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«Redefinition of the σS regulon and new insights into σS protein and promoter dynamics A dissertation submitted to ETH ZÜRICH For the degree of ...»

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DISS. ETH No. 21396

Redefinition of the σS regulon and new insights into σS

protein and promoter dynamics

A dissertation submitted to

ETH ZÜRICH

For the degree of

Doctor of Sciences

presented by

JOHANNES WOLF

Diplom Biotechnologe / Ingénieur en Biotechnologie

Ecole Supérieure de Biotechnologie de Strasbourg (ESBS)

born July 25, 1982

Citizen of Germany

Accepted on the recommendation of Prof. Dr. Thomas Egli, examiner Prof. Dr. Paolo Landini, co-examiner Prof. Dr. Martin Ackermann, co-examiner Prof. Dr. Julia Vorholt, co-examiner Table of content Table of content

Summary

Zusammenfassung

Abbreviations

Chapter I General Introduction

Procaryotes, their environments, and Escherichia coli as a model organism.............. 15 2 Stress response and adaptation to changing environments

2.1 Transcription

General stress response and physiological role of σS

Regulation of σS

4.1 Remark on the importance of sound growth conditions

Transcription of rpoS

4.2 Translation of rpoS

4.3 Stability and degradation of σS

4.4 4.5 Competition for RNA polymerase

4.6 Ambiguous role of different regulators

Mechanisms of promoter recognition by σS

Promoter elements important for recognition by σS

5.1 Transcription factors determining σS specificity

5.2 6 Objectives of this thesis

References

Chapter II Cloning and characterization of a K-12 MG 1655 wild type strain

expressing a His-tagged σS protein

1 Introduction

2 Material and Methods

E. coli strains

2.1

2.2 Cloning

2.3 PCR and sequencing

2.4 Restriction digest

2.5 Batch growth – LB medium

2.6 Batch growth - M9 minimal medium with 300 mM NaCl

2.7 Catalase assay

2.8 Western Blot

2.9 Chromatin Immunoprecipitation (ChIP)

2.10 Quantitative PCR (qPCR)

3 Results

3.1 Selection of clones with wild type growth behaviour

–  –  –

4 Discussion

References

Definition of the direct σS regulon

Chapter III 1 Material and Methods

1.1 ChIP and qPCR

1.2 Qualitative DNA measurements

1.3 Library preparation and sequencing

1.4 Statistical and bioinformatic data analysis

1.5 Relative quantification of gene expression

2 Results

2.1 Successful enrichment of control promoters

2.2 DNA profile of the ChIP samples

2.3 Successful library preparation

2.4 Sequencing led to the identification of 79 binding sites

qPCR of selected targets revealed two new σS-dependent genes

2.5 3 Discussion

References

Chapter IV Influence of transient conditions and nutrient limitations on

sigma factor levels in continuous culture

1 Introduction

2 Material and Methods

2.1 Bacterial strain and media

2.2 Growth conditions

2.3 Determination of cell number

2.4 Calculation of growth rates during transient experiments

2.5 Determination of nutrient concentrations and dry weight

2.6 Western Blot

3 Results

3.1 Biomass patterns

3.2 Specific growth rates

3.3 Dry weight, nutrient concentrations and growth yields

3.4 Cell number

Levels of σS and σFecI change in response to shifts in µ

3.5

3.6 Levels of other sigma factors during shifts in µ

4 Discussion

References

–  –  –

1 Introduction

2 Material and Methods

2.1 Cloning

2.2 Growth conditions

2.3 Preparation of the microfluidic device

2.4 Microscopy

2.5 Data analysis

3 Results

Induction of the rpoS promoter

3.1 4 Discussion

References

Chapter VI Conclusion

Chromatin Immunoprecipitation of σS

2 Influence of D-shifts and different limitations on the physiology in

continuous cultures

Single cell monitoring of rpoS promoter activation in a microfluidic device................ 99 References

Acknowledgements

Curriculum Vitae

–  –  –

The microbial cell is, in contrast to multicellular organisms, directly exposed to environmental stimuli and stresses. This means on one hand that dissolved nutrients can directly and quickly be taken up. On the other hand, this also implies that microbes are directly exposed to unfavourable conditions such as low nutrient concentrations, low or high pH and temperature, or UV light. In order to survive, the cells have to be able to adapt to these conditions. Unfavourable conditions, as well as all environmental changes that require physiological adaptations of the cell, i.e., in gene expression and cellular composition, can be regarded as a stress for the microbial cell. In Escherichia coli a wide range of responses to these stresses is governed by the alternative sigma factor σS. These responses include the expression of catalases that detoxify reactive oxygen species, proteins (dpS) that protect the DNA from oxygen radicals, and the production of intracellular trehalose as osmoprotectant.





