Low concentrations of bile salts induce stress responses and reduce motility in Bacillus cereus ATCC 14579 [corrected]

Abstract

Tolerance to bile salts was investigated in forty Bacillus cereus strains, including 17 environmental isolates, 11 dairy isolates, 3 isolates from food poisoning outbreaks, and 9 other clinical isolates. Growth of all strains was observed at low bile salt concentrations, but no growth was observed on LB agar plates containing more than 0.005% bile salts. Preincubation of the B. cereus type strain, ATCC 14579, in low levels of bile salts did not increase tolerance levels. B. cereus ATCC 14579 was grown to mid-exponential growth phase and shifted to medium containing bile salts (0.005%). Global expression patterns were determined by hybridization of total cDNA to a 70-mer oligonucleotide microarray. A general stress response and a specific response to bile salts were observed. The general response was similar to that observed in cultures grown in the absence of bile salts but at a higher (twofold) cell density. Up-regulation of several putative multidrug exporters and transcriptional regulators and down-regulation of most motility genes were observed as part of the specific response. Motility experiments in soft agar showed that motility decreased following bile salts exposure, in accordance with the transcriptional data. Genes encoding putative virulence factors were either unaffected or down-regulated.

FIG. 1.

Growth and pH curves of B. cereus ATCC 14579 in the presence or absence of bile salts. After 3 h of growth the culture was shifted to fresh medium with or without bile salts added. Triangles represent the start culture, circles represent the control culture, and squares represent the bile salt culture (0.005%). The diamonds and dotted line represent the cell density in a 0.01% bile salt culture. Solid lines indicate the cell density, and the dashed lines represent the pH. The error bars indicate the standard deviation of at least three separate experiments. (A) Growth from start to stationary phase. (B) Magnification of the time period in which samples were taken for RNA isolation.

FIG. 2.

Number of regulated genes, 15 min after the shift to bile salt medium, belonging to each COG class. The red bars represent the number of up-regulated genes, and the green bars represent the number of down-regulated genes.

FIG. 3.

Expression profiles of selected genes relative to the cell density at the time of harvest. The y axis displays the regulation given as the log₂ ratio, and the x axis displays the cell density of the culture at the time of harvest. The expression of each gene is given as the log₂ ratio compared to the reference RNA (C0), which is set to 0 by default. The solid lines and filled symbols represent the bile salt cultures, and the dashed lines and open symbols represent the control culture. (A) Expression trends of mRNA for drug resistance transporters and marR family regulator relative to the cell density at the time of harvesting. •, BC4000 emrB/qacA family drug resistance transporter; ▪, BC4568 emrB/qacA family drug resistance transporter; and ⧫, BC0657, marR family regulator. No signals were obtained for BC4568 in control cultures at 15 and 30 min, and this line is therefore represented by dots. (B) Expression trends of stress genes relative to the cell density at the time of harvesting. •, BC4272 superoxide dismutase; ▪, BC4859 cspD; ▴, BC0294 groES; and ⧫, BC5251 clpP. (C) Expression trends of virulence genes relative to the cell density at the time of harvest. •, BC3103 HBL; ▪, BC5101 perfingolysin O; ▴, BC1331 internalin; and ⧫, BC5350 plcR. (D) Expression trends of genes involved in metabolism of sugars relative to the cell density at the time of harvest. •, BC0668 butanediol dhg; ▪, BC4599 pyruvate kinase; ⧫, BC1376 flavodoxin; ▴, BC5320 PTS, glucose specific.

FIG. 4.

Regulation of a 45-kb region of the B. cereus ATCC 14579 genome containing motility-associated genes in bile salt culture (red and green bars; BS15) and control culture (open bars; C15) 15 min after the shift compared to the reference isolated immediately after the shift (C0). The bars represent the log₂ up-regulation (red) or down-regulation (green) of the displayed genes. Some of the genes listed may have a P value higher than 0.05. These are included because they belong to an operon which has significantly regulated genes or because they are only slightly regulated. UDP-N-Ace, UDP-N-acetylenolpyruvoylglucosamine reductase; Hook 1, -2, -3, flagellar hook-associated protein 1, -2, and -3; ATP synth, flagellum-specific ATP synthase; −, located on the negative strand. Other genes are designated by their Refseq gene names (NC_004722).

FIG. 5.

(A) Aerobic motility test. B. cereus ATCC 14579 was incubated aerobically for 24 h in LB-soft agar with or without bile salts added. Tubes show no inoculate (left), culture without bile salts (middle), and culture with bile salts (right). No changes in motility were observed after 24 h. (B) Anaerobic motility test. B. cereus ATCC 14579 was incubated anaerobically for 24 h in LB-soft agar with or without bile salts added. Tubes show no inoculate (left), culture without bile salts (middle), and culture with bile salts (right). No changes in motility were observed after 24 h.