## 3 Results
Interestingly, fluorescence from mVenus (Figure 3C) was apparent in cultures of both ATCC31749 and C58 around 30 h, although a weaker basal level of mVenus fluorescence was apparent slightly earlier, at 24 h for ATCC31749. Since C58 did not produce curdlan under these conditions, the data suggests that the expression of the genes _crdASC_ was too low to support curdlan production. Alternatively, the critical step controlling the onset of curdlan production lies elsewhere, so that the expression of these genes is not the deciding factor for curdlan production in this strain. This was not the case for ATCC31749, where the onset of strong _crdASC_ gene expression (at about 30 h) correlated relatively well with the onset of curdlan accumulation (as would be expected if curdlan production was controlled by _crdASC_ expression). Thus, curdlan accumulation correlated with _crdA_ gene expression in ATCC31749, but not in C58. Altogether, the newly developed assay appeared to be reliable for sensitively detecting curdlan accumulation and gene expression at a high throughput level. Figure 3: 96-well plate assay reveals a temporal profile of the onset of curdlan production. _Agrobacteriumum tundaciens_ C58 (C58 WT) and _agrobacteriumum_ sp. 31749 (31758 WT) were grown in a 96-well plate (8 wells for each strain). For the medium and other details of this setup, see the materials and methods. For the detection of curdlan, the medium contained 0.1 mg/mL aniline blue (0.01%). Additionally, a translational fusion between _crdA_ and the gene for mVenus was inserted into the chromosome, allowing detection of _crdA_ expression. Growth (**A**), fluorescence from aniline blue/curdlan (**B**) and fluorescence from mVenus (**C**) of the cultures was measured once every three hours for 60 h using an automated Tecan reader setup. Black arrows at approximately 33 h indicate the onset of curdlan production. The _R. sphaeroides_ promoter PphaP is much stronger than Pcrd in _Agrobacteriumium_. Since curdlan production correlates with _crdASC_ gene expression, one possibility for enhancing curdlan production is the enhanced expression of the _crdASC_ genes. For this goal, the replacement of the native _crdASC_ promoter (Pcrd) with a constitutively active promoter could suffice. I came across an obvious candidate promoter during investigations on transcription regulation in the Alphaproteobacteria. This candidate was PphaP from _R. sphaeroides_[52], which was very active in _A. tumefaciens_ C58. To compare the activity of the two promoters, PphaP and Pcrd, in _agrobacteriumium_, the reporter gene for mVenus was fused to each of the promoters on the low copy plasmid pPHU231 (see materials and methods for details). The _agrobacteriumium_ strains C58 and 31749 carrying the KO plasmid were used, since this avoided curdlan production and the resulting floc formation, which tends to interfere with fluorescence measurements and increase standard deviation. Furthermore, a variety of media was used. PY medium contains peptone and yeast extract (a rich source of nitrogen), while the MOPS medium contains a defined, growth limiting level of nitrogen. Apple extract (10%) represents a cheap and locally available source of nutrients. For details see materials and methods. In C58, the activity of Pcrd was low during growth in PY (Figure 4A) and also in MOPS medium. In contrast, the PphaP promoter activity was much stronger in all media (Figure 4B). A similar pattern was observed with the Pcrd and PphaP promoters in 31749 (Figure 4C,D). It is unknown why the presence of glucose or apple extract stimulated PphaP activity in PY medium. Also unexpected was the observation that Pcrd was rather weak in strain C58. This is probably related to the setup conditions, where 100 uL of liquid medium was used.