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.

由于极生鞭毛和侧生鞭毛的作用，巴西Azospirillum细菌会游动并蜂拥而至。它的主要表面糖聚合物由脂多糖（LPS）和结合钙氟的多糖（Cal ⁺表型）组成。运动性和表面糖聚合物对于植物相关细菌与植物的相互作用很重要。兼性植物内生菌巴西种Sp245产生两个抗原不同的LPS，LpsI和LpsII，包含相同的O-多糖。以前，使用载体pJFF350进行随机的Omegon-Km诱变，我们构建了一个名为KM018的Sp245突变体，该突变体尽管瘫痪了，但仍然拥有鞭毛。该突变体不再能够产生结合钙荧光的多糖和LpsII。由于在偶氮螺旋藻中表面糖聚合物的产生和鞭毛运动的遗传方面的实验数据有限，所以本研究的目的是鉴定和更详细地研究在突变体中失活的菌株Sp245的编码序列。我们发现pJFF350被整合到位于Sp245第六个质粒中的功能未知的推定整合膜蛋白（AZOBR_p60025）的编码序列中。为了阐明推定蛋白质的作用，我们在表达载体pRK415中克隆了AZOBR_p60025，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显示，KM018菌株（pRK415-p60025）中存在野生型LpsI⁺ LpsII ⁺配置文件已还原。互补的突变体具有Cal ⁺表型，并且能够游泳和成群运动。因此，AZOBR_p60025编码的蛋白质会显着影响主要细胞表面糖聚合物的组成以及偶氮螺旋藻的单细胞运动和社交活动。