Create a 3 pages page paper that discusses molecular identification of dms-producing bacteria isolated from marine algae. Molecular Identification of DMS-Producing Bacteria Isolated from Marine Algae Rami Aldagrer Background Dimethylsulfide (DMS) is recognized as the predominant form of sulfur liberated from the oceans to the atmosphere, representing a fundamental compound to the global sulfur cycle (Johnston et al., 2007. Johnston et al., 2008. Steinke et al., 2011). Several studies have indicated that DMS emission demonstrates a significant effect on the magnitude of the earth’s cloud cover, influencing the planet’s climate in the process (Charlson et al., 1987. Watson and Liss, 1998). Specifically, this gas acts as a nucleus for cloud formation which reduces the amount of radiation from the sun that reaches the earth’s surface. In addition, DMS also serves as a chemo-attractant to some species of crustaceans, copepods, seabirds, and marine mammals (Nevitt and Bonadonna, 2005). DMS is derived from dimethylsulfopropionate (DMSP), a secondary metabolite produced by many marine algae and phytoplankton (Johnston et al., 2011). DMSP is converted to DMS by virtue of the lyase and CoA-transferase pathways exhibited by many fungi and bacteria (Wolfe et al., 2000. Yoch et al., 2001a). In fact, according to Kiene and Taylor (1988), catabolic activities by marine bacteria account for most of the DMS released by the oceans. Rhodobacter sphaeroides, Roseovarius nubinhibens, Thiocapsa roseopersicina, and Methylophaga sulfidovorans are just some of the known bacteria capable of converting DMSP to DMS (Zwart et al., 1996. Jonkers et al., 1998. Johnston et al., 2009). Since the role of marine bacteria is central to in the dynamics of the DMS pool is indispensable, molecular identification and phylogenetic description of these organisms is an essential part of DMS studies. Molecular identification through 16s rRNA gene amplification has been the gold standard in phylogenetic studies of bacteria (Yoch et al., 2001b. Cowan et al., 2003. Qian et al., 2009). The 16s rRNA is a constituent of a subunit of prokaryotic ribosome. Since it plays an important role in cellular metabolism, the 16s rRNA gene is conserved across many genera of prokaryotes which, according to Kirkpatrick and Kuske (1992), normally show at least 70% gene sequence similarity. This characteristic specifically makes 16s rRNA gene a valuable taxonomic marker. This study proposes to determine the identity of DMS-producing bacteria isolated from phytoplankton cultures and grown on minimal medium with DMSP as a carbon source. Specifically, the study will seek to amplify the 16s rRNA gene through polymerase chain reaction (PCR), sequence the amplified gene, and compare the obtained sequence against existing sequences deposited in GenBank. Objectives The main objective of the study is to identify unknown DMS-producing bacteria using 16s rRNA gene sequence. The specific objectives are: 1. Extract genomic DNA from cultures of DMS-producing bacteria 2. Amplify the 16s rRNA gene of the obtained bacterial DNA using generic primers 3. Sequence the 16s rRNA gene of the bacteria 4. Compare the obtained 16s rRNA sequence with the sequences in the National Center for Biotechnology Information GenBank database to determine the taxonomic position and/or identity of the unknown organism. 5. Construct a phylogenetic tree of the unknown bacteria based on the 16s rRNA sequence Methods DNA extraction DMS-producing bacteria have already been isolated from selected phytoplankton cultures and will be re-isolated in minimal medium with DMSP. Then, selected clonal isolates will be grown in marine broth for 18-24 hours and subcultured into 50 mL of fresh medium. Then, the bacterial suspension will be incubated at 37 °C for 5 h in a shaker incubator (Yoch et al., 2001b). The cells will be harvested by centrifugation and genomic DNA will be extracted from the pellets using QIAamp® DNA Mini Kit (Qiagen Inc., Valencia, Calif.) Procedures for the DNA extraction will be adapted from the protocol provided by the manufacturer. Agarose Gel Electrophoresis Electrophoresis will be done after DNA extraction to verify the presence and quality of DNA. Agarose gel electrophoresis (AGE) will be done according to the procedure suggested by Voytas (2000). 