A physiological analysis of the role of the truB gene in Escherichia coli

• Main Author: Kinghorn, Seonag Mary
• Published: University of Aberdeen 2002
• Subjects: 572; tRNA
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.248619

The <i>truB</i> gene of <i>Escherichia coli </i>encodes the pseudouridine (ψ)-55 synthase and is responsible for modifying all tRNA molecules in the cell at the U55 position. The aim of this project was to investigate the physiological role of the truB gene and ψ55 tRNA modification. A <i>truB</i> null mutant created in this work, grew normally on all growth media tested, but exhibited a competitive disadvantage in extended co-culture with its wild-type progenitor. The mutant phenotype could be complemented by both the cloned <i>truB</i> gene and by a D48C, catalytically-inactive, allele of <i>truB. </i> The proteome of the <i>truB</i> mutant also contained altered levels of intermediates involved in biogenesis of the outer membrane proteins (OMP), OmpA and OmpX. In addition, the <i>truB</i> mutation reduced the basal expression from two s^E promoters, <i>degP </i>and <i>rpoH</i>P3 although this could not be complemented with cloned <i>truB</i>. The <i>truB</i> mutant also exhibited a defect in survival of rapid transfer from 37^oC to 50^oC. This mutant phenotype could be complemented by the cloned <i>truB</i> gene but not by a D48C, catalytically-inactive, allele of <i>truB</i>. The temperature-sensitivity of <i>truB</i> mutants could be enhanced by combination with a mutation in the <i>trmA</i> gene, encoding the enzyme modifying the universal U54 tRNA nucleoside, but not by mutations in <i>trmH</i> , encoding the enzyme catalysing the formation of Gm18. Three novel aspects to the phenotype of <i>truB </i>mutants have been identified. Importantly the data support the hypothesis that TruB-effected ψ55 modification of tRNA is not essential but contributes to thermal stress tolerance in <i>E. coli</i>, possibly by optimising the stability of the tRNA population at high temperatures.