hlavová M., Hendrychová J., Umysová D., Gálová E., Ševčovičová A.,
Zachleder V., Bišová K.: CDKA and CDKB kinases from Chlamydomonas reinhardtii are able to complement cdc28 temperature-sensitive mutants of Saccharomyces cerevisiae. Protoplasma 232, 183–191
(2008).
Dziadkowiec D., Krasowska A., Liebner A., Sigler K.: Protective role of mitochondrial superoxide dismutase
against high osmolarity, heat and metalloid stress in S. cerevisiae. Folia Microbiol. 52, 120-126 (2007).
Gabriel P., Dienstbier M., Matoulková D., Kosař K., Sigler K.: Optimized acidification power test of yeast
vitality and its use in brewing practice. J. Inst. Brew. 114, 270-276 (2008).
Gabriel P., Dienstbier M., Sladky P., Sigler K.: A new method of optical detection of yeast acidification power.
Folia Microbiol. 53, 527-533 (2008).
Hendrych T., Kodedová M., Sigler K., Gášková D.: Characterization of the kinetics and mechanisms of inhibition of drugs interacting with the S. cerevisiae multidrug resistance pumps Pdr5p and Snq2p. BBA Biomembranes 1788, 717-723 (2009).
Hlaváček O., Kučerová H., Harant K., Palková Z., Váchová L.: Putative role for ABC multidrug exporters in
yeast quorum sensing. FEBS Lett. 583, 1107-13 (2009).
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Holoubek A., Večeř J., Sigler K.: Monitoring of the proton electrochemical gradient in reconstituted vesicles:
Quantitative measurements of both transmembrane potential and intravesicular pH by ratiometric fluorescent probes. J. Fluoresc. 17, 201-213 (2007).
Košin P., Šavel J., Brož A., Sigler K.: Control and prediction of brewery fermentations by gravimetric analysis.
Folia Microbiol. 53, 451-456 (2008).
Krasowska A. Piasecki A., Murzyn A., Sigler K.: Assaying the antioxidant and radical scavenging properties of
aliphatic mono- and di-N-oxides in a test with SOD-deficient yeast and by a chemiluminescence test.
Folia Microbiol. 52, 45-51 (2007).
Krasowska A., Sigler K.: Cell-protective and antioxidant activity of two groups of synthetic amphiphilic antioxidants – phenolics and amine N-oxides: a review. Folia Microbiol. 52(6),585-592 (2007).
Kubrycht J., Sigler K.: Length of hypermutation motif DGYW/WRCH in the focus of statistical limits. J. Theor.
Biol. 255, 8-15 (2008).
Palková Z., Váchová L., Gášková D., Kučerová H.: Synchronous plasma membrane electrochemical potential
oscillations during yeast colony development and aging. Mol Membr Biol. 26, 228-235 (2009).
Řezanka T., Nedbalová L., Cajthaml T., Sigler K.: Very-long-chain anteiso branched fatty acids identified in
N-acylphosphatidylethanolamines from Calothrix. Phytochem. 70, 655-663 (2009).
Řezanka T., Nedbalová L., Sigler K., Cepák V.: Identification of astaxanthin diglucoside diesters from snow
alga Chlamydomonas nivalis by liquid chromatography–atmospheric pressure chemical ionization mass
spectrometry. Phytochem. 69, 479-490 (2008).
Řezanka T., Nedbalová L., Sigler K.: Unusual medium chain polyunsaturated fatty acids from the snow alga
Chloromonas brevispina. Microbiol. Res. 163, 373-379 (2008).
Řezanka T., Nedbalová L., Sigler K.: Unusual medium chain polyunsaturated fatty acids from the snow alga
Chloromonas brevispina identified by atmospheric pressure chemical ionization liquid chromatography–
mass spectrometry. Phytochem. 69, 2849-2855 (2008).
Řezanka T., Nedbalová N., Sigler K.: Identification of very long chain polyunsaturated fatty acids from Amphidinium carterae by atmospheric pressure chemical ionization liquid chromatography-mass spectroscopy.
Phytochem. 69, 2391-2399 (2008).
Řezanka T., Olšovská J., Sobotka M., Sigler K.: The use of APCI-MS with HPLC and other separation techniques for identification of carotenoids and related compounds. Curr. Anal. Chem. 5, 1-25 (2009).
Řezanka T., Prell A., Sigler K.: Identification of odorous compounds from nine fermentor-cultivated Streptomyces strains. Folia Microbiol. 53, 315-318 (2008).
Řezanka T., Řezanka P., Sigler K.: A biaryl xanthone derivative having axial chirality from Penicillium vinaceum. J. Natl. Prod. 71, 820-823 (2008).
Řezanka T., Řezanka P., Sigler K.: Glycosides of arylnaphthalene lignans from Acanthus mollis having axial
chirality. Phytochem. 70, 1049-1054 (2009).
Řezanka T., Řezanka P., Sigler K.: Glycosides of benzodioxole-indol alkaloids from Narcissus having axial
chirality. Phytochem. 2009 in press.
Řezanka T., Řezanka P., Sigler K.: Structural analysis of a polysaccharide from the green alga Chlorella kessleri
by means of gas chromatography - mass spectrometry of its saccharide alditols. Folia Microbiol. 52, 246252 (2007).
Řezanka T., Sigler K.: Antiviral sesqui-, di- and sesterterpenoids. Anti-Infect. Agents in Medicinal Chem. 8, 169192 (2009).
