Biology subjects

Cosentino Lagomarsino, M.

Publications and source records attributed to Cosentino Lagomarsino, M..

28 records · Page 2Linked to original sources

bioRxiv2020

Epistasis, aneuploidy, and gain-of-function mutations underlie the evolution ofresistance to induced microtubule depolymerization

Microtubules, polymers of alpha- and beta-tubulin, are essential cellular components. When microtubule polymerization is hindered, cells are delayed in mitosis, but eventually they manage to proliferate with massive chromosome missegregation. Several studies have analyzed the first cell division upon microtubules impairing conditions. Here, we asked how cells cope on the long term. Taking advantage of mutations in beta-tubulin, we evolved in the lab for [~]150 generations 24 populations of yeast cells unable to properly polymerize microtubules. At the end of the evolution experiment, cells re-gained the ability to form microtubules, and were less sensitive to microtubule depolymerizing drugs. Whole genome sequencing allowed us to identify genes recurrently mutated (tubulins and kinesins) as well as the pervasive duplication of chromosome VIII. We confirmed that mutations found in these genes and disomy of chromosome VIII allow cells to compensate for the original mutation in beta-tubulin. The mutations we identified were mostly gain-of-function, likely re-allowing the proper use of the mutated form of beta-tubulin. When we analyzed the temporal order of mutations leading to resistance in independent populations, we observed multiple times the same series of events: disomy of chromosome VIII followed by one additional adaptive mutation in either tubulins or kinesins. Analyzing the epistatic interactions among different mutations, we observed that some mutations benefited from the disomy of chromosome VIII and others did not. Given that tubulins are highly conserved among eukaryotes, our results are potentially relevant for understanding the emergence of resistance to drugs targeting microtubules, widely used for cancer treatment.

cell biology↗

bioRxiv2020

An evolutionary model identifies the main selective pressures for the evolution of genome-replication profiles.

Recent results comparing the temporal program of genome replication of yeast species belonging to the Lachancea clade support the scenario that the evolution of replication timing program could be mainly driven by correlated acquisition and loss events of active replication origins. Using these results as a benchmark, we develop an evolutionary model defined as birth-death process for replication origins, and use it to identify the selective pressures that shape the replication timing profiles. Comparing different evolutionary models with data, we find that replication origin birth and death events are mainly driven by two evolutionary pressures, the first imposes that events leading to higher double-stall probability of replication forks are penalized, while the second makes less efficient origins more prone to evolutionary loss. This analysis provides an empirically grounded predictive framework for quantitative evolutionary studies of the replication timing program.

evolutionary biology↗

bioRxiv2020

Threshold accumulation of a constitutive protein explains E. coli cell division behavior in nutrient upshifts.

Despite of a boost of recent progress in dynamic single-cell measurements and analyses in E. coli, we still lack a mechanistic understanding of the determinants of the decision to divide. Specifically, the debate is open regarding the processes linking growth and chromosome replication to division, and on the molecular origin of the observed "adder correlations", whereby cells divide adding roughly a constant volume independent of their initial volume. In order to gain insight into these questions, we interrogate dynamic size-growth behavior of single cells across nutrient upshifts with a high-precision microfluidic device. We find that the division rate changes quickly after nutrients change, much before growth rate goes to a steady state, and in a way that adder correlations are robustly conserved. Comparison of these data to simple mathematical models falsifies proposed mechanisms where replication-segregation or septum completion are the limiting step for cell division. Instead, we show that the accumulation of a putative constitutively expressed "P-sector divisor" protein explains the behavior during the shift. Significance statementThe mechanism leading to cell division in the bacterium E. coli is unknown, but we know that it results in adding a roughly constant size every cell cycle, regardless of size at birth. While most available studies try to infer information on cell division from steadily dividing cells in constant nutrient conditions, this study leverages on a high-resolution device to monitor single-cell growth division upon nutrient changes. Comparing these data with different mathematical models, the authors are able to discriminate among fundamentally different mechanisms of cell division control, and they show that the data support a model where an unregulated protein accumulates to a threshold and triggers division.

