Post-translational Modification In Bacteria Research Articles

Global Acetylomics of Campylobacter jejuni Shows Lysine Acetylation Regulates CadF Adhesin Processing and Human Fibronectin Binding.

Lysine acetylation (KAc) is a reversible post-translational modification (PTM) that can alter protein structure and function; however, specific roles for KAc are largely undefined in bacteria. Acetyl-lysine immunoprecipitation and LC-MS/MS identified 5567 acetylated lysines on 1026 proteins from the gastrointestinal pathogen Campylobacter jejuni (∼63% of the predicted proteome). KAc was identified on proteins from all subcellular locations, including the outer membrane (OM) and extracellular proteins. Label-based LC-MS/MS identified proteins and KAc sites during growth in 0.1% sodium deoxycholate (DOC, a component of gut bile salts). 3410 acetylated peptides were quantified, and 784 (from 409 proteins) were differentially abundant in DOC growth. Changes in KAc involved multiple pathways, suggesting a dynamic role for this PTM in bile resistance. As observed elsewhere, we show KAc is primarily nonenzymatically mediated via acetyl-phosphate; however, the deacetylase CobB also contributes to a global elevation of this modification in DOC. We observed several multiply acetylated OM proteins and altered DOC abundance of acetylated peptides in the fibronectin (Fn)-binding adhesin CadF. We show KAc reduces CadF Fn binding and prevalence of lower mass variants. This study provides the first system-wide lysine acetylome of C. jejuni and contributes to our understanding of KAc as an emerging PTM in bacteria.

Dual incorporation of non-canonical amino acids enables production of post-translationally modified selenoproteins.

Post-translational modifications (PTMs) can occur on almost all amino acids in eukaryotes as a key mechanism for regulating protein function. The ability to study the role of these modifications in various biological processes requires techniques to modify proteins site-specifically. One strategy for this is genetic code expansion (GCE) in bacteria. The low frequency of post-translational modifications in bacteria makes it a preferred host to study whether the presence of a post-translational modification influences a protein's function. Genetic code expansion employs orthogonal translation systems engineered to incorporate a modified amino acid at a designated protein position. Selenoproteins, proteins containing selenocysteine, are also known to be post-translationally modified. Selenoproteins have essential roles in oxidative stress, immune response, cell maintenance, and skeletal muscle regeneration. Their complicated biosynthesis mechanism has been a hurdle in our understanding of selenoprotein functions. As technologies for selenocysteine insertion have recently improved, we wanted to create a genetic system that would allow the study of post-translational modifications in selenoproteins. By combining genetic code expansion techniques and selenocysteine insertion technologies, we were able to recode stop codons for insertion of N ε-acetyl-l-lysine and selenocysteine, respectively, into multiple proteins. The specificity of these amino acids for their assigned position and the simplicity of reverting the modified amino acid via mutagenesis of the codon sequence demonstrates the capacity of this method to study selenoproteins and the role of their post-translational modifications. Moreover, the evidence that Sec insertion technology can be combined with genetic code expansion tools further expands the chemical biology applications.

An emerging field: Post-translational modification in microbiome.

Post-translational modifications (PTMs) play an essential role in most biological processes. PTMs on human proteins have been extensively studied. Studies on bacterial PTMs are emerging, which demonstrate that bacterial PTMs are different from human PTMs in their types, mechanisms and functions. Few PTM studies have been done on the microbiome. Here, we reviewed several studied PTMs in bacteria including phosphorylation, acetylation, succinylation, glycosylation, and proteases. We discussed the enzymes responsible for each PTM and their functions. We also summarized the current methods used to study microbiome PTMs and the observations demonstrating the roles of PTM in the microbe-microbe interactions within the microbiome and their interactions with the environment or host. Although new methods and tools for PTM studies are still needed, the existing technologies have made great progress enabling a deeper understanding of the functional regulation of the microbiome. Large-scale application of these microbiome-wide PTM studies will provide a better understanding of the microbiome and its roles in the development of human diseases.

