Research Interests
The Gerdes laboratory focusses on two main research areas:
1. Bacterial Stress Responses and Antibiotic Multidrug Tolerance (Persistence)
2. Bacterial Cell Division and Shape
In area (1), we focus on:
1a. Molecular Mechanisms behind Bacterial Persistence
1b. Biological Functions of Toxin – Antitoxin (TA) Genes
In area (2), we focus on:
2a. Coordination of Cell Wall Growth and Cell Division
2b. Molecular Mechanisms of DNA Segregation
1. Bacterial Stress Responses and Antibiotic Multidrug Tolerance
Most bacteria live in constantly changing environments and, accordingly, have evolved highly sophisticated regulatory mechanisms that allow them to withstand stressful conditions. For example, amino acid starvation elicits the stringent response that reprograms cellular metabolism from rapid growth to slow growth or dormancy. The effector molecule of the stringent response, (p)ppGpp increases in concentration in response to nutrient limitations such as amino acid starvation and profoundly influences gene expression such that the cell can readily adapt to the limited nutrient supply. Importantly, rRNA synthesis is severely curtailed while transcription of amino acid biosynthetic operons is stimulated. Thus, a primary role of (p)ppGpp is to adjust the size of the protein synthesizing apparatus, and thereby cell growth, to the available nutrient resources. However, (p)ppGpp also affects replication, transcription and protein turnover. Remarkably, (p)ppGpp stimulates the accumulation of inorganic polyphosphate (PolyP), a ubiquitous molecule that consists of long chains of phosphates linked via high-energy phosphodiester bonds. For a long time it was thought that the primary role of PolyP was to function as an energy reservoir. However, recent research has shown that PolyP functions as a signalling molecule that control protein turnover and transcription of certain operons. The house-keeping Lon protease degrades mis-folded proteins during heat shock and other environmental stresses. During nutrient limitations, PolyP binds to and reprograms Lon to degrade ribosomal proteins such that the released free amino acids can be used to de novo synthesis of proteins such as e.g. amino acid biosynthetic enzymes that are required to survive nutrient limitations.
1a. Molecular Mechanisms behind Bacterial Persistence.
Persisters are bacterial cells that a multidrug tolerant because they are slow-growing or dormant. For example, it is well known that penicillin, which inhibits cell wall synthesis, kills growing bacteria. However, non-growing bacteria are not killed by penicillin because these cells have an intact cell wall. Almost all bacteria form slow-growing variants that exhibit multidrug tolerance and such bacteria pose a health threat because they are not eradicated by antibiotics. We discovered recently that Lon and TA loci are required for persistence of E. coli. This model organism has at least eleven Type 2 TA loci, all of which encode inhibitors of cell growth (Fig. 1A). The “toxins” are activated when theantitoxins are degraded. Interestingly all eleven Type 2 antitoxins of E. coli are degraded by Lon. Consistently, Lon is required for persistence of E. coli. This is consistent with the fact that the antitoxins are stable in a strain that lacks Lon. We study how Lon is regulated, both at the level of cell cultures and at the level of single cells, using the most advanced microscopic techniques.
TA loci are ubiquitous in free-living bacteria, thus raising the possibility that TA loci are required for persistence of major pathogens such as for example Mycobacterium tuberculosis that has at least 88 Type 2 TA loci (Fig. 1B).
Figure 1. Toxin – Antitoxin loci of E. coli K-12 and M. tuberculosis H37Rv. Mtb has a large expansion of TA loci.
1b. Biological Functions of Toxin – Antitoxin Genes. Type 2 TA gene cassettes were discovered on plasmids because they confer plasmid stabilization by killing or inhibiting the growth of plasmid-free cells. However, mysteriously, TA loci are also abundant on bacterial and archaeal chromosomes. Type 2 TA loci encode a protein antitoxin that combines with and neutralizes a toxin by direct protein-protein contact. Fig. 2 shows the components and interactions of the best-studied TA locus, relBE of E. coli that encodes RelE toxin and RelB antitoxin.
RelB and RelE combine very efficiently and form non-toxic RelB2-RelE and RelB2-RelE2 complexes. Two RelB dimers bind to operators in the 
relBE promoter region and represses transcription. The RelB2E complex binds cooperatively and stronger to the promoter region and represses transcription more efficiently than a RelB2 dimer. Thus, during rapid growth (when [RelE] < [RelB2]), the relBE promoter is strongly repressed. However, during slow growth, such as e.g. during amino starvation, the RelB/RelE ratio shifts such that [RelE] > [RelB2]. Surprisingly, this shift in TA ratio induces the relBE promoter. We showed that an extra RelE molecule strips off the RelB2-RelE complex by invading the complex and generating a RelB2-RelE2 complex that has a low affinity for operator DNA. Mathematical modelling shows that the conditional stimulation of relBE transcription may serve to prevent fortuitous induction of RelE activity and furthermore may secure fast quenching of RelE activity when starvation conditions and dormancy are terminated.
