BL-11C Micro-MX: a high-flux microfocus macromolecular-crystallography beamline for micrometre-sized protein crystals at Pohang Light Source II.

BL-11C, a new protein crystallography beamline, is an in-vacuum undulator-based microfocus beamline used for macromolecular crystallography at the Pohang Accelerator Laboratory and it was made available to users in June 2017. The beamline is energy tunable in the range 5.0-20 keV to support conventional single- and multi-wavelength anomalous-dispersion experiments against a wide range of heavy metals. At the standard working energy of 12.659 keV, the monochromated beam is focused to 4.1 µm (V) × 8.5 µm (H) full width at half-maximum at the sample position and the measured photon flux is 1.3 × 1012 photons s-1. The experimental station is equipped with a Pilatus3 6M detector, a micro-diffractometer (MD2S) incorporating a multi-axis goniometer, and a robotic sample exchanger (CATS) with a dewar capacity of 90 samples. This beamline is suitable for structural determination of weakly diffracting crystalline substances, such as biomaterials, including protein, nucleic acids and their complexes. In addition, serial crystallography experiments for determining crystal structures at room temperature are possible. Herein, the current beamline characteristics, technical information for users and some recent scientific highlights are described.

Identification and structural investigation of potential novel drug candidates against lethal human pathogen.

Neisseria meningtidis is responsible for causing meningococcal meningitis along with acute septicaemia in human beings. Functional genomics strategies proved cruciality of certain genes/proteins in Neisseria meningitidis pathogenesis. During the present studies, three important Neisseria meningitidis proteins i.e., Dead box RNA-Helicase, Polyribonucleotide nucleotidyl-transferase PNPase and Ribonuclease-III were targeted for homology modeling and protein-ligand docking studies not only to determine their three dimensional architectures but also to identify their potential novel inhibitors. The Biscoumarin, malonitrile and indole derivatives showed the best inhibitory mode against all of the three enzymes. Since, these enzymes are assembled in Gram-negative bacteria to form RNA degradosome assembly therefore their inhibition will definitely shut off the degradosome assembly and ultimately the decay of RNA, which is an essential life process. This is the first ever structural investigation of these drug targets along with identification of potential novel drug candidates. We believe that these small chemical compounds will be proved as better drugs and will provide an excellent barrier towards Neisseria meningitidis pathogenesis.

Mix-and-Match System for the Enzymatic Synthesis of Enantiopure Glycerol-3-Phosphate-Containing Capsule Polymer Backbones from Actinobacillus pleuropneumoniae, Neisseria meningitidis, and Bibersteinia trehalosi.

Capsule polymers are crucial virulence factors of pathogenic bacteria and are used as antigens in glycoconjugate vaccine formulations. Some Gram-negative pathogens express poly(glycosylglycerol phosphate) capsule polymers that resemble Gram-positive wall teichoic acids and are synthesized by TagF-like capsule polymerases. So far, the biotechnological use of these enzymes for vaccine developmental studies was restricted by the unavailability of enantiopure CDP-glycerol, one of the donor substrates required for polymer assembly. Here, we use CTP:glycerol-phosphate cytidylyltransferases (GCTs) and TagF-like polymerases to synthesize the poly(glycosylglycerol phosphate) capsule polymer backbones of the porcine pathogen Actinobacillus pleuropneumoniae, serotypes 3 and 7 (App3 and App7). GCT activity was confirmed by high-performance liquid chromatography, and polymers were analyzed using comprehensive nuclear magnetic resonance studies. Solid-phase synthesis protocols were established to allow potential scale-up of polymer production. In addition, one-pot reactions exploiting glycerol-kinase allowed us to start the reaction from inexpensive, widely available substrates. Finally, this study highlights that multidomain TagF-like polymerases can be transformed by mutagenesis of active site residues into single-action transferases, which in turn can act in trans to build-up structurally new polymers. Overall, our protocols provide enantiopure, nature-identical capsule polymer backbones from App2, App3, App7, App9, and App11, Neisseria meningitidis serogroup H, and Bibersteinia trehalosi serotypes T3 and T15. IMPORTANCE Economic synthesis platforms for the production of animal vaccines could help reduce the overuse and misuse of antibiotics in animal husbandry, which contributes greatly to the increase of antibiotic resistance. Here, we describe a highly versatile, easy-to-use mix-and-match toolbox for the generation of glycerol-phosphate-containing capsule polymers that can serve as antigens in glycoconjugate vaccines against Actinobacillus pleuropneumoniae and Bibersteinia trehalosi, two pathogens causing considerable economic loss in the swine, sheep, and cattle industries. We have established scalable protocols for the exploitation of a versatile enzymatic cascade with modular architecture, starting with the preparative-scale production of enantiopure CDP-glycerol, a precursor for a multitude of bacterial surface structures. Thereby, our approach not only allows the synthesis of capsule polymers but might also be exploitable for the (chemo)enzymatic synthesis of other glycerol-phosphate-containing structures such as Gram-positive wall teichoic acids or lipoteichoic acids.

