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 efforts 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. Consistently use superscript 'T' when referring to the type strains.
Reply: Thanks for your kind suggestions, and all the type strains in the manuscript were corrected using
superscripy ‘T’
Comment 2. L49. Use correct terminology when referring to colony macromorphology; what is the
meaning 'colonial'?
Reply: We are sorry for the incorrect writing, and the word “colonial” in the original sentence of “ In our
studies on the diversity of the microbial community in graveyard soil from the No. 1 pit soil of Emperor
Qin's Terra-cotta Warriors, one representative isolate, FJAT-13831T, was found to have typical colonical
features of Bacillus genus (Liu et al. 2012)” has been changed into “ morphology”.
Comment 3. L52. Spell out 'ANI'.
Reply: The full name of ANI is average nucleotide identity, and has been spelled out in line 55.
Comment 4. L55. Provide details on the isolation procedures, dates of the isolation; use correct
terminology.
Reply: The details on the isolation procedures and dates of the isolation were as followed: the source of the
isolates was from the soil taken in 2010 by Dr. Liu in the pit of Emperor Qin's Terra-cotta Warriors in Xi’an
province, the northwest part of China. For the isolation of the strain, 10 gram of soil was placed into 90 ml
sterile water, vortexed, diluted, then spread onto a solid medium of nutrient agar (NA) (Atlas, 1993) with
0.5% NaCl solution, and incubated at 30 ºC for 48 h. The isolated strains were subcultured several times to
obtain a purified culture, and were then further characterized, and this part has been added in the
manuscript, as shown in line 58-62.
Comment 5. L68. Add reference.
1
Reply: Experiment of halotolerance performed on Nutrient broth (NB) was conducted as the reference of
(Atlas, 1993) and this reference has added in the manuscript, as shown in line 83.
Comment 6. L125. What is meaning of class in the Bacillus context?
Reply: We have to apology for our mistake and “class” in the sentence of the phylogenetic analysis
positioned strain FJAT-13831T in the Bacillus class with the closely related species B. pseudomycoides
DSM 12442T (99.72% sequence similarity) (Fig. 1) has been change in to “genus”, as highlighted in line
140
Comment 7. L130. Delete 'sp.nov'
Reply: Thanks for your suggestion, we have deleted 'sp.nov' which is in the sentence of its affiliation to the
novel species Bacillus safensis sp. nov. has been determined based on its 91.2% gyrB sequence similarity
with B. pumilus and other classification tests (Satomi et al. 2006), as shown in line 145
Comment 8. L139. This statement is not accurate.
Reply: The sentence “Stackebrandt and Ebers (2006) reported that bacterial strains with a difference in the
gyrB gene sequence of less than 5% cannot be classified as the same species” was rephrased as
“ Stackebrandt and Ebers (2006) reported that bacterial strains with a difference in the gyrB gene sequence
of more than 5% cannot be classified as the same species”, as shown in line 153-155.
Comment 9. L148. Add strain numbers, justify the range of the strains used and their source. Note, some
of these strains are not mentioned L57.
Reply: Strain numbers of Lysinibacillus sphaericus and lysinibacillus fusiformis, were CCUG7428 and
CCUG28888, respectively. These two strain along with Bacillus thuringiensis ATCC 10792T, Bacillus
weihenstephanensis KBAB4, Bacillus anthracis ATCC 14578, Bacillus megaterium DSM 319T and
Bacillus aquimaris DSM 16205T were all obtain from DSMZ (Deutsche Sammlung von Mikroorganismen
und Zellkulturen, Braunschweig, Germany) and all selected for DDH experiment, 16S rRNA and gryB gene
analysis. And the details could be found in line 65-71.
Comment 10. L149. Include standard deviation values for the DDH experiment. Extend DDH experiments
according to current requirements for species description - including three other species which showed
more than 97% of 16S rRNA gene sequence similarities.
Reply: We have to apology for our careless. Total eleven reference strains used in DDH experiment and
the relative information have been redescribed in the section of DNA-DNA hybridization, as shown in line
113-114
Comment 11. L152-158. Revise the text, carefully use the wording to avoid overstatements, e.g., 'clearly'
is not relevant in this context. Further, L159 - 36.5 mol% is within the previously described values; taken
into account standard deviation it is a well fit value.
Reply: Modifications have made in the revised manuscript accordingly, as shown in line 161-167, and the
standard deviation of GC content is ± 0.08, which have been added in the manuscript.
Comment 12. L172-183. Revise the text using accurate wording: all 7 species formed a robust
phylogenetic cluster.
2
Reply: The text has been rephrased as “A phylogenetic analysis revealed that all seven strains formed a
robust phylogenetic cluster, indicating the strain FJAT-13831T belong to the bacillus genus, as highlighted
in line 188-190.
Comment 13. L196-198. Compare with other relevant species.