Protein post-translational modifications in bacteria.

Over the past decade the number and variety of protein post-translational modifications that have been detected and characterized in bacteria have rapidly increased. Most post-translational protein modifications occur in a relatively low number of bacterial proteins in comparison with eukaryotic proteins, and most of the modified proteins carry low, substoichiometric levels of modification; therefore, their structural and functional analysis is particularly challenging. The number of modifying enzymes differs greatly among bacterial species, and the extent of the modified proteome strongly depends on environmental conditions. Nevertheless, evidence is rapidly accumulating that protein post-translational modifications have vital roles in various cellular processes such as protein synthesis and turnover, nitrogen metabolism, the cell cycle, dormancy, sporulation, spore germination, persistence and virulence. Further research of protein post-translational modifications will fill current gaps in the understanding of bacterial physiology and open new avenues for treatment of infectious diseases.

Quantitative Analyses Reveal Novel Roles for N-Glycosylation in a Major Enteric Bacterial Pathogen.

In eukaryotes, glycosylation plays a role in proteome stability, protein quality control, and modulating protein function; however, similar studies in bacteria are lacking. Here, we investigate the roles of general protein glycosylation systems in bacteria using the enteropathogen Campylobacter jejuni as a well-defined example. By using a quantitative proteomic strategy, we were able to monitor changes in the C. jejuni proteome when glycosylation is disrupted. We demonstrate that in C. jejuni, N-glycosylation is essential to maintain proteome stability and protein quality control. These findings guided us to investigate the role of N-glycosylation in modulating bacterial cellular activities. In glycosylation-deficient C. jejuni, the multidrug efflux pump and electron transport pathways were significantly impaired. We demonstrate that in vivo, fully glycosylation-deficient C. jejuni bacteria were unable to colonize its natural avian host. These results provide the first evidence of a link between proteome stability and complex functions via a bacterial general glycosylation system.IMPORTANCE Advances in genomics and mass spectrometry have revealed several types of glycosylation systems in bacteria. However, why bacterial proteins are modified remains poorly defined. Here, we investigated the role of general N-linked glycosylation in a major food poisoning bacterium, Campylobacter jejuni The aim of this study is to delineate the direct and indirect effects caused by disrupting this posttranslational modification. To achieve this, we employed a quantitative proteomic strategy to monitor alterations in the C. jejuni proteome. Our quantitative proteomic results linked general protein N-glycosylation to maintaining proteome stability. Functional analyses revealed novel roles for bacterial N-glycosylation in modulating multidrug efflux pump, enhancing nitrate reduction activity, and promoting host-microbe interaction. This work provides insights on the importance of general glycosylation in proteins in maintaining bacterial physiology, thus expanding our knowledge of the emergence of posttranslational modification in bacteria.

Ancient Regulatory Role of Lysine Acetylation in Central Metabolism.

ABSTRACTLysine acetylation is a common protein post-translational modification in bacteria and eukaryotes. Unlike phosphorylation, whose functional role in signaling has been established, it is unclear what regulatory mechanism acetylation plays and whether it is conserved across evolution. By performing a proteomic analysis of 48 phylogenetically distant bacteria, we discovered conserved acetylation sites on catalytically essential lysine residues that are invariant throughout evolution. Lysine acetylation removes the residue’s charge and changes the shape of the pocket required for substrate or cofactor binding. Two-thirds of glycolytic and tricarboxylic acid (TCA) cycle enzymes are acetylated at these critical sites. Our data suggest that acetylation may play a direct role in metabolic regulation by switching off enzyme activity. We propose that protein acetylation is an ancient and widespread mechanism of protein activity regulation.

