scholarly journals Scaling between DNA and cell size governs bacterial growth homeostasis and resource allocation

2021 ◽  
Author(s):  
Boyan Li ◽  
Songyuan Zhang ◽  
Le Zhang ◽  
Xiaoying Qiao ◽  
Yiqiang Shi ◽  
...  
Keyword(s):  
Cell Size ◽  
Mechanistic Model ◽  
Replication Initiation ◽  
Cellular Growth ◽  
Plasmid Copy ◽  
Escherichia Coli Cells ◽  
Copy Numbers ◽  
Dna Contents ◽  
Growth Laws ◽  
Novel Strategy

Bacteria maintain a stable cell size and a certain DNA content through proliferation as described by classic growth laws. How cells behave when this inherent scaling is broken, however, has rarely been interrogated. Here we engineered Escherichia coli cells with extremely low DNA contents using a tunable synthetic tool CRISPRori that temporarily inhibited chromosome replication initiation. A detailed mechanistic model coupling DNA replication, cell growth, and division revealed a fundamental DNA-centric growth law, which was validated by two observations. First, lineage dynamics were robust to large CRISPRori perturbations with division cycles rapidly restoring through a timer mechanism rather than the adder rule. Second, cellular growth transitioned into a linear regime at low DNA-cytoplasm ratios. Experiments and theory showed that in this regime, cellular resource was redirected to plasmid-borne gene expression. Together with the ability of CRISPRori to bi-directionally modulate plasmid copy numbers, these findings suggest a novel strategy for bio-production enhancement.

scholarly journals Chlamydia trachomatis intra-bacterial and total plasmid copy number in clinical urogenital samples

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
J. A. M. C. Dirks ◽  
K. Janssen ◽  
C. J. P. A. Hoebe ◽  
T. H. B. Geelen ◽  
M. Lucchesi ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Diagnostic Value ◽  
Plasmid Copy Number ◽  
Extracellular Dna ◽  
Chromosomal Dna ◽  
Plasmid Copy ◽  
Clinical Sensitivity ◽  
Copy Numbers ◽  
Vaginal Swabs ◽  
And Gender

AbstractChlamydia trachomatis (CT) increases its plasmid numbers when stressed, as occurs in clinical trachoma samples. Most CT tests target the plasmid to increase the test sensitivity, but some only target the chromosome. We investigated clinical urogenital samples for total plasmid copy numbers to assess its diagnostic value and intra-bacterial plasmid copy numbers to assess its natural variation. Both plasmid and chromosome copies were quantified using qPCR, and the plasmid:chromosome ratio (PCr) calculated in two cohorts: (1) 383 urogenital samples for the total PCR (tPCr), and (2) 42 vaginal swabs, with one half treated with propium-monoazide (PMA) to prevent the quantification of extracellular DNA and the other half untreated to allow for both tPCr and intra-bacterial PCr (iPCr) quantification. Mann–Whitney U tests compared PCr between samples, in relation to age and gender. Cohort 1: tPCr varied greatly (1–677, median 16). Median tPCr was significantly higher in urines than vaginal swabs (32 vs. 11, p < 0.001). Cohort 2: iPCr was more stable than tPCr (range 0.1–3 vs. 1–11). To conclude, tPCr in urogenital samples was much more variable than previously described. Transport time and temperature influences DNA degradation, impacting chromosomal DNA more than plasmids and urine more than vaginal samples. Data supports a plasmid target in CT screening assays to increase clinical sensitivity.

scholarly journals Large cells activate global protein degradation to maintain cell size homeostasis

2021 ◽  
Author(s):  
Shixuan Liu ◽  
Ceryl Tan ◽  
Chloe Melo-Gavin ◽  
Kevin G. Mark ◽  
Miriam Bracha Ginzberg ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Protein Degradation ◽  
Cell Size ◽  
Cell Cycle Progression ◽  
Mapk Pathway ◽  
Cellular Growth ◽  
Small Cells ◽  
Animal Cells ◽  
Size Dependent

