Harald Nothaft

Protein glycosylation in bacteria: sweeter than ever

Investigations into bacterial protein glycosylation continue to progress rapidly. It is now established that bacteria possess both N-linked and O-linked glycosylation pathways that display many commonalities with their eukaryotic and archaeal counterparts as well as some unexpected variations. In bacteria, protein glycosylation is not restricted to pathogens but also exists in commensal organisms such as certain Bacteroides species, and both the N-linked and O-linked glycosylation pathways can modify multiple proteins. Improving our understanding of the intricacies of bacterial protein glycosylation systems should lead to new opportunities to manipulate these pathways in order to engineer glycoproteins with potential value as novel vaccines.

The sugar phosphotransferase system of Streptomyces coelicolor is regulated by the GntR‐family regulator DasR and links N‐acetylglucosamine metabolism to the control of development

Members of the soil‐dwelling, sporulating prokaryotic genus Streptomyces are indispensable for the recycling of the most abundant polysaccharides on earth (cellulose and chitin), and produce a wide range of antibiotics and industrial enzymes. How do these organisms sense the nutritional state of the environment, and what controls the signal for the switch to antibiotic production and morphological development? Here we show that high extracellular concentrations of N‐acetylglucosamine, the monomer of chitin, prevent Streptomyces coelicolor progressing beyond the vegetative state, and that this effect is absent in a mutant defective of N‐acetylglucosamine transport. We provide evidence that the signal is transmitted through the GntR‐family regulator DasR, which controls the N‐acetylglucosamine regulon, including the pts genes ptsH, ptsI and crr needed for uptake of N‐acetylglucosamine. Deletion of dasR or the pts genes resulted in a bald phenotype. Binding of DasR to its target genes is abolished by glucosamine 6‐phosphate, a central molecule in N‐acetylglucosamine metabolism. Extracellular complementation experiments with many bld mutants showed that the dasR mutant is arrested at an early stage of the developmental programme, and does not fit in the previously described bld signalling cascade. Thus, for the first time we are able to directly link carbon (and nitrogen) metabolism to development, highlighting a novel type of metabolic regulator, which senses the nutritional state of the habitat, maintaining vegetative growth until changing circumstances trigger the switch to sporulation. Our work, and the model it suggests, provide new leads towards understanding how microorganisms time developmental commitment.

l-Fucose utilization provides Campylobacter jejuni with a competitive advantage

Campylobacter jejuni is a prevalent gastrointestinal pathogen in humans and a common commensal of poultry. When colonizing its hosts, C. jejuni comes into contact with intestinal carbohydrates, including l-fucose, released from mucin glycoproteins. Several strains of C. jejuni possess a genomic island (cj0480c–cj0490) that is up-regulated in the presence of both l-fucose and mucin and allows for the utilization of l-fucose as a substrate for growth. Strains possessing this genomic island show increased growth in the presence of l-fucose and mutation of cj0481, cj0486, and cj0487 results in the loss of the ability to grow on this substrate. Furthermore, mutants in the putative fucose permease (cj0486) are deficient in fucose uptake and demonstrate a competitive disadvantage when colonizing the piglet model of human disease, which is not paralleled in the colonization of poultry. This identifies a previously unrecorded metabolic pathway in select strains of C. jejuni associated with a virulent lifestyle.

Bacterial Protein N-glycosylation: New Perspectives and Applications

Protein glycosylation is widespread throughout all three domains of life. Bacterial protein N-glycosylation and its application to engineering recombinant glycoproteins continue to be actively studied. Here, we focus on advances made in the last 2 years, including the characterization of novel bacterial N-glycosylation pathways, examination of pathway enzymes and evolution, biological roles of protein modification in the native host, and exploitation of the N-glycosylation pathways to create novel vaccines and diagnostics.