A Sir2 family protein Rv1151c deacetylates HU to alter its DNA binding mode in Mycobacterium tuberculosis

Till recently, knowledge about epigenetic regulation in bacterial world confined largely to DNA methylation. Lysine acetylation/deacetylation of histones is a major contributor for chromatin dynamics in eukaryotes. However, little is known about such epigenetic changes brought about by post-translational modifications in bacteria. Here, we describe an example of such mechanism occurring in a histone like protein, HU from Mycobacterium tuberculosis (Mtb). Previously, we demonstrated the interaction and acetylation of Mtb HU (MtHU) by one of the acetyl transferases, Eis. In this work, we demonstrate the deacetylation of acetylated HU (MtHUAc) by Rv1151c, the only Sir2 like protein discovered in Mtb. The DNA binding properties of MtHU are significantly altered upon acetylation but reversed consequent to deacetylation by the deacetylase. Deacetylated HU (MtHUdAc) bound to relaxed DNA leading to the formation of looped and dense molecules as compared to open structures formed by its acetylated form. Interaction of MtHUdAc with linear DNA modifies its organization leading to formation of highly bridged compact structures while binding of MtHUAc leads to the formation of stiff and straight rods. That a nucleoid associated protein can undergo acetylation/deacetylation to alter its DNA binding and architectural role opens up a new dimension of investigation of epigenetic regulation in mycobacteria.

The Bacterial Two-Hybrid System Uncovers the Involvement of Acetylation in Regulating of Lrp Activity in Salmonella Typhimurium.

N𝜀-lysine acetylation is an abundant and important Post-translational modification in bacteria. We used the bacterial two-hybrid system to screen the genome library of the Salmonella Typhimurium to identify potential proteins involved in acetyltransferase Pat – or deacetylase CobB-mediated acetylation. Then, the in vitro (de)acetylation assays were used to validate the potential targets, such as STM14_1074, NrdF, RhaR. Lrp, a leucine-responsive regulatory protein and global regulator, was shown to interact with Pat. We further demonstrate that Lrp could be acetylated by Pat and deacetylated by NAD+-dependent CobB in vitro. Specifically, the conserved lysine residue 36 (K36) in helix-turn-helix (HTH) DNA-binding domain of Lrp was acetylated. Acetylation of K36 impaired the function of Lrp through altering the affinity with the target promoter. The mutation of K36 in chromosome mimicking acetylation enhanced the transcriptional level of itself and attenuated the mRNA levels of Lrp-regulated genes including fimA, which was confirmed by yeast agglutination assay. These findings demonstrate that the acetylation regulates the DNA-binding activity of Lrp, suggesting that acetylation modification of transcription factors is a conserved regulatory manner to modulate gene expression in bacteria and eukaryotes.

Temporal Regulation of the Bacillus subtilis Acetylome and Evidence for a Role of MreB Acetylation in Cell Wall Growth.

The past decade highlighted Nε-lysine acetylation as a prevalent posttranslational modification in bacteria. However, knowledge regarding the physiological importance and temporal regulation of acetylation has remained limited. To uncover potential regulatory roles for acetylation, we analyzed how acetylation patterns and abundances change between growth phases in B. subtilis. To demonstrate that the identification of cell growth-dependent modifications can point to critical regulatory acetylation events, we further characterized MreB, the cell shape-determining protein. Our findings led us to propose a role for MreB acetylation in controlling cell width by restricting cell wall growth.

Regulatory potential of post-translational modifications in bacteria.

