Catechol 2,3-dioxygenase-assisted cleavage of aromatics by “anaerobic” termite gut spirochetes and genomic evidence of a complete meta-pathway

The termite hindgut microbial ecosystem functions like a miniature lignocellulose-metabolizing natural bioreactor, has significant implications to nutrient cycling in the terrestrial environment, and represents an array of microbial metabolic diversity. Deciphering the intricacies of this microbial community to obtain as complete a picture as possible of how it functions as a whole, requires a combination of various traditional and cutting-edge bioinformatic, molecular, physiological, and culturing approaches. Isolates from this ecosystem, including Treponema primitia str. ZAS-1 and ZAS-2 as well as T. azotonutricium str. ZAS-9, have been significant resources for better understanding the termite system. While not all functions predicted by the genomes of these three isolates are demonstrated in vitro, these isolates do have the capacity for several metabolisms unique to spirochetes and critical to the termite system’s reliance upon lignocellulose. In this thesis, work culturing, enriching for, and isolating diverse microorganisms from the termite hindgut is discussed. Additionally, strategies of members of the termite hindgut microbial community to defend against O₂-stress and to generate acetate, the “biofuel” of the termite system, are proposed. In particular, catechol 2,3-dioxygenase and other meta-cleavage catabolic pathway genes are described in the “anaerobic” termite hindgut spirochetes T. primitia str. ZAS-1 and ZAS-2, and the first evidence for aromatic ring cleavage in the phylum (division) Spirochetes is also presented. These results suggest that the potential for O₂-dependent, yet nonrespiratory, metabolisms of plant-derived aromatics should be re-evaluated in termite hindgut communities. Potential future work is also illustrated.

I. Spectroscopic evidence for slow vibrational relaxation in excited electronic states of diatomics in rare gas solids. II. Static crystal field effects in the electronic spectra of isotopically mixed benzene crystals

Part I

Studies of vibrational relaxation in excited electronic states of simple diatomic molecules trapped in solid rare-gas matrices at low temperatures are reported. The relaxation is investigated by monitoring the emission intensity from vibrational levels of the excited electronic state to vibrational levels of the ground electronic state. The emission was in all cases excited by bombardment of the doped rare-gas solid with X-rays.

The diatomics studied and the band systems seen are: N₂, Vegard-Kaplan and Second Positive systems; O₂, Herzberg system; OH and OD, A ²Σ⁺ - X²II_i system. The latter has been investigated only in solid Ne, where both emission and absorption spectra were recorded; observed fine structure has been partly interpreted in terms of slightly perturbed rotational motion in the solid. For N₂, OH, and OD emission occurred from v' > 0, establishing a vibrational relaxation time in the excited electronic state of the order, of longer than, the electronic radiative lifetime. The relative emission intensity and decay times for different v' progressions in the Vegard-Kaplan system are found to depend on the rare-gas host and the N₂ concentration, but are independent of temperature in the range 1.7°K to 30°K.

Part II

Static crystal field effects on the absorption, fluorescence, and phosphorescence spectra of isotopically mixed benzene crystals were investigated. Evidence is presented which demonstrate that in the crystal the ground, lowest excited singlet, and lowest triplet states of the guest deviate from hexagonal symmetry. The deviation appears largest in the lowest triplet state and may be due to an intrinsic instability of the ³B_1u state. High resolution absorption and phospho- rescence spectra are reported and analyzed in terms of site-splitting of degenerate vibrations and orientational effects. The guest phosphorescence lifetime for various benzene isotopes in C₆D₆ and sym-C₆H₃D₃ hosts is presented and discussed.

Ancillary ligand effects : from zirconium(IV)-catalyzed homogeneous propylene polymerization to platinum(II)-mediated C-H bond activation

A series of C_s- and C₁-symmetric doubly-linked ansa-metallocenes of the general formula {1,1'-SiMe₂-2,2'-E-('ƞ⁵-C₅H₂-4-R¹)-(ƞ⁵-C₅H-3',5'-(CHMe₂)₂)}ZrC₂ (E = SiMe₂ (1), SiPh₂ (2), SiMe₂ -SiMe₂ (3); R¹ = H, CHMe₂, C₅H₉, C₆H₁₁, C₆H₅) has been prepared. When activated by methylaluminoxane, these are active propylene polymerization catalysts. 1 and 2 produce syndiotactic polypropylenes, and 3 produces isotactic polypropylenes. Site epimerization is the major pathway for stereoerror formation for 1 and 2. In addition, the polymer chain has slightly stronger steric interaction with the diphenylsilylene linker than with the dimethylsilylene linker. This results in more frequent site epimerization and reduced syndiospecificity for 2 compared to 1.

