A New Pillar in Pilus Assembly.

Pilus assembly in bacteria typically occurs by one of four pathways. In the study by Xu et al., the structures of 20 pilin subunits of human oral and gut Bacteroidales are elucidated, revealing a new pilin superfamily, assembled into pili by a distinct fifth pathway.

A general O-glycosylation system important to the physiology of a major human intestinal symbiont.

The Bacteroides are a numerically dominant genus of the human intestinal microbiota. These organisms harbor a rare bacterial pathway for incorporation of exogenous fucose into capsular polysaccharides and glycoproteins. The infrequency of glycoprotein synthesis by bacteria prompted a more detailed analysis of this process. Here, we demonstrate that Bacteroides fragilis has a general O-glycosylation system. The proteins targeted for glycosylation include those predicted to be involved in protein folding, protein-protein interactions, peptide degradation as well as surface lipoproteins. Protein glycosylation is central to the physiology of B. fragilis and is necessary for the organism to competitively colonize the mammalian intestine. We provide evidence that general O-glycosylation systems are conserved among intestinal Bacteroides species and likely contribute to the predominance of Bacteroides in the human intestine.

Mobile Type VI secretion system loci of the gut Bacteroidales display extensive intra-ecosystem transfer, multi-species spread and geographical clustering.

The human gut microbiota is a dense microbial ecosystem with extensive opportunities for bacterial contact-dependent processes such as conjugation and Type VI secretion system (T6SS)-dependent antagonism. In the gut Bacteroidales, two distinct genetic architectures of T6SS loci, GA1 and GA2, are contained on Integrative and Conjugative Elements (ICE). Despite intense interest in the T6SSs of the gut Bacteroidales, there is only a superficial understanding of their evolutionary patterns, and of their dissemination among Bacteroidales species in human gut communities. Here, we combine extensive genomic and metagenomic analyses to better understand their ecological and evolutionary dynamics. We identify new genetic subtypes, document extensive intrapersonal transfer of these ICE to Bacteroidales species within human gut microbiomes, and most importantly, reveal frequent population fixation of these newly armed strains in multiple species within a person. We further show the distribution of each of the distinct T6SSs in human populations and show there is geographical clustering. We reveal that the GA1 T6SS ICE integrates at a minimal recombination site leading to their integration throughout genomes and their frequent interruption of genes, whereas the GA2 T6SS ICE integrate at one of three different tRNA genes. The exclusion of concurrent GA1 and GA2 T6SSs in individual strains is associated with intact T6SS loci and with an ICE-encoded gene. By performing a comprehensive analysis of mobile genetic elements (MGE) in co-resident Bacteroidales species in numerous human gut communities, we identify 74 MGE that transferred to multiple Bacteroidales species within individual gut microbiomes. We further show that only three other MGE demonstrate multi-species spread in human gut microbiomes to the degree demonstrated by the GA1 and GA2 ICE. These data underscore the ubiquity and dissemination of mobile T6SS loci within Bacteroidales communities and across human populations.

Nanaerobic growth enables direct visualization of dynamic cellular processes in human gut symbionts.

Mechanistic studies of anaerobic gut bacteria have been hindered by the lack of a fluorescent protein system to track and visualize proteins and dynamic cellular processes in actively growing bacteria. Although underappreciated, many gut "anaerobes" are able to respire using oxygen as the terminal electron acceptor. The oxygen continually released from gut epithelial cells creates an oxygen gradient from the mucus layer to the anaerobic lumen [L. Albenberg et al., Gastroenterology 147, 1055-1063.e8 (2014)], with oxygen available to bacteria growing at the mucus layer. Here, we show that Bacteroides species are metabolically and energetically robust and do not mount stress responses in the presence of 0.10 to 0.14% oxygen, defined as nanaerobic conditions [A. D. Baughn, M. H. Malamy, Nature 427, 441-444 (2004)]. Taking advantage of this metabolic capability, we show that nanaerobic growth provides sufficient oxygen for the maturation of oxygen-requiring fluorescent proteins in Bacteroides species. Type strains of four different Bacteroides species show bright GFP fluorescence when grown nanaerobically versus anaerobically. We compared four different red fluorescent proteins and found that mKate2 yields the highest red fluorescence intensity in our assay. We show that GFP-tagged proteins can be localized in nanaerobically growing bacteria. In addition, we used time-lapse fluorescence microscopy to image dynamic type VI secretion system processes in metabolically active Bacteroides fragilis The ability to visualize fluorescently labeled Bacteroides and fluorescently linked proteins in actively growing nanaerobic gut symbionts ushers in an age of imaging analyses not previously possible in these bacteria.

