The CasKR two-component system is required for the growth of mesophilic and psychrotolerant Bacillus cereus strains at low temperatures

Abstract

The different strains of Bacillus cereus can grow at temperatures covering a very diverse range. Some B. cereus strains can grow in chilled food and consequently cause food poisoning. We have identified a new sensor/regulator mechanism involved in low-temperature B. cereus growth. Construction of a mutant of this two-component system enabled us to show that this system, called CasKR, is required for growth at the minimal temperature (Tmin). CasKR was also involved in optimal cold growth above Tmin and in cell survival below Tmin. Microscopic observation showed that CasKR plays a key role in cell shape during cold growth. Introducing the casKR genes in a ΔcasKR mutant restored its ability to grow at Tmin. Although it was first identified in the ATCC 14579 model strain, this mechanism has been conserved in most strains of the B. cereus group. We show that the role of CasKR in cold growth is similar in other B. cereus sensu lato strains with different growth temperature ranges, including psychrotolerant strains.

FIG 1

Growth curves of B. cereus wt and ΔBC_2216-17 strains at various temperatures. Growth of the wt (black) and ΔBC_2216-17 (gray) strains was performed in 100 ml LB at the indicated temperature under shaking. Three biological replicates are shown for each condition (different symbols represent different replicates). During the lag phase at 10°C, cells sometimes formed temporary small aggregates which caused a decrease in the measured OD. y axes indicate the ODs at 600 nm; x axes indicate times (in hours).

FIG 2

Microscopic observations of B. cereus wt and ΔBC_2216-17 strains grown at 12 and 37°C until the stationary phase. Microscopic observations were made under a phase-contrast microscope at a magnification of ×1,000 (A), and under a TEM at magnifications of ×7,000 and ×14,000 (B).

FIG 3

Survival of B. cereus wt and ΔcasKR strains at 4°C. B. cereus wt (black symbols) and ΔcasKR (gray symbols) strains in bacterial suspensions were incubated in LB at 4°C. CFU enumeration was performed every day. Different symbols represent data from 3 different replicates. The curves represent theoretical data calculated as described in Materials and Methods.

FIG 4

Complementation of ΔcasKR restores the ability to grow at low temperature. Five microliters of serial dilutions of the B. cereus wt, ΔcasKR, and ΔcasKR/pHT-casKR strains (grown in liquid medium at 30°C and adjusted to 10⁸ CFU/ml) was spotted on LB agar, and the strains were incubated at 10°C for 20 days.

FIG 5

Growth at various temperatures of wt and ΔcasKR strains from phylogenetic groups II, III, and V of B. cereus sensu lato. Growth of wt and ΔcasKR strains belonging to phylogenetic group II (strain MM3) (A, D, F), group III (strain AH187) (B, G), and group V (strain Rock 3-28) (C, E, H) was performed in an automated turbidimeter with shaking at 37°C (A to C), at a low temperature of 13°C (D) or 10°C (E), or at a temperature close to the T_min of the strains of 8°C (F, H) or 12°C (G). y axes indicate the OD at 600 nm; x axes indicate time (in hours). Mean values ± SDs of three biological replicates are shown for each condition. Black curves, wt strains; gray curves, ΔcasKR mutants.