Dual adhesive unipolar polysaccharides synthesized by overlapping biosynthetic pathways in Agrobacterium tumefaciens.

Agrobacterium tumefaciens is a member of the Alphaproteobacteria that pathogenises plants and associates with biotic and abiotic surfaces via a single cellular pole. A. tumefaciens produces the unipolar polysaccharide (UPP) at the site of surface contact. UPP production is normally surface-contact inducible, but elevated levels of the second messenger cyclic diguanylate monophosphate (cdGMP) bypass this requirement. Multiple lines of evidence suggest that the UPP has a central polysaccharide component. Using an A. tumefaciens derivative with elevated cdGMP and mutationally disabled for other dispensable polysaccharides, a series of related genetic screens have identified a large number of genes involved in UPP biosynthesis, most of which are Wzx-Wzy-type polysaccharide biosynthetic components. Extensive analyses of UPP production in these mutants have revealed that the UPP is composed of two genetically, chemically, and spatially discrete forms of polysaccharide, and that each requires a specific Wzy-type polymerase. Other important biosynthetic, processing, and regulatory functions for UPP production are also revealed, some of which are common to both polysaccharides, and a subset of which are specific to each type. Many of the UPP genes identified are conserved among diverse rhizobia, whereas others are more lineage specific.

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

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.