## 4 Discussion  
The work here shows, firstly, that curdlan is produced by _agrobacteriumum_ strains and deposited as a capsule that is responsible for strong cell-cell aggregation and floc formation in liquid cultures. Secondly, the work reveals a genetic engineering approach that enables approximately 8-fold higher curdlan yields from the commercially useful strain _agrobacteriumum_ sp. 31749, and forces the wild type strain _A. tumefaciens_ C58 to produce significant levels of curdlan where it previously produced none. Curdlan-producing _agrobacteriumum_ colonies grown on agar medium with a sufficient carbon source and aniline blue produce curdlan as a stable, intensely blue-staining layer which may be stripped, along with the cells, from the colony surface [14]. This suggests close proximity between cells and curdlan, and that curdlan is somehow adhesive and involved in attachment between cells. However, the relationship between curdlan and the bacterial cell surface had not been clearly defined. Scanning electron microscopy of the Gram positive curdlan producing bacterium _cellulomonas_ indicated that curdlan may be deposited as a capsule [16]. However, staining with aniline blue could not support  
Figure 6: PphaP increases curdlan production from apple extract (AE) and in defined medium with glucose. (**A**), the modified strains of _agrobacteriumia_ were grown on 1% agar with 0.05% yeast extract and 1% tryptone containing 0.005% aniline blue with varying levels of AE or glucose as indicated. (**B**), bacteria were grown in ADB broth (4% glucose) Cultures (50 mL) were shaken (150 rpm) in 500 mL flasks at 30 \({}^{\circ}\)C. Curdlan was harvested after 3 days (see materials and methods for details). Error bars indicate variation as standard deviation from three biological replicates. C58 did not produce any detectable curdlan. a capsular formation by _cellulomonas_ since the native form of curdlan produced by this bacterium does not stain with aniline blue. It must first undergo alkaline extraction before staining, indicating a fundamentally different native form to curdlan produced by _agrobacteriumium_[15]. In this study, it was shown that _agrobacteriumium_ deposits curdlan as a capsule, using the (1\(\rightarrow\)3)-\(\beta\)-glucan-specific aniline blue fluorochrome (ABF). Additionally, curdlan was negatively-stained by uranyl acetate and thereby visible using TEM. Both ABF-induced fluorescence images and TEM showed that the curdlan capsule is tightly associated with the cell surface. Neither of these features was observed with a curdlan deficient mutant (_crdS_-) or in cultures of strains grown in medium that does not elicit curdlan production, i.e., Luria broth. Cell aggregates depend upon curdlan production, and these aggregates appear to be via interactions between curdlan-encapsulated cells. The data is consistent with a scenario where cells produce a capsule, whereupon these encapsulated cells are recruited to the aggregate on the basis of curdlan-curdlan interactions. In other words, after producing curdlan, the cell randomly encounters a neighboring cell that has also produced curdlan. The two become attached and then encounter additional cells that have produced curdlan, and in this way the aggregates grow larger through cell recruitment over time (Figure S1). Studies on the soil-dwelling gram-positive C. _flavigena_ also found a correlation between aggregation and curdlan production. Furthermore, on subculturing C. _flavigena_ on a nitrogen-rich medium lacking a carbohydrate source, disaggregation occurs and the curdlan disappears, suggesting that the curdlan is mobilized as a carbohydrate source [15; 16]. Consumption of curdlan by _agrobacteriumium_ has not been observed. _A. tumefaciens_ is considered one of the more important, well-studied plant pathogens, being a tumorigenic pathogen responsible for crown gall disease with a wide host range [62]. So far, there is no indication that curdlan is necessary for pathogenesis. _Agrobacteriumia_ strains are endemic in soils.