16s rRNA amplification Amplification of 16s rRNA gene via polymerase chain reaction (PCR) will be optimized first. PCR conditions such as annealing temperature and extension time and denaturation time will be varied until the right combination will produce a desirable result. Using the optimized PCR conditions for 16s rRNA amplification, PCR reagents including the primers, polymerase, and dNTPs will be mixed with the DNA extracted previously. The extracted DNA will serve as the template for the amplification. The primers that will be used to amplify the gene are 27F (5’-AGA GTT TGA TCC TGG CTC AG-3’) and 1492R (5’-GGT TAC CTT GTT ACG ACT T-3’) After amplification, PCR products will be verified using AGE. Purification of PCR products After the presence of PCR amplicon has been confirmed, the PCR products will be purified using QIAquick PCR purification kit (Qiagen Inc., Valencia, Calif.) (Yoch et al., 2001b). Purification procedure will be conducted according to the protocol suggested by the manufacturer. Sequencing and phylogenetic analysis The purified PCR products will be sent to a sequencing company for sequencing. The obtained sequence will then be compared to existing information found in the National Center for Biotechnology Information GenBank database using a sequence alignment tool, BLAST. Molecular Evolutionary Genetic Algorithm (MEGA) software will be used to construct the phylogenetic tree. Workplan Objectives Expected Output Activities Week 1 2 3 4 5 6 7 8 9 10 11 12 DNA extraction from DMS-producing bacteria Genomic DNA of DMS-producing bacteria Culture of bacteria in marine broth DNA extraction Agarose gel electrophoresis PCR amplification of 16s rRNA gene 16s rRNAamplicon Optimization of PCR conditions Actual PCR of 16s rRNA gene Agarose gel electrophoresis of the PCR product Purification of the PCR product Sequencing of 16s rRNA gene 16s rRNA sequences Sending off the purified PCR product for sequencing Analysis of the 16s rRNA sequence Identity of the unknown bacteria Phylogenetic tree of the organism Sequence alignment using data from GenBank Construction of phylogenetic tree References Charlson, R., Lovelock, J., Andreae, M., and Warren, S., 1987. Oceanic phytoplankton, atmospheric sulfur, cloud albedo and climate. Nature 326 (6114): 655–661. Cowan, D., Baker, G., and Smith, J. 2003. Review and re-analysis of domain-specific 16S primers. Journal of Microbiological Methods 55: 541–555. Johnston, A., Sullivan, M., Curson, A., Shearer, N., Todd, J., and Green, R. 2011. Unusual regulation of a leaderless operon involved in the catabolism of dimethylsulfoniopropionate in Rhodobacter sphaeroides PloS One 6 (1): 1–11. Johnston, A., Todd, J., Curson, A., Dupont, C., and Nicholson, P. 2009. The dddP gene, encoding a novel enzyme that converts dimethylsulfoniopropionate into dimethyl sulfide, is widespread in ocean metagenomes and marine bacteria and also occurs in some Ascomycete fungi. Environmental Microbiology 11(6): 1376–1385. Johnston, A., Todd, J., Sun, L., Nikolaidou-Katsaridou, M., Curson, A., and Rogers, R. 2008. Molecular diversity of bacterial production of the climate changing gas, dimethyl sulphide, a molecule that impinges on local and global symbioses. Journal of Experimental Botany 59 (5): 1059–1067. Jonkers, H., Bruin, S., and van Gemerden, H. 1998. Turnover of dimethylsulfoniopropionate (DMSP) by the purple sulfur bacterium Thiocapsa roseopersicina M11: ecological implications. FEMS Microbiology Ecology 27: 281–290. Kirkpatrick, B., and Kuske, C. 1992. Phylogenetic relationships between the Western Aster Yellows Mycoplasma-like organism and other prokaryotes established by 16s rRNA gene sequence. International Journal of Systematic Bacteriology 42(2): 226–233. Li, Y., Todd, J., Rogers, R., Wexler, M., Bond, P., Sun, L., Curson, A., Malin, G., Steinke, M., and Johnston, A. 2007. Structural and regulatory genes required to make the gas dimethyl sulfide in bacteria. Science.315: 66–669. Nevitt G.A., Bonadonna F. 2005. Sensitivity to dimethyl sulphide suggests a mechanism for olfactory navigation by seabirds. Biology Letters1: 303–305. Qian, P., Ki, J., and Zhang, W. 2009.Discovery of marine Bacillus species by 16S rRNA and rpoB comparisons and their usefulness for species identification. Journal of Microbiological Methods 77: 48–57. Steinke, M., Exton, D., and McGenity, T. 2011. Challenges to the bio(geo)chemist: marine gases. The Biochemical Society 1–7. Voytas, D. 2000. Current Protocols in Molecular Biology. John Wiley & Sons, Inc. pp. 2.5A.1-2.5A.9 Watson, A., and Liss, P. 1998.Marine biological controls on climate via the carbon and sulphur geochemical cycles. Phil. Trans. R. Soc. Lond. B. 353(1365): 41–51. Wolfe, G., Levasseur, M., Cantin, G., and Michaud, S. 2000.DMSP and DMS dynamics and microzooplankton grazing in the Labrador Sea: application of the dilution technique. Deep-Sea Research I 47: 2243–2264. Yoch, D., Carraway, R., Friedman, R and Kulkarni, N. 2001a. Dimethylsulfide (DMS) production from dimethylsulfoniopropionate by freshwater river sediments: phylogeny of Gram-positive DMS-producing isolates. FEMS Microbiology Ecology.37: 31–37. Yoch, D., Ansede, J., and Friedman, R. 2001b. Phylogenetic analysis of culturable dimethyl sulfide-producing bacteria from a Spartina-dominated salt marsh and estuarine water. Applied and Environmental Microbiology 67(3): 1210–1217 Zwart, J., Nilesse, P., and Kuenen, G. 1996. Isolation and characterization of Methylophaga sulfidouorans sp. nov.: an obligately methylotrophic, aerobic, dimethylsulfide oxidizing bacterium from a microbial mat. FEMS Microbiology Ecology 20: 261–270.
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