pBBR1MCS-5

Acetic acid bacteria (AAB) Gluconobacter oxidans incompletely oxidize a wide range of carbohydrates and are therefore used industrially for oxidative bioconversion. For G. oxydans, no system allows plasmid-based regulatable expression. The researchers found that the L-arabinose-inducible PBAD promoter and transcriptional regulator AraC from E. coli MC4100 performed well in G. oxydans. The respective pBBR1-based plasmids showed very low basal expression of the reporter genes β-glucuronidase and mNeonGreen, were induced up to 480-fold with 1% L-arabinose, and were tunable between 0.1% and 1% L-arabinose.

However, during the stationary phase, the oxidation product arabic acid strongly promoted the acidification of the growth medium in 621H cultures, resulting in a sharp decrease in reporter gene activity in 621H (pH 3.3), but not in BP.6 cultures (pH 4.4). These activities were rapidly and strongly increased simply by culturing fixed cells in D-mannitol-free medium adjusted to pH 6, suggesting that the reporter molecules were barely degraded but instead became inactive. In pH-controlled bioreactors, these reporter gene activities remained high in the stationary phase (pH 6). Finally, the researchers constructed the araC-PBAD polyclonal vector based on pBBR1MCS-5. Together their results demonstrate the superior functionality and good tunability of the AraC-PBAD system in G. oxydans , which may also be used for other AABs.

In this study, FACS analysis was performed on G. oxydans strains 621H and BP.6 harboring plasmid pBBR1MCS-5-araC-PBAD-mNG. For strain 621H, 8 h after induction, 91% of all cells exhibited high fluorescence signals within the selected gate, indicating high population homogeneity near the end of the exponential growth phase (Figure 1a). After 26 hours of induction, when the pH of the culture medium was 3.3±0.1, the overall fluorescence decreased. Transferring 621H cells to D-mannitol-free and L-arabinose-free medium adjusted to pH 6 resulted in recovery of mNG fluorescence. For G. oxydans BP.6, strong induction and fluorescence with high uniformity were also observed after 8 h. These signals showed little reduction in the stationary phase after 26 h (pH 4.4±0.1) and were not affected by transfer to fresh medium adjusted to pH 6 (Figure 2b). These single-cell results are consistent with the hypothesis that additional acidification of the growth medium results in stronger intracellular acidification during stationary phase. Furthermore, single-cell data demonstrate that the AraC-PBAD system is capable of achieving highly uniform induction responses in cells.

Figure 1. FACS analysis of the G. oxydans strains 621H (a) and BP.6 (b) carrying plasmid pBBR1MCS-5-araC-PBAD-mNG. (Fricke, Philipp Moritz, et al., 2020)