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.