C₁-Symmetric ansa-zirconocenes [1,1 '-SiMe₂-(C₅H₄)-(3-R-C₅H₃)]ZrCl₂ (4), [1,1 '-SiMe₂-(C₅H₄)-(2,4-R₂-C₅H₂)]ZrCl₂ (5) and [1,1 '-SiMe₂-2,2 '-(SiMe₂-SiMe₂)-(C₅H₃)-( 4-R-C₅H₂)]ZrCl₂ (6) have been prepared to probe the origin of isospecificity in 3. While 4 and 3 produce polymers with similar isospecificity, 5 and 6 give mostly hemi-isotactic-like polymers. It is proposed that the facile site epimerization via an associative pathway allows rapid equilibration of the polymer chain between the isospecific and aspecific insertion sites. This results in more frequent insertion from the isospecific site, which has a lower kinetic barrier for chain propagation. On the other hand, site epimerization for 5 and 6 is slow. This leads to mostly alternating insertion from the isospecific and aspecific sites, and consequently, a hemi-isotactic-like polymers. In comparison, site epimerization is even slower for 3, but enchainment from the aspecific site has an extremely high kinetic barrier for monomer coordination. Therefore, enchainment occurs preferentially from the isospecific site to produce isotactic polymers.

A series of cationic complexes [(ArN=CR-CR=NAr)PtMe(L)]⁺[BF₄]⁺ (Ar = aryl; R = H, CH₃; L = water, trifluoroethanol) has been prepared. They react smoothly with benzene at approximately room temperature in trifluoroethanol solvent to yield methane and the corresponding phenyl Pt(II) cations, via Pt(IV)-methyl-phenyl-hydride intermediates. The reaction products of methyl-substituted benzenes suggest an inherent reactivity preference for aromatic over benzylic C-H bond activation, which can however be overridden by steric effects. For the reaction of benzene with cationic Pt(II) complexes, in which the diimine ligands bear 3,5-disubstituted aryl groups at the nitrogen atoms, the rate-determining step is C-H bond activation. For the more sterically crowded analogs with 2,6-dimethyl-substituted aryl groups, benzene coordination becomes rate-determining. The more electron-rich the ligand, as reflected by the CO stretching frequency in the IR spectrum of the corresponding cationic carbonyl complex, the faster the rate of C-H bond activation. This finding, however, does not reflect the actual C-H bond activation process, but rather reflects only the relative ease of solvent molecules displacing water molecules to initiate the reaction. That is, the change in rates is mostly due to a ground state effect. Several lines of evidence suggest that associative substitution pathways operate to get the hydrocarbon substrate into, and out of, the coordination sphere; i.e., that benzene substitution proceeds by a solvent- (TFE-) assisted associative pathway.

Spectroscopic studies of gas-phase molecular clusters

Spectroscopic investigations of hydrogen-bonding and van der Waals' interactions m molecular clusters were studied by the techniques of infrared predissociation and resonance-enhanced multiphoton ionization spectroscopies (REMPI). Ab initio calculations were applied in conjunction for data interpretation.

The infrared predissociation spectroscopy of CN^-•(H_2O)_n (n = 2 - 6) clusters was reported in the region of 2950 - 3850 cm^(-1). The hydrogen bondings for the C-site and N-site binding, and among the water molecules were identified for n = 2 to 4. A spectral transition was observed for n = 5 and 6, implying that the anion was surface-bound onto the water aggregates in larger clusters.