Development of a non-biased, high-throughput ELISA for the rapid evaluation of immunogenicity and cross-reactivity.

Traditional ELISA-based protein analysis has been predicated on the assumption that proteins bind randomly to the solid surface of the ELISA plate polymer (polystyrene or polyvinyl chloride). Random adherence to the plate ensures equal access to all faces of the protein, an important consideration when evaluating immunogenicity of polyclonal serum samples as well as when examining the cross-reactivity of immune serum against different antigenic variants of a protein. In this study we demonstrate that the soluble form of the surface lipoprotein transferrin binding protein B (TbpB) from three different bacterial pathogens (Neisseria meningitidis, Actinobacillus pleuropneumoniae, and Mannheimia haemolytica) bind the ELISA plate in a manner that consistently obscures the transferrin binding face of the proteins' N-lobe. In order to develop a non-biased ELISA where all faces of the protein are accessible, the strong interaction between biotin and avidin has been exploited by adding a biotin tag to these proteins during Escherichia coli-based cytoplasmic expression and utilizing streptavidin or neutravidin coated ELISA plates for protein capture and display. The use of avidin coated ELISA plates also allows for rapid purification of biotin-tagged proteins from crude E. coli lysates, removing the requirement of prior affinity purification of each protein to be included in the ELISA-based analyses. In proof of concept experiments we demonstrate the utility of this approach for evaluating immunogenicity and cross-reactivity of serum from mice and pigs immunized with TbpBs from human and porcine pathogens.

Predominant phosphorylation patterns in Neisseria meningitidis lipid A determined by top-down MS/MS.

Among the virulence factors in Neisseria infections, a major inducer of inflammatory cytokines is the lipooligosaccharide (LOS). The activation of NF-κB via extracellular binding of LOS or lipopolysaccharide (LPS) to the toll-like receptor 4 and its coreceptor, MD-2, results in production of pro-inflammatory cytokines that initiate adaptive immune responses. LOS can also be absorbed by cells and activate intracellular inflammasomes, causing the release of inflammatory cytokines and pyroptosis. Studies of LOS and LPS have shown that their inflammatory potential is highly dependent on lipid A phosphorylation and acylation, but little is known on the location and pattern of these posttranslational modifications. Herein, we report on the localization of phosphoryl groups on phosphorylated meningococcal lipid A, which has two to three phosphate and zero to two phosphoethanolamine substituents. Intact LOS with symmetrical hexa-acylated and asymmetrical penta-acylated lipid A moieties was subjected to high-resolution ion mobility spectrometry MALDI-TOF MS. LOS molecular ions readily underwent in-source decay to give fragments of the oligosaccharide and lipid A formed by cleavage of the ketosidic linkage, which enabled performing MS/MS (pseudo-MS3). The resulting spectra revealed several patterns of phosphoryl substitution on lipid A, with certain species predominating. The extent of phosphoryl substitution, particularly phosphoethanolaminylation, on the 4'-hydroxyl was greater than that on the 1-hydroxyl. The heretofore unrecognized phosphorylation patterns of lipid A of meningococcal LOS that we detected are likely determinants of both pathogenicity and the ability of the bacteria to evade the innate immune system.

Synthesis of orthogonally protected and functionalized bacillosamines.

2,4-Diamino-2,4,6-trideoxyglucose (bacillosamine) is a monosaccharide found in many pathogenic bacteria, variation in the functionalities appended to the amino groups occurs depending on the species the sugar is derived from. We here report the first synthesis of bacillosamine synthons that allow for the incorporation of two different functionalities at the C-2-N-acetyl and C-4-amines. We have developed chemistry to assemble a set of conjugation ready Neisseria meningitidis C-2-N-acetyl bacillosamine saccharides, carrying either an acetyl or (R)- or (S)-glyceroyl at the C-4 amine. The glyceroyl bacillosamines have been further extended at the C-3-OH with an α-d-galactopyranose to provide structures that occur as post-translational modifications of N. meningitidis PilE proteins, which make up the bacterial pili.

