Genetic analysis of Agrobacterium tumefaciens unipolar polysaccharide production reveals complex integrated control of the motile‐to‐sessile switch

Abstract

Summary Many bacteria colonize surfaces and transition to a sessile mode of growth. The plant pathogen Agrobacterium tumefaciens produces a unipolar polysaccharide (UPP) adhesin at single cell poles that contact surfaces. Here we report that elevated levels of the intracellular signal cyclic diguanosine monophosphate (c-di-GMP) lead to surface-contact-independent UPP production and a red colony phenotype due to production of UPP and the exopolysaccharide cellulose, when A. tumefaciens is incubated with the polysaccharide stain Congo Red. Transposon mutations with elevated Congo Red staining identified presumptive UPP negative regulators, mutants for which were hyperadherent, producing UPP irrespective of surface contact. Multiple independent mutations were obtained in visN and visR, activators of flagellar motility in A. tumefaciens, now found to inhibit UPP and cellulose production. Expression analysis in a visR mutant and isolation of suppressor mutations, identified three diguanylate cyclases inhibited by VisR. Null mutations for two of these genes decrease attachment and UPP production, but do not alter cellular c-di-GMP levels. However, analysis of catalytic site mutants revealed their GGDEF motifs are required to increase UPP production and surface attachment. Mutations in a specific presumptive cyclic diguanosine monophosphate phosphodiesterase also elevate UPP production and attachment, consistent with c-di-GMP activation of surface-dependent adhesin deployment.

Agrobacterium-Mediated Plant Transformation: Biology and Applications

“…The unipolar polysaccharide helps A. tumefaciens cells attach to the plant surfaces in a polar fashion because the UPP is mainly produced at the cell pole. The UPP is rarely observed in A. tumefaciens planktonic cells or in colonies on solid media, suggesting that the production of UPP is a signature of A. tumefaciens permanent attachment (Li et al, 2011;Xu et al, 2012;Xu et al, 2013). The UPP consists of at least two types of sugars, N-acetylglucosamine and N-acetylgalactosamine (Tomlinson and Fuqua, 2009;Xu et al, 2012).…”

Spermidine Inversely Influences Surface Interactions and Planktonic Growth in Agrobacterium tumefaciens

“…In A. tumefaciens, elevated intracellular c-di-GMP promotes surface attachment and stimulates production of adhesive polysaccharides, including cellulose and the polar adhesin described as the unipolar polysaccharide (UPP) (21). Overexpression of either cellulose or the UPP or both in A. tumefaciens colonies resulted in dark-red colony pigmentation on ATGN medium with 80 g/ml Congo red (ATGN-CR).…”

“…2A), which is consistent with the original transposon mutant phenotype. Our prior studies have established a strong correlation between the ECR colony phenotype and increased biofilm formation in A. tumefaciens (21). Based on a time course in a static biofilm assay in ATGN minimal medium with no added polyamines, the ⌬odc mutant displayed a growth deficiency in the planktonic phase, with a lag and a lower growth yield (ϳ60%) than that of the wild type (Fig.…”

“…In order to identify genes that were specifically involved in the negative regulation of cellulose production, we screened for the ECR phenotype in roughly 25,000 transposon mutants of an A. tumefaciens mutant strain in which the UPP and other known exopolysaccharides were genetically disabled (EPS Ϫ Cel ϩ strain C58-YW2). Several transposon mutants that formed dark-red colonies on ATGN-CR medium due to cellulose overproduction were identified, and the mutant genes included genes involved in transcriptional regulation and synthesis of c-di-GMP, already known to regulate cellulose synthesis in A. tumefaciens (21,22). Among these mutants, two independent isolates had unique transposon insertions in the Atu3196 gene, annotated as an ornithine decarboxylase gene (odc) presumptively required for the biosynthesis of polyamines, such as putrescine and spermidine.…”

Microbial Surface Colonization and Biofilm Development in Marine Environments

“…For example, Ruegeria mobilis, a member of the MRC, employs the c-di-GMP regulatory pathway to modulate biofilm formation and antibiotic production (483). In Agrobacterium tumefaciens, c-di-GMP activates, in a surface contact-dependent manner, enhanced production of the unipolar polysaccharide adhesin, which is functionally equivalent to a holdfast, for integrated control of the switch from a motile to a sessile lifestyle (484,485). The environmental bacterium Shewanella oneidensis MR-1, which harbors a number of respiratory pathways, including the anaerobic reduction of iron(III), manganese(IV), and uranium(VI), forms biofilms on mineral surfaces through a process controlled by c-di-GMP (486).…”