A Positive Selection for Nucleoside Kinases in E. coli

Abstract

Engineering heterologous nucleoside kinases inside E. coli is a difficult process due to the integral role nucleosides play in cell division and transcription. Nucleoside analogs are used in many kinase screens that depend on cellular metabolization of the analogs. However, metabolic activation of these analogs can be toxic through disruptions of DNA replication and transcription because of the analogs' structural similarities to native nucleosides. Furthermore, the activity of engineered kinases can be masked by endogenous kinases in the cytoplasm, which leads to more difficulties in assessing target activity. A positive selection method that can discern a heterologous kinases' enzymatic activity without significantly influencing the cell's normal metabolic systems would be beneficial. We have developed a means to select for a nucleoside kinase's activity by transporting the kinase to the periplasmic space of an E. coli strain that has its PhoA alkaline phosphatase knocked out. Our proof-of-principle studies demonstrate that the herpes simplex virus thymidine kinase (HSV-TK) can be transported to the periplasmic space in functional form by attaching a tat-signal sequence to the N-terminus of the protein. HSV-TK phosphorylates the toxic nucleoside analog 3'-azido-3'-deoxythymidine (AZT), and this charged, monophosphate form of AZT cannot cross the inner membrane. The translocation of HSV-TK provides significant resistance to AZT when compared to bacteria lacking a periplasmic HSV-TK. However, resistance decreased dramatically above 40 μg/ml AZT. We propose that this threshold can be used to select for higher activity variants of HSV-TK and other nucleoside kinases in a manner that overcomes the efficiency and localization issues of previous selection schemes. Furthermore, our selection strategy should be a general strategy to select or evaluate nucleoside kinases that phosphorylate nucleosides such as prodrugs that would otherwise be toxic to E. coli.

Fig 1. Positive selection for HSV-TK activation of AZT.

A) In typical cells, AZT penetrates the outer membrane, periplasm and inner membrane to be metabolized in to its triphosphate form to generate genotoxicity towards E. coli. B) When HSV-TK is exported to the periplasm AZT is phosphorylated in the periplasm; however, PhoA counteracts by dephosphorylating the AZT-MP, allowing AZT to penetrate the inner membrane causing genotoxicity. C) In cells lacking PhoA but containing periplasmic HSV-TK, AZT is phosphorylated and AZT-MP cannot cross the inner membrane. Thus, periplasmic HSV-TK provides AZT resistance to phoA^–strains.

Fig 2. Export of active HSV-TK using the tat signal sequence.

A) 2500 CFUs of E. coli BW14012 cells with tat-hsvtk, pelB-hsvtk or dsbA-hsvtk were plated on LB agar plates containing 50 μg/mL streptomycin, 2.5 mM ATP, 1mM IPTG, and the presence (bottom row) or absence (top row) of 10 μg/mL AZT. The plates were incubated for 20 hours at 37°C. B) Western blot using anti-HSV-TK antibodies of periplasmic (“P”) and cytoplasmic (“C”) fractions of cells expressing the indicated proteins. The expect size of HSV-TK is 41 kDa. A control using anti-GroEL antibodies was used to confirm there was no cytoplasmic protein contamination in the periplasmic fractions.

Fig 3. Viability of E. coli BW14012 cells expressing tatHSV-TK (blue circles) or tatHSV-TKD (red squares) as a function of AZT concentrations.

Cell viability is expressed relative to the number of colonies at 0 μg/mL AZT for each strain type.

Fig 4. PCR assay demonstrating at least a 10,000-fold enrichment for active tatHSV-TK over an inactive tatHSV-TKΔ.

BW14012 cells expressing one of the two proteins were mixed at a 1:10,000 ratio (tatHSV-TK:tatHSV-TKD) ratio. Twenty colonies formed when 500,000 CFUs (determined under non-selective conditions) were plated on 40 μg/ml AZT. PCR-amplified hsvtk genes from these 20 colonies indicated that all twenty contained tatHSV-TK. Expected band sizes of tatHSV-TK and tatHSV-TKD in this PCR assay are 1226 bp and 1160 bp, respectively.