Myxococcus xanthus moves by gliding motility powered by Type IV pili (S-motility) and a second motility system, A-motility, whose mechanism remains elusive despite the identification of ∼40 A-motility genes. In this study, we used biochemistry and cell biology analyses to identify multi-protein complexes associated with A-motility. Previously, we showed that the N-terminal domain of FrzCD, the receptor for the frizzy chemosensory pathway, interacts with two A-motility proteins, AglZ and AgmU. Here we characterized AgmU, a protein that localized to both the periplasm and cytoplasm. On firm surfaces, AgmU-mCherry colocalized with AglZ as distributed clusters that remained fixed with respect to the substratum as cells moved forward. Cluster formation was favoured by hard surfaces where A-motility is favoured. In contrast, AgmU-mCherry clusters were not observed on soft agar surfaces or when cells were in large groups, conditions that favour S-motility. Using glutathione-S-transferase affinity chromatography, AgmU was found to interact either directly or indirectly with multiple A-motility proteins including AglZ, AglT, AgmK, AgmX, AglW and CglB. These proteins, important for the correct localization of AgmU and AglZ, appear to be organized as a motility complex, spanning the cytoplasm, inner membrane and the periplasm. Identification of this complex may be important for uncovering the mechanism of A-motility.

SUMMARY: In bacteria, motility is important for a wide variety of biological functions such as virulence, fruiting body formation, and biofilm formation. While most bacteria move by using specialized appendages, usually external or periplasmic flagella, some bacteria use other mechanisms for their movements that are less well characterized. These mechanisms do not always exhibit obvious motility structures.Myxococcus xanthusis a motile bacterium that does not produce flagella but glides slowly over solid surfaces. HowM. xanthusmoves has remained a puzzle that has challenged microbiologists for over 50 years. Fortunately, recent advances in the analysis of motility mutants, bioinformatics, and protein localization have revealed likely mechanisms for the twoM. xanthusmotility systems. These results are summarized in this review.

Directional motility in the gliding bacterium Myxococcus xanthus requires controlled cell reversals mediated by the Frz chemosensory system. FrzCD, a cytoplasmic chemoreceptor, does not form membrane-bound polar clusters typical for most bacteria, but rather cytoplasmic clusters that appear helically arranged and span the cell length. The distribution of FrzCD in living cells was found to be dynamic: FrzCD was localized in clusters that continuously changed their size, number, and position. The number of FrzCD clusters was correlated with cellular reversal frequency: fewer clusters were observed in hypo-reversing mutants and additional clusters were observed in hyper-reversing mutants. When moving cells made side-to-side contacts, FrzCD clusters in adjacent cells showed transient alignments. These events were frequently followed by one of the interacting cells reversing. These observations suggest that FrzCD detects signals from a cell contact-sensitive signaling system and then re-localizes as it directs reversals to distributed motility engines.

Significance: The type IV pilus (Tfp) is a multipurpose machine found on bacterial surfaces that works by cycles of synthesis/retraction of a pilin fiber. During surface (twitching) motility, the coordinated actions of multiple Tfps at the cell pole promotes single cells and synchronized group movements. Here, directly observing polar Tfp machines in action during motility of Myxococcus xanthus , we identified the mechanism underlying pole-specific Tfps activation. In this process, the Ras-like protein MglA targets a novel essential Tfp-activator, SgmX, to the pole, ensuring both the unipolar activation of Tfps and its switching to the opposite pole when cells reverse their movement. Thus, a dynamic cascade of polar activators regulates multicellular movements, a feature that is likely conserved in other twitching bacteria.