Osmotic stress-induced genetic rearrangements in Escherichia coli H10407 detected by randomly amplified polymorphic DNA analysis

Abstract

Randomly amplified polymorphic DNA (RAPD) analysis is a DNA polymorphism assay commonly used for fingerprinting genomes. After optimizing the reaction conditions, samples of Escherichia coli H10407 DNA were assayed to determine the influence of osmotic and/or oligotrophic stress on variations in RAPD banding patterns. Genetic rearrangements or DNA topology variations could be detected as changes in agarose gel electrophoresis banding profiles. A new amplicon generated using DNA extracted from bacteria prestarved by an osmotic stress and resuscitated in rich medium was observed. Enrichment improved recovery of mutator cells and allowed them to be detected in samples, suggesting that DNA modifications, such as stress-induced alterations and supercoiling phenomena, should be taken into consideration before beginning RAPD analyses.

FIG. 1

Survival curves of exponential- and stationary-phase cells of E. coli H10407, starved in ASW. AODC for stationary or exponential phase (▴), DVC for stationary or exponential phase (♦), and plate counts on Trypticase soy agar for stationary phase (▪) and exponential phase (●) are shown.

FIG. 2

Electrophoretic agarose gel analysis of RAPD products from total DNA extracted from E. coli H10407. The DNA was extracted from bacteria in different physiological states: exponential phase (A) and early stationary phase (B). Lanes 1, MVII DNA ladder; lanes 2, cells grown in BHI broth; lanes 3 and 5, bacteria stressed in ASW for 1 and 18 h, respectively; lanes 4 and 6, bacteria stressed in DW for 1 and 18 h, respectively; lanes 7 and 8, Bacteria stressed in ASW for 18 h then resuscitated and grown to the exponential and stationary phases respectively; lanes 9, MVIII DNA ladder.