scholarly journals Genome-wide phenotypic analysis of growth, cell morphogenesis and cell cycle events in Escherichia coli.

2017 ◽  
Author(s):  
Manuel Campos ◽  
Sander K Govers ◽  
Irnov Irnov ◽  
Genevieve S Dobihal ◽  
Francois Cornet ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Growth Rate ◽  
Cell Division ◽  
Cell Size ◽  
Gene Knockout ◽  
Single Gene ◽  
Cell Morphogenesis ◽  
Phenotypic Analysis ◽  
New Genes

Cell size, cell growth and the cell cycle are necessarily intertwined to achieve robust bacterial replication. Yet, a comprehensive and integrated view of these fundamental processes is lacking. Here, we describe an image-based quantitative screen of the single-gene knockout collection of Escherichia coli, and identify many new genes involved in cell morphogenesis, population growth, nucleoid (bulk chromosome) dynamics and cell division. Functional analyses, together with high-dimensional classification, unveil new associations of morphological and cell cycle phenotypes with specific functions and pathways. Additionally, correlation analysis across ~4,000 genetic perturbations shows that growth rate is surprisingly not predictive of cell size. Growth rate was also uncorrelated with the relative timings of nucleoid separation and cell constriction. Rather, our analysis identifies scaling relationships between cell size and nucleoid size and between nucleoid size and the relative timings of nucleoid separation and cell division. These connections suggest that the nucleoid links cell morphogenesis to the cell cycle.

scholarly journals Dynamics of Escherichia coli Chromosome Segregation during Multifork Replication

2007 ◽  
Vol 189 (23) ◽  
pp. 8660-8666 ◽  
Author(s):  
Henrik J. Nielsen ◽  
Brenda Youngren ◽  
Flemming G. Hansen ◽  
Stuart Austin
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Growth Rate ◽  
Cell Division ◽  
Chromosome Segregation ◽  
Replication Forks ◽  
Daughter Cells ◽  
Initiation Of Replication ◽  
Replication Terminus ◽  
Multiple Replication

ABSTRACT Slowly growing Escherichia coli cells have a simple cell cycle, with replication and progressive segregation of the chromosome completed before cell division. In rapidly growing cells, initiation of replication occurs before the previous replication rounds are complete. At cell division, the chromosomes contain multiple replication forks and must be segregated while this complex pattern of replication is still ongoing. Here, we show that replication and segregation continue in step, starting at the origin and progressing to the replication terminus. Thus, early-replicated markers on the multiple-branched chromosomes continue to separate soon after replication to form separate protonucleoids, even though they are not segregated into different daughter cells until later generations. The segregation pattern follows the pattern of chromosome replication and does not follow the cell division cycle. No extensive cohesion of sister DNA regions was seen at any growth rate. We conclude that segregation is driven by the progression of the replication forks.

scholarly journals Integrated biphasic growth rate, gene expression, and cell-size homeostasis behaviour of singleB. subtiliscells

2019 ◽  
Author(s):  
Niclas Nordholt ◽  
Johan H. van Heerden ◽  
Frank J. Bruggeman
Keyword(s):  
Cell Cycle ◽  
Growth Rate ◽  
Cell Division ◽  
Protein Expression ◽  
Cell Size ◽  
Cell Cycle Progression ◽  
Bacterial Cells ◽  
Cycle Progression ◽  
Average Cell ◽  
Cycle Dependent

ABSTRACTThe growth rate of single bacterial cells is continuously disturbed by random fluctuations in biosynthesis rates and by deterministic cell-cycle events, such as division, genome duplication, and septum formation. It is not understood whether, and how, bacteria reject these disturbances. Here we quantified growth and constitutive protein expression dynamics of singleBacillus subtiliscells, as a function of cell-cycle-progression. Variation in birth size and growth rate, resulting from unequal cell division, is largely compensated for when cells divide again. We analysed the cell-cycle-dynamics of these compensations and found that both growth and protein expression exhibited biphasic behaviour. During a first phase of variable duration, the absolute rates were approximately constant and cells behaved as sizers. In the second phase, rates increased and growth behaviour exhibited characteristics of a timer-strategy. This work shows how cell-cycle-dependent rate adjustments of biosynthesis and growth are integrated to compensate for physio-logical disturbances caused by cell division.IMPORTANCEUnder constant conditions, bacterial populations can maintain a fixed average cell size and constant exponential growth rate. At the single cell-level, however, cell-division can cause significant physiological perturbations, requiring compensatory mechanisms to restore the growth-related characteristics of individual cells toward that of the average cell. Currently, there is still a major gap in our understanding of the dynamics of these mechanisms, i.e. how adjustments in growth, metabolism and biosynthesis are integrated during the bacterial cell-cycle to compensate the disturbances caused by cell division. Here we quantify growth and constitutive protein expression in individual bacterial cells at sub-cell-cycle resolution. Significantly, both growth and protein production rates display structured and coordinated cell-cycle-dependent dynamics. These patterns reveal the dynamics of growth rate and size compensations during cell-cycle progression. Our findings provide a dynamic cell-cycle perspective that offers novel avenues for the interpretation of physiological processes that underlie cellular homeostasis in bacteria.

