FEDS: a Novel Fluorescence-Based High-Throughput Method for Measuring DNA Supercoiling In Vivo

Abstract

DNA supercoiling (DS) is essential for life because it controls critical processes, including transcription, replication, and recombination. Current methods to measure DNA supercoiling in vivo are laborious and unable to examine single cells. Here, we report a method for high-throughput measurement of bacterial DNA supercoiling in vivoFluorescent evaluation of DNA supercoiling (FEDS) utilizes a plasmid harboring the gene for a green fluorescent protein transcribed by a discovered promoter that responds exclusively to DNA supercoiling and the gene for a red fluorescent protein transcribed by a constitutive promoter as the internal standard. Using FEDS, we uncovered single-cell heterogeneity in DNA supercoiling and established that, surprisingly, population-level decreases in DNA supercoiling result from a low-mean/high-variance DNA supercoiling subpopulation rather than from a homogeneous shift in supercoiling of the whole population. In addition, we identified a regulatory loop in which a gene that decreases DNA supercoiling is transcriptionally repressed when DNA supercoiling increases.IMPORTANCE DNA represents the chemical support of genetic information in all forms of life. In addition to its linear sequence of nucleotides, it bears critical information in its structure. This information, called DNA supercoiling, is central to all fundamental DNA processes, such as transcription and replication, and defines cellular physiology. Unlike reading of a nucleotide sequence, DNA supercoiling determinations have been laborious. We have now developed a method for rapid measurement of DNA supercoiling and established its utility by identifying a novel regulator of DNA supercoiling in the bacterium Salmonella enterica as well as behaviors that could not have been discovered with current methods.

Keywords: DNA gyrase; DNA topology; feedback loop; gene transcription.

FIG 1

Map of pSupR, a supercoiling reporter plasmid for enterobacteria. It includes one constitutive promoter (red, left side) and one promoter exclusively repressed by DNA supercoiling (green, right side), each transcribing either of two genes coding for different fluorescent proteins, the origin of replication from plasmid pMB1 and the bla gene conferring resistance to ampicillin.

FIG 2

A scoring system to identify promoters responding exclusively to DNA supercoiling. (A) Genes in the S. enterica serovar Typhimurium 14028s genome were ranked according to their expression properties (as measured by RNA-seq). The amplitude score (maximum of 3 points) and fitting score (maximum of 1 point) rewarded genes that vary strongly and predictably with DNA supercoiling, respectively, and the mean expression score (maximum of 1 point) penalized genes whose expression is weak. The overall score (i.e., the sum of the three individual scores) was used to rank genes that were desirable as supercoiling reporters. (B) Distribution of scores depending on the regression type used. A cutoff value of 2.9 was chosen empirically.

FIG 3

Expression of the ydeJ gene is inversely correlated with DNA supercoiling. S. enterica serovar Typhimurium wild-type strain 14028s or isogenic mutants bearing plasmid pSupR or pJV were grown in a variety of media. Strains bearing pJV were used to measure DNA supercoiling by the agarose/chloroquine gel method. Strains bearing pSupR were used to measure the fluorescence ratios. Blue data points indicate conditions corresponding to the 11 conditions used in the RNA-seq experiment; red data points indicate conditions that alter DNA supercoiling but that were not used in the RNA-seq experiment. A full description of the study conditions is available in Table S1B.

FIG 4

pSupR responds to DNA supercoiling in vitro. In vitro transcription was performed using supercoiled pSupR, relaxed pSupR (treated with topoisomerase I), or linear pSupR (linearized by restriction digestion). Transcripts were quantified by qPCR, and the ratio of gfp transcripts to tdtomato transcripts is presented. *, P < 0.05 (Student's t test, n = 3).

FIG 5

RdsA inhibits DNA supercoiling. (A) DNA supercoiling of wild-type (14028s) and rdsA (AAD219) Salmonella was measured in 96-well plates using FEDS. (B) DNA supercoiling of the strains described for panel A was measured using the classical agarose/chloroquine gel method. (C) The higher level of DNA supercoiling of the mutant observed with both methods indicates that RdsA is involved in a double-negative-feedback loop with DNA supercoiling. ***, P < 0.001 (Student's t test).

FIG 6

FEDS recapitulates known DNA supercoiling behaviors. (A) DNA supercoiling of wild-type E. coli strain MG1655/pSupR grown in LB in 96-well plates. DNA supercoiling was measured every 12 min using the green/red fluorescence ratio. Data are represented as means (solid lines) ± standard deviations (SD) (dashed lines) of results from 3 replicates. Data representing the average growth rate (dotted gray line) and OD₆₀₀ values (“Growth”; indicated in arbitrary units [AU]) are also plotted. Raw green and red fluorescence data are presented in Fig. S3. (B) DNA supercoiling of E. coli MG1655/pSupR grown in HH800 minimal medium, with NaCl or H₂O₂ (where indicated), or in LB broth in 96-well plates as indicated. Conditions were compared when the OD₆₀₀ reached 30% of the maximum OD₆₀₀. Data are represented as means (solid bars) ± SD (error bars) of results from 6 replicates. ns, not significant; ***, P < 0.001 (Tukey’s HSD). (C) DNA supercoiling of wild-type Salmonella (14028s)/pSupR grown in HH800 minimal medium, with NaCl or H₂O₂ (where indicated), or in LB broth in 96-well plates as indicated. Conditions were compared when the OD₆₀₀ reached 30% of the maximum OD₆₀₀. Data are represented as means (solid bars) ± SD (error bars) of results from 6 replicates. **, P < 0.01; ***, P < 0.001 (Tukey’s HSD).

FIG 7

Growth-phase-regulated changes in subpopulations exhibiting different DNA supercoiling behaviors. (A) Density profiles of the wild-type S. Typhimurium (14028s)/pSupR strain in LB at different time points. DNA supercoiling was evaluated using the green/red fluorescence ratio produced by the organisms harboring plasmid pSupR. (B) Overlay of density profiles in late exponential phase in HH800 (wild-type strain and isogenic speE oat mutant strain AAD58) or HH plus H₂O₂ (wild-type strain only). (C) Correlation between population mean and standard deviation across all time points and conditions (14028s/pSupR in LB, HH800, HH, or HH plus H₂O₂; isogenic speE oat mutant in HH800). Flow cytometry profiles of the populations were recorded every 90 min for 7.5 h.