Peptide Deformylase (def) is essential in Mycobacterium smegmatis, but the essentiality is compensated by inactivation of methionine formylation

Abstract

Background: Co-translational processes in bacteria are attractive drug targets, but while some processes are essential, others are not. The essentiality of Peptide Deformylase (PDF, def) for vitality of mycobacteria was speculated, but never unequivocally proven.

Results: Here we show by targeted deletion experiments that def can only be deleted from M. smegmatis when an additional copy is present; that prior deletion of tRNA^fMet-Formyl Transferase (FMT, encoded by fmt) renders def completely dispensable; and that re-introduction of fmt into a Δdef mutant is not possible - constituting a definitive proof for the essentiality of def in mycobacteria.

Conclusions: Peptide deformylase is essential in M. smegmatis, but the fact that inactivation of fmt renders the gene completely dispensable, and thus any inhibitor of def useless, casts doubt on the usefulness of PDF as a drug-target in mycobacteria.

Keywords: Co-translation; Formyl Transferase; Mycobacteria; Peptide Deformylase; Protein synthesis.

Fig. 1

def cannot be deleted from wt M. smegmatis. On the right, colonies after a confirmed second recombination event were checked by PCR to differentiate def-deletion (resulting in 1.2 kb product) from reversal to wt (1.8 kb fragment). 13 colonies are shown, out of 60 examined. On the left – wt control, and a true Δdef mutant (obtained as described in section 3.2)

Fig. 2

With pre-complementation with an additional copy of def, a deletion of the native gene is easily obtainable. Right – 12 colonies after second recombination. 4 of them have the PCR product obtained from a Δdef genotype (1200 Bp). Left – wt control (1800 bp)

Fig. 3

On a Δfmt background, deletion of def is highly efficient. Left: 10 candidate colonies. Right: wt control. The leftmost colony was later re-named mDB224

Fig. 4

A functional fmt is incompatible with a Δfmt/Δdef genotype. mDB224 (Δfmt/Δdef) was electroporated with a control vector (left), control+fmt^tuberculosis (middle), and control+fmt^smegmatis (right). The picture was taken after 4 days incubation, but plates were kept for 10 days before discarding, not to miss slow-growing mutant