Řezanka T., Sigler K.: Biologically active compounds of semi-metals, in: Studies in Natural Products Chemistry. (Ed. Atta-Ur-Rahman) Vol. 35, pp. 835-921 (2008).
Řezanka T., Sigler K.: Biologically active compounds of semi-metals. Phytochem. 69(3), 585-606 (2008).
Řezanka T., Sigler K.: Hirtusneanoside, a new unsymmetrical dimeric tetrahydroxanthone from the lichen Usnea
hirta. J. Natl. Prod. 70, 1487-1491 (2007).
Řezanka T., Sigler K.: Identification of eight-membered heterocycles - hicksoanes A-C - from the gorgonian
Subergorgia hicksoni. Eur. J. Org. Chem. 2008, 1265-1270 (2008).
Řezanka T., Sigler K.: Identification of very long chain unsaturated fatty acids from Ximenia oil by atmospheric
pressure chemical ionization liquid chromatography-mass spectroscopy. Phytochemistry 68, 925-934
(2007).
Řezanka T., Sigler K.: Odd-numbered very long chain fatty acids in the microbial, plant and animal kingdoms.
Progr. Lipid Res. 48, 206-238 (2009).
Řezanka T., Sigler K.: Sinaicinone, a complex adamantanyl derivative from Hypericum sinaicum. Phytochemistry 68, 1272-1276 (2007).
Řezanka T., Sigler K.: Sterols and triterpenoids with antiviral activity. Anti-Infect. Agents in Medicinal Chem. 8,
193-210 (2009).
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Řezanka T., Sigler K.: The use of atmospheric pressure chemical ionization mass spectrometry with high performance liquid chromatography and other separation techniques for identification of triacylglycerols.
Curr. Anal. Chem. 3, 252-271 (2007).
Řezanka T., Siřišťová L., Melzoch K., Cajthaml T., Sigler K.: N-acylated bacteriohopanehexol-mannosamides
from the thermophilic bacterium Alicyclobacillus acidoterrestris. Eur. J. Org. Chem.2009, in press.
Řezanka T., Siřišťová L., Melzoch K., Sigler K.: Identification of (S)-11-cycloheptyl-4-methylundecanoic acid
in acylphosphatidylglycerol from Alicyclobacillus acidoterrestris. Chem. Phys. Lipids 159, 104-113
(2009).
Řezanka T., Sobotka M., Prell A., Sigler K.: Relationship between volatile odor substances and production of
avermectins by Streptomyces avermitilis. Folia Microbiol. 52, 26-30 (2007).
Řezanka T., Spížek J., Sigler K.: Medicinal use of lincosamides and microbial resistance to them. Anti-Infective
Agents in Medicinal Chem. 6, 133-144 (2007).
Řezanka T., Syřišťová L., Melzoch K., Sigler K.: Direct ESI-MS analysis of O-acyl glycosylated cardiolipins
from the thermophilic bacterium Alicyclobacillus acidoterrestris. Chem. Phys. Lipids 161, 115-121
(2009).
Řičicová M., Kučerová H., Váchová L., Palková Z.: Association of putative ammonium exporters Ato with
detergent-resistant compartments of plasma membrane during yeast colony development: pH affects
Ato1p localisation in patches. Biochim. Biophys. Acta-Biomembranes, 1768:1170-8 (2007).
Sigler K., Matoulková D., Dienstbier M., Gabriel P.: Net effect of wort osmotic pressure on fermentation course,
yeast vitality, beer flavor and haze. Appl. Microbiol. Biotechnol. 82, 1027-1035 (2009).
Sigler K., Matoulková D., Gabriel P., Dienstbier M., Gášková D.: Yeast and stress: from the laboratory to the
brewery. Kvasný průmysl 2009, in press.
Šavel J., Košin P., Brož A., Sigler K.: Convenient monitoring of the course of brewery fermentation by refractometry. Kvasný prům. 55, 94-99 (2009).
Škaloudová B., Zemek R., Křivan V.: The effect of predation risk on an acarine system. Anim. Behav. 74, 813821 (2007).
Váchová L., Chernyavskiy O., Strachotová D., Bianchini P., Burdíková Z., Ferčíková I., Kubínová L., Palková
Z.: Architecture of developing multicellular yeast colony: spatio-temporal expression of Ato1p ammonium exporter. Environ. Microbiol. 11, 1866-1877 (2009).
Váchová L., Kučerová H., Devaux F., Úlehlová M., Palková Z.: Metabolic diversification of cells during the
development of yeast colonies. Environ. Microbiol. 11, 494-504 (2009).
Váchová L., Palková Z.: Caspases in yeast apoptosis-like death: facts and artefacts. FEMS Yeast Res. 7,12-21
(2007).
Vopálenská I., Šťovíček V., Janderová B., Váchová L., Palková Z.: Role of distinct dimorphic transitions in
territory colonizing and formation of yeast colony architecture. Environ. Microbiol, in press;
doi:10.1111/j.1462-2920.2009.02067.x. (2009).
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Laboratory 125
MOLECULAR BIOLOGY OF BACTERIAL PATHOGENS
Head
Peter Šebo, PhD.
Scientific staff:
Irena Adkins, PhD.
Ladislav Bumba, PhD.
Jana Holubova-Hejnová, PhD.
Irena Linhartová, PhD.
Jiří Mašin, PhD.
Radim Osička, PhD.
Marek Basler, PhD.
Part-time lab members
Ivo Konopásek, PhD., Assoc.Prof., Charles University,
Faculty of Science
Radek Fišer, MSc., Charles University, Faculty of Science
Marcela Šimšová, PhD.
