Gut Commensal Bacteroidetes Encode a Novel Class of Vitamin B12-Binding Proteins.

Human gut commensal Bacteroidetes rely on multiple transport systems to acquire vitamin B12 and related cobamides for fitness in the gut. In addition to a set of conserved transport proteins, these systems also include a diverse repertoire of additional proteins with unknown function. Here, we report the function and structural characterization of one of these proteins, BtuH, which binds vitamin B12 directly via a C-terminal globular domain that has no known structural homologs. This protein is required for efficient B12 transport and competitive fitness in the gut, demonstrating that members of the heterogeneous suite of accessory proteins encoded in Bacteroides cobamide transport system loci can play key roles in vitamin acquisition. IMPORTANCE The gut microbiome is a complex microbial community with important impacts on human health. One of the major groups within the gut microbiome, the Bacteroidetes, rely on their ability to capture vitamin B12 and related molecules for fitness in the gut. Unlike well-studied model organisms, gut Bacteroidetes genomes often include multiple vitamin B12 transport systems with a heterogeneous set of components. The role, if any, of these components was unknown. Here, we identify new proteins that play key roles in vitamin B12 capture in these organisms. Notably, these proteins are associated with some B12 transport systems and not others (even in the same bacterial strain), suggesting that these systems may assemble into functionally distinct machines to capture vitamin B12 and related molecules.

Human Rad51 protein can form homologous joints in the absence of net strand exchange.

The eukaryotic homologs of RecA protein are central enzymes of recombination and repair, and notwithstanding a high degree of conservation they differ sufficiently from RecA to offer insights into mechanisms and biological roles. The yield of DNA strand exchange reactions driven by both Escherichia coli RecA protein and its human homolog HsRad51 protein was inversely related to the GC content of oligonucleotide substrates, but at any given GC composition, HsRad51 promoted less exchange than RecA. When 40% of bases were GC pairs, the rate constant for strand exchange by HsRad51 was unmeasurable, whereas the rate constants for homologous pairing were unaltered relative to more AT-rich DNA. The ability of HsRad51 to form joints in the absence of net strand exchange was confirmed by experiments in which heterologous blocks at both ends of linear duplex oligonucleotides produced joints that instantly dissociated upon deproteinization. These findings suggest that HsRad51 acting alone on human DNA in vivo is a pairing protein that cannot form extensive heteroduplex DNA.

Rapid exchange of A:T base pairs is essential for recognition of DNA homology by human Rad51 recombination protein.

Human Rad51 belongs to a ubiquitous family of proteins that enable a single strand to recognize homology in duplex DNA, and thereby to initiate genetic exchanges and DNA repair, but the mechanism of recognition remains unknown. Kinetic analysis by fluorescence resonance energy transfer combined with the study of base substitutions and base mismatches reveals that recognition of homology, helix destabilization, exchange of base pairs, and initiation of strand exchange are integral parts of a rapid, concerted mechanism in which A:T base pairs play a critical role. Exchange of base pairs is essential for recognition of homology, and physical evidence indicates that such an exchange occurs early enough to mediate recognition.

Determination of molecular masses of proteins in solution: Implementation of an HPLC size exclusion chromatography and laser light scattering service in a core laboratory.

Size exclusion chromatography (SEC) coupled with "on-line" laser light scattering (LS), refractive index (RI), and ultraviolet (UV) detection provides an elegant approach to determining the molecular weights of proteins and their complexes in solution. SEC serves solely as a fractionation step to minimize the ambiguity that otherwise can result from the fact that light scattering provides the weight-average molecular weight (MW) of all species in solution. Our goal is to establish realistic expectations for MW determination using LS coupled with SEC, define sample requirements, and identify possible limitations of SEC/LS analysis. Analyses of 14 protein standards that range from 12 to 475 kd suggest that the molecular weights of native proteins may be determined in a single SEC/LS experiment with an accuracy of +/-5%. The MW determination depends only on the downstream LS and RI detectors, and it is independent of elution position. Unusual elution because of nonglobular shape or interaction with the SEC support has no impact on the MW determination by SEC/LS. With the instrument configuration that was used, the optimal amount of protein needed for SEC/LS is about 50 g for proteins with molecular weight greater than 40 kd. However, analyses of ovalbumin and transferrin demonstrate that even 10 g is sufficient to determine the MW with an error of less than +/-6%. Although SEC/LS has some limitations, such as proteins that contain chromophores whose absorption spectrum overlaps that of the emission spectrum of the laser, it represents a fast and robust approach to determining MW and to monitoring protein oligomerization in solution.

RecA tests homology at both pairing and strand exchange.

