Investigation of glycopolymer assembly systems in lactococcus lactis
University College Cork
The lactic acid bacterium Lactococcus lactis is the most widely employed species in dairy fermentations, and due to this economic significance extensive research pertaining to its functionality, physiology and interaction with its environment has been performed (1). Since the discovery of their disruptive impact on fermentations, extensive scientific efforts have been directed to elucidate the molecular interactions between L. lactis and its infecting bacteriophages (phages). The lactococcal cell wall consists of a thick peptidoglycan layer as well as its associated secondary polymers. It represents a formidable barrier for phages, and for this reason deserves thorough and focused investigation. In particular, several components of the cell wall have either been conclusively determined to act as receptors for phage recognition and attachment during infection, or are at least suspected to play a role in this process. In particular, polysaccharides associated with the cell wall play a crucial role in the recognition/attachment process of many phages. In this thesis, the biosynthesis of such glycopolymers as well as their contribution to phage-host interactions will be extensively discussed. A major component of the L. lactis cell wall is the cell wall-associated polysaccharide (CWPS), which consists of a rhamnosyl polysaccharide, known as the rhamnan, and a phosphopolysaccharide (PSP), known as the pellicle. Previous work has established the importance of the so-called cwps gene cluster for the assembly of this glycopolymer (2) as well as its importance in establishing phage/host interactions (3). Following an extensive investigation of the L. lactis genome, seven additional genes were identified based on their homology to previously identified genes in Gram positive bacteria, encoding enzymes involved in glycopolymer assembly. Of these seven genes, six are found as three pairs, termed csdAB, csdCD, and csdEF, in the lactococcal genome. The first gene of each pair (csdA, csdC, and csdE) is presumed to encode an undecaprenyl-phosphate (C55-P) activating glycosyltransferase (C55-P GT), while the second gene (csdB, csdC, and csdE) encodes a putative polytopic glycosyltransferase (PolM GT). Lastly, a flippase-encoding gene, termed cflA, was also identified. Each pair of the GT-encoding genes along with the flippase-encoding gene could putatively encode for a three-component glycosylation system previously encountered in several Gram-negative and Gram-positive bacteria (4). The specific function of each of the three csd gene pairs was investigated through a mutational approach, whereby a non-sense mutation was introduced into the above-mentioned genes either individually or in combination using single stranded DNA (ssDNA) recombineering. The effect of these mutations on various saccharidic structures associated with the lactococcal cell wall was investigated by means of high performance anion exchange chromatography (HPAEC), matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and gas-chromatography mass-spectrometry (GC-MS). Our analysis was successful in determining the functionality of each of the three gene pairs (csdAB, csdCD, csdEF). More specifically, CsdA/CsdB are required for the glucosylation of the rhamnan component of the CWPS, while the CsdC/CsdD proteins are involved in PSP glucosylation. The products of csdEF do not appear to alter the structure of CWPS, yet were shown to galactosylate lipoteichoic acid (LTA) moieties. We present evidence that the presumed flippase, CflA, is responsible for rhamnan and, though partially, PSP glucosylation. Finally, glucosylation of lactococcal rhamnan and PSP, as imparted by CsdAB and CsdCD respectively, was shown to have an impact on phage/host interactions. No such association was identified for the galactosylation of LTA by CsdEF, however, preliminary evidence for the importance of LTA glycosylation in the cell’s intrinsic bacteriocin sensitivity was observed. A similar mutational strategy to that described above was employed for the functional genomic dissection of the cwps gene cluster. An extensive structural analysis of the extracted CWPS from these individual cwps mutants provided insights into the various functions of the enzymes encoded by the mutated cwps genes. From the structural analysis of the PSP, it was shown that certain mutations exerted a more detrimental impact on PSP production than others: mutations in wpsJ, wpsA, and wpsC appear to cause complete elimination of PSP production, while mutations in the remaining genes, (wpsBDEFHI) still allowed for the production of reduced levels of (sometimes incomplete) PSP. This difference in PSP production (i.e. no or reduced levels of PSP) of these cwps mutants also had an impact on their phage sensitivity profiles. More specifically, it was observed that all cwps mutants were resistant to infection by phages that are able to infect the wild type host L. lactis NZ9000. However, upon sustained exposure of certain cwps mutants to such phages, escape mutants could be isolated that were able to infect cwps mutants producing reduced or incomplete PSP, whereas no such escape mutants could be obtained against strains carrying cwps mutations that cause complete elimination of PSP production. In tandem with the functional characterization of cwps genes, the transcriptional organisation of the cluster was investigated through promoter mapping and primer extension analysis. These results confirmed previous speculation on the division of the gene cluster into three distinct transcriptional units in which the 5’-end operon is dedicated to rhamnan assembly and the 3’-end operon is dedicated to PSP assembly. The work described in this thesis has identified three distinct glycosylation systems which are variably present within members of the L. lactis species, and which are employed for the modification of different cell wall-associated glycopolymers. Together with the extensive characterisation of the cwps gene cluster, it has provided further insight into the discrete biosynthetic steps required for the assembly of complex glycopolymers such as the CWPS and LTA in L. lactis. Although we obtained a working model on the mode of assembly of these structures, the importance of their observed structural diversity on bacterial phenotypic differences has been far from resolved and further investigations will be required to understand these relationships and associated biological significance.
Lactococcus lactis , Glycobiology , Cell wall polysaccharides , Glycosyltransferase
Theodorou, I. 2019. Investigation of glycopolymer assembly systems in lactococcus lactis. PhD Thesis, University College Cork.