A novel plasmid-transcribed regulatory sRNA, QfsR, controls chromosomal polycistronic mRNAs in Agrobacterium tumefaciens

AbstractPlasmids are mobile DNAs that adjust host cell functions for their own amplification and dissemination. We identified QfsR, a small RNA, transcribed from the Ti plasmid in the phytopathogen Agrobacterium fabrum. QfsR is widely conserved throughout RepABC plasmids carried by Rhizobiaceae. Target prediction, expression analysis and site-direct mutagenesis experiments show that QfsR directly pairs within polycistronic mRNAs transcribed from chromosomes (involved in flagella apparatus and succinoglycan biosynthesis) and Ti plasmid (involved in conjugative transfer). QfsR leads to a coordinated expression of whole polycistronic mRNA molecules. Whereas a lack of QfsR induces motility and reduces succinoglycan production, its overproduction increases the quorum sensing signal accumulation and the Ti plasmid conjugative transfer. Based on these observations, we propose QfsR as a hub connecting regulatory networks of motility, succinoglycan biosynthesis and plasmid conjugative transfer. To our knowledge, QfsR is the first example of a plasmid-encoded sRNA that controls chromosomal polycistronic mRNAs.SignificancePlasmids represent an important cost for the hosting cell although some are beneficial under certain circumstances. Agrobacterium tumefaciens harboring Tumor inducing plasmid (pTi) are able to infect plants and to use specific resources produced by the infected cells. We characterized QfsR, a novel small RNA (sRNA) from pTi, that directly regulates plasmid polycistronic mRNA but also chromosomal ones. QfsR contributes to a fine-tuned regulation of bacterial motility, exopolysaccharide biosynthesis and conjugative dissemination of pTi. Our results report the first plasmid-encoded sRNA able to modify and coordinate cellular behaviour probably for the benefit of the plasmid dissemination and tight crosstalk between plasmid and chromosome. This could be widespread since QfsR homologs were predicted in other plasmids of Rhizobiaceae symbionts and pathogens.

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Co-Dependent and Interdigitated: Dual Quorum Sensing Systems Regulate Conjugative Transfer of the Ti Plasmid and the At Megaplasmid in Agrobacterium tumefaciens 15955

10.1101/2020.12.15.422598 ◽

2020 ◽

Author(s):

Ian S Barton ◽

Justin L Eagan ◽

Priscila A Nieves-Otero ◽

Ian P Reynolds ◽

Thomas G Patt ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Quorum Sensing ◽

Homoserine Lactone ◽

Ti Plasmid ◽

Conjugative Transfer ◽

Gene Promoters ◽

Transcription Activator ◽

Conjugation System ◽

Direct Production ◽

Sensing Systems

Members of the Rhizobiaceae, often carry multiple secondary replicons in addition to the primary chromosome with compatible repABC-based replication systems. Unlike secondary chromosomes and chromids, repABC-based megaplasmids and plasmids can undergo copy number fluctuations and are capable of conjugative transfer in response to environmental signals. Several Agrobacterium tumefaciens lineages harbor three secondary repABC-based replicons, including a secondary chromosome (often linear), the Ti (tumor-inducing) plasmid and the At megaplasmid. The Ti plasmid is required for virulence and encodes a conjugative transfer (tra) system that is strictly regulated by a subset of plant-tumor released opines and a well-described acyl-homoserine lactone (AHL)-based quorum-sensing mechanism. At plasmids are generally not required for virulence, but carry genes that enhance rhizosphere survival, and these plasmids are often conjugatively proficient. We report that the At megaplasmid of the octopine-type strain A. tumefaciens 15955 encodes a quorum-controlled conjugation system that directly interacts with the paralogous quorum sensing system on the co-resident Ti plasmid. Both the pAt15955 and pTi15955 plasmids carry homologues of a TraI-type AHL synthase, a TraR-type AHL-responsive transcription activator, and a TraM-type anti-activator. The traI genes from both pTi15955 and pAt15955 can direct production of the inducing AHL (3-octanoyl-L-homoserine lactone) and together contribute to the overall AHL pool. The TraR protein encoded on each plasmid activates AHL-responsive transcription of target tra gene promoters. The pAt15955 TraR can cross-activate tra genes on the Ti plasmid as strongly as its cognate tra genes, whereas the pTi15955 TraR preferentially biased towards its own tra genes. Putative tra box elements are located upstream of target promoters, and comparing between plasmids, they are in similar locations and share an inverted repeat structure, but have distinct consensus sequences. The two AHL quorum sensing systems have a combinatorial effect on conjugative transfer of both plasmids. Overall, the interactions described here have implications for the horizontal transfer and evolutionary stability of both plasmids and, in a broad sense, are consistent with other repABC systems that often have multiple quorum-sensing controlled secondary replicons.

