Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 13 Prospekt Entuziastov, Saratov, Russia, 410049.
Folia Microbiol (Praha). 2020 Dec;65(6):963-972. doi: 10.1007/s12223-020-00805-5. Epub 2020 Jun 30.
The bacterium Azospirillum brasilense can swim and swarm owing to the work of polar and lateral flagella. Its major surface glycopolymers consist of lipopolysaccharides (LPS) and Calcofluor-binding polysaccharides (Cal phenotype). Motility and surface glycopolymers are important for the interactions of plant-associated bacteria with plants. The facultative plant endophyte A. brasilense Sp245 produces two antigenically different LPS, LpsI, and LpsII, containing identical O-polysaccharides. Previously, using vector pJFF350 for random Omegon-Km mutagenesis, we constructed a mutant of Sp245 named KM018 that still possessed flagella, although paralyzed. The mutant was no longer able to produce Calcofluor-binding polysaccharides and LpsII. Because of the limited experimental data on the genetic aspects of surface glycopolymer production and flagellar motility in azospirilla, the aim of this study was to identify and examine in more detail the coding sequence of strain Sp245, inactivated in the mutant. We found that pJFF350 was integrated into a coding sequence for a putative integral membrane protein of unknown function (AZOBR_p60025) located in the sixth plasmid of Sp245. To clarify the role of the putative protein, we cloned AZOBR_p60025 in the expression vector pRK415 and used it for the genetic complementation of mutant KM018. The SDS-PAGE, immunodiffusion, and linear immunoelectrophoresis analyses showed that in strain KM018 (pRK415-p60025), the wild-type LpsI LpsII profile was restored. The complemented mutant had a Cal phenotype and it was capable of swimming and swarming motility. Thus, the AZOBR_p60025-encoded protein significantly affects the composition of the major cell-surface glycopolymers and the single-cell and social motility of azospirilla.
巴西固氮螺菌能够游动和群集，这要归功于极性和侧向鞭毛的作用。其主要表面糖聚合物由脂多糖（LPS）和Calcofluor 结合多糖（Cal 表型）组成。运动性和表面糖聚合物对于与植物相关的细菌与植物的相互作用很重要。兼性植物内生菌巴西固氮螺菌 Sp245 产生两种具有不同抗原性的 LPS，LpsI 和 LpsII，它们含有相同的 O-多糖。先前，使用载体 pJFF350 进行随机 Omegon-Km 诱变，我们构建了一个名为 KM018 的 Sp245 突变体，该突变体仍然具有鞭毛，尽管瘫痪了。该突变体不再能够产生 Calcofluor 结合多糖和 LpsII。由于在游动螺菌的表面糖聚合物产生和鞭毛运动的遗传方面的实验数据有限，本研究的目的是鉴定并更详细地研究突变体中失活的 Sp245 菌株的编码序列。我们发现，pJFF350 整合到位于 Sp245 第六个质粒中的一个未知功能的假定整合膜蛋白（AZOBR_p60025）的编码序列中。为了阐明假定蛋白的作用，我们将 AZOBR_p60025 克隆到表达载体 pRK415 中，并将其用于突变体 KM018 的遗传互补。SDS-PAGE、免疫扩散和线性免疫电泳分析表明，在菌株 KM018（pRK415-p60025）中，恢复了野生型 LpsI LpsII 图谱。互补突变体具有 Cal 表型，并且能够游动和群集运动。因此，AZOBR_p60025 编码的蛋白显著影响主要细胞表面糖聚合物的组成以及游动螺菌的单细胞和社会运动性。
