A physiological analysis of the role of the truB gene in Escherichia coli
The truB gene of Escherichia coli 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 truB 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 truB gene and by a D48C, catalytically-inactive, allele of truB. The proteome of the truB mutant also contained altered levels of intermediates involved in biogenesis of the outer membrane proteins (OMP), OmpA and OmpX. In addition, the truB mutation reduced the basal expression from two sE promoters, degP and rpoHP3 although this could not be complemented with cloned truB. The truB mutant also exhibited a defect in survival of rapid transfer from 37oC to 50o C. This mutant phenotype could be complemented by the cloned truB gene but not by a D48C, catalytically-inactive, allele of truB. The temperature-sensitivity of truB mutants could be enhanced by combination with a mutation in the trmA gene, encoding the enzyme modifying the universal U54 tRNA nucleoside, but not by mutations in trmH , encoding the enzyme catalysing the formation of Gm18. Three novel aspects to the phenotype of truB 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 E. coli, possibly by optimising the stability of the tRNA population at high temperatures.