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.
Highlights
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
The conserved, 5Ј-positioned region of the cwps cluster was proposed to encode the biosynthetic abilities for the production of the rhamnan component of the CWPS, whereas the more variable 3Ј-located region was proposed to encode for the biosynthesis of the polysaccharide pellicle (PSP) component [11, 15]
The biosynthesis pathway proposed here for L. lactis CWPS accounts for the high structural diversity encountered in these glycopolymers among strains
Summary
A dual-chain assembly pathway generates the high structural diversity of cell-wall polysaccharides in Lactococcus lactis. Ilias Theodorou‡§, Pascal Courtin¶, Simon Palussiere¶, Saulius Kulakauskas¶, X Elena Bidnenko¶, Christine Pechouxʈ, X Francois Fenaille**, Christophe Penno‡§, Jennifer Mahony‡§1, Douwe van Sinderen‡§2,3, and X Marie-Pierre Chapot-Chartier¶2,4 From the ‡School of Microbiology and §APC Microbiome Ireland, University College Cork, Western Road, Cork, Ireland, ¶Micalis Institute, INRA, AgroParisTech, Universite Paris-Saclay, 78350 Jouy-en-Josas, France, ʈINRA, UMR 1313 Genetique Animale et Biologie Integrative (GABI), Plate-forme MIMA2, 78350 Jouy-en-Josas, France, and **CEA, Institut Joliot, Service de Pharmacologie et d’Immunoanalyse, UMR 0496, Laboratoire d’Etude du Metabolisme des Medicaments, MetaboHUB-Paris, Universite Paris-Saclay, 91191 Gif-sur-Yvette, France
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