Article,
Species-dependent phenotypes of replication-temperature-sensitive trfA mutants of plasmid RK2: a codon-neutral base substitution stimulates temperature sensitivity by leading to reduced levels of trfA expression.
J. Bacteriol., 180 (15): 3793-8 (1998)
Abstract
TrfA is the only plasmid-encoded protein required for initiation of
replication of the broad-host-range plasmid RK2. Here we describe
the isolation of four trfA mutants temperature sensitive for replication
in Pseudomonas aeruginosa. One of the mutations led to substitution
of arginine 247 with cysteine. This mutant has been previously described
to be temperature sensitive for replication, but poorly functional,
in Escherichia coli. The remaining three mutants were identical,
and each of them carried two mutations, one leading to substitution
of arginine 163 with cysteine (mutation 163C) and the other a codon-neutral
mutation changing the codon for glycine 235 from GGC to GGU (mutation
235). Neither of the two mutations caused a temperature-sensitive
phenotype alone in P. aeruginosa, and the effect of the neutral mutation
was caused by its ability to strongly reduce the trfA expression
level. The double mutant and mutant 163C could not be stably maintained
in E. coli, but mutant 235 could be established and, surprisingly,
displayed a temperature-sensitive phenotype in this host. Mutation
235 strongly reduced the trfA expression level also in E. coli. The
glycine 85 codon in trfA mRNA is GGU, and a change of this to GGC
did not significantly affect expression. In addition, we found that
wild-type trfA was expressed at much lower levels in E. coli than
in P. aeruginosa, indicating that this level is a key parameter in
the determination of the temperature-sensitive phenotypes in different
species. The E. coli lacZ gene was translationally fused at the 3'
end and internally in trfA, in both cases leading to elimination
of the effect of mutation 235 on expression. We therefore propose
that this mutation acts through an effect on mRNA structure or stability.
Tags
- *dna
- *escherichia
- *plasmids
- *point
- ;
- acid
- aeruginosa/*genetics/metabolism
- amino
- arginine
- bacterial
- beta-galactosidase/biosynthesis
- biosynthesis
- coli
- coli/*genetics/metabolism
- conjugation,
- cysteine
- escherichia
- fusion
- genetic
- gov't
- mutagenesis,
- mutation
- non-u.s.
- phenotype
- protein
- proteins
- proteins/*biosynthesis/*genetics
- proteins/biosynthesis
- pseudomonas
- recombinant
- replication
- research
- site-directed
- species
- specificity
- substitution
- support,
- temperature