The infrared predissociation spectroscopy of Br^-•(NH_3) and I^-•(NH_3)_n (n =1-3) clusters was reported in the region of 3050-3450 cm^(-1). For the Br^-•(NH_3) complex, a dominating ionic NH stretch appeared at 3175 cm^(-1), and the weaker free NH stretch appeared at 3348 cm^(-1). The observed spectrum was consistent to the structure in which there was one nearly linear hydrogen bond between Br^- and the NH_3 moiety. For the I^- •(NH_3) complex, five distinct IR absorption bands were observed in the spectrum. The spectrum was not consistent with basic frequency patterns of three geometries considered in the ab initio calculations - complex with one, two and three hydrogen bondings between I^- and the NH_3 moiety. Substantial inhomogenous broadening were displayed in the spectra for I^-•(NH_3)_n (n =2-3), suggesting the presence of multiple isomers.

The REMPI spectroscopy of the bound 4p ^2П 1/2 and ^2П 3/2 states, and the dissociative 3d ^2Σ^+ 1/2 state in the Al•Ar complex was reported. The dissociative spectrum at Al^+ channel suggested the coupling of the 4p ^2П 1/2,3/2 states to the repulsive 3d ^2Σ^+ 1/2 state. The spin-electronic coupling was further manifested in the dissociative Al^+ spectrum of the 3d ^2Σ^+ 1/2 state. Using the potential energy curves obtained from ab initio calculations, a bound → continuum Franck-Condon-intensity simulation was performed and compared with the one-photon 3d ^2Σ^+ 1/2 profile. The agreement provided evidence for the petturbation above the Al(3d)Ar dissociation limit, and the repulsive character of the 3d ^2Σ^+ 1/2 state.

Studies of the serotonin type 3A receptor and the chemical preparation of tRNA

This thesis describes studies surrounding a ligand-gated ion channel (LGIC): the serotonin type 3A receptor (5-HT₃AR). Structure-function experiments using unnatural amino acid mutagenesis are described, as well as experiments on the methodology of unnatural amino acid mutagenesis. Chapter 1 introduces LGICs, experimental methods, and an overview of the unnatural amino acid mutagenesis.

In Chapter 2, the binding orientation of the clinically available drugs ondansetron and granisetron within 5-HT₃A is determined through a combination of unnatural amino acid mutagenesis and an inhibition based assay. A cation-π interaction is found for both ondansetron and granisetron with a specific tryptophan residue (Trp183, TrpB) of the mouse 5-HT₃AR, which establishes a binding orientation for these drugs.

In Chapter 3, further studies were performed with ondansetron and granisetron with 5-HT₃A. The primary determinant of binding for these drugs was determined to not include interactions with a specific tyrosine residue (Tyr234, TyrC2). In completing these studies, evidence supporting a cation-π interaction of a synthetic agonist, meta-chlorophenylbiguanide, was found with TyrC2.

In Chapter 4, a direct chemical acylation strategy was implemented to prepare full-length suppressor tRNA mediated by lanthanum(III) and amino acid phosphate esters. The derived aminoacyl-tRNA is shown to be translationally competent in Xenopus oocytes.

Appendix A.1 gives details of a pharmacological method for determining the equilibrium dissociation constant, K_B, of a competitive antagonist with a receptor, known as Schild analysis. Appendix A.2 describes an examination of the inhibitory activity of new chemical analogs of the 5-HT₃A antagonist ondansetron. Appendix A.3 reports an organic synthesis of an intermediate for a new unnatural amino acid. Appendix A.4 covers an additional methodological examination for the preparation of amino-acyl tRNA.

A Cerenkov-ΔΕ-Cerenkov detector for high energy cosmic ray isotopes and an accelerator study of ^(40)Ar & ^(56)Fe fragmentation

This thesis has two major parts. The first part of the thesis will describe a high energy cosmic ray detector -- the High Energy Isotope Spectrometer Telescope (HEIST). HEIST is a large area (0.25 m²sr) balloon-borne isotope spectrometer designed to make high-resolution measurements of isotopes in the element range from neon to nickel (10 ≤ Z ≤ 28) at energies of about 2 GeV/nucleon. The instrument consists of a stack of 12 NaI(Tl) scintilla tors, two Cerenkov counters, and two plastic scintillators. Each of the 2-cm thick NaI disks is viewed by six 1.5-inch photomultipliers whose combined outputs measure the energy deposition in that layer. In addition, the six outputs from each disk are compared to determine the position at which incident nuclei traverse each layer to an accuracy of ~2 mm. The Cerenkov counters, which measure particle velocity, are each viewed by twelve 5-inch photomultipliers using light integration boxes.

