Download Untitled - Biologie in Kaiserslautern

content
Bad Bergzabern- map ................................................................................................................. 3
Program ...................................................................................................................................... 4
Poster Presentations.................................................................................................................... 8
Oral Presentations-Abstracts .................................................................................................... 12
Special Lectures .................................................................................................................... 12
Session 1: Quorum Sensing .................................................................................................. 14
Session 2: Regulation of Metabolism ................................................................................... 16
Session 3: Second Messengers, bacterial differentiation, and antibiotic production ........... 22
Session 4: Regulation by RNA ............................................................................................. 25
Session 5: Regulation in Biofilms ........................................................................................ 27
Session 6: Stress responses and Signal Transduction ........................................................... 29
Session 7: Diverse Regulators .............................................................................................. 34
Poster Presentations-Abstracts ................................................................................................. 39
2
Bad Bergzabern- map
3
Program
Wednesday, 28.09. 2016
12:00 – 14:00 Arrival and Registration
14:30
Welcome
Session 1: Quorum Sensing
Chair:
14:45
Ralf Heermann, Ludwig-Maximilians-Universität München
Silent chats - Communication among entomopathogenic Photorhabdus
bacteria.
15:25
Nina Jagmann; Universität Münster
Quorum sensing and pyocyanin production by Pseudomonas aeruginosa in a
co-culture with Aeromonas hydrophila are co-regulated by the stringent
response and other metabolic influences
15:45
coffee break
Session 2: Regulation of Metabolism
16:10
Fabian Commichau, Universität Göttingen
The Bacillus subtilis glutamate dehydogenases RocG and GudB play a double
game.
16:50
Dominik Tödter, Universität Göttingen
The interplay between an Asp23 protein family member and acetyl-CoA
carboxylase in Bacilus subtilis
17:10
Miriam Dormeyer, Universität Göttingen
Compensation for glutamate auxotrophy of a Bacillus subtilis gltC mutant
by three independent mutational events
17:30
Andreas Schwentner, Universität Stuttgart
Metabolic engineering to direct evolution in Corynebacterium glutamicum
17:50
Bingyao Zhu, Universität Göttingen
SubtiWiki, an integrated database for model organism Bacillus subtilis
18:10
Jörg Stülke, Universität Göttingen
Large-scale reduction of the Bacillus subtilis genome: Consequences for the
transcriptional network, resource allocation, and metabolism
4
18:30
Dinner
20:00
Special Lecture I
Josef Deutscher, Unité Expression Génétique Microbienne, CNRS, Paris
The role of PTS components in catabolite repression of Listeria
monocytogenes virulence genes
21:00
Poster and Wine
Thursday, 29.09.2016
Session 3: Second Messengers, bacterial differentiation, and antibiotic
production
8:30
Natalia Tschowri, Humboldt-Universität Berlin
Cyclic di-nucleotide signalling in bacterial differentiation and antibiotic
production
9:10
Ilka Bischofs, Universität Heidelberg
Spore memory couples entry and exit from bacterial dormancy in Bacillus
subtilis.
9:30
Carsten Volz, Helmholtz-Institut für Pharmazeutische Forschung,
Saarbrücken
Regulation of Secondary Metabolite Gene Clusters in Social Myxobacteria
9:50
coffee break
Session 4: Regulation by RNA
10:20
Kai Papenfort, Ludwig-Maximilians-Universität München
From Strings of Nucleotides to Collective Behavior: Lessons from Vibrio
cholerae
11:00
Bernhard Remes, Universität Gießen
Surprising small RNA features in Rhodobacter sphaeroides
Session 5: Regulation in Biofilms
Chair:
11:20
Ákos T. Kovács, Universität Jena
Exploitation of the biofilm matrix to colonize a surface
12:00
Ramses Gallegos-Monterrosa, Universität Jena
Cell-cell communications in Bacillus subtilis mixed-species biofilms
12:30
Lunch
5
14:00
Excursion to Burg Berwartstein, Erlenbach
18:15
Dinner
20:00
Special Lecture II
t
Erhard Bremer, Universität Marburg
Osmotic forces at work – Stress responses to the front!
21:00
Poster / Flammkuchen/ New Wine
Friday, 30.09.2016
Session 6: Stress Responses and Signal Transduction
Chair:
8:30
Geraldine Laloux, Université catholique de Louvain, Brussels,
Connecting cell wall homeostasis and a major envelope stress response in
Escherichia coli
9:10
Emina Ćudić, Universität Osnabrück
The Cpx-system of Escherichia coli analyzed by SRM and Superresolution Microscopy
9:30
Bianca Warmbold, Universität Marburg
Uncovering the regulatory circuits of the glycine betaine synthesizing pathway
in Bacillus subtilis
9:50
Diana Wolf, Technische Universität Dresden
Characterization of the ABC-Transporter Associated Two-Component System
YxdJK in Bacillus subtilis as Biosensor for Eukaryotic Antimicrobial Peptides
10:10
coffe break
Session 7: Diverse Regulators
Chair:
10:30
Kim Julia Kraxner, Forschungszentrum Jülich
A new piece in the big puzzle of cell division: The transcriptional regulator
FtsR regulates FtsZ in Corynebacterium glutamicum
10:50
Eugen Pfeifer, Forschungszentrum Jülich
Silencing of cryptic prophages in Corynebacterium glutamicum
11:10
Aathmaja A. Rangarajan, Universität Köln
Inverse correlation between the transcription
repression in Escherichia coli
rate
and
H-NS/StpA
6
11:30
Susann M. Fragel, Universität Köln
Characterization of structural features controlling activity of LeuO, a
pleiotropic transcriptional regulator and H-NS antagonist
11:50
Hannes Breddermann, Universität Köln
Feedback control of leuO encoding a pleiotropic regulator and H-NS
antagonist in Escherichia coli
12:10
Poster Price, Concluding Remarks
12:30
Farewell lunch
7
Poster Presentations
Poster 1:
Computational prediction of the regulatory interactions for an ECF sigma factor group
with fused C-terminal domain and co-factor
Hao Wu and Georg Fritz.
Poster 2:
Implementation, analysis and mathematical modeling of ECF sigma factor-based
synthetic gene cascades in E.coli and B.subtilis
Marco Mauri1, Daniela Pinto2, Stefano Vecchione1, Hao Wu1, Thorsten Mascher2, Georg
Fritz1.
Poster 3:
Modulation of the behaviour of an Extracytoplasmic Function sigma factor (ECF)-based
genetic switch
Daniela Pinto1, Franziska Dürr1, Dayane Araújo1,2, Qiang Liu1,2 and Thorsten Mascher1.
Poster 4:
Theoretical models and FRET-assays for signal transduction via switchable allosteric
modulator proteins (SAMPs) in Bacillus subtilis
Heiko Babel and Ilka B. Bischofs.
Poster 5:
Regulation of gene expression by small sRNA
Dipl.-Ing. Lyubov Kyselova
Poster 6:
Regulatory interactions between Corynebacterium glutamicum and the prophage CGP3
Max Hünnefeld, Eugen Pfeifer and Julia Frunzke.
Poster 7:
Deciphering the Response of Corynebacterium glutamicum to Oxygen Deprivation
Julian Lange1, Tobias Busche2, Jörn Kalinowski2, Ralf Takors1, Bastian Blombach1
Poster 8:
From substrate specificity to promiscuity: molecular analysis of a hybrid ABC
transporter
L. Teichmann, C. Chen & E. Bremer
Poster 9:
The cellular function and localization of the cyclic-di-GMP phosphodiesterase PdeL
Cihan Yilmaz and Karin Schnetz
Poster 10:
Elucidation of the metabolic pathway for SDS degradation and its regulation in
Pseudomonas aeruginosa
Gianna Panasia and Bodo Philipp.
8
Poster 11:
The role of the phosphodiesterase NbdA in NO-induced dispersal of Pseudomonas
aeruginosa
Martina Rüger1, Sabrina Heine2, Michael Entian2, Yi Li3, Karin Sauer3 and Nicole
Frankenberg-Dinkel1, 2
Poster 12:
Osmotic responsive transcription of ectoine biosynthetic genes from Pseudomonas
stutzeri is transferable to a non-ectoine producing surrogate host
Laura Czech, Philipp Hub, Florian Kindinger, Oliver Dähn, Nadine Stöveken & Erhard
Bremer
Poster 13:
A special role for acetate kinase AckA in the regulation of CiaR activity in the absence
of the cognate kinase CiaH.
Anne Sexauer and Reinhold Brückner.
Poster 14:
Transport and regulation by the alternative anaerobic C4-dicarboxylate-transporters
DcuA, DcuB and DcuC in Escherichia coli
Alexander Strecker and Gottfried Unden
Poster 15:
The DxxxQ phosphatase motif in the O2 sensor kinase NreB of Staphylococcus carnosus
Ann-Katrin Kretzschmar and Gottfried Unden
Poster 16:
The function of the ExxN motif of the C4-dicarboxylate sensor kinase DcuS of
Escherichia coli in signal transduction
Stefaniya Gencheva, Sebastian Wörner and Gottfried Unden
Poster 17:
Analysis of quinone mutants in respect to ArcA phosphorylation and product formation
Annika Nitzschke and Katja Bettenbrock
Poster 18:
Analysis of the signal transduction by the heme-based sensor kinase MsmS from
Methanosarcina acetivorans
Fiege, K.1,2, Molitor, B.2, Blasius, L.1, Querebillo, C.3,4, Hildebrandt, P.3, Laurich, C.5, Lubitz,
Poster 19:
A TCS is involved in the regulation of the organohalide respiration in Sulfurospirillum
spp.
Jens Esken1, Tobias Goris1, Cynthia Sharma2, Torsten Schubert1, and Gabriele Diekert1.
9
Poster 20:
Biochemical characterization of the iron responsive regulator RirA from
Dinoroseobacter shibae
Maren Behringer, Elisabeth Härtig and Dieter Jahn
Poster 21:
Adrenochrome – oxidation product of adrenaline and bacterial effector molecule
Charlotte Toulouse, Kristina Metesch, Pit Engling, Bernd Michel and Julia Steuber.
Poster 22:
Mechanism and function of non-standard circadian clock systems in cyanobacteria
Christin Köbler1, Anja Dörrich2, Anika Wiegard3, Annegret Wilde1
Poster 23:
Characteristics of a SoxR-based single cell NADPH biosensor in Escherichia coli
Alina Spielmann, Meike Baumgart and Michael Bott
10
11
Oral Presentations-Abstracts
Special Lectures
The role of PTS components in catabolite repression of Listeria monocytogenes virulence
genes
Josef Deutscher
Unité Expression Génétique Microbienne, Centre National de la Recherche Scientifique,
13, Rue Pierre et Marie Curie, F-75005 Paris, France
Listeria monocytogenes is a saprophyte well adapted to growth in the soil on decaying plants and
other organic material. For this purpose, L. monocytogenes contains a large number of carbohydrate
transport systems, which allow the bacterium to utilize the numerous carbon sources produced during
plant decay. However, this bacterium has a dual lifestyle, because it is also a foodborne human
pathogen causing the disease listeriosis (1). The infection process by L. monocytogenes has been
intensively studied. Infection usually occurs via the digestive tract after ingestion of contaminated
food. The pathogen is able to actively invade several types of human cells and to enter the
bloodstream, where it can cause sepsis and after crossing the blood-brain barrier meningitis. It can also
cross the blood-placental barrier and if women are infected during pregnancy, this can lead to abortion.
Infection by L. monocytogenes requires a set of virulence factors, which have been intensively studied.
These proteins allow the pathogen to adhere to and to enter into host cells via phagocytosis, to escape
from the phagocyte, to proliferate in the cytoplasm of the host cell, to move within the host cell and to
spread from one epithelial cell to another.
Most of the genes encoding these virulence factors are located on two pathogenicity islands. The
expression of these virulence genes is controlled by PrfA, a Crp-like transcription activator. A recent
study revealed that if the virulence genes would be expressed when the bacterium is roaming in the
environment, this would significantly reduce the fitness of L. monocytogenes and its competitiveness.
L. monocytogenes therefore developed mechanisms aimed at repressing the expression of its virulence
genes when it is present in the soil.
One of these mechanisms is based on sensing the temperature of the environment. It has been
observed that while the virulence genes are strongly expressed at 37°C, a relatively small shift to 30°C
already strongly reduces their expression and therefore the synthesis of the virulence factors. This
mechanism is based on a thermoswitch of a secondary structure formed by the RNA preceding the
prfA gene. At temperatures at 30°C or lower, formation of this secondary structure prevents the access
to the ribosome binding site preceding the prfA mRNA and hence synthesis of the transcription
activator of virulence genes.
A second virulence gene repression mechanism responds to the presence of efficiently metabolized
carbon sources. Inside host cells, L. monocytogenes encounters glycerol and some glucose-6-P as
carbon sources, which are less efficient than glucose and therefore allow the expression of its
virulence genes. However, when exposed to glucose, fructose, cellobiose or other efficiently utilized
carbon sources found in decaying plants, the virulence genes are strongly repressed. Repressing sugars
are taken up by the phosphoenolpyruvate:sugar phosphotransferase system (PTS), which
phosphorylates its substrates before they enter the cytoplasm. Components of the PTS are involved in
the general carbon catabolite repression mechanism. Certain components of a cellobiose-specific PTS
and their state of phosphorylation play also a role in L. monocytogenes virulence gene repression.
When an efficiently metabolized PTS sugar is present, the PTS components are dephosphorylated and
one of them specifically inhibits PrfA activity by a yet unknown mechanism. Deletion of this
component leads to a general relief from virulence gene repression by carbon sources.
References
12
1. Freitag, N.E., Port, G.C. and Miner, M.D. (2009) Listeria monocytogenes - from saprophyte to
intracellular pathogen. Nat. Rev. Microbiol. 7, 623-628.
Osmotic forces at work – Stress responses to the front!
Erhard Bremer
Laboratory for Microbiology, Department of Biology, Philipps-University Marburg, Karlvon-Frisch Str. 8,
D-35043 Marburg, Germany [[email protected]]
The development of a semi-permeable cytoplasmic membrane through which water can pass
freely, but ions, nutrients and waste products cannot, was a key event in the evolution of
microbial proto-cells. Changes in the external osmolarity will inevitably trigger water fluxes
along the osmotic gradient in or out of the microbial cell. As a consequence, the magnitude of
vital turgor will be affected and the ensuing osmotic stress will negatively impact cell growth
and integrity. Water influx at low external osmolarity will prompt a raise in turgor that
eventually results in cell rupture. Conversely, high external osmolarity will trigger water
efflux and thereby causes dehydration of the cytoplasm; growth will be arrested. It is obvious
that if these negative consequences of fluctuations in the external osmolarity would not be
counteracted, microbial cells will be placed under considerable strain and will eventually die.
I will present an overview of the stress reactions of the ubiquitously distributed Grampositive bacterium Bacillus subtilis, which allows the cells to survive exposures to either low
or high osmolarity souroundings. I will describe the synthesis pathways for compatible solutes
(L-proline and glycine betaine), the structure of importers for these osmostress protectants and
their operation at high salinity, and address the release of water-attracting ions and compatible
solutes via MscS- and MscL-type mechanosensitive channels upon an osmotic down-shock. I
will also focus on the genetics of osmostress-regulated gene expression. Finally, I will present
data connecting the osmotic stress responses of free-living cells with key regulatory factors
controlling biofilm formation.
It will become clear from my presentation that the B. subtilis cell has to engage into a
highly coordinated and systems-wide fight in order to survive osmotic fluctuations in its
varied habitats. These stress responses encompass the SigB-controlled general stress response,
adjustment processes that deal with management of water fluxes in or out of the cell, and
genetic and cellular amendments that will allow life of B. subtilis in a biofilm.