RecA is a 38-kDa protein from Escherichia coli that polymerizes on single-stranded DNA, forming a nucleoprotein filament that pairs with homologous duplex DNA and carries out strand exchange in vitro. To observe the effects of mismatches on the kinetics of the RecA-catalyzed recombination reaction, we used assays based upon fluorescence energy transfer that can differentiate between the pairing and strand displacement phases. Oligonucleotide sequences that produced 2-14% mismatches in the heteroduplex product of strand exchange were tested, as well as completely homologous and heterologous sequences. The equilibrium constant for pairing decreased as the number of mismatches increased, which appeared to result from both a decrease in the rate of formation and an increase in the rate of dissociation of the intermediates. In addition, the rate of strand displacement decreased with increasing numbers of mismatches, roughly in proportion to the number of mismatches. The equilibrium constant for pairing and the rate constant for strand displacement both decreased 6-fold as the heterology increased to 14%. These results suggest that discrimination of homology from heterology occurs during both pairing and strand exchange.

Base-catalysis of imino proton exchange in DNA: effects of catalyst upon DNA structure and dynamics.

Characterization of the kinetics and energetics of base-pair opening in nucleic acids relies upon measurements of the rates of exchange of imino protons with water protons at high concentrations of the exchange catalyst. Under these conditions, the exchange catalyst may affect structural or dynamic properties of the nucleic acid molecule and thus, limit the significance of the exchange data. To address this problem, we have used NMR spectroscopy to characterize the effects of a catalyst of imino proton exchange, namely, ammonia upon the structure and dynamics of the self-complementary DNA dodecamer [d(CGCAGATCTGCG)]2. The changes in structure were monitored in proton NOESY and DQF-COSY experiments and in phosphorus spectra at 15 degrees C and at ammonia concentrations ranging from 0.002 to 0.5 M. The results indicate that ammonia induces subtle changes in the solution conformation of the dodecamer, but the overall structure is maintained close to the B-type DNA structure. However, the relaxation rates (i.e., transverse, longitudinal, and cross-relaxation rates) of several non-exchangeable protons were found to increase by approximately 50% upon changing ammonia concentration from 0.002 to 0.5 M. The increases were comparable for all protons investigated suggesting that they originate from an ammonia-induced increase in the overall correlation time of the DNA dodecamer. Numerical analysis revealed that the catalyst-induced enhancements in proton relaxation can alter significantly the calculated values of the exchange rates of imino protons, especially those obtained from measurements of the line widths of these proton resonances.

Energetics of base pair opening in a DNA dodecamer containing an A3T3 tract.

Nuclear magnetic resonance spectroscopy has been used to characterize the kinetics and energetics of opening of base pairs in the DNA dodecamer [d(CGCAAATTTGCG)]2. The dodecamer contains an A3T3 tract that induces intrinsic curvature of the helix axis. Previous studies from this and other laboratories have shown that the kinetics of base pair opening in AnTn tracts is unique: the opening rates of the A.T base pairs in the interior of the tract are much lower than that of the A.T base pair at the 5'-end of the tract. In the present work, we have investigated the energetics of the pathways for opening of the A.T base pairs in the A3T3 tract. The energetic parameters of the activated state(s) are obtained from the temperature dependence of the opening rate constants. The lower opening rates for the A.T base pairs situated in the interior of the tract are shown to originate from higher activation enthalpies which are compensated, in part, by increases in the activation entropies. We have also obtained an energetic characterization of the open state(s) of the A.T base pairs in the dodecamer by measuring the equilibrium constants for base pair opening and their temperature dependence. The results suggest that the transitions from closed to open state(s) in the A.T base pairs of the A3T3 tract are energetically similar.

Sequence dependence of base-pair opening in a DNA dodecamer containing the CACA/GTGT sequence motif.

Proton nuclear magnetic resonance spectroscopy is used to characterize the kinetics and energetics of base-pair opening in two self-complementary DNA dodecamer duplexes: [d(CGCACATGTGCG)]2 and [d(CGCAGATCTGCG)]2. The first dodecamer contains two symmetrical CACA/GTGT motifs; in the second dodecamer, each motif is interrupted by a change of the central C.G base pair to a G.C base pair. The opening rates and the equilibrium constants for formation of the open state of each base pair are obtained from the dependence of the imino proton exchange rates on the concentration of ammonia catalyst. The results indicate that the opening rates of the central three base pairs in the CACA/GTGT motif are 3-8-fold larger than the corresponding ones in the CAGA/GTCT sequence. The activation enthalpies and entropies, and the standard enthalpy and entropy changes for formation of the open state, are obtained from the temperature dependence of the opening rates and equilibrium constants, respectively. The results reveal that enthalpy/entropy compensation exists, for all base pairs in both dodecamers, in activation as well as in the equilibria between closed and open states. As a result, the opening rates and equilibrium constants for opening are maintained, in both dodecamers, within a relatively narrow range of values. Nevertheless, large sequence-induced variations are observed for the activation enthalpies and the standard enthalpy changes for opening. The A.T base pair located between the C.G base pairs in the CACA/GTGT motif has a negative enthalpy change for formation of the activated state during opening. This is the first case in which a negative activation enthalpy is observed for opening of a Watson-Crick base pair in DNA.(ABSTRACT TRUNCATED AT 250 WORDS)