Characteristics and Application of a Novel Species of Bacillus: Bacillus

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Characteristic and application of a novel species of Bacillus: Bacillus velezensis Miao Ye, Xiangfang Tang, Ru Yang, Hongfu Zhang, Fangshu Li, Fangzheng Tao, Fei Li, and Zaigui Wang ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00874 • Publication Date (Web): 08 Jan 2018 Downloaded from http://pubs.acs.org on January 9, 2018

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Characteristic and application of a novel species of Bacillus: Bacillus velezensis Miao Ye, Xiangfang Tang, Ru Yang, Hongfu Zhang, Fangshu Li, Fangzheng Tao, Fei Li, Zaigui Wang* Miao Ye and Xiangfang Tang contributed equally to the work. Institution: 1, College of Life Science, Anhui Agricultural University, Anhui, Hefei, 230036, the Peoples’ Republic of China 2, Institute of Animal Science, Chinese Academy of Agricultural Sciences, State Key Laboratory of Animal Nutrition of China, Beijing, 100081, the People's Republic of China. First author: Miao Ye, E-mail: [email protected], Department of Life Science, Anhui Agriculture University, 230036, No. 130, Changjiang Road, Hefei, Anhui, the People's Republic of China. Co-first author: Xiangfang Tang, E-mail: [email protected], Institute of Animal Science, Chinese Academy of Agricultural Sciences, State Key Laboratory of Animal Nutrition of China, Beijing, 100081, the People's Republic of China. Ru Yang, E-mail: [email protected], Department of Life Science, Anhui Agriculture University, 230036, No. 130, Changjiang Road, Hefei, Anhui, the People's Republic of China. Hongfu Zhang, E-mail: [email protected], Institute of Animal Science, Chinese Academy of Agricultural Sciences, State Key Laboratory of Animal Nutrition of China, Beijing, 100081, the People's Republic of China. Fangshu Li, E-mail: [email protected], Department of Life Science, Anhui Agriculture University, 230036, No. 130, Changjiang Road, Hefei, Anhui, the People's Republic of China. Fangzheng Tao, E-mail: [email protected], Department of Life Science, Anhui Agriculture University, 230036, No. 130, Changjiang Road, Hefei, Anhui, the People's Republic of China. 1


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Fei Li, E-mail: [email protected], Department of Life Science, Anhui Agriculture University, 230036, No. 130, Changjiang Road, Hefei, Anhui, the People's Republic of China. *Corresponding author: Zaigui Wang, E-mail: [email protected], Tel: +86-551-6578-6232, Fax: +86-551-6578-6232, Department of Life Science, Anhui Agriculture University, 230036, No. 130, Changjiang Road, Hefei, Anhui, the People's Republic of China. Running Head: The Characteristic and application of Bacillus velezensis

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Abstract: Bacillus velezensis has been investigated and applied more and more widely because it can inhibit fungi and bacteria, and become a potential biocontrol agent recently. In order to provide more clear and comprehensive understanding of B. velezensis for researchers, we collected the recent relevant articles systematically and reviewed the discovery and taxonomy, secondary metabolites, characteristic and application, gene function and molecular research of B. velezensis. This article will give some directions to the research and application of this strain for future. Keywords: Bacillus velezensis, taxonomy, secondary metabolites, gene function and molecular research.

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Some Bacillus have played an increasingly important role in many areas such as agriculture and fermentation industry. Bacillus velezensis, as a novel species, is widely distributed in nature which can be easily separated and cultured. It is harmless to human and animal, and does not pollute the environment. Its metabolites are abundant with broad spectrum antibacterial activity and strong ability to resist stress. This bacteria has good characteristic of growing fast and stability. As a result, there are an increasingly number of researches on its properties and application. In order to facilitate the later application and on B. velezensis, we will summarize the recent-research advancement of B. velezensis. 1.