Reply: According to the reference of Schleifer and Kandler, 1972 (Bacteriol Rev 36: 407–477) and Ahmed
et al., 2007 (Int J Syst Evol Microbiol 57: 1117–1125), the cell-wall peptidoglycan composition and
predominant menaquinone type of strain FJAT 13381T was compared with other bacillus species,
presenting that the cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic cell wall
diamino acid, corresponding with the same peptidoglycan type of the majority bacillus species except the
species of B. fusiformis DSM 2898T (Schleifer & Kandler, 1972) and the strain FJAT-13831T contained
MK-7 (89%) which also occurred in other bacillus species, such as Lysinibacillus fusiformis and
Lysinibacillus sphaericus (Ahmed et al., 2007), and the detail descriptions was shown in line 213-219 .
Comment 14. L200-215. Revise the text to remove repeated in species description information; summarize
the differential characteristics.
Reply: We have deleted the repeated in species description information, and added the differential
characteristics by comparing with the reference strains (B. pseudomycoides DSM 12442T B. mycoides DSM
2408T B. cereus DSM 31T), and the details was as follow: morphologically, the colony features of strain
FJAT-13831T were greyish-white, nearly circular, and opaque, with a dry surface and a wrinkled margin
(Fig. 3a), which were obvious different from the other three reference strain, because B. pseudomycoides
DSM 12442T B. mycoides DSM 2408T B. cereus DSM 31T were all pale yellow in color, irregularly shape
and with branching (Fig. 3b), dentate (Fig. 3c), and smooth margins (Fig. 3d), respectively. Cells of the
strain FJAT-13831T were cylindrical (1.6 - 3.3 x 1.1 - 1.8 μm), motile, aerobic, and Gram-positive (Fig. 4a),
appearing the marked differences compared with the other three bacillus species (Fig.4). Furthermore, the
tested bacillus species could grow at the range of 20-40 ◦C, however, unlike other bacillus species, no
growth of strain FJAT-13831T occurred below 20 ◦C and above 40 ◦C (Table 1). They also were tolerant to
different NaCl concentrations, strain FJAT-13831T up to 4%, B. pseudomycoides DSM 12442T up to 2 %,
B. mycoides DSM 2408T up to 6 % and B. cereus DSM 31T up to 8% (Table 1). The different pH range for
growth between strain FJAT-13831T and other reference strain was observed difference, coupling with 4-10
(strain FJAT-13831T), 5-9 (B. pseudomycoides DSM 12442T), 6-10 (Bacillus mycoides DSM 2408T) and
6-10 (Bacillus cereus DSM 31T) (Table 1), as shown in line 215-228.
Comment 15. L223-226. Make sure the species epithet is correctly explained.
Reply: Bacillus bingmayongensis (bing. ma. yong. en'sis. Chinese phonetic alphabet n. Bīng Mǎ Yǒng,
literally "military servants") (Terra-cotta Warriors and Horses, a collection of 8099 life-size terra-cotta
figures of warriors and horses located in the Mausoleum of the First Qin Emperor from more than two
thousand years ago in China); N.L. masc. adj. bingmayongensis, belonging to Bīng Mǎ Yǒng, a
mausoleum in Xi'an City, China, the source of the isolates.
Comment 16. Improve Table 1, add other species of B.cereus group, delete characteristics which are '+' or
'-' for all species and mention these in the footnotes.
Reply: Thanks, table 1 has been improved accordingly. The mark of “+”, “-” and “w” means growth, no
growth and weak growth, which have been mentioned in the footnotes, as shown in the new Table1.
Comment 17. L417. Data is Latin, plural.
3
Reply: Thanks, “is” has been revised as “are”.
Comment 18. Table 3. Add strain numbers.
Reply: Strain Lysinibacillus fusiformis and Lysinibacillus sphaericus have been added the strain numbers
of CCUG28888 and CCUG7428, respectively, as shown in the new Table 3.
Comment 19. Improve figures legends. Move Fig.3 to Supplementary. Revise Fig.4.
Reply: Figures legends have been improved, and we showed the new species characterics of isolate FJAT
13381T from the morphology by adding the colony and SEM of the reference strains, B. pseudomycoides
DSM 12442T, B. mycoides DSM 2408T and B. cereus DSM 31T, which were shown in the new Fig. 3 and
Fig. 4.
2. Comments from Reviewer #1
The paper describes new species of the genus Bacillus isolated from the pit soil of Emperor Qin's
Terracotta Warriors in China. This is an interesting paper that describes the new species based on some
biochemical characteristics and whole genome sequence analyses. However, I believe there are several
revisions need to be made prior to final acceptance.
Comment1: -Major points
*The authors have proposed a new species based on the data from only one strain. All of the taxonomic
studies as described in this paper only pertain to the strain FJAT-13831, and not any other strains of this
species are examined, which is likely insufficient to describe a new species. The authors should
demonstrate and effort to collect more strains belonging to this species.
Reply: Strain FJAT-13831 was proposed as a new species based on the its different characteristics, and all
taxonomic studies were designed according to the references of Montero-Calasanz et al., 2013(Antonie van
Leeuwenhoek. 103: 449-456) and Goodfellow et al., 2012 (Antonie van Leeuwenhoek. 101:185-193), that
both only one representative strain, isolate CF6/1T, from arid desert soil and isolate AB-18-032T, from a
marine sediment, respectively, was selected in the taxonomic studies.
