Chromosome and plasmid-borne PLacO3O1 promoters differ in sensitivity to critically low temperatures

Abstract

Temperature shifts trigger genome-wide changes in Escherichia coli’s gene expression. We studied if chromosome integration impacts on a gene’s sensitivity to these shifts, by comparing the single-RNA production kinetics of a PLacO3O1 promoter, when chromosomally-integrated and when single-copy plasmid-borne. At suboptimal temperatures their induction range, fold change, and response to decreasing temperatures are similar. At critically low temperatures, the chromosome-integrated promoter becomes weaker and noisier. Dissection of its initiation kinetics reveals longer lasting states preceding open complex formation, suggesting enhanced supercoiling buildup. Measurements with Gyrase and Topoisomerase I inhibitors suggest hindrance to escape supercoiling buildup at low temperatures. Consistently, similar phenomena occur in energy-depleted cells by DNP at 30 °C. Transient, critically-low temperatures have no long-term consequences, as raising temperature quickly restores transcription rates. We conclude that the chromosomally-integrated PLacO3O1 has higher sensitivity to low temperatures, due to longer-lasting super-coiled states. A lesser active, chromosome-integrated native lac is shown to be insensitive to Gyrase overexpression, even at critically low temperatures, indicating that the rate of escaping positive supercoiling buildup is temperature and transcription rate dependent. A genome-wide analysis supports this, since cold-shock genes exhibit atypical supercoiling-sensitivities. This phenomenon might partially explain the temperature-sensitivity of some transcriptional programs of E. coli.

Positive supercoiling buildup is a trigger of E. coli’s short-term response to cold shock

“…In agreement, a recent study [Oliveira et al, 2019] tracked RNA production at the molecular level by synthetic variants of the Lac promoter. It was shown that, at low temperatures, RNA production kinetics is weaker and noisier when the gene is chromosome integrated than when it is plasmid borne (in plasmids, supercoiling buildup should be much slower due to the annihilation of positive and negative supercoils [Liu et al, 1987]).…”

“…To our knowledge, temperature sensitive PSB is the first identified physical mechanism of how an E. coli gene can be CS responsive, and evidence suggests that it is present in nearly half of E. coli ’s CSR genes. This finding, first hypothesized in [Oliveira et al, 2019], opens an avenue for the engineering of future synthetic, temperature sensitive and temperature resistant gene regulatory circuits, whose functioning could be tuned by the adaptive regulation of Gyrase activity. Further, we expect that it will contribute to learning how the short- and long-term transcriptional programs of E. coli responsive to CS have evolved.…”

“…As such, they heterogeneously influence transcription at a genome-wide level. Further, evidence suggests that the efficiency of Gyrase and Topoisomerase is temperature sensitive [Drlica 1992; Wang et al, 1985; Oliveira et al, 2019].…”

“…This diversity of single-gene responses may be explained by the likely existence of multiple causes for their alterations in expression rates during CS. For example, studies using synthetic gene constructs suggest that temperature can affect the kinetics of rate-limiting steps in transcription initiation, such as the closed and open complex formations [Oliveira et al, 2016], and such effects can differ between promoters [Oliveira et al, 2019]. Other studies showed that temperature affects chromosomal DNA compaction [Goldstein et al, 1984, López-García et al, 2000], which is associated with supercoiling buildup [Stuger et al, 2002; Holmes et al, 2000].…”

Alteration of DNA supercoiling serves as a trigger of short-term cold shock repressed genes ofE. coli

“…This diversity of single-gene responses may be explained by the likely existence of multiple causes for their alterations in expression rates during cold shock. For example, studies using synthetic gene constructs suggest that temperature can affect the kinetics of rate-limiting steps in transcription initiation, such as the closed and open complex formations (4), and such effects can differ between promoters (16). Other studies showed that temperature affects chromosomal DNA compaction (17)(18)(19), which is associated with supercoiling buildup (19,20).…”