Differential involvement of the five RNA helicases in adaptation of Bacillus cereus ATCC 14579 to low growth temperatures

Abstract

Bacillus cereus ATCC 14579 possesses five RNA helicase-encoding genes overexpressed under cold growth conditions. Out of the five corresponding mutants, only the ΔcshA, ΔcshB, and ΔcshC strains were cold sensitive. Growth of the ΔcshA strain was also reduced at 30°C but not at 37°C. The cold phenotype was restored with the cshA gene for the ΔcshA strain and partially for the ΔcshB strain but not for the ΔcshC strain, suggesting different functions at low temperature.

FIG. 1.

Growth at 30°C (A and B) and 10°C (C and D) of B. cereus ATCC 14579 (▪) and the ΔcshA (□), ΔcshB (▵), ΔcshC (×), ΔcshD (○), and ΔcshE (⋄) mutants in LB broth. Changes in OD₆₀₀ (A and C) and numeration of bacterial cells in log CFU ml⁻¹ (B and D) are presented. Independent cultures were performed in triplicate; error bars indicate standard deviations.

FIG. 2.

Morphology of cells grown at low temperatures of B. cereus WT (A and E) and the ΔcshA (B and F), ΔcshB (C and G), and ΔcshC (D and H) strains. Cells grown at 12°C (WT, ΔcshB, and ΔcshC) or 15°C (ΔcshA) and harvested at an OD₆₀₀ of 1.0 were observed under phase-contrast microscopy at a magnification of ×1,000 (left-hand panel) or after staining with a LIVE/DEAD bacterial viability test (Invitrogen) (right-hand panel). Red and green cells were counted in 10 random microscope fields.

FIG. 3.

Transmission electron micrographs of B. cereus WT (A), ΔcshA (B), ΔcshB (C), and ΔcshC (D) cells grown at low temperatures (×18,000 magnification). Cells were grown until late exponential phase and prepared as described previously (26). Bars indicate 500 nm. Cells in the presented pictures were representative of the majority of the bacteria observed.

FIG. 4.

Complementation assays with the cshA gene at 12°C of B. cereus ΔcshA strain. Horizontal lines represent the promoter region of cshA (solid lines) or the Pxyl promoter (dashed line); boxes are putative cold-boxes. Colonies on LB agar plates were grown for 7 days at 12°C from 10-fold serially diluted bacterial suspensions.