Mechanisms and regulation of surface interactions and biofilm formation in Agrobacterium

Abstract

For many pathogenic bacteria surface attachment is a required first step during host interactions. Attachment can proceed to invasion of host tissue or cells or to establishment of a multicellular bacterial community known as a biofilm. The transition from a unicellular, often motile, state to a sessile, multicellular, biofilm-associated state is one of the most important developmental decisions for bacteria. Agrobacterium tumefaciens genetically transforms plant cells by transfer and integration of a segment of plasmid-encoded transferred DNA (T-DNA) into the host genome, and has also been a valuable tool for plant geneticists. A. tumefaciens attaches to and forms a complex biofilm on a variety of biotic and abiotic substrates in vitro. Although rarely studied in situ, it is hypothesized that the biofilm state plays an important functional role in the ecology of this organism. Surface attachment, motility, and cell division are coordinated through a complex regulatory network that imparts an unexpected asymmetry to the A. tumefaciens life cycle. In this review, we describe the mechanisms by which A. tumefaciens associates with surfaces, and regulation of this process. We focus on the transition between flagellar-based motility and surface attachment, and on the composition, production, and secretion of multiple extracellular components that contribute to the biofilm matrix. Biofilm formation by A. tumefaciens is linked with virulence both mechanistically and through shared regulatory molecules. We detail our current understanding of these and other regulatory schemes, as well as the internal and external (environmental) cues mediating development of the biofilm state, including the second messenger cyclic-di-GMP, nutrient levels, and the role of the plant host in influencing attachment and biofilm formation. A. tumefaciens is an important model system contributing to our understanding of developmental transitions, bacterial cell biology, and biofilm formation.

Keywords: Agrobacterium; attachment; biofilm; cyclic-di-GMP; motility; polarity.

FIGURE 1

Key steps in attachment and biofilm formation by Agrobacterium tumefaciens. Motile planktonic cells approach and physically interact with potential attachment substrates. Initial surface interactions are reversible and may depend on physiochemical forces at the interface of the surface with the local medium. Following these initial surface interactions the unipolar polysaccharide (UPP) is secreted by the bacterium at the point of surface contact. This irreversible surface attachment establishes a site for microcolony formation through continued growth and cell division of attached bacteria as well as aggregation of neighboring microcolonies. During and following this period cells secrete matrix components, including cellulose. As the biofilm matures cells may differentiate into various metabolic and reproductive states as the local environment within the biofilm changes. Dispersal from the biofilm may be initiated by an internal developmental cue or by an extracellular factor, as well as through release of motile daughter cells from attached mother cells. Note that in this cartoon only the outer membrane of the Gram-negative cell envelope is depicted.

FIGURE 2

Multiple inputs regulate attachment and biofilm formation by Agrobacterium tumefaciens. Depicted in the image are the known factors regulating attachment and biofilm formation and discussed in the text. Solid black arrows and bars indicate direct positive or negative regulation, respectively. Hashed arrows and bars indicate regulation that is indirect or where the molecular mechanism has not been defined. Note that the cell envelope is represented only by the outer (red) and inner (black) membranes, and the periplasmic peptidoglycan is not shown.

FIGURE 3

The second messenger cyclic-di-GMP. Cyclic diguanylate monophosphate, or cyclic-di-GMP (c-di-GMP) is a common second messenger in prokaryotic systems. C-di-GMP is generated from two molecules of guanosine triphosphate (GTP) by diguanylate cyclases (DGC) and degraded by phosphodiesterases (PDE) to the linear form, 5′-phosphoguanylyl-guanosine (pGpG), and ultimately to two molecules of guanosine monophosphate (GMP). In many bacteria, including Agrobacterium tumefaciens, c-di-GMP levels reciprocally regulate the transition between motility and attachment. In A. tumefaciens globally or locally increased c-di-GMP levels positively regulate attachment and biofilm formation while negatively regulating motility. The effect of c-di-GMP on virulence in A. tumefaciens has not been described, although in many bacteria virulence is negatively regulated by elevated c-di-GMP levels. The chemical structure of c-di-GMP is included in the center of the figure.

FIGURE 4

Agrobacterium tumefaciens generates and maintains multiple developmental asymmetries. (A) Shown are morphological features of A. tumefaciens known to localize primarily to one pole of the bacterium, including multiple flagella, the unipolar polysaccharide (UPP), Vir pilus and type IV secretion system (T4SS). (B) Cell division in A. tumefaciens occurs by a polar budding mechanism. The cell division protein FtsZ (green) appears at the site of early constriction, from which the daughter cell buds and at which cytokinesis occurs. The rate of budding indicated is for growth in defined medium (ATGN) on agar pads. (C) The coordination of division and development (CDD) regulatory pathway. Proteins for which genetic and/or phenotypic data confirm the suggested pathway architecture are in bold typeface. Italicized proteins are present in the A. tumefaciens genome but do not have experimental support; placement of these proteins in the pathway is based on data from other model systems. Histidine kinases are colored orange, response regulators are colored blue, and a single Hpt phosphotransferase (ChpT) is in black text. The location of PdhS1 and PdhS2 is suggested by current data, but not confirmed. Note that the bacterial cell envelope in all panels is depicted as described in Figure 3.