cell biology↗

bioRxiv2020

ppGpp is a bacterial cell size regulator

Growth and division are central to cell size. Bacteria achieve size homeostasis by dividing when growth has added a constant size since birth, termed the "adder" principle, by unknown mechanisms [1-4]. Growth is well known to be regulated by ppGpp, which controls anything from ribosome production to metabolic enzyme activity and replication initiation, and whose absence or excess can induce the stress response, filamentation, and yield growth-arrested miniature cells [5-8]. These observations raise unresolved questions about the relation between ppGpp and size homeostasis mechanisms during normal exponential growth. Here, to untangle effects of ppGpp and nutrients, we gained control of cellular ppGpp by inducing the synthesis and hydrolysis enzymes RelA and Mesh1. We found that ppGpp not only exerts control over the growth rate, but also over cell division and hence the steady state cell size. The added size responds rapidly to changes in the ppGpp level, aided by transiently accelerated or delayed divisions, and establishes its new constant value while the growth rate still adjusts. Moreover, the magnitude of the added size and resulting steady-state birth size correlate consistently with the ppGpp level, rather than with the growth rate, which results in cells of different size that grow equally fast. Our findings suggest that ppGpp serves as a critical regulator that coordinates cell size and growth control.

microbiology↗

bioRxiv2020

Taxonomic classification method for metagenomics based on core protein families with Core-Kaiju

Characterizing species diversity and composition of bacteria hosted by biota is revolutionizing our understanding of the role of symbiotic interactions in ecosystems. However, determining microbiomes diversity implies the classification of taxa composition within the sampled community, which is often done via the assignment of individual reads to taxa by comparison to reference databases. Although computational methods aimed at identifying the microbe(s) taxa are available, it is well known that inferences using different methods can vary widely depending on various biases. In this study, we first apply and compare different bioinformatics methods based on 16S ribosomal RNA gene and whole genome shotgun sequencing for taxonomic classification to three small mock communities of bacteria, of which the compositions are known. We show that none of these methods can infer both the true number of taxa and their abundances. We thus propose a novel approach, named Core-Kaiju, which combines the power of shotgun metagenomics data with a more focused marker gene classification method similar to 16S, but based on emergent statistics of core protein domain families. We thus test the proposed method on the three small mock communities and also on medium- and highly complex mock community datasets taken from the Critical Assessment of Metagenome Interpretation challenge. We show that Core-Kaiju reliably predicts both number of taxa and abundance of the analysed mock bacterial communities. Finally we apply our method on human gut samples, showing how Core-Kaiju may give more accurate ecological characterization and fresh view on real microbiomes.

bioinformatics↗

bioRxiv2019

Early fate of exogenous promoters in E. coli

Gene gain by horizontal gene transfer is a major pathway of genome innovation in bacteria. The current view posits that acquired genes initially need to be silenced and that a bacterial chromatin protein, H-NS, plays a role in this silencing. However, we lack direct observation of the early fate of a horizontally transferred gene to prove this theory. We combine sequencing, flow cytometry and sorting, and microscopy to monitor gene expression and its variability after large-scale random insertions of a reporter gene in a population. We find that inserted promoters have a wide range of gene-expression variability related to their location. We find that high-expression clones carry insertions that are not correlated with H-NS binding. Conversely, binding of H-NS correlates with silencing. Finally, while most promoters show a common level of extrinsic noise, some insertions show higher noise levels. Analysis of these high-noise clones supports a scenario of switching due to transcriptional interference from divergent ribosomal promoters. Altogether, our findings point to evolutionary pathways where newly-acquired genes are not necessarily silenced, but may immediately explore a wide range of expression levels to probe the optimal ones.

genomics↗

bioRxiv2019

A decrease in transcription capacity limits growth rate upon translation inhibition

AbstractIn bacterial cells, inhibition of ribosomes by sublethal concentrations of antibiotics leads to a decrease in growth rate despite an increase in ribosome content. The limitation of ribosomal activity results in an increase in the level of expression from ribosomal promoters; this can deplete the pool of RNA polymerase (RNAP) that is available for the expression of non-ribosomal genes. However, the magnitude of this effect remains to be quantified. Here, we use the change in the activity of constitutive promoters with different affinities for RNAP to quantify the change in the concentration of free RNAP. The data are consistent with a significant decrease in the amount of RNAP available for transcription of both ribosomal and non ribosomal genes. Results obtained with different reporter genes reveal an mRNA length dependence on the amount of full-length translated protein, consistent with the decrease in ribosome processivity affecting more strongly the translation of longer genes. The genes coding for the {beta} and {beta} subunits of RNAP are amongst the longest genes in the E. coli genome, while the genes coding for ribosomal proteins are among the shortest genes. This can explain the observed decrease in transcription capacity that favors the expression of genes whose promoters have a high affinity for RNAP, such as ribosomal promoters.\n\nImportanceExposure of bacteria to sublethal concentrations of antibiotics can lead to bacterial adaptation and survival at higher doses of inhibitors, which in turn can lead to the emergence of antibiotic resistance. The presence of sublethal concentrations of antibiotics targeting translation results in an increase in the amount of ribosomes per cell and a decrease in the cells growth rate. In this work, we have found that inhibition of ribosome activity can result in a decrease in the amount of free RNA polymerase available for transcription, thus limiting the protein expression rate via a different pathway than what was expected. This result can be explained by our observation that long genes, such as those coding for RNA polymerase subunits, have a higher probability of premature translation termination in the presence of ribosome inhibitors, while expression of short ribosomal genes is affected less, consistent with their increased concentration.

