Network analysis of the transcriptional pattern of young and old cells of Escherichia coli during lag phase

Background: The aging process of bacteria in stationary phase is halted if cells are subcultured and enter lag phase and it is then followed by cellular division. Network science has been applied to analyse the transcriptional response, during lag phase, of bacterial cells starved previously in stationary phase for 1 day (young cells) and 16 days (old cells). Results: A genome scale network was constructed for E. coli K-12 by connecting genes with operons, transcription and sigma factors, metabolic pathways and cell functional categories. Most of the transcriptional changes were detected immediately upon entering lag phase and were maintained throughout this period. The lag period was longer for older cells and the analysis of the transcriptome revealed different intracellular activity in young and old cells. The number of genes differentially expressed was smaller in old cells (186) than in young cells (467). Relatively, few genes (62) were up- or down-regulated in both cultures. Transcription of genes related to osmotolerance, acid resistance, oxidative stress and adaptation to other stresses was down-regulated in both young and old cells. Regarding carbohydrate metabolism, genes related to the citrate cycle were up-regulated in young cells while old cells up-regulated the Entner Doudoroff and gluconate pathways and down-regulated the pentose phosphate pathway. In both old and young cells, anaerobic respiration and fermentation pathways were down-regulated, but only young cells up-regulated aerobic respiration while there was no evidence of aerobic respiration in old cells.Numerous genes related to DNA maintenance and replication, translation, ribosomal biosynthesis and RNA processing as well as biosynthesis of the cell envelope and flagellum and several components of the chemotaxis signal transduction complex were up-regulated only in young cells. The genes for several transport proteins for iron compounds were up-regulated in both young and old cells. Numerous genes encoding transporters for carbohydrates and organic alcohols and acids were down-regulated in old cells only. Conclusion: Network analysis revealed very different transcriptional activities during the lag period in old and young cells. Rejuvenation seems to take place during exponential growth by replicative dilution of old cellular components

Non-essential genes form the hubs of genome scale protein function and environmental gene expression networks in Salmonella enterica serovar Typhimurium

Background Salmonella Typhimurium is an important pathogen of human and animals. It shows a broad growth range and survives in harsh conditions. The aim of this study was to analyze transcriptional responses to a number of growth and stress conditions as well as the relationship of metabolic pathways and/or cell functions at the genome-scale-level by network analysis, and further to explore whether highly connected genes (hubs) in these networks were essential for growth, stress adaptation and virulence. Results De novo generated as well as published transcriptional data for 425 selected genes under a number of growth and stress conditions were used to construct a bipartite network connecting culture conditions and significantly regulated genes (transcriptional network). Also, a genome scale network was constructed for strain LT2. The latter connected genes with metabolic pathways and cellular functions. Both networks were shown to belong to the family of scale-free networks characterized by the presence of highly connected nodes or hubs which are genes whose transcription is regulated when responding to many of the assayed culture conditions or genes encoding products involved in a high number of metabolic pathways and cell functions. The five genes with most connections in the transcriptional network (wraB, ygaU, uspA, cbpA and osmC) and in the genome scale network (ychN, siiF (STM4262), yajD, ybeB and dcoC) were selected for mutations, however mutagenesis of ygaU and ybeB proved unsuccessful. No difference between mutants and the wild type strain was observed during growth at unfavorable temperatures, pH values, NaCl concentrations and in the presence of H2O2. Eight mutants were evaluated for virulence in C57/BL6 mice and none differed from the wild type strain. Notably, however, deviations of phenotypes with respect to the wild type were observed when combinations of these genes were deleted. Conclusion Network analysis revealed the presence of hubs in both transcriptional and functional networks of S. Typhimurium. Hubs theoretically confer higher resistance to random mutation but a greater susceptibility to directed attacks, however, we found that genes that formed hubs were dispensable for growth, stress adaptation and virulence, suggesting that evolution favors non-essential genes as main connectors in cellular networks

Functional studies on BolA and related genes: increasing the understanding of a protein with pleiotropic effects