Technical staff
Hana Kubínová
Ilona Krupičková
Soňa Charvátová
Doctoral students
Jawid N. Ahmad, Jana Kamanová, MSc., Martina Kosová, MSc., Zuzana Marčeková,
MSc., Lenka Sadílková, MSc., Ondřej Staněk, MSc., Tomáš Wald, MSc.
The team worked on topics related to molecular mechanisms of host-pathogen interactions and mechanisms of action of bacterial protein toxins. The main research focus was on
(i) analyzing structure-function relationships and mechanisms underlying the action and target
cell penetration of the adenylate cyclase toxin (ACT) of Bordetella pertussis; (ii) mechanisms
of cAMP signaling underlying subversion of host phagocyte functions by ACT and (iii)
design and exploitation of a genetically detoxified form of ACT (dACT) as a novel “molecular injection device” for delivery of antigens into dendritic cells.
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Further research projects comprised (iv) studies on the use of the self-processing module of the RTX protein FrpC from Neisseria meningitidis as a novel self-processing affinity
purification tag for recombinant antigens. More recently, we initiated a project aiming at
(v) design and evaluation of a novel antigen delivery tools for detection of latent infections
and (vi) design of tools for use in nanoimmunosensors for cytokines.
Molecular mechanisms and structure–function relationships underlying the subversive
action of Bordetella pertussis adenylate cyclase toxin (ACT) on myeloïd phagocytic cells
Bordetella ACT is an RTX family leukotoxin that binds the αMβ2 integrin CD11b/CD18
(Mac-1 or CR3) and penetrates into the cytosol of myeloïd phagocytic cells, such as macrophages and neutrophils or dendritic cells. There ACT binds calmodulin and catalyzes dissipation of ATP into cAMP, paralyzing bactericidal capacities of host phagocytes. In parallel, the
toxin permeabilizes cell membrane by forming small cation-selective membrane channels
capable of causing cell lysis.
We have shown that the toxin interacts with its receptor by primarily recognizing Nlinked oligosaccharide glycan chains of the CD11b/CD18 heterodimer. Moreover, we could
show that this represents a paradigm for interaction of the whole family of RTX cytolytic
toxins with their β2 integrin receptors.
Previously, we dissected the complex mechanism of ACT interaction with the
CD11b/CD18 receptor and the ensuing cytotoxic action of ACT into individual contributions
of its enzymatic and channel-forming activities by using a battery of ACT mutants with selective defects in distinct steps of toxin action. Analyzing the impact of the mutations in the
pore-forming domain of ACT, residues playing a central role in membrane translocation of
the AC domain into cells (Glu509 and Glu570) and in controlling the size (Glu516), ion selectivity (Glu570) and half-life (Glu581) of ACT channels were identified.
Furthermore, a third biological activity of ACT was discovered, consisting in the capacity of its AC domain to participate in formation of a novel and transiently opened path for
calcium ion influx into cells. Curiously enough, we could show that this was not due to cAMP
signalig of the toxin or its pore-forming activity, where the calcium influx was found to occur
concomitantly with AC domain polypeptide insertion and translocation across the cytoplasmic membrane of cells.
The capacity to promote ACT-mediated calcium influx into cells has now been shown
to play a pivotal role in toxin penetration across membrane, allowing its mobilization into the
cholesterol-enriched membrane microdomains.
In a parallel project, focusing on the mechanism by which toxin-induced cAMP signaling interferes with phagocytic functions of macrophages and dendritic cells, we could show
that low ACT doses provoke rapid and unproductive, subversive, actin cytoskeleton rearrangements, which cause massive ruffling of myeloïd cell membrane (Fig. 19). This was
shown to result from transient and cAMP-signaled inactivation of the small GTPase protein
RhoA, perturbing the regulatory circuitry of the actin cytoskeleton hoemostasis. As a result,
ACT action almost instantaneously ablates the complement-mediated phagocytic capacity of
cells, disabling a key defense mechanism of the innate immune system of naïve hosts and
likely facilitating bacterial colonization.
ACT as a ‘molecular syringe’ for vaccine delivery into dendritic cells
Our laboratory has over the years contributed to the establishment of the genetically detoxified dACT as a potent novel and non-replicative carrier capable of delivering passenger
antigens into dendritic cells for processing and presentation on MHC molecules. As dACT
can target both MHC class I and II-restricted pathways in parallel, thereby allowing simulta-
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neous induction of antigen-specific CD8+ cytotoxic (CTL) and CD4+ T lymphocyte immune
responses, this could be exploited for both preventative as well as immunotherapeutic experimental vaccination against viruses and tumor growth. Recently, prime/boost immunization with dACT and live attenuated Salmonella vaccines delivering circumsporozoïte antigen
was shown to induce potent and specific Th1-polarized CD8+ T-lymphocyte immune responses against Plasmodium berghei that could be shown to protect mice against a lethal
challenge by the parasite.
Fig. 19. ACT induces pronounced transient alteration of macrophage cytoskeleton and cell morphology that manifests as massive membrane ruffling of macrophages in resonse ro cytosolic cAMP elevation by the toxin. J774A.1 cells were treated with CyaA (10 ng/ml) for 5 minutes fixed by 4 % paraformaldehyde , stained for F-actin with FITC-phalloidin (green) and examined with Olympus BX60
fluorescence microscope. Cell nuclei were stained blue with DAPI.
