PREreview of Type VI secretion system killing by commensal Neisseria is influenced by the spatial dynamics of bacteria

In this manuscript by Custodio and coworkers, the authors describe for the first time the antibacterial activity of a T6SS in Neisseria spp. - N. cinerea, which is a commensal of the human respiratory tract. It is well known that T6SSs mediated bacterial killing; however, the fact that strains expressing a type 4 pilus (TfP) are more susceptible to T6SS-mediated killing is a new finding. The N. cinerea T6SS structural cluster is encoded in a plasmid together with six effector/immunity pair. The N. cinerea T6SS is constitutively active in the conditions tested. The commensal N. cinerea outcompete pathogenic Neisseria species, such as N. meningitidis and N. gonorrhoeae, in co-cultures. The authors also describe that the presence of a capsule can protect prey cells against T6SS attack, and that the expression of Tfp is a disadvantage to the prey cells. The article is well written and contains all the proper controls. I have some comments.   

1-    Line 82: microcolonies instead of micocolonies

2-    Have the authors tried to mobilize the Neisseria plasmid containing the T6SS to other bacteria? If yes, please briefly describe de results.

3-    Figure 2. Was the TssB-GFP contraction observed when attacking cells were not in contact with prey cells? Please include a graph showing number of TssB contraction events when attacker cells were or were not in contact with prey cells.

4-    Figure 4. Have the authors searched the genome of N. cinerea for additional toxin/immunity proteins other than the six pairs contained in the plasmid?

5-    Figure 5A-B. It was not clear to me whether N. meningitidis and N. gonorrhoeae do not encode a T6SS or whether the T6SSs in these species are not active under the conditions tested. Please clarify.

6-    Figure 5A,C. The attacker:prey ratio of 100:1 seems too high. What was the result obtained if the ratio was used 10:1 or 2:1? Please comment and describe results in the manuscript.

7-    Figure 6. Can the Tfp from a prey cell induce the attack by an T6SS+ attacker cell in a similar mechanism as the one described as tit-for-tat (Basler 2013 Cell)? Please include a graph showing number of TssB contraction events comparing Tfp+ and Tfp- prey cells. If the Tfp is triggering more T6SS firing events than the explanation for the phenotype might change.