Date of Award

2-2011

Degree Type

Thesis

Degree Name

Master of Science (MSc)

Department

Biology

Supervisor

T.M. Finan

Language

English

Abstract

The bacterium Sinorhizobium meliloti forms N2-fixing nodules on the roots of alfalfa and some other leguminous plants. A gene cluster smc04260-smc04251 was previously found to be expressed when S.meliloti is grown in media containing cellobiose, gentiobiose, salicin and arbutin. These monosaccharides of these sugars are connected via glucosidic bonds of β (1-4) or β (1-6) configuration. The gene cluster smc04260-smc04251 appears to include an ABC-type uptake system as well as genes for catabolism of these sugars. Data from growth curves with strains carrying plasmid co-integrants suggested that the smc04260-smc04251 gene region were tested for their ability to grow on cellobiose, gentiobiose, salicin or arbutin as sole sources of carbon. In order to study the effects of specific gene deletions on the growth of S.meliloti, smc04259, manB, smc04254 were deleted in-frame and growth curves with these strains revealed that smc04259, manB and smc04254 were all important for growth of aryl-glycosides such as salicin and arbutin.

ManB protein, a putative β-mannosidase enzyme, was overexpressed, purified and then tested in protein assays to determine its activity with different substrates. In one assay, 4-nitrophenyl-β-D-glucopyranoside and 4-nitrophenyl-β-D-mannopyranoside were used as substrates and it was determined that ManB was only active on 4-nitrophenyl-β-D-mannopyranoside. Glucose oxidase assay was used with cellobiose, gentiobiose, mannobiose, salicin and arbutin to study the activity of ManB with each substrate, and it was found that ManB only had significant activity when mannobiose was used as a substrate.

Transposon mutagenesis with transposon Tn5-B20 was used to select strains that could not grow or grew poorly when cellobiose and arbutin were used as sole sources of sugar. The mutants had transposon insertions within smc04260-smc04251 gene cluster as well as the smc03160-smc03165 gene cluster. Five mutants were mapped and tested for their ability to grow on cellobiose, gentiobiose, mannobiose, arbutin and salicin. These mutants were later complemented with pLAFR1 cosmid library. A complete understanding of metabolic capabilities of S.meliloti can be beneficial for studying similar systems in soil organisms. The ability to break down a variety of sugars increases the fitness of the organisms and widens the scope of understanding of such systems, which have some potential in biotechnology.

McMaster University Library

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