Structural Basis of Ca2+-Dependent Self-Processing Activity of Repeat-in-Toxin Proteins.

The posttranslational Ca2+-dependent "clip-and-link" activity of large repeat-in-toxin (RTX) proteins starts by Ca2+-dependent structural rearrangement of a highly conserved self-processing module (SPM). Subsequently, an internal aspartate-proline (Asp-Pro) peptide bond at the N-terminal end of SPM breaks, and the liberated C-terminal aspartyl residue can react with a free ε-amino group of an adjacent lysine residue to form a new isopeptide bond. Here, we report a solution structure of the calcium-loaded SPM (Ca-SPM) derived from the FrpC protein of Neisseria meningitidis The Ca-SPM structure defines a unique protein architecture and provides structural insight into the autocatalytic cleavage of the Asp-Pro peptide bond through a "twisted-amide" activation. Furthermore, in-frame deletion of the SPM domain from the ApxIVA protein of Actinobacillus pleuropneumoniae attenuated the virulence of this porcine pathogen in a pig respiratory challenge model. We hypothesize that the Ca2+-dependent clip-and-link activity represents an unconventional strategy for Gram-negative pathogens to adhere to the host target cell surface.IMPORTANCE The Ca2+-dependent clip-and-link activity of large repeat-in-toxin (RTX) proteins is an exceptional posttranslational process in which an internal domain called a self-processing module (SPM) mediates Ca2+-dependent processing of a highly specific aspartate-proline (Asp-Pro) peptide bond and covalent linkage of the released aspartyl to an adjacent lysine residue through an isopeptide bond. Here, we report the solution structures of the Ca2+-loaded SPM (Ca-SPM) defining the mechanism of the autocatalytic cleavage of the Asp414-Pro415 peptide bond of the Neisseria meningitidis FrpC exoprotein. Moreover, deletion of the SPM domain in the ApxIVA protein, the FrpC homolog of Actinobacillus pleuropneumoniae, resulted in attenuation of virulence of the bacterium in a pig infection model, indicating that the Ca2+-dependent clip-and-link activity plays a role in the virulence of Gram-negative pathogens.

A simple and rapid pipeline for identification of receptor-binding sites on the surface proteins of pathogens.

Ligand-receptor interactions play a crucial role in the plethora of biological processes. Several methods have been established to reveal ligand-receptor interface, however, the majority of methods are time-consuming, laborious and expensive. Here we present a straightforward and simple pipeline to identify putative receptor-binding sites on the pathogen ligands. Two model ligands (bait proteins), domain III of protein E of West Nile virus and NadA of Neisseria meningitidis, were incubated with the proteins of human brain microvascular endothelial cells immobilized on nitrocellulose or PVDF membrane, the complex was trypsinized on-membrane, bound peptides of the bait proteins were recovered and detected on MALDI-TOF. Two peptides of DIII (~916 Da and ~2003 Da) and four peptides of NadA (~1453 Da, ~1810 Da, ~2051 Da and ~2433 Da) were identified as plausible receptor-binders. Further, binding of the identified peptides to the proteins of endothelial cells was corroborated using biotinylated synthetic analogues in ELISA and immunocytochemistry. Experimental pipeline presented here can be upscaled easily to map receptor-binding sites on several ligands simultaneously. The approach is rapid, cost-effective and less laborious. The proposed experimental pipeline could be a simpler alternative or complementary method to the existing techniques used to reveal amino-acids involved in the ligand-receptor interface.

A novel enantioselective SGNH family esterase (NmSGNH1) from Neisseria meningitides: Characterization, mutational analysis, and ester synthesis.

