doi: 10.15389/agrobiology.2020.4.816eng
UDC: 579.62:579.852.11:615.33:575.113
Acknowledgements:
Supported financially from the Russian Foundation for Basic Research, grant No. 19-316-90041 “Whole-genome sequencing of bacilli strains isolated from the cicatricial contents of various ruminants”
GENOMIC AND PHENOTYPICAL POTENTIAL OF ANTIMICROBIAL ACTIVITY OF A BACILLUS STRAIN Bacillus megaterium В-4801
G.Yu. Laptev1, E.A. Yildirim2, T.P. Dunyashev1, L.A. Ilyina2, D.G. Tyurina2, V.A. Filippova2, E.A. Brazhnik2, N.V. Tarlavin2, A.V. Dubrovin2, N.I. Novikova2, V.K. Melikidi2, S.N. Bikonya2
1Saint Petersburg State Agrarian University, 2, lit A, Peterburgskoe sh., St. Petersburg—Pushkin, 196601 Russia, e-mail laptev@biotrof.ru (corresponding author ✉), timur@biotrof.ru;
2JSC «Biotrof+», 19, korp. 1, Zagrebskii bulv., St. Petersburg, 192284 Russia, e-mail deniz@biotrof.ru, ilina@biotrof.ru, bea@biotrof.ru, dumova@biotrof.ru, novikova@biotrof.ru, tiurina@biotrof.ru, tarlav1995@biotrof.ru, dubrovin@biotrof.ru, veronika@biotrof.ru, svetlana@biotrof.ru
ORCID:
Laptev G.Yu. orcid.org/0000-0002-8795-6659
Brazhnik E.A. orcid.org/0000-0003-2178-9330
Yildirim E.A. orcid.org/0000-0002-5846-4844
Tarlavin N.V. orcid.org/0000-0002-6474-9171
Dunyashev T.P. orcid.org/0000-0002-3918-0948
Dubrovin A.V. orcid.org/0000-0001-8424-4114
Ilyina L.A. orcid.org/0000-0003-2490-6942
Novikova N.I. orcid.org/0000-0002-9647-4184
Tyurina D.G. orcid.org/0000-0001-9001-2432
Melikidi V.K. orcid.org/0000-0002-2883-3974
Filippova V.A. orcid.org/0000-0001-8789-9837
Bikonya S.N. orcid.org/0000-0002-3900-6341
Received June 13, 2020
The genetic determinants of bacterial strains Bacillus sp., which determine the possibility of biosynthesis of various antimicrobial compounds, are of particular scientific interest, since thanks to them these microorganisms are widely used as the basis of probiotics. An important stage in the systemic analysis of the mechanisms of probiotic action, in particular the antimicrobial activity of microorganisms, is the reconstruction of its metabolic map, that is, the collection and visualization of all potential cell processes. In this work, for the first time, the potentially inherent genetic mechanisms for the synthesis of a number of biologically active substances in the bacterial strain Bacillus megaterium are described, in particular, the possibility of synthesizing canosamine, a bacteriocin belonging to the aminoglycoside group, which can play an important role in the implementation of probiotic properties due to its pronounced antimicrobial activity. Our goal was to study the antimicrobial activity of the strain Bacillus megaterium B-4801 against pathogenic and opportunistic bacteria, as well as to search for genes associated with antimicrobial activity based on whole genome sequencing. The B. megaterium B-4801 strain deposited in the collection of OOO BIOTROF+, possesses a pronounced probiotic activity. Its antimicrobial activity against Staphylococcus aureus, Candida tropicalis, Clostridium sp., and Escherichia coli was assessed by the method of delayed antagonism using wells. A DNA library for whole genome sequencing was generated using Nextera XT kit (Illumina, Inc., USA). Nucleotide sequences were determined using a MiSeq instrument (Illumina, Inc., USA) and MiSeq Reagent Kit v3 (300-cycle) (Illumina, Inc., USA). Invalid sequences and adapters were removed using the Trimmomatic-0.38 program. Filtered in length from 50 to 150 bp pair-terminal sequences were assembled de novo using genomic assembler SPAdes-3.11.1. Functional annotation of the genome was performed with PROKKA 1.12 and RAST 2.0 programs. The pool of genes associated with antimicrobial activity was assessed and the metabolic map was constructed using the KEGG Pathway database (http://www.genome.jp/kegg/). The antagonistic activity of B. megaterium B-4801 against