Table 1.
Primers used for rpoN mutant construction and complementation, in 5’ to 3’ orientation.
Restriction sites are in bold.
Fig 1.
Colonies of B. cereus ATCC 14579 WT (a), its ΔrpoN mutant derivative (b) and the complemented mutant (ΔrpoN-comp) (c) cultivated on BHI plates for 24 h at 30°C.
Fig 2.
Cells of the WT and its ΔrpoN mutant.
SEM images of aerobically grown overnight cultures of B. cereus ATCC 14579 WT (a) and its ΔrpoN mutant derivative (b) in BHI at 30°C.
Fig 3.
Growth of B. cereus ATCC 14579 WT, ΔrpoN and ΔrpoN-comp in BHI in three conditions with different oxygen availability a) aerated, b) static and c) anaerobic at 30°C.
Fig 4.
Number of spores and total viable counts in BHI formed by B. cereus ATCC 14579 WT, ΔrpoN and ΔrpoN-comp in BHI following aerated growth at 30°C for 48 h. The asterisk indicates significant difference (student’s t test, p<0.05) of the ΔrpoN compared to both the WT and ΔrpoN-comp.
Fig 5.
Growth of B. cereus ATCC 14579 WT, ΔrpoN and ΔrpoN-comp at 12°C. a) Aerated growth in BHI monitored up to 96 h, b) growth on BHI agar plate, picture was taken on day 12.
Fig 6.
Motility and biofilm formation.
Motility, presence of flagella and biofilm formation by B. cereus ATCC 14579 WT, ΔrpoN and ΔrpoN-comp. (a-c) Motility on swimming BHI plates with 0.3% agar after 48 h, (d-f) flagella staining of cells taken from the swimming plate, (g-i) Static biofilm formation on stainless steel coupons partly submerged in BHI after 48 h at 30°C. Biofilms were stained with crystal violet.
Fig 7.
Production of non-hemolytic toxin (NheA) measured in BHI at 30°C with aeration at mid-exponential (OD 600 nm = 0.2) and end-exponential (OD 600 nm = 1) growth phases, and at mid-exponential (OD 600 nm = 0.2) phase during static growth.
Fig 8.
Overrepresented metabolic pathways significantly down regulated in ΔrpoN. Outcome of FIVA analysis of significantly affected genes. Categories with asterisk (*) were restored to close to WT condition by complementation.
Fig 9.
Affected processes in the cell.
Schematic model of cellular processes, based on observed phenotypes combined with affected and complemented genes in the rpoN mutant. a) Aerated growth mid-exponential phase, b) aerated growth end-exponential phase, c) static growth mid-exponential phase. The numbers in (brackets) are the number of genes affected in the given category. Asterisk (*) means the gene was not complemented.