Pilus Biogenesis in Lactococcus lactis: Molecular Characterization and Role in Aggregation and Biofilm Formation

The genome of Lactococcus lactis strain IL1403 harbors a putative pilus biogenesis cluster consisting of a sortase C gene flanked by 3 LPxTG protein encoding genes (yhgD, yhgE, and yhhB), called here pil. However, pili were not detected under standard growth conditions. Over-expression of the pil operon resulted in production and display of pili on the surface of lactococci. Functional analysis of the pilus biogenesis machinery indicated that the pilus shaft is formed by oligomers of the YhgE pilin, that the pilus cap is formed by the YhgD pilin and that YhhB is the basal pilin allowing the tethering of the pilus fibers to the cell wall. Oligomerization of pilin subunits was catalyzed by sortase C while anchoring of pili to the cell wall was mediated by sortase A. Piliated L. lactis cells exhibited an auto-aggregation phenotype in liquid cultures, which was attributed to the polymerization of major pilin, YhgE. The piliated lactococci formed thicker, more aerial biofilms compared to those produced by non-piliated bacteria. This phenotype was attributed to oligomers of YhgE. This study provides the first dissection of the pilus biogenesis machinery in a non-pathogenic Gram-positive bacterium. Analysis of natural lactococci isolates from clinical and vegetal environments showed pili production under standard growth conditions. The identification of functional pili in lactococci suggests that the changes they promote in aggregation and biofilm formation may be important for the natural lifestyle as well as for applications in which these bacteria are used.

乳酸乳球菌（Lactococcus lactis）IL1403菌株的基因组中携带一个推定的菌毛生物合成基因簇：该簇由被3个LPxTG蛋白编码基因（yhgD、yhgE与yhhB）侧翼环绕的C型分选酶（sortase C）基因组成，本文中将其命名为pil。然而，在标准培养条件下并未检测到菌毛的存在。对该pil操纵子进行过表达后，乳球菌表面成功产生并展示了菌毛。针对菌毛生物合成装置的功能分析显示：菌毛轴由YhgE菌毛蛋白的寡聚体构成，菌毛帽由YhgD菌毛蛋白形成，而YhhB则为负责将菌毛纤维锚定至细胞壁的基底菌毛蛋白。菌毛蛋白亚基的寡聚化过程由C型分选酶催化，而菌毛向细胞壁的锚定则由A型分选酶（sortase A）介导。携带菌毛的乳酸乳球菌在液体培养物中表现出自聚集表型，该表型归因于主要菌毛蛋白YhgE的聚合作用。与无菌毛的细菌相比，携带菌毛的乳球菌可形成更厚实、更具气生性的生物被膜，这一表型同样由YhgE的寡聚体所介导。本研究首次解析了非致病性革兰氏阳性菌中的菌毛生物合成机制。对临床与植物环境中分离得到的天然乳球菌菌株进行分析后发现，其在标准培养条件下可产生菌毛。在乳球菌中鉴定出功能性菌毛，表明菌毛所介导的聚集与生物被膜形成变化，可能对其天然生存方式以及该细菌的各类应用场景均具有重要意义。