13
Session 1: Quorum Sensing
Silent chats - Communication among entomopathogenic Photorhabdus bacteria
Ralf Heermann
Ludwig-Maximilians-Universität München, Biozentrum, Bereich Mikrobiologie,
Großhaderner Str. 2-4, 82152 Martinsried/München, Germany
email: [email protected]
It is well understood that bacteria communicate to coordinate their behaviour, a process
termed quorum sensing (QS). Thereby, bacteria do not use voice, but a silent way for
“chatting”: small diffusible molecules. The best understood way of bacterial communication
is the use of acylated homoserine lactones (AHLs) as “language”. The prototypical of AHL
using communication systems consists of a LuxI-like AHL synthase and a cognate LuxR-type
receptor that senses the signal. However, many proteobacteria lack any LuxI-type synthase,
and thus they cannot communicate via AHLs. Nevertheless, most of them have LuxR-type
receptors, which are referred to as LuxR orphans or solos. Entomopathogenic bacteria of the
genus Photorhabdus all harbor an extreme high number of LuxR solos, more than any other
bacteria known so far. We have identified two novel ways for bacterial communication in
Photorhabdus species to date, which the bacteria use for regulation of pathogenicity. P.
luminescens and P. temperata
-pyrones named photopyrones (PPYs)
instead of AHLs, which are produced by the pyrone synthase PpyS and recognized by the
LuxR solo PluR.[1] P. asymbiotica, a closely related insect and human pathogen, uses
dialkylresorcinols (DARs) for communication, which are produced by the DarABC pathway
and recognized by the LuxR solo PauR.[2] Upon sensing the specific signaling molecule, PluR
as well as PauR activate expression of the pcf operon in P. luminescens and P. asymbiotica,
respectively. This leads to cell clumping, which then contributes to the overall pathogenicity
of the bacteria against insects. The PpyS/PluR as well as the DarABC/PauR systems are the
first two examples of LuxR solo-based QS systems, which do not use AHLs as “language”.
Since the different PPYs and DARs derivatives activate the respective QS response with
different strength, these molecules as well as the different AHLs have been regarded as
bacterial “dialects”.[3] In summary, our studies reveal that bacterial “silent chats” go far
beyond AHL-signaling in nature.
References
[1] A.O. Brachmann, S. Brameyer, D. Kresovic, I. Hitkova, Y. Kopp, C. Manske, K.
Schubert, H.B. Bode, R. Heermann (2013). Pyrones as bacterial signaling molecules, Nature
Chem. Biol. 9(9):573-578.
[2] S. Brameyer, D. Kresovic, H.B. Bode, R. Heermann (2015). Dialkyresorcinols as bacterial
signaling molecules. PNAS 112(2):572-577.
[3] S. Brameyer, H.B. Bode, R. Heermann (2015). Languages and dialects: bacterial
communication beyond homoserine lactones. Trends Microbiol. 23(9):521-523.
14
Quorum sensing and pyocyanin production by Pseudomonas aeruginosa in a co-culture
with Aeromonas hydrophila are co-regulated by the stringent response and other
metabolic influences
Nina Jagmann and Bodo Philipp
Institute for Molecular Microbiology and Biotechnology, University of Münster,
Corrensstr. 3, D-48149 Münster, Germany
The opportunistic pathogen P. aeruginosa is a metabolically versatile bacterium that can
adapt to different environments because of a complex regulatory network that is involved in
sensing and responding to different environmental cues. Key regulatory elements of P.
aeruginosa are its quorum sensing (QS) systems that control the production of virulence
factors. Besides cell density, QS in P. aeruginosa is co-regulated by metabolic influences like
nutrient limitation (1).
Previously, we established a co-culture model system consisting of P. aeruginosa and the
chitinolytic bacterium A. hydrophila with chitin as sole growth substrate, in which parasitic
growth of P. aeruginosa is strictly dependent on the QS-controlled production of the
virulence factor pyocyanin (2). This redox-active metabolite inhibits the enzyme aconitase of
A. hydrophila through the formation of reactive oxygen species causing a block of the citric
acid cycle and, thus, a massive release of acetate by A. hydrophila, which supports substantial
growth of P. aeruginosa.
The stringent response is a regulatory mechanism mediated by the alarmone (p)ppGpp,
which leads to physiological adaptations to environmental stresses like nutrient deprivation.
We could show that activation of QS under co-culture conditions is dependent on the stringent
response as a relAspoT double mutant of P. aeruginosa did not produce pyocyanin in coculture anymore.
To identify further genes that are involved in the co-regulation of QS by metabolic
influences, we employed transposon mutagenesis and identified the gene gbuA encoding a
guanidinobutyrase (3). Deletion of gbuA leads to a loss of pyocyanin production in cocultures and to a reduced pyocyanin production in single cultures. This is likely caused by an
accumulation of 4-guanidinobutyrate (4-GB), the natural substrate of GbuA, as addition of 4GB to the mutant strain enhances the negative effect on pyocyanin production in single
cultures. The gbuA mutant shows a reduced transcription of the pqsABCDE operon, which is
part of the alkylquinolone-mediated QS system of P. aeruginosa controlling pyocyanin
production. Production of pyocyanin by the gbuA mutant can be restored by the addition of
alkylquinolone signal molecules and PqsE overexpression.
The strong effect of gbuA deletion on the QS-controlled pyocyanin production in cocultures shows the value of this approach for the discovery of novel gene functions linking
metabolism and QS in P. aeruginosa.
References
1. Mellbye, B. and Schuster, M. (2014) Physiological framework for the regulation of quorum
sensing-dependent public goods in Pseudomonas aeruginosa, J Bacteriol 196, 1155-1164
2. Jagmann, N., Brachvogel, H. P., and Philipp, B. (2010) Parasitic growth of Pseudomonas
aeruginosa in co-culture with the chitinolytic bacterium Aeromonas hydrophila, Environ
Microbiol 12, 1787-1802
3. Jagmann, N., Bleicher, V., Busche, T., Kalinowski, J., and Philipp, B. (2016) The
guanidinobutyrase GbuA is essential for the alkylquinolone-regulated pyocyanin production
during parasitic growth of Pseudomonas aeruginosa in co-culture with Aeromonas
hydrophila, Environ Microbiol, doi: 10.1111/1462-2920.13419.
15
Session 2: Regulation of Metabolism
The Bacillus subtilis glutamate dehydrogenases RocG and GudB play a double game
Fabian M. Commichau
Department of General Microbiology, University of Göttingen,
Grisebach-Str. 8, D-37077 Göttingen, Germany
Key metabolic intersections in the central metabolism of an organism have to be tightly
regulated to make the most out of nutrients that are available in a given environment. The
enzymatic reactions involved in glutamate synthesis and degradation constitute an important
metabolic intersection because they connect carbon to nitrogen metabolism in the Grampositive model bacterium Bacillus subtilis and in other organisms (1). During growth with the
carbon source glucose and ammonium as a source of nitrogen, the transcription factor GltC
activates the expression of the glutamate synthase genes. By contrast, in the absence of
glucose and in the presence of a source of glutamate, the glutamate synthase genes are not
expressed, and glutamate is converted to ammonium and 2-oxoglutarate of which the latter is
fed into carbon metabolism. Recently, we found that B. subtilis possesses the glutamate
dehydrogenases RocG and GudB that are both trigger enzymes, active in glutamate
degradation and in controlling gene regulation (2,3). We could show that the enzymes
function as sensors that depending on the intracellular glutamate concentration control
glutamate synthesis through an inhibitory interaction with the transcription factor GltC (2).
Here, I will discuss the recent findings on the control of a key metabolic intersection in B.
subtilis and provide an overview about the evolutionary stages of trigger enzymes.
References
1. Gunka, K. and Commichau, F.M. (2012) Control of glutamate homeostasis in Bacillus
subtilis: a complex interplay between ammonium assimilation, glutamate biosynthesis and
degradation. Mol Microbiol 85, 213-224.
2. Stannek, L., Thiele, M.J., Ischebeck, T., Gunka, K., Hammer, E., Völker, U., Commichau,
F.M. (2015) Evidence for synergistic control of glutamate biosynthesis by glutamate
dehydrogenases and glutamate in Bacillus subtilis. Environ Microbiol 17, 3379-3390.
3. Commichau, F.M., Stülke, J. (2015) Trigger enzymes: coordination of metabolism and
virulence gene expression. Microbiol Spectr 3, doi: 10.1128/microbiolspec.MBP-00102014
16
The interplay between an Asp23 protein family member and acetyl-CoA carboxylase in
Bacilus subtilis
Dominik Tödter and Jörg Stülke
Department of General Microbiology, University of Göttingen, Germany
Grisebachstr. 8, 37077 Göttingen
More than 20% of the proteins in the PFAM database belong to domain of unknown
function (DUF) families, indicating that a large fraction of proteins is in need of further
investigation. This is especially the case for the Asp23 protein family (DUF322). The name
giving alkaline shock protein 23 (Asp23) of Staphylococcus aureus is one of the most
abundant proteins and members of this family are both highly conserved and highly expressed
in Gram-positive bacteria (1). Despite the obvious importance of these proteins, almost
nothing is known about the functions of Asp23 proteins. The aim of our work is the
characterization of the so far unknown proteins YqhY and YloU, the representatives of the
Asp23 protein family in Bacillus subtilis. In contrast to the previously reported essentiality of
yqhY (2), we were able to delete the yqhY gene. The deletion of yqhY resulted in the rapid
acquisition of suppressor mutations that affect the subunits of the acetyl-CoA carboxylase
(AccABCD). This protein complex catalyzes the first committed step in fatty acid
biosynthesis, the conversion of acetyl-CoA to malonyl-CoA. The observed genetic link
between YqhY and the acetyl-CoA carboxylase as well as the location of the yqhY gene in the
strongly conserved accBC yqhY operon suggest an involvement of YqhY in fatty acid
synthesis. On the other hand, in some cases the suppressor mutations lead to the deletion of
ctsR, a transcription regulator of clpC, clpE, clpP and clpX. These genes encode the proteases
responsible for protein quality control by degrading unfolded or aggregated proteins. This
indicates a participation of YqhY in protein degradation as well. The results of ongoing
investigations about a possible interplay between the acetyl-CoA carboxylase, Clp-dependent
protein degradation and YqhY will be discussed.
References
1 Müller et al., 2014. Mol. Microbiology. 93: 1259–1268
2 Thomaides et al., 2007. J. Bacteriol. 189: 591-602.
17
Compensation for glutamate auxotrophy of a Bacillus subtilis gltC mutant by three
independent mutational events
Miriam Dormeyer, Anastasia L. Lübke and Fabian M. Commichau
Department of General Microbiology, University of Göttingen,
Grisebach-Str. 8, D-37077 Göttingen, Germany
Glutamate is the most abundant metabolite in all organisms and it fulfills a variety of
important functions such as the supply of nitrogen for anabolic reactions (1,2). The Grampositive model bacterium Bacillus subtilis can either use exogenous glutamate provided by
the medium or synthesize it from glutamine and 2-oxoglutarate using a glutamate synthase,
which is encoded by the gltAB genes (2). In the absence of exogenous glutamate, the LysRtype transcription factor GltC activates the expression of the GOGAT-encoding genes to meet
the need for glutamate, and to achieve high growth rates. Thus, it is not surprising that a gltC
mutant strain is auxotrophic for glutamate (3). Using a genetic screening system, we have
isolated several mutants that had acquired the ability to synthesize glutamate, independent of
GltC. Whole genome re-sequencing analyses revealed (i) mutations in the gltR gene, encoding
the LysR-type transcription factor GltR (ii) mutations in the promoter of the gltAB genes and
in a gene of unknown function, and (iii) massive amplification of the genomic locus
containing the gltAB genes. Thus, high genome flexibility is the key to relieve glutamate
auxotrophy of a B. subtilis gltC mutant. Currently, we are investigating how and to which
extent the various mutations enable the bacteria to synthesize glutamate.
References
1. Park, J.O., Rubin, S.A., Xu, Y.F., Amador-Noguez, D., Fan, J., Shlomi, T., Rabinowitz,
J.D. (2016) Metabolite concentrations, fluxes and free energies imply efficient enzyme usage.
Nat Chem Biol 12, 482-489.
2. Gunka, K. and Commichau, F.M. (2012) Control of glutamate homeostasis in Bacillus
subtilis: a complex interplay between ammonium assimilation, glutamate biosynthesis and
degradation. Mol Microbiol 85, 213-224.
3. Bohannon, D.E. and Sonenshein, A.L. (1989) Positive regulation of glutamate
biosynthesis in Bacillus subtilis. J Bacteriol 171, 4718-4727.
4. Belitsky, B.R. and Sonenshein, A.L. (1997) Altered transcription activation of a mutant
form of Bacillus subtilis GltR, a LysR family member. J Bacteriol 179, 1035-1043.
18
Metabolic engineering to direct evolution in Corynebacterium glutamicum
Andreas Schwentner1, E. Hoffart1, T. Busche2, C. Rückert2, J. Kalinowski2, R. Takors1, B.
Blombach1
1
2
University of Stuttgart, Institute of Biochemical Engineering, Stuttgart, Germany
Bielefeld University, Center for Biotechnology (CeBiTec), Bielefeld, Germany
Metabolic engineering to direct evolution (MEDE) is a novel evolutionary approach,
which enables the identification and evolution of new targets for improving microbial
producer strains. It consists of a first step of metabolic engineering, whereby evolutionary
pressure is put upon the strain of choice, a second step of evolutionary growth phase,
eventually yielding an evolutionary event, and a third step of comparative whole genome
sequencing (WGS), to identify evolutionary targets. Growth as straightforward screening
method and the minimal amount of mutations make MEDE a time-saving and easy to handle
evolutionary method.
In an applied approach, the genes ppc and pyc, encoding the anaplerotic enzymes
phosphoenolpyruvate carboxylase and pyruvate carboxylase, were deleted in C. glutamicum
ATCC 13032. The resulting strain C. glutamicum Δppc Δpyc showed strongly impaired
growth and was cultivated in minimal medium containing 40 g l-1 glucose and 1 g l-1 yeast
extract. Cells were sequentially transferred every third day for 14 days including concomitant
screening for faster growing mutants. After an evolutionary event, WGS was performed to
identify relevant mutations. In contrast to the initial strain C. glutamicum Δppc Δpyc, which
showed a growth rate of 0.17 h-1, three independently evolved mutants yielded growth rates of
about 0.32 h-1, indicating mutational events. Interestingly, the intersection of the mutations
obtained by WGS revealed isocitrate dehydrogenase (ICD) as consistent target in these
strains. Upon re-engineering in the basis strain, said point mutations led to diminished ICD
activities and an activation of the glyoxylate shunt enzymes isocitrate lyase and malate
synthase. Both enzymes are typically repressed during growth on glucose as sole carbon
source (1). Product suitability of the re-engineered strains was demonstrated by introducing
plasmid pJC4ilvBNCE (2), ensuring overexpression of the L-valine biosynthesis genes. These
strains accumulated up to 8.1 ± 1.0 g l-1 L-valine (3.4 times more than the reference C.
glutamicum pJC4ilvBNCE), corresponding to a product yield of 0.16 ± 0.01 g L-valine per g
glucose. This proofs ICD mutations as a potent alternative to a pyruvate dehydrogenase
complex with reduced activity (3) for the production of L-valine.
References
1. Gerstmeir, R., Wendisch, V.F., Schnicke, S., Ruan, H., Farwick, M., Reinscheid, D.,
and Eikmanns, B.J. (2003). J. Biotechnol. 104, 99–122.
2. Radmacher, E., Vaitsikova, A., Burger, U., Krumbach, K., Sahm, H., and Eggeling, L.
(2002). Appl Environ Microbiol 68, 2246–2250.
3. Buchholz, J., Schwentner, A., Brunnenkan, B., Gabris, C., Grimm, S., Gerstmeir, R.,
Takors, R., Eikmanns, B.J., and Blombach, B. (2013). Appl Environ Microbiol 79,
5566–5575.