The Discovery and Taxonomy of Bacillus velezensis Bacillus velezensis, firstly isolated from the mouth of the river Ve´lez in Ma´laga (Southern Spain),

has been originally described by Ruiz-Garcıá et al., as a Gram positive bacterium which grew within the temperature scope of 15 to 45℃ and pH scope of 5.0 to 10.0(1). The 16S rRNA gene sequences of B. velezensis shared 99% similarity with Bacillus amyloliquefaciens. Based on the DNA-DNA relatedness values, Wang et al. proposed that B. velezensis is a later heterotypic synonym of Bacillus amyloliquefaciens(2). However, comparative genomics and DNA-DNA relatedness calculations of B. velezensis NRRL B-41580T showed that B. velezensis is not a synonym of B. amyloliquefaciens. And comparative genomic analysis of B. velezensis NRRL B-41580T with that of B. amyloliquefaciens subsp. plantarum FZB42T only showed that there were slight differences from each other. Furthermore, the DNA-DNA hybridization values (﹥84%, calculated by comparisons between the B. velezensis NRRL B-41580T and the B. amyloliquefaciens subsp. plantarum FZB42T）is far above the species threshold of 70%. Latest analysis of morphology, physiology, chemical taxonomy and phylogenetic showed that the two strains had the phenotype and genotype consistency. And, Dunlap et al. proposed that B. velezensis, B. amyloliquefaciens subsp plantarum, should be reclassified as later heterotypic 4



synonym of B. velezensis(3). However, most researchers still considered the FZB42 should be classed as “Bacillus amyloliquefaciens”. Therefore, Fan et al. compared the TETRA (tetranucleotide), ANI (average nucleotide identity), AAI (average amino acid identity) and dDDH (digital DNA-DNA hybridization) values of B. amyloliquefaciens, with those of conspecific group including B. amyloliquefaciens plantarum FZB42, B. methylotrophicus, and B. velezensis, showing that FZB42 is not B. amyloliquefaciens which should be instead by B. velezensis(4). According to microbial genomic taxonomy delineation, same bacterial species are defined as a group of strains sharing DDH similarity( > 70%), ∆Tm ( < 5°C), G+C difference of total genomic DNA ( < 5%), 16S rRNA identity( > 98% )(5). From the results by Dunlap et al. and Fan et al., we can see that TETRA, ANI, AAI, DDH 16S rRNA values of FZB42 compared to B. velezensis are 0.9991%, 98.30%, 99.11%, 87.10% and 99% respectively, which is far above the species threshold in the light of the microbial genomic taxonomy standard. And because the valid publication date of B. velezensis preceded the publication of the FZB42(1, 6), we agree that FZB42 is B. velezensis rather than B. amyloliquefaciens. 2.

The Secondary Metabolites with Biocontrol Function B. velezensis FZB42 was isolated from the soil nearby maize roots. In the past decade, the

researchers have carried out a series of studies on the biological properties and molecular mechanisms which made it mostly clear that the biological roles could improve plant growth by promoting rhizobacterium (PGPR). It was reported that B. velezesis FZB42 had an impressive capacity to produce secondary metabolites with antimicrobial activities, including antibiotic lipopeptide (surfactin, fengycin and bacillomycin D), polyketides (macrolactin, bacillaene and difficidin or oxydifficidin), and peptide 5


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(plantazolicin, amylocyclicin and bacilysin)(7). The genes, gene clusters and functions of these secondary metabolic organisms are shown in Table 1. The Characteristic and Application of Bacillus velezensis 3.1. Azoreductase and dye detoxification activities B. velezensis AB was isolated basing on its ability to decolorize the azo dye Direct Red 28 (DR28) which showed it has Azoreductase (60 kDa) and dye detoxification activity. The Azoreductase required nicotinamide adenine dinucleotide (NADH) as a cofactor and was not sensitive to oxygen. The B. velezensis AB had ability to degrade the DR28 to benzidine and 4-aminobiphenyl, which were potent mutagens. Yet, these two compounds could be further degraded over a short period of incubation that can lead to reducing dye toxicity and mutagenicity. Therefore, because of their safe disposal, the culture appeared to be an appropriate candidate for further study of the decolorization and detoxification of azo dyes(23). 3.2. Enzyme activity and cell growth of Bacillus velezensis B. velezensis P11, isolated from the Brazilian Amaon basin, had remarkable keratinolytic activity, proteolytic activity, and de-hairing activity(24). Additionally, the cellulolytic potential of B. velezensis P11 was also investigated by Congo red method, it showed that the strain could produce high activity cellulase(25). Another microorganism named B. velezensis A-68 producing carboxymethylcellulase (CMCase) was isolated from seawater. The optimal conditions for the cell growth and the production of CMCase in a flask were investigated by the methodology of response surface(26, 27). And the factors associated with dissolved oxygen in 7 and 100 L bioreactors were optimized for the pilot-scale production of CMCase(28). The results were showed in Table 2. 6



