Biodegradation of Pyrethroids by a Hydrolyzing Carboxylesterase EstA from Bacillus cereus BCC01
Round 1
Reviewer 1 Report
Interesting study with applciation potential. I have following comments:
The abstract is not very clear and should contain more data.
Lines 218-220. There is temperature in two factors (X1 and X2). Is that correct?
Line 221 - what do you mean by "term". Is it a "factor"?
Line 231 - I think more than one value is neeed to call the model "reliable".
Lines 230-232 The sentence starting "Its growth..." is duplicated.
Author Response
Please find attached.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Hu et al investigated a very interesting and relevant topic, however, I have some major concerns about the manuscript.
I don't recognize any added value in the mathematical modeling part. It surely is very trendy , but in this case, the beautiful graphs do not tell us much - the experimental part is limited only to one medium and three different variables, the inoculum size is very unspecific - we don't know how many bacteria are in 9.6% inoculum, what the OD is, what is the spore/vegetative cell ratio, how does the growth phase influence the degradation etc.
The supplementary figures are missing, so I cannot fully comment on the results.
The literature review is very modest.
I am afraid that this journal is not a good fit for this manuscript. The manuscript does not push the frontiers, nor presents advanced knowledge or cutting edge methodology/technology/ideas (however, the number of analyzed colonies is breathtaking!)
methodological details missing. Materials and Methods could be reorganized.
I recommend rejection.
Here are some minor remarks:
"highlights" are dispensable.
L33-47: the statements contradict each other. is it toxic for humans or not?
L58 - catalyze pesticides? in what way? Clarification needed. Maybe modify?
L66 - the statement is slightly too strong. You determined only conditions for the specific medium.
L72 - enzyme, in singular.
L79-81: structural formulas of these compounds would be appreciated.
L96-112 - better explanation and justification needed so people who are not strong in computational biology/modeling can understand (e.g. what values does each code represent?). To be perfectly honest, the modeling looks nice, but I don't see any added value to your manuscript.
L138 - HPLC details missing
L162- what is homogeneous plating? please clarify
L156-167 - certain details missing (restriction, ligation). did you do blue/white screening? If yes, Did you use IPTG/X-gal? Did you select the clones on LB supplemented with antibiotics? if yes, please clarify.
L165 - please define "good growth conditions"
L169-170 - what primers did you use for sequencing?
L185 - please clarify what exactly was analyzed by SDS-PAGE? PAGE details missing
L187 - please clarify that BSA was used for calibration curve.
L191 - homogenized how? mixed? vortexes? Please clarify
throughout - please use non-dividing space between a value and a unit.
L192 Please rewrite the sentence to avoid confusion.
195 - empty lines missing.
L227 - it is very unclear to me - what does 9.6% inoculum mean? How many cells is this? What OD600? What is the ratio between vegetative cells and spores? Maybe I missed it, but what was used for pH adjustments?
Figure 2 - controls missing. Fig2A - is beta-cypermethrin the only carbon source in the medium? or is this LB supplemented with BC? Clarification needed. The symbols are not very clear, so a color reminder would be greatly appreciated.
Author Response
Please find attached.
Author Response File: Author Response.pdf
Reviewer 3 Report
The manuscript submitted by Hu an coworkers summarises results related to the biodegradation of pyrethroids by a hydrolyzing 2 carboxylesterase EstA from Bacillus cereus BCC01. The topic is of high interest mainly for those researchers involved in "green chemistry", "bioremediation", etc.
The manuscript is in general well written, clear, concise and conclusions are supported by results.
The work is closely related to the topics include in this journal.However, some minor items must be fully addressed before its publication.
Some comments have been embedded throughout the manuscript in order to help the authors to get an improved version.
Comments for author File: Comments.pdf
Author Response
Please find attached.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
The authors improved the manuscript, however, it still needs major proofreading by a native speaker. Please see below.
Comments to your answers:
"We renewed the review in the introduction part."
The literature review is still very modest (40 references)
"The part of “Materials and Methods” was reorganized and the details were added in the manuscript and marked blue."
There are still missing parts. please see below.
Point 12: There are still certain things that need clarification. Please see below.
Point 17: please include M13 primer sequences in the manuscript.
Point 24: control still missing. See below.
L21 - tautology "inoculated by adding the inocula". Please reformulate the section. It still does not make sense.
L25 - putification - typo
L25-26 - clarification needed. Soluble in what? Catalytic activity against what?
L27 - suggestion: "... pH values (6.5-9)"
L27-28 - Please clarify. Could provide?
