3. EMBL-Umeá University

EMBL-Umeå University
EMBL-Umeå University

Cell wall plasticity by extracellular metabolites in Vibrionaceae

Our investigations discovered non canonical D-amino acids (NCDAAs) as a new type of communication system that a great variety of microorganisms use to adapt to changing environments. Though NCDAAs were initially found in V. cholerae stationary growth phase supernatants, we have shown that very diverse bacteria also release high concentrations of NCDAAs to the extracellular media. Because the accumulation of NCDAAs coincides with the transition into stationary phase and down-regulates cell wall synthesis, D-amino acids enable coordination of metabolic slowing in cell wall and cytoplasmic compartments when resources become scarce. A critical aspect is that NCDAAs govern cell wall metabolism through their incorporation in the bacterial peptidoglycan (PG). Since NCDAAs appears to be a general trait amongst many Bacteria, we are currently expanding our investigations in NCDAAs signalling to other microorganisms to properly understand bacterial relations with neighbouring organisms at all levels, from commensalism to pathogenesis.
Additionally, based that extracellular, actively released, metabolites have a great impact on PG composition and structure, the Cava Lab aims at a comprehensive characterization of murein diversity in the Bacteria Kingdom. Investigation of bacterial murein under natural conditions will lead to the identification of novel imprints and to provide a trustworthy picture of bacterial cell wall variability in composition and architecture in nature. Gathered data will be the basis of the first extensive murein database, a precious instrument to promote collaborative networks amongst fairly unconnected disciplines in life sciences towards addressing fundamental questions on vibrios adaptation to challenging conditions (i.e. during an infection). One of the main goals of our research is to use this tool for the development of a next generation class of vibrio-specific antimicrobials.

Specific aims:
1. Characterization of NCDAAs as regulatory agents of the bacterial cell wall in Vibrio cholerae.
2. Investigation of novel modes of cell wall growth in Bacteria
3. Comprehensive characterization of murein diversity and plasticity in Vibrionaceae

Here are some of the techniques that we use in our lab:

Microbiology: Cultivation of a large diversity of bacteria; mutagenesis (allelic exchange, transposon based), conjugation, transformation, biofilm analysis, MIC determination, plate reader- fitness assays...
Analytical chemistry: We are pioneers implementating the state-of-the-art Ultra Performance Liquid Chromatography coupled to Mass Spectrometry for cell wall analysis. We can make dedicated methods for small metabolites analyses.
Molecular Biology: General molecular biology techniques including DNA engineering, PCR, RT-PCR, PCR-RFLP, DNA footprint, Primer extension, Two Hybrid System, b-galactosidase assays, thermo-stabilization of proteins, RNAP recruitment assays, In vitro transcription assays, Microarrays and RNA-seq analysis, quantitative PCR SYBR/Taqman, RACE, etc.
Microscopy: Bacterial time lapses and time courses using optical and confocal microscopy. Use of fluorescent proteins reporters. Use of FLDAA, FL-Vancomycin and click chemistry to label bacterial PG growth and dynamics, labelling by FL-lipid derivatives (i.e. FM1-64) or succinimidyl esters (i.e. Oregon Green, Texas Red). Electron Microscopy (TEM and SEM), D-Cysteine method for PG inert labelling, etc.
Biochemistry: Amongst standard DNA/RNA and Protein techniques we have strong expertise in advanced enzymology including on PG-enzymes, HPLC/MS analysis of PG, Bocillin-FL gels, etc

In addition, Umeå University has outstanding  infrastructures in Metabolomics, NMR, Chemi­cal synthesis and screening facilities, Computational Life Science Cluster (CLiC) and Bioimaging (including STORM, spinning disk confocal microscopy...)

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