The Effect of the flhB Plasmid Gene of the Flagellar Export Component on Flagellation and Motility in Azospirillum Bacteria

Pub Date : 2023-09-01 DOI:10.3103/s0891416823030060

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Abstract

The effect was analyzed of the flhB2 gene, which is located on the AZOBR_p4 (AZOBR_p410073 gene) plasmid in Azospirillum baldaniorum Sp245 and on the ABSP7_p3 (AMK58_26270 gene) plasmid in A. brasilense Sp7 and codes for the FlhB protein, a flagellar export component that ensures flagellin assembly, flagellation, and motility in these bacteria. We used A. baldaniorum strain Sp245, its Fla^– Laf^– mutant Sp245.1063 (Sp245-flhB1::Omegon-Km), and A. brasilense Sp7. Mutants defective in the flhB2 gene were generated by site-directed mutagenesis. Bacterial morphology and motility were characterized by electron and phase-contrast microscopy. An A. baldaniorum Sp245 mutant, Sp245-flhB2::Km, was generated that had a cloned kanamycin resistance gene in the coding sequence (CDS) AZOBR_p410073. In contrast to the Fla^– Laf^– mutant Sp245-flhB1::Omegon-Km, the flhB1 chromosomal gene of which is inactivated (AZOBR_150177 gene), strain Sp245-flhB2::Km retained the synthesis of a functioning polar flagellum (Fla), but the synthesis and functioning of lateral flagella (Laf) was impaired and the movement and spreading rates of swarming cells in semiliquid agarized media were decreased. Inactivation of the AMK58_26270 plasmid gene in Sp7, which is homologous to the AZOBR_p410073 gene (97% identity), resulted in a similar Laf^– phenotype in the corresponding mutant. Two putative flhB genes are present in the genome of strains Sp245 and Sp7. These genes are located in the chromosome (flhB1) and on the AZOBR_p4 or ABSP7_p3 plasmid (flhB2), respectively. Expression of the flhB2 gene is required for Laf assembly. Transcription of flhB2 is regulated by a mechanosignal, the perception and generation of which is ensured by the functioning Fla, apparently with the involvement of the FlhB protein encoded by the flhB1 chromosomal gene.

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鞭毛出口元件的 flhB 质粒基因对氮青霉鞭毛和运动的影响

摘要分析了flhB2基因的影响，该基因位于Azospirillum baldaniorum Sp245的AZOBR_p4（AZOBR_p410073基因）质粒和A. brasilense Sp7的ABSP7_p3（AMK58_26270基因）质粒上，编码FlhB蛋白，FlhB蛋白是一种鞭毛输出成分，可确保这些细菌中鞭毛蛋白的组装、鞭毛和运动。我们使用了 A. baldaniorum 菌株 Sp245、其 Fla- Laf- 突变体 Sp245.1063 （Sp245-flhB1::Omegon-Km）和 A. brasilense Sp7。通过定点诱变产生了 flhB2 基因缺陷突变体。通过电子显微镜和相位对比显微镜鉴定了细菌的形态和运动特性。产生了一种 A. baldaniorum Sp245 突变体 Sp245-flhB2::Km，其编码序列（CDS）AZOBR_p410073 中克隆了卡那霉素抗性基因。与Fla- Laf-突变体Sp245-flhB1::Omegon-Km（其flhB1染色体基因被灭活（AZOBR_150177基因））相比，菌株Sp245-flhB2::Km保留了功能正常的极鞭毛（Fla）的合成，但侧鞭毛（Laf）的合成和功能受损，在半液体琼脂培养基中蜂拥细胞的移动和扩散速度下降。Sp7 中的 AMK58_26270 质粒基因与 AZOBR_p410073 基因同源（相同度为 97%），该基因的失活导致相应突变体出现类似的 Laf 表型。Sp245 株和 Sp7 株的基因组中有两个假定的 flhB 基因。这些基因分别位于染色体（flhB1）和 AZOBR_p4 或 ABSP7_p3 质粒（flhB2）上。Laf 组装需要 flhB2 基因的表达。flhB2 的转录受机械信号的调控，而机械信号的感知和产生则由功能正常的 Fla 来确保，这显然与 flhB1 染色体基因编码的 FlhB 蛋白有关。

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