ABB  Vol.3 No.7 , November 2012
Isolation, cytotoxic activity and phylogenetic analysis of Bacillus sp. bacteria associated with the red sea sponge Amphimedon ochracea
ABSTRACT
Most of marine sponges harbor dense and diverse microbial communities of bioactivity importance. Four Gram positive bacterial cultures (HA-21, HA-68, HA- MS-105 and HA-MS-119) were isolated from the sponge Amphimedon ochracea, collected from the Red Sea coast of Egypt. Bacterial species were identified based on the phylogenetic analysis of the nucleotide sequences of their 16S rDNA genes. The Sequences similarity values of 98% - 100% to other strains in the NCBI database showed strong similarities with the 16S rDNA genes of firmicutes (Bacillus sp.). The four bacterial species were submitted to the GenBank database and had accession numbers of: HA-21 [JQ-768238]; HA-68 [JQ751264]; HA-MS-105 [JQ768239]; HAMS-119 [JQ768240]. The cytotoxic activities of the bacterial isolates were tested against three established human cancer cell lines; HepG2 (hepatocellular carcinoma), HCT (colon carcinoma) and MCF-7 (breast carcinoma). The inhibitory effect on these cell lines, measured by MTT cell assay protocol, revealed promising cytotoxic activity of the four isolates (IC50 values (μg/mL) were: HA-21: 13.2, 9.3 and 12.2; HA-68: 10.42, 4.3 and 5.5; HA-MS-105: 46.9, 28.6 and 21.3; HAMS-119: 10.42, 6.3 and 22.1; respectively). The recovery of bacterial strains with cytotoxic activity suggests that marine invertebrates remain a rich source for the isolation of culturable isolates capable of producing novel bioactive secondary metabolites.

Cite this paper
Aboul-Ela, H. , Shreadah, M. , Abdel-Monem, N. , Yakout, G. and Soest, R. (2012) Isolation, cytotoxic activity and phylogenetic analysis of Bacillus sp. bacteria associated with the red sea sponge Amphimedon ochracea. Advances in Bioscience and Biotechnology, 3, 815-823. doi: 10.4236/abb.2012.37101.
References
[1]   Newman, D.J. and Cragg, G.M. (2004) Marine natural products and related compounds in clinical and advanced preclinical trials. Journal of Natural Products, 67, 1216-1238. doi:10.1021/np040031y

[2]   Simmons, T.L., Andrianasolo, E., McPhail, K., Flatt, P.M. and Gerwick, W.H. (2005) Marine natural products as anticancer drugs. Molecular Cancer Therapeutics, 4, 333-342.

[3]   Thoms, C. and Shrupp, P. (2005) Biotechnological potential of marine sponges and their associated bacteria as producers of new pharmaceuticals. Journal of International Biotechnology Law, 2, 257-264.

[4]   Simmons, T.L., Coates, R.C., Clark, B.R., Eugene, N., Gonzalez, D., Esquenazi, E., et al. (2008) Biosynthetic origin of natural products isolated from marine organisms-invertebrate assemblages. Proceedings of the National Academy of Sciences of the United States of America, 105, 4587-4594. doi:10.1073/pnas.0709851105

[5]   Livett, B.G., Gaylera, K.R. and Khalilb, Z. (2004) Drugs from the sea: Conopeptides as potential therapeutics. Current Medicinal Chemistry, 11, 1715-1723. doi:10.2174/0929867043364928

[6]   Faulkner, D.J. (2000) Marine natural products. Natural Products Reports, 17, 7-55. doi:10.1039/a809395d

[7]   Taylor, M.W., Radax, R., Steger, D. and Wagner, M. (2007) Sponge-associated microorganisms: Evolution, ecology, and biotechnological potential. Microbiology and molecular biology reviews, 71, 295-347.

