A lytic transglycosylase connects bacterial focal adhesion complexes to the peptidoglycan cell wall

Reviewer #1 (Public Review):

Summary:

This manuscript nicely outlines a conceptual problem with the bFAC model in A-motility, namely, how is the energy produced by the inner membrane AglRQS motor transduced through the cell wall into mechanical force on the cell surface to drive motility? To address this, the authors make a significant contribution by identifying and characterizing a lytic transglycosylase (LTG) called AgmT. This work thus provides clues and a future framework work for addressing mechanical force transmission between the cytoplasm and the cell surface.

Strengths:

(1) Convincing evidence shows AgmT functions as an LTG and, surprisingly, that mltG from E. coli complements the swarming defect of an agmT mutant.

(2) Authors show agmT mutants develop morphological changes in response to treatment with a -lactam antibiotic, mecillinam.

(3) The use of single-molecule tracking to monitor the assembly and dynamics of bFACs in WT and mutant backgrounds.

(4) The authors understand the limitations of their work and do not overinterpret their data.

Weaknesses:

(1) A clear model of AgmT's role in gliding motility or interactions with other A-motility proteins is not provided. Instead, speculative roles for how AgmT enzymatic activity could facilitate bFAC function in A-motility are discussed.

(2) Although agmT mutants do not swarm, in-depth phenotypic analysis is lacking. In particular, do individual agmT mutant cells move, as found with other swarming defective mutants, or are agmT mutants completely nonmotile, as are motor mutants?

(3) The bioinformatic and comparative genomics analysis of agmT is incomplete. For example, the sequence relationships between AgmT, MltG, and the 13 other LTG proteins in M. xanthus are not clear. Is E. coli MltG the closest homology to AgmT? Their relationships could be addressed with a phylogenetic tree and/or sequence alignments. Furthermore, are there other A-motility genes in proximity to agmT? Similarly, does agmT show specific co-occurrences with the other A-motility genes across genera/species?

(4) Related to iii, what about the functional relationship of the endogenous 13 LTG genes? Although knockout mutants were shown to be motile, presumably because AgmT is present, can overexpression of them, similar to E. coli MltG, complement an agmT mutant? In other words, why does MltG complement and the endogenous LTG proteins appear not to be relevant?

(5) Based on Figure 2B, overexpression of MltG enhances A-motility compared to the parent strain and the agmT-PAmCh complemented strain, is this actually true? Showing expanded swarming colony phenotypes would help address this question.

(6) Cell flexibility is correlated with gliding motility function in M. xanthus. Since AgmT has LTG activity, are agmT mutants less flexible than WT cells and is this the cause of their motility defect?

Reviewer #2 (Public Review):

The manuscript by Carbo et al. reports a novel role for the MltG homolog AgmT in gliding motility in M. xanthus. The authors conclusively show that AgmT is a cell wall lytic enzyme (likely a lytic transglycosylase), its lytic activity is required for gliding motility, and that its activity is required for proper binding of a component of the motility apparatus to the cell wall. The data are generally well-controlled. The marked strength of the manuscript includes the detailed characterization of AgmT as a cell wall lytic enzyme, and the careful dissection of its role in motility. Using multiple lines of evidence, the authors conclusively show that AgmT does not directly associate with the motility complexes, but that instead its absence (or the overexpression of its active site mutant) results in the failure of focal adhesion complexes to properly interact with the cell wall.

An interpretive weakness is the rather direct role attributed to AgmT in focal adhesion assembly. While their data clearly show that AgmT is important, it is unclear whether this is the direct consequence of AgmT somehow promoting bFAC binding to PG or just an indirect consequence of changed cell wall architecture without AgmT. In E. coli, an MltG mutant has increased PG strain length, suggesting that M. xanthus's PG architecture may likewise be compromised in a way that precludes AglR binding to the cell wall. However, this distinction would be very difficult to establish experimentally. MltG has been shown to associate with active cell wall synthesis in E.c oli in the absence of protein-protein interactions, and one could envision a similar model in M. xanthus, where active cell wall synthesis is required for focal adhesion assembly, and MltG makes an important contribution to this process.