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

Abstract

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 (β-1,3 glucan), β-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.

Keywords: Attachment; Biofilms; Cell surface structures; Regulation.

Figure 1.. General structure of the bacterial flagellum.

Diagrammatic representation of a bacterial flagellum structure based on flagella from Salmonella enterica serovar Typhimurium (Salmonella typhimurium), alongside a flagellum structure determined by cryoelectron microscopy of the flagellum from Treponema primitia. Combined figure adapted with permissions from Nat Rev Microbiol (Pallen and Matzke, 2006) and Curr. Biol. (DeRosier 2006).

Figure 2.. Genetic basis of Ctp pili and model for assembly.

Predicted localization and assembly mechanism for Ctp proteins based on the general model for Tad pilus assembly (Tomich et al. 2007). The CtpA pilin is processed by CtpB cleavage and incorporated into the emerging pilus. CtpG hydrolyzes ATP to drive pilus assembly. Protein names indicated in the figure. Gene map indicates the ctp gene names above the gene arrows, and gene names in the generalized Tad-type pilus system are provided below. Gene colors match protein colors in the diagram. OM, outer membrane; PG, peptidoglycan; IM, inner membrane.

Figure 3.. Cellulose: Genetic basis and biosynthesis model.

Predicted localization and mechanism of biosynthesis for cellulose in A. tumefaciens based on the generalized model (Römling and Galperin, 2015). Cellulose strand depicted as linked green hexagons. Cel protein names indicated in the figure. For CelA, GT is the predicted glycosyl transferase domain and PilZ is the cdGMP-binding domain. Black squiggle on CelH is a predicted lipid linkage. Gene colors match protein colors in the diagram; Cel names are above with corresponding Bcs nomenclature below. OM, outer membrane; PG, peptidoglycan; IM, inner membrane, Ac, acetyl groups; Glc-6-P, glucose-6-phosphate; Glc-1-P, glucose-1-phosphate; UDP-Glc, uridyl diphosphate glucose.

Figure 4.. Wzx/Wzy polysaccharide pathway: Genetic basis and biosynthesis model for UPP.

Predicted localization and mechanism of biosynthesis for UPP in A. tumefaciens based on Wzx-Wzy generalized model (Cuthbertson et al. 2009). Branched green filament of diamonds is meant to depict the polysaccharide strand. The black square on some residues is putative acetylation. The Wzb/c protein may hydrolyze ATP to power polymerization. Upp protein names and the general Wzx-Wzy components are indicated in the figure. Black squiggle on the polysaccharide subunit is the undecaprenyl phosphate. Gene colors match protein colors in the diagram; upp gene names above and corresponding Wzx-Wzy components below (AcTrans – acetyltransferase; GT glycosyl transferase). Assembled UPP structure is represented by green cross-hatched oval outside cell, and the diamond headed lines are putative linkages to the cell pole surface. OM, outer membrane; PG, peptidoglycan; IM, inner membrane.

Figure 5.. Diguanylate cyclases and phosphodieterases of A. tumefaciens.

(A) DGC proteins, (B) DGC-PDE proteins and PDE/HD-GYP only proteins. Atu gene numbers and if assigned, genetic names are provided for each protein. Predicted transmembrane domains are indicated by vertical lines and any recognizable functional domains are indicated. Protein domains. REC - two-component-type receiver domain; GAF - cGMP-specific phosphodiesterases, Adenylyl cyclases and FhlA; HAMP - Histidine kinases, Adenyl cyclases, Methyl-accepting proteins and Phosphatases; PAS - Per, ARNT, Sim; CBS - Cystathionine-Beta-Synthase; CHASE4, predicted ligand binding module; MHYT, 6-transmembrane domain; DISM, 7-transmembrane domain, Diverse Intracellular Signaling Modules.

Figure 5.. Diguanylate cyclases and phosphodieterases of A. tumefaciens.

(A) DGC proteins, (B) DGC-PDE proteins and PDE/HD-GYP only proteins. Atu gene numbers and if assigned, genetic names are provided for each protein. Predicted transmembrane domains are indicated by vertical lines and any recognizable functional domains are indicated. Protein domains. REC - two-component-type receiver domain; GAF - cGMP-specific phosphodiesterases, Adenylyl cyclases and FhlA; HAMP - Histidine kinases, Adenyl cyclases, Methyl-accepting proteins and Phosphatases; PAS - Per, ARNT, Sim; CBS - Cystathionine-Beta-Synthase; CHASE4, predicted ligand binding module; MHYT, 6-transmembrane domain; DISM, 7-transmembrane domain, Diverse Intracellular Signaling Modules.