Bacteria are often viewed as simple organisms, with very basic and robust cellular regulation, optimized for rapid growth. While they certainly fit that description, bacteria also possess an amazing capacity for adaptation, and diversity of survival strategies, including variations of cell morphology, size, mode of growth and developmental behavior. Post-translational modifications (PTMs) of proteins contribute significantly to bacterial adaptability and cell cycle control. Research on PTMs in bacteria started with the assumption that they lack many features regularly found in more complex organisms. However, ongoing investigation keeps revealing new types of PTMs of bacterial proteins. This research topic gathers a number of articles discussing the advanced methods for systematic study of bacterial PTMs, approaches to utilize PTMs for biotechnological purposes, and revealing new cellular functions controlled by PTMs. Most bacterial PTMs are dynamic and reversible. This allows the cell to exploit them as regulatory devices. It also means that the full understanding of the cellular roles of different PTMs necessitates global, quantitative and time-resolved studies. One seminal paper in this topic reports a novel proteomics approach for such quantitative studies: the Intensity Based Absolute Quantitation (iBAQ). Using this approach, the authors have quantified the expression and the occupancy of various PTM sites in the proteome of Escherichia coli (Soufi et al., 2015). The authors report remarkable differences in expression and occupancy of PTMs sites under different growth conditions. The dataset comprised 2300 proteins, which is close to 90% of the expressed proteome. This is an important landmark, since proteomics in general has not yet attained the same level of coverage that global transcriptome studies can achieve. Among different bacterial PTMs, protein phosphorylation is the most extensively studied one, and in this topic it features prominently. Several papers present global studies of protein phosphorylation in bacteria (Spat et al., 2015), including important bacterial pathogens (Fortuin et al., 2015; Nakedi et al., 2015). The focus on pathogens is understandable, since protein phosphorylation, and several other PTMs, are heavily involved in different infection strategies displayed by bacterial pathogens (Michard and Doublet, 2015). In addition to phosphoproteome studies, interactomics is also featured as a useful approach to chart the phosphorylation-based regulatory networks. It enables researchers to trace the connections among protein kinases, phosphatases, and their substrates (Shi et al., 2014a). This approach for reconstructing phosphorylation networks highlights the capacity of bacterial proteins kinases from different families to interact with, and phosphorylate, each other (Shi et al., 2014b). Protein-tyrosine kinases, and their various roles in bacterial physiology were in the focus of several review and opinion articles in this topic (Barak, 2014; Gerwig and Stulke, 2014; Mijakovic and Deutscher, 2015). Other topic contributions highlight the broad spectrum of PTMs involved in key cellular processes. In particular, novel modifications are discussed: redox regulation via reversible S-thiolation (Loi et al., 2015), post-translational hydroxylation (van Staalduinen and Jia, 2015) and the role of citrullination for the interaction between bacteria and human mucosal surfaces (Sofat et al., 2015). Several experimental papers report studies on post-translationally modified antimicrobial peptides known as lantibiotics. These are ribosomally synthesized peptides which can efficiently inhibit the growth of Gram-positive bacteria. A study by Zhou et al. (2015) described an engineering strategy to make the lantibiotics more effective in inhibiting several important bacterial pathogens. In another study, Khusainov et al. (2015) describe the active site of the nisin dehydratase. This enzyme, essential for the production of the lantibiotic nisin, converts serines, and threonines, to dehydroalanine and dehydrobutyrine residues, respectively. Interestingly, bacterial PTM systems, such as the N-glycosylation machinery, are also being exploited and engineered to facilitate the production of recombinant vaccines (Garcia-Quintanilla et al., 2014). Another focal point of the topic are the PTMs targeting the PII proteins, which are the key signal transduction proteins involved in the control of nitrogen metabolism in bacteria and archaea (Radchenko et al., 2014; Merrick, 2015). Finally, Stannek et al. (2015) contributed a study on a regulatory mechanism involving arginine phosphorylation and regulated proteolysis. In conclusion, the contributions in this topic reflect the diversity of bacterial PTMs. Several studies highlight one important emergent feature of PTM systems: their capacity to interact with each other, creating an additional level of complexity in the cellular regulation. This is one of the key features of bacterial PTM systems and a challenge which future studies will have to address.

Structural, kinetic and proteomic characterization of acetyl phosphate-dependent bacterial protein acetylation.