[8]   Faulkner, D.J., Harper, M.K., Haygood, M.G., Salomon, C.E. and Schmidt, E.W. (2000) Symbiotic bacteria in sponges: Sources of bioactive substances. In: Fusetani, N., Ed., Drugs from the Sea, Karger, Basel, 107-119.

[9]   Sipkema, D., Franssen, M.C.R., Osinga, R., Tramper, J. and Wijffels, R.H. (2005) Marine sponges as pharmacy. Marine Biotechnology, 7, 142-162. doi:10.1007/s10126-004-0405-5

[10]   Faulkner, D.J. (2002) Marine natural products. Natural Products Reports, 19, 1-48. doi:10.1039/b009029h

[11]   Friedrich, A.B., Merkert, H., Fendert, T., Hacker, J., Proksch, P. and Hentschel, U. (1999) Microbial diversity in the marine sponge Aplysina cavernicola (formerly Verongia cavernicola) analyzed by ?uorescence in-situ hybridization (FISH). Marine Biology, 134, 461-470. doi:10.1007/s002270050562

[12]   Vacelet, J. and Donadey, C. (1977) Electron microscope study of the association between some sponges and bacteria. Journal of Experimental Marine Biology and Ecolology, 30, 301-314. doi:10.1016/0022-0981(77)90038-7

[13]   Taylor, M.W., Radax, R., Steger, D. and Wagner, M. (2007) Sponge-associated microorganisms: Evolution, ecology, and biotechnological potential. Microbiology and Molecular Biology Reviews, 71, 295-347. doi:10.1128/MMBR.00040-06

[14]   Bewley, C.A., Holland, N.D. and Faulkner, D.J. (1996) Two classes of metabolites from Theonella swinhoei are localized in distinct populations of bacterial symbionts. Experientia, 52, 716-722. doi:10.1007/BF01925581

[15]   Flowers, A.E., Garson, M.J., Webb, R.I., Dumdei, E.J. and Charan, R.D. (1998) Cellular origin of chlorinated diketopiperazines in the dictyoceratid sponge Dysidea herbacea (Keller). Cell and Tissue Research, 292, 597- 607doi:10.1007/s004410051089

[16]   Proksch, P., Edrada, R.A. and Ebel, R. (2002) Drugs from the seas? Current status and microbiological implications. Applied Microbiology and Biotechnology, 59, 125-134. doi:10.1007/s00253-002-1006-8

[17]   Schmidt, E.W., Obraztsova, A.Y., Davidson, S.K., Faulkner, D.J. and Haygood, M.G. (2000) Identi?cation of the antifungal peptidecontaining symbiont of the marine sponge Theonella swinhoei as a novel Deltaproteobacterium, BC and idatus entotheonella palauensis. Marine Biology, 136, 969-977. doi:10.1007/s002270000273

[18]   Unson, M.D., Holland, N.D. and Faulkner, D.J. (1994) A brominated secondary metabolite synthesized by the cyanobacterial symbiont of a marine sponge and accumulation of the crystalline metabolite in the sponge tissue. Marine Biology, 119, 1-11. doi:10.1007/BF00350100

[19]   Oclarit, J.M., Okada, H., Ohta, S., Kaminura, K., Yamaoka, Y., Iizuka, T., Miyashiro, S. and Ikegami, S. (1994) Anti-bacillus substance in the marine sponge, Hyatella species produced by an associated Vibrio species bacterium. Microbios, 78, 7-16.

[20]   Shigemori, H., Bae, M.A., Yazawa, K., Sasaki, T. and Kobayashi, J. (1992) Alteramide A, a new tetracyclic alkaloid from a bacterium Alteromonas sp. associated with the marine sponge Halichondria okadai. The Journal of Organic Chemistry, 57, 4317-4320. doi:10.1021/jo00041a053

[21]   Stierle, A.C., Cardellina, J.H. and Singleton, F.L. (1988) A marine Micrococcus produces metabolites ascribed to the sponge Tedania ignis. Cellular and Molecular Life Sciences, 44, 1021. doi:10.1007/BF01939910

[22]   Weiner, R.M., Segall, A.M. and Colwell, R.R. (1985) Characterization of a marine bacterium associated with Crassostrea virginica (the eastern oyster). Applied and Environmental Microbiology, 49, 83-90.

