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Phage-host interaction

Bacteriophages, short ‘phages’, are viruses preying on bacteria. Phages represent the most abundant biological entities on this planet and are estimated to outnumber their host by a factor of ten. Consequently, life in all ecosystems- from the environment to the human body - is shaped by the predation of viruses. However, the immense genetic resources and diversity of phage genomes still remain almost unexplored.

We are interested in how phages target key regulatory hubs of the host cell and how bacteria protect themselves against this attack on the cellular and multicellular level. As model system, we are focusing on phages infecting Actinobacteria – representing a phylum of outstanding biotechnological and medical importance (Fig. 1).

Picture Streptomyces coelicolor coloniesFigure 1: Streptomyces coelicolor colonies, TEM picture of a S. coelicolor phage, and S. coelicolor plaques. Adapted from Hardy et al., 2020

Temperate bacteriophages are able to integrate into the host genome and maintain as prophages in a long-term association with their host. Consequently, prophages represent an ubiquitous element of bacterial genomes. Illustrated by the development of mutually beneficial traits, this close interaction between host and virus has significantly shaped bacterial evolution.

A current research focus of our group lies on the regulatory interaction and integration of viral DNA (prophages) in the networks of the host. We recently identified a prophage-encoded xenogeneic silencing protein (CgpS, Fig. 2) playing a key role in the silencing of cryptic prophage islands and in the maintenance of the lysogenic state. Insights are harnessed for the development of novel tools and approaches for metabolic engineering as exemplified by the design of transcription factor-based biosensors or synthetic regulatory circuits.

Graphic Model for functions of phage-encoded silencers and counter-silencersFigure 2: Model for functions of phage-encoded silencers and counter-silencers. Virulent phages might employ XS-like proteins as a weapon to interfere with host XS proteins or to repress other host defense mechanisms. Binding of a Transcription factor (TF) within CgpS silenced promoters can lead to counter-silencing and the binding site position determines counter-silencing efficiencies. Further details are presented in Wiechert et al., 2020, doi: 10.1128/mBio.02273-19.

Selected publications

Hardy A, Sharma V, Kever L, and Frunzke J (2020) Genome Sequence and Characterization of Five Bacteriophages Infecting Streptomyces Coelicolor and Streptomyces Venezuelae: Alderaan, Coruscant, Dagobah, Endor1 and Endor2. Viruses, doi:

Wiechert J, Filipchyk A, Hünnefeld M, Gätgens C, Brehm J, Heermann R and Frunzke J, (2020) Deciphering the Rules Underlying Xenogeneic Silencing and Counter-Silencing of Lsr2-like Proteins Using CgpS of Corynebacterium glutamicum as a Model. mBio, doi: 10.1128/mBio.02273-19

Pfeifer E, Michniewski S, Gätgens C, Münch E, Müller F, Polen T, Millard A, Blombach B, and Frunzke J (2019) Generation of a Prophage-Free Variant of the Fast-Growing Bacterium Vibrio natriegens. Appl Environ Microbiol, doi 10.1128/AEM.00853-19

Pfeifer E, Hünnefeld M, Popa O, and Frunzke J (2019) Impact of Xenogeneic Silencing on Phage-Host Interactions. J Mol Biol, doi: 10.1016/j.jmb.2019.02.011

Pfeifer E, Hünnefeld M, Popa O, Polen T, Kohlheyer D, Baumgart M, and Frunzke J (2016) Silencing of cryptic prophages in Corynebacterium glutamicum. Nucleic Acids Res, doi: 10.1093/nar/gkw692

Transcription factor-based biosensors and metabolic engineering

Living organisms have evolved a plethora of sensing systems for the intra- and extracellular detection of small molecules, ions and physical parameters. We exploit and incorporate these sensory mechanisms in synthetic circuits to devise genetically-encoded biosensors, which are highly valuable for a wide range of biotechnological applications (Fig. 3). Transcription factor-based (TF) biosensors are applied to translate cellular metabolite production into an easily detectable and measureable output (e.g. fluorescence).

Our group is interested in the design and application of TF-based biosensors for high-throughput screening approaches, single-cell analysis, and dynamic pathway regulation. TF-based biosensors are also used for the establishment of biosensor-driven adaptive laboratory evolution strategies to improve microbial small molecule production and to enhance our understanding of biological systems.
Projects focus on the biotechnological platform organism Corynebacterium glutamicum and the fast growing bacterium Vibrio natriegens, representing an emerging host for molecular biology and biotechnology.

Graphic Versatile applications of TF-based biosensorsFigure 3: Versatile applications of TF-based biosensors. Biosensors with an optical readout, e.g. production of an autofluorescent protein (AFP), are efficient tools for a multitude of applications. Taken from Mahr et al., 2016, doi: 10.1007/s00253-015-7090-3.