19
SubtiWiki, an integrated database for model organism Bacillus subtilis
Bingyao Zhu and Jörg Stülke
Department of General Microbiology, University of Göttingen, Germany
Grisebachstr. 8, 37077 Göttingen
Information collecting and sharing has always been an important aspect of biological
research. In this internet era, online platforms, including databases and other kind of online
resources, are more preferred to the traditional paper-based publishing. SubtiWiki
(http://subtiwiki.uni-goettingen.de) (Michna et al., 2016), as one of those online resources,
has its main focus on the model organism Bacillus subtilis. It is a free collection of manually
curated annotation and provides access to public. In SubtiWiki, information of individual
genes/proteins is the center of our focus. Moreover, knowledge of association among genes
and proteins, in form of interaction, regulation, and biochemical fluxes are also gathered and
presented in diagrams. Furthermore, gene and protein expression data are compiled and
displayed in interactive charts. With our iOS and Android App (available in App Store and
Google Play Store), mobile access to our data is guaranteed. Along with the constant update
of data, the database structure has also undergone significant changes. A new database layout
is applied. Upon that, a PHP framework is developed to meet the needs to organize data. The
source code of the framework is scheduled to go open source under MIT license. With the
new database layout, better organized information, clear visualization of data, we will
continue assisting the researchers who focus on B. subtilis and other Gram-positive bacteria.
References
Michna et al., 2016. Nucleic Acids Res. 44(D1):D654-62.
20
Large-scale reduction of the Bacillus subtilis genome: Consequences for the
transcriptional network, resource allocation, and metabolism
Daniel Reuß1, Josef Altenbuchner2, Ulrike Mäder3, Hermann Rath3, Till Ischebeck4, Bingyao
Zhu1, Stefan Klumpp5, Fabian Commichau1, Uwe Völker3 and Jörg Stülke1
1 Dept. of General Microbiology, University of Göttingen, Grisebachstr. 8, 37077 Göttingen
2 Institute of Industrial Genetics, University of Stuttgart
3 Institute for Genetics and Functional Genomics, University Medicine Greifswald
4 Dept. of plant Biochemistry, University of Göttingen
5 Institute for Nonlinear Dynamics, University of Göttingen
Understanding cellular life requires a comprehensive knowledge of the essential cellular
functions, the components involved, and their interactions. Minimized genomes are an
important tool to gain this knowledge. We have constructed strains of the model bacterium
Bacillus subtilis whose genomes have been reduced by about 36%. These strains are fully
viable and their growth rates in complex medium are comparable to those of wild type strains.
An in-depth multi-omics analysis of the genome reduced strains revealed how the deletions
affect the transcription regulatory network of the cell, translation resource allocation, and
metabolism. A comparison of gene counts and resource allocation demonstrates drastic
differences in the two parameters, with 50% of the genes using as little as 10% of translation
capacity whereas the 6% essential genes require 57% of the translation resources. Taken
together, the results are a valuable resource on gene dispensability in B. subtilis, and they
suggest the roads to further genome reduction to approach the final aim of a minimal cell in
which all functions are understood.
21
Session 3: Second Messengers, bacterial differentiation, and antibiotic
production
Cyclic di-nucleotide signalling in bacterial differentiation and antibiotic production
Natalia Tschowri1, Maria A. Schumacher2, Susan Schlimpert3, Naga babu Chinnam2, Richard
G. Brennan2 and Mark J. Buttner3
1
Institut für Biologie - Mikrobiologie, Humboldt-Universität zu Berlin, Philippstraße 13,
10115 Berlin
2
Department of Biochemistry, Duke University School of Medicine, Durham, NC, USA
3
Department of Molecular Microbiology, John Innes Centre, Norwich Research Park,
Norwich NR4 7UH, UK
The multi-talented bacteria Streptomyces are „Microbe of the Year 2016“ (VAAM) and have
been awarded the Nobel Prize twice (1952 and 2015) for their exceptional ability to produce
diverse medically-useful natural products. The synthesis of these secondary metabolites is
genetically and temporally tightly interlinked with the developmental life cycle of
Streptomycetes, and facing the urgent need for new antibiotics it is of particular significance
to understand the signals and pathways that control development in these bacteria.
In our recent study, we have shown that the bacterial second messenger cyclic di-GMP
(c-di-GMP), which is produced by GGDEF-type diguanylate cyclases and degraded by EALor HD-GYP-type phosphodiesterases, determines the timing of differentiation initiation in S.
venezuelae by regulating the activity of the highly conserved developmental master regulator
BldD. Our structural and biochemical analyses revealed that a tetrameric form of c-di-GMP
activates BldD DNA-binding by driving a unique form of protein dimerization, leading to
repression of the BldD regulon of sporulation genes during vegetative growth (1, 2, 3).
Currently, we aim to understand which of the 10 putative c-di-GMP-metabolising
enzymes encoded by S. venezuelae contribute to c-di-GMP pool(s) sensed by BldD and how
the BldD-c-di-GMP complex is assembled. Our initial data indicate that a distinct set of
GGDEF / EAL proteins influences the developmental programme progression in S.
venezuelae and that loading of BldD with tetrameric c-di-GMP is a two-step process.
Altogether, our work will greatly improve our understanding of Streptomyces
physiology and c-di-GMP signalling in multicellular differentiation and secondary metabolite
production and can contribute to a better exploitation of genetic engineering in Streptomyces
for the production of antibiotics.
References
1. Tschowri N, Schumacher MA, Schlimpert S, Chinnam NB, Findlay KC, Brennan RG,
Buttner MJ. (2014) Tetrameric c-di-GMP Mediates Effective Transcription Factor
Dimerization to Control Streptomyces Development. Cell, 2014 Aug 28;158(5):1136-47
2. Bush MJ, Tschowri N, Schlimpert S, Flärdh K, Buttner MJ. c-di-GMP signalling and the
regulation of developmental transitions in streptomycetes. (2015) Review. Nature Reviews
Microbiology, 2015 Dec; 13(12):749-60
3. Tschowri N. (2016) Cyclic dinucleotide-controlled regulatory pathways in Streptomyces.
Review. Journal of Bacteriology, 2016 Jan;198 (1):47-54
22
Spore memory couples entry and exit from bacterial dormancy in Bacillus subtilis
Alper Mutlu, Stephanie Trauth, Marika Ziesack, Sonja Schulmeister and Ilka Bischofs
Bioquant Center, University of Heidelberg
Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany
In bacteria, entry into and exit from dormancy are controlled by regulatory networks with
little known overlap, indicating that the two processes operate independently from each other.
Here we show that both processes are linked by phenotypic memory. Using B. subtilis as a
model we developed an advanced time-lapse microscopy assay and a fluorescent marker that
reports on a spore’s differentiation history to study the effect of variable sporulation timing on
nutrient-induced spore revival. Early spores were kinetically favored over late spores on
different levels of the revival pathway and in response to different stimuli. We furthermore
show that spore outgrowth is controlled by phenotypic memory and can be reprogrammed
with alanine dehydrogenase. As a consequence of the simple coupling provided by phenotypic
memory, genetic changes that affect sporulation timing also affect the spore revival
properties. We therefore suggest that phenotypic memory contributes to the emergence of
complex adaptive traits.
23
Regulation of Secondary Metabolite Gene Clusters in Social Myxobacteria
Carsten Volz and Rolf Müller.
Department Microbial Natural Products, Helmholtz-Institute for Pharmaceutical Research
Saarland,
University Campus E8 1, D-66123 Saarbrücken, Germany
Motile predatory myxobacteria are producers of multiple secondary metabolites and, on
starvation, undergo concerted cellular differentiation to form multicellular fruiting bodies.
These abilities demand myxobacterial genomes to encode sophisticated regulatory networks
which are not satisfactorily understood. One approach in our efforts to identify and
characterize new natural compounds exhibiting interesting biological activities is to elucidate
the transcriptional regulation of the respective secondary metabolite gene clusters. The
genetic manipulation of such regulatory components should enable an increased production of
interesting natural compounds or should enable the induction of the production of unknown
secondary metabolites. We here give an overview on interesting new regulatory mechanisms
we have been able to elucidate but also on the drawback of such an approach working with
myxobacteria.
24
Session 4: Regulation by RNA
From Strings of Nucleotides to Collective Behavior: “Lessons from Vibrio
cholerae”
Kai Papenfort
Faculty of Biology I, Ludwig-Maximilians-University, Munich
Quorum-sensing (QS), is a process of bacterial cell-to-cell communication that relies on the
production, release, and population-wide detection of extracellular signal molecules.
Processes
controlled by QS are unproductive when undertaken by an individual bacterium but become
effective when undertaken by the group. QS controls many important microbial processes
including bioluminescence, secretion of virulence factors, competence and biofilm formation.
In this study, we identified and characterized of a novel bacterial communication system
present in Vibrio cholerae. This system consists of a transcriptional regulator, VqmA and a
small regulatory RNA (sRNA), VqmR.
VqmR is activated by VqmA and functions as a trans-acting regulator through base-pairing
with multiple target mRNAs. Among these targets are key factors for biofilm formation and
virulence factor expression in V. cholerae indicating that VqmA/R could participate in the
regulation of complex behaviors. Indeed, our results show that VqmA binds to and is
activated by an extracellular signal, which we determined as the novel autoinducer molecule,
DPO. DPO is a new molecule to biology and is produced by diverse pro- and eukaryotes.
Further, we obtained evidence that the signaling molecule is produced by commensal species
of the host microbiota and that VqmA/R plays an important role during V. cholerae
pathogenesis and the communication with other bacteria.
25
Surprising small RNA features in Rhodobacter sphaeroides
Bernhard Remes, Katrin Müller, Lennart Weber and Gabriele Klug.
Department of Microbiology and Molecular Biology, University of Gießen,
Heinrich-Buff-Ring 26-32, 35390 Gießen, Germany
Among the strategies developed by bacteria to adapt to environmental changes is the use
of sRNAs, which usually act at the post-transcriptional level via base-pairing with the targeted
mRNA. Since trans-encoded sRNAs are located in another chromosomal location and are
only partially complementary to their target mRNAs, most of them require Hfq for their
stability in the cell and their regulatory function [1, 2].
In Rhodobacter sphaeroides RNAseq-based studies identified amongst others the sRNAs
RSs0827 [3] and UpsM [4]. RSs0827 is the gene with the highest increase in expression after
60 h of stationary phase (Remes et al., submitted). UpsM is the most abundant orphan sRNA
of R. sphaeroides and represents about 60% of all Hfq bound sRNAs [5]. Upon several stress
conditions, RNase E cleaves UpsM in an Hfq- and target-dependent manner (Weber et al.,
submitted). The sRNA is located in the 5’ UTR of mraZ, the first gene of the dcw (division
and cell wall) gene cluster. Transcription of mraZ depends on the UpsM promoter, which
implicates that the terminator of UpsM allows read-through in order to guarantee transcription
of mraZ. However, an answer for the regulatory function of the two sRNAs or the target
responsible for processing of UpsM remained unclear.
Using in vitro and in vivo experiments, we characterized UpsM as an atypical target for
RSs0827. Indeed, RSs0827 base pairs in the 5’ region of upsM and triggers the decay of
UpsM, henceforth renamed StsR (sRNA targeting sRNA). Moreover, cells lacking StsR
showed increased read-through of the UpsM terminator, leading to mraZ expression and in
turn to continuous growth even in late stationary phase. Collectively, we present the first
interaction between two sRNAs and further widen our knowledge about the interplay between
sigma factors, Hfq and sRNAs, as well as their role in controlling cell division in response to
external stresses.
References
1. Vogel, J. and B.F. Luisi, Hfq and its constellation of RNA. Nat Rev Microbiol, 2011. 9(8):
p. 578-89.
2. Valentin-Hansen, P., M. Eriksen, and C. Udesen, The bacterial Sm-like protein Hfq: a key
player in RNA transactions. Mol Microbiol, 2004. 51(6): p. 1525-33.
3. Remes, B., et al., Role of oxygen and the OxyR protein in the response to iron limitation in
Rhodobacter sphaeroides. BMC Genomics, 2014. 15: p. 794.
4. Berghoff, B.A., et al., Photooxidative stress-induced and abundant small RNAs in
Rhodobacter sphaeroides. Mol Microbiol, 2009. 74(6): p. 1497-512.
5. Berghoff, B.A., et al., Contribution of Hfq to photooxidative stress resistance and global
regulation in Rhodobacter sphaeroides. Mol Microbiol, 2011. 80(6): p. 1479-95.
26
Session 5: Regulation in Biofilms
Exploitation of the biofilm matrix to colonize a surface
Ákos T. Kovács
Terrestrial Biofilms Group, Institute of Microbiology, Friedrich Schiller University Jena,
Neugasse 23, D-07743 Jena, Germany
Multicellular biofilm formation and surface motility are bacterial behaviors considered as
mutually exclusive. Moreover, the basic decision to move over or stay attached to a surface is
poorly understood in bacteria. It is well established that flagellum based individual cell based
motility is required for founding an air-liquid interface biofilm (1). In Bacillus subtilis, the
key sporulation- and biofilm-controlling transcription factor, Spo0A~P governs the flagellaindependent mechanism of social sliding motility (2). Microarray experiments and subsequent
genetic characterization revealed that the machineries of sliding and biofilm formation, share
the same secreted components (i.e. surfactin, the hydrophobin BslA, and exopolysaccharide).
Sliding proficiency is transduced by the Spo0A-phosphorelay histidine kinases KinB and
KinC, while potassium is the specific sliding-activating signal through a cytosolic domain of
KinB, which resembles the selectivity filter sequence of potassium channels. Interestingly, the
gradual increase in Spo0A~P orchestrates the sequential activation of sliding, followed by
sessile biofilm formation and finally sporulation in the same population (2,3).
Similar to sliding, the secreted matrix benefits colony biofilm expansion, while its
production is costly for the individuals. Mutant strains lacking matrix production have a
higher fitness under well mixed planktonic conditions. However, matrix producers have an
advantage when cultivated in a spatially structured environment (4). The density of cells at the
onset of biofilm growth on a solid surface affects pattern formation and high assortment
facilitates cooperation during biofilm growth (4,5).
In sum, I will present how multicellular behaviors (sessile biofilm development and surface
spreading) are coordinately activated and highlight the impact of spatial assortment (6) and
diffusion on privatization of secreted components prerequisite for sliding.
References
1. Hölscher, T., Bartels, B., Lin, Y.-C., Gallegos-Monterrosa, R., Price-Whelan, A., Kolter, R.,
Dietrich, L.E.P. and Kovács, Á.T. (2015) Motility, chemotaxis and aerotaxis contribute to
competitiveness during bacterial pellicle biofilm development. Journal of Molecular Biology 427,
3695-3708.
2. Grau, R., de Oña, P., Kunert, M., Leñini, C., Gallegos-Monterrosa, R., Mhatre, E., Vileta, D.,
Donato, V., Hölscher, T., Boland, W., Kuipers, O.P. and Kovács, Á.T. (2015) A duo of potassiumresponsive histidine kinases govern the multicellular destiny of Bacillus subtilis. mBio 6, e00581-15.
3. Kovács, Á.T. (2016) Bacterial differentiation via gradual activation of global regulators. Current
Genetics 62, 125-128.
4. van Gestel, J., Weissing, F.J., Kuipers, O.P. and Kovács, Á.T. (2014) Density of founder cells
affects spatial pattern formation and cooperation in Bacillus subtilis biofilms. ISME Journal 8, 2069–
2079.
5. Kovács, Á.T. (2014) Impact of spatial distribution on the development of mutualism in microbes.
Frontiers in Microbiology 5, 649.
6. Hölscher, T., Dragoš, A., Gallegos-Monterrosa, R., Martin, M., Mhatre, E., Richter, A. and Kovács,
Á.T. (2016) Monitoring spatial segregation in surface colonizing microbial populations. Journal of
Visualized Experiments e54752.