L40 - which pests?
L38-49. -the sentences could be joined.
L47 - space after reproduction
L 67 - please proofread or rewrite the sentence. what does "its" refer to?
L76 - is one enzyme sufficient? what is the toxicity of EstA metabolic product?
L87 - please specify that the strain has previously been identified
L92-94 - please check if commas are placed correctly.
L98-99 - please rewrite and clarify the sentence
L107-109 - I still don't understand - what values do -1, 0 and 1 correspond to?
L127 - pH adjustment should be mentioned in the "media" section, not here
Section 2.4 - please reformulate the section and make it clearer
L129 - w/ shaking or w/o? please specify
L136. -it does not make sense. How did you transfer 9.6% of individual colony?
L134 - if you inoculated 30 mL of the medium, how did you sample 30 mL? it does not make sense.
L137 - please use spaces between values and units
L137-138 - I don't understand. Please clarify the sentence.
L152 - are these final concentrations?
L157 - please use non-breaking space between values and units
Section 2.5 - how were the metabolites identified? did you use standards? Database? please clarify
L167 - please correct the name of the enzyme (no spaces)
L 171 - non-breaking space
L171 - spots=colonies?
Section 2.7 - 2D protein structure missing - or is this considered "physical property"?
L195 - please clarify "inoculated products"
L204 - SDS-PAGE details still missing - gel percentage etc.
L204 - did you additionally purify the protein to remove the bovine enterokinase?
L208 - Ten uL
L210 - solution in what?
L210 and throughout - please unify the symbol for degrees
L211-214 - please proofread, re-write and make it clearer.
L213 - did you use the same protocol for HPLC as described above? if yes, please specify.
L218 - non-breaking spaces, unify the symbol for degrees.
L228 - please include reference for SAS
Figure 2B - control (medium without cells) missing. Please demonstrate that there was no spontaneous degradation.
Fig2A - please specify the medium in the caption (black triangles)
L278. -identification not included in Materials and methods
L312 - did you confirm the pI? Does the proposed size correspond the band on the gel?
L331-335 - please fix the alignment. Please proofread it again. there are some errors (species not italicized, no space after period etc)
L332 - which protein marker?
L333 - what does the symbol after degree celsius mean?
I might have missed it - what is the degradation product of EstA?
Supplementary
FigS1- the resolution of images is poor, the numbers and the details cannot be seen. What is the red frame in S1A?
FigS2 caption- what tool was used for secondary structure prediction? Details in Materials and Methods missing. Or is this considered "physical property"?
FigS3 caption - please proofread and make it clearer. I cannot see "boxed" motif.
Fig S4 caption - Callibration curve.
Table S1 caption - please explain the acronym (CAS)
Table S2 caption - Please explain Pr>F.
Table S3 - this is not really a table. Please reconsider the caption if you decide keeping it. I would rather suggest depositing the sequence in the database and providing the accession number in the manuscript.
Author Response
Response to Reviewer 2 Comments
Point 1: Comments to your answers: "We renewed the review in the introduction part." The literature review is still very modest (40 references)
Response 1: Thank you very much for your suggestion. We already summarized the process in the field, we do think the current version could reflect the reason why we need to carry out this experiment. And 23 references to list some of the related works could be adequate in this current study as far as we concerned.
Point 2: Point 17: please include M13 primer sequences in the manuscript.
Response 2: Thank you very much, and we added the M13 primer sequences in the manuscript.
Point 3: Point 24: control still missing. See below.
Response 3: Figure 2 stands for the growth of BCC01 at different culture condition with or without beta-cypermethrin. The experiment was carried out by using the growth condition of BCC01 at LB media to serve as control. Normally, this kind of experiment does not have a “real control”, please refer to other references (Chen et al., Biodegradation of beta-cypermethrin and 3-phenoxybenzoic acid by a novel Ochrobactrum lupini DG-S-01. Journal of Hazardous material 187 (2011) 433-440- Figure 2. Li et al., Biofilm-inspired encapsulation of probiotics for the treatment of complex infection. Advanced materials. 30 (2018) 1803925-Figure 1. Liu., et al.. Bioluminescent nanopaper for rapid screening of toxic substances. Nano research. 11 (2018) 114-125-Figure 1a. The growth tendency of bacteria has all been measured in these papers, and none of them has “control”.
Point 4: L21 - tautology "inoculated by adding the inocula". Please reformulate the section. It still does not make sense.
Response 4: Thank you very much for your suggestion, and we revised the sentence in the manuscript.
