Three distinct glycosylation pathways are involved in the decoration of Lactococcus lactis cell wall glycopolymers

Abstract

Extracytoplasmic sugar decoration of glycopolymer components of the bacterial cell wall contributes to their structural diversity. Typically, the molecular mechanism that underpins such a decoration process involves a three-component glycosylation system (TGS) represented by an undecaprenyl-phosphate (Und-P) sugar-activating glycosyltransferase (Und-P GT), a flippase, and a polytopic glycosyltransferase (PolM GT) dedicated to attaching sugar residues to a specific glycopolymer. Here, using bioinformatic analyses, CRISPR-assisted recombineering, structural analysis of cell wall–associated polysaccharides (CWPS) through MALDI-TOF MS and methylation analysis, we report on three such systems in the bacterium Lactococcus lactis. On the basis of sequence similarities, we first identified three gene pairs, csdAB, csdCD, and csdEF, each encoding an Und-P GT and a PolM GT, as potential TGS component candidates. Our experimental results show that csdAB and csdCD are involved in Glc side-chain addition on the CWPS components rhamnan and polysaccharide pellicle (PSP), respectively, whereas csdEF plays a role in galactosylation of lipoteichoic acid (LTA). We also identified a potential flippase encoded in the L. lactis genome (llnz_02975, cflA) and confirmed that it participates in the glycosylation of the three cell wall glycopolymers rhamnan, PSP, and LTA, thus indicating that its function is shared by the three TGSs. Finally, we observed that glucosylation of both rhamnan and PSP can increase resistance to bacteriophage predation and that LTA galactosylation alters L. lactis resistance to bacteriocin.

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

“…The glucosylated rhamnan structure of SK11 is consistent with the presence of homolog of csdAB and the flippase-encoding gene in the genome of SK11 (Theodorou et al, 2020). Downstream of rmlA-D are the rhamnosyltransferase-encoding genes, which appear less TA B L E 1 Chemical structures and characteristics of the CWPS components of selected lactococcal strains…”

“…The rhamnan subunit of the C‐type strain SK11 is similar to those of all other analyzed C‐type strains, though it contains a stoichiometric Glc side‐chain decoration (Table 1). The transfer of a glucose side‐chain to the rhamnan structure was recently shown to be mediated by a three‐component glycosylation system (TGS) incorporating two glycosyltransferases (CsdAB, of which the corresponding genes are located outside of the cwps locus) and a flippase‐encoding gene (Theodorou et al ., 2020). The glucosylated rhamnan structure of SK11 is consistent with the presence of homolog of csdAB and the flippase‐encoding gene in the genome of SK11 (Theodorou et al ., 2020).…”

“…Ultimately, this will permit the prediction of the CWPS structure of uncharacterized, yet sequenced L. lactis strains, which in turn may provide information on phage sensitivity of such strains. Beyond the cwps loci, two distinct, so‐called three component glycosylation systems (TGSs) have been shown to add a glucose moiety to either the rhamnan or PSP component of lactococcal CWPS structures (Theodorou et al ., 2020), and thus, contribute to CWPS structural diversity. A TGS is composed of three proteins: an Und‐P‐sugar activating glycosyltransferase (Und‐P GT), a flippase transferring the sugar moiety outside the cytoplasmic membrane, and a polytopic glycosyltransferase (PolM GT) dedicated to attaching sugar residues to a specific glycopolymer.…”

Cell wall homeostasis in lactic acid bacteria: threats and defences

“…Recently, the genes involved in Gal transfer onto TA chains were identified and shown to encode a three component glycosylation system, allowing extra-cytoplasmic modification of glycopolymers (Theodorou et al . 2020 ). In such a model, Gal would be first transferred from UDP-Gal onto undecaprenyl-phosphate, then flipped to the outside of the cytoplasmic membrane with the help of a four transmembrane segment flippase, and finally transferred onto TA chains by an integral membrane glycosyltranferase with a GT-C fold (Mann and Whitfield 2016 ).…”

“…Nevertheless, additional structural diversity of PSP subunits or rhamnan results from the activity of glycosyltranferases located outside the cwps gene cluster that are part of three component mechanisms, which catalyze the addition of single Glc as side chains of PSP subunits or rhamnan (Theodorou et al . 2020 ). In L. lactis , CWPS act as receptors for numerous bacteriophages and their structural diversity explains, at least partially, the narrow host range of a number of these phages (Mahony, Cambillau and van Sinderen 2017a ).…”