27
Cell-cell communications in Bacillus subtilis mixed-species biofilms
Ramses Gallegos-Monterrosa1, Stefanie Kankel1, Sebastian Götze2, Pierre Stallforth2, Ákos T.
Kovács1
1
Terrestrial Biofilms Group, Friedrich Schiller University Jena. Neugasse 23, 07743 Jena,
Germany
2
Leibniz Institute for Natural Product Research and Infection Biology. Beutenbergstraße
11/a, 07745 Jena, Germany
Biofilm development in diverse bacteria has been shown to response to multiple
environmental signals like small molecules secreted by microorganisms. These signals have
been usually considered to be self-generated, i.e. quorum-sensing, but can also be produced
by other organisms living in the vicinity, thus creating an interspecies communication
network. Bacillus subtilis is a Gram-positive model bacterium for studying biofilm formation.
It differentiates into several subpopulations of specialized cell types in response to different
environmental cues thus making it an ideal model for studying complex signaling networks.
Multiple soil bacteria can produce small signaling molecules that influence biofilm formation
by B. subtilis. We aim to identify such organisms and to characterize the chemical nature and
signaling pathway of novel signaling molecules that are able to modify the architecture of B.
subtilis biofilms.
Bacteria isolated from soil samples were screened for their ability to produce signaling
molecules able to modify the structure of B. subtilis biofilms. Five soil isolates were selected
for further characterization due to their ability to modify B. subtilis complex colony
structures. These bacteria were identified through 16S rDNA sequencing. Four of the isolates
were identified as either Lysinibacillus sp. or Bacillus pumilus, which are closely related to B.
subtilis and thus may share similar signaling mechanisms. The culture supernatants of
selected bacteria were submitted to chromatography, enzymatic, and biochemical analysis to
discern the nature of the signaling molecules. The purine derivative hypoxanthine was
identified as a signaling molecule produced by one of the Lysinibacillus sp. isolates.
B. subtilis is able to recognize and respond to several signaling molecules produced
mainly by members of closely related genus but also from non-related organisms that may
share the same ecological niche. Hypoxanthine is one such molecule that has shown to
increase wrinkle formation in complex colony biofilms of B. subtilis. The signalling pathway
of hypoxanthine on B. subtilis is being studied through the use of knock-out mutants and
fluorescent gene-expression reporter fusions.
28
Session 6: Stress responses and Signal Transduction
Connecting cell wall homeostasis and a major envelope stress response in E.coli
Geraldine Laloux
Institut de Duve, Université catholique de Louvain
Avenue Hippocrate, 75 - B1.75.08, 1200 Bruxelles
The envelope of Gram-negative bacteria is an essential compartment that constitutes a
protective and permeability barrier between the cell and its environment. The envelope also
hosts the cell wall, a mesh-like structure made of peptidoglycan (PG) that determines cell
shape and provides osmotic protection. Since the PG must grow and divide in a cell-cyclesynchronized manner, its synthesis and remodeling are tightly regulated. Here, we discovered
that PG homeostasis is intimately linked to the levels of activation of the Cpx system, an
envelope stress response system traditionally viewed as being involved in protein quality
control in the envelope. We first show that Cpx is activated when PG integrity is challenged
and that this activation provides protection to cells exposed to antibiotics inhibiting PG
synthesis. By rerouting the outer membrane lipoprotein NlpE, a known Cpx activator, to a
different envelope subcompartment, we managed to manipulate Cpx activation levels. We
found that Cpx overactivation leads to aberrant cellular morphologies, to an increased
sensitivity to b-lactams, and to dramatic division and growth defects, consistent with a loss of
PG homeostasis. Remarkably, these phenotypes were largely abrogated by the deletion of
ldtD, a Cpx-induced gene involved in noncanonical PG cross-linkage, suggesting that this
transpeptidase is an important link between PG homeostasis and the Cpx system. Altogether
our data show that fine-tuning of an envelope quality control system constitutes an important
layer of regulation of the highly organized cell wall structure.
29
The Cpx-system of Escherichia coli analyzed by SRM and Super-resolution Microscopy
Emina Ćudić1, Kristin Surmann2, Rainer Kurre3, Elke Hammer2 and Sabine Hunke1.
1
Department of Microbiology, University of Osnabrueck, Barbarastraße 11, D-49076
Osnabrueck, Germany
2
Department of Functional Genomics, University of Greifswald, Friedrich-Ludwig-Jahn
Straße 15A, D-17475 Greifswald, Germany
3
Department of Biophysics, University of Osnabrueck, Barbarastraße 11, D-49076
Osnabrueck, Germany
Bacteria rely on two-component systems (TCS) in order to sense and response to
environmental changes and thus to different stimuli [1]. These systems make use of a
phosphorylation cascade from a transmembrane sensor kinase (SK) to a cytoplasmic response
regulator (RR) and are set back to the initial state via desphosphorylation of the RR [1]. For a
better understanding of the functionality and the dynamics of a TCS it is important to know
the absolute amounts of the proteins and their localization within living cells. Here, we used
the Cpx-envelope stress TCS as a model. It consists of the inner membrane-spanning SK
CpxA, the cytosolic RR CpxR and the periplasmic accessory protein CpxP, which inhibits the
autophosphorylation activity of CpxA [2, 3]. We investigated absolute protein amounts by
single reaction monitoring (SRM) and the localization of CpxA and CpxP by super-resolution
microscopy. We could determine absolute amounts for CpxA, CpxR and CpxP being 41
molecules/cell (CpxA), 393 molecules/cell (CpxR) and 36 molecules/cell (CpxP) under Cpxnon affecting conditions [4]. However, although it is known that CpxA and CpxP interact in
order to keep the Cpx-TCS in an OFF-state [5], the affinity between CpxP and CpxA is very
low [6]. In combination with low absolute amounts per cell we asked how CpxA and CpxP
might physically interact in living cells to promote inhibition of the Cpx-TCS. Therefore, we
investigated the localization of CpxA and CpxP under different conditions in living cells and
analyzed whether CpxA and CpxP co-localize as a prerequisite for functional interaction. We
generated chromosomal fusions of CpxA with the SNAP-Tag® and CpxP with the
HaloTag®. These fusions enable native protein levels and covalent staining with different
combinations of fluorescent dyes for detection by Total Internal Refraction Fluorescence
(TIRF)-Microscopy. We quantified co-localization, close-together localization and no colocalization events and found the biggest shift from close-together localization to colocalization after inhibition of the Cpx-TCS by cpxP-overexpression. Altogether, our results
demonstrate the importance of a combination of in vitro and in vivo studies to get a deeper
insight into the function of a TCS.
References
1. Stock, A. M.; Robinson, V. L. and Goudreau, P. N. (2000). Annu. Rev. Biochem. 69, p.
183-215.
2. Fleischer, R; Heermann, R.; Jung, K, and Hunke, S. (2007). J. Biol. Chem. 282, p. 85838593.
3. Hunke, S.; Keller, R., and Müller, V.S. (2012). FEMS Microbiol. 326, p. 12–22.
4. *Surmann, K.; *Ćudić, E.; Hammer, E., and Hunke, S. (2016). MicrobiologyOpen
doi: 10.1002/mbo3.353
5. Tschauner, K.; Hörnschemeyer, P.; Müller, V.S.; Hunke, S. (2014) PLoS ONE 9(9).
e107383. doi:10.1371/journal.pone.0107383
30
6. Hörnschemeyer, P.; Liss, V.; Heermann, R.; Jung, K. and Hunke, S. (2016) PLoS ONE
11(2). e0149187. doi:10.1371/journal.pone.0149187
Uncovering the regulatory circuits of the glycine betaine synthesizing pathway in
Bacillus subtilis
Bianca Warmbold, Stefanie Ronzheimer, Tamara Hoffmann , Erhard Bremer.
Laboratory for Microbiology, Department of Biology, Philipps-University Marburg,
Karl-von-Frisch Str. 8, D-35043 Marburg, Germany
Confronted with hyperosmotic stress, the soil bacterium Bacillus subtilis accumulates
compatible solutes to maintain cell turgor, and to sustain growth under osmotically
unfavorable circumstances. Glycine betaine is such a compatible solute and it can either be
taken up from the environment via several Opu-transporters or it can be synthesized from the
precursor choline. Oxidation of choline is mediated by the dehydrogenases GbsB and GbsA,
whose structural genes are transcribed as an operon (1, 2). Upstream of the gbsAB gene
cluster, the gbsR gene is located which encodes a choline-responsive repressor regulating the
expression of the gbsAB operon as well as the opuB operon, which encodes a substratespecific ABC transporter for choline (3).
Bioinformatic analysis of the DNA-region upstream of the gbsAB operon and of the opuB
operon revealed a palindromic repeat within the in silico predicted GbsR binding site (4). We
showed that mutations targeting these regions abolish GbsR-mediated repression as shown by
gbsA-treA and opuB-treA reporter fusion studies. Binding of the ligand choline to the GbsR
regulator relieves repression of the gbsAB operon. An in silico model of the GbsR protein
structure hints that four phenylalanines arranged in an aromatic cage probably form a binding
pocket for the inducer choline. We constructed mutants of the GbsR homologue OpuAR of B.
infantis NRRL B-14911 and determined their binding affinity for choline by fluorescence
spectroscopy. With this approach we were able to show that indeed each of the phenylalanine
residues is involved in choline binding.
Since GbsR acts as repressor of the opuB but not of the closely related opuC operon (it
encodes an ABC transporter for various osmostress protectants, including choline), we studied
the networks involved in the regulation of both gene clusters at the transcriptional level. This
study revealed a striking difference in the expression pattern between the two operons.
Whereas the expression of the opuB operon increases with rising salt concentrations, the opuC
operon shows the strongest expression at moderate salt concentrations. However, they share
the essential activation by the biofilm activator RemA (5) and the regulation through the
MarR-type repressor OpuCR. We uncovered the physiological role of OpuCR, since we were
able to show that it is involved in the re-establishment of opuC repression under high salt
concentrations, a function in agreement with the salt-induced expression of the opuCR gene.
Taken together, our findings highlight a complex regulatory circuit of the pathways
leading to the formation of the cellular pool of the cytoprotectant glycine betaine in
osmotically stressed B. subtilis cells.
References
1. Boch, J. et al. (1996) J. Bacteriol. 178:5121-56129
2 .Boch, J. et al. (1997) Arch. Microbiol. 168:282-289
3. Nau-Wagner, G. et al. (2012) J. Bacteriol. 194:2703-2714
4. Leyn, S. et al. (2012) J. Bacteriol. 195:2463-2473
31
5. Winkelman, J.T. et al. (2013) Mol. Microbiol. 88:984-997
32
Characterization of the ABC-Transporter Associated Two-Component System YxdJK
in Bacillus subtilis as Biosensor for Eukaryotic Antimicrobial Peptides
Katharina Sievers, Diana Wolf and Thorsten Mascher.
Institute of Microbiology, Technical University Dresden, Zellescher Weg 20 b, Dresden,
Germany
The cell envelope of bacteria represents the primary target of many antimicrobial
substances, especially antimicrobial peptides (AMPs). Therefore, an intact cell envelope
integrity is crucial for survival of the cell. Bacteria evolved designated cell envelope stress
response (CESR) mechanisms to constantly monitor and maintain the cell envelope integrity.
The main part of CESRs is mechanistically mediated by signal transduction via twocomponent systems (TCS) that links extracellular signal perception by a histidine kinase to a
corresponding cellular response realized by a response regulator [1]. In B. subtilis, the CESRmediating TCSs are mostly associated to ABC-transporters which confer resistance to
antimicrobial compounds. The Bce-system that responds highly specific to bacitracin and
several other structurally related AMPs has been investigated very well [2]. The genome of B.
subtilis contains two further Bce-like systems, the Psd- and the Yxd-system which are also
involved in mediating resistance to peptide antibiotics. Regarding the Yxd-system, the
expression of the ABC-transporter YxdLM controlled by the response regulator YxdJ that
binds to PyxdL after induction caused by the human immune peptide LL-37 has already been
described [3,4]. Recently, we observed a 1000 to 10000-fold activation of the Yxd-system
after treatment with larvae extracts of the black soldier fly Hermetia illucens. Microarray
studies emphasized the expression of diverse AMPs in H. illucens larvae. Accordingly, we
investigated the Yxd-system induced with H. illucens larvae extracts in more detail and found
that the system confer resistance to larvae extracts. Furthermore on basis that the Yxd-system
responds specifically to AMPs produced by eukaryotes, an application of the system as a
powerful biosensor for the identification of eukaryotic AMPs is feasible.
References
[1] Schrecke et al., 2012, In: Gross R, Beier D (eds) Two component systems in bacteria.
Horizon Scientific Press, Hethersett, Norwich, UK, pp. 199-229
[2] Rietkötter et al., 2008, Mol Microbiol 68(3):768-85
[3] Joseph et al., 2004, Microbiology 150(8):2609-2617
[4] Pietiäinen et al., 2005, Microbiology 151(5):1577-1592
33
Session 7: Diverse Regulators
A new piece in the big puzzle of cell division: The transcriptional regulator FtsR
regulates FtsZ in Corynebacterium glutamicum
Kim Julia Kraxner, Meike Baumgart, Michael Bott.
IBG-1: Biotechnology, Institute of Bio- and Geosciences
Forschungszentrum Jülich, Germany
Corynebacterium glutamicum is a non-pathogenic, aerobic, Gram-positive soil bacterium
which is used for the large scale production of several L-amino acids and other industrially
relevant compounds [1]. Moreover, it is a useful model organism for the Corynebacteriales,
including pathogenic species such as Corynebacterium diphtheriae and Mycobacterium
tuberculosis. The inhibition of microbial cell division serves as an attractive target for the
development of new antimicrobial drugs. Whereas the key steps in cell division and their
regulation are well understood in other model bacteria such as E. coli and B. subtilis,
knowledge about positive and negative regulators of cytokinesis in Actinobacteria is very
limited [2].
In our studies we analyzed FtsR, a so far uncharacterized transcriptional regulator of
C. glutamicum. A ftsR deletion mutant showed growth defects and a drastically altered cell
morphology, suggesting a malfunction of cell division or cell wall synthesis. The wild-type
phenotype could be restored by re-integrating ftsR at a different position in the chromosome.
Additionally, full complementation of the growth defect and of the morphological phenotype
was achieved by plasmid-based heterologous expression of the ftsR homolog from
C. diphtheriae.
To determine potential target genes of FtsR, chromatin affinity purification with
subsequent next generation sequencing (ChAP-Seq) was performed, which revealed a region
upstream of ftsZ as a major target. Using the ChAP-Seq results and the motif-based sequence
analysis tool MEME-ChIP, a putative DNA-binding motif could be identified for FtsR. DNA
microarray experiments revealed significantly reduced ftsZ mRNA levels in the ftsR mutant
compared to the wildtype. Furthermore, electrophoretic mobility shift assays (EMSAs) and
reporter gene studies confirmed ftsZ to be an FtsR target gene.
In summary, a novel transcriptional regulator of C. glutamicum was identified, which
serves as an activator of ftsZ expression and is required for normal growth and cell
morphology.
References
1. Eggeling, L., Bott, M. (2005) Handbook of Corynebacterium glutamicum. CRC Press,
Boca Raton, USA
2. Donovan C., Bramkamp M. (2014) Cell division in Corynebacterianeae. Front Microbiol
5:132
34
Silencing of cryptic prophages in Corynebacterium glutamicum
Eugen Pfeifer1, Max Hünnefeld1, Ovidiu Popa2, Meike Baumgart1, Tino Polen1, Dietrich
Kohlheyer1 and Julia Frunzke1.