The chitobiose-binding protein, DasA, acts as a link between chitin utilization and morphogenesis in Streptomyces coelicolor

Streptomycetes are mycelial soil bacteria that undergo a developmental programme that leads to sporulating aerial hyphae. As soil-dwelling bacteria, streptomycetes rely primarily on natural polymers such as cellulose, xylan and chitin for the colonization of their environmental niche and therefore these polysaccharides may play a critical role in monitoring the global nutritional status of the environment. In this work we analysed the role of DasA, the sugar-binding component of the chitobiose ATP-binding cassette transport system, in informing the cell of environmental conditions, and its role in the onset of development and in ensuring correct sporulation. The chromosomal interruption of dasA resulted in a carbon-source-dependent vegetative arrest phenotype, and we identified a second DasR-dependent sugar transporter, in addition to the N-acetylglucosamine phosphotransferase system (PTS(GlcNAc)), that relates primary metabolism to development. Under conditions that allowed sporulation, highly aberrant spores with many prematurely produced germ tubes were observed. While GlcNAc locks streptomycetes in the vegetative state, a high extracellular concentration of the GlcNAc polymer chitin has no effect on development. The striking distinction is due to a difference in the transporters responsible for the import of GlcNAc, which enters via the PTS, and of chitin, which enters as the hydrolytic product chitobiose (GlcNAc(2)) through the DasABC transporter. A model explaining the role of these two essentially different transport systems in the control of development is provided.

The permease gene nagE2 is the key to N‐acetylglucosamine sensing and utilization in Streptomyces coelicolor and is subject to multi‐level control

The availability of nutrients is a major determinant for the timing of morphogenesis and antibiotic production in the soil‐dwelling bacterium Streptomyces coelicolor. Here we show that N‐acetylglucosamine transport, the first step of an important nutrient signalling cascade, is mediated by the NagE2 permease of the phosphotransferase system, and that the activity of this permease is linked to nutritional control of development and antibiotic production. The permease serves as a high‐affinity transporter for N‐acetylglucosamine (Km of 2.6 µM). The permease complex was reconstituted with individually purified components. This showed that uptake of N‐acetylglucosamine requires a phosphoryl group transfer from phosphoenolpyruvate via the phosphotransferases EI, HPr and IIACrr to NagF, which in turn phosphorylates N‐acetylglucosamine during transport. Transcription of the nagF and nagE2 genes is induced by N‐acetylglucosamine. Nutrient signalling by N‐acetylglucosamine that triggers the onset of development was abolished in the nagE2 and nagF mutants. nagE2 is subject to multi‐level control by the global transcription factor DasR and the activator AtrA that also stimulates genes for antibiotic actinorhodin biosynthesis. Hence, it is apparent that streptomycetes tightly control the nutritional state in a complex manner to ensure the correct timing for the developmental programme.

Identification and Quantification of Glycoproteins Using Ion-Pairing Normal-phase Liquid Chromatography and Mass Spectrometry

Glycoprotein structure determination and quantification by MS requires efficient isolation of glycopeptides from a proteolytic digest of complex protein mixtures. Here we describe that the use of acids as ion-pairing reagents in normal-phase chromatography (IP-NPLC) considerably increases the hydrophobicity differences between non-glycopeptides and glycopeptides, thereby resulting in the reproducible isolation of N-linked high mannose type and sialylated glycopeptides from the tryptic digest of a ribonuclease B and fetuin mixture. The elution order of non-glycopeptides relative to glycopeptides in IP-NPLC is predictable by their hydrophobicity values calculated using the Wimley-White water/octanol hydrophobicity scale. O-linked glycopeptides can be efficiently isolated from fetuin tryptic digests using IP-NPLC when N-glycans are first removed with PNGase. IP-NPLC recovers close to 100% of bacterial N-linked glycopeptides modified with non-sialylated heptasaccharides from tryptic digests of periplasmic protein extracts from Campylobacter jejuni 11168 and its pglD mutant. Label-free nano-flow reversed-phase LC-MS is used for quantification of differentially expressed glycopeptides from the C. jejuni wild-type and pglD mutant followed by identification of these glycoproteins using multiple stage tandem MS. This method further confirms the acetyltransferase activity of PglD and demonstrates for the first time that heptasaccharides containing monoacetylated bacillosamine are transferred to proteins in both the wild-type and mutant strains. We believe that IP-NPLC will be a useful tool for quantitative glycoproteomics.

Identification of novel carbohydrate modifications on Campylobacter jejuni 11168 flagellin using metabolomics-based approaches.

