University of Ljubljana, Biotechnical Faculty, Department of Microbiology, Ljubljana, Slovenia.
University of Ljubljana, Chair of Microprocess Engineering and Technology - COMPETE, Ljubljana, Slovenia.
Appl Environ Microbiol. 2022 Sep 22;88(18):e0087122. doi: 10.1128/aem.00871-22. Epub 2022 Sep 12.
Microorganisms in nature form multicellular groups called biofilms. In biofilms, bacteria embedded in the extracellular matrix (ECM) interact intensely due to their proximity. Most studies have investigated genetically homogeneous biofilms, leaving a gap in knowledge on genetically heterogeneous biofilms. Recent insights show that a Gram-positive model bacterium, Bacillus subtilis, discriminates between strains of high (kin) and low (nonkin) genetic similarity, reflected in merging (kin) and boundaries (nonkin) between swarms. However, it is unclear how kinship between interacting strains affects their fitness, the genotype assortment, and incorporation of the mutant lacking the main structural ECM polysaccharide (EpsA-O) into floating biofilms (pellicles). We cultivated Bacillus subtilis strains as mixtures of isogenic, kin, and nonkin strain combinations in the biofilm-promoting minimal medium under static conditions, allowing them to form pellicles. We show that in nonkin pellicles, the dominant strain strongly reduced the frequency of the other strain. Segregation of nonkin mixtures in pellicles increased and invasion of nonkin EpsA-O-deficient mutants into pellicles decreased compared to kin and isogenic floating biofilms. Kin and isogenic strains had comparable relative frequencies in pellicles and showed more homogenous cell mixing. Overall, our results emphasize kin discrimination as a social behavior that shapes strain distribution, spatial segregation, and ECM mutant ability to incorporate into genetically heterogenous biofilms of B. subtilis. Biofilm communities have beneficial and harmful effects on human societies in natural, medical, and industrial environments. Bacillus subtilis is a biotechnologically important bacterium that serves as a model for studying biofilms. Recent studies have shown that this species engages in kin discriminatory behavior during swarming, which may have implications for community assembly, thus being of fundamental importance. Effects of kin discrimination on fitness, genotype segregation, and success of extracellular matrix (ECM) polysaccharide (EpsA-O) mutant invasion into biofilms are not well understood. We provide evidence that kin discrimination depends on the antagonism of the dominant strain against nonkin by using environmental strains with determined kin types and integrated fluorescent reporters. Moreover, this antagonism has important implications for genotype segregation and for when the bacteria are mixed with ECM producers. The work advances the understanding of kin-discrimination-dependent bacterial sociality in biofilms and its role in the assembly of multicellular groups.
自然界中的微生物形成称为生物膜的多细胞群体。在生物膜中，嵌入细胞外基质 (ECM) 中的细菌由于彼此接近而强烈相互作用。大多数研究都调查了遗传同质的生物膜，而在遗传异质的生物膜方面存在知识空白。最近的研究结果表明，革兰氏阳性模式细菌枯草芽孢杆菌能够区分遗传相似度高（亲缘关系）和低（非亲缘关系）的菌株，这反映在群集（亲缘关系）和群集之间的边界（非亲缘关系）中。然而，亲缘关系菌株之间的相互作用如何影响它们的适应性、基因型组合以及缺乏主要结构 ECM 多糖 (EpsA-O) 的突变体融入浮式生物膜（菌膜）尚不清楚。我们在促进生物膜形成的最小培养基中，在静态条件下培养枯草芽孢杆菌作为同基因、亲缘关系和非亲缘关系菌株组合的混合物，使其形成菌膜。我们表明，在非亲缘关系的菌膜中，优势菌株强烈降低了其他菌株的频率。与亲缘关系和同基因浮式生物膜相比，非亲缘关系混合物在菌膜中的分离增加，非亲缘关系 EpsA-O 缺陷突变体对菌膜的入侵减少。亲缘关系和同基因菌株在菌膜中的相对频率相当，并且细胞混合更均匀。总体而言，我们的结果强调了亲缘关系识别作为一种社交行为，它塑造了枯草芽孢杆菌遗传异质生物膜中菌株的分布、空间分离和 ECM 突变体的整合能力。生物膜群落对自然、医学和工业环境中的人类社会既有有益影响，也有有害影响。枯草芽孢杆菌是一种具有生物技术重要性的细菌，可用作研究生物膜的模型。最近的研究表明，该物种在群集时会进行亲缘关系的辨别行为，这可能对群落组装产生影响，因此具有重要意义。亲缘关系识别对适应性、基因型分离以及细胞外基质 (ECM) 多糖 (EpsA-O) 突变体入侵生物膜的影响尚不清楚。我们使用具有确定亲缘关系类型的环境菌株和整合荧光报告基因，提供了证据表明，亲缘关系识别取决于优势菌株对非亲缘关系菌株的拮抗作用。此外，这种拮抗作用对基因型分离以及当细菌与 ECM 生产者混合时具有重要意义。这项工作推进了对生物膜中基于亲缘关系识别的细菌社会性及其在多细胞群体组装中的作用的理解。