Publications
Basler M., Knapp O., Masin J., Fiser R., Maier E., Benz R., Sebo P., Osicka R.: Segments crucial for membrane
translocation and pore-forming activity of Bordetella adenylate cyclase toxin. J. Biol. Chem. 282, 1241912429 (2007).
Connell T.G., Shey M.S., Seldon R., Ranggaka M.X., van Cutsem G., Simsova M., Marcekova Z., Sebo P.,
Curtis N., Diwakar L., Meintjes G.A., Leclerc C., Wilkinson R.J., Wilkinson K.A.: Enhanced Ex vivo
stimulation of Mycobacterium tuberculosis-specific T cells in HIV1-infected persons via antigen delivery
by the Bordetella pertussis adenylate cyclase vector. Clin. Vacc. Immunol. 14, 847-854 (2007).
Fiser R., Masin J., Basler M., Krusek J., Spulakova V., Konopasek I., Sebo P.: A third activity of Bordetella
adenylate cyclase toxin-hemolysin: Membrane translocation of AC domain polypeptide promotes calcium
influx into CD11b+ monocytes independently of the catalytic and hemolytic activities. J. Biol. Chem.
282, 2808-2820 (2007).
Kamanova J., Kofronova O., Masin J., Genth H., Vojtova J., Linhartova I., Benada O., Just I., Sebo P.: Adenylate cyclase toxin subverts phagocyte function by RhoA inhibition and unproductive ruffling. J. Immunol. 181, 5587-5597 (2008).
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Knapp O., Maier E., Masin J., Sebo P., Benz R.: Pore formation by the Bordetella adenylate cyclase toxin in
lipid bilayer membranes: Role of voltage and pH. BBA Biomembr. 1778, 260-269 (2007).
Marcekova, Z., Psikal, I., Kosinova, E., Benada, O., Sebo, P., Bumba L.: Heterologous expression of full-length
capsid protein of porcine circovirus 2 in Escherichia coli and its potential use for detection of antibodies.
J. Virol. Meth. 2009 Aug 5. [Epub ahead of print].
Morova J., Osicka R., Masin J., Sebo P.: RTX cytotoxins recognize β2 integrin receptors through N-linked
oligosaccharides. Proc. Natl. Acad. Sci. USA 105, 5355-5360 (2008).
Sadilkova L., Osicka R., Sulc M., Linhartova I., Novak P., Sebo P.: Single-step affinity purification of recombinant proteins using a self-excising module from Neisseria meningitidis FrpC. Protein Sci. 17, 1834-1843
(2008).
Sewald X., Gebert-Vogl B., Prassl S., Weiss E., Fabbri M., Osicka R., Schiemann M., Busch D.H., Semmrich
M., Holzmann B., Sebo P., Haas R.: CD18 is the T-lymphocyte receptor of the Helicobacter pylori
vacuolating cytotoxin. Cell Host & Microbe 3, 20-29 (2008).
Tartz S., Rüssmann H., Kamanova J., Sebo P., Sturm A., Heussler V., Fleischer B., Jacobs T.: Complete protection against P. berghei malaria upon heterologous prime/boost immunization using recombinant Salmonella and Bordetella adenylate cyclase vaccines. Vaccine 26, 5935-5943 (2008).
Vojtova-Vodolanova, J., Basler, M., Osicka, R., Knapp, O., Maier., E., Cerny, J., Benada, O., Benz, R., Sebo,
P.: Oligomerization is involved in pore formation by Bordetella adenylate cyclase toxin. FASEB J. 23,
2381-2843 (2009).
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Laboratory 126
BIOINFORMATICS
Head
Jiří Vohradský, PhD.
Scientific staff
Josef Pánek, PhD.
Jan Bobek, PhD.
Technician
Alice Ziková, Bc.
Doctoral students
Eva Straková, MSc., Aleš Ulrych, MSc.
The laboratory focuses on the computational biology of cell regulatory processes, employing the –omics methods, bioinformatics of protein and DNA sequences, and studies on
small noncoding RNAs.
Computational biology of cell regulatory processes
Our main objective is to analyze, model, and simulate regulatory processes in the cell,
and provide bioinformatics support for measured data. We develop computerized models of
regulation of gene expression and use statistics and artificial intelligence for analysis of proteomic and other large scale – omics data.
The computerized model of gene expression was based on the concept of genetic network which is in our case formalized to a principle of recurrent neural network. We assume
that the rate of protein expression of each individual protein is a result of combinatorial action
of all molecules which influence expression of the given gene. The influence of each molecule is expressed in a weight matrix. This principle has been formalized to a set of timedependent differential equations. Their solution allows for simulation of behavior of the given
system over time. Inverse problem – reconstruction of the weight matrix from experimentally
77
measured time series has been applied to the prediction of part of the genetic network controlling cell cycle in S. cerevisiae. The reconstructed weight matrix then allowed drawing of
mutual interaction maps among regulators and their target.
For the reconstruction of genetic networks from experimentally measured time series of
gene expression, we used a known part of genetic network as a training set. This partial network allowed extracting the principal features of the regulator-target gene interaction. These
features were coded to a kernel matrix using genetic programming and later used for prediction of interactions of knew genes with the genes of the training network. Such scheme allowed
an expansion of the training network by addition of other genes having the same principle of
interaction as the genes of the training network. This scheme proved to be successful in prediction of the network controlling cell cycle in S. cerevisiae.