1) Institute of Bio- und Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich,
52425 Jülich, Germany
2) Quantitative and Theoretical Biology, Heinrich-Heine-Universität Düsseldorf
40225, Düsseldorf, Germany
Viral DNA and prophage-like elements can account for up to 20% of the entire bacterial
genome and may significantly contribute to their host’s fitness [1]. However, acquisition of
new genetic elements bears risks for their hosts since their activation or gene expression may
cause high metabolic costs and can even lead to cell death by lysis. To enable controlled
integration of newly acquired DNA into the host’s regulatory network organisms rely on the
action of small nucleoid-associated proteins, which function as xenogeneic silencers of
foreign DNA elements [2].
In our studies, we use the Gram-positive soil bacterium Corynebacterium glutamicum
ATCC 13032 as model system to study prophage-host interactions. The genome of ATCC
13032 comprises three cryptic prophages (CGP1-3), of which CGP3 was shown to undergo
spontaneous activation in a small fraction of cells [3]. However, the molecular factors
controlling CGP3 activity are currently not known.
Recently, we identified a small nucleoid-associated protein, named CgpS, which is
encoded on the CGP3 prophage island and shares sequence similarity with the mycobacterial
xenogeneic silencer Lsr2. In our studies, we could show that CgpS is an essential gene due to
its function as a silencer of cryptic phage elements in C. glutamicum. Genome-wide binding
analyses displayed the preferred association to AT-rich elements, especially to the CGP3
prophage, but also to other regions which have likely been acquired by horizontal gene
transfer. Bioinformatical analysis revealed orthologous proteins in almost all Actinomycetes,
but remarkably, also in several phage and prophage genomes. Our results emphasize CgpS as
a key factor for the control of CGP3 activity and highlight the importance of small nucleoidassociated proteins for the control of foreign DNA in bacterial host strains.
References
[1] Nanda, A.M., K. Thormann & J. Frunzke, (2015) Impact of spontaneous prophage
induction on the fitness of bacterial populations and host-microbe interactions. J Bacteriol
197: 410-419.
[2] Pfeifer, E, M. Hünnefeld, O. Popa, T. Polen, D. Kohlheyer, M. Baumgart & J. Frunzke
(2016). Silencing of cryptic prophages in Corynebacterium glutamicum. Nucleid Acid Res In
Revision.
[3] Helfrich, S., E. Pfeifer, C. Kramer, C.C. Sachs, W. Wiechert, D. Kohlheyer, K. Noh & J.
Frunzke, (2015) Live cell imaging of SOS and prophage dynamics in isogenic bacterial
populations. Mol Microbiol 98: 636-650.
35
Inverse correlation between the transcription rate and H-NS/StpA repression in
Escherichia coli
Aathmaja A. Rangarajan and Karin Schnetz.
Institut für Genetik, Universität zu Köln,
Zülpicher Str. 47a, 50674 Cologne, Germany.
The nucleoid-associated protein H-NS is an enterobacterial global repressor that inhibits
transcription by forming extended DNA stiffening or bridging complexes. H-NS represses
transcription at the level of initiation either by excluding RNA polymerase or by trapping it at
promoters. StpA is a H-NS paralogue that presumably acts similarly as H-NS. H-NS (and
StpA) mediated repression can be relieved locus specifically by binding of specific
transcription factors, by perturbations of the DNA structure, and other mechanisms [1].
However, it is an open question whether H-NS (and StpA) complexes also interfere with
transcription elongation. In vitro, at conditions that favour bridging, H-NS has been shown to
enhance RNA polymerase pausing and to promote Rho-dependent termination [2].
Complementarily, it was shown that inhibition of Rho-mediated termination that results in
increased transcription reduces H-NS binding [3]. Furthermore, our previous data revealed an
inverse correlation between the efficiency of repression by H-NS when binding within the
transcription unit and the strength of the promoter that is directing transcription across the HNS-bound DNA tract [4].
In this project we analyzed the effect of transcription elongation on repression by H-NS.
In our experimental system we inserted a cassette consisting of a constitutive promoter and
conditional transcriptional terminator (λtR1) upstream of H-NS (and StpA) loci, and varied
the transcription rate by expressing anti-terminator protein λN. Our data show that
transcription elongation across H-NS-bound DNA tracts relieves repression of H-NS and
H-NS/StpA repressed promoters. In addition we observed a linear inverse correlation of
transcription across the H-NS-bound DNA tracts and H-NS-mediated repression. The data
suggest that the transcribing RNA polymerase is able to remodel the H-NS (and StpA)
complex and/or dislodge H-NS (and StpA) from the DNA and thus relieve repression, while
at low transcription rates the H-NS repression complex is stable.
36
Characterization of structural features controlling activity of LeuO, a pleiotropic
transcriptional regulator and H-NS antagonist
Susann M. Fragel and Karin Schnetz.
Universität zu Köln, Institut für Genetik, Zülpicher Str. 47a, 50674 Köln
LeuO is a conserved LysR-type transcriptional regulator of global function, controlling
more than 100 genes in Escherichia coli and Salmonella enterica. LeuO is required in the
control of pathogenicity, stress adaptation, and biofilm formation in various enterobacterial
species. A vast majority of the LeuO regulated genes are regulated together with the nucleoidstructuring and global repressor protein H-NS and LeuO is considered a global H-NS
antagonist. LeuO function and leuO gene regulation have been addressed in depth.
Nonetheless, open questions are which signals induce leuO expression and whether the LeuO
protein activity is modulated by a co-effector, similar to the control of other regulators of the
LysR-family.
In this project, we characterize structural features that control LeuO activity. In a first
step, we analyzed the control of LeuO target promoters, among which the CRISPR-associated
cas promoter turned out to be the target that is most specifically regulated by LeuO in E. coli.
Using the cas promoter as reporter, we then performed site-directed and random mutagenesis
screens for LeuO mutants with changed activity. By this approach we identified amino acid
residue exchanges that render LeuO hyper-active. Mapping of these residues onto a predicted
structure of the C-terminal effector-binding domain of LeuO suggests that these residues are
all surface exposed, with two of them mapping at the entrance of the presumptive co-effector
binding cleft. Additional substitutions of amino acid residues, located within the presumptive
co-effector binding cleft, inactivate the LeuO protein. Preliminary data obtained of the crystal
structure of the C-terminal effector-binding domain of LeuO support this interpretation. Our
structural and functional characterization of LeuO suggests that LeuO activity is modulated
by a co-effector. This is relevant in understanding the molecular mechanism of transcriptional
regulation by LeuO, and the role of LeuO in the bacterial stress response, CRISPR-cas
mediated immunity, and pathogenicity.
37
Feedback control of leuO encoding a pleiotropic regulator and H-NS antagonist in
Escherichia coli
Hannes Breddermann and Karin Schnetz.
Institut für Genetik, Universität zu Köln,
Zülpicher Str. 47a, 50674 Köln, Germany
The enterobacterial LeuO protein is a pleiotropic LysR-type transcriptional regulator that
is conserved in Enterobacteriaceae and plays an important role in pathogenicity, stress
adaptation and the CRISPR/Cas immunity system. At standard growth conditions, expression
of leuO is silenced by the master regulator H-NS and by its paralogue StpA. Expression of
leuO can be activated by BglJ-RcsB and involves a double-positive feedback loop regulation.
The leuO gene is activated by BglJ-RcsB [2], and LeuO activates expression of bglJ, encoded
within the H-NS repressed yjjQ-bglJ operon [1]. Activation of leuO by BglJ-RcsB is in
addition antagonistically controlled by LeuO [2] suggesting that the double-positive feedback
regulation of leuO is tightly controlled. The activation dynamics of the leuO promoter by the
antagonistic action of LeuO and BglJ-RcsB were characterized by a leuO promoter
fluorescence reporter fusion in dependence of ectopically expressed LeuO and BglJ. The leuO
promoter activity was analyzed by flow cytometry. Results suggest that the antagonistic
control of the leuO promoter activity by LeuO and BglJ is controlled in dependence of their
relative concentration. H-NS and StpA mediated silencing probably keeps leuO in an OFF
state and dominates positive feedback regulation of leuO and bglJ at standard laboratory
growth conditions. The data are in agreement with a straightforward model of antagonistic
regulation by the two regulators that act independently of each other. Furthermore, screening
for additional activators of leuO revealed LrhA as further regulator. LrhA activates a third and
a fourth leuO promoter and shows direct DNA-binding to the leuO promoter region. The
obtained data suggest a coregulation of the leuO promoter by BglJ-RcsB, LeuO and LrhA,
reminiscent of complex regulation of leuO expression. This tightly controlled and complex
regulation is likely to be important in the response to specific, virulence-related environments.
References
1. Stratmann, T., Madhusudan, S., & Schnetz, K. Regulation of the yjjQ-bglJ operon,
encoding LuxR-type transcription factors, and the divergent yjjP gene by H-NS and LeuO. J.
Bacteriol. 190, 926-935 (2008).
2. Stratmann, T., Pul, Ü. Wurm, R., Wagner, R., & Schnetz, K. RcsB-BglJ activates the
Escherichia coli leuO gene, encoding an HNS antagonist and pleiotropic regulator of
virulence determinants. Mol. Microbiol., 83, 1109–1123 (2012).
38
Poster Presentations-Abstracts
Uneven numbers please be at your poster Wednesday, even numbers Thursday.
Poster 1:
Computational prediction of the regulatory interactions for an ECF sigma factor group
with fused C-terminal domain and co-factor
Hao Wu and Georg Fritz.
LOEWE Center for Synthetic Microbiology, Philipps University Marburg
Hans-Meerwein Str. 6, D-35032 Marburg, Germany
Evolutionary co-variation of amino acid residues has been extensively exploited to predict
conserved intra- and inter-domain interaction of proteins. Some groups of extracytoplasmic
function (ECF) sigma-factors contain a large C-terminal extension that might function as a
fused anti-sigma factor. A previous study suggested a rather complex regulatory role of this
C-terminal domain. Here we focus on ECF sigma factors of group 42, which, on top of a
fused C-terminal domain, feature a highly conserved gene encoding a YCII-related protein in
its genomic context. To gain first insights into potential regulatory interactions between these
players, we here use a computational method to predict protein-protein contacts among the
sigma-domain, the C-terminal domain and the YCII-related protein. Our results suggest a
cluster of interactions interfacing between the sigma4 domain of ECF42 and the first alpha
helix in the fused C-terminal domain. Moreover the YCII-related protein is also predicted to
contact a number of residues at the same interface, suggesting a function as a co-factor to the
regulation of the sigma factor. With this, our computational analysis provides intriguing hints
to the regulatory mechanism employed by these underexplored signaling devices.
39
Poster 2:
Implementation, analysis and mathematical modeling of ECF sigma factor-based
synthetic gene cascades in E.coli and B.subtilis
Marco Mauri1, Daniela Pinto2, Stefano Vecchione1, Hao Wu1, Thorsten Mascher2, Georg
Fritz1.
1
LOEWE Center for Synthetic Microbiology, Philipps University Marburg,
Hans-Meerwein-Str. 6C, 35043 Marburg, Germany
2
Institut für Mikrobiologie, Technische Universität Dresden
Zellescher Weg 20b, 01217 Dresden, Germany
Functionality of synthetic gene networks is often restricted by cross-reactions between
network components and physiological processes within the host. In addition, to date most
synthetic biology applications rely on a limited set of building blocks consisting of a handful
of transcriptional regulators. To overcome these restrictions, we use Extracytoplasmic
function sigma factors (ECFs) to build natural switches.
ECFs are the largest group of alternative sigma factors in bacteria and represent ideal building
blocks for synthetic network design because they are modular, universal and highly promotersequence specific.
Here, we present a first implementation of gene cascade based on ECFs in E.coli and
B.subtilis. After a quantitative study of simple ECF switches, we use a computational
modeling approach to predict the function of more complex networks. We show that our ECFbased gene cascade sequentially activates a series of target genes with a defined time delay
both in E.coli and in B.subtilis.
The characterization and the theoretical modeling of such constructs serve as rational design
principle for universal ECF-based switches in bacterial cells.
40
Poster 3:
Modulation of the behaviour of an Extracytoplasmic Function sigma factor (ECF)-based
genetic switch
Daniela Pinto1, Franziska Dürr1, Dayane Araújo1,2, Qiang Liu1,2 and Thorsten Mascher1.
1
Institüt für Mikrobiologie, Technische Universität Dresden, 01062 Dresden, Germany
Department Biology I, Ludwig-Maximilians-Universität München, Großhaderner Str. 2-4,
82152 Planegg-Martinsried, Germany
2
Bacteria rely on distinct signal transducing systems to monitor their environment and
mount fast and adequate responses. These systems can be divided into three groups: onecomponent systems, two-component systems and extracytoplasmic function sigma factors
(ECFs). While the first two systems have been extensively explored as tools for synthetic
biology, ECFs have only been slightly exploited (1).
Our starting point was a Bacillus subtilis ECF genetic switch based on the ECF41 of
Bacillus licheniformis (2). We have systematically altered both the ECF as well as its cognate
promoter and determined the behaviour of the switch. We have altered the copy number of the
ECF and/or its target promoter, the nature of the inducible promoter driving the expression of
the ECF, the stability of the ECF and the size of the DNA fragment containing the promoter.
Additionally, we have explored the effects of antisense transcription driven by constitutive
promoters of different strengths.
In order to expand the toolbox of available ECF-based switches in B. subtilis we have
additionally attempted to implement ECFs and cognate promoters from other sources. With
the aim of covering a wide taxonomical range we focused on ECFs from Bacillus cereus,
Escherichia coli, Sinorhizobium meliloti and Streptomyces venezuelae.
Collectively, our efforts generated a collection of switches with different behaviours, e.g.,
different levels of maximal activity, background activity or activation thresholds.
Additionally, this work highlighted a previously unknown limitation: that only ECFs from
closely related organisms, e.g. Firmicutes, can be implemented into B. subtilis without further
manipulation.
References
1.
Rhodius V.A., Segall-Shapiro T.H., Sharon B.D., Ghodasara A., Orlova E., Tabakh H.,
Burkhardt D.H., Clancy K., Peterson T.C., Gross C. A. and Voigt C. A. (2013), Design
of orthogonal genetic switches based on a crosstalk map of σs, anti-σs, and promoters.
Mol. Syst. Biol. 9:702.
2.
Wecke T., Halang P., Staroń A., Dufour Y.S., Donohue T.J. and Mascher T. (2012),
Extracytoplasmic function σ factors of the widely distributed group ECF41 contain a
fused regulatory domain. Microbiologyopen 1:194–213.
41
Poster 4:
Theoretical models and FRET-assays for signal transduction via switchable allosteric
modulator proteins (SAMPs) in Bacillus subtilis
Heiko Babel and Ilka B. Bischofs.
BioQuant, University of Heidelberg,
Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany
Allosteric regulation is a common motif in prokaryotic signal transduction networks. Not only
receptor-proteins are regulated allosterically by a molecular signal but also modulators can be
subject to allosteric regulation. Proteins of the Rap-modulator family in Bacillus subtilis are
regulated by a molecular peptide-signal; they are so-called switchable allosteric modulator
proteins (SAMPs). The Phr-peptide signal, which is co-expressed with a cognate Rapmodulator, is processed by an import-export circuit and finally inhibits its Rap protein.
Theoretically, the Phr-peptide could act as a Quorum-sensing signal and the Rap-modulator as
its Quorum-sensing receptor. However it is currently not known how signals are processed via
SAMP-based receptors.
To address this issue we developed a mathematical model to systematically identify possible
influencing parameters of SAMP-signal processing1. In enzymatic SAMPs two allosteric
modes determine signal-transduction. They can implement diverse switching-behaviors that
differ in amplitude, Hill-coefficient and half-maximal signal-concentration. Interestingly
optimal information-processing in SAMPs also depends on an optimal modulator-to-regulator
stoichiometry which is shaped by the pathway activity and the allosteric modes of the
modulator.