pathogenic and opportunistic microorganisms was revealed by cultural methods. The growth inhibition zones of the test strains ranged from 2±0.15 to 25±1.4 mm. The genome of the B. megaterium B-4801 strain is a single circular chromosome with a size of 6,113,972 bp, containing 37.5 % GC pairs. More than 45 % of B. megaterium B-4801 genes are involved in the transport and metabolism of amino acids, transcription, translation, transport and metabolism of carbohydrates and proteins. The key genetic loci that determine the synthesis of antimicrobial metabolites have been identified. The sequenced genome of the strain contains genes (FabD, FabF, FabG, FabZ, FabI, etc.) associated with the production of proteins involved in the synthesis of aliphatic unsaturated C3-C18 carboxylic acids, in particular, butyric, nylon, caprylic, capric, lauric, myristic, palmitic, stearic, oleic. According to the information accumulated by world science, all these substances have pronounced antimicrobial properties. The whole-genome sequencing also discovered a cluster of genes (Asm22-24, Asm43-45, and Asm47) associated with the biosynthesis of bacteriocin kanosamin, which belongs to the aminoglycoside group, and polyketide ansamycin antibiotics from the macrolide group. The established probiotic potential indicates the role of the investigated strain as a potential probiotic candidate, in particular for use in animal husbandry. The performed genomic analysis revealed new systems of operons that control the metabolic pathways for the synthesis of antimicrobial substances, which were not previously described for B. megaterium.
Keywords: whole-genome sequencing, Bacillus megaterium, acid biosynthesis, bacteriocins, antimicrobial activity, canosamine, ansamycin antibiotics, probiotics.
REFERENCES
- Hong H.A., Duc L.H., Cutting S.M. The use of bacterial spore formers as probiotics. FEMS Microbiology Reviews, 2010, 29(4): 813-835 CrossRef
- Il'ina L.A. Izuchenie mikroflory rubtsa krupnogo rogatogo skota na osnove molekulyarno-biologicheskogo metoda T-RFLP s tsel'yu razrabotki sposobov ee optimizatsii. Kandidatskaya dissertatsiya [Study of the microflora of the cattle rumen based on the molecular biological method T-RFLP in order to develop methods for its optimization. PhD Thesis]. St. Petersburg, 2012 (in Russ.).
- Caulier S., Nannan C., Gillis A., Licciardi F., Bragard C., Mahillon J. Overview of the antimicrobial compounds produced by members of the Bacillus subtilis. Frontiers in Microbiology, 2019, 10: 302 CrossRef
- Stein T. Bacillus subtilis antibiotics: structures, syntheses and specific functions. Molecular Microbiology, 2005, 56(4): 845-857 CrossRef
- Cotter P.D., Hill C., Ross R.P. What's in a name? Class distinction for bacteriocins. Nature Reviews Microbiology, 2006, 4(2): 160 CrossRef
- Gabrielsen C., Brede D.A., Nes I.F., Diep D.B. Circular bacteriocins: biosynthesis and mode of action. Applied and Environmental Microbiology, 2014, 80(22): 6854-6862 CrossRef
- Begley M., Cotter P.D., Hill C., Ross R.P. Identification of a novel two-peptide lantibiotic, lichenicidin, following rational genome mining for LanM proteins. Applied and Environmental Microbiology, 2009, 75(17): 5451-5460 CrossRef
- Saising J., Dube L., Ziebandt A.-K., Voravuthikunchai S.P., Nega M., Götz F. Activity of gallidermin on Staphylococcus aureus and Staphylococcus epidermidis biofilms. Antimicrobial Agents and Chemotherapy, 2012, 56(11): 5804-5810 CrossRef
- Field D., Cotter P.D., Hill C., Ross R.P. Bioengineering lantibiotics for therapeutic success. Frontiers in Microbiology, 2015, 6: 1363 CrossRef
- Abriouel H., Franz C.M., Ben Omar N., Gálvez A. Diversity and applications of Bacillus bacteriocins. FEMS Microbiology Reviews, 2011, 35(1): 201‐232 CrossRef
