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

Abstract

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.

Keywords: conjugative transfer; gene regulation; plasmids; quorum sensing (QS); repABC system.

Figure 1

Octopine-induced activation of P_traA and P_traI from pAt and pTi in WT A. tumefaciens 15955. WT A. tumefaciens 15955 carrying a plasmid-borne copy of either P_traA (top) or P_traI (bottom) from pTi15955 (green) or pAt15955 (blue) translationally fused at their start codons to lacZ were spotted on 0.2 μm cellulose acetate filter discs placed on ATGN with or without 3.25 mM octopine (Oct.) and/or 0.1 μM N-3-oxo-C8-HSL (AHL) and incubated at 28°C for 48 h. Bars with no, light, medium, or dark fill indicate no addition, addition of AHL, addition of octpine, or addition of both octopine and AHL, respectively. Cells were resuspended and promoter activities were calculated as Miller Units (Methods). Bars under the same bracket are not significantly different, unless they are marked with an asterisk (p < 0.05). Bars under brackets that have the same number above them (1, 2, 3) in each panel (A,B) denote a significant difference in averages (p < 0.05) between the bars under the similarly numbered brackets.

Figure 2

Qualitative and quantitative AHL assays of A. tumefaciens strains ectopically expressing traI genes, and traI mutant derivatives. (A) AHL production by A. tumefaciens NTL4 (Ti plasmidless, AHL⁻) ectopically expressing traI genes from pAt15955 and pTi15955 expressed from P_lac (top streaks). These strains were used in a plate-based AHL production diffusion assays (McLean et al., 1997) with NTL4(pCF218)(pCF372), (AHL reporter, bottom streak). The traI designations are listed above. Media was ATGN agar supplemented with IPTG (400 μM) and X-Gal (40 ug/ml). (B) A. tumefaciens 15955 or derivatives with clean deletions in traI from pTi15955 (ΔtraI^Ti), pAt15955 (ΔtraI^At_1), or both (ΔtraI^Ti; ΔtraI^At_1) (top streak) were used in plate-based AHL production assays with A. tumefaciens NTL4(pCF218)(pCF372) (AHL reporter, bottom streak). (C) Cell-free supernatants from A. tumefaciens 15955 (WT, mixed green/blue bar) and derivatives mutated for traI on pTi15955 (ΔtraI^Ti, blue bar), pAt15955 (ΔtraI^At_1, green bar), or both (ΔtraI^Ti; ΔtraI^At_1, open bar) were added to subcultures of the AHL biosensor strain, A. tumefaciens NTL4(pCF218)(pCF372), and allowed to grow until mid-exponential phase. Standard β-gal assays were then conducted on reporter strain cultures to determine relative AHL levels in the cell-free supernatant, calculated as β-gal activity (Methods). AHL- indicates cell-free supernatant from the biosensor strain A. tumefaciens NTL4(pCF218)(pCF372) (gray bar). Assays performed as three biological replicates, and bars marked with asterisks are significantly different from wild type (p < 0.05).

Figure 3

Stimulation of pTi15955 and pAt15955 conjugation by traR expression is dependent on pAt15955- and pTi15955-encoded traI. A tumefaciens 15955 derivatives with a GmR cassette integrated at a neutral location on pTi15955 (green bars) or a KmR cassette similarly integrated on pAt15955 (blue bars) harboring either a plasmid-borne copy of P_lac-traR from pTi15955 (light green and light blue bars) or pAt15955 (dark green and dark blue bars) were mixed 1:1 with a plasmidless recipient (A. tumefaciens ERM52, SpR) and spotted on ATGN agar containing 400 μM IPTG. After 24 h incubation, transconjugants and donors were enumerated and conjugation frequencies were calculated as transconjugants per output donor (Methods). Green and blue bars depict pTi15955 and pAt15955 conjugation frequencies, respectively. Asterisks denote significant difference between averages relative to wild type (p < 0.05).

Figure 4

AHL production induced by ectopic expression of traR^Ti and traR^At in A. tumefaciens 15955. A. tumefaciens 15955 expressing a plasmid-borne copy of (A) traR^Ti or (B) traR^At with or without in-frame deletions in traI^Ti, traI^At_1, or both (top streaks) were used as AHL donors for A. tumefaciens NTL4(pCF218)(pCF372) (bottom streaks) on ATGN media supplemented with XGal, with or without 400 μM IPTG (top and bottom plates, respectively). Genotypes of A. tumefaciens 15955 AHL donor strain relative to traI indicated above.

Figure 5

Mutational analysis of traR gene with traI reporter fusions. WT A. tumefaciens 15955 and mutant derivatives (ΔtraR^Ti and ΔtraR^At), and (ΔtraR^TiΔtraR^At) carrying a plasmid-borne copy of either P_traI from pTi15955 (A, green bars) or from pAt15955 (B, blue bars) translationally fused to lacZ were spotted on 0.2 μm cellulose acetate filter discs placed on ATGN on its own (open bars), with 0.1 μM N-3-oxo-C8-HSL (AHL, light-fill bars), with 3.25 mM octopine (Oct., medium-fill bars), and with both (dark-fill bars) Cells were resuspended and promoter activities were calculated as Miller Units (Methods). Bars are standard deviation. All bars that differ by 10-fold or more are significant (p < 0.05). Bars that differ by <10-fold, that are however significantly different (p < 0.05) are designated with a bracketed asterisk.

Figure 6

Differential activation of pAt15955 and pTi15955 P_traA and P_traI by traR. Plasmid-borne copies of P_traA-lacZ or P_traI-lacZ from pTi15955 (green) or pAt15955 (blue) and P_lac-traR with traR genes from either pAt15955 (At) or pTi15955 (Ti) were moved into A. tumefaciens NTL4 and β-galactosidase activity (MU) of P_traA (A) or P_traI (B) were determined in the presence or absence of 0.1 μM 3-oxo-C8-HSL (+ AHL and – AHL, respectively). Bars with the same numbers possess significant difference in averages (p < 0.05).

Figure 7

Model of Dual Quorum Sensing Systems in A. tumefaciens 15955. Green shaded symbols representing proteins and genes from pTi15955, and blue shaded symbols represent proteins and genes from pAt15955. Solid arrows indicate translation of specific genes, dotted arrows represent enzymatic synthesis of AHL, and dashed arrows are regulatory impacts of each system. The thinner arrows from TraR^Ti to the pAt15955 target genes reflect the observed weaker effect of this protein on their expression.