HEIST-2 determines the mass of individual nuclei by measuring both the change in the Lorentz factor (Δγ) that results from traversing the NaI stack, and the energy loss (ΔΕ) in the stack. Since the total energy of an isotope is given by Ε = γM, the mass M can be determined by M = ΔΕ/Δγ. The instrument is designed to achieve a typical mass resolution of 0.2 amu.

The second part of this thesis presents an experimental measurement of the isotopic composition of the fragments from the breakup of high energy ⁴⁰Ar and ⁵⁶Fe nuclei. Cosmic ray composition studies rely heavily on semi-empirical estimates of the cross-sections for the nuclear fragmentation reactions which alter the composition during propagation through the interstellar medium. Experimentally measured yields of isotopes from the fragmentation of ⁴⁰Ar and ⁵⁶Fe are compared with calculated yields based on semi-empirical cross-section formulae. There are two sets of measurements. The first set of measurements, made at the Lawrence Berkeley Laboratory Bevalac using a beam of 287 MeV/nucleon ⁴⁰Ar incident on a CH₂ target, achieves excellent mass resolution (σ_m ≤ 0.2 amu) for isotopes of Mg through K using a Si(Li) detector telescope. The second set of measurements, also made at the Lawrence Berkeley Laboratory Bevalac, using a beam of 583 MeV/nucleon ⁵⁶FeFe incident on a CH₂ target, resolved Cr, Mn, and Fe fragments with a typical mass resolution of ~ 0.25 amu, through the use of the Heavy Isotope Spectrometer Telescope (HIST) which was later carried into space on ISEE-3 in 1978. The general agreement between calculation and experiment is good, but some significant differences are reported here.

Observation and interpretation of tectonic strain release mechanisms

In four chapters various aspects of earthquake source are studied.

Chapter I

Surface displacements that followed the Parkfield, 1966, earthquakes were measured for two years with six small-scale geodetic networks straddling the fault trace. The logarithmic rate and the periodic nature of the creep displacement recorded on a strain meter made it possible to predict creep episodes on the San Andreas fault. Some individual earthquakes were related directly to surface displacement, while in general, slow creep and aftershock activity were found to occur independently. The Parkfield earthquake is interpreted as a buried dislocation.

Chapter II

The source parameters of earthquakes between magnitude 1 and 6 were studied using field observations, fault plane solutions, and surface wave and S-wave spectral analysis. The seismic moment, M_O, was found to be related to local magnitude, M_L, by log M_O = 1.7 M_L + 15.1. The source length vs magnitude relation for the San Andreas system found to be: M_L = 1.9 log L - 6.7. The surface wave envelope parameter AR gives the moment according to log M_O = log AR₃₀₀ + 30.1, and the stress drop, τ, was found to be related to the magnitude by τ = 0.54 M - 2.58. The relation between surface wave magnitude M_S and M_L is proposed to be M_S = 1.7 M_L - 4.1. It is proposed to estimate the relative stress level (and possibly the strength) of a source-region by the amplitude ratio of high-frequency to low-frequency waves. An apparent stress map for Southern California is presented.

Chapter III

Seismic triggering and seismic shaking are proposed as two closely related mechanisms of strain release which explain observations of the character of the P wave generated by the Alaskan earthquake of 1964, and distant fault slippage observed after the Borrego Mountain, California earthquake of 1968. The Alaska, 1964, earthquake is shown to be adequately described as a series of individual rupture events. The first of these events had a body wave magnitude of 6.6 and is considered to have initiated or triggered the whole sequence. The propagation velocity of the disturbance is estimated to be 3.5 km/sec. On the basis of circumstantial evidence it is proposed that the Borrego Mountain, 1968, earthquake caused release of tectonic strain along three active faults at distances of 45 to 75 km from the epicenter. It is suggested that this mechanism of strain release is best described as "seismic shaking."

