Rigorous science demands support of transgender scientists.

To build a just, equitable, and diverse academy, scientists and institutions must address systemic barriers that sex and gender minorities face. This Commentary summarizes (1) critical context informing the contemporary oppression of transgender people, (2) how this shapes extant research on sex and gender, and (3) actions to build an inclusive and rigorous academy for all.

Bye bye, linearity, bye: quantification of the mean for linear CRNs in a random environment.

Molecular reactions within a cell are inherently stochastic, and cells often differ in morphological properties or interact with a heterogeneous environment. Consequently, cell populations exhibit heterogeneity both due to these intrinsic and extrinsic causes. Although state-of-the-art studies that focus on dissecting this heterogeneity use single-cell measurements, the bulk data that shows only the mean expression levels is still in routine use. The fingerprint of the heterogeneity is present also in bulk data, despite being hidden from direct measurement. In particular, this heterogeneity can affect the mean expression levels via bimolecular interactions with low-abundant environment species. We make this statement rigorous for the class of linear reaction systems that are embedded in a discrete state Markov environment. The analytic expression that we provide for the stationary mean depends on the reaction rate constants of the linear subsystem, as well as the generator and stationary distribution of the Markov environment. We demonstrate the effect of the environment on the stationary mean. Namely, we show how the heterogeneous case deviates from the quasi-steady state (Q.SS) case when the embedded system is fast compared to the environment.

Temperature-dependence of the single-cell variability in the kinetics of transcription activation in Escherichia coli.

From in vivo single-cell, single-RNA measurements of the activation times and subsequent steady-state active transcription kinetics of a single-copy Lac-ara-1 promoter in Escherichia coli, we characterize the intake kinetics of the inducer (IPTG) from the media, following temperature shifts. For this, for temperature shifts of various degrees, we obtain the distributions of transcription activation times as well as the distributions of intervals between consecutive RNA productions following activation in individual cells. We then propose a novel methodology that makes use of deconvolution techniques to extract the mean and the variability of the distribution of intake times. We find that cells, following shifts to low temperatures, have higher intake times, although, counter-intuitively, the cell-to-cell variability of these times is lower. We validate the results using a new methodology for direct estimation of mean intake times from measurements of activation times at various inducer concentrations. The results confirm that E. coli's inducer intake times from the environment are significantly higher following a shift to a sub-optimal temperature. Finally, we provide evidence that this is likely due to the emergence of additional rate-limiting steps in the intake process at low temperatures, explaining the reduced cell-to-cell variability in intake times.

The precision of the symmetry in Z-ring placement in Escherichia coli is hampered at critical temperatures.

Cell division in Escherichia coli is morphologically symmetric due to, among other things, the ability of these cells to place the Z-ring at midcell. Studies have reported that, at sub-optimal temperatures, this symmetry decreases at the single-cell level, but the causes remain unclear. Using fluorescence microscopy, we observe FtsZ-GFP and DAPI-stained nucleoids to assess the robustness of the symmetry of Z-ring formation and positioning in individual cells under sub-optimal and critical temperatures. We find the Z-ring formation and positioning to be robust at sub-optimal temperatures, as the Z-ring's mean width, density and displacement from midcell maintain similar levels of correlation to one another as at optimal temperatures. However, at critical temperatures, the Z-ring displacement from midcell is greatly increased. We present evidence showing that this is due to enhanced distance between the replicated nucleoids and, thus, reduced Z-ring density, which explains the weaker precision in setting a morphologically symmetric division site. This also occurs in rich media and is cumulative, i.e. combining richer media and critically high temperatures enhances the asymmetries in division, which is evidence that the causes are biophysical. To further support this, we show that the effects are reversible, i.e. shifting cells from optimal to critical, and then to optimal again, reduces and then enhances the symmetry in Z-ring positioning, respectively, as the width and density of the Z-ring return to normal values. Overall, our findings show that the Z-ring positioning in E. coli is a robust biophysical process under sub-optimal temperatures, and that critical temperatures cause significant asymmetries in division.

Polar Localization of the Serine Chemoreceptor of Escherichia coli Is Nucleoid Exclusion-Dependent.

We studied whether nucleoid exclusion contributes to the segregation and retention of Tsr chemoreceptor clusters at the cell poles. Using live time-lapse, single-cell microscopy measurements, we show that the single-cell spatial distributions of Tsr clusters have heterogeneities and asymmetries that are consistent with nucleoid exclusion and cannot be explained by the diffusion-and-capture mechanism supported by Tol-Pal complexes at the poles. Also, in cells subjected to ampicillin, which enhances relative nucleoid lengths, Tsr clusters locate relatively closer to the cell extremities, whereas in anucleated cells (deletion mutants for mukB), the Tsr clusters are closer to midcell. In addition, we find that the fraction of Tsr clusters at the poles is smaller in deletion mutants for Tol-Pal than in wild-type cells, although it is still larger than would be expected by chance. Also in deletion mutants, the distribution of Tsr clusters differs widely between cells with relatively small and large nucleoids, in a manner consistent with nucleoid exclusion from midcell. This comparison further showed that diffusion-and-capture by Tol-Pal complexes and nucleoid exclusion from the midcell have complementary effects. Subsequently, we subjected deletion mutants to suboptimal temperatures that are known to enhance cytoplasm viscosity, which hampers nucleoid exclusion effects. As the temperature was lowered, the fraction of clusters at the poles decreased linearly. Finally, a stochastic model including nucleoid exclusion at midcell and diffusion-and-capture due to Tol-Pal at the poles is shown to exhibit a cluster dynamics that is consistent with the empirical data. We conclude that nucleoid exclusion also contributes to the preference of Tsr clusters for polar localization.