The image of the current state of gene expression can be monitored mainly by transcriptomics and proteomics. We have focused on the proteomic analysis of gene expression during
development of streptomycetes (S. coelicolor), which are industrially important producers of
antibiotics with complex developmental cycle. We have been analyzing quantitative changes
in the proteome during different stages of development. We use proteomic and transcriptomic
data together with statistics and artificial intelligence for identification of global trends in
gene expression, principles of transition between different developmental phases, and identification of proteins and protein functional groups involved in the process.
Proteomic data have been stored in the proteomic server developed and maintained by
our lab, originally in collaboration with Biozentrum, Univ. Basel (http://proteom.biomed.
cas.cz). The server hosts the proteomic databases created in our department, and is equipped
with graphical interface and search engines which allow the retrieval of relevant information.
The databases are linked with other data sources as GeneBank, and KEGG metabolic pathway
server.
Protein sequence bioinformatics
A novel alignment-free method for computing functional similarity of membrane proteins was devised. The method was based on the features of hydropathy distribution. The
approach was applied to two classes of membrane proteins: secondary transporters and orphan secondary neurotransmitter transporters with uknown functions. In both cases, the approach predicted successfully function attributes of the transporters that allowed for
prediction of their functions. In general, the approach showed that analysis of hydropathy
distribution can be used for function prediction of membrane proteins.
Small noncoding RNAs
The first systematic study of small non-coding RNAs (sRNA, ncRNA) in Streptomyces
was conceived. This study was based on sequence conservation in intergenic regions of Streptomyces, localization of transcription termination factors, and genomic arrangement of genes
flanking the predicted sRNAs. Thirty-two potential sRNAs in Streptomyces were predicted.
Of these, expression of 20 was detected by microarrays and RT-PCR. The prediction was
validated by a structure-based computational approach. Two predicted sRNAs were found to
be terminated by transcription termination factors different from the Rho-independent terminators. One predicted sRNA was identified computationally with high probability as a Streptomyces 6S RNA. Out of the 32 predicted sRNAs, 24 were found to be structurally dissimilar
from known sRNAs. Streptomyces is the largest genus of Actinomyces, whose sRNAs have
not been studied. The Actinomyces is a group of bacterial species with unique genomes and
phenotypes. Therefore, in Actinomyces, new unique bacterial sRNAs may be identified. The
sequence and structural dissimilarity of the predicted Streptomyces sRNAs demonstrated by
this study serve as the first evidence of the uniqueness of Actinomyces sRNAs.
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In further study made in cooperation with the laboratory of Dr. Marie Elliot, McMaster
University, Canada, bioinformatic and experimental approach enabled the identification and
characterization of nine novel sRNAs in S. coelicolor, including a cis-encoded antisense
sRNA. We examined sRNA expression throughout the S. coelicolor developmental cycle,
which progresses from vegetative mycelium formation, to aerial mycelium formation and
finally sporulation. We further determined the effects of growth medium composition (rich
versus minimal medium) on sRNA gene expression, and compared wild-type sRNA expression profiles with those of four developmental mutants. All but two of the sRNAs exhibited
some degree of medium dependence, with three sRNAs being expressed exclusively during
growth on one medium type. Unlike most sRNAs characterized thus far, several sRNA genes
in S. coelicolor were expressed constitutively (except for during late sporulation), suggesting
a possible housekeeping role for these transcripts. Others were expressed at specific developmental stages, and their expression profiles were altered in response to developmental mutations.
Transfer-messenger RNA (tmRNA) is a bacterial RNA having both tRNA and mRNA
properties and playing an essential role in recycling of ribosomes that are stalled on defective
mRNA. Streptomycetes undergo complex developmental cycle and are naturally exposed to
various physical and chemical stresses where the trans-translational system is thought to be
crucial for bacterial survival. A major challenge in understanding the regulation of the function of tmRNA is the definition of protein interactions. Proteins from various phases of development of Streptomyces aureofaciens associated with tmRNA were identified. Our data also
show that the ribosomal protein SS1 is required for translation of the tmRNA tag-reading
frame.
The lab is a member of the consortium ActinoGEN of the EU 6th framework program.
Publications
Mikulík K., Palečková P., Felsberg J., Bobek J., Zídková J., Halada P.: SsrA genes of streptomycetes and
association of proteins to the tmRNA during development and cellular differentiation. Proteomics 8, 14291441 (2008).
Palečková P., Bobek J. Mikulik K.: tmRNA of Streptomyces collinus and Streptomyces griseus during the
growth and in the presence of antibiotics. Microbial Biotechnol. 2, 114-122 (2009).
Palečková P., Felsberg J., Bobek J., Mikulík K.: tmRNA abundance in Streptomyces aureofaciens, S. griseus
and S. colinus under stress-inducing conditions. Folia Microbiol 52, 463-470 (2007a).
Palečková P., Kontrová F., Kofroňová O., Bobek J., Benada O., Mikulík K.: Effect of protein kinase inhibitors
on protein phosphorylation and germination of aerial spores from Streptomyces coelicolor. Folia Microbiol
52, 215-222 (2007b).
Pánek J., Bobek J., Mikulík K., Basler M., Vohradský J.: Biocomputational prediction of small non-coding
RNAs in Streptomyces. BMC Genomics 9, 217 (2008).
Pánek J., Eidhammer I., Aasland R.: Using hydropathy features for function prediction of membrane proteins.
Mol. Membr. Biol. 24, 304-312 (2007).
Pánek J.: A comparative computational analysis of protein sequences and literature mining classify 'orphan'
neurotransmitter transporters. J. Theor. Biol. 254, 301-307 (2008).
