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.

The chemotaxis signal transduction network regulates the biased random walk of many bacteria in favorable directions and away from harmful ones through modulating the frequency of directional reorientations. In mutants of diverse bacteria lacking the chemotaxis response, migration in classic motility agar, which constitutes a fluid-filled porous medium, is compromised; straight-swimming cells unable to tumble become trapped within the agar matrix. Spontaneous mutations that restore spreading have been previously observed in the enteric bacterium Escherichia coli, and recent work in other bacterial species has isolated and quantified different classes of nonchemotacting mutants exhibiting the same spreading phenotype. We present a theoretical description of bacterial diffusion in a porous medium-the natural habitat for many cell types-which elucidates how diverse modifications of the motility apparatus resulting in a nonzero tumbling frequency allows for unjamming of otherwise straight-swimming cells at internal boundaries and leads to net migration. A unique result of our analysis is increasing diffusive spread with increasing tumbling frequency in the small pore limit, consistent with earlier experimental observations but not captured by previous models. Our theoretical results, combined with a simple model of bacterial diffusion and growth in agar, are compared with our experimental measurements of swim ring expansion as a function of time, demonstrating good quantitative agreement. Our results suggest that the details of the cellular tumbling process may be adapted to enable bacteria to propagate efficiently through complex environments. For engineered, self-propelled microswimmers that navigate via alternating straight runs and changes in direction, these results suggest an optimal reorientation strategy for efficient migration in a porous environment with a given microarchitecture.

Bacterial locomotion driven by flagella is given directionality by the chemotaxis signal transduction network. In the classic plate assays of migration in porous motility agar, efficient motility is compromised in chemotaxis mutants of diverse bacteria. Nonchemotactic mutants become trapped within the agar matrix. Suppressor mutations that prevent this entanglement but do not restore chemotaxis, a phenomenon designated pseudotaxis, were first reported to arise for Escherichia coli. In this study, novel mechanisms of pseudotaxis have been identified for the plant-pathogenic alphaproteobacterium Agrobacterium tumefaciens. Mutants with chemotaxis mutation suppressor (cms) mutations that impart enhanced migration in motility agar compared to that of their straight-swimming, nonchemotactic parent were isolated. We find that pseudotaxis in A. tumefaciens occurs most commonly via mutations in the D1 domain of the flagellar hook protein, FlgE, but it can also be found less frequently to be due to mutations in the hook length regulator, FliK, or in the motor protein, MotA. Single-cell-tracking studies of cms mutants in bulk medium clearly reveal frequent changes in the direction of swimming, similar to the swimming of strains that are proficient for chemotaxis, but independent of a sensory mechanism. Our results suggest that the tumbling process can be tuned through mutation and evolution to optimize migration through complex, porous environments.

Rotary flagella propel bacteria through liquid and across semisolid environments. Flagella are composed of the basal body that constitutes the motor for rotation, the curved hook that connects to the basal body, and the flagellar filament that propels the cell. Flagellar filaments can be composed of a single flagellin protein, such as in , or made up of multiple flagellins, such as in The four distinct flagellins FlaA, FlaB, FlaC, and FlaD produced by wild-type are not redundant in function but have specific properties. FlaA and FlaB are much more abundant than FlaC and FlaD and are readily observable in mature flagellar filaments, when either FlaA or FlaB is fluorescently labeled. Cells producing FlaA with any one of the other three flagellins can generate functional filaments and thus are motile, but FlaA alone cannot constitute a functional filament. In mutants that manifest swimming deficiencies, there are multiple ways by which these mutations can be phenotypically suppressed. These suppressor mutations primarily occur within or upstream of the flagellin gene or in the transcription factor regulating flagellin expression. The helical conformation of the flagellar filament appears to require a key asparagine residue present in FlaA and absent in other flagellins. However, FlaB can be spontaneously mutated to render helical flagella in the absence of FlaA, reflecting their overall similarity and perhaps the subtle differences in the specific functions they have evolved to fulfill. Flagellins are abundant bacterial proteins comprising the flagellar filaments that propel bacterial movement. Several members of the alphaproteobacterial group express multiple flagellins, in contrast to model systems, such as with , which has one type of flagellin. The plant pathogen has four flagellins, the abundant and readily detected FlaA and FlaB, and lower levels of FlaC and FlaD. Mutational analysis reveals that FlaA requires at least one of the other flagellins to function, as mutants produce nonhelical flagella and cannot swim efficiently. Suppressor mutations can rescue this swimming defect through mutations in the remaining flagellins, including structural changes imparting helical shape to the flagella, and putative regulators. Our findings shed light on how multiple flagellins contribute to motility.

31Rotary flagella propel bacteria through liquid and across semi-solid environments. 32Flagella are composed of the basal body that constitutes the motor for rotation, the 33 curved hook that connects to the basal body, and the flagellar filament that propels the 34 cell. Flagellar filaments can be comprised of a single flagellin protein such as in 35 Escherichia coli or with multiple flagellins such is in Agrobacterium tumefaciens. The 36 four distinct flagellins FlaA, FlaB, FlaC and FlaD produced by wild type A. tumefaciens, 37 are not redundant in function, but have specific properties. FlaA and FlaB are much 38 more abundant than FlaC and FlaD and are readily observable in mature flagellar 39 filaments, when either FlaA or FlaB is fluorescently labeled. Cells having FlaA with any 40 one of the other three flagellins can generate functional filaments and thus are motile, 41 but FlaA alone cannot constitute a functional filament. In flaA mutants that manifest 42 swimming deficiencies, there are multiple ways by which these mutations can be 43 phenotypically suppressed. These suppressor mutations primarily occur within or 44 upstream of the flaB flagellin gene or in the transcriptional factor sciP regulating flagellar 45 expression. The helical conformation of the flagellar filament appears to require a key 46 asparagine residue present in FlaA and absent in other flagellins. However, FlaB can be 47 Importance 51 Flagellins are abundant bacterial proteins comprising the flagellar filaments that propel 52 bacterial movement. Several members of the Alphaproteobacterial group express 53 multiple flagellins, in contrast to model systems such as Escherichia coli that has only 54 one flagellin protein. The plant pathogen Agrobacterium tumefaciens has four flagellins, 55 the abundant and readily detected FlaA and FlaB, and lower levels of FlaC and FlaD. 56 Mutational analysis reveals that FlaA requires at least one of the other flagellins to 57 function -flaA mutants produce non-helical flagella and cannot swim efficiently. 58 Suppressor mutations can rescue this swimming defect through mutations in the 59 remaining flagellins, including structural changes imparting flagellar helical shape, and 60 putative regulators. Our findings shed light on how multiple flagellins contribute to 61 motility. 62 63 sphaeroides has one flagellum (laterally positioned on the cell) that is made up of a 87 single flagellin protein (9, 10). On the other hand, examples of bacteria with filaments 88 made of multiple flagellins include Caulobacter crescentus, which has a single polar 89 flagellum, but remarkably six flagellin proteins (11). Within the Rhizobiaceae family 90 Sinorhizobium meliloti, Rhizobium leguminosarum, Agrobacterium sp. H13-3, and 91 Agrobacterium tumefaciens all encode multiple flagellins. A. tumefaciens mutated in 92 three of its four flagellin genes is reduced in virulence by about 38% and Agrobacterium 93 sp. H13-3 lacking all of its three flagellin genes is resistant to flagella-specific phage 94 infection (12-16). F...