Analysis of Effector and Immunity Proteins of the GA2 Type VI Secretion Systems of Gut Bacteroidales.

Three distinct genetic architectures (GAs) of Type VI secretion systems (T6SSs) have been described in gut Bacteroidales species, each with unique genes and characteristics. Unlike the GA3 T6SSs, potent antagonism has not yet been demonstrated for the GA1 or GA2 T6SSs. We previously showed that the GA2 T6SS loci are contained on integrative and conjugative elements and that there are five subtypes. Collectively, GA2 are the most prevalent Bacteroidales T6SSs in the human populations analyzed. In this study, we provide a comprehensive bioinformatic analysis of the three variable regions of GA2 T6SS loci, which encode toxic effector and immunity proteins. In total, we identified 63 distinct effectors encoded within 31 nonredundant GA2 loci, 18 of which do not have described motifs or predicted functions. We provide experimental evidence for toxin activity for four different GA2 effectors, showing that each functions only when present in the periplasm, and experimentally confirm their cognate immunity proteins. Our data demonstrate that each GA2 locus encodes at least three distinct effectors with targets in both the cytoplasm and the periplasm. The data also suggest that the effectors of a given locus are loaded onto the tube by different mechanisms, which may allow all three effectors encoded within a single GA2 locus with distinct antibacterial activity to be loaded onto a single T6 tube, increasing the antagonistic effect. IMPORTANCE Humans are colonized with many gut Bacteroidales species at high density, allowing for extensive opportunities for contact-dependent antagonism. To begin to understand the antagonistic potential of the GA2 T6SSs of the gut Bacteroidales, we performed bioinformatic and experimental analyses of the three divergent regions containing the toxin effector and immunity genes. We show that each GA2 T6SS locus encodes at least three distinct toxic effectors including toxins linked to Rhs and Hcp with cytoplasmic targets, and unlinked effectors with targets in the periplasm. The diversity and modality of effectors exceeds that of the GA1 or GA3 T6SS loci (M. J. Coyne, K. G. Roelofs, and L. E. Comstock, BMC Genomics 17:58, 2016, https://doi.org/10.1186/s12864-016-2377-z) and suggests that these T6SSs have the potential to be potent antibacterial weapons in the human gut.

Analysis of a phase-variable restriction modification system of the human gut symbiont Bacteroides fragilis.

The genomes of gut Bacteroidales contain numerous invertible regions, many of which contain promoters that dictate phase-variable synthesis of surface molecules such as polysaccharides, fimbriae, and outer surface proteins. Here, we characterize a different type of phase-variable system of Bacteroides fragilis, a Type I restriction modification system (R-M). We show that reversible DNA inversions within this R-M locus leads to the generation of eight specificity proteins with distinct recognition sites. In vitro grown bacteria have a different proportion of specificity gene combinations at the expression locus than bacteria isolated from the mammalian gut. By creating mutants, each able to produce only one specificity protein from this region, we identified the R-M recognition sites of four of these S-proteins using SMRT sequencing. Transcriptome analysis revealed that the locked specificity mutants, whether grown in vitro or isolated from the mammalian gut, have distinct transcriptional profiles, likely creating different phenotypes, one of which was confirmed. Genomic analyses of diverse strains of Bacteroidetes from both host-associated and environmental sources reveal the ubiquity of phase-variable R-M systems in this phylum.

METHOD COMPARISON FOR MEASUREMENT OF SYMMETRIC DIMETHYLARGININE IN TIGERS (PANTHERA TIGRIS).