Swiercz J.P., Hindra, Bobek J., Haiser H.J., Di Berardo C., Tjaden B., Elliot M.A.: Small non-coding RNAs in
Streptomyces coelicolor. Nucleic Acids Res 36, 7240-7251 (2008).
To C.C., Vohradský J.: A parallel genetic algorithm for single class pattern classification and its application for
gene expression profiling in Streptomyces coelicolor. BMC Genomics 8 49 (2007).
To C.C., Vohradský J.: Supervised inference of gene-regulatory networks. BMC Bioinformatics 9(1), 2 (2008).
Vohradský J., Branny P., Li X.M., Thompson C.J.: Effect of protein degradation on spot M(r) distribution in
2-D gels--a case study of proteolysis during development of Streptomyces coelicolor cultures. Proteomics 8,
2371-2375 (2008).
79
Vohradský J., Branny P., Thompson C.J.: Comparative analysis of gene expression on mRNA and protein level
during development of Streptomyces cultures by using singular value decomposition. Proteomics 7), 38533866 (2007).
Vu T.T., Vohradsky J.: Inference of active transcriptional networks by integration of gene expression kinetics
modeling and multisource data. Genomics 93, 426-433 (2009).
Vu T.T., Vohradský J.: Nonlinear differential equation model for quantification of transcriptional regulation
applied to microarray data of Saccharomyces cerevisiae. Nucleic Acids Res. 35, 279-287 (2007).
80
Laboratory 127
CELL SIGNALING
Head
Pavel Branny, PhD.
Scientific staff
Linda Nováková, PhD.
Tomáš Vomastek, PhD.
Karel Mikulík, DSc.
Doctoral students
Jana Goldová, MSc., Zuzana Sušická, MSc., Miloslava Maninová, MSc.,
Aleš Ulrych, MSc.
ZuzanaVacková, Michaela Štechová, Eliška Malíková, Eva Kučerová
The research in the laboratory focuses on the molecular mechanisms that bacteria use
for intra- and intercellular communication. Our goal is to understand how bacteria detect
multiple environmental cues, and how the integration and processing of this information
provided by eukaryotic-type Ser/Thr protein kinases results in the precise regulation of gene
expression. Through reversible protein phosphorylation, protein kinases and phosphates
provide the fundamental machinery for environmental sensing and physiological signalling.
Second area of interest focuses on the role of extracellular signal regulated kinase (ERK)
cascade in regulation of different cellular processes.
A new signalling pathway in Streptococcus pneumoniae
involving Ser/Thr protein kinase StkP
We recently characterized the biochemical properties of both the protein kinase StkP
and the protein phosphatase PhpP of S. pneumoniae and showed that StkP and PhpP could
operate as functional pair in vivo. Analysis of phosphoproteome maps of both wild-type and
81
stkP null mutant strains revealed that one of StkP endogenous substrates is phosphoglucosamine mutase GlmM. Therefore, phosphorylation of GlmM by protein kinase StkP in S. pneumoniae could be a factor regulating the activation of GlmM and consequently the flow of
metabolites in the cell wall biosynthetic pathways.
In addition to GlmM, we have identified the α-subunit of RNA-polymerase (RNAP) as
another substrate of StkP in in vivo labeling experiments although the significance of its
modification is unclear.
Due to the transmembrane topology of StkP and the presence of an extracellular sensor
domain containing reiterated PASTA signature sequences (Penicillin-binding protein And
Ser/Thr protein kinase Associated domain), we hypothesized that StkP could transmit environmental cues into the cell. By using genetic and biochemical approaches we demonstrated
that both the transmembrane and extracellular domains are efficient dimerization motifs.
Analysis of growth characteristics of a S. pneumoniae stkP mutant under different stress
conditions revealed that stkP mutation confers a phenotype that is sensitive to many stressors
including elevated temperature, oxidizing agents, osmotic pressure, and acidic conditions.
These results suggest that expression of StkP can contribute to the resistance of S. pneumoniae to environmental stresses.
Fig. 20. Active ERK localization in migrating cell. Rat fibroblast cell was stained using
an antibody against active ERK (red) and focal adhesion marker Paxillin (green) and examined by immunofluorescence microscopy. Co-localization of active ERK with paxillin
in focal adhesions apear as yelow. In addition to focal adhesions active ERK displays
strong staining at the cell periphery and in the nucleus.
To identify the genes that are controlled by protein kinase StkP we examined the transcription profile of stkP loss-of-function mutant prepared by PCR ligation mutagenesis and
allelic exchange. This analysis revealed that the stkP mutation is broadly pleiotropic and
affects transcription of several sets of important genes. Several functional gene categories
have been identified that could account for a reduced stress response and attenuated virulence.
82
These genes are involved in the processes as different as cell wall biosynthesis, purine and
pyrimidine metabolism, iron uptake, antibiotics efflux, transcription regulation, and genetic
competence development. Furthermore, we show that StkP of S. pneumoniae plays a role in
the maintenance of low expression levels of competence genes under conditions that do not
support competence development. To identify the substrate(s) of protein kinase cultures of the
wild type as well as stkP null mutant strains were labelled in vivo with [33P]-orthophosphate
and soluble proteins were separated by two-dimensional gel electrophoresis
Functional role of the MAPK/ERK signaling pathway in mammalian cell proliferation
and migration (Supervisor T. Vomastek)
Our laboratory also works on mechanism by which mammalian cells respond to a broad
spectrum of extracellular signals and transmit these signals in a multitude of contextdependent responses. We focus on the role the extracellular signal regulated kinase (ERK)
cascade plays in the regulation of a diverse array of cellular programs including cell growth
and division, cell death, cell differentiation and cell movement. We are particularly interested
in ERK subcellular localization (Fig. 20) as an important regulatory element in directing ERK
activity towards specific targets.