scholarly journals Genomewide phenotypic analysis of growth, cell morphogenesis, and cell cycle events in Escherichia coli

2018 ◽  
Vol 14 (6) ◽  
Author(s):  
Manuel Campos ◽  
Sander K Govers ◽  
Irnov Irnov ◽  
Genevieve S Dobihal ◽  
François Cornet ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Cell Morphogenesis ◽  
Phenotypic Analysis

scholarly journals Genome-Wide Screening Identifies Six Genes That Are Associated with Susceptibility to Escherichia coli Microcin PDI

2015 ◽  
Vol 81 (20) ◽  
pp. 6953-6963 ◽  
Author(s):  
Zhe Zhao ◽  
Lauren J. Eberhart ◽  
Lisa H. Orfe ◽  
Shao-Yeh Lu ◽  
Thomas E. Besser ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Atp Synthase ◽  
Disulfide Bonds ◽  
Gene Knockout ◽  
Single Gene ◽  
Site Directed Mutagenesis ◽  
Content Type ◽  
E Coli ◽  
Synthase Inhibitor ◽  
Loss Of Susceptibility

ABSTRACTThe microcin PDI inhibits a diverse group of pathogenicEscherichia colistrains. Coculture of a single-gene knockout library (BW25113;n= 3,985 mutants) against a microcin PDI-producing strain (E. coli25) identified six mutants that were not susceptible (ΔatpA, ΔatpF, ΔdsbA, ΔdsbB, ΔompF, and ΔompR). Complementation of these genes restored susceptibility in all cases, and the loss of susceptibility was confirmed through independent gene knockouts inE. coliO157:H7 Sakai. Heterologous expression ofE. coliompFconferred susceptibility toSalmonella entericaandYersinia enterocoliticastrains that are normally unaffected by microcin PDI. The expression of chimeric OmpF and site-directed mutagenesis revealed that the K47G48N49region within the first extracellular loop ofE. coliOmpF is a putative binding site for microcin PDI. OmpR is a transcriptional regulator forompF, and consequently loss of susceptibility by the ΔompRstrain most likely is related to this function. Deletion of AtpA and AtpF, as well as AtpE and AtpH (missed in the original library screen), resulted in the loss of susceptibility to microcin PDI and the loss of ATP synthase function. Coculture of a susceptible strain in the presence of an ATP synthase inhibitor resulted in a loss of susceptibility, confirming that a functional ATP synthase complex is required for microcin PDI activity. Intransexpression ofompFin the ΔdsbAand ΔdsbBstrains did not restore a susceptible phenotype, indicating that these proteins are probably involved with the formation of disulfide bonds for OmpF or microcin PDI.

scholarly journals Cell growth dilutes the cell cycle inhibitor Rb to trigger cell division

2018 ◽  
Author(s):  
Evgeny Zatulovskiy ◽  
Daniel F. Berenson ◽  
Benjamin R. Topacio ◽  
Jan M. Skotheim
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Cell Growth ◽  
Cell Size ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Inverse Correlation ◽  
Cell Types ◽  
Similar Amount ◽  
Cell Cycle Inhibitor

