Dominant Bacterial Phyla from the Human Gut Show Widespread Ability To Transform and Conjugate Bile Acids.

Gut bacteria influence human physiology by chemically modifying host-synthesized primary bile acids. These modified bile acids, known as secondary bile acids, can act as signaling molecules that modulate host lipid, glucose, and energy metabolism and affect gut microbiota composition via selective antimicrobial properties. However, knowledge regarding the bile acid-transforming capabilities of individual gut microbes remains limited. To help address this knowledge gap, we screened 72 bacterial isolates, spanning seven major phyla commonly found in the human gut, for their ability to chemically modify unconjugated bile acids. We found that 43 isolates, representing 41 species, were capable of in vitro modification of one or more of the three most abundant unconjugated bile acids in humans: cholic acid, chenodeoxycholic acid, and deoxycholic acid. Of these, 32 species have not been previously described as bile acid transformers. The most prevalent bile acid transformations detected were oxidation of 3α-, 7α-, or 12α-hydroxyl groups on the steroid core, a reaction catalyzed by hydroxysteroid dehydrogenases. In addition, we found 7α-dehydroxylation activity to be distributed across various bacterial genera, and we observed several other complex bile acid transformations. Finally, our screen revealed widespread bacterial conjugation of primary and secondary bile acids to glycine, a process that was thought to only occur in the liver, and to 15 other amino acids, resulting in the discovery of 44 novel microbially conjugated bile acids. IMPORTANCE Our current knowledge regarding microbial bile acid transformations comes primarily from biochemical studies on a relatively small number of species or from bioinformatic predictions that rely on homology to known bile acid-transforming enzyme sequences. Therefore, much remains to be learned regarding the variety of bile acid transformations and their representation across gut microbial species. By carrying out a systematic investigation of bacterial species commonly found in the human intestinal tract, this study helps better define the gut bacteria that impact composition of the bile acid pool, which has implications in the context of metabolic disorders and cancers of the digestive tract. Our results greatly expand upon the list of bacterial species known to perform different types of bile acid transformations. This knowledge will be vital for assessing the causal connections between the microbiome, bile acid pool composition, and human health.

Fracture toughness of modified dental resin systems.

This study compared the relative fracture toughness of a Bis-GMA//TEGDMA (50:50 wt%)-based resin system modified by 5, 10, and 15 wt% of a methacrylate-terminated poly(butadiene-acrylonitrile-acrylic acid) terpolymer toughening agent. After storage in distilled water at 37 +/- 2 degrees C for 7 days, plane strain fracture toughness (KIC) was determined on an Instron testing machine at a 0.5-mm min-1 displacement rate. The glass transition temperature (Tg) in degrees C was determined after 7 days (dry and wet) storage by thermomechanical analysis. The results of this study showed significantly improved fracture toughness and lowered water sorption with the modified resin systems which was indicated by higher wet glass transition temperatures.

The heme pocket afforded by Gly117 is crucial for proper heme ligation and activity of CooA.

CooA, a CO-sensing homodimeric transcription activator from Rhodospirillum rubrum, undergoes a conformational change in response to CO binding to its heme prosthetic group that allows it to bind specific DNA sequences. In a recent structural study (Lanzilotta, W. N., Schuller, D. J., Thorsteinsson, M. V., Kerby, R. L., Roberts, G. P., and Poulos, T. L. (2000) Nat. Struct. Biol. 7, 876-880), it was suggested that CO binding to CooA results in a modest repositioning of the C-helices that serve as the dimer interface. Gly(117) is one of the residues on the C-helix within 7 A of the heme iron on the Pro(2) side of the heme in CooA. Analysis of a series of Gly(117) variants revealed altered CO-sensing function and heme ligation states dependent on the size of the substituted amino acid at this position; bulky substitutions perturbed CooA both spectrally and functionally. A combination of spectroscopic and mutagenic studies showed that a representative Gly(117) variant, G117I CooA, was specifically perturbed in its Pro(2) ligation in both Fe(III) and Fe(II) forms, but comparison with other CooA variants indicated that perturbation of Pro(2) ligation is not the basis for the lack of CO response in G117I CooA. These results have led to the hypothesis that (i) the heme and the C-helix region move toward each other following CO binding and the interaction of the heme with the C-helix is crucial for CooA activation, and (ii) this event occurs only when a properly sized heme pocket is afforded.