Renal disease is well documented in nondomestic felids and is monitored and diagnosed by serum concentration of blood urea nitrogen, creatinine, and phosphorous. Symmetric dimethylarginine (SDMA) has proven to be an earlier and more sensitive biomarker for the assessment of glomerular filtration rate. Although SDMA is commonly measured in nondomestic felids, information concerning the validity of the assay is lacking. The purpose of the study was to perform a method comparison between high-throughput immunoassay and the reference method, liquid chromatography-tandem mass spectrometry (LC-MS/MS), to quantify SDMA concentrations in tiger blood samples. Concentrations of SDMA were measured for 81 individual tiger samples. The SDMA immunoassay demonstrated excellent correlation to the LC-MS/MS reference method. A Passing and Bablok linear regression analysis had a slope of 1.03 (95% CI, 0.99-1.11), an intercept of 1.64 (95% CI, 0.46-2.34), and a Pearson R= 0.99. The mean bias was 1.53 µg/dl (95% CI, 0.63-2.42 µg/dl), and the limit of agreement was ±7.96 µg/dl. The degree of bias is within established acceptance criteria of 1-3 µg/dl for the immunoassay. Although this study provides good evidence of the utility of the immunoassay to measure SDMA in tiger serum and plasma, further assay validation is recommended.

Identification of a Fifth Antibacterial Toxin Produced by a Single Bacteroides fragilis Strain.

Bacteroidales are the most abundant Gram-negative bacteria of the healthy human colonic microbiota, comprising nearly 50% of the colonic bacteria in many individuals. Numerous species and strains of gut Bacteroidales are present simultaneously at high concentrations in this ecosystem. Studies are revealing that gut Bacteroides has numerous antibacterial weapons to antagonize closely related members. In this study, we identify a new diffusible antibacterial toxin produced by Bacteroides fragilis 638R, designated BSAP-4. This is the fifth antibacterial toxin produced by this strain and the second toxin of this strain with a membrane attack complex/perforin domain (MACPF). We identify the target molecule of sensitive cells as a ß-barrel outer membrane protein (OMP) with calycin-like domains. As with other MACPF toxins, the gene encoding the target in sensitive strains is in the same genetic region as bsap-4 in producing strains. A comparison of B. fragilis strains showed there are two sensitive variants of this OMP that are 87% similar to each other and 50% similar to the resistant OMP. Unlike other MACPF toxins, there are numerous B. fragilis strains that harbor the resistant OMP without bsap-4 Several OMP variants from strains that are BSAP-4 resistant under the conditions of our assay confer BSAP-4 sensitivity to Bacteroides thetaiotaomicron when constitutively expressed. Using a reporter assay, we show that the BSAP-4 receptor gene is differentially expressed in sensitive and resistant strains leading to apparent BSAP-4 resistance under the conditions of our assay, despite harboring the BSAP-4 target gene.IMPORTANCE The intestinal microbiota is a diverse microbial ecosystem that provides numerous benefits to humans. The factors that govern its establishment and stability are just beginning to be elucidated. Identification and characterization of antimicrobial toxins produced by its members and their killing range are essential to understanding the role of antagonism in community composition and stability. Here, we identify a fifth antimicrobial toxin produced by a single Bacteroides fragilis strain and identify its target. The finding of such a large number of toxins that antagonize competing members suggests that this feature substantially contributes to the fitness of these bacteria. In addition, these toxins may have applications in genetically engineered gut bacteria to allow engraftment or to antagonize a potentially pathogenic member.

Type VI secretion systems of human gut Bacteroidales segregate into three genetic architectures, two of which are contained on mobile genetic elements.

BACKGROUND: Type VI secretion systems (T6SSs) are contact-dependent antagonistic systems employed by Gram negative bacteria to intoxicate other bacteria or eukaryotic cells. T6SSs were recently discovered in a few Bacteroidetes strains, thereby extending the presence of these systems beyond Proteobacteria. The present study was designed to analyze in a global nature the diversity, abundance, and properties of T6SSs in the Bacteroidales, the most predominant Gram negative bacterial order of the human gut. RESULTS: By performing extensive bioinformatics analyses and creating hidden Markov models for Bacteroidales Tss proteins, we identified 130 T6SS loci in 205 human gut Bacteroidales genomes. Of the 13 core T6SS proteins of Proteobacteria, human gut Bacteroidales T6SS loci encode orthologs of nine, and an additional five other core proteins not present in Proteobacterial T6SSs. The Bacteroidales T6SS loci segregate into three distinct genetic architectures with extensive DNA identity between loci of a given genetic architecture. We found that divergent DNA regions of a genetic architecture encode numerous types of effector and immunity proteins and likely include new classes of these proteins. TheT6SS loci of genetic architecture 1 are contained on highly similar integrative conjugative elements (ICEs), as are the T6SS loci of genetic architecture 2, whereas the T6SS loci of genetic architecture 3 are not and are confined to Bacteroides fragilis. Using collections of co-resident Bacteroidales strains from human subjects, we provide evidence for the transfer of genetic architecture 1 T6SS loci among co-resident Bacteroidales species in the human gut. However, we also found that established ecosystems can harbor strains with distinct T6SS of all genetic architectures. CONCLUSIONS: This is the first study to comprehensively analyze of the presence and diversity of T6SS loci within an order of bacteria and to analyze T6SSs of bacteria from a natural community. These studies demonstrate that more than half of our gut Bacteroidales, equivalent to about » of the bacteria of this ecosystem, encode T6SSs. The data reveal several novel properties of these systems and suggest that antagonism between or distributed defense among these abundant intestinal bacteria may be common in established human gut communities.