Initially, protein levels of σS were found to rise upon entry into stationary phase and exposure to stresses. Later, it was found that σS levels inversely correlate with growth rate in chemostat cultures meaning that the slower the cells grow, the higher σS levels are.

Surprisingly, it is still not clear, if different stresses all enhance σS, or if different stresses in general reduce the specific growth rate, which then in turn upregulates σS and thereby triggers the general stress response. Thus, specific growth rate might be the signal integrator for the cell and not σS itself. Besides this stress response sigma factor, Escherichia coli has six further sigma factors, among them σD, which is the housekeeping sigma factor and responsible for most vegetative functions and essential genes. Surprisingly, σS and σD exhibit similar promoter recognition sequences, even though they control genes of very different nature. Therefore, analysing promoter sequences does not allow to elucidate whether a gene is σS-, or σD-dependent.

The first part of this thesis was dedicated to determine the direct σS regulon, i.e., which promoters are directly bound by the σS protein in vivo. Therefore, we established a chromatin immunoprecipitation (ChIP) protocol that allows specific purification of promoter fragments bound to the σS protein. Since the only commercially available σS antibody is not suitable for ChIP assays, we constructed a mutant that carried the rpoS gene fused to a His

<

Summary

tag coding sequence. We proved that this genetic alteration neither affected general physiological parameters such as specific growth rate nor did it change σS levels and σSdependent stress response induction such as katE. Hence, the mutant strain behaves very similar to the wild type and allows sound experiments. Using formaldehyde fixation a “snapshot” of σS distribution on the genome can be obtained. After the ChIP procedure, the fragments obtained were directly sequenced (ChIP-seq) and 79 σS binding sites were identified. Among these binding sites some 40 % corresponded to promoters of genes that were already known to be σS-dependent, be it directly or indirectly. This result is very important as it is a proof that the technique yields reliable results. Semi-quantitative PCR was then used to test expression of some candidate genes found by ChIP-seq. This experiment revealed two new directly σS-dependent genes, ydbK and ybiI. However, the majority of the genes analysed by qPCR did not show different expression levels. It is possible that induction of these genes does not lead to transcript levels that are high enough for validation by qPCR. Moreover, some σS binding sites were identified within genes and their role is still to be elucidated. One reason could be that these are promoters for unannoted genes, or it might represent a way of negative regulation of the respective genes by blocking the proceeding of the polymerase.

Furthermore, we investigated how steady-state cultures react to sudden dilution rate changes under carbon-, phosphorus- or nitrogen-limitation. In the environment, sudden and short-term availability of nutrients followed by another phase of famine might represent a common condition the microbial cell has to quickly adapt to in order to benefit from increased nutrient availability. Thus, we grew cells under the mentioned limitations in steady-state cultures at a low dilution rate, and imposed a sudden dilution rate increase, followed by switching back to the original dilution rate. Throughout the experiments, physiological parameters and sigma factor levels were quantified and it was found that different limitations exhibit very different adaptation patterns to an increase in specific growth rate.

The last part of the thesis consisted of combining an rpoS promoter GFP fusion mutant with a microfluidic device also referred to as “mother machine”, which allows to follow rpoS promoter activity at a single cell level. The constructed mutant exhibited a GFP signal when the rpoS promoter was activated. The cells were grown at different growth rates, which led

Summary

to the induction of the rpoS promoter. Specific growth rates and promoter activities for a single cell, which was retained at the bottom of a channel, were recorded and analysed. This technique provides the possibility to study not only rpoS promoter activity, but also, combined with downstream reporter genes, the impact of the σS protein on target genes.

Concluding, this thesis provides new insight into the σS regulon, σS dynamics during transient growth conditions in continuous culture and the possibility of studying rpoS transcription dynamics at a single cell level following the fate of individual cells.

