## 3 Results
The previous setup used in Figure 3 involved only 20 uL of liquid culture overlaying 100 uL of agar, likely providing better access of the bacterial culture to oxygen, which is known to be important for curdlan production [27], and is likely more critical for strain C58 than for the industrial strain 31749. Also in 31749, the Pcrd promoter was virtually inactive in PY medium, while in the MOPS medium, the Pcrd promoter was not active at 21 h, but did show some activity at the 45 and 72 h time points (Figure 4C). This fits well with the data in Figure 3C (the main difference being that the _crdAS_:mVenus fusion was carried on the chromosome in Figure 3C, and on a low copy plasmid in Figure 4). This also fits with the knowledge that the expression of the _crdASC_ genes is strongly activated upon N depletion [37]. In summary, these data demonstrate that PphaP was far more active than Pcrd in all the growth media tested. PphaP activates curdlan production. At this point, I was interested in seeing how the PphaP promoter affected curdlan accumulation. Specifically, would the use of PphaP to drive the expression of _crdASC_ lead to a higher production of curdlan? To test this the 96-well plate assay was used. The data shows that the C58 WT (Figure 5A) did not produce curdlan (as was also shown in Figure 3B), while the insertion of PphaP resulted in curdlan production. Also in 31749 the presence of PphaP clearly enhanced curdlan production so that curdlan accumulation was apparent much earlier, at around 10 h of incubation, while in the 31749 WT, the onset of curdlan accumulation was apparent after 30 h of incubation (Figure 5B). Fluorescence reached maximum levels at around 400 fluorescence units in 31749, possibly because of exhaustion of the carbon source (mannitol). Curdlan production requires both the expression of the _crdASC_ genes and a suitable carbon source. Does the insertion of PphaP mean that curdlan production is independent upon N availability? When grown on PY agar with aniline blue but without any additional carbon source, the colonies did not stain blue, indicating the absence of curdlan production (Figure 6A). However, when glucose was added (1% final concentration), both the strains carrying PphaP, but not the WT strains, stained blue during growth on agar, indicating curdlan production (Figure 6A). This result implies that curdlan production requires at least two criteria: high expression of the _crdASC_ genes as well as the presence of a suitable carbon source. Only one of these two conditions does not lead to curdlan production. Importantly, when Pcrd was replaced with PphaP, the bacteria appeared capable of curdlanproduction regardless of whether nitrogen was limiting, provided that a suitable source of carbon was available. Apple extract is a suitable carbon source for curdlan production. Given that one of the major costs associated with curdlan production is the carbon source, I looked for realistic alternatives to the typical carbon sources. Apple juice (100%) was obtained from a local apple juice factory, sterilized via autoclaving and added to PY agar in increasing concentrations. At 1% (final concentration), a low level of curdlan production was apparent judging by the aniline blue stain (Figure 6A). Production was improved with 3%, 10% and 30% apple extract, with 10% supporting the most intense blue color formation. Figure 4: PphaP is more active than Pcrd. _Agrobacteriumium_ strains ((**A,B**), C58, (**C,D**) 31749) incapable of curdlan production (via the pK-KO suicide vector) carrying the low copy plasmid pPHU231 containing the Pcrd::mVenus fusion (**A,C**) or the PphaP::mVenus fusion (**B,D**) were grown in a 96-well plate in various media. PY, peptone yeast, Glu, glucose, AE, apple extract (10%), MOPS, buffered minimal defined medium. Cultures were measured for mVenus fluorescence at the times indicated, and fluorescence per \(\mu\)L of bacterial culture is presented. Error bars indicate standard deviation from 8 well cultures.