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Co-dependent and Interdigitated: Dual Quorum Sensing Systems Regulate Conjugative Transfer of the Ti Plasmid and the At Megaplasmid in Agrobacterium tumefaciens 15955

Frontiers in Microbiology ◽

10.3389/fmicb.2020.605896 ◽

2021 ◽

Vol 11 ◽

Author(s):

Ian S. Barton ◽

Justin L. Eagan ◽

Priscila A. Nieves-Otero ◽

Ian P. Reynolds ◽

Thomas G. Platt ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Quorum Sensing ◽

Homoserine Lactone ◽

Ti Plasmid ◽

Conjugative Transfer ◽

Gene Promoters ◽

Transcription Activator ◽

Conjugation System ◽

Direct Production ◽

Sensing Systems

Members of the Rhizobiaceae, often carry multiple secondary replicons in addition to the primary chromosome with compatible repABC-based replication systems. Unlike secondary chromosomes and chromids, repABC-based megaplasmids and plasmids can undergo copy number fluctuations and are capable of conjugative transfer in response to environmental signals. Several Agrobacterium tumefaciens lineages harbor three secondary repABC-based replicons, including a secondary chromosome (often linear), the Ti (tumor-inducing) plasmid and the At megaplasmid. The Ti plasmid is required for virulence and encodes a conjugative transfer (tra) system that is strictly regulated by a subset of plant-tumor released opines and a well-described acyl-homoserine lactone (AHL)-based quorum-sensing mechanism. The At plasmids are generally not required for virulence, but carry genes that enhance rhizosphere survival, and these plasmids are often conjugatively proficient. We report that the At megaplasmid of the octopine-type strain A. tumefaciens 15955 encodes a quorum-controlled conjugation system that directly interacts with the paralogous quorum sensing system on the co-resident Ti plasmid. Both the pAt15955 and pTi15955 plasmids carry homologs of a TraI-type AHL synthase, a TraR-type AHL-responsive transcription activator, and a TraM-type anti-activator. The traI genes from both pTi15955 and pAt15955 can direct production of the inducing AHL (3-octanoyl-L-homoserine lactone) and together contribute to the overall AHL pool. The TraR protein encoded on each plasmid activates AHL-responsive transcription of target tra gene promoters. The pAt15955 TraR can cross-activate tra genes on the Ti plasmid as strongly as its cognate tra genes, whereas the pTi15955 TraR is preferentially biased toward its own tra genes. Putative tra box elements are located upstream of target promoters, and comparing between plasmids, they are in similar locations and share an inverted repeat structure, but have distinct consensus sequences. The two AHL quorum sensing systems have a combinatorial effect on conjugative transfer of both plasmids. Overall, the interactions described here have implications for the horizontal transfer and evolutionary stability of both plasmids and, in a broad sense, are consistent with other repABC systems that often have multiple quorum-sensing controlled secondary replicons.

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The BlcC (AttM) Lactonase of Agrobacterium tumefaciens Does Not Quench the Quorum-Sensing System That Regulates Ti Plasmid Conjugative Transfer

Journal of Bacteriology ◽

10.1128/jb.01304-08 ◽

2008 ◽

Vol 191 (4) ◽

pp. 1320-1329 ◽

Cited By ~ 42

Author(s):

Sharik R. Khan ◽

Stephen K. Farrand

Keyword(s):

Agrobacterium Tumefaciens ◽

Quorum Sensing ◽

Homoserine Lactone ◽

Ti Plasmid ◽

Mutation Induction ◽

Conjugative Transfer ◽

Wild Type ◽

In Planta ◽

Sensing System ◽

Quorum Sensing System

ABSTRACT The conjugative transfer of Agrobacterium plasmids is controlled by a quorum-sensing system consisting of TraR and its acyl-homoserine lactone (HSL) ligand. The acyl-HSL is essential for the TraR-mediated activation of the Ti plasmid Tra genes. Strains A6 and C58 of Agrobacterium tumefaciens produce a lactonase, BlcC (AttM), that can degrade the quormone, leading some to conclude that the enzyme quenches the quorum-sensing system. We tested this hypothesis by examining the effects of the mutation, induction, or mutational derepression of blcC on the accumulation of acyl-HSL and on the conjugative competence of strain C58. The induction of blc resulted in an 8- to 10-fold decrease in levels of extracellular acyl-HSL but in only a twofold decrease in intracellular quormone levels, a measure of the amount of active intracellular TraR. The induction or mutational derepression of blc as well as a null mutation in blcC had no significant effect on the induction of or continued transfer of pTiC58 from donors in any stage of growth, including stationary phase. In matings performed in developing tumors, wild-type C58 transferred the Ti plasmid to recipients, yielding transconjugants by 14 to 21 days following infection. blcC-null donors yielded transconjugants 1 week earlier, but by the following week, transconjugants were recovered at numbers indistinguishable from those of the wild type. Donors mutationally derepressed for blcC yielded transconjugants in planta at numbers 10-fold lower than those for the wild type at weeks 2 and 3, but by week 4, the two donors showed no difference in recoverable transconjugants. We conclude that BlcC has no biologically significant effect on Ti plasmid transfer or its regulatory system.

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