Publications
Hercík K., Hášová V., Janeček J., Branny P.: Molecular evidence of Bartonella DNA in ixodid ticks in Czechia.
Folia Microbiol. 52, 503-509 (2007).
Mikulík K., Palečková P., Felsberg J., Bobek J., Zídková J., Halada P.: SsrA genes of streptomycetes and
association of proteins to the tmRNA during development and cellular differentiation. Proteomics 8,
1429-1441 (2008).
Pallová P., Hercík K., Sasková L., Nováková L., Branny P.: A eukaryotic-type serine/threonine protein kinase
StkP of Streptococcus pneumoniae acts as a dimer in vivo. Biochem. Biophys. Res. Commun. 355, 526530 (2007).
Pánek J., Bobek J., Mikulík K., Basler M., Vohradský J.: Biocomputational prediction of small non-coding
RNAs in Streptomyces. BMC Genomics 9, 217 (2008).
Sasková L., Nováková L., Basler M., Branny P.: Microarray-based identification of genes regulated by Ser/Thr
protein kinase StkP of Streptococcus pneumoniae. J. Bacteriol. 189, 4168-4179 (2007).
Vohradský J., Branny P., Li X.M., Thompson C.J.: Effect of protein degradation on spot M(r) distribution in 2D gels – a case study of proteolysis during development of Streptomyces coelicolor cultures. Proteomics
8, 2371-2375 (2008).
Vohradský J., Branny P., Thompson C.J.: Comparative analysis of gene expression on mRNA and protein level
during development of Streptomyces cultures by using singular value decomposition. Proteomics 7, 38533866 (2007).
Vomastek T., Iwanicki M.P., Burack W.R., Tiwari D., Kumar D., Parsons J.T., Weber M.J., Nandicoori V.K.:
Extracellular signal-regulated kinase 2 (ERK2) phosphorylation sites and docking domain on the nuclear
pore complex protein Tpr cooperatively regulate ERK2-Tpr interaction. Mol. Cell. Biol. 28, 6954-66
(2008).
Vomastek, T., Iwanicki, M., Schaeffer, H-J., Tarcsafalvi, A., Parsons, J.T., Weber M.J.: RACK1 targets the
ERK/MAP kinase pathway to link integrin engagement with focal adhesion disassembly and cell motility.
Mol. Cell. Biol. 27, 8296-8305 (2007).
83
Laboratory 128
REGULATION OF GENE EXPRESSION
Head
Leoš Valášek, PhD.
Technical staff
Olga Krýdová
Doctoral students
Lucie Cuchalová, MSc., István Dányi, MSc., Anna Herrmannová MSc.,
Martina Janošková, MSc., Tomáš Kouba, MSc., Vanda Munzarová, MSc.,
Susan Wagner, MSc.
Translation is one of the fundamental processes of cell biology and as such its regulation represents a critical aspect of cellular homeostasis. Translational control is the key regulatory mechanism of gene expression mainly under conditions where transcriptional
regulation cannot act promptly, for example during initial response to stresses. Since most
regulation occurs at the initiation stage, our lab has a long-term interest in uncovering molecular details of functions of several initiation factors including eIF3 during general translation initiation and of their contribution to translational control.
The N-terminal domain of eIF3a promotes 40S-binding by eIF3 and is critically required for reinitiation
Translation initiation in eukaryotes is masterminded by numerous proteins called eukaryotic initiation factors (eIFs). Among them, eIF3 is the most complex factor composed of
6 subunits in the yeast S. cerevisiae (Fig. 21). Given such a complexity, it is not surprising
that eIF3 was demonstrated to promote nearly all initiation steps including recruitment of the
eIF2•Met-tRNAiMet•GTP ternary complex (TC) and mRNA to the 40S ribosomal subunit
(40S) as well as the following scanning and AUG recognition processes (Fig. 22A–D). All of
these activities are facilitated by other eIFs that make direct contacts with eIF3 (Fig. 21).
84
In our study published last year in Genes & Development, we demonstrated that the Nterminal domain (NTD) of yeIF3a forms an important intermolecular bridge between eIF3
and the 40S, most probably via its previously identified binding partner in the small ribosomal
protein RPS0A. RPS0A is located near the mRNA exit channel where the main body of eIF3
was proposed to reside. Strikingly, we also found that deletion of the NTD of yeIF3a severely
blocked the up-regulation of GCN4 translation that occurs via reinitiation (REI).
Fig. 21. A 3-D model of the Multifactor complex composed of eIFs 3, 1, 5, and the ternary complex
(eIF2•Met-tRNAiMet•GTP) based on a comprehensive analysis of the mutual subunit interactions. The
labeled protein subunits are shown roughly in proportion to their molecular weights. The degree of
overlap between two different subunits depicts the extent of their interacting surfaces. The segments
with solid shading represent the domains proposed to play a critical role in eIF3-association with the
40S ribosome. ntd, N-terminal domain; ctd, C-terminal domain; hld, HCR1-like domain; rrm, RNA
recognition motif.
