Characterisation of the interaction of the cytoskeleton protein BacM and the Ras GTPase MglA of Myxococcus xanthus

Abstract

MglA is the master regulator of A- and S-motility, and one of three RasGTPases, MXAN_1925 (MglA), MXAN_6703 (SofG), and MXAN_2495 of the soil bacterium Myxococcus xanthus. Besides motility, MglA plays key roles in cell division, polarity, fruiting body formation, and sporulation. Recent research has revealed that MglA and SofG specifically interact with bactofilin cytoskeletal proteins, suggesting that this interaction has important functional implications. Using a novel luciferase-based protein-protein two-hybrid system, it was shown that two out of four tested MglA-BacM pairs from the non-myxobacterial prokaryotes, Leptothrix mobilis and Bdellovibrio bacteriovorus, interacted specifically with each other in M. xanthus. This indicates that RasGTPase binding to bactofilins is a broadly conserved function. Importantly, a previously reported SofG-BacP interaction could not be confirmed, as SofG only bound to BacM in the luciferase assay. To understand how MglA controls these different processes, its cellular localization was studied using differently tagged versions of the protein. Unfortunately, N- and C-terminal eYFP-tagged MglAs were either unable to complement the mglA phenotype or only able to restore motility at single-cell but not colony level. Full complementation, including colony level motility, was only observed using MglA tagged with the small six-amino-acid-long TC tag. Using fluorescence light microscopy, the TC-tagged MglA also showed the previously described characteristic intracellular distribution, a large unipolar cluster with smaller lateral clusters. Expression of eYFP-MglA in yeast cells resulted in mitochondrial staining, suggesting that MglA may bind to cardiolipin-containing membranes, which are also found at the poles of M. xanthus cells. Various bacterial and eukaryotic MglA homologues were used to test their ability to restore motility, colony expansion, fruiting body formation, and sporulation in mglA. With the exception of the 99% identical MglAstigm from the closely related myxobacterium S. aurantiaca, no other tested pro- or eukaryotic homologue, including mutated versions, was able to complement mglA. Importantly, in contrast to an earlier report, Sar1 from yeast was unable to rescue fruiting body spore formation, but instead formed glycerol spores upon starvation. Finally, mutated MglA was unsuccessfully used to detect potential post translational modifications, indicating that other methods such as mass spectrometry may have to be employed in the future.