Proliferating animal cells maintain a stable size distribution over generations despite fluctuations in cell growth and division size. This tight control of cell size involves both cell size checkpoints (e.g., delaying cell cycle progression for small cells) and size-dependent compensation in rates of mass accumulation (e.g., slowdown of cellular growth in large cells). We previously identified that the mammalian cell size checkpoint is mediated by a selective activation of the p38 MAPK pathway in small cells. However, mechanisms underlying the size-dependent compensation of cellular growth remain unknown. In this study, we quantified global rates of protein synthesis and degradation in naturally large and small cells, as well as in conditions that trigger a size-dependent compensation in cellular growth. Rates of protein synthesis increase proportionally with cell size in both perturbed and unperturbed conditions, as well as across cell cycle stages. Additionally, large cells exhibit elevated rates of global protein degradation and increased levels of activated proteasomes. Conditions that trigger a large-size-induced slowdown of cellular growth also promote proteasome-mediated global protein degradation, which initiates only after growth rate compensation occurs. Interestingly, the elevated rates of global protein degradation in large cells were disproportionately higher than the increase in size, suggesting activation of protein degradation pathways. Large cells at the G1/S transition show hyperactivated levels of protein degradation, even higher than similarly sized or larger cells in S or G2, coinciding with the timing of the most stringent size control in animal cells. Together, these findings suggest that large cells maintain cell size homeostasis by activating global protein degradation to induce a compensatory slowdown of growth.

scholarly journals Dynamics in Copy Numbers of Five Plasmids of a DairyLactococcus lactisStrain under Dairy-Related Conditions Including Near-Zero Growth Rates

2018 ◽  
Vol 84 (11) ◽  
Author(s):  
Oscar van Mastrigt ◽  
Marcel M. A. N. Lommers ◽  
Yorick C. de Vries ◽  
Tjakko Abee ◽  
Eddy J. Smid
Keyword(s):  
Nutrient Limitation ◽  
Lactococcus Lactis ◽  
Growth Rates ◽  
Copy Number ◽  
Plasmid Copy ◽  
Content Type ◽  
Copy Numbers ◽  
Wide Range ◽  
Zero Growth ◽  
The Impact

ABSTRACTLactic acid bacteria can carry multiple plasmids affecting their performance in dairy fermentations. The expression of plasmid-borne genes and the activity of the corresponding proteins are severely affected by changes in the numbers of plasmid copies. We studied the impact of growth rate on the dynamics of plasmid copy numbers at high growth rates in chemostat cultures and down to near-zero growth rates in retentostat cultures. Five plasmids of the dairy strainLactococcus lactisFM03-V1 were selected, and these varied in size (3 to 39 kb), in replication mechanism (theta or rolling circle), and in putative (dairy-associated) functions. The copy numbers ranged from 1.5 to 40.5, and the copy number of theta-type replicating plasmids was negatively correlated to the plasmid size. Despite the extremely wide range of growth rates (0.0003 h−1to 0.6 h−1), the copy numbers of the five plasmids were stable and only slightly increased at near-zero growth rates, showing that the plasmid replication rate was strictly controlled. One low-copy-number plasmid, carrying a large exopolysaccharide gene cluster, was segregationally unstable during retentostat cultivations, reflected in a complete loss of the plasmid in one of the retentostat cultures. The copy number of the five plasmids was also hardly affected by varying the pH value, nutrient limitation, or the presence of citrate (maximum 2.2-fold), signifying the stability in copy number of the plasmids.IMPORTANCELactococcus lactisis extensively used in starter cultures for dairy fermentations. Important traits for the growth and survival ofL. lactisin dairy fermentations are encoded by genes located on plasmids, such as genes involved in lactose and citrate metabolism, protein degradation, oligopeptide uptake, and bacteriophage resistance. Because the number of plasmid copies could affect the expression of plasmid-borne genes, it is important to know the factors that influence the plasmid copy numbers. We monitored the plasmid copy numbers ofL. lactisat near-zero growth rates, characteristic for cheese ripening. Moreover, we analyzed the effects of pH, nutrient limitation, and the presence of citrate. This showed that the plasmid copy numbers were stable, giving insight into plasmid copy number dynamics in dairy fermentations.

scholarly journals Thermodynamic Limits and Optimality of Microbial Growth

Entropy ◽  
2020 ◽  
Vol 22 (3) ◽  
pp. 277
Author(s):  
Nima Saadat ◽  
Tim Nies ◽  
Yvan Rousset ◽  
Oliver Ebenhöh
Keyword(s):  
Microbial Growth ◽  
Black Box ◽  
Cellular Growth ◽  
Modeling Approaches ◽  
Theoretical Approaches ◽  
Growing Cell ◽  
Limiting Nutrient ◽  
Growth Laws ◽  
Thermodynamic Limits ◽  
Optimality Principles

