Carbohydrate polymers drive microbial diversity in the human gut microbiota. It is unclear, however, whether bacterial consortia or single organisms are required to depolymerize highly complex glycans. Here we show that the gut bacterium Bacteroides thetaiotaomicron utilizes the most structurally complex glycan known; the plant pectic polysaccharide rhamnogalacturonan-II, cleaving all but one of its 21 distinct glycosidic linkages. We show that rhamnogalacturonan-II side-chain and backbone deconstruction are coordinated, to overcome steric constraints, and that degradation reveals previously undiscovered enzyme families and novel catalytic activities. The degradome informs revision of the current structural model of RG-II and highlights how individual gut bacteria orchestrate manifold enzymes to metabolize the most challenging glycans in the human diet.

Metals are needed by at least one-quarter of all proteins. Although metallochaperones insert the correct metal into some proteins, they have not been found for the vast majority, and the view is that most metalloproteins acquire their metals directly from cellular pools. However, some metals form more stable complexes with proteins than do others. For instance, as described in the Irving-Williams series, Cu(2+) and Zn(2+) typically form more stable complexes than Mn(2+). Thus it is unclear what cellular mechanisms manage metal acquisition by most nascent proteins. To investigate this question, we identified the most abundant Cu(2+)-protein, CucA (Cu(2+)-cupin A), and the most abundant Mn(2+)-protein, MncA (Mn(2+)-cupin A), in the periplasm of the cyanobacterium Synechocystis PCC 6803. Each of these newly identified proteins binds its respective metal via identical ligands within a cupin fold. Consistent with the Irving-Williams series, MncA only binds Mn(2+) after folding in solutions containing at least a 10(4) times molar excess of Mn(2+) over Cu(2+) or Zn(2+). However once MncA has bound Mn(2+), the metal does not exchange with Cu(2+). MncA and CucA have signal peptides for different export pathways into the periplasm, Tat and Sec respectively. Export by the Tat pathway allows MncA to fold in the cytoplasm, which contains only tightly bound copper or Zn(2+) (refs 10-12) but micromolar Mn(2+) (ref. 13). In contrast, CucA folds in the periplasm to acquire Cu(2+). These results reveal a mechanism whereby the compartment in which a protein folds overrides its binding preference to control its metal content. They explain why the cytoplasm must contain only tightly bound and buffered copper and Zn(2+).

Methanobactins (mbs) are a class of copper-binding peptides produced by aerobic methane oxidizing bacteria (methanotrophs) that have been linked to the substantial copper needs of these environmentally important microorganisms. The only characterized mbs are those from Methylosinus trichosporium OB3b and Methylocystis strain SB2. M. trichosporium OB3b produces a second mb (mb-Met), which is missing the C-terminal Met residue from the full-length form (FL-mb). The as-isolated copper-loaded mbs bind Cu(I). The absence of the Met has little influence on the structure of the Cu(I) site, and both molecules mediate switchover from the soluble iron methane mono-oxygenase to the particulate copper-containing enzyme in M. trichosporium OB3b cells. Cu(II) is reduced in the presence of the mbs under our experimental conditions, and the disulfide plays no role in this process. The Cu(I) affinities of these molecules are extremely high with values of (6-7) × 10(20) M(-1) determined at pH ≥ 8.0. The affinity for Cu(I) is 1 order of magnitude lower at pH 6.0. The reduction potentials of copper-loaded FL-mb and mb-Met are 640 and 590 mV respectively, highlighting the strong preference for Cu(I) and indicating different Cu(II) affinities for the two forms. Cleavage of the disulfide bridge results in a decrease in the Cu(I) affinity to ∼9 × 10(18) M(-1) at pH 7.5. The two thiolates can also bind Cu(I), albeit with much lower affinity (∼ 3 × 10(15) M(-1) at pH 7.5). The high affinity of mbs for Cu(I) is consistent with a physiological role in copper uptake and protection.

Methane-oxidizing bacteria are nature's primary biological mechanism for suppressing atmospheric levels of the second-most important greenhouse gas via methane monooxygenases (MMOs). The copper-containing particulate enzyme is the most widespread and efficient MMO. Under low-copper conditions methane-oxidizing bacteria secrete the small copper-binding peptide methanobactin (mbtin) to acquire copper, but how variations in the structures of mbtins influence copper metabolism and species selection are unknown. Methanobactins have been isolated from Methylocystis strains M and hirsuta CSC1, organisms that can switch to using an iron-containing soluble MMO when copper is limiting, and the nonswitchover Methylocystis rosea. These mbtins are shorter, and have different amino acid compositions, than the characterized mbtin from Methylosinus trichosporium OB3b. A coordinating pyrazinedione ring in the Methylocystis mbtins has little influence on the Cu(I) site structure. The Methylocystis mbtins have a sulfate group that helps stabilize the Cu(I) forms, resulting in affinities of approximately 10 21 M −1. The Cu(II) affinities vary over three orders of magnitude with reduction potentials covering approximately 250 mV, which may dictate the mechanism of intracellular copper release. Copper uptake and the switchover from using the ironcontaining soluble MMO to the copper-containing particulate enzyme is faster when mediated by the native mbtin, suggesting that the amino acid sequence is important for the interaction of mbtins with receptors. The differences in structures and properties of mbtins, and their influence on copper utilization by methaneoxidizing bacteria, have important implications for the ecology and global function of these environmentally vital organisms. C opper is an essential protein cofactor involved in many important cellular processes (1, 2), and copper-trafficking systems have been extensively studied (1,(3)(4)(5)(6)(7)(8). Although copper uptake by eukaryotes is well defined (1, 4, 9), acquisition of this metal by prokaryotes remains poorly understood. Methane-oxidizing bacteria secrete the small copper-binding molecule methanobactin (mbtin) when copper is limiting (10-18), presumably for sequestration of this metal. These organisms have conditionally high requirements for copper (19), primarily for the active site (20) of the particulate methane monooxygenase (pMMO). Almost all known methane-oxidizing bacteria use pMMO for the consumption of methane (19), an important greenhouse gas. A subclass of "switchover" organisms exists that can also produce a less efficient iron-containing soluble MMO (sMMO) under copperdeficient conditions, with pMMO expression up-regulated in response to an increase in the copper-to-cell ratio (15, 21).Methanobactin production has been examined in a number of methane-oxidizing bacteria (22-24), but mbtins from only two organisms have been characterized (13,18). The mbtin (two forms) from Methylosinus trichosporium OB3b (a switchover organism) is the most extensively studi...