Dissertation presented to obtain a Doctoral degree in Biology by Instituto de Tecnologia Química e BiológicaBolA is a protein that is able to change bacterial shape, confer resistance against large antibiotic molecules and detergents, reduce permeability, change the equilibrium of the outer membrane porins, and it is even involved in biofilm formation. This protein has such pleiotropic effects, that its function has been very difficult to unravel. This was the starting point for the work of this dissertation. If bolA is responsible for global cellular changes that confer resistance to a multitude of stresses, it is imperative to obtain more molecular insights to increase the understanding of the role of BolA in cell physiology and survival.(...)Inês Batista e Guinote was the recipient of a Doctoral Fellowship from Fundação para a Ciência e Tecnologia (FCT): PhD grant – SFRH/BD/ 31758/2006. The work was suspended for 5 months for maternity leave

Functional Genomics and physiology of growth initiation in Salmonella

Abstract Lag phase is a period of bacterial adaptation that occurs prior to cell division. The aim of this project was to characterise the processes used by Salmonella enterica serovar Typhimurium to escape from lag phase, and determine whether these processes are dependent on the bacterial ‘physiological history’. The lag phase transcriptomic response at 25 °C of stationary phase cells that had been held for twelve days at 2 °C was compared with that of stationary phase cells not subjected to this cold storage treatment. Cold-stored cells showed significant changes in expression of 78 % genes during lag phase, with 875 genes altering their expression ≥2-fold within the first four minutes of inoculation into fresh medium. Functional categories of genes that were significantly up-regulated included those encoding systems involved with metal ion uptake, stress resistance, phosphate uptake, ribosome synthesis and cellular metabolism. Genes in the OxyR regulon were induced earlier in cold-stored cells, a response coupled with a delay in the expression of Fe2+ acquisition genes, and down-regulation of genes encoding central metabolic enzymes. Together, these findings with physiological tests demonstrated that Salmonella held in cold storage exhibited an increased sensitivity to oxidative stress in midlag phase, although the lag time was not increased. Despite an oxidative stress response at the transcriptomic level during lag phase under both experimental conditions, deletion of the OxyR and SoxRS systems did not lead to an increased lag time during aerobic growth at 25 °C. The intracellular concentration of metal ions was quantified using ICP-MS, and changes observed during lag phase confirmed the transcriptomic data. Metal ions specifically accumulated during lag phase included Mn2+, Fe2+, Cu2+ and Ca2+, with the latter being the most abundant metal ion. The intracellular concentration of Zn2+ and Mg2+ remained the same as for stationary phase cells, and Ni2+, Mo2+ and Co2+ were expelled from the cell during lag phase. Metal homeostasis was determined to be a critical process, highlighted by growth in the presence of a chelator causing an extended lag time. Overall, lag phase was found to be a robust and reproducible adaptation period which was not perturbed by the mutagenesis approaches utilised in this study

Evolucija bakterija tijekom stacionarne faze rasta

Metagenomics and advances in molecular biology methods have enhanced knowledge of microbial evolution, metabolism, functions, their interactions with other organisms and their environment. The ability to persist and adapt to changes in their environment is a common lifestyle of 1 % of the known culturable bacteria. Studies in the variety of species have identified an incredible diversity of bacterial lifespan. The holy grail of molecular biology is to understand the integrated genetic and metabolic patterns of prokaryotic organisms like the enteric bacterium Escherichia coli. The usual description of E. coli life cycle comprises four phases: lag, logarithmic, stationary, and death phase, omitting their persistence and evolution during prolonged stationary phase. During prolonged stationary/starvation period, in batch bacterial culture, selected mutants with increased fitness express growth advantage in stationary phase (GASP), which enables them to grow and displace the parent cells as the majority population. The analyses of growth competition of Gram-negative and/or Gram-positive mixed bacterial cultures showed that GASP phenomenon can result in four GASP phenotypes: strong, moderate, weak or abortive. Bacterial stress responses to starvation include functions that can increase genetic variability and produce transient mutator state, which is important for adaptive evolution.Metagenomika i suvremene metode molekularne biologije omogućili su razumijevanje evolucije, metabolizma i funkcije mikroorganizama te njihovih interakcija s drugim organizmima u okolišu. Otpornost i prilagodba na promjene u okolišu uobičajeni su za 1 % poznatih bakterija što se mogu uzgajati u laboratoriju. Istraživanjem različitih bakterijskih vrsta uočena je njihova velika raznolikost. Escherichia coli je „sveti gral“ molekularne biologije u razumijevanju genetike i metaboličkih modela. Životni se ciklus E. coli sastoji od četiri faze: lag, logaritamske, stacionarne i faze odumiranja, zanemarujući bakterijsku postojanost i evoluciju tijekom produljene stacionarne faze. U šaržnoj bakterijskoj kulturi, tijekom produljene stacionarne faze ili vremena izgladnjivanja, preživjele stanice mutanata brže rastu (engl. growth advantage in stationary phase - GASP), pa prerastaju i zamjenjuju većinu roditeljskih stanica. Analiza kompetitivnoga rasta Gram-pozitivnih i/ili Gram-negativnih bakterija, tijekom produljene stacionarne faze u mješovitim kulturama, pokazala je postojanje četiriju GASP fenotipova: jaki, umjereni, slabi i nerazvijeni. Bakterijski odgovor na izgladnjivanje obuhvaća stanične funkcije koje mogu povećati genetičku raznolikost i stvarati mutator stanice bitne za adaptivnu evoluciju bakterija