An antimicrobial protein of the gut symbiont Bacteroides fragilis with a MACPF domain of host immune proteins.

Bacteroidales are the most abundant Gram-negative bacteria of the human intestinal microbiota comprising more than half of the bacteria in many individuals. Some of the factors that these bacteria use to establish and maintain themselves in this ecosystem are beginning to be identified. However, ecological competition, especially interference competition where one organism directly harms another, is largely unexplored. To begin to understand the relevance of this ecological principle as it applies to these abundant gut bacteria and factors that may promote such competition, we screened Bacteroides fragilis for the production of antimicrobial molecules. We found that the production of extracellularly secreted antimicrobial molecules is widespread in this species. The first identified molecule, described in this manuscript, contains a membrane attack complex/perforin (MACPF) domain present in host immune molecules that kill bacteria and virally infected cells by pore formation, and mutations affecting key residues of this domain abrogated its activity. This antimicrobial molecule, termed BSAP-1, is secreted from the cell in outer membrane vesicles and no additional proteins are required for its secretion, processing or immunity of the producing cell. This study provides the first insight into secreted molecules that promote competitive interference among Bacteroidales strains of the human gut.

Phylum-wide general protein O-glycosylation system of the Bacteroidetes.

The human gut symbiont Bacteroides fragilis has a general protein O-glycosylation system in which numerous extracytoplasmic proteins are glycosylated at a three amino acid motif. In B. fragilis, protein glycosylation is a fundamental and essential property as mutants with protein glycosylation defects have impaired growth and are unable to competitively colonize the mammalian intestine. In this study, we analysed the phenotype of B. fragilis mutants with defective protein glycosylation and found that the glycan added to proteins is comprised of a core glycan and an outer glycan. The genetic region encoding proteins for the synthesis of the outer glycan is conserved within a Bacteroides species but divergent between species. Unlike the outer glycan, an antiserum raised to the core glycan reacted with all Bacteroidetes species tested, from all four classes of the phylum. We found that diverse Bacteroidetes species synthesize numerous glycoproteins and glycosylate proteins at the same three amino acid motif. The wide-spread conservation of this protein glycosylation system within the phylum suggests that this system of post-translational protein modification evolved early, before the divergence of the four classes of Bacteroidetes, and has been maintained due to its physiological importance to the diverse species of this phylum.

Inflammation and bacteriophages affect DNA inversion states and functionality of the gut microbiota.

Reversible genomic DNA inversions control the expression of numerous gut bacterial molecules, but how this impacts disease remains uncertain. By analyzing metagenomic samples from inflammatory bowel disease (IBD) cohorts, we identified multiple invertible regions where a particular orientation correlated with disease. These include the promoter of polysaccharide A (PSA) of Bacteroides fragilis, which induces regulatory T cells (Tregs) and ameliorates experimental colitis. The PSA promoter was mostly oriented "OFF" in IBD patients, which correlated with increased B. fragilis-associated bacteriophages. Similarly, in mice colonized with a healthy human microbiota and B. fragilis, induction of colitis caused a decline of PSA in the "ON" orientation that reversed as inflammation resolved. Monocolonization of mice with B. fragilis revealed that bacteriophage infection increased the frequency of PSA in the "OFF" orientation, causing reduced PSA expression and decreased Treg cells. Altogether, we reveal dynamic bacterial phase variations driven by bacteriophages and host inflammation, signifying bacterial functional plasticity during disease.

A pervasive large conjugative plasmid mediates multispecies biofilm formation in the intestinal microbiota increasing resilience to perturbations.

