Another Brick in the Wall: a Rhamnan Polysaccharide Trapped inside Peptidoglycan of Lactococcus lactis

Abstract

Polysaccharides are ubiquitous components of the Gram-positive bacterial cell wall. In Lactococcus lactis, a polysaccharide pellicle (PSP) forms a layer at the cell surface. The PSP structure varies among lactococcal strains; in L. lactis MG1363, the PSP is composed of repeating hexasaccharide phosphate units. Here, we report the presence of an additional neutral polysaccharide in L. lactis MG1363 that is a rhamnan composed of α-l-Rha trisaccharide repeating units. This rhamnan is still present in mutants devoid of the PSP, indicating that its synthesis can occur independently of PSP synthesis. High-resolution magic-angle spinning nuclear magnetic resonance (HR-MAS NMR) analysis of whole bacterial cells identified a PSP at the surface of wild-type cells. In contrast, rhamnan was detected only at the surface of PSP-negative mutant cells, indicating that rhamnan is located underneath the surface-exposed PSP and is trapped inside peptidoglycan. The genetic determinants of rhamnan biosynthesis appear to be within the same genetic locus that encodes the PSP biosynthetic machinery, except the gene tagO encoding the initiating glycosyltransferase. We present a model of rhamnan biosynthesis based on an ABC transporter-dependent pathway. Conditional mutants producing reduced amounts of rhamnan exhibit strong morphological defects and impaired division, indicating that rhamnan is essential for normal growth and division. Finally, a mutation leading to reduced expression of lcpA, encoding a protein of the LytR-CpsA-Psr (LCP) family, was shown to severely affect cell wall structure. In lcpA mutant cells, in contrast to wild-type cells, rhamnan was detected by HR-MAS NMR, suggesting that LcpA participates in the attachment of rhamnan to peptidoglycan.

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).…”

“…Additional portions of the OS were prepared by extraction of the cell debris with the hot 0.01 HCl (Sadovskaya et al, 2017). The deproteinated HCl extract (100 mg) was treated with HF (48%, 24 hr, 4°C), evaporated, and fractionated on a Sephadex G-50 column (1 × 40 cm).…”

“…to be covalently attached to a second CWPS, a peptidoglycanembedded rhamnan, which was shown to be a polymer of a neutral rhamnose-containing trisaccharide subunit in the model strain MG1363 (Sadovskaya et al, 2017). The PSP exhibits structural diversity between strains which appears as a major factor explaining the narrow specificity of lactococcal phages.…”

N-Deacetylases required for muramic-δ-lactam production are involved in Clostridium difficile sporulation, germination, and heat resistance

“…Spore measurements were obtained using the spore preparation protocol described above. Samples were prepared as described in Sadovskaya et al (58). Staining and examinations were done by the GABI-MIMA2 TEM Platform at INRA, Jouy-en-Josas, France.…”

Genetics of Lactococci

“…Second, a pellicle-like carbohydrate structure was visualized on the L. lactis MG1363 surface and characterized biochemically (137). A third structure, described as trapped within the peptidoglycan, corresponds to another pellicle-like carbohydrate thin outer layer surrounding L. lactis strain MG1363 (138).…”