福建省农业科学院
Fujian Academy of Agricultural Sciences, P. R. China
Tel: 0086-591-87864601 Fax: 0086-591-87884262 Email:
fzliubo@163.com
Notes for the paper corrected
according to the reviewers and editor
Dear Mr. Prof. Dr. Editor,
We sincerely thank for your great effort in the paper correction. According to the reviewers and
editor's comments the paper is conscientiously corrected. The notes show as follow.
1. Comments from Editor
Comment 1. Editors’ comment: are genome sequences available for any of the closest relatives of strain
FJAT-14515T? If so it would be useful to include genomic comparisons (e.g. ANI and digital DDH values).
Reply: There are not complete genome sequences of the reference strains close to strain FJAT-14515T
found in the NCBI GenBank database, between which ANI and digital DDH values cannot be calculated. It
was difficult to get the genome sequence of two reference strains.
2. Comments from Reviewer #1
Antonie van Leeuwenhoek paper ANTO-D-14-00123 by Liu et al., describes a new
species within a genus Bacillus based on the polyphasic characterization of single strain
FJAT-14515T isolated from a soil sample. Some parts of the paper could be improved prior to
its publication.
(1) General comments:
Comment 1. The introduction is very poor, and some expressions were not accurate, such as
"their cell walls contain peptidoglycan of the meso-diaminopimelic type", because some
Bacillus strains have other peptidoglycan types;
Reply: Thank you very much for the comment that the introduction is really poor. We has rewritten the
introduction to summarize the bacillus-like species found in Taiwan. The details were showed in the text.
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Comment 2. The author should state clearly how did they get the strain? Why did they
choose this isolate as a candidate novel species? What is the geographic information of the
sampling site? How the soil sample was sampled and transported to Fujian where the research
was performed. Is it legal to get the soil sample from Taiwan to Fujian?
Reply: Thanks, this is a very good question. Strain FJAT-14515T was isolated from the plant
rhizosphere soil (20 cm) of Acacia confusa (121°17'89.03''E and 24°50'45.73''N) at the Cihu
area of Taoyuan County in Taiwan in Aug. 2013 by Prof. Dr. Ming-Kuang Wang, who is a soil
scientist working in Department of Agricultural Chemistry, National Taiwan University. In May 2012 he
has been officially hired as a scientific advisor for Fujian Academy of Agricultural Sciences (FAAS) in
conjunction on researches of soil microorganism between FAAS and Taiwan University. He donates a
serial of soil samples to FAAS for comparison of soil researches. He is one of the authors being legal to get
the soil samples from Taiwan to Fujian. From which we isolated Bacillus strains, namely FJAT-14514,
FJAT-14515T, and FJAT-14516. The method for isolation is by using the standard dilution-plating
technique on nutrient agar (NA) (Atlas 1993) at 30 °C for 48 h. Through the 16S analysis of three strains,
one of them, FJAT-14515 showed 97.6% 16S rRNA similarity closely to Bacillus muralis, which was
selected to do further testing in the paper.
Table 1 16S rRNA similarities of isolated Bacillus strains in the sample from Taiwan
Isolates
Closest species
Identified (%)
FJAT-14514
Bacillus aryabhattai
100.0
FJAT-14515T
Bacillus cihuensis
97.6
FJAT-14516
Bacillus pseudomyciodes
100.0
The co-author, Prof. Dr. Ming-Kuang Wang gives the statement showed as follow:
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Comment 3. How to determine cell wall peptidoglycan?
Reply: The cell wall peptidoglycan was analyzed by TLC as described by Schleifer (1985) that executed
in China Center of Industrial Culture Collection (CICC), China.
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Comment 4. The data about the mobile ability conflict each other: "non-motile" in line 170,
but "motile" in line 211 and "mobile" in Table 1. Please clarify. Can you provide a
transmission electron micrograph exhibiting flagella?
Reply: Sorry, it is our writing error, the strain FJAT-14515T is motile. The motile ability of strain
FJAT-14515T was determined by motility agar (Chen et al. 2007). The cell morphology was observed by
scanning electric and the flagella was not observed.
Comment 5. Table 1, temperature, pH and NaCl, Please just give the range and optimum.
Reply: Ok, the Table 1 is rewrited. The range and optimum of temperature, pH and NaCl of strain
FJAT-14515T and reference strain B. muralis DSM 16288T and B. simplex DSM 1321T were added. The
details were showed in the rewriting Table 1.