Understanding microbial growth with the use of mathematical models has a long history that dates back to the pioneering work of Jacques Monod in the 1940s. Monod’s famous growth law expressed microbial growth rate as a simple function of the limiting nutrient concentration. However, to explain growth laws from underlying principles is extremely challenging. In the second half of the 20th century, numerous experimental approaches aimed at precisely measuring heat production during microbial growth to determine the entropy balance in a growing cell and to quantify the exported entropy. This has led to the development of thermodynamic theories of microbial growth, which have generated fundamental understanding and identified the principal limitations of the growth process. Although these approaches ignored metabolic details and instead considered microbial metabolism as a black box, modern theories heavily rely on genomic resources to describe and model metabolism in great detail to explain microbial growth. Interestingly, however, thermodynamic constraints are often included in modern modeling approaches only in a rather superficial fashion, and it appears that recent modeling approaches and classical theories are rather disconnected fields. To stimulate a closer interaction between these fields, we here review various theoretical approaches that aim at describing microbial growth based on thermodynamics and outline the resulting thermodynamic limits and optimality principles. We start with classical black box models of cellular growth, and continue with recent metabolic modeling approaches that include thermodynamics, before we place these models in the context of fundamental considerations based on non-equilibrium statistical mechanics. We conclude by identifying conceptual overlaps between the fields and suggest how the various types of theories and models can be integrated. We outline how concepts from one approach may help to inform or constrain another, and we demonstrate how genome-scale models can be used to infer key black box parameters, such as the energy of formation or the degree of reduction of biomass. Such integration will allow understanding to what extent microbes can be viewed as thermodynamic machines, and how close they operate to theoretical optima.

scholarly journals Biogenesis of Developmental Master Regulatory 27nt-RNAs in Stylonychia—Can Coding RNA Turn into Non-Coding?

Genes ◽  
2019 ◽  
Vol 10 (11) ◽  
pp. 940
Author(s):  
Postberg ◽  
Weil ◽  
Pembaur
Keyword(s):  
Mechanistic Model ◽  
Histone Variants ◽  
Rdrp Activity ◽  
Copy Numbers ◽  
Somatic Genome ◽  
Chromosome Formation ◽  
Heterochromatin Structure ◽  
Rna Complexes ◽  
Dependent Pathway ◽  
Specific Sequences

In the ciliate Stylonychia, somatic macronuclei differentiate from germline micronuclei during sexual reproduction, accompanied by developmental sequence reduction. Concomitantly, over 95% of micronuclear sequences adopt a heterochromatin structure characterized by the histone variant H3.4 and H3K27me3. RNAi-related genes and histone variants dominate the list of developmentally expressed genes. Simultaneously, 27nt-ncRNAs that match sequences retained in new macronuclei are synthesized and bound by PIWI1. Recently, we proposed a mechanistic model for ‘RNA-induced DNA replication interference’ (RIRI): during polytene chromosome formation PIWI1/27nt-RNA-complexes target macronucleus-destined sequences (MDS) by base-pairing and temporarily cause locally stalled replication. At polytene chromosomal segments with ongoing replication, H3.4K27me3-nucleosomes become selectively deposited, thus dictating the prospective heterochromatin structure of these areas. Consequently, these micronucleus-specific sequences become degraded, whereas 27nt-RNA-covered sites remain protected. However, the biogenesis of the 27nt-RNAs remains unclear. It was proposed earlier that in stichotrichous ciliates 27nt-RNA precursors could derive from telomere-primed bidirectional transcription of nanochromosomes and subsequent Dicer-like (DCL) activity. As a minimalistic explanation, we propose here that the 27nt-RNA precursor could rather be mRNA or pre-mRNA and that the transition of coding RNA from parental macronuclei to non-coding RNAs, which act in premature developing macronuclei, could involve RNA-dependent RNA polymerase (RDRP) activity creating dsRNA intermediates prior to a DCL-dependent pathway. Interestingly, by such mechanism the partition of a parental somatic genome and possibly also the specific nanochromosome copy numbers could be vertically transmitted to the differentiating nuclei of the offspring.

scholarly journals Cell size sensing in animal cells coordinates anabolic growth rates and cell cycle progression to maintain cell size uniformity