Lyme disease is a multisystem disorder caused by the spirochete Borrelia burgdorferi. A common late-stage complication of this disease is oligoarticular arthritis, often involving the knee. In ∼10% of cases, arthritis persists after appropriate antibiotic treatment, leading to a proliferative synovitis typical of chronic inflammatory arthritides. Here, we provide evidence that peptidoglycan (PG), a major component of the B. burgdorferi cell envelope, may contribute to the development and persistence of Lyme arthritis (LA). We show that B. burgdorferi has a chemically atypical PG (PGBb) that is not recycled during cell-wall turnover. Instead, this pathogen sheds PGBb fragments into its environment during growth. Patients with LA mount a specific immunoglobulin G response against PGBb, which is significantly higher in the synovial fluid than in the serum of the same patient. We also detect PGBb in 94% of synovial fluid samples (32 of 34) from patients with LA, many of whom had undergone oral and intravenous antibiotic treatment. These same synovial fluid samples contain proinflammatory cytokines, similar to those produced by human peripheral blood mononuclear cells stimulated with PGBb. In addition, systemic administration of PGBb in BALB/c mice elicits acute arthritis. Altogether, our study identifies PGBb as a likely contributor to inflammatory responses in LA. Persistence of this antigen in the joint may contribute to synovitis after antibiotics eradicate the pathogen. Furthermore, our finding that B. burgdorferi sheds immunogenic PGBb fragments during growth suggests a potential role for PGBb in the immunopathogenesis of other Lyme disease manifestations.

Chlamydiae are important pathogens and symbionts, with unique cell biology features. They lack the cell-division protein FtsZ, which functions in maintaining cell shape and orchestrating cell division in almost all other bacteria. In addition, the existence of peptidoglycan (PG) in chlamydial cell envelopes has been highly controversial. Using electron cryotomography, mass spectrometry and fluorescent labeling dyes, here we show that some environmental chlamydiae have cell-wall sacculi consisting of an unusual PG type. Treatment with fosfomycin (a PG synthesis inhibitor) leads to lower infection rates and aberrant cell shapes, suggesting that PG synthesis is crucial for the chlamydial life cycle. Our findings demonstrate for the first time the presence of PG in a member of the Chlamydiae. They also present a unique example of a bacterium with a PG sacculus but without FtsZ, challenging the current hypothesis that it is the absence of a cell wall that renders FtsZ non-essential.

Modification of essential bacterial peptidoglycan (PG) containing cell walls can lead to antibiotic resistance, for example β-lactam resistance by L,D-transpeptidase activities. Predatory Bdellovibrio bacteriovorus are naturally antibacterial and combat infections by traversing, modifying and finally destroying walls of Gram-negative prey bacteria, modifying their own PG as they grow inside prey. Historically, these multi-enzymatic processes on two similar PG walls have proved challenging to elucidate. Here, with a PG labelling approach utilizing timed pulses of multiple fluorescent D-amino acids (FDAAs), we illuminate dynamic changes that predator and prey walls go through during the different phases of bacteria:bacteria invasion. We show formation of a reinforced circular port-hole in the prey wall; L,D-transpeptidaseBd mediated D-amino acid modifications strengthening prey PG during Bdellovibrio invasion and a zonal mode of predator-elongation. This process is followed by unconventional, multi-point and synchronous septation of the intracellular Bdellovibrio, accommodating odd- and even-numbered progeny formation by non-binary division.

Glycans are major nutrients for the human gut microbiota (HGM). Arabinogalactan proteins (AGPs) comprise a heterogenous group of plant glycans in which a β1,3-galactan backbone and β1,6-galactan side chains are conserved. Diversity is provided by the variable nature of the sugars that decorate the galactans. The mechanisms by which nutritionally relevant AGPs are degraded in the HGM are poorly understood. Here we explore how the HGM organism Bacteroides thetaiotaomicron metabolises AGPs. We propose a sequential degradative model in which exo-acting glycoside hydrolase (GH) family 43 β1,3-galactanases release the side chains. These oligosaccharide side chains are depolymerized by the synergistic action of exo-acting enzymes in which catalytic interactions are dependent on whether degradation is initiated by a lyase or GH. We identified two GHs that establish two previously undiscovered GH families. The crystal structures of the exo-β1,3-galactanases identified a key specificity determinant and departure from the canonical catalytic apparatus of GH43 enzymes. Growth studies of Bacteroidetes spp. on complex AGP revealed three keystone organisms that facilitated utilisation of the glycan by 17 recipient bacteria, which included B. thetaiotaomicron . A surface endo-β1,3-galactanase, when engineered into B. thetaiotaomicron , enabled the bacterium to utilise complex AGPs and act as a keystone organism.