福建省农业科学院
Fujian Academy of Agricultural Sciences, P. R. China
Tel:86-13905917339 Fax: 86-591-87884262
Email: fzliubo@163.com
IJSEM-D-14-00024
Bacillus radicibacter sp. nov., a bacterium isolated from Oxytropis ochrocephala Bunge root nodule
International Journal of Systematic and Evolutionary Microbiology
Dear Prof. Liu,
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I would greatly appreciate your help in reviewing the following paper for International Journal of
Systematic and Evolutionary Microbiology.
The abstract for this paper is:
Abstract, A gram-positive, rod-shaped, endospore-forming bacterium, designated 53-2T, was
isolated from the root nodule of Oxytropis ochrocephala bunge, was studied to determine its taxonomic
status. The BLAST search hit based on 16S rRNA gene sequence indicated that it was closely related to
Bacillus acidicola 105-2(T) (95.64%), Bacillus kribbensis BT080T (95.43%), Bacillus soli LMG 21838T
(95.39%) and Bacillus vireti LMG 21834T (95.35%). Comparative 16S rRNA gene sequence analysis
showed that the strain 53-2T formed a distinct phyletic line within the genus Bacillus. Chemotaxonomic
data (major isoprenoid quinone, MK7; DNA G+C content , 37.78%; polar lipid, diphosphatidylglycerol,
phosphatidylglycerol, unknown phospholopid and polar lipid, a small quantity of
aminoglycophospholipid; fatty acid, anteiso-C15: 0 and anteiso-C17: 0) supported the affiliation of the
isolate to the genus Bacillus. On the basis of physiological, biochemical and phylogenetic properties,
strain 53-2T represents a novel species within Bacillus genus, for which the name Bacillus radicibacter is
proposed, the type strain is 53-2T (=DSM27302T =ACCC06115T= CCNWQLS5).
Thank you very much for your time and we look forward to hearing from you.
Yours sincerely
Alejandro P. Rooney
Editor
International Journal of Systematic and Evolutionary Microbiology
Dear Prof. Alejandro P. Rooney
Thank you for your invitation for review of Bacillus radicibacter sp. nov., a bacterium isolated from
Oxytropis ochrocephala Bunge root nodule. I write the comment about the manuscript to you.
Comment in general
In the paper of Bacillus radicibacter sp. nov., a bacterium isolated from Oxytropis ochrocephala
Bunge root nodule, the authors indicate that this strain is most closely related to Bacillus acidicola
105-2(T) at 95.64%16S rRNA gene sequence similarity, and the DNA-DNA relatedness with the closest
relatives species (Bacillus acidicola 105-2(T), Bacillus kribbensis BT080T, Bacillus soli LMG 21838T, and
Bacillus vireti LMG 21834T) has not been done. It seems that the species would be a noval species. But
many problems are in the paper. Only when whole the questions are solved the new species would be
published. I will write the problems one by one.
Comment 1 About reference strains
In the paper there is no mention where the reference strains come from, are Bacillus acidicola
105-2T, Bacillus kribbensis BT080T, Bacillus soli LMG 21838T, and Bacillus vireti LMG 21834T the
reference strains, please note that. If any of the strains used for compared is not the reference strain,
the paper is directly refused.
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Comment 2 About DNA–DNA hybridization
The DNA–DNA relatedness between the isolate and the reference strain has not been done. It is
very importance of DNA–DNA hybridization for identifying a noval species, especially when the puzzle
reference strains used. It is not enough with the citation to guess DNA–DNA hybridization according to
the reports of E. Stackebrandt and B. M. Goebel, it is unlikely that 53-2T and other related Bacillus spp.
have more than 60-70% DNA similarity and hence that they are related at the species level
(Stackebrandt et al., 1994). It must be done with DNA–DNA hybridization between the isolate and the
reference strain before it publishes.
Comment 3 About methods
1 Abstract should be rewrite as the writing forms of this journal. Please add growth conditions
and optimal growth conditions for the isolate, Where did the isolate come from? Data of DNA-DNA
relatedness between the isolate and its closest phylogenetic neighbors must be cited, and so on.
2 Introduction was very poor. Pleas rewrite it.
3 Lines 75-79, you should provide the storage methods of the strains, and where did you get the
reference strains? Line 87-89, How did you determine the optimum growth condition of strain 53-2T ?
NA is abbreviation of nutrient agar, what is the meaning of ‘NA liquid medium’？
4 Line 114-116. Please determine this result again, or change the writing of this result. Line
116-119, What is the meaning of this sentence? Which substrate has the result like you described in the
text? Lines 139-141, In the text, “constructed using the neighbor-joining, maximum-parsimony and
maximum-likelihood methods implemented in the software package MEGA version 5.0 (Tamura et al.,
2011)”, but there was no any data in the text or in supplementary files. Please add the phylogenetic
trees constructed by maximum-parsimony and maximum-likelihood methods as supplementary Figures.
Please include Bacillus subtilis type strain data of 16S rRNA gene in phylogenetic analysis.
5 Line 141-142, Here, the closely reference strains did not agree with the above. Lines 129-146,
This paragraph needs to be rewritten. How does the sequence aligned before constructing phylogenetic
trees? Please add the cited references how the phylogenetic trees were constructed in this paragraph.
Lines 147-149, This paragraph should be rephrased. There was no any DNA-DNA hybridization data in
the text, you should provide the DNA-DNA relatedness between 53-2T and its closest reference strains?