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Miriam Bracha Ginzberg ◽  
Nancy Chang ◽  
Heather D'Souza ◽  
Nish Patel ◽  
Ran Kafri ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Size ◽  
Growth Rates ◽  
Cell Cycle Progression ◽  
Cellular Growth ◽  
Small Cells ◽  
Live Cells ◽  
Control Mechanisms ◽  
Animal Cells ◽  
Size Uniformity

Cell size uniformity in healthy tissues suggests that control mechanisms might coordinate cell growth and division. We derived a method to assay whether cellular growth rates depend on cell size, by monitoring how variance in size changes as cells grow. Our data revealed that, twice during the cell cycle, growth rates are selectively increased in small cells and reduced in large cells, ensuring cell size uniformity. This regulation was also observed directly by monitoring nuclear growth in live cells. We also detected cell-size-dependent adjustments of G1 length, which further reduce variability. Combining our assays with chemical/genetic perturbations confirmed that cells employ two strategies, adjusting both cell cycle length and growth rate, to maintain the appropriate size. Additionally, although Rb signaling is not required for these regulatory behaviors, perturbing Cdk4 activity still influences cell size, suggesting that the Cdk4 pathway may play a role in designating the cell’s target size.

scholarly journals Struggle To Survive: the Choir of Target Alteration, Hydrolyzing Enzyme, and Plasmid Expression as a Novel Aztreonam-Avibactam Resistance Mechanism

mSystems ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Ke Ma ◽  
Yu Feng ◽  
Alan McNally ◽  
Zhiyong Zong
Keyword(s):  
Antimicrobial Resistance ◽  
Antisense Rna ◽  
Antimicrobial Agents ◽  
Binding Protein ◽  
Resistance Mechanism ◽  
Resistance Mechanisms ◽  
Penicillin Binding Protein ◽  
Plasmid Copy ◽  
Copy Numbers

ABSTRACT Aztreonam-avibactam is a promising antimicrobial combination against multidrug-resistant organisms, such as carbapenemase-producing Enterobacterales. Resistance to aztreonam-avibactam has been found, but the resistance mechanism remains poorly studied. We recovered three Escherichia coli isolates of an almost identical genome but exhibiting varied aztreonam-avibactam resistance. The isolates carried a cephalosporinase gene, blaCMY-42, on IncIγ plasmids with a single-nucleotide variation in an antisense RNA-encoding gene, inc, of the replicon. The isolates also had four extra amino acids (YRIK) in penicillin-binding protein 3 (PBP3) due to a duplication of a 12-nucleotide (TATCGAATTAAC) stretch in pbp3. By cloning and plasmid-curing experiments, we found that elevated CMY-42 cephalosporinase production or amino acid insertions in PBP3 alone mediated slightly reduced susceptibility to aztreonam-avibactam, but their combination conferred aztreonam-avibactam resistance. We show that the elevated CMY-42 production results from increased plasmid copy numbers due to mutations in inc. We also verified the findings using in vitro mutation assays, in which aztreonam-avibactam-resistant mutants also had mutations in inc and elevated CMY-42 production compared with the parental strain. This choir of target modification, hydrolyzing enzyme, and plasmid expression represents a novel, coordinated, complex antimicrobial resistance mechanism and also reflects the struggle of bacteria to survive under selection pressure imposed by antimicrobial agents. IMPORTANCE Carbapenemase-producing Enterobacterales (CPE) is a serious global challenge with limited therapeutic options. Aztreonam-avibactam is a promising antimicrobial combination with activity against CPE producing serine-based carbapenemases and metallo-β-lactamases and has the potential to be a major option for combatting CPE. Aztreonam-avibactam resistance has been found, but resistance mechanisms remain largely unknown. Understanding resistance mechanisms is essential for optimizing treatment and developing alternative therapies. Here, we found that either penicillin-binding protein 3 modification or the elevated expression of cephalosporinase CMY-42 due to increased plasmid copy numbers does not confer resistance to aztreonam-avibactam, but their combination does. We demonstrate that increased plasmid copy numbers result from mutations in antisense RNA-encoding inc of the IncIγ replicon. The findings reveal that antimicrobial resistance may be due to concerted combinatorial effects of target alteration, hydrolyzing enzyme, and plasmid expression and also highlight that resistance to any antimicrobial combination will inevitably emerge.

scholarly journals A study of SeqA subcellular localization in Escherichia coli using photo-activated localization microscopy

