Pseudomonas response regulators produced in an E. coli heterologous expression host exhibit host-derived post-translational phosphorylation

In this report, we systematically characterize 32 response regulators (RRs) from a metal tolerant groundwater isolate, Pseudomonas stutzeri RCH2 to assess the impact of host-derived post-translational phosphorylation. As observed by distinct shifted bands in a phos-tag gel, 12 of the 24 detected RRs show homogenous mixtures of phosphorylated proteins or heterogenous mixtures of unphosphorylated and phosphorylated proteins. By evaluating the phosphorylation state of CzcR and CopR II under varying assay parameters, we found that changes to pH and exogenous addition of phospho-donors (e.g. acetyl phosphate) have little to no effect on phosphorylation state. By applying protein production conditions that decrease the pool of intracellular acetyl-phosphate in E. coli, we found a reduction in the phosphorylated population of CopR II when magnesium was added to the medium, but observed no change in phosphorylated population when CopR II is expressed in E. coli BL21 (DE3) ∆pta, a mutant with a metabolic disruption to the acetyl-phosphate pathway. Therefore, the specific mechanism of post-translational phosphorylation of RRs in E. coli remains obscure. These findings show the importance of characterizing the phosphorylation state of proteins when heterologously expressed, since their biochemical and physiological properties can be dependent on post-translational modification.

SF2: Quantification for systematic characterization of phosphorylation state of heterologously expressed RRs graphical quantification of bands in 12% or phos-tag gels for each RR characterized from P. stutzeri RCH2. Blue heatmap shows quantification for non-phosphorylated bands (RR), red heatmap shows quantification for shifted, phosphorylated bands (RR-P). Plots labeled as RR-P 1 and RR-P 2 show the quantification of two distinctive shifted bands identified as two subpopulations of phosphorylated RRs that did not appear in the 12% gel. Paralogous RRs referenced in text are indicated by yellow bars. SF3: Recombinant CopR II has a partially phosphorylated population when cultivated in auto-induction media Recombinant CopR II was purified from expression strains cultivated in auto-induction media. Phospho-donors were added exogenously to purified protein with buffer pH 7.5 or 8.5. Proteins were resolved with (A) 12% (B) phos-tag gels. Quantitation can be referred to in ST1.
SF4 Recombinant CopR II and CzcR has a partially phosphorylated population when cultivated in TB media (A) Recombinant CopR II and CzcR were purified from expression strains cultivated in TB media. Phospho-donors were added exogenously to purified protein with buffer pH 7.5 or 8.5. Proteins were resolved with phos-tag gels. (B) Recombinant CopR II, CzcR D51A and CzcR were purified from expression strains cultivated in TB media. Phospho-donors were added exogenously to purified protein with buffer pH 7.5. Proteins were resolved with phos-tag gels. CzcR D51A does not have a phosphorylated population represented by a band shift. Quantitation can be referred to in Figure 2 and ST2.

SF5
The effect of exogenously added phospho-donors on CzcR and CopR II Quantitation of unphosphorylated (blue) and phosphorylated (red) bands for CzcR and CopR II with and without phospho-donors AcP or CP. Bars and error bars represent the averages and standard errors between the average of two technical replicates.
SF6 Recombinant CopR II is dephosphorylated when under denaturing conditions Recombinant CopR II was purified from expression strains cultivated in TB media. Phospho-donors or dephosphorylation reagents were added exogenously to purified protein with buffer pH 7.5. For heat conditions, recombinant proteins were denatured at 100˚C for 10 minutes. Proteins were resolved with phos-tag gels. Quantitation can be referred to in ST3.
SF7 Cultivation with copper chloride does not impact the phosphorylated population of recombinant CopR II phos-tag western blot (A) and quantitation by total and percent absorbance (B) for CopR II cultivated in TB media with and without CuCl 2 . The first two lanes of CopR II and CopR II D51A were cultivated in a prior batch without CuCl 2 and were stored at cryogenic temperatures for greater than 2 weeks before electrophoretic separation. Bars and error bars represent the averages and standard errors between the average of two technical replicates.

ST1:
Recombinant CopR II has a partially phosphorylated population when cultivated in auto-induction media Recombinant CopR II was purified from expression strains cultivated in auto-induction media. Phospho-donors were added exogenously to purified protein with buffer pH 7.5 or 8.5. Proteins were resolved with 12% or phos-tag gels. Absorbance and % total absorbance are reported for phosphorylated (P) and unphosphorylated (U) bands. Annotated images can be referred to in SF3.