Although horizontal gene transfer is pervasive in the intestinal microbiota, we understand only superficially the roles of most exchanged genes and how the mobile repertoire affects community dynamics. Similarly, little is known about the mechanisms underlying the ability of a community to recover after a perturbation. Here, we identified and functionally characterized a large conjugative plasmid that is one of the most frequently transferred elements among Bacteroidales species and is ubiquitous in diverse human populations. This plasmid encodes both an extracellular polysaccharide and fimbriae, which promote the formation of multispecies biofilms in the mammalian gut. We use a hybridization-based approach to visualize biofilms in clarified whole colon tissue with unprecedented 3D spatial resolution. These biofilms increase bacterial survival to common stressors encountered in the gut, increasing strain resiliency, and providing a rationale for the plasmid's recent spread and high worldwide prevalence.

Comprehensive analyses of a large human gut Bacteroidales culture collection reveal species and strain level diversity and evolution.

Species of the Bacteroidales order are among the most abundant and stable bacterial members of the human gut microbiome with diverse impacts on human health. While Bacteroidales strains and species are genomically and functionally diverse, order-wide comparative analyses are lacking. We cultured and sequenced the genomes of 408 Bacteroidales isolates from healthy human donors representing nine genera and 35 species and performed comparative genomic, gene-specific, mobile gene, and metabolomic analyses. Families, genera, and species could be grouped based on many distinctive features. However, we also show extensive DNA transfer between diverse families, allowing for shared traits and strain evolution. Inter- and intra-specific diversity is also apparent in the metabolomic profiling studies. This highly characterized and diverse Bacteroidales culture collection with strain-resolved genomic and metabolomic analyses can serve as a resource to facilitate informed selection of strains for microbiome reconstitution.

Distant relatives of a eukaryotic cell-specific toxin family evolved a complement-like mechanism to kill bacteria.

Cholesterol-dependent cytolysins (CDCs) comprise a large family of pore-forming toxins produced by Gram-positive bacteria, which are used to attack eukaryotic cells. Here, we functionally characterize a family of 2-component CDC-like (CDCL) toxins produced by the Gram-negative Bacteroidota that form pores by a mechanism only described for the mammalian complement membrane attack complex (MAC). We further show that the Bacteroides CDCLs are not eukaryotic cell toxins like the CDCs, but instead bind to and are proteolytically activated on the surface of closely related species, resulting in pore formation and cell death. The CDCL-producing Bacteroides is protected from the effects of its own CDCL by the presence of a surface lipoprotein that blocks CDCL pore formation. These studies suggest a prevalent mode of bacterial antagonism by a family of two-component CDCLs that function like mammalian MAC and that are wide-spread in the gut microbiota of diverse human populations.

Comprehensive analyses of a large human gut Bacteroidales culture collection reveal species- and strain-level diversity and evolution.

Species of the Bacteroidales order are among the most abundant and stable bacterial members of the human gut microbiome, with diverse impacts on human health. We cultured and sequenced the genomes of 408 Bacteroidales isolates from healthy human donors representing nine genera and 35 species and performed comparative genomic, gene-specific, metabolomic, and horizontal gene transfer analyses. Families, genera, and species could be grouped based on many distinctive features. We also observed extensive DNA transfer between diverse families, allowing for shared traits and strain evolution. Inter- and intra-species diversity is also apparent in the metabolomic profiling studies. This highly characterized and diverse Bacteroidales culture collection with strain-resolved genomic and metabolomic analyses represents a valuable resource to facilitate informed selection of strains for microbiome reconstitution.

"Cross-glycosylation" of proteins in Bacteroidales species.

While it is now evident that the two Bacteroidales species Bacteroides fragilis and Tannerella forsythia both have general O-glycosylation systems and share a common glycosylation sequon, the ability of these organisms to glycosylate a protein native to the other organism has not yet been demonstrated. Here, we report on the glycosylation of heterologous proteins between these two organisms. Using genetic tools previously developed for Bacteroides species, two B. fragilis model glycoproteins were expressed in the fastidious anaerobe T. forsythia and the attachment of the known T. forsythia O-glycan to these proteins was demonstrated by liquid chromatography electrospray ionization tandem mass spectrometry. Likewise, two predominant T. forsythia glycoproteins were expressed in B. fragilis and glycosylation with the B. fragilis O-glycan was confirmed. Purification of these proteins from B. fragilis allowed the preliminary characterization of the previously uncharacterized B. fragilis protein O-glycan. Based on mass spectrometric data, we show that the B. fragilis protein O-glycan is an oligosaccharide composed of nine sugar units. Compositional and structural similarities with the T. forsythia O-glycan suggest commonalities in their biosynthesis. These data demonstrate the feasibility of exploiting these organisms for the design of novel glycoproteins.