It is well known that the flagellin of Campylobacter jejuni is extensively glycosylated by pseudaminic acid and the related acetamindino derivative, in addition to flagellin glycosylation being essential for motility and colonization of host cells. Recently, the use of metabolomics permitted the unequivocal characterization of unique flagellin modifications in Campylobacter, including novel legionaminic acid sugars in Campylobacter coli, which had been impossible to ascertain in earlier studies using proteomics‐based approaches. To date, the precise identities of the flagellin glycosylation modifications have only been elucidated for C. jejuni 81‐176 and C. coli VC167 and those present in the first genome‐sequenced strain C. jejuni 11168 remain elusive due to lability and respective levels of individual glycan modifications. We report the characterization of the carbohydrate modifications on C. jejuni 11168 flagellin using metabolomics‐based approaches. Detected as their corresponding CMP‐linked precursors, structural information on the flagellin modifications was obtained using a combination of MS and NMR spectroscopy. In addition to the pseudaminic acid and legionaminic acid sugars known to be present on Campylobacter flagellin, two unusual 2,3‐di‐O‐methylglyceric acid modifications of a nonulosonate sugar were identified. By performing a metabolomic analysis of selected isogenic mutants of genes from the flagellin glycosylation locus of this pathogen, these novel CMP‐linked precursors were confirmed to be di‐O‐methylglyceric acid derivatives of pseudaminic acid and the related acetamidino sugar. This is the first comprehensive analysis of the flagellar modifications in C. jejuni 11168 and structural elucidation of di‐O‐methylglyceric acid derivatives of pseudaminic acid on Campylobacter flagellin.

Study of free oligosaccharides derived from the bacterial N-glycosylation pathway

The food-borne pathogen Campylobacter jejuni is one of the leading causes of bacterial gastroenteritis worldwide and the most frequent antecedent in neuropathies such as the Guillain-Barré and Miller Fisher syndromes. C. jejuni was demonstrated to possess an N-linked protein glycosylation pathway that adds a conserved heptasaccharide to >40 periplasmic and membrane proteins. Recently, we showed that C. jejuni also produces free heptasaccharides derived from the N-glycan pathway reminiscent of the free oligosaccharides (fOS) produced by eukaryotes. Herein, we demonstrate that C. jejuni fOS are produced in response to changes in the osmolarity of the environment and bacterial growth phase. We provide evidence showing the conserved WWDYG motif of the oligosaccharyltransferase, PglB, is necessary for fOS release into the periplasm. This report demonstrates that fOS from an N-glycosylation pathway in bacteria are potentially equivalent to osmoregulated periplasmic glucans in other Gram-negative organisms.

A New Piece of an Old Jigsaw: Glucose Kinase Is Activated Posttranslationally in a Glucose Transport-Dependent Manner in Streptomyces coelicolor A3(2)

Members of the soil-dwelling prokaryotic genus Streptomyces are indispensable for the recycling of complex polysaccharides, and produce a wide range of natural products. Nutrient limitation is likely to be a major signal for the onset of their development, resulting in spore formation by specialized aerial hyphae. Streptomycetes grow on numerous carbon sources, which they utilize in a preferential manner. The main signaling pathway underlying this phenomenon is carbon catabolite repression, which in streptomycetes is totally dependent on the glycolytic enzyme glucose kinase (Glk). How Glk exerts this fascinating dual role (metabolic and regulatory) is still largely a mystery. We show here that while Glk is made constitutively throughout the growth of Streptomyces coelicolor A3(2), its catalytic activity is modulated in a carbon source-dependent manner: while cultures growing exponentially on glucose exhibit high Glk activity, mannitol- grown cultures show negligible activity. Glk activity was directly proportional to the amount of two Glk isoforms observed by Western blot analysis. The activity profile of GlcP, the major glucose permease, correlated very well with that of Glk. Our data are consistent with a direct interaction between Glk and GlcP, suggesting that a Glk-GlcP permease complex is required for efficient glucose transport by metabolic trapping. This is supported by the strongly reduced accumulation of glucose in glucose kinase mutants. A model to explain our data is presented.