Linking the Peptidoglycan Synthesis Protein Complex with Asymmetric Cell Division during Bacillus subtilis Sporulation

Round 1

Reviewer 1 Report

Major comments:

1) If authors have E. coli strain which overproduce protein of interest (GpsB, SpoIIE whole or its fragments) why its interaction is show in whole protein extract? It cannot be ruled out, that by unspecific interaction with E. coli protein(s) my influence observed.The experiment with precisely chosen concentration of protein of interest may be more informative.

2) It would strengthen the article if in the experiment show at Figure 1B presence of GpsB protein will be shown, for example, by using anti-his antibody.

3) Photographs showed at Figure 4A should be magnified. The fluorescence signal indicated by the arrows is barely visible in the photo.

Minor comments:

1) The world “gram” should be written by capital letter (page 2, line24; page 3, line 2).

2) If the Table 1 refer sporulation efficiency to the wild type strain in the lines with gpsB and ezrA mutants should be 100%. In the current presentation method, the sporulation efficiency of the wild strain is unknown

3) The bacterial species in the literature section should be written in italics style.

Author Response

We would like to thank reviewer for his/her comments with clear aim to improve the manuscript and also for positive evaluation of our study.

Reviewer1:

Comments and Suggestions for Authors

Major comments:

1) If authors have E. coli strain which overproduce protein of interest (GpsB, SpoIIE whole or its fragments) why its interaction is show in whole protein extract? It cannot be ruled out, that by unspecific interaction with E. coli protein(s) my influence observed. The experiment with precisely chosen concentration of protein of interest may be more informative.

His-GpsB was purified using chromatography on Ni Sepharose HP column and eluted with 1M imidazole. We can clearly exclude unspecific interaction with E. coli protein(s) as signal using anti S-tag antibody is very specific and we did not observe any signal in our control when we pulled down E. coli lysate containing only empty pETDuet vector.

2) It would strengthen the article if in the experiment show at Figure 2B presence of GpsB protein will be shown, for example, by using anti-his antibody.

We included the suggested Western blot into Fig. 2. Accordingly, we changed the text and Figure2 description – pages 7 and 8 in revised ms. We also changed corresponding text in Materials and methods -page19 line 411 in revised ms.

3) Photographs showed at Figure 4A should be magnified. The fluorescence signal indicated by the arrows is barely visible in the photo.

We made the suggested change to Fig. 4.

Minor comments:

1) The world “gram” should be written by capital letter (page 2, line24; page 3, line 2).

We changed the gram-positive to Gram-positive – page 3 line 78, page4 line 106 in revised ms.

2) If the Table 1 refer sporulation efficiency to the wild type strain in the lines with gpsB and ezrA mutants should be 100%. In the current presentation method, the sporulation efficiency of the wild strain is unknown.

We changed mentioned sporulation efficiency to 100% -table1 and page16 in revised ms.

3) The bacterial species in the literature section should be written in italics style.

We wrote the bacterial species in the literature in the italics style in the revised version of ms.

Reviewer 2 Report

This is a very nicely written paper, representing a key microbiological topic. 

I only have a couple of very minor comments to make

The Gram stain was named after Dr Gram. Because of that Gram needs to be written as a name, hence Gram with a capital G

pg 2, ln 24: Firmicutes phylum in italics

pg 1, ln 33: insert the word 'the' between that and division 

Don't start a conclusion with the phrase "To summarise ...". Don't summarise your work; highlight the main conclusions.

Author Response

We would like to thank reviewer for the comments with clear aim to improve the manuscript and also for positive evaluation of our study.

Reviewer2:

Comments and Suggestions for Authors

This is a very nicely written paper, representing a key microbiological topic.

I only have a couple of very minor comments to make

The Gram stain was named after Dr Gram. Because of that Gram needs to be written as a name, hence Gram with a capital G

We changed the gram-positive to Gram-positive - page 3 line 78, page4 line 106 in revised ms.

pg 2, ln 24: Firmicutes phylum in italics

We changed the style of Firmicutes to italics – page3 line77 in revised ms.

pg 1, ln 33: insert the word 'the' between that and division

We inserted “the” as suggested – page2 line40.

Don't start a conclusion with the phrase "To summarise ...". Don't summarise your work; highlight the main conclusions.

We changed the first sentence of conclusions as suggested by the reviewer- page 20 line423 in revised ms.