PREreview of Killing of Gram-negative and Gram-positive bacteria by a bifunctional cell wall-targeting T6SS effector

In this manuscript, the authors followed up on their previous work, which analyzed the contribution of noncanonical D-amino acids (NCDAA) on T6SS-mediated competition (Le et al., 2020 Sci Adv). Previously, authors have shown that production, secretion and incorporation of D-Lys into the producer cell peptidoglycan cell wall protected the bacteria from an T6SS-mediated attack by a competitor species. In the present study, authors claim that the secreted D-Lys has an additional function and increases the extracellular pH in the surrounding space, which will increase the enzymatic activity of a specific antibacterial effector (Tse4) against target cells. The manuscript also shows for the first time an T6SS antibacterial activity against Gram-positives and claim to have characterized the first bifunctional antibacterial toxin, which has two enzymatic activities (lytic transglycosylase and endopeptidase). The article is original and brings some novelty to the field. I have a few comments and questions described below:

 1.    It is not quite certain to me that the phenotype observed in the ΔracK is due to a direct effect of D-Lys on the extracellular pH. How can the authors rule out that this mutation does not affect the metabolism of the cells and therefore the T6SS-killing capacity?

2.    The hypothesis that the extracellular D-Lys and L-Arg would increase the surrounding pH and the Tse4 enzymatic activity is poorly supported by experimental evidence. Can the authors measure the pH in the proximity of the cells using fluorescent probes? This is very important for the proposed mechanism.

3.    The experiments in Fig. 4 in which authors claim there is a T6SS-dependent killing of Gram-positives (G+) in new to the field. This conclusion appears to be overstated as the experimental evidence provided only demonstrate G+ species do not grow as well in the presence of WT Acinetobacter compared to ΔtssM, there is no experiment actually showing the cells were killed as stated in the text. Authors should perform live-imaging experiment to show bacteria in contact and target cell death.

4.    Also related to the previous comment, I believe authors should repeat the experiments using different concentrations of D-Lys and pH using G+ as target cells. As multiple groups were unable to detect a T6SS-killing effect against G+ for several years, it is important to make sure the phenotype reported in this manuscript is reproducible in these conditions, and the proposed D-Lys/pH/Tse4 is the same.

5.    Lines 116-118, authors hypothesize the Δ123 strain secretes increased amounts of Tse4. This could be measured by western blot using endogenous Tse4 tagged protein.

6.    In Fig 5, authors could include two protein versions in the enzymatic assay as controls: each version should contain a point mutation specific to each catalytic domain, either M23 or Lyz-like. This would strength the claim that Tse4 is a bifunctional effector.