Effects of desferrioxamine on Rhodococcus growth

Rhodococcus opacus 1CP is known for its resilience and ability to grow in the presence of compounds that are toxic to many other organisms. To determine whether desferrioxamine B (DFOB) affects the growth of R. opacus 1CP, we conducted growth curve experiments. These experiments aimed to determine whether DFOB facilitates iron uptake, thereby promoting growth, or inhibits iron availability, thus restricting growth. Growth was assessed in the presence of saturated DFOB, DFOB purified with ascorbic acid, and unpurified DFOB representing the likely state of DFOB accumulation in an expression culture.In all cases, the presence of DFOB did not impair the growth rate of R. opacus 1CP (Fig. 1). However, the presence of unpurified and saturated DFOB resulted in a shortened stationary phase, with a noticeable decline in cell density observed at the 60-hour mark. In contrast, the control and purified DFOB samples entered the stationary phase at 48 hours and 36 hours, respectively, and maintained stable growth until the end of the measurement period. After 60 hours of incubation, the DFO mix sample appeared to enter a decline phase, followed by a renewed growth phase at 72 hours. This apparent reduction may be due to a measurement error, as the deviation aligns with both the preceding and subsequent measurements.

Figure 1 Growth curves of Rhodococcus opacus 1CP in LB media (Control) under the presence of unpurified DFOB (DFO mix), with iron saturated DFOB (DFO saturated) and with ascorbic acid purified DFOB (DFOB clean).

DFO quantification via CAS-Assay

The primary objective of our team was to introduce the necessary genes (DesA–DesD) into Rhodococcus opacus 1CP to enable more efficient production of desferrioxamine B (DFOB). A key consideration in this context is the effect of DFOB on the growth rate of R. opacus 1CP. To investigate this, DFOB was produced using Streptomyces coelicolor in preparation for growth curve experiments.

Several methods were tested to remove iron ions bound to DFOB. The DFOB was purified by incubating XAD-4 and XAD-16 resins in the supernatant of a one-week-old S. coelicolor culture at 4 °C for 24 hours. Elution was performed by stepwise addition of methanol to the resin while rigorously vortexing.

The concentration of DFOB in the eluate was quantified using the Chrome Azurol S (CAS) assay with various dilutions. An EDTA standard curve was used for calibration. Dilution factors were accounted for when calculating iron ion free DFOB concentrations. However, when the EDTA standard was diluted beyond a 1:10 ratio, the measured DFOB concentrations began to decrease unexpectedly.

Subsequently, the DFOB eluate was dried and resuspended in ddH₂O. To remove iron ions bound to DFOB, another CAS assay was performed after treating the DFOB solution with ascorbic acid, sodium carbonate, and sodium hydroxide.

All three treatments were effective in removing iron ions. However, control reactions containing only ascorbic acid, sodium carbonate, or sodium hydroxide (without DFOB) also caused a color change in the CAS assay. This suggests that the CAS assay may not be fully reliable for assessing the efficiency of iron-ion removal under these conditions.

Figure 2 Iron ion free DFOB concentrations determined by the CAS assay using diluted elution samples. Concentrations were calculated based on an EDTA standard curve.

Figure 3 Purification of DFOB assessed using the CAS assay with varying concentrations of ascorbic acid, sodium carbonate, and sodium hydroxide. Concentrations were quantified using an EDTA standard curve.

Split-GFP system

To monitor gene cluster activity during the transfer of the desferrioxamine B (DFOB) biosynthesis genes (DesA–DesD) into Rhodococcus opacus 1CP, we aimed to implement a split-GFP system. For our split GFP system to be compatible with Rhodococcus opacus 1CP, we first needed to transfer the relevant genes into pNit vector systems. We successfully cloned the GFP1-10 fragment into the pNit_QC1 vector and the GFP11ß into the pNit_QT1 resulting in the plasmids pNit_QC1_GFP1-10 and pNit_QT1_GFP11ß. Both plasmids were verified via sequencing. Unfortunately we were not able to test the split GFP system in Rhodococcus opacus 1CP due to time constraints. Testing in E.coli was also not feasible since pNit_QT1 and pNit_QC1 cannot simultaneously be transformed into E. coli since they share the same origin of replication and ampicillin resistance.