Studies on polar cell wall growth and antibiotic susceptibility of Corynebacterium glutamicum

Corynebacterium glutamicum is a Gram positive soil bacterium with
high industrial importance in ton scale production of amino acids.
Apart from that, it becomes more and more important for medical
studies, where it serves as model organism due to its close
relation to bacteria causing several pathogens such as
tuberculosis, diphtheria and leprosy. C. glutamicum, like
Mycobacterium tuberculosis, has a distinct cell wall which is
composed of a peptidoglycan layer (murein) with covalently bound
polysaccharide layers that are capped with mycolic acids. In
addition, both organisms have a polar cell wall synthesis machinery
which is spatially regulated by DivIVA (Wag31 in M. tuberculosis).
The present study shows that DivIVA regulates cell wall synthesis
upon direct interaction with the lipid II flippase RodA. RodA
determines morphology and growth in C. glutamicum and is localized
to the poles and septa. The absence of rodA results in growth
defects and cell shape alterations as well as altered lipid II
proliferation of the poles (polar cell growth is sustained). DivIVA
is furthermore involved in chromosome segregation upon direct
interaction with the partitioning ParB protein, which binds to parS
sites on the chromosome, thus tethering the replicated nucleoids to
the cell poles. Interactions of DivIVA with ParB and RodA were
identified in a synthetic in vivo protein-protein interaction assay
where fluorescently labeled proteins of interest are expressed in
E. coli cells and interaction is analyzed microscopically. A
decisive improvement of this assay is the application of FRET,
which is more sensitive and allows quantification of interaction.
In order to test whether ParB and RodA compete for the same
interaction site in DivIVA, we mapped interaction sites of both
proteins. It turned out that ParB binds to a middle region of
DivIVA, whereas RodA binds to the N-terminal domain of DivIVA where
one lysine residue is essential for interaction. To fight bacterial
infections, that cause thousands of casualties each year, it is
mandatory to understand mechanisms in cellular processes, such as
cell division and growth, to find new targets for antibiotic
intervention. Unfortunately, bacteria are able to develop
resistances against many antibiotics. The mycolic acid or arabinan
layer and synthesis machinery are good candidates for new
antibiotics. Amongst others, two of them have emerged as useful
drugs against M. tuberculosis, ethambutol (EMB) and BTZ043. In this
study, we investigated the modes of action and antibiotic
susceptibility of C. glutamicum after EMB and BTZ043 treatment. We
found that both antibiotics, which target the arabinan synthesis
pathway, affect exclusively polar elongation growth, as
demonstrated in different staining assays. Interestingly, only 10%
of the cells were killed and cells in stationary phase were not
affected by EMB or BTZ043. Moreover, we used a chromosomal
DivIVA-mCherry fusion and found that DivIVA protein level is
drastically increased. The cells show asymmetric recovery after
treatment, in which one daughter cell acquires the excess DivIVA
whereas the other daughter cell exhibits normal cell growth.