## 4 Discussion
Many are not necessarily pathogenic [63], and may exhibit long term survival even in the absence of host colonization [64]. Probably the ability to produce curdlan is related to long term survival, although this has never been demonstrated. Indeed, the _A. tumefaciens_ C58 strain used in this study did not produce curdlan under standard laboratory conditions. Interestingly, when C58 was grown on apple extract, some curdlan production was apparent. Importantly, when C58 carried PphaP, strong curdlan accumulation was apparent (Figure 6A). It is likely that curdlan production is more highly regulated in C58 than in 31749 and that, in addition to nitrogen limitation, other environmental conditions (such as desiccation, or plant-derived molecules) are needed for the activation of curdlan production in C58. The curdlan dependent aggregation phenomenon described here appear to enable the formation of microcolonies or flocs and biofilms in these nutritionally stressful situations. The curdlan capsule formed under these conditions may play a role in protection from desiccation by virtue of the impressive water-holding capacity of curdlan capsule, i.e., the encapsulated cell may act as a'spore', as suggested in the case of cellulose-producing bacteria [11]. Moreover, curdlan could protect not only the producing cells, but also the entire community of cells from desiccation as well as soil predators such as phagocytic protozoans [65] and parasitic bdellovibrio species [66]. Interestingly, not all cells in the curdlan producing cultures were encapsulated. This heterogeneity within the population is somewhat similar for the production of another exopolysaccharide, galactoglucan, by another member of the family rhizobiacea, _sinorthizobium meliloti_[67]. The ability of clonal populations to display multiple phenotypes has been noted [68], and provides a number of advantages for survival such as bet-hedging and division of labor. Without knowing the role of curdlan in survival, it is difficult to say which of these options applies to curdlan. For example, curdlan forms a capsule, which may provide an advantage for the producing cell (e.g., protection against predation or dehydration). This could be a bet-hedging strategy, which can be viewed as a preadaptation of a subpopulation to a danger before it is encountered. However, curdlan also promotes cell-cell aggregation and formation of flocs, and could therefore be important for the formation of a biofilm (which benefits the whole population). In such a scenario, curdlan production by a subpopulation could be a form of division of labor. Both scenarios are not mutually exclusive, and more experiments are needed to clarify the role (if any) of heterogeneity in curdlan producing cultures. Relevant to the goal of maximal curdlan production, such heterogeneity likely has the undesired effect of decreasing curdlan production, since not all individuals contribute. A more desirable outcome would be that all cells contribute to curdlan production. The use here of a strong, constitutively active promoter such as PphaP to drive the _crdASC_ gene expression may well have contributed to the increase in curdlan production by removing the heterogeneic pattern of production, i.e., by forcing all cells to contribute. The _phaP_ promoter (PphaP) from _rhodobacter__shareoides_ controls the expression of the phasin gene _phaP_. Previous work on this gene found that it was strongly active in the exponential growth phase of _R. sphaeroides_[52]. Interestingly, upon heat shock, transcription of the _phaP_ gene increased by over 20-fold [53], making the promoter of _phaP_ one of the most active promoters that I have ever observed. Indeed, PphaP showed very high activity in _Agrobacteriumrium_ strains (Figure 4). In comparison, the native _crd_ promoter (Pcrd) was not only much weaker than PphaP, it was also highly dependent upon the growth medium (e.g., nitrogen content). For example, the PY medium (containing peptone and yeast extract) did not support Pcrd activity, while PphaP was relatively strong.