In Neisseria sp., SGNH family esterases are involved in bacterial pathogenesis as well as cell wall peptidoglycan maturation. Here, a novel enantioselective SGNH family esterase (NmSGNH1) from Neisseria meningitidis, which has sequence similarity to carbohydrate esterase (CE3) family, was catalytically characterized and functionally explored. NmSGNH1 exhibited a wide range of substrate specificities including naproxol acetate, tert-butyl acetate, glucose pentaacetate as well as p-nitrophenyl esters. Deletion of C-terminal residues (NmSGNH1Δ11) led to the altered substrate specificity, reduced catalytic activity, and increased thermostability. Furthermore, a hydrophobic residue of Leu92 in the substrate-binding pocket was identified to be critical in catalytic activity, thermostability, kinetics, and enantioselectivity. Interestingly, immobilization of NmSGNH1 by hybrid nanoflowers (hNFs) and crosslinked enzyme aggregates (CLEAs) showed increased level of activity, recycling property, and enhanced stability. Finally, synthesis of butyl acetate, oleic acid esters, and fatty acid methyl esters (FAMEs) were verified. In summary, this work provides a molecular understanding of substrate specificities, catalytic regulation, immobilization, and industrial applications of a novel SGNH family esterase from Neisseria meningitidis.

Structures of the Neisseria meningitides methionine-binding protein MetQ in substrate-free form and bound to l- and d-methionine isomers.

The bacterial periplasmic methionine-binding protein MetQ is involved in the import of methionine by the cognate MetNI methionine ATP binding cassette (ABC) transporter. The MetNIQ system is one of the few members of the ABC importer family that has been structurally characterized in multiple conformational states. Critical missing elements in the structural analysis of MetNIQ are the structure of the substrate-free form of MetQ, and detailing how MetQ binds multiple methionine derivatives, including both l- and d-methionine isomers. In this study, we report the structures of the Neisseria meningitides MetQ in substrate-free form and in complexes with l-methionine and with d-methionine, along with the associated binding constants determined by isothermal titration calorimetry. Structures of the substrate-free (N238A) and substrate-bound N. meningitides MetQ are related by a "Venus-fly trap" hinge-type movement of the two domains accompanying methionine binding and dissociation. l- and d-methionine bind to the same site on MetQ, and this study emphasizes the important role of asparagine 238 in ligand binding and affinity. A thermodynamic analysis demonstrates that ligand-free MetQ associates with the ATP-bound form of MetNI ∼40 times more tightly than does liganded MetQ, consistent with the necessity of dissociating methionine from MetQ for transport to occur.

Quantitation of residual sodium dodecyl sulfate in meningococcal polysaccharide by gas chromatography-mass spectrometry.

Sodium dodecyl sulfate (SDS) is a commonly used surfactant in protein solubilization and also during the polysaccharide purification. A GC-MS method has been developed to quantitate residual SDS in meningococcal polysaccharide serogroups A,C,W,Y and X circumventing the need of spectroscopic assays and HPLC based methods which are either unstable or requires the confirmation by MS. The developed method is based on quantitative conversion of SDS to 1-dodecanol at elevated temperature. Meningococcal polysaccharides and SDS standards were treated with methanolic-HCl and extracted in n-Hexane. The conversion of SDS to 1-dodecanol was confirmed by mass spectra and separation was achieved using a DB-5ms column. The mass spectral analysis of 1-dodecanol showed characteristic ions at m/z 168, 140 and 125. The GC-MS method validation performed on intermediate and purified meningococcal polysaccharides showed linearity with r2 > 0.99 over the concentration range of 2.5-200 µg/ml with LOD and LOQ of 1.27 and 3.85 respectively. The method was found to be precise, robust and accurate with spike recovery ranging 83-117%. The GC-MS method can be used in the quantitation of residual SDS during polysaccharide purification and provides valuable information about consistency of polysaccharide manufacturing process for development of pentavalent meningococcal conjugate vaccine.

Meningococcal polysaccharides identification by NIR spectroscopy and chemometrics.

Near-infrared (NIR) spectroscopy is an attractive tool for pharmaceutical analyses. The main purpose of this study was to assess the potential of NIR spectroscopy coupled with different multivariate classification tools for the identification of meningococcal polysaccharide serogroups A and C. Moreover, it sought to determine, if the models established on production batches, could be used to correctly identify National Institute for Biological Standards and Control standards. Two different classification tools were investigated: soft independent modeling of class analogy (SIMCA) and partial least squares discriminant analysis (PLS-DA). Models' performance was evaluated by external validation. Although both models were able to correctly classify 100% of meningococcal polysaccharides from serogroups A and C, they performed differently in the presence of similar non-target serogroups W135 and Y. These results demonstrate that NIR spectroscopy, coupled with either SIMCA or PLS-DA, provides a method suitable for the identification of meningococcal polysaccharides A and C.

