Hi Naresh,

Dont worry. Simple doubts are just as relevant as complex ones - And I just hope I can help clear out some of your questions.

1. No, the elegant work by Justin Silpe and Bonnie Bassler cannot be used to explain our scenario. The overall phenotype is simply opposite. The find highest induction at HCD, while we find it at LCD.

2. In V. cholerae (and some other Vibrios) it well-studied that biofilm formation is most prominent at low-cell-density state. You can for example read the paper by Praveen Singh et al. 2017 in Current Biology. If you then add the work by Jun Zhu & John Mekalanos (Developmental cell 2003) you will find why this make sense from an infectious perspective. Biofilm in the early stage is important for infection and likely also for surviving the journey through the acid environment of many host before reaching the intestines.
Regarding to microcolonies, biofilm, aggregates etc. it is a constant debate. The way I see it, is that they present different stages of a continuum, but where they separate from one another can be difficult to define. The reason why we did it this way, is that Crystal Violet is a fine tool for screening, but not exactly accurate - So we wanted to support the Crystal Violet quantification with another quantification method that is more accurate. As you rightly mention, biofilms is complex mixture of various matrix components including eDNA and dead cells. You can stay these various components of interest (See the work of Thomas Neu for example), but in many cases staining the bacteria will be sufficient to find the size of the "biofilm/aggregate". As mentioned, the definitions are intertwined, and we saw in the microscope that cell aggregates on the bottom where nicely separated and not laying as a blanket. Agreed that the number 28um3 is quite arbitrary, but we decided that this was the size where we think an aggregate becomes "relevant" or "real" aggregation. And in this case you shouldn't put too much into the terms, here aggregate and biofilm cover the same phenotype, but because the latter analysis is made in microscope you can distinguish that biofilms are formed in aggregates rather than blanket, and hence we thought that word would be most suited.

3. I would hope the purpose would be visible in the introduction, and from the behind the paper text. In short we had an interest in phage H20, because Kalatzis et al. 2017 found it distributed worldwide, without any clear and obvious reasons on the genome annotation. So we made a prophage mutant and started searching for interesting phenotypes - and found that the answer was more complex than +/- prophage, but also took QS into account - Which in my humble opinion is super interesting from an evolutionary perspective.

I hope you find these answers useful. Good luck with entering this research field - phage-bacteria-interactions are great and interesting, and I hope you'll make some nice discoveries.

Mads Frederik Hansen

Hi again Naresh,
In this case we have to separate things. One phenomenom doesen't nescessarily rule out the other, but can create an artefact in the quantification. It is quite well-investigated that phage defenses are QS regulated (citations in the paper) and among these are downregulation of surface receptors at HCD. S. Abedon has published a hypothesis on "spatial vulnerability", where he talk about the risk of being phage targeting when you are in a group. He refers to spatial arrangements like biofilms, but you can argue that it also applies to the mere situation of being a dense population - So from an evolutionary perspective it makes a lot of sense to reduce phage receptors when you are a large phage target and vulnerable.
All right, we do not know which specific receptor is targeted by phage H20? So we cannot make direct quantifications of the regulation, but we can make an indirect analysis by measuring adsorption of the phage in the different mutants. This is important because it could influence how we quantify prophage induction - if there is a high level of adsorption at one QS state compared to another, it could affect the quantification of free, induced prophages as we measured. This doesn't affect the QS-prophage-induction-mechanism per se, but potentially hinder correct induction quantification. However, when we did the adsorption assay, although we found a slight difference, we did not find a significant difference in adsorption between mutants. And even if we take the adsorption-difference into account, adsorption is actually 'lowest' or 'slowest" at HCD (As the vulnerability hypothesis would suggest), which would mean that even if it creates a bias in the quantification, it only supports our claim; That there are more free induced phages at LCD.

It was quite long, but I hope it make sense to you.

Mads Frederik Hansen

Dear Naresh Kumar,
Thank you for your interest. We did had a working hypothesis; That VanT would bind upstream of the CI-like repressor gene at HCD and upregulate CI expression, and thereby strength lysogeny / prevent induction. This idea was initiated by the identification of a motif upstream of the CI gene, which was similar to a known binding motif of HapR in cholerae (Tsou et al. NAR 2009). So we had some ideas about investigating this molecular mechanism, but never got around to do so unfortunately. But I think Assoc. Prof. Svenningsen is partly looking into this mechanism, so we might know more in near future.
I hope this answered your question.
Mads Frederik Hansen