Selected publications

Wiechert J, Gätgens C, Wirtz A, and Frunzke J (2020) Inducible expression systems based on xenogeneic silencing and counter-silencing and design of a metabolic toggle switch. ACS Synth Biol, doi: 10.1021/acssynbio.0c00111

Stella RG, Wiechert J, Noack S, and Frunzke J (2019) Evolutionary engineering of Corynebacterium glutamicum. Biotechnol J, doi: 10.1002/biot.201800444

Pfeifer E, Gätgens C, Polen T, and Frunzke J (2017) Adaptive laboratory evolution of Corynebacterium glutamicum towards higher growth rates on glucose minimal medium. Sci Rep, doi: 10.1038/s41598-017-17014-9

Huber I, Palmer DJ, Ludwig KN, Brown IR, Warren MJ, and Frunzke J (2017) Construction of Recombinant Pdu Metabolosome Shells for Small Molecule Production in Corynebacterium glutamicum. ACS Synth Biol, doi: 10.1021/acssynbio.7b00167

Mahr R and Frunzke J (2016) Transcription factor-based biosensors in biotechnology: current state and future prospects. Appl Microbiol Biotechnol, doi: 10.1007/s00253-015-7090-3

Mahr R, Gätgens C, Gätgens J, Polen T, Kalinowski J, and Frunzke J (2015) Biosensor-driven adaptive laboratory evolution of l-valine production in Corynebacterium glutamicum. Metab Eng, doi: 10.1016/j.ymben.2015.09.017

Mustafi N, Grünberger A, Mahr R, Helfrich S, Nöh K, Blombach B, Kohlheyer D, and Frunzke J (2014) Application of a Genetically Encoded Biosensor for Live Cell Imaging of L-Valine Production in Pyruvate Dehydrogenase Complex-Deficient Corynebacterium glutamicum Strains. PLoS One, doi: 10.1371/journal.pone.0085731

Mustafi N, Grünberger A, Kohlheyer D, Bott M, and Frunzke J (2012) The development and application of a single-cell biosensor for the detection of l-methionine and branched-chain amino acids. Metab Eng, doi: 10.1016/j.ymben.2012.02.002

Gene Regulatory Networks

Microorganisms are living in a complex and varying environment and, thus, must be able to rapidly adapt to changing conditions, such as nutrient availability, physical stresses or the presence of friends or foes. The micronutrient iron is essential for almost all organisms and is often a growth-limiting factor in aerobic and microaerobic environments due to the extremely low solubility of Fe3+. Consequently, organisms have evolved sophisticated regulatory networks tightly controlling uptake and storage, while avoiding the accumulation of toxic intracellular levels.

We are studying gene regulatory networks and signal transduction cascades controlling iron and heme homeostasis in the Gram-positive soil bacterium Corynebacterium glutamicum (Fig. 4). We are further interested in how intercellular microbial interactions affect intracellular networks and vice versa. To decipher network hierarchy and dynamics, we are combining classical molecular microbiology methods with state-of-the art genome-wide profiling (ChIP-Seq), omics techniques (RNA-Seq) and mathematical modelling.

Model of heme-responsive control of components of the respiratory chain by HrrSAFigure 4. Model of heme-responsive control of components of the respiratory chain by HrrSA. The results of this study reveal HrrSA as a global regulator of heme homeostasis coordinating the expression of genes involved in heme biosynthesis, oxidative stress responses, glucose uptake and cell envelope remodelling. Taken from Keppel et al., 2020, doi: 10.1093/nar/gkaa415.

Selected publications

Keppel M, Hünnefeld M, Filipchyk A, Viets U, Davoudi C-F, Krüger A, Mack C, Pfeifer E, Polen T, Baumgart M, Bott M, and Frunzke J (2020) HrrSA orchestrates a systemic response to heme and determines prioritization of terminal cytochrome oxidase expression. Nucleic Acids Res, doi: 10.1093/nar/gkaa415

Hünnefeld M, Persicke M, Kalinowski J, and Frunzke J (2019) The MarR-Type Regulator MalR Is Involved in Stress-Responsive Cell Envelope Remodeling in Corynebacterium glutamicum. Front Microbiol, doi: 10.3389/fmicb.2019.01039

Keppel M, Piepenbreier H, Gätgens C, Fritz G, and Frunzke J (2019) Toxic but tasty - Temporal dynamics and network architecture of heme-responsive two-component signalling in Corynebacterium glutamicum. Mol Microbiol, doi: 10.1111/mmi.14226

Keppel M, Davoudi E, Gätgens C, and Frunzke J (2018) Membrane Topology and Heme Binding of the Histidine Kinases HrrS and ChrS in Corynebacterium glutamicum. Front Microbiol, doi: 10.3389/fmicb.2018.00183