microbiology↗

bioRxiv2018

Dissecting the control mechanisms for DNA replication and cell division in E. coli

Understanding how single E. coli cells coordinate the timing of cell division with genome replication would unlock a classic problem of biology, and open the way to address cell-cycle progression at the single-cell level. Several recent studies produced new data and proposed different models, based on the hypothesis that replication-segregation is the bottleneck process for cell division. However, due to the apparent contrast in both experimental results and proposed mechanisms, the emerging picture is fragmented and unclear. In this work, we re-evaluate jointly available data and models, and we show that, while each model contains useful insights, none of the proposed models, as well as generalizations based on the same assumptions, correctly describes all the correlation patterns observed in data. This analysis leads us to conclude that the assumption that replication is the bottleneck process for cell division is too restrictive. Instead, we propose that two concurrent cycles responsible for division and initiation of DNA replication together set the time of cell division. This framework correctly captures available data and allows us to select a nearly constant added size per origin between subsequent initiations as the most likely mechanism setting initiation of replication.

cell biology↗

bioRxiv2018

Concurrent processes set E. coli cell division

A cell can divide only upon completion of chromosome segregation, or its daughters would lose genetic material [1, 2]. In E. coli bacteria, the prevalent view is that cells divide a fixed amount of time after they start to copy the chromosomes [3, 4], and a known pathway prevents cells from dividing if the chromosomes interfere with the cytokinesis machinery [5]. However, whether completion of segregation is typically the bottleneck process for the decision to divide has never been stringently tested on single cells. We show how key trends in single-cell data lead to challenge the classic idea of replication-segregation limiting cell division. Instead, the data agree with a model where two concurrent processes (setting replication initiation and inter-division time) set cell division on competing time scales. During each cell cycle, division is set by the slowest process (an \" ...

cell biology↗

bioRxiv2018

A bHLH-PAS protein regulates light-dependent rhythmic processes in the marine diatom Phaeodactylum tricornutum

Periodic light-dark cycles govern the timing of basic biological processes in organisms inhabiting land as well as the sea, where life evolved. Although prominent marine phytoplanktonic organisms such as diatoms show robust diurnal rhythms in growth, cell cycle and gene expression, the molecular foundations controlling these processes are still obscure. By exploring the regulatory landscape of diatom diurnal rhythms, we unveil the function of a Phaeodactylum tricornutum bHLH-PAS protein, PtbHLH1a, in the regulation of light-dependent diurnal rhythms. Peak expression of PtbHLH1a mRNA occurs toward the end of the light period and it adjusts to photoperiod changes. Ectopic over-expression of PtbHLH1a results in lines showing a phase shift in diurnal cell fluorescence, compared to the wild-type cells, and with altered cell cycle progression and gene expression. Reduced oscillations in gene expression are also observed in overexpression lines compared to wild-type in continuous darkness, showing that the regulation of rhythmicity by PtbHLH1a is not directly dependent on light inputs and cell division. PtbHLH1a homologs are widespread in diatom genomes which may indicate a common function in many species. This study adds new elements to understand diatom biology and ecology and offers new perspectives to elucidate timekeeping mechanisms in marine organisms belonging to a major, but underinvestigated branch of the tree of life.\n\nSIGNIFICANCE STATEMENTMost organisms experience diurnal light-dark changes and show rhythms of basic biological processes such that they occur at optimal times of the day. The ocean harbours a huge diversity of organisms showing light-dependent rhythms, but their molecular foundations are still largely unknown. In this study, we discover a novel protein, PtbHLH1a that regulates cell division, gene expression and the diurnal timing of these events in the marine diatom Phaedoactylum tricornutum. The identification of PtbHLH1a-like genes in many diatom species suggests a conserved function in diurnal rhythm regulation in the most species-rich group of algae in the ocean. This study unveils critical features of diatom biology and advances the field of marine rhythms and their environmental regulation.

molecular biology↗