mraY is an essential gene for cell growth in Escherichia coli

Abstract

The synthesis of the murein precursor lipid I is performed by MraY. We have shown that mraY is an essential gene for cell growth. Cells depleted of MraY first swell and then lyse. The expression of mraY DNA in vitro produces a 40-kDa polypeptide detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

FIG. 1

Organization of the mra region and the plasmids constructed for the purpose of this study. The upper part shows the ORFs in the mra region to scale in kilobase pairs. The boxed letters refer to the genes within this region: w, mraW; z, mraZ; L, ftsL; I, ftsI; E, murE; F, murF; Y, mraY; D, murD; W, ftsW; G, murG; C, murC; B, ddlB; Q, ftsQ; A, ftsA; Z, ftsZ; and eA, envA. Also shown are the cloned fragments with direction of expression as indicated by arrowheads. The types of promoter used to express these ORFs are listed at the left. P, PvuII; K, KpnI; Ev, EcoRV; N∗, NdeI; X, XmnI; and B, BglII. The NdeI site was introduced by PCR-directed mutagenesis.

FIG. 2

Southern blot analysis of the genomic DNA from the partial diploid strains DSB1 (mraY wild type, lane 2) and DBYC1 (mraY::cat mutant, lane 3). Lanes 1 and 4 contain control DNAs mraY::cat (pDYC1) and mraY wild type (pDEG1), respectively. All of the DNAs were restricted with EcoRV, electrophoresed through agarose, and Southern blotted onto a nylon membrane. The blot was first probed with mraY (A) to show mraY::cat at 3.5 kb and mraY at 2.7 kb (lanes 1 and 4, respectively). Lane 2 shows strain DSB1 to have only wild-type mraY at 2.7 kb. Lane 3 shows both the wild-type and disrupted forms of mraY from DBYC1 DNA. Probing the same blot with cat (B) shows that only pDYC1 (lane 1) and DBYC1 (lane 3) contain the mraY::cat fragment at 3.5 kb.

FIG. 3

Induction and repression of P_BAD::mraY in the mraY-null mutant strain DBYC3/pBAY1 cultured in LB plus kanamycin supplemented with either 0.2% (vol/vol) arabinose (□) or 0.2% (vol/vol) glucose (◊) at 37°C. O.D., optical density.

FIG. 4

Micrographs of DBYC3/pBAY1 grown in LB supplemented with either arabinose or glucose. (A and C) Cells from the arabinose-containing culture sampled at 0 and 180, respectively. (B and D) Cells from the glucose-containing culture sampled at 0 and 180 min, respectively. Bar, 5 μm.

FIG. 5

SDS–10% PAGE analysis of the in vitro translation products (radiolabelled) produced by pJFY3c (lane 1) and pJF118EH (lane 2) after induction with 0.5 M isopropyl-β-d-thiogalactopyranoside. In lane 1 a unique polypeptide migrates as a 40-kDa peptide. This band is absent from the pJF118EH sample and is therefore presumed to be MraY.