Because it is still unclear how Phr-peptides are processed in vivo, we developed a novel
FRET-sensor for the RapA-modulator of B. subtilis. The sensor is able to detect the PhrAsignal selectively. In conjunction with the mathematical model, we find that the RapAmodulator forms a trimolecular complex with the PhrA-signal and the Spo0F-regulator. Also
RapA seems to employ only one allosteric mode, which agrees with biochemical studies 2 and
possibly could influence the optimal modulator-regulator ratio.
References
1.
Babel, H. & Bischofs, I. B. Molecular and Cellular Factors Control Signal
Transduction via Switchable Allosteric Modulator Proteins (SAMPs). BMC Syst. Biol.
(2016).
2.
Diaz, A. R. et al. Bacillus subtilis RapA phosphatase domain interaction with its
substrate Spo0F~P and inhibitor PhrA peptide. J. Bacteriol. 194, 1378–88 (2012).
42
Poster 5:
Regulation of gene expression by small sRNA
Dipl.-Ing. Lyubov Kyselova.
Max Planck Institute for Dynamics of Complex Technical Systems,
Research Group: Experimental Systems Biology
Sandtorstr. 1, 39106 Magdeburg, Germany
Many genes are essential for the cell growth and biosynthesis. Their deletion may lead to
strong or complete growth inhibition. sRNA (small regulatory RNA) allow the dynamic
switch off of genes. Small RNAs interact with the mRNA by direct base pairing, which leads
in most cases to the destabilization of the mRNA and to the inhibition of translation. A major
advantage of this approach in comparison to the chromosomal gene deletion is that sRNA
genes can be regulated at any point in time, allowing dynamic process control [1].
Synthetic sRNA is cloned into an inducible plasmid [2]. The construct consists of the target
sequence (reverse complementary to the target mRNA), a MicC scaffold sequence and a
terminator site. By adding the inducer, the promoter is activated and the small RNA is
synthesized. Several target sequences can be cloned in the plasmid, so that various genes can
be controlled simultaneously.
The gene lacZ was selected as target, because its activity is easy to determine. The activity of
lacZ after induction was significantly smaller. This method was used to block translation of
gene ackA in a next step. The expression of ackA was 4 times smaller in induced strain.
Therefore the described method is a good possibility to decrease gene expression. Further task
is to apply this method in respect to production of succinate in E.coli [3].
References
1. Na et al. Metabolic engineering of Escherichia coli using synthetic small RNAs. Nature
Biotechnol. 31 (2):170-174), 2013
2. Yoo et al. Design and use of synthetic regulatory small RNAs to control gene expression in
Escherichia coli. Nature Protocols 8, 1694-1707, 2013
3. Sánchez et al. Novel pathway engineering design of the anaerobic central metabolic
pathway in Escherichia coli to increase succinate yield and productivity. Metabolic
engineering 7, 229-239, 2005
43
Poster 6:
Regulatory interactions between Corynebacterium glutamicum and the prophage CGP3
Max Hünnefeld, Eugen Pfeifer and Julia Frunzke.
Institute of Bio- und Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich,
52425 Jülich, Germany
Virus-derived DNA represents a predominant cause for strain-specific differences within a
bacterial species. However, the integration of these DNA elements into the genome and into
host regulatory circuits requires a stringent regulation.
The genome of the Gram-positive soil bacterium Corynebacterium glutamicum
ATCC 13032 contains three prophages (CGP1-3). Among those, the large, cryptic prophage
element CGP3 covers almost 6 % of the entire genome (~187 kbp) and is still inducible [1].
Prophage activation can be triggered both spontaneously and in an SOS-dependent manner
[2]. Hitherto, current studies focus on the investigation of the molecular mechanisms
underlying the control of prophage induction in C. glutamicum and its regulatory interaction
with the host.
In recent studies we identified the small nucleoid-associated protein CgpS (CgpS:
C. glutamicum prophage silencer), which was shown to act as an essential silencer of cryptic
prophage elements in C. glutamicum [3]. ChAP-Seq experiments in combination with EMSA
studies revealed that CgpS binds to AT-rich DNA and represses gene expression of mainly
horizontally acquired genomic regions. Counteraction of CgpS activity by overexpression of
the N-terminal oligomerization domain resulted in a severe growth defect and a highly
increased frequency of CGP3 induction leading to cell death.
In recent attempts, we aimed at the identification of further transcriptional regulators
interacting with CGP3. Interestingly, DNA affinity chromatography using promoter regions
of various prophage genes revealed several host regulatory proteins binding to the CGP3
element. Among those, we identified prominent regulators of global stress responses and
central carbon metabolism. These proteins illustrate the tight regulatory interaction of the host
and its prophage. Current studies are aiming at a further functional analysis of selected
candidates and their impact on CGP3 control.
References
1.
2.
3.
Helfrich, S., et al., Live cell imaging of SOS and prophage dynamics in isogenic
bacterial populations. Mol Microbiol, 2015. 98(4): p. 636-50.
Nanda, A.M., K. Thormann, and J. Frunzke, Impact of spontaneous prophage
induction on the fitness of bacterial populations and host-microbe interactions. J
Bacteriol, 2015. 197(3): p. 410-9.
Pfeifer, E., et al., Silencing of cryptic prophages in Corynebacterium glutamicum. in
revision, 2016.
44
Poster 7:
Deciphering the Response of Corynebacterium glutamicum to Oxygen Deprivation
Julian Lange1, Tobias Busche2, Jörn Kalinowski2, Ralf Takors1, Bastian Blombach1.
1
2
Institute of Biochemical Engineering, University of Stuttgart, Stuttgart, Germany
Center for Biotechnology (CeBiTec), University of Bielefeld, Bielefeld, Germany
Background. Bacteria encounter varying oxygen concentrations in manifold situations e.g.
in their natural habitat and especially in large scale industrial processes evoking viability and
production deficiencies (1). These fluctuations range from aerobic via micro-aerobic to
anaerobic conditions. To adapt to the changing environment bacteria have to remodel their
entire metabolism (2, 3). Despite its relevance for pathogenicity and pharmaceutical and biobased production processes, the molecular events during these transitions are poorly
understood. To address this question, we systematically investigate the adaptation of the
industrially relevant Corynebacterium glutamicum to such altering conditions.
Methods. A “triple-phase” batch bioprocess with C. glutamicum was established that
depicts the three successive phases (aerobiosis, micro-aerobic interface and anaerobiosis) in a
single bioreactor. Throughout the process, samples were withdrawn and analyzed for substrate
consumption, organic acid production, enzyme activities and intracellular metabolites and
additionally used for whole transcriptome analysis by RNA-sequencing.
Results. The bacterium’s physiological changes delineated a decreasing growth rate at
increasing oxygen limitation. L-lactic acid, succinic acid and acetic acid were the main
fermentation products secreted to the culture supernatant, interestingly in a manner, that their
respective differential product yields clearly bordered each process phase. RNA Sequencing
analysis revealed differential expression of 1421 genes. Notably, under anaerobic conditions
translation as well as transcription itself was tightly downregulated, which was not yet
prominent under micro-aerobiosis. Pentose phosphate pathway activity was found to be
regulated not on a transcriptional but presumably solely on a metabolic level. Differential
expression of 34 transcriptional regulators may include yet unknown oxygen sensors.
Conclusion. The established bioprocess is an elegant approach for the systematic
understanding of C. glutamicum’s adaptation to a progressive oxygen deprivation. Deeper
analysis of the RNA-sequencing data especially focusing on regulators and the network’s
hierarchy could reveal novel targets for strain optimization or even pharmaceutical
applications.
References
1. Blombach B, Riester T, Wieschalka S, Ziert C, Youn J-W, Wendisch VF, Eikmanns BJ.
2011. Appl. Environ. Microbiol. 77:3300–10.
2. Patschkowski T, Bates DM, Kiley PJ. 2000. p. 61–78. In Storz, G, Hengge-Aronis, R
(eds.), Bacterial stress responses. ASM Press, Washington, D.C.
3. Bunn HF, Poyton RO. 1996. Physiol. Rev. 76:839–885.
45
Poster 8:
From substrate specificity to promiscuity: molecular analysis of a hybrid ABC
transporter
L. Teichmann, C. Chen & E. Bremer.
Laboratory for Microbiology, Department of Biology, Philipps-University Marburg,
Karl-von-Frisch Str. 8, D-35043 Marburg, Germany
The two closely related ABC transporters OpuB and OpuC (osmoprotectant uptake) are
crucial for acquiring a variety of compatible solutes under osmotic and temperature stress
conditions in Bacillus subtilis [1]. Whereas the substrate binding protein OpuCC recognizes a
broad spectrum of compatible solutes, its 70% sequence-identical paralogue OpuBC only
binds choline [2] [3]. This raises the question about the molecular determinants governing the
strikingly different substrate specificities of the OpuB and OpuC systems. In this study we
used molecular “micro-surgery” to genetically substitute the OpuBC substrate binding protein
of the OpuB transporter with that (OpuCC) of the OpuC system in order to analyze whether
the hybrid ABC-transporter OpuB::CC is functional and what its substrate specificity might
be. Physiological experiments showed that the hybrid OpuB::CC ABC system is functional
and able to import all compatible solutes previously transported exclusively by OpuC. Hence,
by implanting the ligand binding protein of an ABC transporter with a broad substrate
spectrum, we were able to synthetically convert OpuB into a promiscuous ABC transporter.
OpuC exhibits high affinity towards glycine betaine (Km= 4.8 µM) and possesses substantial
transport capacity (vmax= 100 nmol min-1 mg protein-1), whereas the hybrid transporter
exhibits a weaker transport capacity for this substrate (vmax = 20.2 nmol min-1 mg protein-1)
but shows the same high affinity (Km= 5.6 µM).
By genetic enrichment, we screened for suppressor mutants that showed increased
transport capacity via the hybrid OpuB::CC system by providing enhanced osmostress
protection of B. subtilis at a very low external glycine betaine concentration. Indeed, several
suppressor mutants were found that exhibited enhanced transport capacity (vmax= 92-99 nmol
min-1 mg protein-1). The corresponding mutations were mapped to the coding region of gbsR,
which encodes a repressor (GbsR) of the opuB operon. The studied mutant gbsR genes encode
GbsR protein variants carrying only single amino acid substitutions. These mutant GbsR
proteins are either not at all, or only partially functional and thereby enhance opuB
transcription through a less tight negative transcriptional control. These mutant variants give
important clues with respect to the functioning of this repressor protein when they were
projected onto an in silico generated model of the GbsR structure.
References
[1] Hoffmann & Bremer (2011) J Bacteriol 193: 1552-1562
[2] Pittelkow et al., (2011) J Mol Biol 411: 53-67
[3] Du et al., (2011) Biochem J 436: 283-289
46
Poster 9:
The cellular function and localization of the cyclic-di-GMP phosphodiesterase PdeL
Cihan Yilmaz and Karin Schnetz.
Institute for Genetics, University of Cologne, Zülpicher Str. 47a, 50674 Cologne
Cyclic-di-GMP is a universal bacterial second messenger for the control of a variety of
lifestyle parameters including the regulation of biofilm formation, motility, and virulence [1].
PdeL is a cyclic-di-GMP phosphodiesterase and putative transcription regulator in E. coli
which harbors a N-terminal FixJ/NarL-type DNA-binding domain (PdeLHTH) and a Cterminal, enzymatically active EAL-domain (PdeLEAL) [2]. So far PdeL has been
characterized in respect of its enzymatic activity and its role in cyclic-di-GMP hydrolysis.
However, deletion of pdeL has no phenotype and does not cause high cellular levels of cyclicdi-GMP, unlike deletion of the major phosphodiesterase pdeH. Further, pdeL expression is
repressed by the global transcription regulator H-NS. We performed a functional analysis of
PdeL by different experimental approaches including analysis of the cellular localization as
well as its role in motility and biofilm formation. We analysed the cellular localization of
PdeL, by plasmidic expression of fusions of full length PdeL and its individual domains to the
fluorescent protein mVenus. This demonstrated that the full length protein, but not the
individual domains, co-localizes with the nucleoid. Nucleoid localization depends on
dimerization of the C-terminal domain for which the native C-terminal domain can be
substituted by another C-terminal dimerization domain to the N-terminal PdeLHTH DNAbinding domain. Alanine mutagenesis of PdeL supports the relevance of PdeLHTH in nucleoid
association while mutagenesis of the PdeLEAL domain had only slight effects. Plasmidic
expression of fluorescent PdeL fusion proteins affected cell growth and size as well as
chromosomal organization. Further, PdeL expression decreases motility and increases biofilm
formation independent of the PdeL phosphodiesterase activity, which is contrary to known
effects that changes in cellular cyclic-di-GMP levels have on motility and biofilm formation
in E. coli [3]. Taken together these findings indicate a possible role of PdeL as a cyclic-diGMP dependent transcription regulator that may control expression of genes involved in
chromosome organization as well as motility and/or biofilm formation.
References
1.
Römling, U., M.Y. Galperin, and M. Gomelsky, Cyclic di-GMP: the First 25 Years of
a Universal Bacterial Second Messenger. Microbiology and Molecular Biology
Reviews, 2013. 77(1): p. 1-52.
2.
Sundriyal, A., et al., Inherent Regulation of EAL Domain-catalyzed Hydrolysis of
Second Messenger Cyclic di-GMP. Journal of Biological Chemistry, 2014. 289(10): p.
6978-6990.
3.
Serra, D.O. and R. Hengge, Stress responses go three dimensional - the spatial order
of physiological differentiation in bacterial macrocolony biofilms. Environ Microbiol,
2014. 16(6): p. 1455-71.
47
Poster 10:
Elucidation of the metabolic pathway for SDS degradation and its regulation in
Pseudomonas aeruginosa
Gianna Panasia and Bodo Philipp.
Institute of Molecular Microbiology and Biotechnology, University of Münster,
Corrensstr. 3, D-48149 Münster, Germany
Pseudomonas aeruginosa is an ubiquitous environmental bacterium that can act as an
opportunistic and nosocomial pathogen. Its metabolic versatility and pronounced resistance
against toxic chemicals enables it to survive and grow in hygienic environments. Thereby, it
can cause outbreaks in clinical settings. In this context, P. aeruginosa is able to use the
common and toxic detergent sodium dodecyl sulfate (SDS) as growth substrate [1]. Previous
studies demonstrated cell aggregation of P. aeruginosa during growth with SDS as a specific
survival strategy [2]. In the current model, the proposed stress sensor SiaA, a putative ser/thr
phosphatase, acts together with SiaD, a di-guanylate cyclase, as a signal transduction module
regulating SDS induced cell aggregation in a c-di GMP and RsmA dependent manner [3,4].
However, little is known about the metabolic pathway for the SDS degradation and its
regulation. Thus, we address the unknown enzymatic steps and the respective gene
expression.
Based on a DNA-microarray analysis comparing SDS- and succinate-grown cells several
genes with a plausible function in SDS degradation were identified [3]. Thereby, a special
alcohol dehydrogenase, encoded by exaA, belonging to the ethanol oxidation system and an
unknown putative oxidoreductase, encoded by PA0364, are induced. Single deletion mutants
of these genes still grow with SDS but have different phenotypes in cell aggregation behavior
in comparison to the wild type. Remarkably, the respective double knockout mutant is not
able to grow with SDS. Complementation with either exaA or PA0364 restored growth with
SDS similar to the respective single mutants. This indicates a significant participation of both
genes in SDS degradation.
In general, the analysis of this catabolic pathway on the transcriptional and posttranslational
level, will give insight in how P. aeruginosa copes to grow with SDS and potentially other
similar toxic compounds.
References
[1] Klebensberger J. et al. (2006). Cell aggregation of Pseudomonas aeruginosa strain
PAO1 as an energy-dependent stress response during growth with sodium dodecyl-sulfate.
Arch. Microbiol. 185:417-2.
[2] Klebensberger J. et al. (2007). Detergent-induced cell aggregation in subpopulations of
Pseudomonas aeruginosa as a preadaptive survival strategy. Environ. Microbiol. 9(9):224759.
