mRNA stability and plasmid copy number effects on gene expression from an inducible promoter system

Plasmids are one of the most commonly used and time-tested molecular biology platforms for genetic engineering and recombinant gene expression in bacteria. Despite their ubiquity, little consideration is given to metabolic effects and fitness costs of plasmid copy numbers on engineered genetic systems. Here, we introduce two systems that allow for the finely-tuned control of plasmid copy number: a plasmid with an anhydrotetracycline-controlled copy number, and a massively parallel assay that is used to generate a continuous spectrum of ColE1-based copy number variants. Using these systems, we investigate the effects of plasmid copy number on cellular growth rates, gene expression, biosynthesis, and genetic circuit performance. We perform single-cell timelapse measurements to characterize plasmid loss, runaway plasmid replication, and quantify the impact of plasmid copy number on the variability of gene expression. Using our massively parallel assay, we find that each plasmid imposes a 0.063% linear metabolic burden on their hosts, hinting at a simple relationship between metabolic burdens and plasmid DNA synthesis. Our plasmid system with tunable copy number should allow for a precise control of gene expression and highlight the importance of tuning plasmid copy number as tool for the optimization of synthetic biological systems.

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Escherichia coli O157:H7 Strains Isolated from High-Event Period Beef Contamination Have Strong Biofilm-Forming Ability and Low Sanitizer Susceptibility, Which Are Associated with High pO157 Plasmid Copy Number

Journal of Food Protection ◽

10.4315/0362-028x.jfp-16-113 ◽

2016 ◽

Vol 79 (11) ◽

pp. 1875-1883 ◽

Cited By ~ 8

Author(s):

RONG WANG ◽

BRANDON E. LUEDTKE ◽

JOSEPH M. BOSILEVAC ◽

JOHN W. SCHMIDT ◽

NORASAK KALCHAYANAND ◽

...

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Biofilm Formation ◽

Copy Number ◽

Plasmid Copy Number ◽

Bacterial Biofilm ◽

Escherichia Coli O157 ◽

Plasmid Copy ◽

E Coli ◽

High Event

ABSTRACT In the meat industry, a high-event period (HEP) is defined as a time period when beef processing establishments experience an increased occurrence of product contamination by Escherichia coli O157:H7. Our previous studies suggested that bacterial biofilm formation and sanitizer resistance might contribute to HEPs. We conducted the present study to further characterize E. coli O157:H7 strains isolated during HEPs for their potential to cause contamination and to investigate the genetic basis for their strong biofilm-forming ability and high sanitizer resistance. Our results show that, compared with the E. coli O157:H7 diversity control panel strains, the HEP strains had a significantly higher biofilm-forming ability on contact surfaces and a lower susceptibility to common sanitizers. No difference in the presence of disinfectant-resistant genes or the prevalence of antibiotic resistance was observed between the HEP and control strains. However, the HEP strains retained significantly higher copy numbers of the pO157 plasmid. A positive correlation was observed among a strain's high plasmid copy number, strong biofilm-forming ability, low sanitizer susceptibility, and high survival and recovery capability after sanitization, suggesting that these specific phenotypes could be either directly correlated to gene expression on the pO157 plasmid or indirectly regulated via chromosomal gene expression influenced by the presence of the plasmid. Our data highlight the potential risk of biofilm formation and sanitizer resistance in HEP contamination by E. coli O157:H7, and our results call for increased attention to proper and effective sanitization practices in meat processing facilities.

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Faculty Opinions recommendation of Increased plasmid copy number is essential for Yersinia T3SS function and virulence.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726475088.793562337 ◽

2019 ◽

Author(s):

Joan Mecsas

Keyword(s):

Copy Number ◽

Plasmid Copy Number ◽

Plasmid Copy

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Segregational Drift and the Interplay between Plasmid Copy Number and Evolvability

Molecular Biology and Evolution ◽

10.1093/molbev/msy225 ◽

2018 ◽

Vol 36 (3) ◽

pp. 472-486 ◽

Cited By ~ 18

Author(s):

Judith Ilhan ◽

Anne Kupczok ◽

Christian Woehle ◽

Tanita Wein ◽

Nils F Hülter ◽

...

Keyword(s):

Copy Number ◽

Plasmid Copy Number ◽

Plasmid Copy

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RNA polyadenylation and its consequences in prokaryotes

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2018.0166 ◽

2018 ◽

Vol 373 (1762) ◽

pp. 20180166 ◽

Cited By ~ 7

Author(s):

Eliane Hajnsdorf ◽

Vladimir R. Kaberdin

Keyword(s):

Gene Expression ◽

State Level ◽

Rna Degradation ◽

Plasmid Copy Number ◽

Plasmid Copy ◽

Protein Coding ◽

Mrna Metabolism ◽

Isolation And Characterization ◽

Polymerase I ◽

The Impact

Post-transcriptional addition of poly(A) tails to the 3′ end of RNA is one of the fundamental events controlling the functionality and fate of RNA in all kingdoms of life. Although an enzyme with poly(A)-adding activity was discovered in Escherichia coli more than 50 years ago, its existence and role in prokaryotic RNA metabolism were neglected for many years. As a result, it was not until 1992 that E. coli poly(A) polymerase I was purified to homogeneity and its gene was finally identified. Further work revealed that, similar to its role in surveillance of aberrant nuclear RNAs of eukaryotes, the addition of poly(A) tails often destabilizes prokaryotic RNAs and their decay intermediates, thus facilitating RNA turnover. Moreover, numerous studies carried out over the last three decades have shown that polyadenylation greatly contributes to the control of prokaryotic gene expression by affecting the steady-state level of diverse protein-coding and non-coding transcripts including antisense RNAs involved in plasmid copy number control, expression of toxin–antitoxin systems and bacteriophage development. Here, we review the main findings related to the discovery of polyadenylation in prokaryotes, isolation, and characterization and regulation of bacterial poly(A)-adding activities, and discuss the impact of polyadenylation on prokaryotic mRNA metabolism and gene expression. This article is part of the theme issue ‘5′ and 3′ modifications controlling RNA degradation’.

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