Oligobacterial physiology is mostly unstudied due to cultivation difficulty. New isolation techniques such as extinction culture have produced cultivable representatives of the aquatic environment namely Sphingopyxis alaskensis. Attempts were made to grow the bacterium in batch cultures using glucose and tyrosine as ideal substrates as determined from growth studies. Differential protein expression from cytoplasmic and membrane fractions of the putative culture were compared so as to identify key proteins involved in substrate uptake and metabolism followed by incorporation of protein quantities into mathematical models of oligotroph growth. However artifactual results from two dimensional gel electrophoresis led to the question of culture purity, which was eventually confirmed by light microscopy, flow cytometry and 16S rRNA gene sequencing. This research gives better insight into the possible problems that can crop up while working with hard to culture marine oligobacteria. I demonstrate the rationale used to identify the contaminant, which was difficult to detect because its slow growth was similar to the target organism. A major achievement was successful cell fractionation as it has never been attempted in oligobacteria due to culturing difficulties and the procedure is different from the routine methods adopted in bacteria and fungi. Also the research demonstrates a complete protocol for eliminating uncertainties in culture purity.
Thesis (M.S.) University of Alaska Fairbanks, 2008
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