[3] Klebensberger J. et al. (2009). SiaA and SiaD are essential for inducing autoaggregation
as a specific response to detergent stress in Pseudomonas aeruginosa. Environ. Microbiol.
11(12):3073-86.
48
[4] Colley B. et al. (2016). SiaA/D interconnects c-di-GMP and RsmA signaling to coordinate
cellular aggregation of Pseudomonas aeruginosa in response to environmental conditions.
Front. Microbiol. 7:179 doi: 10.3389/fmicb.2016.00179.
Poster 11:
The role of the phosphodiesterase NbdA in NO-induced dispersal of Pseudomonas
aeruginosa
Martina Rüger1, Sabrina Heine2, Michael Entian2, Yi Li3, Karin Sauer3 and Nicole
Frankenberg-Dinkel1, 2.
1
Department of Biology, Microbiology, Technical University Kaiserslautern, Kaiserslautern,
Germany
2
Physiology of Microorganisms, Ruhr-University Bochum, Bochum, Germany
3
Department of Biological Sciences, Binghamton University, Binghamton, New York, USA
Pseudomonas aeruginosa is an important opportunistic human pathogen causing a variety of
nosocomial infections including pneumonia, sepsis, catheter and urinary tract infections. The
bacterium has become a model system for biofilm research because of its resistance to
conventional antibiotics, host antimicrobial effector mechanisms and its ability to form
biofilms. Dispersal is considered as the last step of the biofilm life cycle being a process used
by bacteria to transfer from sessil to motile lifestyle. Changes in c-di-GMP levels have been
shown to be associated with biofilm detachment in a number of different bacteria. The
signalling molecule nitric oxide (NO) induces biofilm dispersal through stimulation of c-diGMP (bis-(3‘-5‘)-cyclic dimeric guanosine monophosphate) degrading phosphodiesterase
(PDE) activity. We characterised the membrane-bound proteins MucR and NbdA (NOinduced biofilm dispersion locus A) regarding their role in NO-induced dispersal. Both share
an identical domain organisation consisting of MHYT-GGDEF-EAL. Inactivation of mucR
impaired biofilm dispersal in response to glutamate and NO while deletion of nbdA only
negatively affected biofilm detachment upon exposure to NO. Biochemical analyses of
recombinant protein variants lacking the membrane-anchored MHYT-domain revealed NbdA
being an active PDE. In contrast, MucR showed diguanylate cyclase and PDE activity in vitro
[1]. Interestingly, a P. aeruginosa strain lacking both, nbdA and mucR displayed enhanced
biofilm formation under tested conditions, whereas ΔnbdA and ΔmucR single mutants showed
rather wild type like phenotype. The hyper biofilm formation phenotype of the ΔnbdA ΔmucR
double mutant might be due to highly increased c-di-GMP levels caused by lacking PDE
activity of NbdA and MucR. These results suggest either a functional redundancy or possible
interdependence of both proteins.
References
[1] Li Y, Heine S, Entian M, Sauer K, Frankenberg-Dinkel N. (2013) NO-induced biofilm
dispersion in Pseudomonas aeruginosa is mediated by an MHYT domain-coupled
phosphodiesterase. J Bacteriol. 195:3531-3542.
49
Poster 12:
Osmotic responsive transcription of ectoine biosynthetic genes from Pseudomonas
stutzeri is transferable to a non-ectoine producing surrogate host
Laura Czech, Philipp Hub, Florian Kindinger, Oliver Dähn, Nadine Stöveken & Erhard
Bremer.
Laboratory for Microbiology, Department of Biology, Philipps-University Marburg,
Karl-von-Frisch Str. 8, D-35043 Marburg, Germany
A large number of microorganisms posses the ability to produce and accumulate the
compatible solutes ectoine and 5-hydroxyectoine in response to an osmotic upshift to
maintain vital turgor and to sustain cell growth under osmotically unfavorable conditions. The
production of these chemical chaperones [1] has already been described in the soil bacterium
Pseudomonas stutzeri A1501 and depends on the salt-inducible ectABCD_ask operon [2].
Analysis of an P. stutzeri A1501 ectA-ectB-lacZ-reporter gene fusion in a non-ectoine
producing Escherichia coli heterologous host strain showed a highly dynamic response of
promoter activity, when cells faced step-wise increases in the external salinities. Activity of
the ect promoter could not only be stimulated by high NaCl concentration but also by other
osmolytes, demonstrating that this promoter responds to a true osmotic stress signal. And not
just to salt. This expression pattern is neither modulated by the nucleoid-structuring protein
H-NS, nor by the alternative sigma factor (RpoS) controlling the general stress response of E.
coli.
Successive truncations of the region up-stream of ectA resulted in a reporter gene fusion, that
exhibits a hybrid promoter consisting of a weak -35 region stemming from the backbone of
the plasmid and the original -10 region. This observation, together with sequence comparison
of the regions in front of ectA derived from different Pseudomonas strains allowed the
determination of the promoter consisting of a nearly perfect -35 region, an 18 bp spacer
sequence and a -10 region unusually rich in C-G base pairs. We introduced different point
mutations into the ect promoter in order to analyze its response to changes in external salinity.
Creation of a perfect SigA promoter resulted in the loss of the salt-induction and a 16-fold
increase in the basal activity.
Collectively, our data suggest that the finely tuned osmotic control of the P. stutzeri
ect promoter is dependent on its unusual 18 bp spacer and the abnormal -10 region. Changes
in DNA supercoiling might be a contributing factor as well. Since osmotic control of ect gene
expression also occurs in a non-ectoine producing surrogate host organism E. coli, it seems to
us that no specific regulatory protein is required to drive osmostress-responsive ectABCD_ask
gene expression. A possible mechanism will be discussed.
References
1.
Pastor, J. M. et al. (2010) Biotechnol Adv. 28: 782–801.
2.
Stöveken, N. et al. (2011) J Bacteriol. 193: 4456–4468.
50
Poster 13:
A special role for acetate kinase AckA in the regulation of CiaR activity in the absence
of the cognate kinase CiaH.
Anne Sexauer and Reinhold Brückner.
Department of Microbiology, University of Kaiserslautern,
Paul-Ehrlich Str. 24, D-67663 Kaiserslautern, Germany
The two-component regulatory system CiaRH of Streptococcus pneumoniae is implicated
in competence regulation, ß-lactam resistance, maintenance of cell integrity, bacteriocin
production, host colonization, biofilm formation and virulence. A surface-exposed protease
HtrA and five small noncoding csRNAs, all directly controlled by CiaR, are the major
mediators of these phenotypes (4). Expression analyses indicated that the CiaR system is
highly active under a variety of growth conditions, not showing an on-off switch typical for
many other two-component systems. In addition, depending on the growth conditions, CiaR is
active in the absence of its cognate kinase CiaH, although phosphorylation of CiaR is required
for DNA binding and gene regulation (2). To determine if acetyl phosphate could be the
alternative phosphodonor, genes involved in pyruvate metabolism were disrupted to alter
cellular levels of acetyl phosphate. In a CiaH-deficient strain devoid of pyruvate oxidase
SpxB, phosphotransacetylase Pta, and acetate kinase AckA, very low acetyl phosphate levels
were observed, and in paralell, strongly reduced CiaR-mediated gene expression (3). These
results clearly indicate that alternative phosphorylation of CiaR is dependend on acetyl
phosphate. A surprising synthetic lethality was detected in CiaH-deficient strains producing
high levels of acetyl phosphate. The ackA gene could not be inactivated. Furthermore, a strain
producing half of the acetyl phosphate level of the wild type lacking AckA showed a 13-fold
increase in CiaR-dependent promoter activation. In the absence of AckA, CiaR appears to be
extremely activated, provided acetyl phosphate is present and the CiaH kinase is absent. It
appears therefore, that alternative phosphorylation of CiaR is affected negatively by AckA. In
a first step to determine if this negative regulation is direct, the adenylate reconstituion
Escherichia coli two hybrid system (1) was applied to detect interaction between CiaR and
AckA. The results of these experiments clearly demonstrated contact between these two
proteins. The surprising link of the response regulator CiaR to a metabolic enzyme, AckA,
adds another level of complexity to two-component regulatory system regulation.
References
1. Battesti, A., and E. Bouveret. 2012. The bacterial two-hybrid system based on adenylate
cyclase reconstitution in Escherichia coli. Methods 58:325-334.
2. Halfmann, A., A. Schnorpfeil, M. Müller, P. Marx, U. Günzler, R. Hakenbeck, and R.
Brückner. 2011. Activity of the two-component regulatory system CiaRH in
Streptococcus pneumoniae R6. J. Mol. Microbiol. Biotechnol. 20:96-104.
3. Marx, P., M. Meiers, and R. Brückner. 2015. Activity of the response regulator CiaR in
mutants of Streptococcus pneumoniae R6 altered in acetyl phosphate production. Front.
Microbiol. 5:772.
51
4. Schnorpfeil, A., M. Kranz, M. Kovács, C. Kirsch, J. Gartmann, I. Brunner, S. Bittmann,
and R. Brückner. 2013. Target evaluation of the non-coding csRNAs reveals a link of the
two-component regulatory system CiaRH to competence control in Streptococcus
pneumoniae R6. Mol. Microbiol. 89:334-349.
Poster 14:
Transport and regulation by the alternative anaerobic C4-dicarboxylate-transporters
DcuA, DcuB and DcuC in Escherichia coli
Alexander Strecker and Gottfried Unden.
Institute for Microbiology and Wine Research
Johannes Gutenberg-University Mainz, Germany
Escherichia coli metabolizes C4-dicarboxylates during aerobic and anaerobic growth. Under
anaerobic conditions the uptake of C4-dicarboxylates is catalyzed by the three transporters
DcuA, DcuB and DcuC [1].
DcuB forms a complex with DcuS and functions as a coregulator for DcuSR dependent gene
expression under anaerobic conditions [2]. The deletion of DcuB causes a constitutive
expression of DcuS regulated genes. The homologous transporters DcuA and DcuC show no
regulatory effect on gene expression [3].
Complexome profiling of membrane proteins, mSPINE and BACTH indicates that the DcuA,
DcuB and DcuC transporters interact and form heterocomplexes. A potential role of
DcuA/DcuB or DcuC/DcuB heterocomplexes on DcuS function was characterized.
[1] Unden, G., Strecker, A., Kleefeld, A., & Kim, O. B. (2016) EcoSal Plus, 7(1). doi:
10.1128/ecosalplus.ESP-0021-2015.
[2] Wörner, S., Strecker, A., Monzel, C., Zeltner, M., Witan, J., Ebert‐Jung, A., & Unden, G.
(2016). Environ Microbiol. doi: 10.1111/1462-2920.13418.
[3] Kleefeld, A., Ackermann, B., Bauer, J., Krämer, J., & Unden, G. (2009) J Biol Chem.
284(1):265-75.
52
Poster 15:
The DxxxQ phosphatase motif in the O2 sensor kinase NreB of Staphylococcus carnosus
Ann-Katrin Kretzschmar and Gottfried Unden.
Institute for Microbiology and Wine Research, Johannes Gutenberg-University Mainz
Johann-Joachim-Becherweg 15, D-55128 Mainz, Germany
In Staphylococcus carnosus the anaerobic nitrate respiration is regulated by the O2-sensitive
two component system NreB-NreC and the nitrate sensor NreA [1, 2]. The sensor kinase
NreB is autophosphorylated at His159 and the phosphoryl group is transferred to the response
regulator NreC. NreA modulates NreB activity by nitrate dependent interaction resulting in a
combined oxygen/nitrate sensing complex [1, 2].
NreB contains a DxxxQ motif adjacent to the phospho-accepting His159. In the nitrate sensor
kinase NarX of E. coli this motif is crucial for kinase and phosphatase activity of the sensor
[3, 4]. The significance of the Asp160 and Gln164 residues of the DxxxQ motif was tested in
vivo and in vitro by mutation.
The data suggest that the motif is important for autophosphorylation of NreB and
dephosphorylation of NreC, both in response to O2 and to nitrate regulation by NreA.
References:
[1] Nilkens, S., Koch-Singenstreu, M., Niemann, V., Götz, F., Stehle, T., Unden, G. (2014)
Mol
Microbiol
91:381-393
[2] Niemann, V., Koch-Singenstreu, M., Neu, A., Nilkens, S., Götz, F., Unden, G., Stehle, T.
(2014)
J
Mol
Biol
426:1539-1553
[3] Huynh, T., Noriega, C., Stewart, V. (2010) Proc Natl Acad Sci USA 107: 21140-21145
[4] Hentschel, E., Mack, C., Gätgens, C., Bott, M., Brocker, M., Frunzke, J. (2014) Mol
Microbiol 92: 1326-42
53
Poster 16:
The function of the ExxN motif of the C4-dicarboxylate sensor kinase DcuS of
Escherichia coli in signal transduction
Stefaniya Gencheva, Sebastian Wörner and Gottfried Unden.
Institute for Microbiology and Wine Research
Johannes Gutenberg-University Mainz, Germany
The two component system DcuSR of E. coli is composed of the membrane-bound histidine
kinase DcuS and the cytoplasmic response regulator DcuR. Under anaerobic conditions DcuS
forms a DcuS/DcuB sensor complex with the transporter DcuB. This complex formation is
essential for conversion of DcuS to the C4-dicarboxylate responsive form and the activation of
the kinase domain of DcuS [1, 2].
In vitro studies show a positive effect of non complexed DcuS on DcuR dephosphorylation
[3]. The DHp domain of DcuS contains a conserved ExxN phosphatase motif [4]. In vivo and
in vitro studies were performed with DcuS and ExxN mutants to study the role of the motif in
DcuS phosphorylation and dephosphorylation.
[1] Unden, G., Wörner, S., Monzel, C. (2016): In Adc Microbiol Physiol., Poole, Robert K.,
68:139–167.
[2] Steinmetz, P. A., Worner, S. Unden, G. (2014): Mol Microbiol. 94: 218–229.
[3] Janausch, I. G., Garcia-Moreno, I. Unden, G. (2002):Biochim Biophys Acta.1553: 39-56.
[4] Huynh, T. N., Noriega, C.E., Stewart, V. (2010): Proc Natl Acad Sci U S A. 107: 21140–
21145.
54
Poster 17:
Analysis of quinone mutants in respect to ArcA phosphorylation and product formation
Annika Nitzschke and Katja Bettenbrock.
MPI for dynamics of complex technical systems, Sandtor Str. 1, D-39106 Magdeburg,
Germany
E.coli is able to respond to changes in the oxygen availability through regulation of
metabolism. Two major transcription factors (TF) are responsible for this adaption, the twocomponent system ArcB/A und the global TF FNR. In contrast to FNR, the two-component
system ArcB/A reacts only indirectly to the change in oxygen supply. Rather other signals
seem to have an influence on the ArcB/A activation. The effect of the redox pool of the cell
on the activation of ArcA has already been discussed in literature, whereby the focus has been
on the redox ratio of the quinones (1). These function as electron carriers between the
dehydrogenases and oxidases of the electron transport chain (ETC). E.coli possesses three
different quinone species: ubiquinone (UQ), which is synthesized mainly during aerobic
conditions and demethylmenaquinone (DMK) und menaquinone (MK), which are synthesized
mainly during anaerobic respiration (2).
To study in more detail the function of different quinone species, strains with deletions
preventing UQ synthesis, as well as MK and/or DMK synthesis were cultured under aerobic
as well as anaerobic conditions. A special focus was on deriving a correlation between the
compositions of the quinone pool und the ArcA phosphorylation state. In contrast to the
indirect measurements of ArcA phosphorylation by reporter genes, we determined the relative
phosphorylation state of the TF directly by Phos-tag SDS-PAGE and Western Blot. The
results from the characterization of the mutants compared to the wild type strain MG1655
showed that in contrast to the in vitro results (3), in vivo, no inhibitory effect from the UQ on
the ArcA phosphorylation was observed.