Point 5: L25 - putification - typo
Response 5: Thank you very much for your suggestion, and we are deeply sorry for our negligence. We revised the sentence.
Point 6: L25-26 - clarification needed. Soluble in what? Catalytic activity against what?
Response 6: The recombinant protein was soluble, which suggested after induction by IPTG, this protein was expressed mostly in liquid supernatant and soluble in buffer, instead of expressing in precipitant. The meaning of catalytic activity was that the purified protein exerted a broad-spectrum degrading activity against pyrethroid as suggested in the results part (Line 339-346).
Point 7: L27 - suggestion: "... pH values (6.5-9)"
Response 7: Thank you very much for your suggestion. We revised the sentence.
Point 8: L27-28 - Please clarify. Could provide?
Response 8: Collectively, this study could provide information regarding the degrading mechanism of BCC01, including the optimal culture condition of BCC01, the degrading pathway of BCC01 against beta-cypermethrin, and its degrading enzyme, which could be helpful for the actual application of BCC01 in the environment.
Point 9: L40 - which pests?
Response 9: Thank you very much for your suggestion, pyrethroid insecticides have been widely used in agriculture due to their potent toxic activity against various insect pests, including Lepidoptera (Spodoptera litura Fabricius, Spodoptera exigua Hübner, Hellula undalis Fabricius) and Diptera (Liriomyza trifolii Burgess, Melanagromyza sojae Zehnter) which were reported in the literatures (Vargas, G.; Lastra, LA.; Ramirez, GD.; Solis, MA. The Diatraea Complex (Lepidoptera: Crambidae) in Colombia’s Cauca River Valley. Neotrop Entomol 2018, 47, 395–402.; Viviana M.A.; James M-L.; Claudia E-R.; Michaudc J.P.; Germán V. Host resistance to two parasitoids (Diptera: Tachinidae) helps explain a regional outbreak of novel Diatraea spp. stem borers (Lepidoptera: Crambidae) in Colombia sugarcane. Biological Control 2019, 129, 18-23), we revised this sentence in the manuscript.
Point 10: L47 - space after reproduction
Response 10: Thank you very much for your suggestion. We revised the sentence.
Point 11: L 67 - please proofread or rewrite the sentence. what does "its" refer to?
Response 11: “ITS” refers to the optimal cultivation condition of BCC01, we revised the sentence, and thank you very much for suggestion.
Point 12: L76 - is one enzyme sufficient? what is the toxicity of EstA metabolic product?
Response 12: There is definitely more than one enzyme in the bacteria, and it may take a lot of enzymes to degrade the pesticide. But only one enzyme was screened out by using the way of constructing the genomic library. And we are sorry that we have not done any research on the toxicity of EstA metabolic products. We are focus on studying the molecular degradation of pesticides, and we can carry out the research on the toxicity of degradation products in the future work.
Point 13: L87 - please specify that the strain has previously been identified.
Response 13: Thank you very much for your suggestion, and this stain has been identified in our previous published paper by using physiological identification and 16S rDNA BLAST.
Point 14: L92-94 - please check if commas are placed correctly.
Response 14: Yes, they are correct.
Point 15: L98-99 - please rewrite and clarify the sentence
Response 15: Thank you very much for your suggestion, and we revised this sentence in the manuscript.
Point 16: L107-109 - I still don't understand - what values do -1, 0 and 1 correspond to?
Response 16: The values of -1, 0, and 1 correspond to the lowest, middle, highest coded levels, respectively, which refers to -1 (6), 0 (7.5), +1 (9) for pH; -1 (25 ℃) 0 (30 ℃), +1 (35 ℃) for temperature; -1 (Inoculum (%) (v/v) =5), 0 (Inoculum (%) (v/v) =10), +1 (Inoculum (%) (v/v) =15) for inoculum. Thank you very much for your suggestion, and we added the caption in the Table1.
Point 17: L127-pH adjustment should be mentioned in the "media" section, not here
Response 17: Thank you very much for your suggestion, and we revised the pH adjustment.
Point 18: Section 2.4 - please reformulate the section and make it clearer
Response 18: Thank you very much for your suggestion, and we revised the section.
Point 19: L129 - w/ shaking or w/o? please specify
Response 19: The single colony was cultivated in solid LB media without shaking. After transferred to liquid LB media, the stain was cultivated at 30 ℃ and 170 rpm on a rotary shaker. Thank you very much, and we revised this sentence.
Point 20: L136. -it does not make sense. How did you transfer 9.6% of individual colony?
