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Differential Analysis of Unique Genes Expressed in Stenotrophomonas maltophilia Strain OR02 in Response to Selenite

Moffo, Nathan
http://orcid.org/0000-0001-9592-9522
http://rave.ohiolink.edu/etdc/view?acc_num=ysu15663177454459

Abstract Details

2019, Master of Science in Biological Sciences, Youngstown State University, Department of Biological Sciences and Chemistry.
Stenotrophomonas maltophilia Oak Ridge Strain OR02 (S. maltophilia 02), which was isolated from a heavy metal contaminated site in Oak Ridge, TN, grows in the presences of toxic levels of heavy metals, including selenite. Selenium exists in four different valences as selenate (+6), selenite (+4), elemental selenium (0) and selenide (-2). Selenate (SeO42-) and selenite (SeO32-) are soluble, uncolored forms of selenium and elemental selenium forms an insoluble red precipitate. Microbes that use selenium, import it into the cell as selenite, reduce it to selenide and incorporate it into selenocysteine. When environmental selenite concentrations are higher than required, some bacteria reduce it to insoluble elemental selenium and methylate it to form volatile methyl selenide. When S. maltophilia 02 is growing in the presence of 0.5 mM sodium selenite, it produces an insoluble red precipitate and a stale garlic odor, which are presumably elemental selenium and methyl selenide, respectively. A subtractive hybridization technique was used in an attempt to identify genes that are expressed in response to high concentrations of selenite. S. maltophilia 02 was grown to early log phase and exposed to 0.5 mM selenite. After two hours of selenite exposure, RNA was extracted from untreated and treated cultures. The RNA was converted to double stranded cDNA and cut with the four-base cutter, restriction endonuclease, Sau3AI. After ligating linkers to the digested cDNA, it was amplified by PCR. The PCR reaction on the untreated cDNA was labeled with dCTP and dUTP biotin. The PCR reaction on the treated cDNA did not contain biotin labeled nucleotides. PCR products from both reactions were mixed, heated at 98°C to denature the DNA and allowed to hybridize at 68°C for 24 hours. The hybridization mixture was added to streptavidin magnetic beads and exposed to a magnetic stand to remove the cDNA from RNA that was present under both the treated and untreated conditions. The remaining cDNA from RNA transcribed in response to selenite was PCR amplified and used in further hybridization and subtraction steps. After 3 rounds of subtraction, the final product was cloned and sequenced. If successful, this method was expected to possibly detect genes for proteins involved in glutathione synthesis and maintenance since many bacteria detoxify selenite using glutathione. It was expected to detect genes for proteins involved in methylation, as methylation is another form of detoxification. The subtractive hybridization procedure employed to find these unique genes however encountered difficulties in separating rRNA from mRNA, and due to complications from the amount of rRNA in total RNA, no unique genes were identified. If repeated, exhaustive efforts must be made to ensure that rRNA encoding genes are completely removed before sequencing.
Jonathan Caguiat, PhD (Advisor)
David Asch, PhD (Committee Member)
Xiangjia Min, PhD (Committee Member)
59 p.
Biology; Genetics; Microbiology; Molecular Biology
microbial genetics; selenite; subtractive hybridization; representational differential analysis; stenotrophomonas maltophilia; molecular genetics; heavy metal resistance; gene expression; RNA isolation

Recommended Citations

Citations

  • Moffo, N. (2019). Differential Analysis of Unique Genes Expressed in Stenotrophomonas maltophilia Strain OR02 in Response to Selenite [Master's thesis, Youngstown State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ysu15663177454459

    APA Style (7th edition)

  • Moffo, Nathan. Differential Analysis of Unique Genes Expressed in Stenotrophomonas maltophilia Strain OR02 in Response to Selenite. 2019. Youngstown State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ysu15663177454459.

    MLA Style (8th edition)

  • Moffo, Nathan. "Differential Analysis of Unique Genes Expressed in Stenotrophomonas maltophilia Strain OR02 in Response to Selenite." Master's thesis, Youngstown State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=ysu15663177454459

    Chicago Manual of Style (17th edition)

ysu15663177454459
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This open access ETD is published by Youngstown State University and OhioLINK.