CooA: a heme-containing regulatory protein that serves as a specific sensor of both carbon monoxide and redox state.

CooA, the heme-containing carbon monoxide (CO) sensor from the bacterium Rhodospirillum rubrum, is a transcriptional factor that activates expression of certain genes in response to CO. As with other heme proteins, CooA is unable to bind CO when the Fe heme is oxidized, consistent with the fact that some of the regulated gene products are oxygen-labile. Upon reduction, there is an unusual switch of protein ligands to the six-coordinate heme and the reduced heme is able to bind CO. CO binding stabilizes a conformation of the dimeric protein that allows sequence-specific DNA binding, and transcription is activated through contacts between CooA and RNA polymerase. CooA is therefore a novel redox sensor as well as a specific CO sensor. CooA is a homolog of catabolite responsive protein (CRP), whose transcriptionally active conformation has been known for some time. The recent solution of the crystal structure of the CO-free (transcriptionally inactive) form of CooA has allowed insights into the mechanism by which both proteins respond to their specific small-molecule effectors.

Redox-mediated transcriptional activation in a CooA variant.

CooA, the carbon monoxide-sensing transcription factor from Rhodospirillum rubrum, binds CO at a reduced (Fe(II)) heme moiety with resulting conformational changes that promote DNA binding. In this study, we report a variant of CooA, M124R, that is active in transcriptional activation in a redox-dependent manner. Where wild-type CooA is active only in the Fe(II) + CO form, M124R CooA is active in both Fe(II) + CO and Fe(III) forms. Analysis of the pH dependence of the activity of Fe(III) M124R CooA demonstrated that the activity was also coordination state-dependent with a five-coordinate, high-spin species identified as the active form and Cys(75) as the retained ligand. In contrast, the active Fe(II) + CO forms of both wild-type and M124R CooA are six-coordinate and low-spin with a protein ligand other than Cys(75), so that WT and Fe(III) M124R CooA are apparently achieving an active conformation despite two different heme coordination and ligation states. A hypothesis to explain these results is proposed. This study demonstrates the utility of CooA as a model system for the isolation of functionally interesting heme proteins.

Structure of the CO sensing transcription activator CooA.

CooA is a homodimeric transcription factor that belongs to the catabolite activator protein (CAP) family. Binding of CO to the heme groups of CooA leads to the transcription of genes involved in CO oxidation in Rhodospirillum rubrum. The 2.6 A structure of reduced (Fe2+) CooA reveals that His 77 in both subunits provides one heme ligand while the N-terminal nitrogen of Pro 2 from the opposite subunit provides the other ligand. A structural comparison of CooA in the absence of effector and DNA (off state) with that of CAP in the effector and DNA bound state (on state) leads to a plausible model for the mechanism of allosteric control in this class of proteins as well as the CO dependent activation of CooA.

Characterization of variants altered at the N-terminal proline, a novel heme-axial ligand in CooA, the CO-sensing transcriptional activator.

CooA, the carbon monoxide-sensing transcription factor from Rhodospirillum rubrum, binds CO through a heme moiety resulting in conformational changes that promote DNA binding. The crystal structure shows that the N-terminal Pro(2) of one subunit (Met(1) is removed post-translationally) provides one ligand to the heme of the other subunit in the CooA homodimer. To determine the importance of this novel ligand and the contiguous residues to CooA function, we have altered the N terminus through two approaches: site-directed mutagenesis and regional randomization, and characterized the resulting CooA variants. While Pro(2) appears to be optimal for CooA function, it is not essential and a variety of studied variants at this position have substantial CO-sensing function. Surprisingly, even alterations that add a residue (where Pro(2) is replaced by Met(1)-Tyr(2), for example) accumulate heme-containing CooA with functional properties that are similar to those of wild-type CooA. Other nearby residues, such as Phe(5) and Asn(6) appear to be important for either the structural integrity or the function of CooA. These results are contrasted with those previously reported for alteration of the His(77) ligand on the opposite side of the heme.