–  –  –

Zusammenfassung Im Gegensatz zu multizellulären Organismen ist die mikrobielle Zelle Umwelteinflüssen und Stress direkt ausgesetzt. Einerseits heisst dies, dass gelöste Nährstoffe schnell direkt aufgenommen werden können, bedingt andererseits aber auch, dass Mikroorganismen ungünstigen Bedingungen wie niedrigen Nährstoffkonzentrationen, extremen pH-Werten und Temperaturen oder UV-Strahlung unmittelbar ausgesetzt sind. Um derartige Bedingungen zu überleben, müssen die Zellen in der Lage sein sich diesen anzupassen. Dabei müssen nicht nur ungünstige Umweltbedingungen als Stress für Mikroorganismen angesehen werden, sondern generell jede Veränderung der Umgebung, die eine physiologische Anpassung auf Ebene der Genexpression oder Zellzusammensetzung erfordert. In Escherichia coli wird eine Reihe von Antworten auf Umweltstress über den alternativen Sigma-Faktor σS gesteuert. Diese Antworten umfassen die Expression von Katalasen, die reaktive Sauerstoffspezies abbauen, Proteine wie dpS, welche die DNA vor Sauerstoffradikalen schützen und die Produktion von intrazellulärer Trehalose, die als Schutz vor osmotischem Stress dienen kann. Zunächst wurde entdeckt, dass σS-Niveaus bei Eintritt in die stationäre Phase und bei Stress ansteigen. Später wurde herausgefunden, dass sich σSMengen in Chemostat Kulturen antiproportional zur spezifischen Wachstumsrate verhalten.

D.h. je langsamer die Zellen wachsen, desto mehr σS kann in diesen detektiert werden.

Erstaunlicherweise ist nicht klar, ob verschiedene Stresse σS induzieren oder ob verschiedene Stresse im Allgemeinen die spezifische Wachstumsrate, µ, verringern, was wiederum zur Folge hat, dass σS-Proteinmengen ansteigen, was die allgemeine Stressantwort auslöst. Daher könnte die spezifische Wachstumsrate, und nicht σS selbst, als Signalintegrator wirken. Neben dem Sigma-Faktor der generellen Stressantwort, σS, verfügt Escherichia coli noch über sechs weitere Sigma-Faktoren, unter ihnen σD, welcher der Haushalts-Sigma-Faktor ist und die meisten vegetativen und essentiellen Gene steuert.

Überraschenderweise erkennen σS und σD ähnliche Promotorsequenzen, auch wenn sie Gene sehr verschiedener Funktion kontrollieren. Folglich reicht die Analyse von Promotorsequenzen in diesem Fall nicht aus, um herauszufinden, ob ein Gen σS- oder σDabhängig ist.

Der erste Teil dieser Doktorarbeit befasst sich mit der Identifikation des direkten σSRegulons, d.h. der Promotoren, die direkt von σS in vivo gebunden werden. Hierfür haben wir Zusammenfassung ein Chromatin-Immunopräzipitations (ChIP) etabliert, welches die spezifische Aufreinigung von Promotorfragmenten ermöglicht, die von σS gebunden sind. Da der einzige kommerziell verfügbare σS-Antikörper nicht für ChIP-Experimente geeignet ist, haben wir eine Mutante konstruiert, die eine His-tag kodierende Sequenz am σS Gen trägt und spezifische Aufreinigung durch einen ChIP-kompatiblen Antikörper erlaubt. Wir konnten zeigen, dass diese genetische Veränderung weder physiologische Parameter wie die spezifische Wachstumsrate, noch σS-Niveaus und σS-abhängige Stressantworten, wie z.B. Induktion von katE, verändert hat. Demzufolge verhält sich der konstruierte Stamm ähnlich wie der Wildtyp und macht gründliche Experimente möglich. Um eine Momentaufnahme der Verteilung der σS-Proteine im Genom zu erhalten, wurden die Zellen vor der ChromatinImmunopräzipitation durch Formaldehyd fixiert. Die durch ChIP erhaltenen DNA-Fragmente wurden sequenziert, was zur Identifizierung von 79 σS-Bindungsstellen führte. Unter diesen gehören etwa 40 % zu schon bekannten σS-abhängigen Genen. Dieses Ergebnis ist sehr wichtig, da es zeigt, dass die genannte Technik zu verlässlichen Resultaten führt. Um den Einfluss von σS auf die Expression von einigen Kandidatengenen zu überprüfen, die zuvor durch die Sequenzierung identifiziert worden waren, wurde semi-quantitative PCR

angewendet. Auf diese Weise wurden zwei bisher unbekannte σS-abhängige Gene entdeckt:



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