- Poudel P., Tashiro Y., Miyamoto H., Miyamoto H., Okugawa Y., Sakai K. Direct starch fermentation to L-lactic acid by a newly isolated thermophilic strain, Bacillus sp. Journal of Industrial Microbiology and Biotechnology, 2015, 42: 143-149 CrossRef
- Alakomi H.L., Skyttä E., Saarela M., Mattila-Sandholm T., Latva-Kala K., Helander I.M. Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane. Applied and Environmental Microbiology, 2000, 66(5): 2001‐2005 CrossRef
- Ortiz A., Sansinenea E. Succinic acid production as secondary metabolite from Bacillus megaterium ELI24. The Natural Products Journal, 2020, 10(2): 154-157 CrossRef
- Chuanqing Z., Jiang A.U., Huang A., Qi W. Cao X. Guangxiang studies on the acid-production characteristics of Bacillus megaterium strain P17. AIP Conference Proceedings, 2017, 1839(1): 020054 CrossRef
- Li P., Tian W., Jiang Z., Liang Z., Wu X., Du B. Genomic characterization and probiotic potency of Bacillus sp. DU-106, a highly effective producer of L-lactic acid isolated from fermented yogurt. Frontiers in Microbiology, 2018, 9: 2216 CrossRef
- Khatri I., Sharma G., Subramanian S. Composite genome sequence of Bacillus clausii, a probiotic commercially available as Enterogermina®, and insights into its probiotic properties. BMC Microbiology, 2019, 19(1): 307 CrossRef
- Eppinger M., Boyke B., Mitrick A.J., Janaka N.E., Kirthi K.K., Koenig S.S.K., Creasy H.H., Rosovitz M.J., Riley D.R., Daugherty S., Martin M., Elbourne L.D.H., Paulsen I., Biedendieck R., Braun C., Grayburn S., Dhingra S., Lukyanchuk V., Ball B., Ul-Qamar R., Seibel J., Bremer E., Jahn D., Ravel J., Vary P.S. Genome sequences of the biotechnologically important Bacillus megaterium strains QM B1551 and DSM319. Journal of Bacteriology, 2011, 193(16): 4199-4213 CrossRef
- Vílchez J.I., Tang Q., Kaushal R., Wang W., Suhui L., Danxia H., Zhaoqing C., Heng Z., Renyi L., Huiming Z. Complete genome sequence of Bacillus megaterium strain TG1-E1, a plant drought tolerance-enhancing bacterium. Microbiology Resource Announcements, 2018, 7(12): e00842-18 CrossRef
- Liu L., Li Y., Zhang J., Zou W., Zhou Z., Liu J., Li X., Wang L., Chen J. Complete genome sequence of the industrial strain Bacillus megaterium WSH-002. Journal of Bacteriology, 2011, 193(22): 6389-6390 CrossRef
- Bolger A.M., Lohse M., Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics, 2014, 30(15): 2114‐2120 CrossRef
- Nurk S., Bankevich A., Antipov D., Gurevich A., Korobeynikov A., Lapidus A., Prjibelsky A., Pyshkin A., Sirotkin A., Sirotkin Y., Stepanauskas R., McLean J., Lasken R., Clingenpeel S.R., Woyke T., Tesler G., Alekseyev M.A., Pevzner P.A. Assembling genomes and mini-metagenomes from highly chimeric reads. In: Research in Computational Molecular Biology. RECOMB 2013. Lecture Notes in Computer Science, vol. 7821. Springer, Berlin, Heidelberg, 2013: 158-170 CrossRef
- Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics, 2014, 30(14): 2068‐2069 CrossRef
- Aziz R.K. The RAST server: rapid annotations using subsystems technology. BMC Genomics, 2008, 9(75) CrossRef
- Kanehisa M., Goto S. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Research, 2000, 28(1): 27-30 (doi: 10.1093/nar/28.1.27">CrossRef
- Kanehisa M., Goto S., Sato Y., Furumichi, M. Tanabe M. KEGG for integration and interpretation of large-scale molecular datasets. Nucleic Acids Research, 2012, 40(D1): D109-D114 (doi: 10.1093/nar/gkr988">CrossRef
- Boss R., Cosandey A., Luini M., Artursson K., Bardiau M., Breitenwieser F., Hehenberger E., Lam Th., Mansfeld M., Michel A., Mösslacher G., Naskova J., Nelson S., Podpečan O., Raemy A., Ryan E., Salat O., Zangerl P., Steiner A., Graber H.U. Bovine Staphylococcus aureus: Subtyping, evolution, and zoonotic transfer. Journal of Dairy Science, 2016, 99(1): 515‐528 CrossRef
- Loken K.I., Thompson E.S., Hoyt H.H., Ball R.A. Infection of the bovine udder with Candida tropicalis. Journal of the American Veterinary Medical Association, 1959, 134(9): 401‐403.