Protease, as a type of important enzyme in organism, can effectively hydrolyze protein with the advantages of mild catalytic conditions, high efficiency and producing some functional peptides. It has been widely used in foods, leather, animal feed, medicine, environmental protection, chemical industry, and so on(29). Cellulase produced by this strain has also been used as an important industrial enzyme(30). Currently, protease and cellulase occupy a large proportion of industrial enzymes. However, some series of the known protease and cellulase are insufficient to meet the needs of industry. The production of enzymes by fermentation of microbe is still considered as the most significant production method now. Therefore, the study of the protease and cellulase production from B. velezensis has an extremely important value. So the optimization of the fermentation condition for these two kinds of enzymes from this strain will be investigated by our laboratory. 3.3. Biofilm formation By studying the biofilm formation, a kind of collagen-like protein was found indispensable on the product level(31). In the subsequent trials, an understanding of a set of genes involved in biofilm formation in B. velezensis FZB42 was also provided. In summary, the development of biofilm might be of great significance to the biocontrol-properties of B. velezensis FZB42 in the rhizosphere of host plants. A new discovery, which was gene encoding for the antimicrobial peptide LCI among the most highly expressed genes in both growing states, maybe imply that this peptide produced was conducive to biocontrol capacity development of the strain(32). 3.4. Antifungal effect on plant pathogenic fungi and Biocontrol application Bacillus can resist heat, ultraviolet, electromagnetic radiation and produce some stable substances. The spores not only have good characteristics for preservation, but also can tolerate extreme external environment and long-term survival. There have been many extensively studies on biocontrol agents 7


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due to their excellent ability to plant suppress. At present, some of the dominant strains of Bacillus such as B. velezensis, have been put into the application of biological pesticides to plant diseases and registered as a biological fungicide abroad for the control of powdery mildew, gray mold, sheath blight, sclerotia, late blight which were classified into air-borne or soil-borne fungal diseases(33). Some American researchers found that B. velezensis AH2 strain had a very good resistance to Botrytis cinerea, Pythium, Phytophthora, Sclerotinia, Sclerotinia, Penicillium, Alternaria alternata(34). It showed that there was no significant difference in the control effect of the biological agents from the B. velezensis AH2 strain compared with the chemical drugs by the leaf spray method. The B. velezensis G341 had a strong inhibitory effect on plant diseases such as rice blast, rice sheath blight, pepper anthracnose, tomato gray mold, wheat root rot and barley powdery mildew(35). B. velezensis S3-5 was demonstrated that it had antifungal activities (more than 70% control efficacy) against many plant diseases. The results of this report showed that a recombinant B. velezensis strain might be used to control both pests and plant fungal diseases equally in a crop. Furthermore, both culture broth and harvested cells of this strain could be used as single biological control agents for integrated crop protection(36). The biocontrol activity of B. velezensis RC 218 which could successfully reduce disease severity and deoxynivalenol accumulation was confirmed under the field conditions. Due to the detection of gene clusters of these compounds, the biocontrol activity may be connected with the strain's ability to produce several lipopeptides from the surfactin, fengycin and iturin families(37). B. velezensis CC09 was also verified that it had a great biocontrol capacity to many plant diseases caused by pathogens such as wheat powdery mildew under the molecular basis(38). B. velezensis BAC03 was studied to verify its effect on Streptomyces scabies which indicated that BAC03 obviously could reduce the 8



