Differential Gene Expression for Investigation of Escherichia coli Biofilm Inhibition by Plant Extract Ursolic Acid

Abstract

After 13,000 samples of compounds purified from plants were screened, a new biofilm inhibitor, ursolic acid, has been discovered and identified. Using both 96-well microtiter plates and a continuous flow chamber with COMSTAT analysis, 10 g of ursolic acid/ml inhibited Escherichia coli biofilm formation 6-to 20-fold when added upon inoculation and when added to a 24-h biofilm; however, ursolic acid was not toxic to E. coli, Pseudomonas aeruginosa, Vibrio harveyi, and hepatocytes. Similarly, 10 g of ursolic acid/ml inhibited biofilm formation by >87% for P. aeruginosa in both complex and minimal medium and by 57% for V. harveyi in minimal medium. To investigate the mechanism of this nontoxic inhibition on a global genetic basis, DNA microarrays were used to study the gene expression profiles of E. coli K-12 grown with or without ursolic acid. Ursolic acid at 10 and 30 g/ml induced significantly (P < 0.05) 32 and 61 genes, respectively, and 19 genes were consistently induced. The consistently induced genes have functions for chemotaxis and mobility (cheA, tap, tar, and motAB), heat shock response (hslSTV and mopAB), and unknown functions (such as b1566 and yrfHI). There were 31 and 17 genes repressed by 10 and 30 g of ursolic acid/ml, respectively, and 12 genes were consistently repressed that have functions in cysteine synthesis (cysK) and sulfur metabolism (cysD), as well as unknown functions (such as hdeAB and yhaDFG). Ursolic acid inhibited biofilms without interfering with quorum sensing, as shown with the V. harveyi AI-1 and AI-2 reporter systems. As predicted by the differential gene expression, deleting motAB counteracts ursolic acid inhibition (the paralyzed cells no longer become too motile). Based on the differential gene expression, it was also discovered that sulfur metabolism (through cysB) affects biofilm formation (in the absence of ursolic acid).

Exploring the antimicrobial action of a carbon monoxide-releasing compound through whole-genome transcription profiling of Escherichia coli

“…Biofilm formation was quantified by the crystal violet method (Ren et al, 2005;Zhang et al, 2007). Briefly, overnight LB-grown cultures of wild-type E. coli, DtqsA and DbhsA mutant strains were used to inoculate fresh medium to an OD 600 of 0.05 and then incubated for 6 h, at 37 uC.…”

Antibacterial activity of oleanolic and ursolic acids and their derivatives

“…In a landmark study, Ren and coworkers demonstrated that UA caused differential gene expression in E. coli and inhibited biofilm formation in several bacterial species (Table 2) [85]. In these studies UA was applied at sublethal concentrations, up to 30 mg mL -1 , which did not inhibit the growth of any species tested.…”

Escherichia coli transcription factor YncC (McbR) regulates colanic acid and biofilm formation by repressing expression of periplasmic protein YbiM (McbA)

“…Crystal-violet biofilm assay A static biofilm formation assay was performed in 96-well polystyrene plates as reported previously (Ren et al, 2005). Briefly, cells were inoculated with an initial turbidity at 600 nm of 0.05 in LB or LB glu at 37 1C or 30 1C for 24 h without shaking, then cell density (turbidity at 620 nm) and total biofilm (absorbance at 540 nm) were measured using crystal violet staining.…”