P2 growth restriction on an rpoC mutant is suppressed by alleles of the Rz1 homolog lysC

Abstract

Escherichia coli strain 397c carries a temperature-sensitive mutation, rpoC397, that removes the last 50 amino acids of the RNA polymerase beta' subunit and is nonpermissive for plating of bacteriophage P2. P2 gor mutants productively infect 397c and define a new gene, lysC, encoded by a reading frame that extensively overlaps the P2 lysis accessory gene, lysB. The unusual location of lysC with respect to lysB is reminiscent of the Rz/Rz1 lysis gene pair of phage lambda. Indeed, coexpression of lysB and lysC complemented the growth defect of lambda Rz/Rz1 null mutants, indicating that the LysB/C pair is similar to Rz/Rz1 in both gene arrangement and function. Cells carrying the rpoC397 mutation exhibited an early onset of P2-induced lysis, which was suppressed by the gor mutation in lysC. We propose that changes in host gene expression resulting from the rpoC397 mutation result in changes in the composition of the bacterial cell wall, making the cell more susceptible to P2-mediated lysis and preventing accumulation of progeny phage sufficient for plaque formation.

FIG. 1.

The rpoC397 mutation interferes with bacteriophage P2 growth. A plasmid-encoded RNAP β′ subunit complements the P2 plating defect on a 397c host. Bacteriophage P2 was used to infect lawns of 397c or its rpoC⁺ parent, P90A5c, harboring plasmids expressing wild-type rpoC, rpoC carrying the 397c mutation or control vector plasmid. The results of overnight plating at 30°C are presented.

FIG. 2.

P2 plates on a Ts⁺ pseudorevertant of 397c. A spontaneous revertant of E. coli 397c able to form colonies at 42°C was isolated. (Top panel) Proteins from whole-cell lysates of rpoC⁺ strain P90A5c, the 397c mutant, and the 397c Ts⁺ revertant were resolved by electrophoresis on a 5% sodium dodecyl sulfate gel and visualized by Coomassie staining. The portion of the gel containing the β and β′ subunits is shown. (Bottom panel) Bacteriophage P2 was used to infect lawns of the indicated cells. The results of overnight growth at 30°C are presented.

FIG. 3.

P2 mutants that grow on 397c define a new gene, lysC. (A) Map of the bacteriophage P2 lysis region. The P2 genome coordinates of the EcoRV fragment used to rescue the trl and gor mutations are indicated, as are the relative locations of the genes in this region. The amino acid sequence of the lysC reading frame is shown below the map, and the amino acid sequence changes resulting from the trl and gor mutations are indicated below the sequence. The PP dipeptide characteristic of all Rz1 proteins is underlined. Also shown for comparison is the amino acid sequence of λ Rz1. Gray arrows indicate the putative signal peptidase II cleavage sites in the two proteins. (B) Overexpression of lysC harboring gor or trl mutations, but not wild-type lysC, is sufficient to overcome the plating defect of P2. 397c or P90A5c cells harboring plasmids expressing wild-type lysC, lysC carrying the gor or trl mutations, or control vector plasmid pCYB2 were infected with wild-type P2 (top) or a P2 mutant carrying an amber mutation in lysC (bottom). The results of overnight growth at 30°C are presented.

FIG. 4.

Co-overexpression of bacteriophage P2 lysB and lysC complements the lysis defect caused by bacteriophage λ Rz and/or Rz1 mutations. Logarithmically growing rpoC⁺ P90A5c cells lysogenic for λ Rz⁺/Rz1⁺ or the indicated Rz and/or Rz1 λ mutants were thermally induced for 15 min at 43°C (hatched area) and then transferred to 37°C. Cell lysis was monitored spectrophotometrically. The values presented are mean values from three independent experiments. Different panels represent lysis curves obtained with induced lysogens harboring plasmids expressing lysC (pCYB2lysC) (A), lysB (pCYB2lysB) (B), or coexpressing wild-type lysB and lysC (pCYB2lysB, lysC) (C).

FIG. 5.

lysC gor delays host lysis. Logarithmically growing RW4206 (rpoC⁺) (A) or RW4204 (rpoC397) (B) E. coli cells carrying the ogr expression plasmid pTG257 and either pUCF4 (vector control), pGC160 (lysC⁺), or pGC163 (lysC^gor) plasmids with P2 lysis genes under the control of the P2 pF promoter were induced with IPTG, and cell lysis was monitored spectrophotometrically. Curves representative of the results from six independent experiments are shown.