Novel technologies to study single-cell response to environmental stimuli

Antibiotic tolerant phenotypes, such as persister and viable but non culturable cells (VBNC), are known to be present in isogenic bacterial populations. These phenotypes are now recognised as an important factor in the recalcitrance of infections and the development of antibiotic resistance; which itself is currently a major global health crisis. However, despite their clinical importance, we still know little about the mechanisms behind their formation and the relationship between the two phenotypes. Due to the relatively low abundance of the two phenotypes within the population and, in the case of VBNC cells, their ability to remain dormant for extended periods of time, high throughput single cell approaches currently provide the best opportunities for investigating them; in particular microfluidics has emerged as an exciting platform for investigating phenotypic heterogeneity at the single cell level due to the control it allows of the extracellular environment. Using antibiotic persistence as a proxy, we identify temporal windows in which a growing E. coli population exhibits significant changes in phenotypic heterogeneity and determine highly regulated genes and pathways at the population level. We then develop a high throughput microfluidic protocol, based on the pre-existing Mother Machine device, to investigate persister and VBNC cells before, during and after antibiotic exposure at the single cell level. We then developed the first fully automated image analysis pipeline that is capable of analysing Mother Machine images acquired in both bright field and phase contrast imaging modalities. The combination of our protocol and image analysis software allowed us to investigate the role of the previously identified genes in the formation of antibiotic persister and VBNC cells, where we identify potential biomarkers for these phenotypes before exposure to antibiotic. We then used the microfluidic set up to investigate the relationship between protein aggregation and antibiotic persister and VBNC cells. We find that protein aggregation can be correlated to the expression of exogenous proteins and that cells containing visible protein aggregates are, in turn, more likely to be persister or VBNC cells; providing further evidence that these phenotypes are not distinct and are instead part of one physiological continuum

Fundamental Principles in Bacterial Physiology - History, Recent progress, and the Future with Focus on Cell Size Control: A Review

Bacterial physiology is a branch of biology that aims to understand overarching principles of cellular reproduction. Many important issues in bacterial physiology are inherently quantitative, and major contributors to the field have often brought together tools and ways of thinking from multiple disciplines. This article presents a comprehensive overview of major ideas and approaches developed since the early 20th century for anyone who is interested in the fundamental problems in bacterial physiology. This article is divided into two parts. In the first part (Sections 1 to 3), we review the first `golden era' of bacterial physiology from the 1940s to early 1970s and provide a complete list of major references from that period. In the second part (Sections 4 to 7), we explain how the pioneering work from the first golden era has influenced various rediscoveries of general quantitative principles and significant further development in modern bacterial physiology. Specifically, Section 4 presents the history and current progress of the `adder' principle of cell size homeostasis. Section 5 discusses the implications of coarse-graining the cellular protein composition, and how the coarse-grained proteome `sectors' re-balance under different growth conditions. Section 6 focuses on physiological invariants, and explains how they are the key to understanding the coordination between growth and the cell cycle underlying cell size control in steady-state growth. Section 7 overviews how the temporal organization of all the internal processes enables balanced growth. In the final Section 8, we conclude by discussing the remaining challenges for the future in the field.Comment: Published in Reports on Progress in Physics. (https://doi.org/10.1088/1361-6633/aaa628) 96 pages, 48 figures, 7 boxes, 715 reference

Insights into the versatile metabolism of the Alphaproteobacterium Paracoccus denitrificans

