scholarly journals Exopolysaccharide defects cause hyper-thymineless death in Escherichia coli via massive loss of chromosomal DNA and cell lysis

2020 ◽  
Vol 117 (52) ◽  
pp. 33549-33560
Author(s):  
T. V. Pritha Rao ◽  
Andrei Kuzminov
Keyword(s):  
Escherichia Coli ◽  
Cell Envelope ◽  
Glucose Production ◽  
Cell Lysis ◽  
Chromosomal Dna ◽  
Chromosomal Replication ◽  
Enterobacterial Common Antigen ◽  
Acute Starvation ◽  
Thymineless Death ◽  
Dna Loss

Thymineless death in Escherichia coli thyA mutants growing in the absence of thymidine (dT) is preceded by a substantial resistance phase, during which the culture titer remains static, as if the chromosome has to accumulate damage before ultimately failing. Significant chromosomal replication and fragmentation during the resistance phase could provide appropriate sources of this damage. Alternatively, the initial chromosomal replication in thymine (T)-starved cells could reflect a considerable endogenous dT source, making the resistance phase a delay of acute starvation, rather than an integral part of thymineless death. Here we identify such a low-molecular-weight (LMW)-dT source as mostly dTDP-glucose and its derivatives, used to synthesize enterobacterial common antigen (ECA). The thyA mutant, in which dTDP-glucose production is blocked by the rfbA rffH mutations, lacks a LMW-dT pool, the initial DNA synthesis during T-starvation and the resistance phase. Remarkably, the thyA mutant that makes dTDP-glucose and initiates ECA synthesis normally yet cannot complete it due to the rffC defect, maintains a regular LMW-dT pool, but cannot recover dTTP from it, and thus suffers T-hyperstarvation, dying precipitously, completely losing chromosomal DNA and eventually lysing, even without chromosomal replication. At the same time, its ECA+thyA parent does not lyse during T-starvation, while both the dramatic killing and chromosomal DNA loss in the ECA-deficient thyA mutants precede cell lysis. We conclude that: 1) the significant pool of dTDP-hexoses delays acute T-starvation; 2) T-starvation destabilizes even nonreplicating chromosomes, while T-hyperstarvation destroys them; and 3) beyond the chromosome, T-hyperstarvation also destabilizes the cell envelope.

scholarly journals Thymineless Death inEscherichia coliIs Unaffected by Chromosomal Replication Complexity

2019 ◽  
Vol 201 (9) ◽  
Author(s):  
Sharik R. Khan ◽  
Andrei Kuzminov
Keyword(s):  
Escherichia Coli ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
Functional Limit ◽  
Chromosomal Replication ◽  
Content Type ◽  
E Coli ◽  
Thymineless Death ◽  
Ts Mutant ◽  
Antimicrobial Treatments

ABSTRACTThymineless death (TLD) is a rapid loss of viability of unclear mechanism in cultures ofthyAmutants starved for thymine/thymidine (T starvation). It is accepted that T starvation repeatedly breaks replication forks, while recombinational repair restores them, but when the resulting futile breakage-repair cycle affects the small replication bubbles atoriC, the origin is degraded, killing the cell. Indeed, cells with increased chromosomal replication complexity (CRC), expressed as an elevated origin/terminus (ori/ter) ratio, die more extensively during TLD. Here we tested this logic by elevating the CRC inEscherichia colithyAmutants before T starvation, anticipating exaggerated TLD. Unexpectedly, TLD remained unaffected by a CRC increase to either the natural limit (ori/ter ratio, ∼6) or the functional limit (ori/ter ratio, ∼16). Moreover, when we forced the CRC over the functional limit (ori/ter ratio, ∼30), TLD lessened. Thus, prior overinitiation does not sensitize cells to TLD. In contradiction with the published results, even blocking new replication initiations by thednaA(Ts) defect at 42°C fails to prevent TLD. Using thethyA dnaA(Ts) mutant in a new T starvation protocol that excludes new initiations, we show that at 42°C, the same degree of TLD still occurs when chromosomes are demonstrably nonreplicating. Remarkably, 80% of the chromosomal DNA in these nonreplicating T-starved cells is still lost, by an unclear mechanism.IMPORTANCEThymineless death kills cells of any type and is used in anticancer and antimicrobial treatments. We tested the idea that the more replication forks there are in the chromosome during growth, the more extensive the resulting thymineless death. We varied the number of replication forks in theEscherichia colichromosome, as measured by the origin-to-terminus ratio, ranging it from the normal 2 to 60, and even completely eliminated replication forks in the nonreplicating chromosomes (ori/ter ratio = 1). Unexpectedly, we found that thymineless death is unaffected by the intensity of replication or by its complete absence; we also found that even nonreplicating chromosomes still disappear during thymine starvation. We conclude that thymineless death can killE. coliindependently of chromosomal replication.

