Masters Theses

Date of Award

5-2012

Degree Type

Thesis

Degree Name

Master of Science

Major

Life Sciences

Major Professor

Dr. Gladys Alexandre

Committee Members

Dr. Albrecht von Arnim, Dr. Engin Serpersu

Abstract

Azospirillum brasilense is a class of rhizobacteria capable of nitrogen fixation, root colonization and hence promoting host plant growth. The bacteria posses cell interaction behaviors like clumping and flocculation that contribute the survival of the organism in nutrient limited conditions. Change in the cell surface adhesive properties allows the cells to progress from free swimming to clumping and finally flocculation. Less is known about the genetic regulation of these processes with flcA being the only transcriptional regulator known so far to directly control flocculation. Recent evidence suggesting that Che1, a chemotaxis like signal transduction pathway controls the cell behavior clumping and hence indirectly controlling flocculation,

To understand the genetic regulation of clumping and flocculation in A. brasilense Sp7, the research here focuses on a subset of 27 of these transposon mutants. The objective of this research was to map the insertion by rescue cloning, characterize the mutant for growth, motility and clumping using qualitative and quantitative assays and characterize the effect of the mutations identified on flocculation.

By rescue cloning we mapped the transposon insertion for nine out of the twenty-seven mutations. The insertions were mapped on glycosyltransferases, glucosyltransferase, putative TonB-dependent siderophore receptor, Acyl-CoA thioesterase and sugar phosphatase of the HAD superfamily. All these mutants were characterized for the clumping and flocculation phenotype and while some of these mutants showed severe delay in clumping and/or flocculation indicating changes in cell surface adhesive properties of the mutants compared to the wild type. Further investigations like supplementation of media with different sources of iron provided an insight into understanding the indirect effect of the PM3 mutation on the iron transport pathway. Also, the lectin-binding assay provided insightful information about the changes in the exopolysaccharide (EPS) composition during the different stages of cell aggregation. In conclusion the experiments provided good measure of information about the changes in the cell surface properties specifically with relation to proteins and EPS as an initial investigation. Future work will concentrate on screening the effects of these mutations on the expression of downstream genes and further phenotypic characterization of the selected mutants.

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