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Transcript of content · content Bad Bergzabern ... Martina Rüger1, Sabrina Heine2, Michael Entian2, ... Anne...

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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

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Bad Bergzabern- map

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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

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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

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14:00 Excursion to Burg Berwartstein, Erlenbach t

18:15 Dinner

20:00 Special Lecture II 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 Super-

resolution 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 rate and H-NS/StpA

repression in Escherichia coli

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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

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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 Pinto

2, Stefano Vecchione

1, Hao Wu

1, Thorsten Mascher

2, Georg

Fritz1.

Poster 3:

Modulation of the behaviour of an Extracytoplasmic Function sigma factor (ECF)-based

genetic switch Daniela Pinto

1, Franziska Dürr

1, Dayane Araújo

1,2, Qiang Liu

1,2 and Thorsten Mascher

1.

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 Busche

2, Jörn Kalinowski

2, Ralf Takors

1, Bastian Blombach

1

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.

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Poster 11:

The role of the phosphodiesterase NbdA in NO-induced dispersal of Pseudomonas

aeruginosa

Martina Rüger1, Sabrina Heine

2, Michael Entian

2, Yi Li

3, Karin Sauer

3 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 Esken

1, Tobias Goris

1, Cynthia Sharma

2, Torsten Schubert

1, and Gabriele Diekert

1.

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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 Dörrich

2, Anika Wiegard

3, Annegret Wilde

1

Poster 23:

Characteristics of a SoxR-based single cell NADPH biosensor in Escherichia coli

Alina Spielmann, Meike Baumgart and Michael Bott

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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

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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, Karl-

von-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 Gram-

positive 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.

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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.

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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 co-

culture 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 co-

cultures 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 4-

GB 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 co-

cultures 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.

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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 Gram-

positive 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-0010-

2014

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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.

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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 Gram-

positive 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 LysR-

type 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.

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Metabolic engineering to direct evolution in Corynebacterium glutamicum

Andreas Schwentner1, E. Hoffart

1, T. Busche

2, C. Rückert

2, J. Kalinowski

2, R. Takors

1, B.

Blombach1

1University of Stuttgart, Institute of Biochemical Engineering, Stuttgart, Germany

2Bielefeld 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.

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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.

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Large-scale reduction of the Bacillus subtilis genome: Consequences for the

transcriptional network, resource allocation, and metabolism

Daniel Reuß1, Josef Altenbuchner

2, Ulrike Mäder

3, Hermann Rath

3, Till Ischebeck

4, Bingyao

Zhu1, Stefan Klumpp

5, Fabian Commichau

1, Uwe Völker

3 and Jörg Stülke

1

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.

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22

Session 3: Second Messengers, bacterial differentiation, and antibiotic

production

Cyclic di-nucleotide signalling in bacterial differentiation and antibiotic production

Natalia Tschowri1, Maria A. Schumacher

2, Susan Schlimpert

3, Naga babu Chinnam

2, Richard

G. Brennan2 and Mark J. Buttner

3

1Institut fur Biologie - Mikrobiologie, Humboldt-Universität zu Berlin, Philippstraße 13,

10115 Berlin 2Department of Biochemistry, Duke University School of Medicine, Durham, NC, USA

3Department 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 EAL-

or 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

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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.

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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.

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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.

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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.

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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 flagella-

independent 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 potassium-

responsive 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.

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Cell-cell communications in Bacillus subtilis mixed-species biofilms

Ramses Gallegos-Monterrosa1, Stefanie Kankel

1, Sebastian Götze

2, Pierre Stallforth

2, 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.

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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-cycle-

synchronized 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.

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The Cpx-system of Escherichia coli analyzed by SRM and Super-resolution Microscopy

Emina Ćudić1, Kristin Surmann

2, Rainer Kurre

3, Elke Hammer

2 and Sabine Hunke

1.

1Department of Microbiology, University of Osnabrueck, Barbarastraße 11, D-49076

Osnabrueck, Germany 2Department of Functional Genomics, University of Greifswald, Friedrich-Ludwig-Jahn

Straße 15A, D-17475 Greifswald, Germany 3Department 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 Cpx-

non 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 co-

localization events and found the biggest shift from close-together localization to co-

localization 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. 8583-

8593.