Fig. 22. A yeast model for eukaryotic reinitiation following translation of a short uORF. (A–B) eIF3
association with the scanning 48S PIC is stabilized by supporting contacts with eIFs 1, 1A, 5 and the
TC. (C) Upon subunit joining, eIF3 (and possibly also eIF4F) remains bound to the 80S ribosomes
owing to its strategic position on the solvent-exposed side of the 40S ribosome and the contacts that it
makes with 40S ribosomal components (e.g. yeIF3a-NTD with RPS0A) and presumably also with
mRNA. (D) During the first few rounds of elongation, weakly associated eIF3 gradually dissociates
from the 80S as a function of length and complexity of the translated region. (E–F) After translation of
a short uORF, certain proportion of 80S ribosomes terminating at its stop codon still contains eIF3, the
presence of which is required for resumption of scanning. (E and G) Binding of the yeIF3a-NTD
directly to the specific 5´ enhancer of uORF1 greatly stabilizes association of the post-termination 40S
subunit with mRNA following dissociation of the 60S subunit in the first stage of the ribosome recycling reaction and thereby promotes efficient resumption of scanning and REI downstream. (F and H)
Absence of the stimulatory 5´ enhancer, for example at uORF4, results in completion of ribosomal
recycling by the majority of terminating 80S ribosomes.
85
86
GCN4 is the transcriptional master-regulator of hundreds of starvation-inducible genes.
REI is a mechanism that utilizes short upstream ORFs (uORFs) to down- or up-regulate translation of regulatory proteins such as proto-oncogenes etc. Ribosomes initiate in the normal
way at the first AUG codon, however, at the termination codon where the 60S subunit dissociates, the 40S subunit remains bound to the mRNA, resumes scanning, and initiates again at
a downstream start site.
Detailed genetic analysis revealed that the NTD of yeIF3a functionally interacts with
the specialized 5’ feature occurring only with the first of four uORFs in the GCN4 mRNA
leader that greatly enhances the efficiency of REI for this specific uORF1. This interaction
occurs near the mRNA exit channel when the 80S ribosome is positioned at the uORF1 stop
codon and serves to stabilize association of the post-termination 40S subunit with mRNA
following dissociation of the 60S subunit. Thereby it critically promotes efficient resumption
of scanning for REI downstream (Fig. 22E–H).
How do eIF3j and its conserved binding partner eIF3b affect AUG recognition?
yeIF3j was shown to stimulate eIF3 binding to the 40S and to directly interact with the
RNA-recognition motif (RRM) of yeIF3b. In collaboration with P. J. Lukavsky (MRC, Cambridge), who recently solved the NMR structure of the human eIF3b-RRM, we set out to
examine the long-standing proposal that eIF3j coordinates binding of mRNA and other eIFs
within the ribosomal decoding center.
We first solved the NMR solution structure of the interaction between the human eIF3bRRM and eIF3j. In fact, this is the first structural insight into molecular interactions within
eIF3 from any organism. With help of yeast genetics and molecular biology we then demonstrated that both proteins closely co-operate on the ribosome together with the master AUG
decoder eIF1A to ensure proper establishment of the scanning-arrested conformation required
for stringent AUG recognition. This study is currently under review in Molecular and Cellular
Biology.
Characterization of the two smallest core subunits of eIF3 and their roles in translation
yeIF3i and yeIF3g subunits are essential in yeast, however, there is no information regarding their cellular functions with the exception of the fact that neither yeast nor mammalian i and g are required for two hallmark functions of eIF3 in recruitment of the TC and
mRNA to the 40S. To learn what essential roles these two subunits perform, we decided to
subject them to systematic mutagenesis followed by genetic and biochemical analysis. We
revealed that yeIF3g might have a function not only during initiation but also during posttermination ribosomal recycling, the mechanism of which is only poorly understood.
What is the molecular mechanism of the yeIF3c involvement in AUG recognition?
The NTD of yeIF3c mediates eIF3-binding to eIFs 1 and 5 (Fig. 21) that we previously
implicated in ensuring the stringency of the AUG start codon selection. Mutagenic analysis of
the yeIF3c-NTD interactions led us to propose that the yeIF3c-NTD promotes efficient loading of eIF1 to its initial ‘landing’ site near the A-site of the 40S ribosome. Upon loading,
however, their interaction is swiftly dissolved and eIF1 is released to the P-site where it is
believed to promote formation of the scanning-conducive conformation together with eIF1A.
Publications
Groušl T., Ivanov P., Frýdlová I., Vašicová P., Janda F., Vojtová J., Malínská K., Malcová I., Nováková L.,
Janošková D., Valášek L., Hašek J.: Robust heat shock induces eIF2α-phosphorylation-independent assembly of stress granules containing eIF3 and 40S ribosomal subunits in budding yeast S. cerevisiae.
J. Cell Sci. 122, 2078-88 (2009).
87
Nielsen K.H., Valášek L.: In vivo deletion analysis of the architecture of a multi-protein complex of translation
initiation factors. Methods Enzymol. 431, 15-32 (2007).
Szamecz B., Rutkai E., Cuchalová L., Munzarová V., Herrmannová A., Nielsen K.H., Burela L., Hinnebusch
A.G., Valášek L.: eIF3a cooperates with sequences 5´ of uORF1 to promote resumption of scanning by
post-termination ribosomes for reinitiation on GCN4 mRNA. Genes & Dev. 22, 2414-2425 (2008).
Valášek L., Szamecz B., Hinnebusch A.G., Nielsen K.H.: In vivo stabilization of pre-initiation complexes by
formaldehyde cross-linking. Methods Enzymol. 429, 163-183 (2007).

division of cell and molecular microbiology

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