Comment 2:*line 86. The authors should correct a reference of the universal primer set for 16S rRNA
gene amplification to "Stackebrandt, E., and W. Liesack. 1993. Nucleic acids and classification, p.152-189.
In M. Goodfellow and A. G. O'Donnell (ed.), Handbook of new bacterial systematics. Academic Press,
London, United Kingdom."
Reply: Thanks, the reference, “Yoon JH, Lee JK, Shin YK, Park YH, Lee ST (1997) Reclassification of
Nocardioides simplex ATCC 400 13260, ATCC 19565, and ATCC 19566 as Rhodococcus erythropolis. Int
J Syst Bacteriol 47: 904–907.” was changed into the reference, “Stackebrandt, E., and W. Liesack. 1993.
Nucleic acids and classification, p.152-189. In M. Goodfellow and A. G. O'Donnell (ed.), Handbook of
new bacterial systematics. Academic Press, London, United Kingdom” and the relative revisions have been
done in the section of text and reference, as shown in line 101 and 391-392.
Comment3 *line 199. The authors should check the phenotypic characteristics of B. pseudomycoides
carefully with comparing between the data obtained in this study and that of original description by. In the
table 1, B. pseudomycoides is negative for aerobic growth. Is it meaning this species anaerobic?
Reply: In our study, B. pseudomycoides is the negative for aerobic growth, that means the species
anaerobic, and the result is consistent with the results obtained from Nakamura, 1998
4
Comment4: *Fig.2. Is accession number "CM000740" of B. pseudomycoides correct? This acc.# is for B.
cereus AH1272. The authors should explain the reason that short length of gyrB sequences (approximately
650bp) were used for phylogenetic analysis. The gyrB primer set described in this paper will provide over
1000bp DNA fragment from DNAs of Bacillus spp. on PCR.
Reply: We are sorry for our wrong writing, the accession number of B. pseudomycoides DSM 12442T
should be CM000745 and has been corrected in the Fig. 2. Furthermore, due to the low resolution of 16S
rRNA gene sequences at the intrageneric level and the comparison of concatenated sequences of
housekeeping gene, such as gryB gene, provides a higher resolution than the 16S rRNA gene sequences
(Alperi et al., 2008; Morandi et al., 2005), Therefore, the short length of gyrB sequences were used for
phylogenetic analysis in our study.
3. Comments from Reviewer #2
Comment1: This manuscript reports detailed phenotypic, chemotaxonomic, phylogenetic, DNA-DNA
hybridization, and ANI analyses for a novel bacterial isolate obtained from the pit soil of Emperor Qin's
Terra-cotta Warriors in China. The analyses suggest that the isolate is closely related to, but distinct from,
the bacteria Bacillus pseudomycoides. The authors make a strong case for the distinctiveness of this newly
isolated bacterial linage within the genus Bacillus using a broad range of polyphasic evidence. Based upon
the evidence presented, the proposal for a new species made here is reasonable and this paper deserves
publication. However, recently Bhandari et al. (IJSEM, 63, 2013, 2712-2726) have proposed criteria for the
placement of new species into the genus Bacillus, in an attempt to simplify the currently problematic
phylogeny of this genus. Although, based on the criteria suggested, this new species will be a part of the
genus Bacillus, the author should discuss the assignment of this new species to the genus Bacillus in the
light of the suggested criteria.
Reply: The isolate was obvious different morphology feature (Fig. 3, 4), phenotypic and Chemotaxonomic
characteristics (Table 1-3) from the other bacillus species and the detailed description about difference have
been added in the new manuscript, as highlighted in line 208-230.
5
Fig.3. Colony feature of four Bacillus strains cultured on a solid medium of nutrient agar (NA) (Seveno et al., 2001)
incubated at 30 ºC for 48 h., Bacillus bingmayongensis FJAT-13831T (1a), Bacillus pseudomycoides DSM 12442T (1b),
Bacillus mycoides DSM 2408T (1c), Bacillus cereus DSM 31T (1d).
Fig. 4. Cell characteristics of four Bacillus strains using Scanning Electron Microscope (×10,000). Bacillus bingmayongensis
FJAT-13831T (2a), Bacillus pseudomycoides DSM 12442T (2b), Bacillus mycoides DSM 2408T (2c), Bacillus cereus DSM
31T (2d).
MinorComments
6
Comment2: Line 83-95 The size of the final trimmed alignments used for phylogenetic analysis should be
indicated.
Reply: The size of the final trimmed alignments used for phylogenetic analysis based on 16S rRNA or
gyrB gene sequences were 1441bp and 644bp, respectively, as highlighted in line 105-107.
Comment3: Line 136 "less than 5%" should be "more than 5%"
Reply: “less than 5%" has been revised as "more than 5%", as shown in line 145-146.
Comment4: Line 142-143 The isolate FJAT-13831 and B. pseudomycoides DSM 12442 cluster together in
Figure 2 and every other figure shown.
Reply: It is true that the isolate FJAT-13831 and B. pseudomycoides DSM 12442 cluster together in the
figures, which displayed the closest phylogenetic between them. However, the isolate was different from B.
pseudomycoides DSM 12442 according the morphology, phenotype difference and ANI analysis
7