Response 20: Firstly, the stain was cultivated in solid media, and then single colony was selected and transferred to liquid LB media, this is called inocula. After overnight cultivation, 9.6% (9.6%*30ml) of the culture suspensions from the inocula was then inoculated into 30 mL of LB media. We are sorry for such misunderstanding, and we rephrased this sentence in the manuscript.
Point 21: L134 - if you inoculated 30 mL of the medium, how did you sample 30 mL? it does not make sense.
Response 21: Sorry for such misunderstanding. The samples were collected from the culture suspensions at different intervals, not 30 mL, but 100 µL. We revised this sentence in the manuscript.
Point 22: L137 - please use spaces between values and units
Response 22: Thank you very much for your suggestion. We revised the sentence
Point 23: L137-138 - I don't understand. Please clarify the sentence.
Response 23: Thank you very much, and we revised the sentence.
Point 24: L152 - are these final concentrations?
Response 24: Yes, they are final concentrations.
Point 25: L157 - please use non-breaking space between values and units
Response 25: Thank you very much, and we added the non-breaking space.
Point 26: Section 2.5 - how were the metabolites identified? did you use standards? Database? please clarify
Response 26: Thank you very much, Compounds were identified by comparison of the mass spectrum of each peak with those of authentic standards in a mass spectra library (NIST, 2010) and reported previously in literature.
Point 27: L167 - please correct the name of the enzyme (no spaces)
Response 27: Thank you very much for your suggestion. We corrected the name.
Point 28: L 171 - non-breaking space
Response 28: OK, thank you.
Point 29: L171 - spots=colonies?
Response 29: Yes, we revised this word.
Point 30: Section 2.7 - 2D protein structure missing - or is this considered "physical property"?
Response 30: The “physical property” refers to its molecular mass and its pI as showed in result. And the secondary structure was showed in Figure S2, which displayed 45.3% α-helix, 15% β-sheet and 39.7% random coil.
Point 31: L195 - please clarify "inoculated products"
Response 31: “inoculate products” stands for the culture suspension after induced by IPTG, and we revised the word to avoid misunderstanding.
Point 32: L204 - SDS-PAGE details still missing - gel percentage etc.
Response 32: Thank you very much, 1.5% SDS-PAGE was used in this study, and we added the details in the manuscript.
Point 33: L204 - did you additionally purify the protein to remove the bovine enterokinase?
Response 33: No, we did not remove the bovine enterokinase.
Point 34: L208 - Ten uL
Response 34: Thank you very much for your suggestion. We revised the sentence.
Point 35: L210 - solution in what?
Response 35: The recombinant E.coli BL21 carrying Pet-32a-estA was cultivated in LB media and after induced by IPTG and purification, the purified protein was eluted by elution buffer and stored in PBS. We are sorry for our negligence, and we added in the manuscript.
Point 36: L210 and throughout - please unify the symbol for degrees
Response 36: Thank you very much for your suggestion. We unified all symbols for degrees.
Point 37: L211-214 - please proofread, re-write and make it clearer.
Response 37: Thank you very much, the sentence has been revised.
Point 38: L213 - did you use the same protocol for HPLC as described above? if yes, please specify.
Response 38: Yes, the sample protocol was used, and we clarified in the manuscript.
Point 39: L218 - non-breaking spaces, unify the symbol for degrees.
Response 39: Thank you very much, we unified all symbols for degrees and non-breaking spaces
Point 40: L228 - please include reference for SAS
Response 40: Statistical Analysis System (SAS, version 9.0, SAS Institute Inc., Cary, NC, USA), and we have explained it when we first mentioned (L124).
Point 41: Figure 2B-control (medium without cells) missing. Please demonstrate that there was no spontaneous degradation.
Response 41: Thank you very much for your suggestion. We supplemented the control and renewed the figure. As is shown above, it indicated that there was spontaneous degradation, however, the degrading efficiency against beta-cypermethrin was much more higher with the addition of BCC01 compared with those in the control group.
Point 42: Fig2A - please specify the medium in the caption (black triangles)
Response 42: Thank you very much, and we added the media in the caption.
Point 43: L278.-identification not included in Materials and methods
Response 43: Thank you very much, and we added the methods of identification.
Point 44: L312 - did you confirm the pI? Does the proposed size correspond the band on the gel?
Response 44: No, we did not confirm the pI after prediction. Yes, the propose size was consistent with the band on the gel. The protein was 28.2 kDa with a his and TrxA tag in the vector (28.2+6+11=45.2), which was the same size on the gel.
