A LuxR-type regulator from Agrobacterium tumefaciens elevates Ti plasmid copy number by activating transcription of plasmid replication genes

ABSTRACT The replicator (rep) of the nopaline-type Ti plasmid pTiC58 is located adjacent to the trb operon of this conjugal element. Previous genetic studies of this region (D. R. Gallie, M. Hagiya, and C. I. Kado, J. Bacteriol. 161:1034–1041, 1985) identified functions involved in partitioning, origin of replication and incompatibility, and copy number control. In this study, we determined the nucleotide sequence of a 6,146-bp segment that encompasses the rep locus of pTiC58. The region contained four full open reading frames (ORFs) and one partial ORF. The first three ORFs, oriented divergently from the traI-trb operon, are closely related to the repA, repB, andrepC genes of the octopine-type Ti plasmid pTiB6S3 as well as to other repA, -B, and -C genes from the Ri plasmid pRiA4b and three large plasmids fromRhizobium spp. The fourth ORF and the partial ORF are similar to y4CG and y4CF, respectively, of the Sym plasmid pNGR234a. The 363-bp intergenic region betweentraI and repA contained two copies of thetra box which is the cis promoter recognition site for TraR, the quorum-sensing activator of Ti plasmid conjugal transfer. Expression of the traI-trb operon from thetra box II-associated promoter mediated by TraR and its acyl-homoserine lactone ligand, AAI, was negatively influenced by an intact tra box III. On the other hand, the region containing the two tra boxes was required for maximal expression of repA, and this expression was enhanced slightly by TraR and AAI. Copy number of a minimal repplasmid increased five- to sevenfold in strains expressingtraR but only when AAI also was provided. Consistent with this effect, constitutive expression of the quorum-sensing system resulted in an apparent increase in Ti plasmid copy number. We conclude that Ti plasmid copy number is influenced by the quorum-sensing system, suggesting a connection between conjugal transfer and vegetative replication of these virulence elements.

Download Full-text

Antisense-RNA-mediated plasmid copy number control in pCG1-family plasmids, pCGR2 and pCG1, in Corynebacterium glutamicum

Microbiology ◽

10.1099/mic.0.043745-0 ◽

2010 ◽

Vol 156 (12) ◽

pp. 3609-3623 ◽

Cited By ~ 11

Author(s):

Naoko Okibe ◽

Nobuaki Suzuki ◽

Masayuki Inui ◽

Hideaki Yukawa

Keyword(s):

Corynebacterium Glutamicum ◽

Antisense Rna ◽

Copy Number ◽

Shuttle Vector ◽

Fold Increase ◽

Plasmid Copy Number ◽

Plasmid Replication ◽

Plasmid Copy ◽

Rna Molecules ◽

Target Mrnas

pCGR2 and pCG1 belong to different subfamilies of the pCG1 family of Corynebacterium glutamicum plasmids. Nonetheless, they harbour homologous putative antisense RNA genes, crrI and cgrI, respectively. The genes in turn share identical positions complementary to the leader region of their respective repA (encoding plasmid replication initiator) genes. Determination of their precise transcriptional start- and end-points revealed the presence of short antisense RNA molecules (72 bp, CrrI; and 73 bp, CgrI). These short RNAs and their target mRNAs were predicted to form highly structured molecules comprising stem–loops with known U-turn motifs. Abolishing synthesis of CrrI and CgrI by promoter mutagenesis resulted in about sevenfold increase in plasmid copy number on top of an 11-fold (CrrI) and 32-fold (CgrI) increase in repA mRNA, suggesting that CrrI and CgrI negatively control plasmid replication. This control is accentuated by parB, a gene that encodes a small centromere-binding plasmid-partitioning protein, and is located upstream of repA. Simultaneous deactivation of CrrI and parB led to a drastic 87-fold increase in copy number of a pCGR2-derived shuttle vector. Moreover, the fact that changes in the structure of the terminal loops of CrrI and CgrI affected plasmid copy number buttressed the important role of the loop structure in formation of the initial interaction complexes between antisense RNAs and their target mRNAs. Similar antisense RNA control systems are likely to exist not only in the two C. glutamicum pCG1 subfamilies but also in related plasmids across Corynebacterium species.

Download Full-text

VirA and VirG activate the Ti plasmid repABC operon, elevating plasmid copy number in response to wound-released chemical signals

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0503458102 ◽

2005 ◽

Vol 102 (41) ◽

pp. 14843-14848 ◽

Cited By ~ 52

Author(s):

H. Cho ◽

S. C. Winans

Keyword(s):

Copy Number ◽

Plasmid Copy Number ◽

Chemical Signals ◽

Ti Plasmid ◽

Plasmid Copy

Download Full-text

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.

Download Full-text

Characterization of a Large, Stable, High-Copy-Number Streptomyces Plasmid That Requires Stability and Transfer Functions for Heterologous Polyketide Overproduction

Applied and Environmental Microbiology ◽

10.1128/aem.01888-06 ◽

2006 ◽

Vol 73 (4) ◽

pp. 1296-1307 ◽

Cited By ~ 11

Author(s):

Ryan Fong ◽

Zhihao Hu ◽

C. Richard Hutchinson ◽

Jianqiang Huang ◽

Stanley Cohen ◽

...

Keyword(s):

Copy Number ◽

Transfer Functions ◽

Biosynthetic Gene Cluster ◽

Plasmid Copy Number ◽

High Copy Number ◽

Plasmid Replication ◽

Biosynthetic Genes ◽

Plasmid Copy ◽

Metabolite Production ◽

Copy Numbers

ABSTRACT A major limitation to improving small-molecule pharmaceutical production in streptomycetes is the inability of high-copy-number plasmids to tolerate large biosynthetic gene cluster inserts. A recent finding has overcome this barrier. In 2003, Hu et al. discovered a stable, high-copy-number, 81-kb plasmid that significantly elevated production of the polyketide precursor to the antibiotic erythromycin in a heterologous Streptomyces host (J. Ind. Microbiol. Biotechnol. 30:516-522, 2003). Here, we have identified mechanisms by which this SCP2*-derived plasmid achieves increased levels of metabolite production and examined how the 45-bp deletion mutation in the plasmid replication origin increased plasmid copy number. A plasmid intramycelial transfer gene, spd, and a partition gene, parAB, enhance metabolite production by increasing the stable inheritance of large plasmids containing biosynthetic genes. Additionally, high product titers required both activator (actII-ORF4) and biosynthetic genes (eryA) at high copy numbers. DNA gel shift experiments revealed that the 45-bp deletion abolished replication protein (RepI) binding to a plasmid site which, in part, supports an iteron model for plasmid replication and copy number control. Using the new information, we constructed a large high-copy-number plasmid capable of overproducing the polyketide 6-deoxyerythronolide B. However, this plasmid was unstable over multiple culture generations, suggesting that other SCP2* genes may be required for long-term, stable plasmid inheritance.

Download Full-text