excellent diseases caused by Streptomyces scabies with the effect in controlling scab by using appropriate application method(39). 3.5. Effects of Bacillus velezensis in promoting plant growth Root-colonizing Bacillus spp. was proved with the effect of enhancing plant growth(40). Bacillus velezensis and Bacillus megaterium could produce enough cytokinin(41). Also, volatile organic compounds from some B. velezensis have been verified to have the ability to trigger growth promotion in Arabidopsis by regulating auxin homeostasis(42). Potential growth promotion effect of B. velezensis BAC03 on nine types of plants were tested in greenhouse conditions at a concentration of 105 colony forming unit cm3 potting mix. The results indicated that production of indole-3-acetic acid, ammonia, and the 1-aminocyclopropane-1-car boxylate deaminase activity produced by BAC03 might be related to the promotion of plant growth(43). Besides, B. velezensis FZB42 could also promote plant growth by secreting Indole-3-Acetic Acid (IAA)(44). The rhizosphere activity of the commercially available inoculant B. velezensis FZB42 on the growth and health of lettuce with its effect on the indigenous rhizosphere bacterial community in field and pot experiments was studied. The results revealed that FZB42 could effectively colonize the rhizosphere in the field of lettuce during the growth period(45). Application of B. velezensis FZB42 on lettuce had showed that it could promot plant growth and health by reducing disease severity. Accordingly, this strain has the potential to be used as an plant protection agent commercially without any harm to environment(46, 47). Salmonicida compounds from B. velezensis V4 were also tested. The diversity of these compounds correlated with the versatility of their mode of action was conformed deeply. The most important one was investigated through the inhibition of protein synthesis by cell lysis caused by 9


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membrane-active biosurfactants and oxydifficidin. It showed that B. velezensis V4 was an potent probiotic strain to O. mykiss with obvious potential for controlling furunculosis and for growth promotion in aquaculture animals(48). 4. Gene Function and Molecular Research 4.1. β-1, 3-1, 4-glucanase gene clone In order to understand the mechanism of β-1, 3-1, 4-glucanase from B. velezensis on fungi, the β-1, 3-1, 4-glucanase gene encoding the enzyme was cloned and characterized. The results showed that β-1, 3-1, 4-glucanase could be expressed at high level and purified in great amounts easily. These results illustrated that the purified enzyme with the functions of antifungal activity against plant pathogenic fungi had large applying potential for commercial purposes(49). 4.2. Complete Genome Sequence of Bacillus velezensis The complete genome sequences of B. velezensis have been sequenced by an increasing number of researchers recently with the deeper insights into the genetic, biological and physiological characteristics of the strain to obtain its biocontrol mechanism which reveals an unexpected potential to produce secondary metabolites. Additionally, B. velezensis could enhance plant growth and trigger plant immunity because it contained a series of genes involved in these functions. These genome sequences are shown in Table 3. These bacteria are only subtypes of B. velezensis and not exactly the same bacteria because they were isolated from different sources such as the root of the ginseng (B. velezensis G341), animal dung (B. velezensis CC09), and soil (B. velezensis 3A-25B). In order to adapt themselves to different living environments, they generally have different functions rooted to genome sequence. For example, B. velezensis 3A-25B separated from the grassland soil, compared with the other subtypes such as hosts of rice and animals, has obvious difference in function. Therefore, B. 10



velezensis 3A-25B has less rRNA and tRNA numbers than other subtypes. 5. Future outlook: Application of Bacillus velezensis in Animal feed Antibiotics are a kind of secondary metabolites with anti-pathogens or other activities produced by microorganisms or higher flora and fauna in the process of life, which are mainly used for the treatment of various bacterial infections or pathogenic microbial infections. However, the abuse of antibiotics can cause great harm to mankind and environment. Some European countries and South Korea have banned the use of antibiotics in animal feed. For the prohibition of antibiotics may have adverse effects on animal health and the interests of farmers, people have been looking for a viable substitute for antibiotics. Probiotics are defined as "living organisms, when applied in sufficient quantities, it would give healthy benefits to the host". They had a positive impact on productivity, immune system and antioxidant status of broilers(63). As a probiotic, Bacillus has been widely used in animal feed as microbial additives. Dietary probiotics are beneficial to poultry health because of their ability to improve oxidative damage. Probiotic supplement for animal feed not only can improve animal health and production but also has benefitted diversify and stabilize gastrointestinal flora to the host animals. Bacillus velezensis is widely distributed in nature and is capable to inhibit a variety of fungi and bacteria. Beside these, it is rich in its own metabolites. With the improvement of molecular research technology, the gene function of B. velezensis will be more understood, which may increase the feasibility of the application. The studies of application of B. velezensis to plants have been widely reported before. Nevertheless, there is little research on this strain as probiotic applied to animal feed instead of antibiotics. In previous work in our laboratory, a strain of Bacillus velezensis was isolated from the feces of piglets. During the experiment, it was found that it had a fast growth rate and a strong ability to 11


