Myxobacteria are famous for their capacity for social behavior and natural product biosynthesis. The unique sociality of myxobacteria is not only an intriguing scientific topic but also the main limiting factor for their manipulation. After more than half a century of research, a series of genetic techniques for myxobacteria have been developed, rendering these mysterious bacteria manipulable. Here, we review the advances in genetic manipulation of myxobacteria, with a particular focus on the exploitation of secondary metabolism. We emphasize the necessity and urgency of constructing the myxobacterial chassis for synthetic biology research and the exploitation of untapped secondary metabolism.

Hsp70 is important for organismic cells to maintain proteostasis and the chaperone protein is duplicated in all eukaryotes and many prokaryotes. Although the functioning mechanism of Hsp70 has been clearly illuminated, the chaperone duplication and functional evolution has been less investigated. DnaK is a highly conserved bacterial Hsp70 family. Here we showed that the dnaK gene is present in 98.9% bacteria and 6.4% bacteria possess duplicated dnaK s; the occurrence and duplication is positively corelated to the increase of proteomic complexity. We identified the interactomes of the two DnaK paralogs in Myxococcus xanthus DK1622, which are mostly nonoverlapped, but both preferring the α&β domain proteins. Consistent with the proteomes, the MxDnaK substrates are both significantly size-larger and pI-higher than that of the single E. coli DnaK. MxDnaK1 is heat-shock inducible, prefers to bind cytosolic proteins, while MxDnaK2 is decreased by heat shock, and is more associated with membrane proteins. The nucleotide binding domain and the substrate binding beta domain are responsible for the significant changes of DnaK substrates, and the former also determines the dimerization of MxDnaK2, but not MxDnaK1. Our work highlights that DnaK is duplicated and regionally evolved for the increase of proteomic complexity in bacteria.

The par operon in the sole myxobacterial plasmid pMF1 includes a function-unknown parC gene in front of the classical parA and parB genes. Removal of parC severely reduced plasmid stability, but ex-situ compensations of parC did not restore the par system function. Individual expression of parA formed insoluble proteins, while co-expression of parC before parA produced a soluble ParC-ParA heterodimer. ParA alone had no ATPase activity and no polymerization, while ParC addition aided ParA to restore the activities. Fusing ParC and ParA in different ways all produced soluble proteins and some restored ATPase activity or increased plasmid stability. Protein interaction model analysis and experiments revealed that ParC structurally mimics the N-terminal of Ia-type SopA (ParA), endowing the Myxococcus ParA protein to play functions by shifting of ParC between two sites on ParA surface. The present results highlight that ParC functions as a part of ParA to support its soluble expression and function, and the separation of ParC and ParA into two proteins in structure enables the ParC ‘fragment’ to shift in a larger range around ParA to function during partitioning. Author summary: Our work on ParC here provides a new example for the evolution of multi-domain protein. ParC and ParA are two proteins, but their expression and function act as a whole, which proposes a new regulatory model for bacterial par system, and also provides research ideas and materials for the study of functional coordination and evolution of ParA domains in the future.