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.

Reductive tricarboxylic acid cycle enzymes and reductive amino acid synthesis pathways contribute to electron balance in a Rhodospirillum rubrum Calvin-cycle mutant.

Purple non-sulfur bacteria (PNSB) use light for energy and organic substrates for carbon and electrons when growing photoheterotrophically. This lifestyle generates more reduced electron carriers than are required for biosynthesis, even during consumption of some of the most oxidized organic substrates like malate and fumarate. Reduced electron carriers not used in biosynthesis must still be oxidized for photoheterotrophic growth to occur. Diverse PNSB commonly rely on the CO2-fixing Calvin cycle to oxidize reduced electron carriers. Some PNSB also produce H2 or reduce terminal electron acceptors as alternatives to the Calvin cycle. Rhodospirillum rubrum Calvin-cycle mutants defy this trend by growing phototrophically on malate or fumarate without H2 production or access to terminal electron acceptors. We used 13C-tracer experiments to examine how a Rs. rubrum Calvin-cycle mutant maintains electron balance under such conditions. We detected the reversal of some tricarboxylic acid cycle enzymes, carrying reductive flux from malate or fumarate to αKG. This pathway and the reductive synthesis of αKG-derived amino acids are likely important for electron balance, as supplementing the growth medium with αKG-derived amino acids prevented Rs. rubrum Calvin-cycle-mutant growth unless a terminal electron acceptor was provided. Flux estimates also suggested that the Calvin-cycle mutant preferentially synthesized isoleucine using the reductive threonine-dependent pathway instead of the less-reductive citramalate-dependent pathway. Collectively, our results suggest that alternative biosynthetic pathways can contribute to electron balance within the constraints of a relatively constant biomass composition.

Function and Regulation of Agrobacterium tumefaciens Cell Surface Structures that Promote Attachment.

Agrobacterium tumefaciens attaches stably to plant host tissues and abiotic surfaces. During pathogenesis, physical attachment to the site of infection is a prerequisite to infection and horizontal gene transfer to the plant. Virulent and avirulent strains may also attach to plant tissue in more benign plant associations, and as with other soil microbes, to soil surfaces in the terrestrial environment. Although most A. tumefaciens virulence functions are encoded on the tumor-inducing plasmid, genes that direct general surface attachment are chromosomally encoded, and thus this process is not obligatorily tied to virulence, but is a more fundamental capacity. Several different cellular structures are known or suspected to contribute to the attachment process. The flagella influence surface attachment primarily via their propulsive activity, but control of their rotation during the transition to the attached state may be quite complex. A. tumefaciens produces several pili, including the Tad-type Ctp pili, and several plasmid-borne conjugal pili encoded by the Ti and At plasmids, as well as the so-called T-pilus, involved in interkingdom horizontal gene transfer. The Ctp pili promote reversible interactions with surfaces, whereas the conjugal and T-pili drive horizontal gene transfer (HGT) interactions with other cells and tissues. The T-pilus is likely to contribute to physical association with plant tissues during DNA transfer to plants. A. tumefaciens can synthesize a variety of polysaccharides including cellulose, curdlan (ß-1,3 glucan), ß-1,2 glucan (cyclic and linear), succinoglycan, and a localized polysaccharide(s) that is confined to a single cellular pole and is called the unipolar polysaccharide (UPP). Lipopolysaccharides are also in the outer leaflet of the outer membrane. Cellulose and curdlan production can influence attachment under certain conditions. The UPP is required for stable attachment under a range of conditions and on abiotic and biotic surfaces. Other factors that have been reported to play a role in attachment include the elusive protein called rhicadhesin. The process of surface attachment is under extensive regulatory control and can be modulated by environmental conditions, as well as by direct responses to surface contact. Complex transcriptional and post-transcriptional control circuitry underlies much of the production and deployment of these attachment functions.