Cell size is fundamental to function in different cell types across the human body because it sets the scale of organelle structures, biosynthesis, and surface transport1,2. Tiny erythrocytes squeeze through capillaries to transport oxygen, while the million-fold larger oocyte divides without growth to form the ~100 cell pre-implantation embryo. Despite the vast size range across cell types, cells of a given type are typically uniform in size likely because cells are able to accurately couple cell growth to division3–6. While some genes whose disruption in mammalian cells affects cell size have been identified, the molecular mechanisms through which cell growth drives cell division have remained elusive7–12. Here, we show that cell growth acts to dilute the cell cycle inhibitor Rb to drive cell cycle progression from G1 to S phase in human cells. In contrast, other G1/S regulators remained at nearly constant concentration. Rb is a stable protein that is synthesized during S and G2 phases in an amount that is independent of cell size. Equal partitioning to daughter cells of chromatin bound Rb then ensures that all cells at birth inherit a similar amount of Rb protein. RB overexpression increased cell size in tissue culture and a mouse cancer model, while RB deletion decreased cell size and removed the inverse correlation between cell size at birth and the duration of G1 phase. Thus, Rb-dilution by cell growth in G1 provides a long-sought cell autonomous molecular mechanism for cell size homeostasis.

scholarly journals Fission yeast cell morphogenesis: identification of new genes and analysis of their role during the cell cycle.

1995 ◽  
Vol 131 (6) ◽  
pp. 1529-1538 ◽  
Author(s):  
F Verde ◽  
J Mata ◽  
P Nurse
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Cell Polarity ◽  
Growth Direction ◽  
Temperature Sensitive ◽  
Cell Morphogenesis ◽  
Cell Cycle Genes ◽  
New Genes ◽  
Morphological Mutants ◽  
Mutant Phenotypes

To identify new genes involved in the control of cell morphogenesis in the fission yeast Schizosaccharomyces pombe we have visually screened for temperature-sensitive mutants that show defects in cell morphology. We have isolated and characterized 64 mutants defining 19 independent genes, 10 of which have not been previously described. One class of mutants, defining 12 orb genes, become round and show a complete loss of cell polarity. A second class of mutants exhibits branched or bent morphologies. These mutants show defects in either selection of the growth site, defining two tea genes, or in the maintenance of growth direction, defining five ban genes. Immunofluorescence analysis of these morphological mutants shows defects in the organization of the microtubule and actin cytoskeleton. These defects include shortened, bundled, and asymmetrically localized microtubules and enlarged and mislocalized actin patches. Analysis of the mutant phenotypes has allowed us to order the genes into four groups according to their function during the cell cycle: genes required for the maintenance of cell polarity throughout the cell cycle; genes necessary only for the reestablishment of cell polarity after mitosis and not for maintaining cell polarity once it is established; genes essential for the transition from monopolar to bipolar growth and genes that severe as 'polarity markers'.

scholarly journals Antibiotic Sensitivity Profiles Determined with an Escherichia coli Gene Knockout Collection: Generating an Antibiotic Bar Code

2010 ◽  
Vol 54 (4) ◽  
pp. 1393-1403 ◽  
Author(s):  
Anne Liu ◽  
Lillian Tran ◽  
Elinne Becket ◽  
Kim Lee ◽  
Laney Chinn ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Knockout ◽  
Single Gene ◽  
Antibiotic Sensitivity ◽  
Multidrug Resistant ◽  
Intrinsic Resistance ◽  
Bar Code ◽  
Entire Collection ◽  
Data Points ◽  
Increased Sensitivity

ABSTRACT We have defined a sensitivity profile for 22 antibiotics by extending previous work testing the entire KEIO collection of close to 4,000 single-gene knockouts in Escherichia coli for increased susceptibility to 1 of 14 different antibiotics (ciprofloxacin, rifampin [rifampicin], vancomycin, ampicillin, sulfamethoxazole, gentamicin, metronidazole, streptomycin, fusidic acid, tetracycline, chloramphenicol, nitrofurantoin, erythromycin, and triclosan). We screened one or more subinhibitory concentrations of each antibiotic, generating more than 80,000 data points and allowing a reduction of the entire collection to a set of 283 strains that display significantly increased sensitivity to at least one of the antibiotics. We used this reduced set of strains to determine a profile for eight additional antibiotics (spectinomycin, cephradine, aztreonem, colistin, neomycin, enoxacin, tobramycin, and cefoxitin). The profiles for the 22 antibiotics represent a growing catalog of sensitivity fingerprints that can be separated into two components, multidrug-resistant mutants and those mutants that confer relatively specific sensitivity to the antibiotic or type of antibiotic tested. The latter group can be represented by a set of 20 to 60 strains that can be used for the rapid typing of antibiotics by generating a virtual bar code readout of the specific sensitivities. Taken together, these data reveal the complexity of intrinsic resistance and provide additional targets for the design of codrugs (or combinations of drugs) that potentiate existing antibiotics.

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