- Wohlgemuth K., Knudtson W. Bovine abortion associated with Candida tropicalis. Journal of the American Veterinary Medical Association, 1973, 162(6): 460‐461.
- Porwal S., Lal S., Cheema S., Kalia V.C. Phylogeny in aid of the present and novel microbial lineages: diversity in Bacillus. PLoS ONE, 2009, 4(2): e4438 CrossRef
- Ondov B.D., Treangen T.J., Melsted P., Mallonee A.B., Bergman N.H., Koren S., Phillippy A.M. Mash: fast genome and metagenome distance estimation using MinHash. Genome Biology, 2016, 17: 132 CrossRef
- Wattam A.R., Davis J.J., Assaf R., Boisvert S., Brettin T., Bun C., Conrad N., Dietrich E.M., Disz T., Gabbard J.L., Gerdes S., Henry C.S., Kenyon R.W., Machi D., Mao C., Nordberg E.K., Olsen G.J., Murphy-Olson D.E., Olson R., Overbeek R., Parrello B., Pusch G.D., Shukla M., Vonstein V., Warren A., Xia F., Yoo H., Stevens R.L. Improvements to PATRIC, the all-bacterial bioinformatics database and analysis resource center. Nucleic Acids Research, 2017, 45(D1): D535-D542 CrossRef
- Frye T.M., Williams S.N., Graham T.W. Vitamin deficiencies in cattle. Veterinary Clinics of North America: Food Animal Practice, 1991, 7(1): 217‐275 CrossRef
- Szafranska A.E., Hitchman T.S., Cox R.J., Crosby J., Simpson T.J. Kinetic and mechanistic analysis of the malonyl CoA:ACP transacylase from Streptomyces coelicolor indicates a single catalytically competent serine nucleophile at the active site. Biochemistry, 2002, 41(5): 1421-1427 CrossRef
- Cronan J.E., Thomas J. Bacterial fatty acid synthesis and its relationships with polyketide synthetic pathways. Methods in Enzymology, 2009, 459: 395‐433 CrossRef
- Wang H., Cronan J.E. Functional replacement of the FabA and FabB proteins of Escherichia coli fatty acid synthesis by Enterococcus faecalis FabZ and FabF homologues. Journal of Biological Chemistry, 2004, 279(33): 34489‐34495 CrossRef
- Yu X., Liu T., Zhu F., Khosla C. In vitro reconstitution and steady-state analysis of the fatty acid synthase from Escherichia coli. Proceedings of the National Academy of Sciences, 2011, 108(46): 18643-18648 CrossRef
- Johnson S.L., Daligault H.E., Davenport K.W., Jaissle J., Frey K.G., Ladner J. T., Broomall S.M., Bishop-Lilly K.A., Bruce D.C., Gibbons H.S., Coyne S.R., Lo C.-C., Meincke L., Munk A.C., Koroleva G.I., Rosenzweig C.N., Palacios G.F., Redden C.L., Minogue T.D., Chain. P.S. Complete genome sequences for 35 biothreat assay-relevant Bacillus species. Genome Announcements, 2015, 3(2): e00151-15 CrossRef
- Kovanda L., Zhang W., Wei X., Luo J., Wu X., Atwill E.R., Vaessen S., Li X., Liu Y. In vitro antimicrobial activities of organic acids and their derivatives on several species of gram-negative and gram-positive bacteria. Molecules, 2019, 24(20): 3770 CrossRef
- Huang C.B., Alimova Y., Myers T.M., Ebersole J.L. Short- and medium-chain fatty acids exhibit antimicrobial activity for oral microorganisms. Archives of Oral Biology, 2011, 56(7): 650‐654 CrossRef
- Prabhadevi V., Sahaya S.S., Johnson M., Venkatramani B., Janakiraman N. Phytochemical studies on Allamanda cathartica L. using GC-MS. Asian Pacific Journal of Tropical Biomedicine, 2012, 2(2): S550-S554 CrossRef
- Rahman M.M., Ahmad S.H., Mohamed M.T., Ab Rahman M.Z. Antimicrobial compounds from leaf extracts of Jatropha curcas, Psidium guajava, and Andrographis paniculata. Scientific World Journal, 2014, 2014: 635240 CrossRef
- Umezawa S., Shibahara S., Omoto S., Takeuchi T., Umezawa H. Studies on the biosynthesis of 3-amino-3-deoxy-D-glucose. The Journal of Antibiotics, 1968, 21(8): 485-491 CrossRef
- Milner J.L., Silo-Suh L., Lee J.C., He H., Clardy J., Handelsman J. Production of kanosamine by Bacillus cereus UW85. Applied and Environmental Microbiology, 1996, 62(8): 3061-3065.