Furthermore gene expression analysis under aerobic conditions showed that the ubiquinone
knockouts exhibit a “pseudo” fermentative state independent of the phosphorylation of ArcA.
This indicates that it is a problematic and error-prone to deduce the phosphorylation state of
ArcA from indirect measurements using reporter gene fusions. A comprehensive analysis of
gene expression in the three quinone deletion strains under anaerobic conditions is currently
performed. First results show, that the reduced phosphorylation of ArcA in the ubiquinone
deletion strains AV33 and AV36 influences gene expression under anaerobic conditions.
In addition the mutation preventing UQ synthesis was combined with an arcA deletion. This
strain shows qualities of a production strains with a good glucose consumption rate in spite of
not growth. Sequencing results indicate that this strain frequently acquires secondary
mutations, namely a deletion of about 16 kBp, containing inter alia ubiE and metE. We are
currently testing this strain in more detail.
References
1. Malpica, R. and Franco, B. (2004) Identification of a quinone-sensitive redox switch in the
ArcB sensor kinase (2004), Proc. Natl. Acad. Sci. USA 101(36), 13318-13323.
2. Bekker M. (2009), Respiratory electron transfer in Escherichia coli: components, energetics
and regulation,(2009)
55
3. Georgellis D. and Kwon O. (2001), Quinones as the redox signal for the arc twocomponent system of bacteria (2001), Science 292(5525), 2314-2316.
Poster 18:
Analysis of the signal transduction by the heme-based sensor kinase MsmS from
Methanosarcina acetivorans
Fiege, K.1,2, Molitor, B.2, Blasius, L.1, Querebillo, C.3,4, Hildebrandt, P.3, Laurich, C.5, Lubitz,
W.6, Rother, M.5, Frankenberg-Dinkel, N.1,2.
1
TU Kaiserslautern, Department of Microbiology, Kaiserslautern, Germany
Ruhr University Bochum, Physiology of Microorganisms, Bochum, Germany
3
TU Berlin, Institute for Chemistry, Berlin, Germany
4
School of Analytical Sciences Adlershof, Humboldt-Universität zu Berlin, Berin, Germany
5
TU Dresden, Institute for Microbiology, Dresden, Germany
6
Max-Planck-Institute for Chemical Energy Conversion, Mülheim, Germany
2
The multidomain protein MsmS from Methanosarcina acetivorans is one of the first
examples for the biochemical characterization of an archaeal sensor kinase with
autophosphorylation activity. It consists of two alternating PAS and GAF domains and a Cterminal H_ATPase domain. The second GAF domain of MsmS covalently binds a heme
cofactor via a cysteine residue. For MsmS, the redox state of the heme cofactor was shown to
influence the autophosphorylation activity of the adjacent kinase domain [1]. For the
investigation of the function of this archaeal signal transduction system and its redox sensory
function, the heme coordination structure was analyzed using UV-vis and Resonance Raman
spectroscopy. Therefore, several variants of truncated MsmS were analyzed to identify the
heme coordinating residues. First UV-vis spectroscopic analysis identified a histidine residue
as the proximal ligand for the heme cofactor. The gene msmS is encoded upstream of the
regulator protein MsrG. It is assumed that these both proteins form a two-component system.
Therefore, also the intermolecular interaction with the regulator protein was analysed.
Finally, the presented results will be discussed in the light of the putative cellular function of
the heme-based sensor kinase.
[1] Molitor, B., Stassen, M., Modi, A., El-Mashtoly, S. F., Laurich, C., Lubitz, W., Dawson, J.
H., Rother, M., and Frankenberg-Dinkel, N. (2013) A heme-based redox sensor in the
methanogenic archaeon Methanosarcina acetivorans. J. Biol. Chem. 288, 18458-18472
56
Poster 19:
A TCS is involved in the regulation of the organohalide respiration in Sulfurospirillum
spp.
Jens Esken1, Tobias Goris1, Cynthia Sharma2, Torsten Schubert1, and Gabriele Diekert1.
.
1
Department of Applied and Ecological Microbiology, University of Jena,
Philosophenweg 12, D-07743 Jena, Germany
2
Research Center for Infectious Diseases, Julius Maximilians University Würzburg, Josef
Schneider Str. 2/ D15, 97080 Würzburg, Germany
Organohalide respiration was studied in detail in the tetrachloroethene (PCE)-dechlorinating
Sulfurospirillum multivorans and S. halorespirans. The organisms display an unusual type of
long-term down-regulation of the PCE reductive dehalogenase gene (pceA) expression in the
absence of PCE (1). In close proximity to pceA, open reading frames encoding twocomponent systems (TCS) were identified. As revealed by RNA sequencing, the 40-kbp
organohalide respiration (OHR) gene region, which surrounds pceA, was transcribed only in
the presence of PCE. The expression of eight transcriptional units ceased completely, when
PCE was absent. As an exception, an operon encoding a two-component system (TCS) still
displayed a low transcript level under these conditions. The histidine kinase (HK) of the TCS
is predicted to contain seven transmembrane helices and an N-terminal domain putatively
exposed to the periplasm. This domain might serve as PCE sensor. Since PCE is a very
hydrophobic compound, its detection outside the cytoplasmic membrane appears effective.
Upstream of the gene for the HK, a response regulator is encoded. The respective gene is
disrupted by a transposase in the non-dehalogenating S. multivorans strain N and S. JPD1.
The residual OHR gene region is almost 100% identical to that of S. multivorans and S.
halorespirans. This and the results of the RNA sequencing led to the assumption that the TCS
is involved in PCE-sensing and its malfunction in S. multivorans strain N and S. JPD1 might
be the reason for the non-dehalogenating phenotype.
References
1. John et al. (2009) J Bacteriol. 191:1650-5.
57
Poster 20:
Biochemical characterization of the iron responsive regulator RirA from
Dinoroseobacter shibae
Maren Behringer, Elisabeth Härtig and Dieter Jahn.
Institute of Microbiology, University Braunschweig, Spielmannstrasse 7, D-38106
Braunschweig, Germany;
The rhizobial iron regulator RirA from Dinoroseobacter shibae belongs to the Rrf2- family of
transcription factors and is supposed to coordinate a Fe-S cluster and thereby measure iron
availability. In Rhizobium leguminosarum RirA is acting as a repressor of iron uptake systems
in the presence of iron. RirA of D. shibae was recombinantly produced and anaerobically
purified. Ligation of an Fe-S cluster was detected by UV/Vis spectroscopy. Using electron
paramagnetic spin resonance (EPR) spectroscopy and Fe-content determinations by atom
absorbance spectroscopy (AAS) one [3Fe-4S]1+ cluster as cofactor per RirA molecule was
determined. Supporting cyclic Voltammetry studies revealed, the cluster is not involved in
electron transport and showed no redox potential. DNA binding of the anaerobically purified
RirA wildtype and mutant proteins was analyzed using electro mobility shift assays (EMSA).
The RirA protein of D. shibae contains four cysteine residues which are highly conserved in
other RirA homolog proteins and might be important for Fe-S cluster formation [1]. To
specify the role of these cysteine residues for the coordination of an Fe-S cluster as cofactor,
each of the four conserved cysteine residues of RirA was changed to an alanine residue via
site directed mutagenesis of the corresponding gene. UV/Vis spectroscopy using the
anaerobically purified mutant proteins showed a reduction of the absorption at 420 nm,
indicating a loss of the Fe-S cluster.
To define the RirA regulon transcriptome analyses using DNA microarrays were performed.
First results indicate, that RirA is able to regulate napF gene expression, encoding the
ferredoxin-type protein NapF. Surprisingly, the RirA-dependent regulation was not dependent
on iron. A β-galactosidase enzyme assay was established to proof, which promoters are under
the control of the RirA regulator.
[1] Bhubhanil, S, Niamyim, P, Sukchawalit, R, and S. Mongkolsuk (2013). Cysteine
desulphurase-encoding gene sufS2 is required for the repressor function of RirA and oxidative
resistance in Agrobacterium tumefaciens. Microbiology, 160:79-90
58
Poster 21:
Adrenochrome – oxidation product of adrenaline and bacterial effector molecule
Charlotte Toulouse, Kristina Metesch, Pit Engling, Bernd Michel and Julia Steuber.
Department of Cellular Microbiology, University of Hohenheim,
Garbenstr. 30, D-70599 Stuttgart, Germany
Catecholamines such as adrenaline and noradrenaline are known to stimulate growth and
swarming of some proteobacteria like EHEC [1] or Salmonella enterica Typhimurium [2] and
also, recently shown by our group, Vibrio cholerae [3], the causative agent of the Cholera
disease. Since catecholamines are known to undergo oxidative degradation [4], we
investigated their stability during bacterial cultivation. Catecholamines can oxidize to their
corresponding aminochromes by autoxidation with O2 or by superoxide. Their formation and
effect on growth, swarming and virulence of V. cholerae was investigated.
Adrenochrome was confirmed, by LC-MS, as oxidation product of adrenaline during
cultivation of Vibrio cholerae.
Isolated V. cholerae membranes contribute to the adrenochrome formation. The central
respiratory membrane protein Na+-NADH:quinone oxidoreductase (Na+-NQR) of V. cholerae
enhances the adrenochrome formation upon addition of NADH. We explain this through the
production of superoxide during electron transfer, since no adrenochrome formation takes
place under anaerobic conditions or when superoxide dismutase (SOD) was added.
We suppose that the availability of O2 and particularly reactive oxygen species (ROS) in
solution determines the amount of adrenochrome formed. Hence, not only the concentration
of the signaling molecule adrenaline is affected by the O2 partial pressure during growth of V.
cholerae, but also adrenochrome, a molecule with putative function in signaling, is formed.
We assume that during the respiratory burst of immune cells, aminochrome formation is also
enhanced. Finally, we show that adrenochrome alters swarming and growth of some
proteobacteria including V. cholerae depending on the choice of medium.
References
1.
Clarke, M.B., et al., The QseC sensor kinase: a bacterial adrenergic receptor.
Proceedings of the National Academy of Sciences of the United States of America,
2006. 103(27): p. 10420-5.
2.
Moreira, C.G., D. Weinshenker, and V. Sperandio, QseC mediates Salmonella
enterica serovar typhimurium virulence in vitro and in vivo. Infection and immunity,
2010. 78(3): p. 914-26.
3.
Halang, P., et al., Response of Vibrio cholerae to the Catecholamine Hormones
Epinephrine and Norepinephrine. Journal of bacteriology, 2015. 197(24): p. 3769-78.
4.
Bors, W., et al., The involvement of oxygen radicals during the autoxidation of
adrenalin. Biochimica et biophysica acta, 1978. 540(1): p. 162-72.
59
Poster 22:
Mechanism and function of non-standard circadian clock systems in cyanobacteria
Christin Köbler1, Anja Dörrich2, Anika Wiegard3, Annegret Wilde1.
1
Albert-Ludwigs-University Freiburg, Germany; 2Justus-Liebig-University
Germany; 3Heinrich-Heine-University Duesseldorf, Germany
Giessen,
Through the rotation of the earth, all organisms are subjected to daily environmental
changes. To facilitate their adaption, many organisms generate an internal rhythm with a
period length of around 24 hours, referred to as circadian rhythm. Within cyanobacteria
Synechococcus elongatus PCC7942 (hereafter Synechococcus) functions as model organism
for the circadian clock. Its clock consists of three core proteins: KaiA, KaiB and KaiC.
Phosphorylation and dephosphorylation of KaiC maintains the timing mechanism. The
circadian system is rather well understood in Synechococcus, but can be quite diverse in other
cyanobacteria. Some species lack kai genes, whereas others acquired additional kai homologs.
Synechocystis sp. PCC 6803 (hereafter Synechocystis) for example, contains two additional
homologs of the kaiB and kaiC genes. However, it is suggested that they form other time
keeping mechanisms, but their function is still unknown. Using deletion mutants for each
additional kai gene we want to elucidate their respective phenotype and uncover their
function. Additionally, we want to explore a putative cross talk between the non-canonical
Kai homologs via interaction studies. Furthermore, the core oscillator of Synechocystis seems
to employ a different output signaling pathway than Synechococcus and until now, most of
this pathway remains unsolved. We aim to identify new components of the output signaling
pathway by establishing a fluorescence based gene reporter system for mutant screening, as
well as, screening for the impaired growth of oscillator deficient strains under dark-light
cycles. Finally, we want to determine interaction partners and position of the newly identified
components within the regulatory network of the circadian clock.
60
Poster 23:
Characteristics of a SoxR-based single cell NADPH biosensor in Escherichia coli
Alina Spielmann, Meike Baumgart and Michael Bott
IBG-1: Biotechnology, Institute of Bio- and Geosciences, Forschungszentrum Jülich,
D-52425 Jülich, Germany
NADPH-dependent alcohol dehydrogenases and ketoreductases play an important role in
industrial biotechnology, especially for the production of chiral alcohols. Therefore, the
improvement of these enzymes for in-vivo and in-vitro applications is of high interest.
Typically, screenings of mutagenized libraries of such enzymes involve dedicated assays for a
certain substrate or product. We recently reported an alternative technology for highthroughput in-vivo screening of NADPH-dependent dehydrogenases using suitable
Escherichia coli reporter strains. The screening system is based upon the genetically encoded
NADPH biosensor pSenSox, which exploits the transcriptional regulator SoxR, its target
promoter PsoxS, and the reporter gene eyfp, encoding a fluorescent GFP derivative [1]. SoxR
activity is controlled by the redox status of its [2Fe2S] cluster: in the oxidized state SoxR is
active and triggers eyfp expression, in the reduced state it is inactive. The enzymatic reduction
and inactivation of oxidized SoxR is NADPH dependent. An increased cellular NADPH
demand lowers SoxR reduction and causes increased expression of eyfp. In this way, the
plasmid-based biosensor pSenSox is able to sense intracellular NADPH availability. For
example, E. coli cells expressing an NADPH-dependent alcohol dehydrogenase of
Lactobacillus brevis become fluorescent when the substrate methyl acetoacetate is reduced to
(R)-methyl 3-hydroxybutyrate. Under suitable conditions, the specific fluorescence of the
cells correlates with the activity of the NADPH-dependent dehydrogenase to be analyzed. In
this way, the system allows high-throughput screening of large dehydrogenase libraries using
fluorescence-activated cell sorting (FACS). As a prerequisite for further improvements of the
screening system, the characteristics of pSenSox-based NADPH-sensing were further
characterized. The redox-cycling drugs paraquat and menadion were found to trigger the
pSenSox-based eyfp fluorescence. The proteins RseC and RsxABCDGE were reported to be
involved in NADPH-dependent SoxR reduction [2] and should be relevant for the pSenSoxbased sensor response. Therefore, ΔrseC und ΔrsxABCDGE deletion mutants of E. coli were
constructed as well as strains overexpressing rseC and rsxABCDGE. It could be shown that
the eYFP fluorescence signal was increased in the deletion strains compared to the reference
strain, whereas it was decreased in the overexpression strains. These results support the
finding that SoxR is reduced by RseC and RsxABCDGE and that the levels of these proteins
influence the ratio between oxidized active SoxR and reduced inactive SoxR.
References
1. Siedler, S., Schendzielorz, G., Binder, S., Eggeling, L., Bringer, S., & Bott, M. (2014).
SoxR as a single-cell biosensor for NADPH-consuming enzymes in Escherichia coli. ACS
Synth Biol, 3(1), 41-47.
2. Koo, M.S., Lee, J. H., Rah, S. Y., Yeo, W. S., Lee, J. W., Lee, K. L., Roe, J. H. (2003). A
reducing system of the superoxide sensor SoxR in Escherichia coli. EMBO J, 22(11), 26142622.
61
Organization:
Dr. Reinhold Brückner and Anne Sexauer
Microbiology, University of Kaiserslautern
Paul-Ehrlichstrasse 24
67663 Kaiserslautern
Germany
62