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produce high activity cellulase, but the content of the current has not been compiled into a paper. There is little research on the replacement of antibiotics by Bacillus in animal feed, thus, in the near future, we will add B. velezensis to animal feed to verify whether it can be a good substitute for antibiotics to promote the growth performance of animals. Author Contributions ZGW designed the structure of the article and modified the article. MY collected references and write the article. RY, FSL, FZT, FL collected references. XFT, HFZ modified the article. Interest statements The authors declare no competing financial interest. Acknowledgment This work was supported by the Major Science and Technology Program of Anhui Province (17030701035), Anhui Industry and Technology System of Poultry Science (AHCYTX-10), the National Key R&D Program of China (2016YFD0500501), the Fund of State Key Laboratory of Animal Nutrition (2004DA125184F1725), the Fund of College Natural Science from Anhui Province (2018). References 1. Ruiz-Garcıá, C., Be´jar, V., Martıńez-Checa, F., Llamas, I., and Quesada, E. (2005) Bacillus velezensis sp. nov., a surfactant producing bacterium isolated from the river Ve´ lez in Ma´ laga, southern Spain. Int. J. Syst. Evol. Microbiol. 55, 191-195. 2. Wang, L. T., Lee, F. L., Tai, C. J., and Kuo, H. P. (2008) Bacillus velezensis is a later heterotypic synonym of Bacillus amyloliquefaciens. Int. J. Syst. Evol. Microbiol. 58, 671-675. 3. Dunlap, C. A., Kim, S. J., Kwon, S. W., and Rooney, A. P. (2016) Bacillus velezensis is not a later 12



heterotypic

synonym

of

Bacillus

amyloliquefaciens;

Bacillus

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methylotrophicus,

Bacillus

amyloliquefaciens subsp. plantarum and ‘Bacillusoryzicola’ are later heterotypic synonyms of Bacillus velezensis based on phylogenomics. Int. J. Syst. Evol. Microbiol. 66, 1212-1217. 4. Fan, B., Blom, J., Klenk, H. P., and Borriss, R. (2017) Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus siamensis Form an “Operational Group B. amyloliquefaciens” within the B. subtilis Species Complex. Front. Microbiol. 8, 22. 5. Stackebrandt, E., and Ebers, J. (2006) Taxonomic parameters revisited: tarnished gold standards. Microbiol. Today. 33, 152–155. 6. Borriss, R., Chen, X. H., Rueckert, C., Blom, J., Becker, A., Baumgarth, B., Fan, B., Pukall, R., Schumann, P., Spröer, C., Junge, H., Vater, J., Pühler, A., and Klenk, H. P. (2011) Relationship of Bacillus amyloliquefaciens clades associated with strains DSM 7T and FZB42T: a proposal for Bacillus amyloliquefaciens subsp. amyloliquefaciens subsp. nov. and Bacillus amyloliquefaciens subsp. plantarum subsp. nov. based on complete genome sequence comparisons. Int. J. Syst. Evol. Microbiol. 6, 1786-1801. 7. Chen, X. H., Koumoutsi, A., Scholz, R., and Borriss, R. (2009) More than anticipated - production of antibiotics and other secondary metabolites by Bacillus amyloliquefaciens FZB42. J. Mol. Microbiol. Biotechnol. 16, 14-24. 8. Chen, X. H., Koumoutsi, A., Scholz, R., Schneider, K., Vater, J., Süssmuth, R., Piel, J., and Borriss, R. (2009) Genome analysis of Bacillus amyloliquefaciens FZB42 reveals its potential for biocontrol of plant pathogens. J. Biotechnol. 140, 27-37. 9. Koumoutsi, A., Chen, X. H., Henne, A., Liesegang, H., Hitzeroth, G., Franke, P., Vater, J., and Borriss, R. (2004) Structural and functional characterization of gene clusters directing nonribosomal 13


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Table1 Gens and gene cluster encoding in B. velezensis FZB42 Metabolites

Genes and gene cluster

Size(kb)

