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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.

Bacteria were previously isolated from two separate sites, one contaminated with lead and the other with Polycyclic Aromatic Hydrocarbons (PAHs). Alcaligenes sp. AO22 and Arthrobacter sp. E9 were identified from the lead contaminated site, while Stenotrophomonas maltophilia was identified from the PAH contaminated site. Minimum Inhibitory Concentration assays (MICs) were previously performed on Alcaligenes sp. AO22 and Arthrobacter sp. E9 and they were found to be resistant to varying levels of heavy metals and polymerase chain reactions suggesting the presence of mercury (mer), copper (pco) and cadmium, zinc and cobalt (czc) resistance genes. S. maltophilia VUN 10010, was previously investigated for its ability to degrade pyrene and other HMW PAHs as a sole carbon and energy source. The purpose of the current project was to further characterise the MICs of these isolates, along with biofilm capabilities. The genetic basis of their heavy metal resistance was also investigated.

The genus Gluconobacter is industrially important due to the ability to accomplish unusual and almost complete oxidation reactions (bioconversions) and to contaminate high sugar content products. Following preliminary evidence that some strains of Gluconobacter were resistant to cadmium, and realizing that cadmium resistance among gram-negative organisms is often encoded by an operon which also encodes cobalt and zinc resistance via an efflux mechanism, 10 strains of Gluconobacter were tested for heavy-metal resistance. Three of the 10 representative strains appeared to be resistant to cadmium chloride, and two were also resistant to cobalt- and zinc chloride. These strains, as well as two cadmium-sensitive strains were analyzed using PCR and sequencing to establish gene homology with Ralstonia eutropha, the most frequently studied Gram-negative bacterium exhibiting cadmium resistance. Amplification of two genes from the czc operon, known to encode cadmium, cobalt and zinc resistance in Ralstonia, was attempted in the three resistant and two sensitive strains of Gluconobacter. The gene, czcA, thought to encode the main pump protein of the efflux mechanism, was found in all Gluconobacter strains tested. However, amplification of a regulatory gene czcD, thought to sense the extracellular metal ion concentration, was not possible in the Gluconobacter strains tested. The PCR products were sequenced and analyzed for homology to the czc operon in Ralstonia. From the data gathered, it appears as though some strains of Gluconobacter contain at least a portion of the czc operon , encoding cadmium, cobalt and zinc resistance in Ralstonia eutropha.