Which reference strains were selected to do hybridization experiment? How did you perform this
experiment? Which method you used in this study?
6 Lines 150-153, ‘The DNA G+C content of strain 53-2T was estimated by using the thermal
denaturation method (Marmur et al., 1962).’ This thermal denaturation method has been demonstrated
to inaccurate and these data are now obtained from HPLC analysis or whole genome sequencing data.
The authors do not indicate whether they used control organisms for this method. Did they obtain the
data for the reference strains using the same method and did they get similar values? Line 154,
Respiratory quinones; Line 199, primary lipoquinone; Line 38, major isoprenoid quinone? Are they the
same thing? You should use the same description.
7 Lines 154-188, these sentences were so complicated and need to be rephrased. You should
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provide concise description of the methods of chemotaxonomic analytical procedures and cite which
reference you referred. There was no reference of detecting quinones by HPLC and no data about MK-7.
No data about the component of cell wall of strain 53-2T? Please provide this data. Lines 165-166,
There is no point to report fatty acid data to the second decimal place. These data are variable from run
to run and highly variable between labs. The second decimal place is most likely outside the standard
deviation of these data. How many times did the authors run the fatty acid analysis?
8 Lines 189-190, In this paragraph, you should add several data and description differentiated
strain 53-2T from its reference strains. Please add period at the end of this paragraph. Lines 192-193,
etymology of the species should be described in detail. Line 198, should provide the optimum growth
data of pH, temperature and NaCl.
Comment 4 About English writing
Too many grammar problem and spelling mistakes were in almost every sentence of the
manuscript. Some of them are detailed as follow.
1 Line 11 and 23, Shaanxi should be Shanxi.
2 Line 32, gram positive should be ‘Gram-positive’
3 The sentence ‘Comparative 16S rRNA gene sequence analysis showed that the strain 53-2T
formed a distinct phyletic line within the genus Bacillus.’ should be forward.
4 Line 38 ? the DNA G+C content? the DNA G+C content was not own to chemotaxonomic data.
5 Line 41, 42, 44, Bacillus genus should be ‘the genus Bacillus’
6 Line 44, ?? formed? by Cohn in 1872
7 Lines 49-50, ‘in the past recent years’ should be ‘in the recent years’?
8 Line 50, please rephrase the sentence ‘even more excited’, it is not placed on the first of the
sentence. ‘increasing’ should be instead of ‘promote’ or ‘accelerate’.
9 Lines 53-54, ?? a well-known locoweed that can lead to poisoning in pasturing area when
mistaken as animal fodder,
10 Line 55, ‘Oxytropis genus’ or the genus ‘Oxytropis’?
11 Line 72, Bacillus spp.
12 Line 73, insert space before (Xu, et al., 2014)
13 Line 75, what is mean of this sentence “The isolation and purification were had been
described in Xu’s paper”, the word ‘were’ and ‘had’ could not appear in a sentence at the same time.
14 Line 77, ?”fit for”. Line 81, 195, ??(0.6~0.9 ×1.7~4.0μm) or (0.6~0.9 μm in width and
1.7~4.0μm in long), Please use the same writing.
15 Line 82, ??(Supplementary Fig. 1).
16 Line 85, ??seen occurred? Please rephrease.
17 Line 86, ??Gram-negative? Please rewrite the sentence ‘The cells stained 86 Gram-negative
after following the procedure described by Doetsch 1981’
18 Line 95 ‘the strain’ should be ‘strain’.
19 Line 112, Gelatin should be gelatin.
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20 Line 113, please delete the ‘,’ before ‘and.’
21 Line 126-127, ‘Bacillus soli IDA0086T, but opposite for Bacillus kribbensis BT080T , the
condition for Bacillus acidicola 105-2T and Bacillus vireti IDA3632T’, should be ‘B. soli IDA0086T, but
opposite for B. kribbensis BT080T , the condition for B. acidicola 105-2T and B. vireti IDA3632T’.
22 Lines 127-128, ?? “The result confirmed that strain 53-2T was distinguished from the reference
strains used in this study”??
23 Line 129, should be 53-2T, insert space before (Wilson, 1997)
24 Line 139, ‘fig. 1’ should be ‘Fig. 1’
25 Line 144, 152, ??(Supplementary Table 2).
26 Line 148, here, ‘Bacillus spp.’ should be ‘Bacillus species’.
27 Line 127, 141, 147, 163, 185, 202, 53-2 should be 53-2T.
28 Line 151 insert space before (Marmur et al., 1062)
29 Line 153, ?? Table 1 ?
30 Line 180 ? 53-3T ?
31 Line 201, ?? The DNA G+C content is 37.2 mol%.
32 line 202-212, Intial of Pyruvic Acid, Adenosine, Glycerol, Esculin and so on, should be low
case.
33 Description of Bacillus radibacter should be described in sequence agreeing with the order of
the results. Please add one sentence “The type strain is 53-2T (=DSM 27302T =ACCC 06115T=
CCNWQLS5). Strain53-2T was isolated from ……….. ”at the end of the paragraph of the “Description
of Bacillus radibacter”
34 Please improve the English.
Conclusion
The reference strains are no clear. The DNA–DNA relatedness between the isolate and the
reference strain has not been done. The English writing is very poor with many mistakes in the text. So
suggest: refuse to publish.
Prof. Dr. Liu Bo
Fujian Academy of Agricultural Sciences
Fuzhou, Fujian 350003, PR China
fzliubo@163.com
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