“Expanding the Lactococcal Cell Wall Polysaccharide Paradigm: Novel Structures and Metabolic Pathways in the Emerging Dairy Species <i>Pseudolactococcus laudensis</i> and <i>Pseudolactococcus raffinolactis</i> ”

Cell surface-associated polysaccharides, including cell wall polysaccharides (CWPSs), capsular polysaccharides (CPSs), and exopolysaccharides (EPSs), play vital roles in bacterial interactions with their environment, influencing critical aspects of dairy fermentations, such as phage-host dynamics. Pseudolactococcus laudensis and Pseudolactococcus raffinolactis (formerly Lactococcus laudensis and Lactococcus raffinolactis) are emerging dairy-associated species whose CWPSs remain uncharacterized. This study analyzed the complete genomes of 21 P. laudensis and seven P. raffinolactis strains to investigate the genetic diversity underlying CWPS and EPS production. Eight novel cwps genotypes (E-L) were identified, significantly expanding the known diversity within the dairy-associated (pseudo)lactococci. Notably, E and G genotypes diverge from the classical rhamnan-PSP organization, suggesting a CWPS biosynthesis pathway distinct from the dual-chain assembly found in previously studied Lactococcus. Additionally, eps loci were identified in 25 of the 28 strains, uncovering 11 distinct genotypes (I-XI) with evidence of horizontal gene transfer. Their integration into chromosomal genomic islands highlights their mobility and potential role in evolutionary adaptation. Chemical analysis revealed unprecedented CWPS structures. P. laudensis DSM 28961 (type E) presented a 6-deoxy-α-l-talan polysaccharide and a β-(1,4)-galactan, marking the first instance of d-talose replacing rhamnose and the first homopolysaccharide in (pseudo)lactococcal CWPS, respectively. These were structurally independent, confirming a novel CWPS organization and biosynthetic pathway. Conversely, P. raffinolactis DSM 20443 (type I) exhibited a typical rhamnan-PSP structure, composed of a variably glycosylated rhamnan and a glucose-lactose hexapolysaccharide, respectively. This study provides the first resolved CWPS structures for the Pseudolactococcus genus, expanding the understanding of polysaccharide biosynthesis in Lactic Acid Bacteria.