(2) Minor comments:
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Comment 6. Lines 31-32: please use - instead of "—"
Reply: Ok, in the Lines 31-32, the “-” was used instead of “—” that the details were “The strain grew at
10 − 35 °C (optimum at 30 °C), pH 5.7 − 9.0 (optimum at pH 7.0) and at salinities of 0 − 5% (w/v) NaCl
(optimum at 1%, w/v).” seen in the text.
Comment 7. Lines 35, 53, 133, `36, 156, 163, 235, etc: ? G + C ?
Reply: Ok, in lines 35, 53, 133, `36, 156, 163, 235, “G + C” was revised as “the DNA G+C content”
Comment 8. Line 35: ? The DNA G + C
?
Reply: The revision of Comment 8 was same with Comment 7. In line 35, the sentence “DNA G+C
content was 37.1 mol%” was altered to “the G+C content of strain FJAT-14515T was 37.1 mol%”.
Comment 9. Lines 35-36: the sentence "The strain was catalase-positive and
oxidase-negative" should be deleted or be moved somewhere forward.
Reply: Ok, thank you for your suggestion. The sentence "The strain was catalase-positive and
oxidase-negative." had been moved forward, behind the first sentence “A Gram-positive, moderately
halotolerant,…………isolated from a soil sample in Cihu, Taoyuan County, Taiwan.” 我再看看，
Comment 10. Lines 36-38: ? A phylogenetic analysis based on 16S rRNA gene sequences
indicated that strain FJAT-14515T belongs to the genus Bacillus, and was most closely related
to the type strains of Bacillus muralis (97.6%) and Bacillus simplex (97.5%). Levels of
DNA-DNA relatedness between strain FJAT-14515T and the type strains of B. muralis and B.
simplex were 27.9% ± 3.32 and 44.1% ± 0.57, respectively ?
Reply: Ok, thank you. According to the reviewer's comment, lines 36-38 “The highest level of 16S rRNA
gene sequence similarity was with B. muralis DSM 16288T (97.6%). DNA–DNA similarity of strain
FJAT-14515 T and B. muralis DSM 16288T was 27.9%.”had been revised as “A phylogenetic analysis
based on 16S rRNA gene sequences indicated that strain FJAT-14515T belongs to the genus Bacillus, and
was most closely related to the type strains of B. muralis (97.6%) and B. simplex (97.5%). Levels of
DNA-DNA relatedness between strain FJAT-14515T and the type strains of B. muralis and B. simplex were
27.9% ± 3.32 and 44.1% ± 0.57, respectively.”
Comment 11. Lines 66-69: The sentence "Soil samples were air-dried, ground, passed
through a 2 mm sieve and stored in plastic box for further analysis and isolation of bacterial
strains" should be moved forward to the beginning of the paragraph.
Reply: Ok, according the comment, we decided to delete the sentence "Soil samples were air-dried, ground,
passed through a 2 mm sieve and stored in plastic box for further analysis and isolation of bacterial strains"
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from this paragraph, and insert some words in the first sentence of this paragraph. The detail was showed in
the text.
Comment 12. Lines 69-74: please rephrase; the sentence "showed 97.6% 16S rRNA
similarity to B. muralis DSM 16288T" should not be in the section "Strains and culture
conditions".
Reply: Ok. Thank you for the comment, and this sentence in ‘Materials and Methods’ was deleted from the
text.
Comment 13. Line 77: ? on NA under ?
Reply: Two reference strains were cultured routinely on NA medium under the identical
conditions
Comment 14. Line 84: ?
on motility agar ?
Reply: The sentence “Motility was examined on motility agar (Farmer, 1999).” was mistake, it should be
revised as that “Motility was examined on motility agar (Farmer, 1999).”
Comment 15. Line 93: nitrate reduction can not be tested using API 20E. Please clarify the
methodology.
Reply: Thank you for your comment. The test of nitrate reduction was using the method described as
Smibert and Krieg (1994).
Comment 16. Lines 96-97: The methodology of utilization of carbon and nitrogen sources
was not directly described by Chen et al (2011). Please clarify the methodology.
Reply: The methodology of utilization of carbon and nitrogen sources did not fully directly reference Chen
et al (2011), and was described by Gao et al (1994).We selected several carbon and nitrogen sources as sole
source and observed the utilization of carbon and nitrogen sources of strains by the growth.
Comment 17.
Lines 135-140: How many repeats of the DNA-DNA hybridization?
Reply: Ok. There were three repeats of the DNA-DNA hybridization in the text.