Altering the specificity of CooA, the carbon monoxide-sensing transcriptional activator: characterization of CooA variants that bind cyanide in the Fe(II) form with high affinity.

CooA is a carbon monoxide- (CO-) sensing homodimeric heme protein that activates the transcription of genes required for the anaerobic oxidation of CO to CO(2) in the phototrophic bacterium Rhodospirillum rubrum. In this study, we demonstrate that mutational alteration of the histidine residue (His(77)) that serves as a heme ligand in the Fe(II) form of CooA allows high-affinity binding of cyanide (K(d) approximately 0.4 mM) to the heme. In contrast, neither these same variants in the Fe(III) form nor wild-type CooA in either oxidation state was able to bind cyanide even at high concentrations (50 mM). Examination of the pH dependence of spectral changes upon addition of cyanide suggested that the cyanide anion coordinated the heme iron. In addition, the UV-visible absorption spectrum of H77Y Fe(II) CooA without added effectors is also pH-dependent, suggesting that an ionizable amino acid has become solvent-accessible in the absence of His(77). Finally, we demonstrate that the transcriptional activity of H77Y CooA shows a small (1.4-fold) increase in the presence of cyanide, suggesting that the binding of cyanide to this variant promotes the active conformation of H77Y CooA.

Fracture toughness of resin-based luting cements.

STATEMENT OF PROBLEM: The introduction of resin-modified glass ionomer cements has expanded the choices of luting cements available to the clinician; however, few independent studies are available on the fracture toughness of the currently available resin-modified glass ionomer luting agents compared with the composite cements. PURPOSE: This investigation evaluated the relative fracture toughness (K(IC)) of 3 composite luting cements (Panavia 21, Enforce, and C&B Metabond), 3 resin-modified glass ionomer luting cements (Advance, Vitremer Luting, and Fuji Duet), and a conventional glass ionomer luting cement (Ketac-Cem) at 24-hour and 7-day storage times. MATERIAL AND METHODS: K(IC) was determined by preparing minicompact test specimens (n = 8) with introduced precracks. Specimens were stored in distilled water at 37 degrees C + 2 degrees C until testing. Testing was performed on an Instron testing machine at a displacement rate of 0.5 mm/min. RESULTS: ANOVA (P <.001) and REGW Multiple Range Test (P <.05) demonstrated significant differences among several of the cements tested. The mean fracture toughness values of C&B Metabond at 24 hours and Enforce at both 24 hours and 7 days were significantly greater than use any of the other cements tested. CONCLUSION: The resin-modified glass ionomer cements exhibited improved fracture toughness when compared with the conventional glass ionomer; however, they were still inferior to Enforce and C&B Metabond composite cements.

Electronic absorption, EPR, and resonance raman spectroscopy of CooA, a CO-sensing transcription activator from R. rubrum, reveals a five-coordinate NO-heme.

Electronic absorption, EPR, and resonance Raman spectroscopies revealed that CooA, the CO-sensing transcriptional regulator from Rhodospirillum rubrum, reacts with NO to form a five-coordinate NO-heme. NO must therefore displace both of the heme ligands from six-coordinate, low-spin Fe(II)CooA in forming five-coordinate Fe(II)CooA(NO). CO, in contrast, displaces a single heme ligand from Fe(II)CooA to form six-coordinate Fe(II)CooA(CO). Of a series of common heme-binding ligands, only CO and NO were able to bind to the heme of wild-type CooA; imidazole, azide anion, and cyanide anion had no effect on the heme absorption spectrum. Although NO binds to the heme and displaces the endogenous ligands, NO was not able to induce CooA to bind to its target DNA. The mechanism of CO-dependent activation of CooA is thus more complex than simple displacement of a ligand from the heme iron since NO does not trigger DNA binding. These observations suggest that the CooA heme site discriminates between NO and the biologically relevant signal, CO.