The emerging view of Nε-lysine acetylation in eukaryotes is of a relatively abundant post-translational modification (PTM) that has a major impact on the function, structure, stability and/or location of thousands of proteins involved in diverse cellular processes. This PTM is typically considered to arise by the donation of the acetyl group from acetyl-coenzyme A (acCoA) to the ε-amino group of a lysine residue that is reversibly catalyzed by lysine acetyltransferases and deacetylases. Here, we provide genetic, mass spectrometric, biochemical and structural evidence that Nε-lysine acetylation is an equally abundant and important PTM in bacteria. Applying a recently developed, label-free and global mass spectrometric approach to an isogenic set of mutants, we detected acetylation of thousands of lysine residues on hundreds of Escherichia coli proteins that participate in diverse and often essential cellular processes, including translation, transcription and central metabolism. Many of these acetylations were regulated in an acetyl phosphate (acP)-dependent manner, providing compelling evidence for a recently reported mechanism of bacterial Nε-lysine acetylation. These mass spectrometric data, coupled with observations made by crystallography, biochemistry, and additional mass spectrometry showed that this acP-dependent acetylation is both non-enzymatic and specific, with specificity determined by the accessibility, reactivity and three-dimensional microenvironment of the target lysine. Crystallographic evidence shows acP can bind to proteins in active sites and cofactor binding sites, but also potentially anywhere molecules with a phosphate moiety could bind. Finally, we provide evidence that acP-dependent acetylation can impact the function of critical enzymes, including glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, and RNA polymerase.

Ser/Thr/Tyr phosphoproteome characterization of Acinetobacter baumannii: Comparison between a reference strain and a highly invasive multidrug-resistant clinical isolate

In the current study, the Ser/Thr/Tyr phosphoproteomes of two Acinetobacter baumannii strains, reference (ATCC17978) and highly invasive multidrug-resistant clinical isolate (Abh12O-A2) were analyzed using SCX and TiO2 chromatography followed by high resolution mass spectrometry. We detected a total of 201 unique phosphorylation sites (p-sites), and, after manual validation of peptide spectra, 91 high-confidence phosphorylation events (p-events) could be localized to a specific amino acid residue. The percentage distribution of Ser/Thr/Tyr phosphorylation was 68.9% on serine, 24.1% on threonine and 5.2% on tyrosine in ATCC17978, and 70.8% on serine, 25.2% on threonine and 3.8% on tyrosine in AbH12O-A2. Across all identified p-sites, 11 were identified in ATCC17978 only, while 43 were identified in Abh12O-A2 only, and 37 overlapped between the two strains. Here for the first time we describe the phosphoproteome of A. baumanii, and significance of selected phosphorylation sites is discussed in the context of stress/starvation, pathogenicity and drug resistance. It is now well established that protein phosphorylation on Ser/Thr/Tyr residues is an important post-translational modification in bacteria. Herein we employed SCX and TiO2 chromatographic phosphopeptide enrichment combined with LTQ-Orbitrap mass spectrometric analyses to characterize and establish a qualitative comparison between the Ser/Thr/Tyr phosphoproteomes of two Acinetobacter baumannii strains: a reference strain and a highly invasive multidrug-resistant clinical isolate. We highlight the identification of phosphoproteins with a role in pathogenicity and those involved in drug resistance.

Beyond gene expression: The impact of protein post-translational modifications in bacteria

The post-translational modification (PTM) of proteins plays a critical role in the regulation of a broad range of cellular processes in eukaryotes. Yet their role in governing similar systems in the conventionally presumed 'simpler' forms of life has been largely neglected and, until recently, was thought to occur only rarely, with some modifications assumed to be limited to higher organisms alone. Recent developments in mass spectrometry-based proteomics have provided an unparalleled power to enrich, identify and quantify peptides with PTMs. Additional modifications to biological molecules such as lipids and carbohydrates that are essential for bacterial pathophysiology have only recently been detected on proteins. Here we review bacterial protein PTMs, focusing on phosphorylation, acetylation, proteolytic degradation, methylation and lipidation and the roles they play in bacterial adaptation - thus highlighting the importance of proteomic techniques in a field that is only just in its infancy. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.