scholarly journals Release of Compact Nucleoids with Characteristic Shapes from Escherichia coli

2001 ◽  
Vol 183 (17) ◽  
pp. 5041-5049 ◽  
Author(s):  
Steven B. Zimmerman ◽  
Lizabeth D. Murphy
Keyword(s):  
Escherichia Coli ◽  
Genomic Dna ◽  
Phase Contrast ◽  
Cell Envelope ◽  
Simple Procedure ◽  
Sodium Dodecyl ◽  
Cell Lysis ◽  
Exponential Phase ◽  
Stable Form ◽  
General Shape

ABSTRACT The genomic DNA of bacteria is contained in one or a few compact bodies known as nucleoids. We describe a simple procedure that retains the general shape and compaction of nucleoids from Escherichia coli upon cell lysis and nucleoid release from the cell envelope. The procedure is a modification of that used for the preparation of spermidine nucleoids (nucleoids released in the presence of spermidine) (T. Kornberg, A. Lockwood, and A. Worcel, Proc. Natl. Acad. Sci. USA 71:3189–3193, 1974). Polylysine is added to prevent the normal decompaction of nucleoids which occurs upon cell lysis. Nucleoids retained their characteristic shapes in lysates of exponential-phase cells or in lysates of cells treated with chloramphenicol or nalidixate to alter nucleoid morphology. The notably unstable nucleoids of rifampin-treated cells were obtained in compact, stable form in such lysates. Nucleoids released in the presence of polylysine were easily processed and provided well-defined DNA fluorescence and phase-contrast images. Uniform populations of nucleoids retaining characteristic shapes could be isolated after formaldehyde fixation and heating with sodium dodecyl sulfate.

Chromosomal replication origins (oriC) of Enterobacter aerogenes and Klebsiella pneumoniae are functional in Escherichia coli

1982 ◽  
Vol 152 (3) ◽  
pp. 983-993
Author(s):  
N E Harding ◽  
J M Cleary ◽  
D W Smith ◽  
J J Michon ◽  
W S Brusilow ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Enterobacter Aerogenes ◽  
Replication Origins ◽  
Chromosomal Dna ◽  
Protein Activity ◽  
Chromosomal Replication ◽  
E Coli ◽  
Dna Replication Origins ◽  
Unc Operon

The chromosomal DNA replication origins (oriC) from two members of the family Enterobacteriaceae, Enterobacter aerogenes and Klebsiella pneumoniae, have been isolated as functional replication origins in Escherichia coli. The origins in the SalI restriction fragments of 17.5 and 10.2 kilobase pairs, cloned from E. aerogenes and K. pneumoniae, respectively, were found to be between the asnA and uncB genes, as are the origins of the E. coli and Salmonella typhimurium chromosomes. Plasmids containing oriC from E aerogenes, K. pneumoniae, and S. typhimurium replicate in the E. coli cell-free enzyme system (Fuller, et al., Proc. Natl. Acad. Sci. U.S.A. 78:7370--7374, 1981), and this replication is dependent on dnaA protein activity. These SalI fragments from E. aerogenes and K. pneumoniae carry a region which is lethal to E. coli when many copies are present. We show that this region is also carried on the E. coli 9.0-kilobase-pair EcoRI restriction fragment containing oriC. The F0 genes of the atp or unc operon, when linked to the unc operon promoter, are apparently responsible for the lethality.