Solubility and sorption of resin-based luting cements.

This study compared the seven-day water sorption, water solubility and lactic acid solubility of three composite cements and three resin-modified glass-ionomer cements. Disc-shaped specimens measuring 15 mm x 0.5 mm were prepared according to each manufacturer's specifications and desiccated to a constant mass. Specimens were then placed in distilled water at 37 degrees C for seven days. Acid solubility was performed in 0.01 M lactic acid. The weight changes of the specimens after immersion in distilled water or 0.01 M lactic acid were measured using an electronic analytical balance. A one-way ANOVA followed by the Ryan-Einot-Gabriel-Welsch (REGW) multiple range test was performed on all data. Significant differences (p < 0.05) were found among several cements tested for each of the properties investigated. Due to their hydrophilic nature, all resin-modified glass-ionomer cements showed significantly higher water sorption compared to composite cements.

Identification of two important heme site residues (cysteine 75 and histidine 77) in CooA, the CO-sensing transcription factor of Rhodospirillum rubrum.

The CO-sensing mechanism of the transcription factor CooA from Rhodospirillum rubrum was studied through a systematic mutational analysis of potential heme ligands. Previous electron paramagnetic resonance (EPR) spectroscopic studies on wild-type CooA suggested that oxidized (FeIII) CooA contains a low-spin heme with a thiolate ligand, presumably a cysteine, bound to its heme iron. In the present report, electronic absorption and EPR analysis of various substitutions at Cys residues establish that Cys75 is a heme ligand in FeIII CooA. However, characterization of heme stability and electronic properties of purified C75S CooA suggest that Cys75 is not a ligand in FeII CooA. Mutational analysis of all CooA His residues showed that His77 is critical for CO-stimulated transcription. On the basis of findings that H77Y CooA is perturbed in its FeII electronic properties and is unable to bind DNA in a site-specific manner in response to CO, His77 appears to be an axial ligand to FeII CooA. These results imply a ligand switch from Cys75 to His77 upon reduction of CooA. In addition, an interaction has been identified between Cys75 and His77 in FeIII CooA that may be involved in the CO-sensing mechanism. Finally, His77 is necessary for the proper conformational change of CooA upon CO binding.

Two-body wear resistance and degree of conversion of laboratory-processed composite materials.

PURPOSE: The purpose of this investigation was to evaluate the relative 2-body abrasive wear and degree of conversion of 4 laboratory-processed composites (Targis, Concept, belleGlass, and Artglass) and 2 direct placement composites (Herculite and Heliomolar) after 7 days of storage. MATERIALS AND METHODS: Human enamel was used as a positive control for 2-body abrasive wear, and 10 cylindric specimens (3.5-mm diameter, 8-mm height) of each material were prepared and stored in distilled water at 37 +/- 2 degrees C for wear testing. Relative 2-body abrasive wear rates were determined using a 30-micron diamond disk and a 2-body pin-on-disk apparatus. Subsequently, 3 polymerized specimens that had been stored in sealed polyethylene vials for 7 days were prepared for degree of conversion testing. The degree of conversion was determined on an infrared spectrometer using standard baseline techniques and various internal standards. RESULTS: Statistical analysis using analysis of variance and the Tukey-Kramer multiple range test indicated significant differences between several of the materials tested for both 2-body abrasive wear and degree of conversion. CONCLUSION: Concept exhibited significantly less 2-body abrasive wear compared to the direct and indirect composites (P < 0.01). Concept and belleGlass exhibited a mean degree of conversion that was significantly higher than any of the other composites tested (P < 0.01).

Substitution of valine for histidine 265 in carbon monoxide dehydrogenase from Rhodospirillum rubrum affects activity and spectroscopic states.

In carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum, histidine 265 was replaced with valine by site-directed mutagenesis of the cooS gene. The altered form of CODH (H265V) had a low nickel content and a dramatically reduced level of catalytic activity. Although treatment with NiCl2 and CoCl2 increased the activity of H265V CODH by severalfold, activity levels remained more than 1000-fold lower than that of wild-type CODH. Histidine 265 was not essential for the formation and stability of the Fe4S4 clusters. The Km and KD for CO as well as the KD for cyanide were relatively unchanged as a result of the amino acid substitution in CODH. The time-dependent reduction of the [Fe4S4]2+ clusters by CO occurred on a time scale of hours, suggesting that, as a consequence of the mutation, a rate-limiting step had been introduced prior to the transfer of electrons from CO to the cubanes in centers B and C. EPR spectra of H265V CODH lacked the gav = 1.86 and gav = 1.87 signals characteristic of reduced forms of the active site (center C) of wild-type CODH. This indicates that the electronic properties of center C have been modified possibly by the disruption or alteration of the ligand-mediated interaction between the nickel site and Fe4S4 chromophore.

CooA, a CO-sensing transcription factor from Rhodospirillum rubrum, is a CO-binding heme protein.

Biological sensing of small molecules such as NO, O2, and CO is an important area of research; however, little is know about how CO is sensed biologically. The photosynthetic bacterium Rhodospirillum rubrum responds to CO by activating transcription of two operons that encode a CO-oxidizing system. A protein, CooA, has been identified as necessary for this response. CooA is a member of a family of transcriptional regulators similar to the cAMP receptor protein and fumavate nitrate reduction from Escherichia coli. In this study we report the purification of wild-type CooA from its native organism, R. rubrum, to greater than 95% purity. The purified protein is active in sequence-specific DNA binding in the presence of CO, but not in the absence of CO. Gel filtration experiments reveal the protein to be a dimer in the absence of CO. Purified CooA contains 1.6 mol heme per mol of dimer. Upon interacting with CO, the electronic spectrum of CooA is perturbed, indicating the direct binding of CO to the heme of CooA. A hypothesis for the mechanism of the protein's response to CO is proposed.

In vivo nickel insertion into the carbon monoxide dehydrogenase of Rhodospirillum rubrum: molecular and physiological characterization of cooCTJ.

The products of cooCTJ are involved in normal in vivo Ni insertion into the carbon monoxide dehydrogenase (CODH) of Rhodospirillum rubrum. Located on a 1.5-kb DNA segment immediately downstream of the CODH structural gene (cooS), two of the genes encode proteins that bear motifs reminiscent of other (urease and hydrogenase) Ni-insertion systems: a nucleoside triphosphate-binding motif near the N terminus of CooC and a run of 15 histidine residues regularly spaced over the last 30 amino acids of the C terminus of CooJ. A Gm(r)omega-linker cassette was developed to create both polar and nonpolar (60 bp) insertions in the cooCTJ region, and these, along with several deletions, were introduced into R. rubrum by homologous recombination. Analysis of the exogenous Ni levels required to sustain CO-dependent growth of the R. rubrum mutants demonstrated different phenotypes: whereas the wild-type strain and a mutant bearing a partial cooJ deletion (of the region encoding the histidine-rich segment) grew at 0.5 microM Ni supplementation, strains bearing Gm(r)omega-linker cassettes in cooT and cooJ required approximately 50-fold-higher Ni levels and all cooC insertion strains, bearing polar or nonpolar insertions, grew optimally at 550 microM Ni.

Strength properties of visible-light-cured resin-modified glass-ionomer cements.

A new generation of glass ionomers containing polymerizable methacrylate monomers and/or prepolymers are now available for use as direct esthetic restorative materials. Proper clinical application of these new resin-modified glass ionomers requires an understanding of their benefits and limitations. The purpose of this investigation was to compare the compressive and diametral tensile strength at 1 hour, 24 hours, and 7 days of three visible-light-cured glass-ionomer cements, a polyacid-modified composite resin, and a composite resin core build-up material under both light-cure and dark-cure conditions. Statistical analysis indicated significant differences between several of the cements tested for both compressive and diametral tensile strengths at all three testing times (P > 0.05). Prosthodent composite resin and Vitremer tricure visible-light-cured glass-ionomer cement are significantly greater in both compressive and diametral tensile strength than any of the other materials tested after 7 days.

