Graduate Students Mini-Symposium I 2025
Graduate Students Mini-Symposium
- Date: Feb 17, 2025
- Time: 01:15 PM (Local Time Germany)
- Location: MPI for Terrestrial Microbiology
- Room: Lecture Hall / Hybrid
- Host: IMPRS
- Contact: imprs@mpi-marburg.mpg.de
13:15 h Pia Richter - AG Thanbichler
Functional
characterization of the peptidoglycan recycling pathway in Caulobacter crescentus
Most bacteria possess a peptidoglycan (PG) cell wall
that is required to withstand the turgor pressure and maintain cell shape. The
PG layer needs to be constantly remodeled to enable the cells to elongate, grow
and divide. During this process, small PG fragments are released into the periplasm.
As these fragments can be used as a source for new PG building blocks, many
bacteria take great effort in recycling these degradation products.
Enterobacteria, such as Escherichia coli,
for instance, reuse up to 60 % of released PG fragments. To this end, the
turnover products are transported into the cytoplasm and further degraded by a
set of PG recycling-specific enzymes.
While E. coli and
other Gammaproteobacteria have rather simple shapes, the class of
Alphaproteobacteria shows a variety of different cell shapes. In these
organisms, correct PG remodeling and therefore also the availability of PG
precursors seems to be even more important. One organism is C. crescentus, a crescent-shaped bacterium
that is characterized by a biphasic life cycle involving to morphological
distinct cell types and asymmetric cell division.
Here, we identified that C. crescentus has a functional PG recycling pathway, which is,
although not necessary for general growth, essential to maintain proper cell shape.
Additionally, we observed that PG recycling is needed to maintain the natural
ß-lactam resistance of C. crescentus.
By characterizing the so far unknown PG recycling mechanism in
Alphaproteobacteria and understanding the connection between ß-lactam resistance
and PG recycling, we aim to gain novel insights which can help uncovering
potential treatments against pathogenic Alphaproteobacteria such as Bartonella or Brucella species.
13:45 h Trinetri Goel - AG Bode
Synthesis of structurally diverse peptide natural products from entomopathogenic bacteria for functional characterization
Natural products, such as non-ribosomal peptides and polyketides, are low
molecular weight compounds produced by living organisms to serve complex
functions such as signaling and defense.1,2 These organisms obtain an
evolutionary advantage due to the high chemical diversity of natural
products, inducing unique bioactive functions.1,3 Nevertheless, the
bioactivity and function of many natural products remain unknown until
today.4 Non-ribosomal peptides are a class of natural products
biosynthesized by large multi-enzyme complexes called non-ribosomal
peptide synthetase.1 Since Merrifield's development of the solid-phase
peptide synthesis method in 19635, chemical synthesis has provided an
efficient and streamlined approach to accessing a vast and diverse range
of peptides, overcoming the difficulties associated with producing and
purifying compounds in microbial hosts.6 Chemical synthesis offers
several advantages over biotechnological production. These include a
more straightforward purification process that does not contain media or
cell components and is independent of low-production titers.
Furthermore, synthesizing toxic compounds that would biotechnologically
result in the death of the producer cell is possible. The synthesis,
derivatization, and subsequent analysis of different peptide natural
products derived from entomopathogenic bacteria aims to elucidate their
structure-function relationship and identify novel natural products and
new-to-nature peptides.
14:15 h Max Schreiber - AG Bode
Utilizing A-domains in NRPS engineering to incorporate reactive, non-natural amino acids into non-ribosomal peptides
Non-ribosomal peptides (NRP), produced by non-ribosomal peptide synthetases (NRPS), are a highly complex class of secondary metabolites characterized by a large functional and structural diversity. Due to their highly selective bioactivity, NRP are also increasingly used as drugs. However, they often need further structural modifications for pharmaceutical use. Direct incorporation of non-natural amino acids into NRP circumvent this issue and expand their chemical and structural properties even further.
Here, I present a fast guideline to efficiently identify promiscuous adenylation (A) domains. This strategy is based on in vivo feeding experiments paired with molecular network analysis to quickly identify novel NRP derivatives.
In contrast to in vitro assays such as the ADP/ATP exchange assay, no protein purification is required and in addition to the activation of non-natural amino acids by A domains, their transfer to the downstream T domain is monitored as well. Therefore, the identified A domains are likely to be functional in an engineered, synthetic NRPS. The corresponding A domains are assigned using high-resolution mass spectrometry fragmentation data. To efficiently exploit these promiscuous A domains in NRPS engineering, we utilize a novel evolution-inspired modular engineering strategy suitable for Golden Gate-based assembly of NRPS libraries. This allows us to produce a diverse range of synthetic NRP with novel chemical and structural properties.