Genome-wide map of quantified epigenetic changes during in vitro chondrogenic differentiation of primary human mesenchymal stem cells.

BACKGROUND: For safe clinical application of engineered cartilage made from mesenchymal stem cells (MSCs), molecular mechanisms for chondrogenic differentiation must be known in detail. Changes in gene expression and extracellular matrix synthesis have been extensively studied, but the epigenomic modifications underlying these changes have not been described. To this end we performed whole-genome chromatin immunoprecipitation and deep sequencing to quantify six histone modifications, reduced representation bisulphite sequencing to quantify DNA methylation and mRNA microarrays to quantify gene expression before and after 7 days of chondrogenic differentiation of MSCs in an alginate scaffold. To add to the clinical relevance of our observations, the study is based on primary bone marrow-derived MSCs from four donors, allowing us to investigate inter-individual variations. RESULTS: We see two levels of relationship between epigenetic marking and gene expression. First, a large number of genes ontogenetically linked to MSC properties and the musculoskeletal system are epigenetically prepatterned by moderate changes in H3K4me3 and H3K9ac near transcription start sites. Most of these genes remain transcriptionally unaltered. Second, transcriptionally upregulated genes, more closely associated with chondrogenesis, are marked by H3K36me3 in gene bodies, highly increased H3K4me3 and H3K9ac on promoters and 5' end of genes, and increased H3K27ac and H3K4me1 marking in at least one enhancer region per upregulated gene. Within the 7-day time frame, changes in promoter DNA methylation do not correlate significantly with changes in gene expression. Inter-donor variability analysis shows high level of similarity between the donors for this data set. CONCLUSIONS: Histone modifications, rather than DNA methylation, provide the primary epigenetic control of early differentiation of MSCs towards the chondrogenic lineage.

A ubiquitous mobile genetic element disarms a bacterial antagonist of the gut microbiota.

DNA transfer is ubiquitous in the gut microbiota, especially among species of Bacteroidales. In silico analyses have revealed hundreds of mobile genetic elements shared between these species, yet little is known about the phenotypes they encode, their effects on fitness, or pleiotropic consequences for the recipient's genome. Here, we show that acquisition of a ubiquitous integrative and conjugative element encoding an antagonistic system shuts down the native contact-dependent antagonistic system of Bacteroides fragilis . Despite inactivating the native antagonism system, mobile element acquisition increases fitness of the B. fragilis transconjugant over its progenitor by arming it with a new weapon. This DNA transfer causes the strain to change allegiances so that it no longer targets ecosystem members containing the same element yet is armed for communal defense.

Theoretical and experimental characterization of the scope of protein O-glycosylation in Bacteroides fragilis.

Among bacterial species demonstrated to have protein O-glycosylation systems, that of Bacteroides fragilis and related species is unique in that extracytoplasmic proteins are glycosylated at serine or threonine residues within the specific three-amino acid motif D(S/T)(A/I/L/M/T/V). This feature allows for computational analysis of the proteome to identify candidate glycoproteins. With the criteria of a signal peptidase I or II cleavage site or a predicted transmembrane-spanning region and the presence of at least one glycosylation motif, we identified 1021 candidate glycoproteins of B. fragilis. In addition to the eight glycoproteins identified previously, we confirmed that another 12 candidate glycoproteins are in fact glycosylated. These included four glycoproteins that are predicted to localize to the inner membrane, a compartment not previously shown to include glycosylated proteins. In addition, we show that four proteins involved in cell division and chromosomal segregation, two of which are encoded by candidate essential genes, are glycosylated. To date, we have not identified any extracytoplasmic proteins containing a glycosylation motif that are not glycosylated. Therefore, based on the list of 1021 candidate glycoproteins, it is likely that hundreds of proteins, comprising more than half of the extracytoplasmic proteins of B. fragilis, are glycosylated. Site-directed mutagenesis of several glycoproteins demonstrated that all are glycosylated at the identified glycosylation motif. By engineering glycosylation motifs into a naturally unglycosylated protein, we are able to bring about site-specific glycosylation at the engineered sites, suggesting that this glycosylation system may have applications for glycoengineering.