Transposon Screen of Marinobacter HI15- 87 Mutants in Dissolved Organic Matter

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2019

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University of Hawai'i at Manoa

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Marine dissolved organic matter (DOM) represents the largest carbon source in the world. The consumption of DOM by microorganisms results in the production of large amounts of atmospheric carbon dioxide gas, thus playing a significant role in the global carbon cycle. Although some key enzymes involved in the breakdown of phosphonates in DOM have been identified, the specific biochemical pathways of marine microbes to degrade high molecular weight DOM remains largely unconfirmed. Sample isolates of Marinobacter HI15-87 from the waters of Station Aloha, approximately 100km north of Oahu, underwent transposon mutagenesis with the transposon pSMC194 containing three cut sites for the endonuclease HpyCH4IV within Streptomycin and Spectinomycin resistance genes. Approximately 10,000 individual HI15-87 colonies were selected through growth on marine broth containing Streptomycin and Spectinomycin to confirm successful transposon mutagenesis. The selected isolates that did not grow in hydrolyzed DOM were suspected to have received the pSMC194 transposon in a location of its genome that disrupted adjacent genes or pathways essential to degradation of DOM as a carbon source. DNA extraction, HpyCH4IV digestion, and sequencing point to the host cell genes flanking the transposon insert, thereby indicating the HI15-87 genes likely required for DOM consumption. Identification of these genes may allow for better understanding of marine microbial metabolisms and communities.

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40 pages

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