Genetic and physical map of a P1 miniplasmid

1982 ◽  
Vol 152 (1) ◽  
pp. 63-71
Author(s):  
S Austin ◽  
F Hart ◽  
A Abeles ◽  
N Sternberg
Keyword(s):  
Physical Map ◽  
Plasmid Replication ◽  
Deletion Mutants ◽  
Plasmid Maintenance ◽  
Bacteriophage P1 ◽  
Daughter Cells ◽  
High Copy Number Plasmid ◽  
Stable Plasmid ◽  
Rep Region ◽  
Stable Unit

The prophage form of bacteriophage P1 is a unit-copy plasmid which is maintained with great fidelity in its Escherichia coli host. The plasmid maintenance functions of P1 are clustered in one region of the genome. An 11.5-kilobase fragment from this region has been cloned into a lambda delta att vector and promotes stable unit-copy plasmid maintenance. The properties of the lambda vector facilitated the isolation of deletion mutants affecting the P1 DNA. Twenty-eight deletion mutants were isolated, and their lesions were mapped by physical techniques. The genetic properties of the mutants with respect to plasmid replication, stability of plasmid maintenance, and ability to exert incompatibility effects against P1 and P7 plasmids were determined. These properties, along with those of several subfragments of the P1 insert cloned into high-copy-number plasmid vectors, allow the construction of an unambiguous genetic and physical map of the maintenance functions. A region of less than 3 kilobases, the rep region, is essential for plasmid replication and contains the incA incompatibility determinant within an 800-base-pair segment. Immediately adjacent to rep is a second region of approximately 3 kilobases which is required for stable plasmid maintenance, but not replication. This region, par, contains a second incompatibility element incB which is approximately 1 kilobase in size. The par region appears to specify equipartition of plasmid copies to daughter cells during cell division.

scholarly journals The phasmid as a tool for plasmid genetics: II. Isolation of point mutations that affect replication of a ColE1-related plasmid

1982 ◽  
Vol 40 (3) ◽  
pp. 233-247 ◽  
Author(s):  
Gianni Cesareni ◽  
Luisa Castagnoli ◽  
Sydney Brenner
Keyword(s):  
Copy Number ◽  
Point Mutations ◽  
Plasmid Replication ◽  
Single Base ◽  
Lambdoid Phage ◽  
Copy Number Plasmid ◽  
High Copy Number Plasmid ◽  
Single Base Pair ◽  
Related Plasmid ◽  
Single Base Pair Change

SUMMARYThe insertion of a high-copy-number plasmid into a lambdoid phage chromosome which lacks a functional repressor gene confers on the hybrid ‘phasmid’ the capacity to grow on an immune lysogen. This was found to be due to titration of repressor because of plasmid replication. We have exploited this property in order to isolate mutants that affect plasmid replication. These mutants have been mapped in a region that was previously characterized as necessary for plasmid replication and incompatibility properties. Some of the mutations could revert at frequencies characteristic of single-base-pair change mutations.

scholarly journals Regulation of Epstein-Barr Virus Origin of Plasmid Replication (OriP) by the S-Phase Checkpoint Kinase Chk2

2010 ◽  
Vol 84 (10) ◽  
pp. 4979-4987 ◽  
Author(s):  
Jing Zhou ◽  
Zhong Deng ◽  
Julie Norseen ◽  
Paul M. Lieberman
Keyword(s):  
Dna Replication ◽  
Epstein Barr Virus ◽  
Replication Timing ◽  
S Phase ◽  
Plasmid Replication ◽  
Acceptor Site ◽  
Plasmid Maintenance ◽  
Barr Virus ◽  
Amino Terminal ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) origin of plasmid replication (OriP) is required for episome stability during latent infection. Telomere repeat factor 2 (TRF2) binds directly to OriP and facilitates DNA replication and plasmid maintenance. Recent studies have found that TRF2 interacts with the DNA damage checkpoint protein Chk2. We show here that Chk2 plays an important role in regulating OriP plasmid stability, chromatin modifications, and replication timing. The depletion of Chk2 by small interfering RNA (siRNA) leads to a reduction in DNA replication efficiency and a loss of OriP-dependent plasmid maintenance. This corresponds to a change in OriP replication timing and an increase in constitutive histone H3 acetylation. We show that Chk2 interacts with TRF2 in the early G1/S phase of the cell cycle. We also show that Chk2 can phosphorylate TRF2 in vitro at a consensus acceptor site in the amino-terminal basic domain of TRF2. TRF2 mutants with a serine-to-aspartic acid phosphomimetic substitution mutation were reduced in their ability to recruit the origin recognition complex (ORC) and stimulate OriP replication. We suggest that the Chk2 phosphorylation of TRF2 is important for coordinating ORC binding with chromatin remodeling during the early S phase and that a failure to execute these events leads to replication defects and plasmid instability.

scholarly journals Efficient long fragment editing technique enables rapid construction of genetically stable bacterial strains

2020 ◽  
Author(s):  
Chaoyong Huang ◽  
Liwei Guo ◽  
Jingge Wang ◽  
Ning Wang ◽  
Yi-Xin Huo
Keyword(s):  
Open Reading Frames ◽  
Deletion Mutants ◽  
Dna Fragments ◽  
Bacterial Strains ◽  
Chromosome Engineering ◽  
Knock Out ◽  
Rapid Construction ◽  
Stable Plasmid ◽  
Chromosomal Engineering ◽  
Genomic Editing