Function

Surfactin(8)

srfABCD

32.0

Biofilm

Fengycin(9)

fenABCDE

38.2

Antifungal

Bacillomycin D(10)

bmyCBAD

39.7

Antifungal

Putative peptide

nrsABCDEF

15.0

Unknown

Bacillibactin(11)

dhbABCDEF

12.8

Antifungal

mlnABCDEFGHI

53.9

Direct suppression

74.3

Direct suppression

71.1

Antifungal

bacABCDE,ywfG

6.9

Antifungal

acnBACDEF

4.49

Antifungal

pznFKGHIAJCDBEL

9.96

Antifungal

Lipopetides

Macrolactin (12, 13) Polyketides

Bacillaene(14)

Difficidin(15, 16)

Bacilysin(17, 18)

Peptide

Amylocyclicin (19) Plantazolicin (20-22)

baeBCDE,acpK, baeGHIJLMNRS dfnAYXBCDEFGHIJK LM

22



Page 24 of 25

Table 2. Optimal condition for cell growth and production of Carboxymethylcellulase by Bacillus velezensis A-68 Scales

Optimal

One factor at a time

conditions

experiment Dry cells

CMCase

weight Rice hulls

Response surface method Dry cells

CMCase

weight

50.0

50.0

50.0

50.0

7.5

5.0

5.0

5.0

Initial pH

7.3

7.3

7.3

7.3

Maximal

1.24 g/L

62.0 U/mL

1.22 g/L

6.17 U/mL

7.5

7.5

7.5

7.5

(g/L) Flask

Yeast

scale-1a

extract (g/L)

production K2HPO4 Flask

(g/L) a

scale-2

NaCl (g/L)

2.0

2.0

1.0

3.0

MgSO4

0.25

0.25

0.1

0.1

1.0

1.0

0.9

0.9

30

35

-

-

1.46 g/L

83.8 U/mL

-

-

300

400

323

380

1.5

0.5

1.46

0.54

1.59 g/L

71.9 U/mL

1.49 g/L

88.3 U/mL

0.00

0.04

-

-

1.46 g/L

108.1 U/mL

-

-

(g/L) (NH4)2SO 4 (g/L) Temperatur Flask

e (℃) a

scale-3

Maximal production

Lab-scaled bioreactor

Agitation

b

speed (rmp) Aeration rate (vvm) Maximal production

Pilot-scaled bioreactor

b

Inner pressure (Mpa) Maximal production

a: (26, 27); b: (28).

23


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Table 3. The results of the whole genome sequence comparison.

FZB42(11)

Genome

GC

Protein-co

tRNA

rRNA

size(bp)

content(%)

ding gene

number

number

3,918,589

46.40

3,693

89

29

Isolation source

Accession

Country

numbers The

CP000560

Germany

plant-pathogen-infested soil G341(50)

4,009,746

46.49

3,953

95

30

The root of ginseng

CP011686

Korea

CBMB205

3,929,745

46.50

3,985

86

27

The rhizoplane of rice

CP014838

Korea

4,167,153

46.10

4,021

73

24

The

CP015443

China

(51) CC09(52)

Cinnamomum

camphora leaf tissue M75(53)

4,007,450

46.60

3,921

86

27

The cotton waste

CP016395

Korea

9912D(54)

4,206,167

46.03

4,436

86

27

The sediment sample

CP017775

China

LS69(55)

3,917,761

46.40

3,643

72

21

The rice field

CP015911

China

LM2303

3,989,393

46.68

3,866

86

27

The dung of wild yak

CP018152

China

JTYP2(57)

3,929,789

46.50

3,656

86

27

The

CP020375

China

GH1-13

4,071,980

46.20

3,930

86

27

The rice paddy soil

CP019039

Korea

3,929,772

46.50

3,690

86

27

The rhizosphere soil

CP016371

China

CP021495

China

MLCW00

Mexico

(56) leaves

of

Echeveria laui (58) S3-1(59)

of cucumber GQJK49

3,929,760

46.50

3,921

86

27

(60) 3A-25B

The

rhizosphere

Lycium barbarum L. 4,017,761

46.34

3,786

68

10

The grassland soil

(61) GF610(62)

of

000000 4,291,535

45.91

4,200

84

22

The garden soil

NQXV000 00000

24


USA

Characteristics and Application of a Novel Species of Bacillus: Bacillus

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