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

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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 substrate-

specific 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

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5. Winkelman, J.T. et al. (2013) Mol. Microbiol. 88:984-997

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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 two-

component 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 CESR-

mediating 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

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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

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Silencing of cryptic prophages in Corynebacterium glutamicum

Eugen Pfeifer1, Max Hünnefeld

1, Ovidiu Popa

2, Meike Baumgart

1, Tino Polen

1, Dietrich

Kohlheyer1 and Julia Frunzke

1.

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 nucleoid-

associated 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.

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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 H-

NS-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.

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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 nucleoid-

structuring 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.

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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).

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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.

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Poster 2:

Implementation, analysis and mathematical modeling of ECF sigma factor-based

synthetic gene cascades in E.coli and B.subtilis

Marco Mauri1, Daniela Pinto

2, Stefano Vecchione

1, Hao Wu

1, Thorsten Mascher

2, Georg

Fritz1.

1LOEWE Center for Synthetic Microbiology, Philipps University Marburg,

Hans-Meerwein-Str. 6C, 35043 Marburg, Germany 2Institut 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 promoter-

sequence 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 ECF-

based 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.

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Poster 3:

Modulation of the behaviour of an Extracytoplasmic Function sigma factor (ECF)-based

genetic switch

Daniela Pinto1, Franziska Dürr

1, Dayane Araújo

1,2, Qiang Liu

1,2 and Thorsten Mascher

1.

1Institüt für Mikrobiologie, Technische Universität Dresden, 01062 Dresden, Germany

2Department Biology I, Ludwig-Maximilians-Universität München, Großhaderner Str. 2-4,

82152 Planegg-Martinsried, Germany

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: one-

component 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.

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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 Rap-

modulator, 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 PhrA-

signal selectively. In conjunction with the mathematical model, we find that the RapA-

modulator 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).

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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

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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. Helfrich, S., et al., Live cell imaging of SOS and prophage dynamics in isogenic

bacterial populations. Mol Microbiol, 2015. 98(4): p. 636-50.

2. 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.

3. Pfeifer, E., et al., Silencing of cryptic prophages in Corynebacterium glutamicum. in

revision, 2016.

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Poster 7:

Deciphering the Response of Corynebacterium glutamicum to Oxygen Deprivation

Julian Lange1, Tobias Busche

2, Jörn Kalinowski

2, Ralf Takors

1, Bastian Blombach

1.

1Institute of Biochemical Engineering, University of Stuttgart, Stuttgart, Germany

2Center 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 bio-

based 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.

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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

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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 C-

terminal, 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 cyclic-

di-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 DNA-

binding 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-di-

GMP 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.

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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):2247-

59.

[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.

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[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 Heine

2, Michael Entian

2, Yi Li

3, Karin Sauer

3 and Nicole

Frankenberg-Dinkel1, 2

.

1Department of Biology, Microbiology, Technical University Kaiserslautern, Kaiserslautern,

Germany 2Physiology of Microorganisms, Ruhr-University

Bochum, Bochum, Germany

3Department 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-di-

GMP (bis-(3‘-5‘)-cyclic dimeric guanosine monophosphate) degrading phosphodiesterase

(PDE) activity. We characterised the membrane-bound proteins MucR and NbdA (NO-

induced 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.

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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.

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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.

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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.

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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

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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.

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55

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 two-

component 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)

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3. Georgellis D. and Kwon O. (2001), Quinones as the redox signal for the arc two-

component 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

2 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

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 C-

terminal 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

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Poster 19:

A TCS is involved in the regulation of the organohalide respiration in Sulfurospirillum

spp.

Jens Esken1, Tobias Goris

1, Cynthia Sharma

2, Torsten Schubert

1, and Gabriele Diekert

1.

. 1Department of Applied and Ecological Microbiology, University of Jena,

Philosophenweg 12, D-07743 Jena, Germany 2Research 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 two-

component 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.

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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

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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.

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Poster 22:

Mechanism and function of non-standard circadian clock systems in cyanobacteria

Christin Köbler1, Anja Dörrich

2, Anika Wiegard

3, Annegret Wilde

1.

1Albert-Ludwigs-University Freiburg, Germany;

2Justus-Liebig-University Giessen,

Germany; 3Heinrich-Heine-University Duesseldorf, Germany

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.

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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 high-

throughput 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 pSenSox-

based 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), 2614-

2622.

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Organization:

Dr. Reinhold Brückner and Anne Sexauer

Microbiology, University of Kaiserslautern

Paul-Ehrlichstrasse 24

67663 Kaiserslautern

Germany