Copper(II)-Induced Restructuring of ZnuD, a Zinc(II) Transporter from Neisseria meningitidis.

Cluster 2 (288HDDDNAHAHTH298) from Neisseria meningitidis ZnuD is a flexible loop that captures zinc(II) ions, acting as a "fishing net". We describe its Zn(II) and Cu(II) binding capabilities, focusing on the thermodynamics of such interactions and comparing them with the complexes of the 1MAHHHHHHL9-NH2 region. Copper(II) complexes with the studied ZnuD regions are thermodynamically more stable than the zinc(II) ones-Cu(II) complexes dominate in solution even in close to physiological ratios of the studied metal ions (a 10-fold excess of Zn(II) over Cu(II)). While the binding of native Zn(II) has no significant impact on the structure of its transporter, Cu(II) binding induces a conformational change of cluster 2 to a polyproline II-like helix. To the best of our knowledge, this is the first evidence of a copper(II)-induced formation of a polyproline II-like structure in a sequence that does not contain proline residues. Cu(II) coordination also changes the structure of an intracellular, N-terminal, His-rich region, folding it to an α helix.

Glycosyltransferase Activity Assay Using Colorimetric Methods.

The glycosyltransferases (GTs) are an important subclass of enzymes that catalyze the biosynthesis of glycosidic bonds in oligosaccharides, polysaccharides and glycoconjugates by transferring a sugar residue from a donor substrate to an acceptor substrate. The membrane-associated GTs play a vital role in the biosynthesis of bacterial cell-wall polysaccharides. Characterization and quantification of GT activities is important for studies of biosynthesis of polysaccharides, drug target development, and production of bacterial products. In this chapter, colorimetric assays for the measurement of GT activities will be presented. Assays for GTs acting on monosaccharide-derivatives are based on the cleavage of unreacted glycosyl-p-nitrophenol acceptors followed by detection of p-nitrophenolate. GT reactions coupled with phosphatases and detection of inorganic phosphate are suitable for most GTs. These assays permit convenient quantification of GT activities and kinetics without the use of radioactive sugars.

The Diversity of Mammalian Hemoproteins and Microbial Heme Scavengers Is Shaped by an Arms Race for Iron Piracy.

Iron is an essential micronutrient for most living species. In mammals, hemoglobin (Hb) stores more than two thirds of the body's iron content. In the bloodstream, haptoglobin (Hp) and hemopexin (Hpx) sequester free Hb or heme. Pathogenic microorganisms usually acquire iron from their hosts and have evolved complex systems of iron piracy to circumvent nutritional immunity. Herein, we performed an evolutionary analysis of genes coding for mammalian heme-binding proteins and heme-scavengers in pathogen species. The underlying hypothesis is that these molecules are engaged in a molecular arms race. We show that positive selection drove the evolution of mammalian Hb and Hpx. Positively selected sites in Hb are located at the interaction surface with Neisseria meningitidis heme scavenger HpuA and with Staphylococcus aureus iron-regulated surface determinant B (IsdB). In turn, positively selected sites in HpuA and IsdB are located in the flexible protein regions that contact Hb. A residue in Hb (S45H) was also selected on the Caprinae branch. This site stabilizes the interaction with Trypanosoma brucei hemoglobin-haptoglobin (HbHp) receptor (TbHpHbR), a molecule that also mediates trypanosome lytic factor (TLF) entry. In TbHpHbR, positive selection drove the evolution of a variant (L210S) which allows evasion from TLF but reduces affinity for HbHp. Finally, selected sites in Hpx are located at the interaction surface with the Haemophilus influenzae hemophore HxuA, which in turn displays fast evolving sites at the Hpx-binding interface. These results shed light into host-pathogens conflicts and establish the importance of nutritional immunity as an evolutionary force.

Potential large scale production of meningococcal vaccines by stable overexpression of fHbp in the rice seeds.