Point 45: L331-335 - please fix the alignment. Please proofread it again. there are some errors (species not italicized, no space after period etc)
Response 45: Thank you very much for your suggestion. We fixed the alignment and revised the errors.
Point 46: L332 - which protein marker?
Response 46: The protein marker from takara was used in this study, and the code number is 3450, and we are sorry for our negligence, and we added the description in the manuscript.
Point 47: L333 - what does the symbol after degree celsius mean?
Response 47: Thank you very much for your suggestion. We revised the symbol.
Point 48: I might have missed it - what is the degradation product of EstA?
Response 48: Thank you very much for your suggestion. We provided the pathway of beta-cypermethrin through the biodegradation by strain BC001 including the degradation product in Figure 3.
Supplementary
Point 49: Fig S1- the resolution of images is poor, the numbers and the details cannot be seen.
Response 49: Thank you very much for your suggestion, we revised the Fig.S1 to make it cleaner.
Point 50: Fig S2 caption- what tool was used for secondary structure prediction? Details in Materials and Methods missing. Or is this considered "physical property"?
Response 50: We used the PSIpred online server at the University of Warwick, UK for secondary structure prediction, at: http://bioinf.cs.ucl.ac.uk/psipred/. and we added in the manuscript.
Point 51: FigS3 caption - please proofread and make it clearer. I cannot see "boxed" motif.
Response 51: Thank you very much for your suggestion. We renewed the figure to make it clearer and we revised the "boxed" motif.
Point 52: Fig S4 caption - Callibration curve.
Response 52: Thank you very much for your suggestion. We revised the caption.
Point 53:Table S1 caption - please explain the acronym (CAS)
Response 53: Thank you very much for your suggestion. We explained the CAS NO.
Point 54: Table S2 caption - Please explain Pr>F.
Response 54: Pr>F indicate the model terms are significant. We replaced Pr>F with P Level in the manuscript, and P Level less than 0.05 indicate the model terms are significant.
Point 55: Table S3 - this is not really a table. Please reconsider the caption if you decide keeping it. I would rather suggest depositing the sequence in the database and providing the accession number in the manuscript.
Response 55: Thank you very much for your suggestion. We deleted the Table S3 and provided the accession number: MH588686 in the manuscript.
Round 3
Reviewer 2 Report
After the authors implement my suggestions, I can endorse this manuscript for publication.
L189 - please include appropriate reference for the primers.
Figure 2B - is beta-cypermethrin control medium without bacteria? If yes, please specify (this is the control I was expecting in my previous review)
Response 6 - maybe "remained soluble"?
L28 and throughout - please make sure that hyphens, en dashes and em dashes are used correctly
Response 16 - this is very valuable information. Please include it in the manuscript
L138 - is asterisk a typo?
Point 30 - 2D structure prediction still missing.
Point48: Do you think this enzyme is involved in the first step?
FigS3 caption - capital. Please thicken the box - i can barely see it.
Author Response
Response to Reviewer 2 Comments
Point 1: L189 - please include appropriate reference for the primers.
Response 1: Thank you for your suggestion, and the reference has been included.
Point 2: Figure 2B - is beta-cypermethrin control medium without bacteria? If yes, please specify (this is the control I was expecting in my previous review)
Response 2: Yes, the beta-cypermethrin control medium without bacteria. Thank you very much for your suggestion, and we added the caption in the Figure 2B.
Point 3: Response 6 - may be "remained soluble" ?
Response 3: Yes, remained soluble may be more appropriate.
Point 4: L28 and throughout - please make sure that hyphens, en dashes and em dashes are used correctly.
Response 4: Thank you very much for your suggestion, and we revised en dashes and em dashes correctly.
Point 5: Response 16 - this is very valuable information. Please include it in the manuscript
Response 5: Thank you very much for your suggestion, and we added this section in the manuscript (L112-114).
Point 6: L138 - is asterisk a typo?
Response 6: The asterisk refers to multiply. Thank you very much for your suggestion, and we replaced the asterisk(*) with multiple sign (×) in the manuscript.
Point 7: Point 30 - 2D structure prediction still missing.
Response 7: Thank you very much for your suggestion, here secondary structure was provided with 3D structure was included this time.
Point 8: Point 48: Do you think this enzyme is involved in the first step?
Response 8: Yes, we guess the Esta is the first step in the reaction.
Point 9: FigS3 caption - Capital. Please thicken the box - i can barely see it.
Response 9: Thank you very much for your suggestion. We revised the "boxed" motif to make it clearer.