- Kevany B.M., Rasko D.A., Thomas M.G. Characterization of the complete zwittermicin A biosynthesis gene cluster from Bacillus cereus. Applied and Environmental Microbiology, 2009, 75(4): 1144-1155 CrossRef
- Vetter N.D., Langill D.M., Anjum S., Boisvert-Martel J., Jagdhane R.C., Omene E., Zheng H., van Straaten K.E., Asiamah I., Krol E.S., Sanders D.A., Palmer D.R. A previously unrecognized kanosamine biosynthesis pathway in Bacillus subtilis. Journal of the American Chemical Society, 2013, 135(16): 5970-5973 CrossRef
- Arakawa K., Müller R., Mahmud T., Yu T.W., Floss H.G. Characterization of the early stage aminoshikimate pathway in the formation of 3-amino-5-hydroxybenzoic acid: the RifN protein specifically converts kanosamine into kanosamine 6-phosphate. Journal of the American Chemical Society, 2002, 124(36): 10644-10645 CrossRef
- Kim W.-G., Song N.-K., Yoo I.-D. Trienomycin G, a new inhibitor of nitric oxide production in microglia cells, from Streptomyces sp. 91614. The Journal of Antibiotics, 2002, 55(2): 204-207 CrossRef
- Kang Q., Shen Y., Bai L. Biosynthesis of 3,5-AHBA-derived natural products. Natural Product Reports, 2012, 29: 243-263 CrossRef
- Wrona I.E., Agouridas V., Panek J.S. Design and synthesis of ansamycin antibiotics. Comptes Rendus Chimie, 2008, 11: 1483-1522 CrossRef
- Yu T.W., Bai L., Clade D., Hoffmann D., Toelzer S., Trinh K.Q., Xu J., Moss S.J., Leistner E., Floss H.G. The biosynthetic gene cluster of the maytansinoid antitumor agent ansamitocin from Actinosynnema pretiosum. Proceedings of the National Academy of Sciences, 2002, 99(12): 7968‐7973 CrossRef
- Khatri I., Sharma G., Subramanian S. Composite genome sequence of Bacillus clausii, a probiotic commercially available as Enterogermina®, and insights into its probiotic properties. BMC Microbiology, 2019, 19(1): 307 CrossRef
- Al-Thubiani A.S.A., Maher Y.A., Fathi A., Abourehab M.A.S., Alarjah M., Khan M.S.A., Al-Ghamdi S.B. Identification and characterization of a novel antimicrobial peptide compound produced by Bacillus megaterium strain isolated from oral microflora. Saudi Pharmaceutical Journal, 2018, 26(8): 1089‐1097 CrossRef
- Malanicheva I.A., Kozlov D.G., Sumarukova I.G. Efremenkova O.V. Zenkova V.A, Katrukha G.S., Reznikova M.I., Tarasova O.D., Sineokiĭ S.P., Él'-Registan G.I. Antimicrobial activity of Bacillus megaterium strains. Microbiology, 2012, 81: 178-185 CrossRef