Abstract Background Bacteria are versatile living systems that enhance our understanding of nature and enable biosynthesis of valuable molecules. Long fragment editing techniques are of great importance for accelerating bacterial chromosome engineering to obtain desirable and genetically stable strains. However, the existing genomic editing methods cannot meet the needs of researchers. Results We herein report an efficient long fragment editing technique for complex chromosomal engineering in Escherichia coli. The technique enabled us to integrate DNA fragments up to 12 kb into the chromosome, and to knock out DNA fragments up to 187 kb from the chromosome, with over 95% positive rates. We applied this technique for E. coli chromosomal simplification, resulting in twelve individual deletion mutants and four cumulative deletion mutants. The simplest chromosome lost a 370.6 kb DNA sequence containing 364 open reading frames. In addition, we applied the technique to metabolic engineering and constructed a genetically stable plasmid-independent isobutanol production strain that produced 1.3 g/L isobutanol via shake-flask micro-aerobic fermentation. Conclusions These results suggested that the technique is a powerful chromosomal engineering tool, highlighting its potential to be applied in different fields of synthetic biology.

Complementation of the Saccharomyces cerevisiae srb1-1 mutation: an autoselection system for stable plasmid maintenance

1992 ◽  
Vol 21 (4-5) ◽  
pp. 339-344 ◽  
Author(s):  
Sandra B. Rech ◽  
Lubomira I. Stateva ◽  
Stephen G. Oliver
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasmid Maintenance ◽  
Stable Plasmid

Efficient long fragment editing technique enables large-scale and scarless bacterial genome engineering

2020 ◽  
Author(s):  
Chaoyong Huang ◽  
Liwei Guo ◽  
Jingge Wang ◽  
Ning Wang ◽  
Yi-Xin Huo
Keyword(s):  
Metabolic Engineering ◽  
Large Scale ◽  
Genome Engineering ◽  
Bacterial Genome ◽  
Open Reading Frames ◽  
Deletion Mutants ◽  
Dna Fragments ◽  
E Coli ◽  
Stable Plasmid ◽  
Reading Frames

ABSTRACTBacteria are versatile living systems that enhance our understanding of nature and enable biosynthesis of valuable chemicals. Long fragment editing techniques are of great importance for accelerating bacterial genome engineering to obtain desirable and genetically stable strains. However, the existing genome editing methods cannot meet the needs of engineers. We herein report an efficient long fragment editing method for large-scale and scarless genome engineering in Escherichia coli. The method enabled us to insert DNA fragments up to 12 kb into the genome and to delete DNA fragments up to 186.7 kb from the genome, with positive rates over 95%. We applied this method for E. coli genome simplification, resulting in 12 individual deletion mutants and four cumulative deletion mutants. The simplest genome lost a total of 370.6 kb of DNA sequence containing 364 open reading frames. Additionally, we applied this technique to metabolic engineering and obtained a genetically stable plasmid-independent isobutanol production strain that produced 1.3 g/L isobutanol via shake-flask fermentation. These results suggest that the method is a powerful genome engineering tool, highlighting its potential to be applied in synthetic biology and metabolic engineering.

scholarly journals Discovery of small-molecule inhibitors of multidrug-resistance plasmid maintenance using a high-throughput screening approach

2020 ◽  
Vol 117 (47) ◽  
pp. 29839-29850
Author(s):  
Katelyn E. Zulauf ◽  
James E. Kirby
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Treatment Options ◽  
Antibiotic Resistance Genes ◽  
Multidrug Resistant ◽  
Replication Initiation ◽  
Plasmid Replication ◽  
Complete Suppression ◽  
Fluorescent Reporter ◽  
Plasmid Maintenance

Carbapenem-resistant Enterobacteriaceae (CRE) are multidrug-resistant pathogens for which new treatments are desperately needed. Carbapenemases and other types of antibiotic resistance genes are carried almost exclusively on large, low-copy-number plasmids (pCRE). Accordingly, small molecules that efficiently evict pCRE plasmids should restore much-needed treatment options. We therefore designed a high-throughput screen to identify such compounds. A synthetic plasmid was constructed containing the plasmid replication machinery from a representativeEscherichia coliCRE isolate as well as a fluorescent reporter gene to easily monitor plasmid maintenance. The synthetic plasmid was then introduced into anE. coliK12tolChost. We used this screening strain to test a library of over 12,000 known bioactive agents for molecules that selectively reduce plasmid levels relative to effects on bacterial growth. From 366 screen hits we further validated the antiplasmid activity of kasugamycin, an aminoglycoside; CGS 15943, a nucleoside analog; and Ro 90-7501, a bibenzimidazole. All three compounds exhibited significant antiplasmid activity including up to complete suppression of plasmid replication and/or plasmid eviction in multiple orthogonal readouts and potentiated activity of the carbapenem, meropenem, against a strain carrying the large, pCRE plasmid from which we constructed the synthetic screening plasmid. Additionally, we found kasugamycin and CGS 15943 blocked plasmid replication, respectively, by inhibiting expression or function of the plasmid replication initiation protein, RepE. In summary, we validated our approach to identify compounds that alter plasmid maintenance, confer resensitization to antimicrobials, and have specific mechanisms of action.

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