The effect of plasmid copy number mutations on pT181 replication initiator protein expression

ABSTRACT Replication of the IncB miniplasmid pMU720 requires synthesis of the replication initiator protein, RepA, whose translation is coupled to that of a leader peptide, RepB. The unusual feature of this system is that translational coupling in repBA has to be activated by the formation of a pseudoknot immediately upstream of the repA Shine-Dalgarno sequence. A small antisense RNA, RNAI, controls replication of pMU720 by interacting with repBA mRNA to inhibit expression of repA both directly, by preventing formation of the pseudoknot, and indirectly, by inhibiting translation of repB. The mechanism of translational coupling in repBA was investigated using the specialized ribosome system, which directs a subpopulation of ribosomes that carry an altered anti-Shine-Dalgarno sequence to translate mRNA molecules whose Shine-Dalgarno sequences have been altered to be complementary to the mutant anti-Shine-Dalgarno sequence. Our data indicate that translation of repA involves reinitiation by the ribosome that has terminated translation of repB. The role of the pseudoknot in this process and its effect on the control of copy number in pMU720 are discussed.

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Single-cell measurement of plasmid copy number and promoter activity

Nature Communications ◽

10.1038/s41467-021-21734-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Bin Shao ◽

Jayan Rammohan ◽

Daniel A. Anderson ◽

Nina Alperovich ◽

David Ross ◽

...

Keyword(s):

Protein Expression ◽

Plasmid Dna ◽

Copy Number ◽

Promoter Activity ◽

Plasmid Copy Number ◽

Plasmid Copy ◽

Rna Transcripts ◽

Cell Measurement ◽

Single Living ◽

Living Bacteria

AbstractAccurate measurements of promoter activities are crucial for predictably building genetic systems. Here we report a method to simultaneously count plasmid DNA, RNA transcripts, and protein expression in single living bacteria. From these data, the activity of a promoter in units of RNAP/s can be inferred. This work facilitates the reporting of promoters in absolute units, the variability in their activity across a population, and their quantitative toll on cellular resources, all of which provide critical insights for cellular engineering.

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Determination of protein expression and plasmid copy number from cloned genes inEscherichia coli by flow injection analysis using an enzyme indicator vector

Biotechnology and Bioengineering ◽

10.1002/bit.260340802 ◽

1989 ◽

Vol 34 (8) ◽

pp. 1023-1036 ◽

Cited By ~ 13

Author(s):

F. J. Schendel ◽

E. J. Baude ◽

M. C. Flickinger

Keyword(s):

Protein Expression ◽

Flow Injection ◽

Flow Injection Analysis ◽

Copy Number ◽

Plasmid Copy Number ◽

Inescherichia Coli ◽

Plasmid Copy

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Conjugative plasmid-encoded toxin–antitoxin system PrpT/PrpA directly controls plasmid copy number

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2011577118 ◽

2021 ◽

Vol 118 (4) ◽

pp. e2011577118

Author(s):

Songwei Ni ◽

Baiyuan Li ◽

Kaihao Tang ◽

Jianyun Yao ◽

Thomas K. Wood ◽

...

Keyword(s):

Copy Number ◽

Plasmid Copy Number ◽

Negative Regulator ◽

Conjugative Plasmid ◽

Plasmid Replication ◽

Plasmid Copy ◽

Conjugative Plasmids ◽

Low Copy Number ◽

Replication Initiator ◽

Postsegregational Killing

Toxin–antitoxin (TA) loci were initially identified on conjugative plasmids, and one function of plasmid-encoded TA systems is to stabilize plasmids or increase plasmid competition via postsegregational killing. Here, we discovered that the type II TA system, Pseudoalteromonas rubra plasmid toxin–antitoxin PrpT/PrpA, on a low-copy-number conjugative plasmid, directly controls plasmid replication. Toxin PrpT resembles ParE of plasmid RK2 while antitoxin PrpA (PF03693) shares no similarity with previously characterized antitoxins. Surprisingly, deleting this prpA-prpT operon from the plasmid does not result in plasmid segregational loss, but greatly increases plasmid copy number. Mechanistically, the antitoxin PrpA functions as a negative regulator of plasmid replication, by binding to the iterons in the plasmid origin that inhibits the binding of the replication initiator to the iterons. We also demonstrated that PrpA is produced at a higher level than PrpT to prevent the plasmid from overreplicating, while partial or complete degradation of labile PrpA derepresses plasmid replication. Importantly, the PrpT/PrpA TA system is conserved and is widespread on many conjugative plasmids. Altogether, we discovered a function of a plasmid-encoded TA system that provides new insights into the physiological significance of TA systems.

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