Identification and characterisation of chemoreceptors with NIT domains in Pseudomonas syringae pv. actinidiae

Bacteria move in response to chemical cues to find environments favourable for survival. This behaviour is called chemotaxis and is mediated by arrays of membrane-located chemoreceptors. The sensor domains of the chemoreceptors can be specific for a single chemical or can recognise many different chemicals. Some bacteria are chemotactic to nitrate, which can serve as an electron receptor or source of nitrogen. Recent studies have identified different types of chemoreceptor sensor domain involved in nitrate chemotaxis in different bacteria. In this work, I investigated putative nitrate/nitrite chemoreceptors in the kiwifruit pathogen Pseudomonas syringae pv. actinidiae (Psa) NZ-V13.

I first used a fluorescent reporter screen to survey 39 Psa NZ-V13 chemoreceptors for nitrate and nitrite binding. Two of the chemoreceptors namely IYO_024760 and IYO_028985, showed strong signals to nitrate and nitrite. IYO_028985 had been partially characterised in the past as a nitrate/nitrite sensor. Therefore, I focused on IYO_024760. Structural modelling predicted that this protein contains a NIT (nitrate-nitrite sensing) domain. I purified the sensor domain and tested it for nitrate/nitrite binding using thermal shift assays and isothermal titration calorimetry. Both techniques indicate the sensor domain of IYO_024760 binds nitrate and nitrite, which suggests this chemoreceptor mediates chemotaxis to these two chemicals. I additionally identified that the IYO_024760 chemoreceptor is overrepresented in the pandemic lineage of Psa3 which suggests it could be involved in the virulence of this pathogen. I also investigated the specificity of a predicted NIT-domain containing chemoreceptor that was negative in the fluorescence reporter screen. I purified the sensor domain of IYO_013180, and demonstrated its ability to bind nitrate and nitrite.

Finally, I investigated the swimming behaviour of Psa NZ-V13 towards potassium nitrate under different conditions. I initially found a strong response but was later unable to reproduce this phenotype. To probe the role of the three nitrate-binding chemoreceptors in Psa NZ-V13 I deleted all three from its genome and evaluated the mutants for changes in chemotaxis to potassium nitrate. Surprisingly, I detected low chemotaxis to potassium nitrate in all strains comparable to the wildtype. This study demonstrates the feasibility of using chimeras to screen for chemoreceptor binding and characterises two new nitrate binding chemoreceptors from Psa NZ-V13.