[23]   Shirling, E.B. and Gottlieb, D. (1966) Methods for characterization of Streptomyces species. International Journal of Systematic Bacteriology, 16, 313-340. doi:10.1099/00207713-16-3-313

[24]   Webster, N.S., Wilson, K.J., Blackall, L.L. and Hill, R.T. (2001) Phylogenetic diversity of bacteria associated with the marine sponge Rhopaloeides odorabile. Applied and Environmental Microbiology, 67, 434-444. doi:10.1128/AEM.67.1.434-444.2001

[25]   Lyman, J. and Fleming, R. (1940) Composition of seawater. Journal of Marine Research, 3, 134-146.

[26]   Sambrook, J., Fritsch, E.F. and Maniatis, T. (2001) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory Press, New York.

[27]   Acinas S.G., Anton J. and Rodriguez-Valera F. (1999) Diversity of free-living and attached bacteria in offshore western Mediterranean waters as depicted by analysis of genes encoding 16S rRNA. Applied and Environmental Microbiology, 65, 514-522.

[28]   Thanomsub, B., Poomeechockchai, W., Limtrakul, A., Arunrattiyakorn, P., Petchleelaha, W., Nitida, T. and Kanzaki, H. (2006) Withdrawn: Chemical structures and biological activities of rhamnolipids produced by Pseudomonas aeruginosa B189 isolated from milk factory waste. Bioresource Technology, 98, 1149-1153. doi:10.1016/j.biortech.2005.10.045

[29]   Hentschel, U., Schmid, M., Wagner, M., Fieseler, L., Gernert, C. and Hacker, J. (2001) Isolation and phylogenetic analysis of bacteria with antimicrobial activities from the mediterranean sponges Aplysina aerophoba and Aplysina cavernicola. FEMS Microbiology Ecology, 35, 305-312. doi:10.1111/j.1574-6941.2001.tb00816.x

[30]   Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. (2011) MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28, 2731-2739. doi:10.1093/molbev/msr121

[31]   Kennedy, J., Baker, P., Piper, C., Cotter, P.D., Walsh, M., Mooij, M.J., Bourke, M.B., Rea, M.C., O’Connor, P.M., Ross, R.P., Hill, C., O’Gara, F., Marchesi, J.R. and Dobson, A.D. (2009) Isolation and analysis of bacteria with antimicrobial activities from the marine sponge Haliclona simulans collected from Irish waters. Marine Biotechnology, 11, 384-396. doi:10.1007/s10126-008-9154-1

[32]   Thakur, N.L., Hentschel, U., Krasko, A., Pabel, C.T., Anil, A.C. and Müller, W.E.G. (2003) Antibacterial activity of the sponge Suberites domuncula and its primmorphs: Potential basis for epibacterial chemical defense. Aquatic Microbial Ecology, 31, 77-83. doi:10.3354/ame031077

[33]   Hentschel, U., Schmid, M., Wagner, M., Fieseler, L., Gernert, C. and Hacker, J. (2001) Isolation and phylogenetic analysis of bacteria with antimicrobial activities from the Mediterranean sponges Aplysina aerophoba and Aplysina cavernicola. FEMS Microbiology Ecology, 35, 305-312. doi:10.1111/j.1574-6941.2001.tb00816.x

[34]   Webster, N.S. and Hill, R.T. (2001) The culturable microbial community of the Great Barrier Reef sponge Rhopaloeides odorabile is dominated by an α-Proteo-bacterium. Marine Biology, 138, 843-851. doi:10.1007/s002270000503

[35]   Tamura, K. and Nei, M. (1993) Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution, 10, 512-526.

[36]   Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. (2011) MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution, 28, 2731-2739.

 
 
Top