Comment 18. Lines 146-147: ? The almost-complete 16S rRNA gene sequence (1439 bp) of
the organism was determined (GenBank accession number JX262264). ?
Reply: Ok. We accepted the suggestion. In lines 146-147, the sentence ‘The 16S rRNA gene sequence of
strain FJAT-14515T 146 was a continuous stretch of 1439 bp’, was revised as “The almost-complete 16S
rRNA gene sequence (1439 bp) of the organism was determined (GenBank accession number JX262264).”
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Comment 19. Lines 146-153: please rephrase the phylogenetic analysis results.
Reply: Ok. Lines 146-153 were not clearly expressed and has been rephrased, the detail was showed in the
text.
Comment 20. Lines 153-154: the sentence "To differentiate strain FJAT-14515T from these
closely related species, DNA-DNA hybridization was performed" should be deleted.
Reply: Ok. According to the comment, the sentence "To differentiate strain FJAT-14515T from these
closely related species, DNA-DNA hybridization was performed" had been deleted from the text.
Comment 21. Lines 170-181: This section should be minimum because this is redundant
description of species description.
Reply: Ok. Lines 170-180, this section was so much and redundant. According to the comment, we had
deleted the redundant description of species description, and left the description of difference between
strain FAJT-14515T and reference strains.
Comment 22. Lines 200-204: It is commended to add a subheading "subheading" for this
paragraph, and more details supporting the proposal of strain FJAT-14515T representing a
novel species of the genus Bacillus should be discussed here.
Reply: Ok. We added the subheading “Conclusion” for the “Lines 200-204”, and added the description of
this section. The details were showed in the text.
3 Comments for Reviewer #4:
This manuscript described a novel species in genus Bacillus. The methods used and
the data obtained were solid. It could be published in this journal except the following
major revisions.
Comment 23. The English writing should be improved greatly.
Page 3, line 51-53. The reference should be updated. It is lightly old.
Reply: The reference had been updated in the text.
Comment 24. Page 3, line 74-75, how long are the storage time at 4 or -80 °C, respectively?
Reply: Ok. In our lab, the storage time of strain collection at 4 or -80 °C was three months and more than
three years, respectively.
Comment 25. Page 4, line 96, two nouns, "Determinatives ion utilization", were used
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together wrongly. The sentence should be rewritten.
Reply: Ok. Thank you for the comment. The sentence “Determination utilization of carbon and 97 nitrogen
sources was performed as described by Chen et al. (2011).” has been altered to “The utilization of sole
carbon and nitrogen sources was determined according to the method described by Gao et al. (1994).”
Comment 26. The tree of 16S rRNA gene should be reconstructed by including more type
strains, such as Bacillus endoradicis, and so on.
Reply: According to the suggestion, we have added another two strains (FJAT-14514 and FJAT-14516)
isolated from the same soil (as above) sample and nine Bacillus type species, namely Bacillus megaterium
IAM 13418T (D16273), Bacillus aryabhattai B8W22T (EF114313), Bacillus pseudomycoides DSM 12442T
(ACMX01000133), Bacillus subtilis DSM 10T, Bacillus herbersteinensis DSM 16534T, Bacillus halmapalus
DSM 8723T, Bacillus endoradicis LMG 25492T, Bacillus coahuilensis CECT 7197T , Bacillus koreensis DSM
16467T, to reconstructed the phylogenetic tree by three methods (Neighbour-joining, minimum-likelihood
and maximum-parsimony menthods), the detail was showed in the text.
Comment 27. Page 7, line 190, appendix 1 should be supplementary files…,
Reply: OK. The appendix 1 had been revised as supplementary Fig. S3.
Comment 28. Page 7, line 192, Appendix 2 should be supplementary Fig. S…
Reply: OK. The appendix 2 had been revised as supplementary Fig. S4
Comment 29. Whole genome of the type strain FJAT-14515T has been sequenced and
published. In this manuscript, the authors should cite this manuscript and state clearly in
details what is the main difference or similarity of this strain from others.
Reply: Whole genome of the type strain FJAT-14515T has been sequenced and published. However, the
genome sequence of reference strains has not been found in the NCBI or other database. So, we could not
analysed the main difference or similarity of this strain from others.
Comment 30. In Fig. 2, a is enough. b can be deleted. c is just a photograph, not an optical
micrograph.
Reply: In Fig. 2, b had been deleted. In the text, “Optical micrograph of strain FJAT-14515T showing the
typical morphology (c).” had been changed to “Optical photograph of strain FJAT-14515T showing the
typical morphology (b).”
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