Chapter IV

The changes of apparent stress with depth are studied in the South American deep seismic zone. For shallow earthquakes the apparent stress is 20 bars on the average, the same as for earthquakes in the Aleutians and on Oceanic Ridges. At depths between 50 and 150 km the apparent stresses are relatively high, approximately 380 bars, and around 600 km depth they are again near 20 bars. The seismic efficiency is estimated to be 0.1. This suggests that the true stress is obtained by multiplying the apparent stress by ten. The variation of apparent stress with depth is explained in terms of the hypothesis of ocean floor consumption.

Studies of noble gases in meteorites and in the earth

The isotopic and elemental abundances of noble gases in the solar system are investigated, using simple mixing models and mass-spectrometric measurements of the noble gases in meteorites and terrestrial rocks and minerals.

Primordial neon is modeled by two isotopically distinct components from the interstellar gas and dust. Neon from the gas dominates solar neon, which contains about ten times more ²⁰Ne than ²²Ne. Neon from the dust is represented in meteorites by neon-E, with ²⁰Ne/²²Ne less than 0.6. Isotopic variations in meteorites require neon from both dust and gas to be present. Mixing dust and gas without neon loss generates linear correlation lines on three-isotope and composition-concentration diagrams. A model for solar wind implantation predicts small deviations from linear mixing, due to preferential sputtering of the lighter neon isotopes.

Neon in meteorites consists of galactic cosmic ray spallation neon and at least two primordial components, neon-E and neon-S. Neon was measured in several meteorites to investigate these end- members. Cosmogenic neon produced from sodium is found to be strongly enriched in ²²Ne. Neon measurements on sodium-rich samples must be interpreted with care so not to confuse this source of ²²Ne with neon-E, which is also rich in ²²Ne.

Neon data for the carbonaceous chondrite Mokoia show that the end member composition of neon-Si in meteorites is ²⁰Ne/²²Ne = 13.7, the same as the present solar wind. The solar wind composition evidently has remained constant since before the compaction of Mokoia.

Ca, Al-rich inclusions from the Allende meteorite were examined for correlation between neon-E and oxygen or magnesium isotopic anomalies. ²²Ne and ³⁶Ar enrichments found in some inclusions are attributed to cosmic- ray-induced reactions on Na and Cl, not to a primordial component. Neon-E is not detectably enriched in Allende.

Measurements were made to determine the noble gas contents of various terrestrial rocks and minerals, and to investigate the cycling of noble gases between different terrestrial reservoirs. Beryl crystals contain a characteristic suite of magmatic gases including nucleogenic ²¹Ne and ²²Ne from (α,n) reactions, radiogenic ⁴⁰Ar, and fissiogenic ^131-136Xe from the decay of K and U in the continental crust. Significant concentrations of atmospheric noble gases are also present in beryl.

Both juvenile and atmospheric noble gases are found in rocks from the Skaergaard intrusion. The ratio ⁴⁰Ar/³⁶Ar (corrected for in situ decay of ⁴⁰K) correlates with δ¹⁸O in plagioclase. Atmospheric argon has been introduced into samples that have experienced oxygen-isotope exchange with circulating meteoric hydrothermal fluids. Unexchanged samples contain juvenile argon with ⁴⁰Ar/³⁶Ar greater than 6000 that was trapped from the Skaergaard magma.

Juvenile and atmospheric gases have been measured in the glassy rims of mid-ocean ridge (MOR) pillow basalts. Evidence is presented that three samples contain excess radiogenic ¹²⁹Xe and fission xenon, in addition to the excess radiogenic ⁴⁰Ar found in all samples. These juvenile gases are being outgassed from the upper-mantle source region of the MOR magma. No isotopic evidence has been found here for juvenile primordial noble gases accompanying the juvenile radiogenic gases in the MOR glasses. Large argon isotopic variations in a single specimen provide a clear indication of the late-stage addition of atmospheric argon, probably from seawater.

The Skaergaard data demonstrate that atmospheric noble gases dissolved in ground water can be transferred into crustal rocks. Subduction of oceanic crust altered by seawater can transport atmospheric noble gases into the upper mantle. A substantial portion of the noble gases in mantle derived rocks may represent subducted gases, not a primordial component as is often assumed.