A dual-chain assembly pathway generates the high structural diversity of cell-wall polysaccharides in Lactococcus lactis

Abstract

In Lactococcus lactis, cell-wall polysaccharides (CWPSs) act as receptors for many bacteriophages, and their structural diversity among strains explains, at least partially, the narrow host range of these viral predators. Previous studies have reported that lactococcal CWPS consists of two distinct components, a variable chain exposed at the bacterial surface, named polysaccharide pellicle (PSP), and a more conserved rhamnan chain anchored to, and embedded inside, peptidoglycan. These two chains appear to be covalently linked to form a large heteropolysaccharide. The molecular machinery for biosynthesis of both components is encoded by a large gene cluster, named cwps. In this study, using a CRISPR/Cas-based method, we performed a mutational analysis of the cwps genes. MALDI-TOF MS-based structural analysis of the mutant CWPS combined with sequence homology, transmission EM, and phage sensitivity analyses enabled us to infer a role for each protein encoded by the cwps cluster. We propose a comprehensive CWPS biosynthesis scheme in which the rhamnan and PSP chains are independently synthesized from two distinct lipid-sugar precursors and are joined at the extracellular side of the cytoplasmic membrane by a mechanism involving a membrane-embedded glycosyltransferase with a GT-C fold. The proposed scheme encompasses a system that allows extracytoplasmic modification of rhamnan by complex substituting oligo-/polysaccharides. It accounts for the extensive diversity of CWPS structures observed among lactococci and may also have relevance to the biosynthesis of complex rhamnose-containing CWPSs in other Gram-positive bacteria.

Complete Structure of the Enterococcal Polysaccharide Antigen (EPA) of Vancomycin-Resistant Enterococcus faecalis V583 Reveals that EPA Decorations Are Teichoic Acids Covalently Linked to a Rhamnopolysaccharide Backbone

“…Two different types of cell wall polysaccharides have been reported in L. lactis. Strain MG1363 has been found to produce a polyrhamnose made of linear trisaccharide (2-␣-L-Rha-2-␣-L-Rha-3-␣-L-Rha) repeating units and a polysaccharide pellicle composed of repeating hexasaccharide phosphate units (31,39,40). The structural organization of the rhamnopolysaccharide produced by E. faecalis is different from that of the rhamnopolysaccharide produced by other Gram-positive organisms described to date since TAs are covalently linked to the EPA rhamnan backbone.…”

The CWPS Rubik’s cube: Linking diversity of cell wall polysaccharide structures with the encoded biosynthetic machinery of selected Lactococcus lactis strains

“…As mentioned previously, a model for the predicted biosynthetic pathway of the rhamnan and PSP of the model C-type strain MG1363 has been put forward (Sadovskaya et al, 2017;Theodorou et al, 2019). Given the significant disparities between the genetic YcbG possesses a LicD domain, which is associated with phosphocholine and phosphosugar transferase activities in a number of species (Kuchta et al, 2009).…”

Cell wall homeostasis in lactic acid bacteria: threats and defences

“…Recently, a comprehensive biosynthesis scheme was proposed based on a mutational analysis of the genes with a CRISPR-Cas9 based method combined with structural analysis of the mutant CWPS by mass spectrometry and bioinformatic analysis of the proteins encoded in the gene cluster (Theodorou et al . 2019 ). This scheme was supported also by transmission electron microscopy and phage sensitivity assays.…”

“…Finally, PSP would be linked covalently to rhamnan at the extracellular face of the cytoplasmic membrane (Theodorou et al . 2019 ) by the membrane-embedded glycosyltranferase with a GT-C fold, WpsG (Fig. 2 ).…”

“…Moreover, phage escape mutants, that are able to infect engineered lactococcal strains with altered CWPS, acquired mutations within the baseplate-encoding region, confirming their adaption to new host receptors (Theodorou et al . 2019 ). CWPSs have been unequivocally proven as receptors for several 936 and P335 phages, as well as for the less common 949 and 1358 phage groups (Farenc et al .…”