Aberrant microbiomes are associated with increased antibiotic resistance gene load in hybrid mice.

Antibiotic resistance is a priority public health problem resulting from eco-evolutionary dynamics within microbial communities and their interaction at a mammalian host interface or geographical scale. The links between mammalian host genetics, bacterial gut community, and antimicrobial resistance gene (ARG) content must be better understood in natural populations inhabiting heterogeneous environments. Hybridization, the interbreeding of genetically divergent populations, influences different components of the gut microbial communities. However, its impact on bacterial traits such as antibiotic resistance is unknown. Here, we present that hybridization might shape bacterial communities and ARG occurrence. We used amplicon sequencing to study the gut microbiome and to predict ARG composition in natural populations of house mice (Mus musculus). We compared gastrointestinal bacterial and ARG diversity, composition, and abundance across a gradient of pure and hybrid genotypes in the European House Mouse Hybrid Zone. We observed an increased overall predicted richness of ARG in hybrid mice. We found bacteria-ARG interactions by their co-abundance and detected phenotypes of extreme abundances in hybrid mice at the level of specific bacterial taxa and ARGs, mainly multidrug resistance genes. Our work suggests that mammalian host genetic variation impacts the gut microbiome and chromosomal ARGs. However, it raises further questions on how the mammalian host genetics impact ARGs via microbiome dynamics or environmental covariates.

Regulation of asymmetries in the kinetics and protein numbers of bacterial gene expression.

Genetic circuits change the status quo of cellular processes when their protein numbers cross thresholds. We investigate the regulation of RNA and protein threshold crossing propensities in Escherichia coli. From in vivo single RNA time-lapse microscopy data from multiple promoters, mutants, induction schemes and media, we study the asymmetry and tailedness (quantified by the skewness and kurtosis, respectively) of the distributions of time intervals between transcription events. We find that higher thresholds can be reached by increasing the skewness and kurtosis, which is shown to be achievable without affecting mean and coefficient of variation, by regulating the rate-limiting steps in transcription initiation. Also, they propagate to the skewness and kurtosis of the distributions of protein expression levels in cell populations. The results suggest that the asymmetry and tailedness of RNA and protein numbers in cell populations, by controlling the propensity for threshold crossing, and due to being sequence dependent and subject to regulation, may be key regulatory variables of decision-making processes in E. coli.

Dissecting the stochastic transcription initiation process in live Escherichia coli.

We investigate the hypothesis that, in Escherichia coli, while the concentration of RNA polymerases differs in different growth conditions, the fraction of RNA polymerases free for transcription remains approximately constant within a certain range of these conditions. After establishing this, we apply a standard model-fitting procedure to fully characterize the in vivo kinetics of the rate-limiting steps in transcription initiation of the Plac/ara-1 promoter from distributions of intervals between transcription events in cells with different RNA polymerase concentrations. We find that, under full induction, the closed complex lasts ∼788 s while subsequent steps last ∼193 s, on average. We then establish that the closed complex formation usually occurs multiple times prior to each successful initiation event. Furthermore, the promoter intermittently switches to an inactive state that, on average, lasts ∼87 s. This is shown to arise from the intermittent repression of the promoter by LacI. The methods employed here should be of use to resolve the rate-limiting steps governing the in vivo dynamics of initiation of prokaryotic promoters, similar to established steady-state assays to resolve the in vitro dynamics.

Single-cell kinetics of a repressilator when implemented in a single-copy plasmid.

Synthetic genetic clocks, such as the Elowitz-Leibler repressilator, will be key regulatory components of future synthetic circuits. We constructed a single-copy repressilator (SCR) by implementing the original repressilator circuit on a single-copy F-plasmid. After verifying its functionality, we studied its behaviour as a function of temperature and compared it with that of the original low-copy-number repressilator (LCR). Namely, we compared the period of oscillations, functionality (the fraction of cells exhibiting oscillations) and robustness to internal fluctuations (the fraction of expected oscillations that would occur). We found that, under optimal temperature conditions, the dynamics of the two systems differs significantly, although qualitatively they respond similarly to temperature changes. Exception to this is in the functionality, in which the SCR is higher at lower temperatures but lower at higher temperatures. Next, by adding IPTG to the medium at low and high concentrations during microscopy sessions, we showed that the functionality of the SCR is more robust to external perturbations, which indicates that the oscillatory behaviour of the LCR can be disrupted by affecting only a few of the copies in a cell. We conclude that the SCR, the first functional, synthetic, single-copy, ring-type genetic clock, is more robust to lower temperatures and to external perturbations than the original LCR. The SCR will be of use in future synthetic circuits, since it complements the array of tasks that the LCR can perform.