Functional characterization of a StyS sensor kinase reveals distinct domains associated with intracellular and extracellular sensing of styrene in P. putida CA-3

Abstract

Bacterial two-component systems (TCSs) are of vital importance in the translation of rapidly changing environmental conditions into appropriate cellular regulatory responses enabling adaptation, growth, and survival. The diverse range of environmental signals that TCSs can process, coupled with discrete modular domains within TCS proteins, offers considerable potential for the rational design of bio-sensor and/or bio-reporter strains. In this study we functionally characterize the multi-domain StyS sensor kinase associated with sensing of the aromatic pollutant styrene by Pseudomonas putida CA-3. Deletion analysis of discrete domains was performed and the ability of the truncated StyS sensor proteins to activate a cognate reporter system in an E. coli host assessed. The essential histidine kinase and PAS input domains were identified for StyS dependent activation of the reporter system. However, co-expression of an ABC-transporter protein StyE, previously linked to styrene transport in P. putida CA-3, enabled activation of the reporter system with a StyS construct containing a non-essential PAS input domain, suggesting a novel role for intracellular detection and/or activation. Site directed mutagenesis and amino acid deletions were employed to further characterize the PAS sensing domains of both input regions. The potential implications of these findings in the use of multi-domain sensor kinases in rational design strategies and the potential link between transport and intracellular sensing are discussed.

Keywords: ABC-transporter; PAS domains; Pseudomonas putida; histidine kinase; styrene; two-component.

Figure 1. (A) Domain organization of the StyS sensor protein in Pseudomonas putida CA-3; LZ, Rec, and HK represent Leucine zipper, Receiver, and Histidine Kinase domains, respectively. styR indicates the position of the co-expressed, response regulator gene. (B) Diagrammatic representations of each SoE construct illustrating the deleted functional domain(s). A bar chart presents the styrene monoxygenase (SMO) activities from the indole to indigo reporter assay system harboring SoE constructs (1–6), respectively.

Figure 2. A diagrammatic representation of the indole to indigo assay reporter system. StyS, (star), detects styrene at membrane and activates StyR (triangle) by phosphorelay, which initiates transcription of styABC reporter genes.

Figure 3. (A) Comparsion of the predicted amino acid sequence of the StyS Input 1 domain of (1) Pseudomonas putida CA-3 with PAS domains of: (2) Pseudomonas species Y2 (CAA03998), (3) P. fluorescens (AAC06271), (4) P. putida F1 (CAB43735), (5) P. mendocina (AAL13332), and (6) T. aromatica (AAD12184). Conserved amino acids are in bold, shaded boxes indicate hydrophobic regions. Amino acids targeted for substitution in StyS from P. putida CA-3 are underlined with the proposed substitution indicated by the letter above. (B) SMO: styrene monooxygenase reporter system activity (nmol min⁻¹ mg cell dry weight⁻¹) of StyS wild type WT and StyS Input 1 mutants; F(48)Y, L(51)M, D(129)E, Y(130)H, and S(131)T, respectively.

Figure 4. (A) Comparsion of the predicted amino acid sequence of the StyS Input 2 domain of (1) Pseudomonas putida CA-3 with PAS domains of: (2) Pseudomonas species Y2 (CAA03998), (3) P. fluorescens (AAC06271), (4) P. putida F1 (CAB43735), and (5) P. mendocina (AAL13332). Conserved amino acids are in bold, shaded boxes indicate hydrophobic regions. Amino acids targeted for deletion substitution are indicated by the ♦. (B) SMO: styrene monooxygenase reporter activity (nmol min⁻¹ mg cell dry weight⁻¹) with StyS wild type WT, and StyS Input 2 mutants ∆SVS and ∆DI, respectively.