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Dec. 13, 2017

Research Experiences for Undergraduates


Microbiology in the Post Genome Era

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REU Class of 2009

 

Naglee Allen

Fort Lewis College in Durango, Colorado

Naglee carried out her research on Isolation and Identification of Manganese Oxidizing and Phosphorous Absorbing Bacteria in the laboratory of Dr. Amy J. Pruden at the Department of Civil and Environmental Engineering.

Earl Middlebrook

The microbiology of drinking water and dairy wastewater treatment systems has not been well characterized which stands as a barrier to their optimization. Enhanced biological phosphorus removal (EBPR) is an environmentally friendly, effective, treatment for wastewater that employs the use of phosphorus accumulating organisms (PAOs) that are difficult to culture and therefore have not been well studied. Manganese (II) oxidation by microbial groups in drinking water filtration systems also has not been well studied. In this study bacteria involved in manganese (II) and phosphorus transformations were isolated and identified. We first isolated pure cultures of manganese (II) oxidizing bacteria, performed 16s gene PCR, and finally DNA sequencing for identification. Culturing was circumvented for PAOs by direct 16s gene PCR, cloning, and DNA sequence. Previously identified manganese (II) oxidizing bacteria such as Bacillus sp, and Pseudomonas were identified. We also found various bacteria not previously identified as Mn (II) oxidizers. For PAOs, various Betaproteobacteria, many of which are known PAOs in addition to Rhodocyclaes and Actinobacteria were found.

Naglee's Mentor: Dr. Amy Pruden

 

 

Earl Middlebrook

Fort Lewis College in Durango, Colorado

Earl carried out his research, An Investigation of Serine O-acetyltransferases in Representative Nitrogen Fixing Microorganisms, in the laboratory of Timothy J. Larson at the Biochemisty Department.

Earl Middlebrook

Sulfur metabolism pathways in nitrogen fixing microorganisms are essential to creating sufficient amounts of cysteine to produce the iron-sulfur clusters and cysteine rich enzymes used in diazotropic growth. It was found that the CysE1 class of serine O-acetyltransferase in Azotobacter vinelandii has a Ki value 50 to 100 times greater than the other two serine o-acetyltransferases of the same organism, which confers that it is relatively feedback resistant to cysteine levels. This gene is found in a nitrogen fixation gene cluster and is co-regulated with the adjacent nif genes. The increased need for cysteine when the nif cluster is transcriptionally active will supposedly be fulfilled regardless of cytosolic cysteine concentrations. This would presumably keep the cell from becoming cysteine deficient. In this work, we will test the hypothesis that other CysE1-like enzymes coded for in nitrogen fixation gene clusters will have a similar resistance to inhibition by cysteine as the Azotobacter enzyme, thereby enhancing cysteine biosynthesis needed for production of nitrogenase enzymes. Representatives of y-proteobacteria and cyanobacteria will be chosen and their cysE1-like gene will be amplified and placed in a vector used to transform a strain of E. coli that can be induced to produce the corresponding serine O-acetyltransferase (the organisms to be used include Azotobacter vinelandii, Acidithiobacillus ferrooxidans, Methylococcus capsulatus and Nostoc punctiforme. The proteins will be created with a histidine tag at the N-terminus that will be used to isolate them using metal chelate affinity chromatography. A thrombin cleavage site is encoded between the his-tag and the enzyme of interest that will be used to cleave the two and leave the pure protein in its native state. The purity and kinetic properties of the isolated enzymes will be determined by using electrophoresis and enzymatic activity assays.

Earl's Mentor: Dr. Timothy J. Larson

 

 

Karla Piedl

Virginia Tech in Blacksburg, Virginia

Karla performed her research, Growth Characteristics of Acidobacterium capsulatum, in the laboratory of Dr. Biswarup Mukhopadhyay at the Virginia Bioinformatics Institute at Virginia Tech.

Earl Middlebrook

Acidobacterium capsulatum is an acidophilic bacteria that was initially isolated from an acidic mineral environment. It was characterized by Kishimoto et. al. as being a pink, mesophilic, aerobic bacteria. A. capsulatum is known as a difficult organism to grow. After simplifying the media used to grow the bacteria, we began working on some of its growth characteristics. The need for a buffering system was apparent when the initial and final pHs of a liquid culture were compared. This was achieved by a citric acid/sodium citrate buffer. The organism cannot use citric acid as an energy source, nor does it inhibit growth. A. capsulatum was tested for growth at pH levels between 3 and 5.5 and the best growth appeared between pH 3 and 4. The oxygen concentration threshold of the bacteria was also tested using sealed serum bottles. Oxygen concentrations above atmospheric changed the growth rate of the organism and at the highest concentration (5 atm) inhibited the growth completely. The effect of two common rhizospheric acids, acetic and succinic, were tested for their effects on the growth of A. capsulatum. A preliminary growth curve (not shown) indicated that succinic acid would be inhibitory. However, succinic acid at concentrations of 0.5 mM and lower had no discernable effect on the organism. Levels of acetic acid above 1.0 mM - 2.5 mM showed an inhibitory effect on the organism.

Karla's Mentor: Dr. Biswarup Mukhopadhyay

 

 

Brandon Robinson

Oakwood University in Huntsville, Alabama

Brandon worked in the laboratory of Dr. Zhiyou Wen in the Biological Systems Engineering Department.

Earl Middlebrook

 

 

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Research Experience For Undergraduates (REU) Microbiology is funded by the National Science Foundation Award Number 1156954.