Deletion mutants of the different lysM genes showed that each gene has a significant role during predation in M. xanthus. Bioinformatic and structural analyses using Alphafold modelling show that these proteins are likely to act as peptidoglycan hydrolases, cutting in specific positions on the peptide bonds of peptidoglycans. Predation assays using the forementioned mutants show that each LysM protein exhibits specificity for different prey. Furthermore, a fourth lysM gene, not included in the original proposal (MXAN_7387), and predicted to act as a glycoside hydrolase has also been analyzed, revealing a more generalist role for this protein, acting as a complementary LysM protein aiding in predation against all prey species tested. According to the predicted enzymatic activity for each LysM protein, together with MXAN_7387, the action of the LysM proteins would generate different peptidoglycan fragments in prey species with different peptidoglycan composition.
The lysM genes are expressed at low levels during predation suggesting that, either they encode potent lytic enzymes, or that these proteins foster predation by triggering a wider and more complex response in the predator. In order to test this, purified LysM proteins were used against prey cultures, and the results have shown that they had no potent lytic activity, as previously suspected, when added extracellularly. However, when added in presence of the predator, LysM proteins were able to improve predation in M. xanthus and to restore mutant phenotypes.
Assays using transcriptional fusions of secondary metabolite genes, which are normally silent in absence of prey, indicate that they are induced in presence of different prey as previous transcriptomic data suggested. Expression of these genes was shown to be altered in single mutants for lysM genes, as well as in a quadruple mutant for all four lysM genes (the original 3 subject genes plus the complementary MXAN_7387 gene). However, for some reporters such as those of the myxovirescin gene cluster, mutation of lysM genes results in a basal overexpression that is unsensitive to the presence of prey, suggesting a general dysregulation of predation genes under these conditions. This evidence would support a model where LysM proteins would degrade prey peptidoglycans or peptidoglycan remains to generate specific fragments that would serve as MAMPs that would signal M. xanthus of the presence and type of prey.
We are currently establishing a colorimetric method based on Remazol brilliant blue to easily assess the peptidoglycan hydrolase activity of the LysM proteins for a screening of the protein variants. Similarly, we are working on the identification of a good candidate for a silent gene cluster to act as a reporter to use for screening of their inducer activity after point mutations, since the reporters tested so far are expressed at levels that can only be measured by enzymatic activity assays and not by colorimetric assays.
