Disruption of the gene for Met-tRNA(fMet) formyltransferase severely impairs growth of Escherichia coli

Abstract

In bacteria, as well as in chloroplasts and mitochondria, the free amino group of the methionylated initiator tRNA(fMet) is specifically modified by the addition of a formyl group. The importance of this modification remains unclear. With the availability of pure Escherichia coli 10-formyltetrahydrofolate:L-methionyl-tRNA(fMet) N-formyltransferase, the enzyme catalyzing Met-tRNA(fMet) formylation, the corresponding fmt gene and its flanking regions were cloned and sequenced. The chromosomal fmt gene was disrupted, and strains modified in their formylation activity were constructed. A depletion of the cellular formylation activity was accompanied by a decrease in the growth rate of the bacteria. At 37 degrees C, in a rich medium, the absence of a functional fmt gene reduced the growth rate to 0.28 doubling per h, from 2.3 for the control strain. At 42 degrees C, the studied fmt mutant strain did not grow further.

Reducing the fitness cost of antibiotic resistance by amplification of initiator tRNA genes

“…1). The results for the fmt mutants are in agreement with previous observations in E. coli, showing that formylation of Met-tRNAi is required for efficient translation initiation and rapid growth (9). For three strains, fitness was also determined by performing competition experiments in mice.…”

“…Similarly, when the metZW plasmid was introduced into other actinonin-resistant fmt mutants (DA8326 and DA8340), the generation times were reduced from 78 to 27 and from 67 to 24 min, respectively. This result shows that an increased level of tRNAi is sufficient to confer suppression of the fmt growth defect, similar to what was previously reported for E. coli fmt mutants (9,38).…”

“…Most resistance mutations substantially reduced bacterial fitness during growth in vitro and in mice. For the fmt and folD mutants, the fitness reduction likely results from the decreased rate of formylation and translation initiation (9,38). Functional compensation of the defective formylation and slow translation rate could occur via two different mechanisms: by restoring the capacity to formylate Met-tRNAi or by bypassing the need for formylated Met-tRNAi to initiate translation.…”

“…When FMT activity is absent, protein synthesis has to be initiated with unformylated Met-tRNAi. As a consequence of this abnormal initiation, both translation and growth rates are reduced (9). Such reductions in fitness (reduced growth rate and͞or virulence) are typical for most types of resistance mechanisms and bacterial species (10)(11)(12)(13)(14).…”

Unconventional initiator tRNAs sustain Escherichia coli

“…Formylation does not take place in archaea and eukaryotes, whereas the 3G-C pairs are found across all life forms. Even within eubacteria, formylation is not essential, although its deficiency results in a growth compromise (16). In Pseudomonas, efficient initiation occurs with unformylated tRNA with only a moderate growth defect (17).…”