Cell lysis directed by σ E in early stationary phase and effect of induction of the rpoE gene on global gene expression in Escherichia coli

Microbiology ◽  
2005 ◽  
Vol 151 (8) ◽  
pp. 2721-2735 ◽  
Author(s):  
Md. Shahinur Kabir ◽  
Daisuke Yamashita ◽  
Satoshi Koyama ◽  
Taku Oshima ◽  
Ken Kurokawa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Cell Envelope ◽  
Expression System ◽  
Global Gene Expression ◽  
Cell Lysis ◽  
Specific Cell ◽  
Early Stationary Phase ◽  
Gene Encoding ◽  
Number Of Genes

It has been shown that Escherichia coli cells with increased expression of the rpoE gene encoding σ E exhibit enhanced cell lysis in early stationary phase. Further analysis of the lysis phenomenon was performed using a transient expression system of the rpoE gene and by DNA microarray. The former analysis revealed a σ E-directed cell lysis, specific for early stationary phase but not for the exponential phase. The microarray analysis with RNAs from exponential and early stationary phase cells revealed that a large number of genes were up- or down-regulated when the rpoE gene was induced, and that several genes were induced in a phase-specific manner. The upregulated genes include many previously identified σ E regulon genes, suggesting that a large number of genes are under the control of σ E in this organism. These genes are involved in various cellular activities, including the cell envelope, cellular processes, regulatory functions, transport and translation. Genes that are presumably related to phase-specific cell lysis in E. coli are discussed.

scholarly journals Completion of Replication Map of Saccharomyces cerevisiae Chromosome III

2001 ◽  
Vol 12 (11) ◽  
pp. 3317-3327 ◽  
Author(s):  
Arkadi Poloumienko ◽  
Ann Dershowitz ◽  
Jitakshi De ◽  
Carol S. Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Complete Analysis ◽  
Replication Origins ◽  
Chromosomal Dna ◽  
Chromosomal Replication ◽  
Autonomously Replicating Sequence ◽  
Eukaryotic Chromosome ◽  
Ars Elements ◽  
Chromosome Iii

In Saccharomyces cerevisiae chromosomal DNA replication initiates at intervals of ∼40 kb and depends upon the activity of autonomously replicating sequence (ARS) elements. The identification of ARS elements and analysis of their function as chromosomal replication origins requires the use of functional assays because they are not sufficiently similar to identify by DNA sequence analysis. To complete the systematic identification of ARS elements onS. cerevisiae chromosome III, overlapping clones covering 140 kb of the right arm were tested for their ability to promote extrachromosomal maintenance of plasmids. Examination of chromosomal replication intermediates of each of the seven ARS elements identified revealed that their efficiencies of use as chromosomal replication origins varied widely, with four ARS elements active in ≤10% of cells in the population and two ARS elements active in ≥90% of the population. Together with our previous analysis of a 200-kb region of chromosome III, these data provide the first complete analysis of ARS elements and DNA replication origins on an entire eukaryotic chromosome.

scholarly journals The lytB Gene of Escherichia coli Is Essential and Specifies a Product Needed for Isoprenoid Biosynthesis

2001 ◽  
Vol 183 (24) ◽  
pp. 7403-7407 ◽  
Author(s):  
Sean McAteer ◽  
Andrew Coulson ◽  
Neil McLennan ◽  
Millicent Masters
Keyword(s):  
Escherichia Coli ◽  
Protein Structure ◽  
Cell Lysis ◽  
Isoprenoid Biosynthesis ◽  
Globular Protein ◽  
Limited Growth ◽  
E Coli ◽  
Nonmevalonate Pathway ◽  
Tim Barrel

ABSTRACT LytB and GcpE, because they are codistributed with other pathway enzymes, have been predicted to catalyze unknown steps in the nonmevalonate pathway for isoprenoid biosynthesis. We constructed a conditional Escherichia coli lytB mutant and found that LytB is essential for survival and that depletion of LytB results in cell lysis, which is consistent with a role for this protein in isoprenoid biosynthesis. Alcohols which can be converted to pathway intermediates beyond the hypothesized LytB step(s) support limited growth of E. coli lytB mutants. An informatic analysis of protein structure suggested that GcpE is a globular protein of the TIM barrel class and that LytB is also a globular protein. Possible biochemical roles for LytB and GcpE are suggested.

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