Htc10, the chemoreceptor of the Wsp-like pathway in Halomonas titanicae LHS3

RAMOS RICCIUTTI, FERNANDO; HERRERA SEITZ, K; BELLINZONI, M; LISA, MN; STUDDERT, CA

Virtual

Congreso; Congreso Conjunto SAIB SAMIGE; 2021

SAIB-SAMIGE

The marine bacterium Halomonas titanicae KHS3 encodes a cluster of chemotaxis-like genes that exhibits certain peculiarities highly reminiscent of the Pseudomonas Wsp system. Besides including a diguanylate cyclase (DGC) with a receiver domain that is presumably activated by a chemoreceptor-controlled histidine kinase, it includes two CheW coupling proteins, an extra tetratricopeptide (TPR) domain at the C-terminus of CheR2 methyltransferase, and an extra receiver domain at the C-terminus of the CheA2 kinase. In Pseudomonas, the Wsp pathway is involved in biofilm formation through an increment of the c-di- GMP levels. However, molecular details about how the pathway is controlled are still missing and its activation has only been observed in response to growth on solid surfaces. We were interested in characterizing the role of Htc10, the chemoreceptor encoded within the Wsp-like cluster in H. titanicae KHS3, in the control of the pathway. The overexpression of the methyltransferase CheR2 resulted in a significantly increased ability to form biofilm, suggesting that a higher level of methylation of the receptor correlates with an activation of the pathway that result in higher levels of c-di-GMP. Consistent with that, a mutant with an interrupted htc10 gene did not show any increase in biofilm upon overexpression of CheR2. In the search for stimuli that control the pathway, the ligand binding domain of Htc10 (LBDHtc10) was expressed with a 6X-histidine tag, purified, and subjected to thermal shift assays against a library of compounds. The purine derivatives guanine and hypoxanthine shifted the melting temperature (Tm) of LBDHtc10 by more than 10°C. The LBDHtc10 was crystallized in the presence of hypoxanthine and guanine, and analyzed by X-ray diffraction. The structure could not be solved by molecular replacement, thus, a new crystallization in the presence of selenomethionine was carried out, and the diffraction data from the obtained crystals were used for experimental phasing. Those data were used to solve the structure of the ligand binding domain from a crystal at 2.1 Å resolution. LBDHtc10 has a double Cache structure, and the ligand binds in the distal pocket. Contacts between the residues Y125, N161, D163 and N177 and the ligand were identified. Proteins carrying the mutations Y125F or N161A/D163A were expressed and subjected to thermal shift assays in the presence or absence of hypoxanthine. In the absence of any ligand, the Tm of the three recombinant proteins was around 45°C, indicating that the mutations did not alter the folding in a drastic way. In contrast, whereas the presence of 10 μM of hypoxanthine caused a shift of 15oC in the Tm of the wild-type protein, it hardly changed the Tm of the two tested mutants. We have demonstrated the involvement of Htc10 signaling on biofilm formation, and its specific recognition of purine derivatives. Ongoing studies are aimed to understand the role of ligands on biofilm control.