Transcription-induced barriers to supercoil diffusion in the Salmonella typhimurium chromosome

Abstract

Transcription and replication both influence and are influenced by superhelical changes in DNA. Explaining how supercoil movement is channeled in living chromosomes has been a major problem for 30 years. Transcription of membrane-associated proteins leads to localized hypersupercoiling of plasmid DNA, and this behavior indicates the presence of aberrant supercoil diffusion. Using the lambda Red recombination system, we constructed model domains in the Salmonella typhimurium chromosome to analyze supercoiling dynamics of regions encoding membrane proteins. Regulation of Tn10-derived tetracycline resistance involves a repressor, TetR, and a membrane-bound export pump, TetA. Strains deficient in TetR activity had 60-fold higher transcription levels (from P(A)) than TetR-positive strains. High tetA transcription caused a 10- to 80-fold decrease in the gammadelta resolution efficiency for the domain that includes the Tet module. Replacing tetA with genes encoding cytosolic proteins LacZ and Kan also caused the appearance of supercoil diffusion barriers in a defined region of the chromosome. In strains containing a functional TetR located next to a regulated lacZ reporter (P(R)tetR-P(A)lacZ), induction of transcription with chlortetracycline caused a 5-fold drop in resolution efficiency in the test domain interval. A short half-life resolvase showed that barriers appeared and disappeared over a 10- to 20-min span. These studies demonstrate the importance of transcription in chromosome structure and the plasticity of supercoil domains in bacterial chromosomes.

Fig. 1.

Effect of WT tetR, a mutant tetR, and a tetR deletion on γδ resolution efficiency. (A) Physical and genetic map of a 12-kb interval between zbj8904 and yliI in the 17- to 20-min region of S. typhimurium includes eight ORFs flanked by drug markers and res sites. Two arrangements of selectable drug markers involve kanamycin and tetracycline resistance (Upper) and gentamicin and kanamycin resistance (Lower). (B) Resolution efficiency comparison among different module insertions in the gene yliI in the 12-kb interval in the log phase. Resolution efficiency for a 12-kb interval in the 43- to 45-min region (6) is designated as the expected resolution efficiency.

Fig. 2.

γδ resolution in the 43- to 45-min of S. typhimurium chromosome. (A) Physical map of two intervals. The 14-kb interval from cobP712 to cobT714 is labeled a, and the 28-kb interval from cob-708 and cobT714 is labeled b. (B) Genetic map of res elements placed at the cobT714 location: MudJr2, MudJr2(kan)<>(P_AtetA), and MudJr2(kan)<>(P_AtetA-inv). (C) Resolution efficiencies for different 14-kb (a) and 28-kb (b) intervals.

Fig. 3.

Effect of genes encoding cytoplasmic proteins on the γδ resolution efficiency in the 12-kb interval between zbj8904 and yliI. (A) Genetic map of different protein coding sequences (tetA, lacZ, and kan) fused to tetA promoter P_A. (B) Resolution efficiency comparison among different insertions in the gene yliI in the 12-kb interval in the log phase.

Fig. 4.

Effect of chlortetracycline concentration on β-gal activity and γδ resolution efficiency. NH3492 was subcultured in 0, 0.1, 0.5, 1, 5, 10, and 20 μg/ml chlortetracycline and was grown to a cell density of 50 Klett units. Both β-gal assays (•) and γδ assays (○) were performed on each sample.

Fig. 5.

Barrier appearance and disappearance in response to chlortetracycline. □, overnight cultures of NH3492 subcultured in LB without chlortetracycline. γδ resolution assays were done when cell density reached 50 Klett units. ▪, 5 μg/ml chlortetracycline added at a cell density of 50 Klett units. Samples were taken at various points after addition of chlortetracycline and subjected to γδ resolution assay. ○, NH3492 grown in LB with 5 μg/ml chlortetracycline. γδ resolution assays were initiated when cell density reached 50 Klett units. •, chlortetracycline removed by quickly washing and then resuspending cells in LB. Samples taken at various times were tested for γδ resolution.