The linear double-stranded DNA of phage Bam35 enters lysogenic host cells, but the late phage functions are suppressed

Abstract

Bam35, a temperate double-stranded DNA bacteriophage with a 15-kb linear genome, infects gram-positive Bacillus thuringiensis cells. Bam35 morphology and genome organization resemble those of PRD1, a lytic phage infecting gram-negative bacteria. Bam35 and PRD1 have an outer protein coat surrounding a membrane that encloses the viral DNA. We used electrochemical methods to investigate physiological changes of the lysogenic and nonlysogenic hosts during Bam35 DNA entry and host cell lysis. During viral DNA entry, there was an early temporal decrease of membrane voltage associated with K+ efflux that took place when either lysogenic or nonlysogenic hosts were infected. Approximately 40 min postinfection, a second strong K+ efflux was registered that was proposed to be associated with the insertion of holin molecules into the plasma membrane. This phenomenon occurred only when nonlysogenic cells were infected. Lysogenic hosts rarely were observed entering the lytic cycle as demonstrated by thin-section electron microscopy.

FIG. 1.

Effects of Bam35 infection on TPP⁺ (A and C) and K⁺ (B and D) ion fluxes across the PM of different B. thuringiensis strains and cell lines during the first 10 min of infection. The experiments were carried out at 37°C in LB (pH 8.0) containing 3 μM TPP⁺ (in panels A and C). Approximately 4 × 10⁸ CFU/ml cells were infected with Bam35 using an MOI of 20 (A and B) or as indicated below (C and D). GD was added to a final concentration of 4 μg/ml. Experiments were done with B. thuringiensis strains HER1410 (black, infected; yellow, noninfected) and 4D22 (blue) and cell lines HER1410_L3 (red) and HER1410_R19 (green) (A and B). HER1410 cells were infected with Bam35 using MOIs of 16 (red), 4 (blue), and 2 (green) (C and D). The insert in panel A indicates the decrease in TPP⁺ concentration due to TPP⁺ binding and dilution by the added phage suspension.

FIG. 2.

Changes in B. thuringiensis HER1410 and HER1410_L3 cell energetics during Bam35 infection. The cells were grown in a large volume (400 ml) and infected at time zero (MOI, ∼10). Two 5-ml samples of cell suspension were withdrawn at each time point, and the amounts of intracellular K⁺ and accumulated TPP⁺ were measured (see Materials and Methods). Infected HER1410 cells, A; noninfected HER1410 cells, B; infected HER1410_L3 cells, C; noninfected HER1410_L3 cells, D. Symbols: K⁺, closed circles; TPP⁺, open triangles; and OD, open circles.

FIG. 3.

Thin-section electron micrographs of Bam35-infected (A, C, D, and E) or noninfected (B) B. thuringiensis cells. Lysogenic cell HER1410_L3 with surface-associated virus particles (A). Partially lysed noninfected HER1410_L3 cell; a few virus particles are visible in the cell interior (B). Progeny viruses observed in HER1410_L3 cell at 55 min postinfection (C). Resistant cell HER1410_R19 after Bam35 addition; no bound viruses are seen (D). Partially lysed HER1410 cell with progeny viruses 40 min postinfection (E). Black arrows mark DNA-containing virus particles, and white arrow points to an empty particle. Bar represents 200 nm in all panels.

FIG. 4.

Effect of metabolic inhibitors on infected or noninfected B. thuringiensis strains HER1410 (A), HER1410_R19 (B), and HER1410_L3 (C). Cells were grown to a cell density of ∼2 × 10⁸ CFU/ml (37°C) and infected at time zero using an MOI of ∼20. Noninfected cells: no metabolic inhibitors added (closed circles) or NaN₃ added (open circles). Bam35-infected cells: no metabolic inhibitors added (closed triangles), NaN₃ added (open triangles), KCN added (closed squares), arsenate added (open squares), or NaF added (closed diamonds). The arrows indicate the time point of addition of inhibitors.