Two distinct metabolic modes provide bacteria with energy (i.e., catabolism) and cellular building blocks (i.e., anabolism). At the interface between both lie the central metabolite acetyl-CoA, as well as the amphibolic tricarboxylic acid (TCA) cycle. The fate of acetyl-CoA in catabolism is its complete oxidation in the TCA cycle for the generation of reducing equivalents and energy, whereby the carbon backbone of the metabolite is fully lost to CO2. To assimilate acetyl-CoA into biomass in anabolism, instead, additional help of so-called replenishment (i.e., anaplerotic) pathways is therefore needed. These pathways circumvent the oxidative, CO2-producing steps of the TCA cycle, thereby allow the incorporation of acetyl-CoA into biomass, and ultimately enable bacterial growth on small carbon compounds such as acetate. While the old picture of metabolism has assumed a biochemical unity, according to which each organism possesses the same dedicated metabolic route for the conversion of a certain substrate, multiple distinct replenishment routes have been discovered in bacteria by today. Amongst them are the glyoxylate cycle (GC) and the ethylmalonyl-CoA pathway (EMCP). Most bacterial species possess only one of the two acetyl-CoA assimilation pathways as standalone route. However, the Alphaproteobacterium Paracoccus denitrificans, like only few others, has the genetic potential for both. This raised the questions what the biological purpose behind this apparent functional degeneracy in the metabolism of this organism is and how it is coordinated in the cell. This work shows that both routes the GC and the EMCP are employed by P. denitrificans during different stages of growth on acetate. While the EMCP is constitutively expressed on various substrates and additionally upregulated in the lag phase after growth switch to acetate, the GC is specifically induced on this substrate and only few others that are solely assimilated via acetyl-CoA as well. Each acetyl-CoA assimilation strategy alone confers distinct advantages on the cell. The EMCP allows metabolization of a great variety of carbon substrates and its action results in high growth yields of P. denitrificans on acetate. The GC, in contrast, is specialized for the rapid metabolization of acetyl-CoA and enables fast exponential growth of the bacterium on the carbon source. A fine-tuned genetic regulation controls expression of both pathways in P. denitrificans and thereby mediates dynamic metabolic rewiring between the two acetyl-CoA assimilation routes. This metabolic plasticity provides the organism with the ability to respond to changes in the nature and availability of carbon sources in a highly flexible manner to meet its physiological requirements. Using a combination of genetic, molecular biological, and biochemical methods, this work shows that RamB, a transcription factor of the ScfR family, senses CoA-ester intermediates of the EMCP to activate expression of the GC. This demonstrates a so-far undescribed phenomenon in bacterial metabolism, in which one seemingly degenerate metabolic pathway directly drives expression of the other. In all, this work expands our understanding of microbial metabolism and presents the molecular basis of plasticity in the central carbon metabolism of bacterial cells. Complete elucidation of the underlying mechanisms hereafter may open possibilities to develop new regulatory modules for application in synthetic pathways and metabolic engineering in the future

The Ability of Flux Balance Analysis to Predict Evolution of Central Metabolism Scales with the Initial Distance to the Optimum

The most powerful genome-scale framework to model metabolism, flux balance analysis (FBA), is an evolutionary optimality model. It hypothesizes selection upon a proposed optimality criterion in order to predict the set of internal fluxes that would maximize fitness. Here we present a direct test of the optimality assumption underlying FBA by comparing the central metabolic fluxes predicted by multiple criteria to changes measurable by a 13C-labeling method for experimentally-evolved strains. We considered datasets for three Escherichia coli evolution experiments that varied in their length, consistency of environment, and initial optimality. For ten populations that were evolved for 50,000 generations in glucose minimal medium, we observed modest changes in relative fluxes that led to small, but significant decreases in optimality and increased the distance to the predicted optimal flux distribution. In contrast, seven populations evolved on the poor substrate lactate for 900 generations collectively became more optimal and had flux distributions that moved toward predictions. For three pairs of central metabolic knockouts evolved on glucose for 600–800 generations, there was a balance between cases where optimality and flux patterns moved toward or away from FBA predictions. Despite this variation in predictability of changes in central metabolism, two generalities emerged. First, improved growth largely derived from evolved increases in the rate of substrate use. Second, FBA predictions bore out well for the two experiments initiated with ancestors with relatively sub-optimal yield, whereas those begun already quite optimal tended to move somewhat away from predictions. These findings suggest that the tradeoff between rate and yield is surprisingly modest. The observed positive correlation between rate and yield when adaptation initiated further from the optimum resulted in the ability of FBA to use stoichiometric constraints to predict the evolution of metabolism despite selection for rate