Figure 1. Antibiotic resistance vs persistence.  Mutants resistant to antibiotics have acquired stable genetic changes, which are inherited by their offspring. The resulting population is therefore entirely composed of antibiotic-resistant cells. Phenotypic variants displaying persistence are only transiently insensitive to the action of antibiotics and become susceptible again before recommencing cell division. The resulting population therefore displays a similar fraction of antibiotic-tolerant cells as the original population.

Figure 2. Production of a biofuel is inherently linked to tolerance of the producing organism.  One of the major obstacles in obtaining high yields is the toxicity of the biofuel itself. When production proceeds and biofuel titers rise, further production is gradually inhibited. Therefore, understanding and increasing biofuel tolerance in producing organisms is needed to further improve the production process and increase the competitiveness of renewable fuels over fossil fuels.

Biological nitrogen fixation in the bean - Rhizobium etli symbiosis

Leguminous plants play a crucial role in natural ecosystems as well as in agriculture.

They contribute one third of the dietary protein nitrogen needs of the world’s population and account for over 27% of primary crop production.

Perhaps the most distinguishing feature of legumes is their natural ability to acquire nitrogen from the air through symbiotic interaction with a group of soil bacteria collectively called rhizobia. Nitrogen is the critical limiting element for growth of most plants and is a building block of all life, being part of nucleic acids, amino acids and proteins.

Consequently, the nitrogen-fixing symbiosis confers a clear advantage under nitrogen-limiting conditions and a better

understanding of the process is expected to benefit worldwide food supply as well as the environment.

Rhizobia can trigger host plants to form root nodules, specialized organs that offer the bacteria an exclusive ecological niche in which they reduce atmospheric dinitrogen to ammonia. Ammonia is made available to the plant, which in turn provides carbon sources to the bacteria.

Research within our group is aimed at achieving a better understanding of the bean - R. etli symbiosis in order to improve the efficiency of biological nitrogen fixation. To this end, we focus on several aspects of the symbiotic process. A main area of research comprises molecular and genetic mechanisms that contribute to bacterial stress resistance. Rhizobia encounter stress during free-living growth in the soil, but also during infection of the host as well as within the particular microenvironment provided by the specialized root nodules. We believe that improving bacterial stress resistance is key to increasing symbiotic performance.

Antibiotic-tolerant persister cells

In addition to antibiotic resistance, bacteria have developed another survival strategy to evade extinction by antibiotic treatments

used in infectious disease therapy: persistence.

Every isogenic bacterial population contains specialized cells, so-called “persister cells”, that can sustain high doses of antibiotics. These cells are present even before the event of disaster and, as such, are an insurance for unpredictable stress situations.

As long as the unfavourable conditions endure, persister cells do not divide and are commonly regarded as dormant or inactive.

When the unfavourable conditions subside, these persisters resuscitate and found a new population exhibiting the same antibiotic susceptibility as the original population. Both the fact that persisters do not grow and that they regrow to a population equally

sensitive as the original one indicates that persister cells are not resistant mutants, but possess phenotypic tolerance against antibiotics

(Figure 1). A detailed description of the exact molecular mechanisms underlying persistence is lacking, but recent research

shows that many pathways underlie this dormant phenotype. For a recent review on persistence, see Van den Bergh et al. 2017.

Although persistence was first observed several decades ago, its clinical impact only became clear during the last 10 years. Recently, it was shown that persister cells are the cause of the chronic nature of biofilm-associated infections by Pseudomonas aeruginosa in cystic fibrosis patients and by Candida albicans in immune-compromised cancer patients.

The presence of persister cells in biofilms, bacterial structures associated with some 80% of chronic infections, significantly increases the clinical relevance of this phenotype. The persister cells within the biofilm are shielded from the host immune response by the biofilm structure and are able to survive antibiotic treatments. As such, they pose an enormous threat to future antibacterial therapy of chronic infections. For a review on the clinical implications of persistence, see Fauvart et al. 2011.

Within the Symbiotic and Pathogenic Interactions group, persistence of the opportunistic human pathogen Pseudomonas aeruginosa and the model bacterium Escherichia coli has been studied over many years.

The focus is on a number of key regulators of persistence, both in P. aeruginosa and E. coli, to yield a better understanding of the underlying genetic and molecular mechanisms. The social-evolutionary impact of persistence is also studied within our group to gather a further fundamental understanding of this phenotype. In addition to these fundamental research topics, we also try to combat chronic infections through translational research. For these studies we target P. aeruginosa as model causative agent of chronic infections and aim to develop lead molecules for new antibacterial and anti-persister drugs.

Specific research topics include:

Artificial genetic variation for stress tolerance in bacteria

Solving the energy issue poses one of the greatest challenges of the 21st century. Fossil fuel reserves are declining while energy demands continue to increase. This is especially evident in the transportation sector where globalization and a growing world-wide population dramatically increase the fuel needs. A further continued depletion of fossil fuel reserves to meet these needs will not only reinforce global warming, but will also problematically increase fossil fuel dependency.

To prevent further deterioration of the environment and become self-sufficient in terms of energy, fundamental advances in full-fledged, renewable petrol alternatives are urgently needed. Bacteria are crucial for the production of many valuable chemicals, including biofuels. However, often these organisms severely suffer from the stress caused by increased concentrations of the end products. Therefore, stress tolerance engineering is needed to increase the efficiency of biofuel production.

Indeed, enhancing the tolerance against a biofuel is expected to increase the production capacity of the organism simultaneously (Figure 2).

Over the past decades, multiple efforts have been undertaken to facilitate the switch from fossil fuels to renewable biofuels. Especially bioethanol has gained much attention. It is one of the biofuels that is already widely integrated in our society.

Therefore, ethanol tolerance in E. coli has been extensively studied in the Michiels lab. Ethanol tolerance is a complex phenotype as it is established by a network of interacting genes and pathways. Moreover, ethanol is highly toxic for E. coli imposing near-lethal stress conditions. To study ethanol tolerance we exploited the power of experimental evolution and natural selection.

Our work provides the first insights into the evolutionary dynamics underlying adaptation to such complex and near-lethal stress conditions. Moreover, we identified genetic determinants causative for the observed increased in ethanol tolerance.

Oue dinkins a framework for improving ethanol production specifically and enhancing production of other value-added chemicals in general.

Specific research topics include

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