The Effect of Hybrid Histidine Kinase Gene Inactivation on Cell Morphology in Azospirillum baldaniorum and Azospirillum brasilense

The aim of this study was to test a hypothesis regarding the influence of the chromosomally located HSHK-RR gene, encoding a hybrid multisensory histidine kinase, on the morphology of bacteria of the Azospirillum genus. The strains used in this study were A. baldaniorum Sp245, the Laf – mutant Sp245.1062 (Sp245-HSHKΔRR-Km), and A. brasilense Sp7 and Sp7.10030 (Sp7-HSHKΔRR-Km). Bacterial morphology and motility were characterized using electron microscopy. Relative gene transcription levels were determined using real-time PCR. The hybrid sensor histidine kinase (HSHK-RR) genes in strains Sp245 (AZOBR_150176) and Sp7 (AMK58_10030) are 95% homologous. To obtain a mutant of the Sp7 strain, we replaced the AMK58_10030 gene with the HSHKΔRR-Km construct, created on the basis of AZOBR_150176. Replacement of the internal fragment of the HSHK-RR gene with the kanamycin resistance gene resulted in changes in the morphology and motility of cells in semisolid agar in the Sp245-HSHKΔRR-Km and Sp7-HSHKΔRR-Km mutants. The mutant cells were shorter than the cells of the parental strains Sp245 and Sp7 and moved more slowly in semisolid agar, forming colonies of a significantly smaller diameter. Both mutants retained the synthesis of a functional polar flagellum (Fla). However, in the case of Sp7-HSHKΔRR-Km, up to 45% of cells synthesized sparse or isolated lateral flagella (Laf) on agar media, whereas in Sp245-HSHKΔRR-Km, such bacteria accounted for no more than 15%. It was established that Laf biogenesis is under multistage control, including a two-component signaling system involving HSHK-RR, encoded in Sp245 and Sp7 strains by chromosomal genes AZOBR_150176 and AMK58_10030, respectively. Phenotypic analysis of mutants for the AZOBR_150176 and AMK58_10030 genes revealed that the studied HSHK-RR modulates the motility of Sp245 and Sp7 under mechanical stress, participating in the transcriptional control of the flagellin genes laf1 and components of the lateral flagellar assembly export system.