Factor H binding protein (fHbp) is the most promising vaccine candidate against serogroup B of Neisseria meningitidis which is a major cause of morbidity and mortality in children. In order to facilitate large scale production of a commercial vaccine, we previously used transgenic Arabidopsis thaliana, but plant-derived fHbp is still far away from a commercial vaccine due to less biomass production. Herein, we presented an alternative route for the production of recombinant fHbp from the seeds of transgenic rice. The OsrfHbp gene encoding recombinant fHbp fused protein was introduced into the genome of rice via Agrobacterium-mediated transformation. The both stable integration and transcription of the foreign OsrfHbp were confirmed by Southern blotting and RT-PCR analysis respectively. Further, the expression of fHbp protein was measured by immunoblotting analysis and quantified by ELISA. The results indicated that fHbp was successfully expressed and the highest yield of fHbp was 0.52 ±â€¯0.03% of TSP in the transgenic rice seeds. The purified fHbp protein showed good antigenicity and immunogenicity in the animal model. The results of this experiment offer a novel approach for large-scale production of plant-derived commercial vaccine fHbp.

Are lactoferrin receptors in Gram-negative bacteria viable vaccine targets?

A number of important Gram-negative pathogens that reside exclusively in the upper respiratory or genitourinary tract of their mammalian host rely on surface receptors that specifically bind host transferrin and lactoferrin as a source of iron for growth. The transferrin receptors have been targeted for vaccine development due to their critical role in acquiring iron during invasive infection and for survival on the mucosal surface. In this study, we focus on the lactoferrin receptors, determining their prevalence in pathogenic bacteria and comparing their prevalence in commensal Neisseria to other surface antigens targeted for vaccines; addressing the issue of a reservoir for vaccine escape and impact of vaccination on the microbiome. Since the selective release of the surface lipoprotein lactoferrin binding protein B by the NalP protease in Neisseria meningitidis argues against its utility as a vaccine target, we evaluated the release of outer membrane vesicles, and transferrin and lactoferrin binding in N. meningitidis and Moraxella catarrhalis. The results indicate that the presence of NalP reduces the binding of transferrin and lactoferrin by cells and native outer membrane vesicles, suggesting that NalP may impact all lipoprotein targets, thus this should not exclude lactoferrin binding protein B as a target.

Epitope imprinting of iron binding protein of Neisseria meningitidis bacteria through multiple monomers imprinting approach.

Epitope imprinting is a promising technique for fabrication of novel diagnostic tools. In this study, an epitope imprinted methodology for recognition of target epitope sequence as well as targeted protein infused by bacterial infection in blood samples of patients suffering from brain fever is developed. Template sequence chosen is a ferric iron binding fbp A protein present in Neisseria meningitidis bacteria. To orient the imprinting template peptide sequence on gold surface of electrochemical quartz crystal microbalance (EQCM), thiol chemistry was utilized to form the self-assembled monolayer on EQCM electrode. Here, synergistic effects induced by various noncovalent interactions extended by multiple monomers (3-sulfopropyl methacrylate potassium-salt and benzyl methacrylate) were used in fabricating the imprinting polymeric matrix with additional firmness provided by N,N-methylene-bis-acrylamide as cross-linker and azo-isobutyronitrile as initiator. Extraction of template molecule was carried out with phosphate buffer solution. After extraction of epitope molecules from the polymeric film, epitope molecularly imprinted polymeric films were fabricated on EQCM electrode surface. Nonimprinted polymers were also synthesized in the similar manner without epitope molecule. Detection limit of epitope molecularly imprinted polymers and imprinting factor (epitope molecularly imprinted polymers/nonimprinted polymers) was calculated 1.39 ng mL-1 and 12.27 respectively showing high binding capacity and specific recognition behavior toward template molecule. Simplicity of present method would put forward a fast, facile, cost-effective diagnostic tool for mass health care.

Covalent Probes for Carbohydrate-Active Enzymes: From Glycosidases to Glycosyltransferases.

Covalent probes for glycosidases and glycosyltransferases are of great interest as tool compounds for chemical biology. For glycosidases, a sizable number of such probes have been developed from covalent glycosidase inhibitors. We review selected recent examples and highlight different design strategies, including probes based on photoaffinity labels and mechanism-based inhibitors, as well as their applications in biology and for activity-based protein profiling. In contrast to glycosidases, only a limited number of covalent probes have been reported to date for glycosyltransferases. We describe a new class of covalent probes for the retaining α-1,4-galactosyltransferase LgtC from Neisseria meningitidis. On the basis of these probes, we have developed an operationally simple two-step protocol for the fluorescent labeling of recombinant LgtC both in purified form and in cell lysates. In principle, our approach is also applicable to other bacterial glycosyltransferases. Among other applications, our protocol may therefore be particularly useful for imaging of the differential expression of these enzymes in different bacterial species and strains.