RelE is an mRNase that cleaves mRNA positioned at the ribosomal A-site (Fig. 3). This observation explains why ectopic overproduction of RelE leads to rapid and efficient inhibition of translation. RelE cleaves enzymatically between the 2nd and 3rd nucleotide of the A-site codon and thereby block further translation by the ribosomes that becomes stalled at the end of the damaged, non-stop mRNA. The bi-functional tmRNA then rescues the stalled ribosomes by the trans-translation reaction (Fig. 1). Accordingly, cells that lack tmRNA are more sensitive to inhibition by RelE.
Interestingly, RelE has an RNase fold similar to that of other microbial RNases but lacks catalytic activity because essential catalytic amino acids are missing. However, via binding to the ribosomal A-site and interacting with 16S rRNA and a ribosomal protein, RelE slightly changes conformation and this change activates RelE. Remarkably, RelE cleaves mRNAs at the A-sites of eukaryotic 80S ribosomes and of mitochondrial ribosomes, showing that the configurations of the A-site and of the mRNA codon at the A-site are highly conserved across all domains of life.
The majority of TA loci encode translational inhibitors, such as RelE described above. Thus, activation of a toxin in a TA complex may lead to reduced cell growth and eventually dormancy (persistence) of the bacterium. Therefore, TA loci likely contribute to the persistent state of pathogenic bacteria such as M. tuberculosis, an interesting aspect that may have practical impact for the understanding of mechanisms of chronic, latent and infections. Remarkably, M. tuberculosis has at least 46 vapBC loci. We have recently that enteric VapCs cleave initiator tRNA site-specifically in the anti-codon loop and that VapC overproduction induces persistence. We are now pursuing VapC targets in M. tuberculosis. To understand the molecular mechanisms behind persistence, we are investigating how TA loci are regulated in E. coli and in major pathogenic bacteria.
2. Bacterial Cell Division and Shape.
2a. Coordination of Cell Wall Growth and Cell Division. New microscopic and protein-labelling techniques have uncovered that bacteria are highly organized with multiple intracellular systems that generate localized structures at the sub-cellular level. For example, FtsZ, the essential regulator of bacterial cell division, is a dynamic cytoskeletal protein that forms helices that condense into the Z ring early in the cell cycle. Later in the cell cycle, the Z ring contracts and primes the synthesis of the septal peptidoglycan. The signal that allows the Z ring to contract is not known.
Another cytoskeletal protein, the actin homologue MreB is required for the shape of rod-shaped bacteria, probably because MreB controls the synthesis of the side (lateral) wall peptidoglycan. Thus, two large enzymatic machineries are responsible for lateral and septal peptidoglycan synthesis. We study how the activities of these two large machineries are coordinated.
2b Plasmid segregation mechanisms. Chromosome segregation in eukaryotic cells is a well understood process. However it has been more difficult to understand how prokaryotic cells segregate their chromosomes, mainly due to a considerably smaller cell size. To study DNA segregation, we use bacterial plasmids as model systems. The common Type I par loci that encode ATPases related to the oscillating MinD protein of E. coli, are present on many plasmids and most chromosomes. Besides ParA, par loci encode a DNA binding protein (ParB) and a DNA centromere site (parC). ParA interacts with ParB bound to parC. Remarkably, plasmid-encoded ParA form oscillating, helical structures that move and position plasmids within the bacterial cell while simultaneously tethering the plasmids to the nucleoid (bacterial chromosome) in a regular array see Fig. 4.
We are currently trying to understand how ParA mediates regular positioning of plasmids over the nucleoid, using both cytology of living cells and in vitro approaches.
PRESENT CO-WORKERS
Etienne Maissoneuve, PhD
David Guymer, PhD
Andrew Fenton, PhD
Patrick Scheu, PhD
Kristoffer S. Winther, PhD
Florian Zardenings, BSc
Milena Jaskolska, BSc
Mikhael Manurung, BSc
Research Roles
At the moment, the Gerdes group consists of5 post docs, 2graduate students and 1 MRes student
Postgraduate Supervision
* Bacterial persistence,
* Toxin - antitoxins andtranslation
* Bacterial Cell Division
* DNA segregation and related topics
Funding
Current
2012 - 2017: ERC funded project "Bacterial Persistence". £2,100,000
2010-2013: Wellcome Trust funded project on DNA Segregation Mechanisms in Prokaryotes. £198,000
2009-2013: EC funded grant, partner in the Consortium DIVINOCELL:
Exploiting Gram-negative cell division targets in the test tube to obtain anti-microbial compounds; €351,000
Completed
2008-2011: BBSRC funded grant on the project Bacterial actin MreB in cell morphogenesis £432,556
2008-2011: Wellcome Trust funded grant on the project Nonsense-mediated mRNA decay in prokaryotes - £228,743.
2005 - 2010: a grant from the The Danish Basic Research Foundation of Dkr 2,400,000,- (ca. £220,000).
2005 - 2007: a grant from the Danish Natural Research Council has established The Centre for Bacterial Genetics. The grant was Dkr 6,800,000,- (ca. £620,000) divided equally on three research groups over three years.
From 1990 to 2000: the group received solid financial support from the Danish Governmental Biotechnology Programmes I, II and III.
From 2000 - 2003: grant from the Danish Biotechnology Instrument Center (DABIC) (Dkr 1,500,000,- ? £136,000)
From 1998 to 2001 and from 2002 to 2005: grants from the EC Framework Programmes 4 and 5 (ca. £120,000 and £160,000).
