Abstract
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.
Highlights
Lactococcus lactis belongs to the group of Lactic Acid Bacteria (LAB), which typically live in nutrient-rich ecological niches such as plants, gut mucus and milk
While this SrtA-driven anchoring mechanism leads to cell wall anchoring of proteins as monomers, some LPxTG proteins harboring defined additional amino acids motifs have a different destiny and may polymerize into a pilus anchored at the cell surface of Gram-positive bacteria
While no function has yet been assigned to these putative pilins, a search for functional domains revealed that YhgE harbors two Cna protein B-type domains described in the Staphylococcus aureus collagen-binding surface protein [67]
Summary
Lactococcus lactis belongs to the group of Lactic Acid Bacteria (LAB), which typically live in nutrient-rich ecological niches such as plants, gut mucus and milk. A functional sortase A is present in the non pathogenic bacterium L. lactis and is involved in the anchoring of several proteins that play an important role in the biology of L. lactis, for example the cell wall anchored proteinase that allows growth of lactococci in milk [34] While this SrtA-driven anchoring mechanism leads to cell wall anchoring of proteins as monomers, some LPxTG proteins harboring defined additional amino acids motifs have a different destiny and may polymerize into a pilus anchored at the cell surface of Gram-positive bacteria. The contribution of the different components of the cluster is reported and we show that this yet unreported trait in lactococci provides this bacterium with a different lifestyle both in liquid medium and on solid surfaces, inducing bacterial auto-aggregation and reticulated biofilms, respectively
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