Shear rebond strength of Rexillium III to enamel using resin composite cements.

This investigation evaluated the shear rebond strength of Rexillium III to enamel using various resin composite luting systems (Panavia, Imperva Dual, ABC Enhanced, C&B Metabond, Optibond, and Comspan). Cast Rexillium III cylinders (3.9 x 6.0 mm) were bonded to human molar buccal enamel surfaces (n = 8) with each cement type after etching with 37% phosphoric acid for 30 seconds. Bonded specimens were stored in distilled water for 7 days at 37 degrees C +/- 2 degrees C and thermocycled (1,500 cycles) in 5 degrees C and 55 degrees C water baths (1 minute dwell time). Specimens were randomly tested in shear mode on an Instron Testing Machine at a crosshead speed of 0.5 mm per minute. Debonded specimens were then rebonded after appropriate metal conditioning and re-etching the enamel surface for 30 seconds. Analysis of variance (P < 0.001) and the Ryan-Einot-Gabriel-Welsh multiple range test showed significant differences between several of the resin cements (P < 0.05). Panavia exhibited significantly higher shear rebond strength than any of the other cements tested. Only Imperva Dual exhibited a significantly lower shear rebond strength compared to its initial shear bond strength.

Shear bond strength of Rexillium III to enamel using resin composite cements.

This study evaluated the shear bond strength of Rexillium III (Jeneric Pentron, Wallingford, CT) to enamel using various resin composite luting systems. Cast alloy cylinders (3.9 mm X 6.0 mm) were bonded with each cement to human molar buccal enamel surfaces (n = 8). The enamel was etched using a 35% phosphoric acid solution for 30 seconds. Bonded specimens were stored in distilled water for 7 days at 37 degrees C and thermocycled (1,500 cycles) in 5 degrees C and 55 degrees C water baths (1-minute dwell time). Specimens were randomly tested in shear using a crosshead speed of 0.5 mm per minute. Use of a one-way analysis of variance (P < .001) and Ryan-Einot-Gabriel-Welsh multiple range test showed significant differences between several of the resin cements. Panavia exhibited a significantly higher shear bond strength than any of the other cements tested.

Characterization of the CO-induced, CO-tolerant hydrogenase from Rhodospirillum rubrum and the gene encoding the large subunit of the enzyme.

In the presence of carbon monoxide, the photosynthetic bacterium Rhodospirillum rubrum induces expression of proteins which allow the organism to metabolize carbon monoxide in the net reaction CO + H2O --> CO2 + H2. These proteins include the enzymes carbon monoxide dehydrogenase (CODH) and a CO-tolerant hydrogenase. In this paper, we present the complete amino acid sequence for the large subunit of this hydrogenase and describe the properties of the crude enzyme in relation to other known hydrogenases. The amino acid sequence deduced from the CO-induced hydrogenase large-subunit gene (cooH) shows significant similarity to large subunits of other Ni-Fe hydrogenases. The closest similarity is with HycE (58% similarity and 37% identity) from Escherichia coli, which is the large subunit of an Ni-Fe hydrogenase (isoenzyme 3). The properties of the CO-induced hydrogenase are unique. It is exceptionally resistant to inhibition by carbon monoxide. It also exhibits a very high ratio of H2 evolution to H2 uptake activity compared with other known hydrogenases. The CO-induced hydrogenase is tightly membrane bound, and its inhibition by nonionic detergents is described. Finally, the presence of nickel in the hydrogenase is addressed. Analysis of wild-type R. rubrum grown on nickel-depleted medium indicates a requirement for nickel for hydrogenase activity. However, analysis of strain UR294 (cooC insertion mutant defective in nickel insertion into CODH) shows that independent nickel insertion mechanisms are utilized by hydrogenase and CODH. CooH lacks the C-terminal peptide that is found in other Ni-Fe hydrogenases; in other systems, this peptide is cleaved during Ni processing.