2015 ◽  
Vol 184 ◽  
pp. 425-450 ◽  
Author(s):  
Jacek T. Mika ◽  
Aster Vanhecke ◽  
Peter Dedecker ◽  
Toon Swings ◽  
Jeroen Vangindertael ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Bacterial Genome ◽  
Genetically Engineered ◽  
Replication Initiation ◽  
Cell Protein ◽  
Experimental Conditions ◽  
Localization Microscopy ◽  
E Coli ◽  
Copy Numbers

Escherichia coli (E. coli) cells replicate their genome once per cell cycle to pass on genetic information to the daughter cells. The SeqA protein binds the origin of replication, oriC, after DNA replication initiation and sequesters it from new initiations in order to prevent overinitiation. Conventional fluorescence microscopy studies of SeqA localization in bacterial cells have shown that the protein is localized to discrete foci. In this study we have used photo-activated localization microscopy (PALM) to determine the localization of SeqA molecules, tagged with fluorescent proteins, with a localization precision of 20–30 nm with the aim to visualize the SeqA subcellular structures in more detail than previously possible. SeqA–PAmCherry was imaged in wild type E. coli, expressed from plasmid or genetically engineered into the bacterial genome, replacing the native seqA gene. Unsynchronized cells as well as cells with a synchronized cell cycle were imaged at various time points, in order to investigate the evolution of SeqA localization during the cell cycle. We found that SeqA indeed localized into discrete foci but these were not the only subcellular localizations of the protein. A significant amount of SeqA–PAmCherry molecules was localized outside the foci and in a fraction of cells we saw patterns indicating localization at the membrane. Using quantitative PALM, we counted protein copy numbers per cell, protein copy numbers per focus, the numbers of foci per cell and the sizes of the SeqA clusters. The data showed broad cell-to-cell variation and we did not observe a correlation between SeqA–PAmCherry protein numbers and the cell cycle under the experimental conditions of this study. The numbers of SeqA–PAmCherry molecules per focus as well as the foci sizes also showed broad distributions indicating that the foci are likely not characterized by a fixed number of molecules. We also imaged an E. coli strain devoid of the dam methylase (Δdam) and observed that SeqA–PAmCherry no longer formed foci, and was dispersed throughout the cell and localized to the plasma membrane more readily. We discuss our results in the context of the limitations of the technique.

scholarly journals Escherichia coli Cells with Increased Levels of DnaA and Deficient in Recombinational Repair Have Decreased Viability

2003 ◽  
Vol 185 (2) ◽  
pp. 630-644 ◽  
Author(s):  
Aline V. Grigorian ◽  
Rachel B. Lustig ◽  
Elena C. Guzmán ◽  
Joseph M. Mahaffy ◽  
Judith W. Zyskind
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Replication Fork ◽  
Replication Initiation ◽  
Recombinational Repair ◽  
Replication Forks ◽  
Dna Replication Initiation ◽  
Escherichia Coli Cells ◽  
Repair Pathways ◽  
Stalled Replication Forks

ABSTRACT The dnaA operon of Escherichia coli contains the genes dnaA, dnaN, and recF encoding DnaA, β clamp of DNA polymerase III holoenzyme, and RecF. When the DnaA concentration is raised, an increase in the number of DNA replication initiation events but a reduction in replication fork velocity occurs. Because DnaA is autoregulated, these results might be due to the inhibition of dnaN and recF expression. To test this, we examined the effects of increasing the intracellular concentrations of DnaA, β clamp, and RecF, together and separately, on initiation, the rate of fork movement, and cell viability. The increased expression of one or more of the dnaA operon proteins had detrimental effects on the cell, except in the case of RecF expression. A shorter C period was not observed with increased expression of the β clamp; in fact, many chromosomes did not complete replication in runout experiments. Increased expression of DnaA alone resulted in stalled replication forks, filamentation, and a decrease in viability. When the three proteins of the dnaA operon were simultaneously overexpressed, highly filamentous cells were observed (>50 μm) with extremely low viability and, in runout experiments, most chromosomes had not completed replication. The possibility that recombinational repair was responsible for the survival of cells overexpressing DnaA was tested by using mutants in different recombinational repair pathways. The absence of RecA, RecB, RecC, or the proteins in the RuvABC complex caused an additional ∼100-fold drop in viability in cells with increased levels of DnaA, indicating a requirement for recombinational repair in these cells.

Sign in / Sign up

Export Citation Format

Share Document