Acetyl-Phosphate Is a Critical Determinant of Lysine Acetylation in E. coli

Lysine acetylation is a frequently occurring posttranslational modification in bacteria; however, little is known about its origin and regulation. Using the model bacterium Escherichia coli (E. coli), we found that most acetylation occurred at a low level and accumulated in growth-arrested cells in a manner that depended on the formation of acetyl-phosphate (AcP) through glycolysis. Mutant cells unable to produce AcP had significantly reduced acetylation levels, while mutant cells unable to convert AcP to acetate had significantly elevated acetylation levels. We showed that AcP can chemically acetylate lysine residues in vitro and that AcP levels are correlated with acetylation levels in vivo, suggesting that AcP may acetylate proteins nonenzymatically in cells. These results uncover a critical role for AcP in bacterial acetylation and indicate that most acetylation in E. coli occurs at a low level and is dynamically affected by metabolism and cell proliferation in a global, uniform manner.

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) (1,2), baculovirus-infected insect (S. frugiperda or T. ni) cells (3), and cell-free in vitro translation systems (2,4) have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins (5-9). However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories. Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein (5,10). In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

Phosphoproteomic Analysis Reveals the Multiple Roles of Phosphorylation in Pathogenic Bacterium Streptococcus pneumoniae

Recent phosphoproteomic characterizations of Bacillus subtilis, Escherichia coli, Lactococcus lactis, Pseudomonas putida, and Pseudomonas aeruginosa have suggested that protein phosphorylation on serine, threonine, and tyrosine residues is a major regulatory post-translational modification in bacteria. In this study, we carried out a global and site-specific phosphoproteomic analysis on the Gram-positive pathogenic bacterium Streptococcus pneumoniae. One hundred and two unique phosphopeptides and 163 phosphorylation sites with distributions of 47%/44%/9% for Ser/Thr/Tyr phosphorylations from 84 S. pneumoniae proteins were identified through the combined use of TiO(2) enrichment and LC-MS/MS determination. The identified phosphoproteins were found to be involved in various biological processes including carbon/protein/nucleotide metabolisms, cell cycle and division regulation. A striking characteristic of S. pneumoniae phosphoproteome is the large number of multiple species-specific phosphorylated sites, indicating that high level of protein phosphorylation may play important roles in regulating many metabolic pathways and bacterial virulence.

Extreme Substrate Promiscuity of the Neisseria Oligosaccharyl Transferase Involved in Protein O-Glycosylation

Neisseria meningitidis PglL belongs to a novel family of bacterial oligosaccharyltransferases (OTases) responsible for O-glycosylation of type IV pilins. Although members of this family are widespread among pathogenic bacteria, there is little known about their mechanism. Understanding the O-glycosylation process may uncover potential targets for therapeutic intervention, and can open new avenues for the exploitation of these pathways for biotechnological purposes. In this work, we demonstrate that PglL is able to transfer virtually any glycan from the undecaprenyl pyrophosphate (UndPP) carrier to pilin in engineered Escherichia coli and Salmonella cells. Surprisingly, PglL was also able to interfere with the peptidoglycan biosynthetic machinery and transfer peptidoglycan subunits to pilin. This represents a previously unknown post-translational modification in bacteria. Given the wide range of glycans transferred by PglL, we reasoned that substrate specificity of PglL lies in the lipid carrier. To test this hypothesis we developed an in vitro glycosylation system that employed purified PglL, pilin, and the lipid farnesyl pyrophosphate (FarPP) carrying a pentasaccharide that had been synthesized by successive chemical and enzymatic steps. Although FarPP has different stereochemistry and a significantly shorter aliphatic chain than the natural lipid substrate, the pentasaccharide was still transferred to pilin in our system. We propose that the primary roles of the lipid carrier during O-glycosylation are the translocation of the glycan into the periplasm, and the positioning of the pyrophosphate linker and glycan adjacent to PglL. The unique characteristics of PglL make this enzyme a promising tool for glycoengineering novel glycan-based vaccines and therapeutics.