Phase-dependent dynamics of the lac promoter under nutrient stress.

To survive, a bacterial population must sense nutrient availability and adjust its growth phase accordingly. Few studies have quantitatively analyzed the single-cell behavior of stress and growth phase-related transcriptional changes in Escherichia coli. To investigate the dynamic changes in transcription during different growth phases and starvation, we analyzed the single-cell transcriptional dynamics of the E. coli lac promoter. Cells were grown under different starvation conditions, including glucose, magnesium, phosphate and thiamine limitations, and transcription dynamics was quantified using a single RNA detection method at different phases. Differences in gene expression over conditions and phases indicate that stochasticity in transcription dynamics is directly connected to cell phase and availability of nutrients. Except for glucose, the pattern of transcription dynamics under all starvation conditions appears to be similar. Transcriptional bursts were more prominent in lag and stationary phase cells starved for energy sources. Identical behavior was observed in exponential phase cells starved for phosphate and thiamine. Noise measurements under all nutrient exhaustion conditions indicate that intrinsic noise is higher than extrinsic noise. Our results, obtained in a relA1 mutational background, which led to suboptimal production of ppGpp, suggest that the single-cell transcriptional changes we observed were largely ppGpp-independent. Taken together, we propose that, under different starvation conditions, cells are able to decrease the trend in cell-to-cell variability in transcription as a common means of adaptation.

Transcription closed and open complex dynamics studies reveal balance between genetic determinants and co-factors.

In E. coli, promoter closed and open complexes are key steps in transcription initiation, where magnesium-dependent RNA polymerase catalyzes RNA synthesis. However, the exact mechanism of initiation remains to be fully elucidated. Here, using single mRNA detection and dual reporter studies, we show that increased intracellular magnesium concentration affects Plac initiation complex formation resulting in a highly dynamic process over the cell growth phases. Mg2+ regulates transcription transition, which modulates bimodality of mRNA distribution in the exponential phase. We reveal that Mg2+ regulates the size and frequency of the mRNA burst by changing the open complex duration. Moreover, increasing magnesium concentration leads to higher intrinsic and extrinsic noise in the exponential phase. RNAP-Mg2+ interaction simulation reveals critical movements creating a shorter contact distance between aspartic acid residues and Nucleotide Triphosphate residues and increasing electrostatic charges in the active site. Our findings provide unique biophysical insights into the balanced mechanism of genetic determinants and magnesium ion in transcription initiation regulation during cell growth.

Origins of transcriptional transition: balance between upstream and downstream regulatory gene sequences.

UNLABELLED: By measuring individual mRNA production at the single-cell level, we investigated the lac promoter's transcriptional transition during cell growth phases. In exponential phase, variation in transition rates generates two mixed phenotypes, low and high numbers of mRNAs, by modulating their burst frequency and sizes. Independent activation of the regulatory-gene sequence does not produce bimodal populations at the mRNA level, but bimodal populations are produced when the regulatory gene is activated coordinately with the upstream and downstream region promoter sequence (URS and DRS, respectively). Time-lapse microscopy of mRNAs for lac and a variant lac promoter confirm this observation. Activation of the URS/DRS elements of the promoter reveals a counterplay behavior during cell phases. The promoter transition rate coupled with cell phases determines the mRNA and transcriptional noise. We further show that bias in partitioning of RNA does not lead to phenotypic switching. Our results demonstrate that the balance between the URS and the DRS in transcriptional regulation determines population diversity. IMPORTANCE: By measuring individual mRNA production at the single-cell level, we investigated the lac promoter transcriptional transition during cell growth phases. In exponential phase, variation in transition rate generates two mixed phenotypes producing low and high numbers of mRNAs by modulating the burst frequency and size. Independent activation of the regulatory gene sequence does not produce bimodal populations at the mRNA level, while it does when activated together through the coordination of upstream/downstream promoter sequences (URS/DRS). Time-lapse microscopy of mRNAs for lac and a lac variant promoter confirm this observation. Activation of the URS/DRS elements of the promoter reveals a counterplay behavior during cell phases. The promoter transition rate coupled with cell phases determines the mRNA and transcriptional noise. We further show that bias in partitioning of RNA does not lead to phenotypic switching. Our results demonstrate that the balance between URS and DRS in transcription regulation is determining the population diversity.

Antimicrobial activity and molecular analysis of azoderivatives of ß-diketones.

The emergence and increase in the number of multidrug resistant microorganisms have highly increased the need of therapeutic trials, necessitating a deep exploration on novel antimicrobial response tactics. This study is intended to screen and analyze the activity of a novel set of azoderivatives of ß-diketones and their known analogs for antimicrobial properties. The compounds were analyzed to determine their minimum inhibitory concentration. Hit compounds 5-(2-(2-hydroxyphenyl)hydrazono)pyrimidine-2,4,6(1H,3H,5H)-trione (C5), 5-chloro-3-(2-(4,4-dimethyl-2,6-dioxocyclohexylidene)hydrazinyl)-2-hydroxybenzenesulfonic acid (C8), 2-(2-carboxyphenylhydrazo)malononitrile (C11) were then considered in evaluating their effect on transcription, translation and cellular oxidation impact. All three compounds were found to have in vitro inhibitory action on E.coli cell growth. The study also revealed that those compounds have a notable impact on cellular activities. It is determined that the newly synthesized azoderivative of barbituric acid (C8) have maximum growth inhibitory activity among the three compounds considered, characterized by a MIC50 of 0.42mg/ml. The MS2 reporter system was used to detect the transcriptional response of the bacteria to the treatment with the selected drugs. All three compounds are found to down regulate the transcriptional pathway. The novel compound, C8, showed maximum inhibition of transcription mechanism, followed by C5 and C11. The effect of the compounds on translation was analyzed using a Yellow Fluorescent protein reporter system. All the compounds displayed reductive impact on translation of which C8 was found to the best, exhibiting 8.5-fold repression followed by C5 and C11, respectively. Fluctuations of the Reactive Oxygen Species (ROS) concentrations were investigated upon incubation in hit compounds using ROS sensor protein. All the three compounds were found to contribute to oxidative pathway. C8 is found to have the best oxidative effect than C5 and C11. All experiments were repeated at least twice, the results being verified to be significant using statistical analysis.

Effects of temperature on the dynamics of the LacI-TetR-CI repressilator.

We studied the behaviour of the repressilator at 28 °C, 30 °C, 33 °C, and 37 °C. From the fluorescence in each cell over time, we determined the period of oscillations, the functionality (fraction of cells exhibiting oscillations) and the robustness (fraction of expected oscillations that occur) of this circuit. We show that the oscillatory dynamics differs with temperature. Functionality is maximized at 30 °C. Robustness decreases beyond 30 °C, as most cells exhibit 'failed' oscillations. These failures cause the distribution of periods to become bimodal, with an 'apparent period' that is minimal at 30 °C, while the true period decreases with increasing temperature. Based on previous studies, we hypothesized that the failures are due to a loss of functionality of one protein of the repressilator, CI. To test this, we studied the kinetics of a genetic switch, formed by the proteins CI and Cro, whose expression is controlled by PRM and PR, respectively. By probing the activity of PRM by in vivo detection of MS2-GFP tagged RNA, we find that, beyond 30 °C, the production of the CI-coding RNA changes from sub-Poissonian to super-Poissonian. Given this, we suggest that the decrease in efficiency of CI as a repressor with temperature hinders the robustness of the repressilator beyond 30 °C. We conclude that the repressilator is sensitive but not robust to temperature. Replacing CI for a less temperature-dependent protein should enhance robustness.