This thesis describes the pathway of sulphur metabolism in Paracoccus denitrificans (NCIB 89MJ-) The compounds involved in the sulphur metabolic pathway were determined in growth experiments (Chapter 2) and time-course and pulse-chase experiments, using radioactively labelled sulphate (Chapter 3). P. denitrificans is an assimilatory sulphate-reducing organism with a pathway of sulphate reduction involving in- organic intermediates. P. denitrificans can not utilise cysteine as a sole sulphur; cysteine inhibits amino acid metabolism at low concentrations and respiration at higher concentrations. Sulphate is taken up by P. denitrificans against a concentration gradient (Chapter 4.) . The mechanism of sulphate uptake was investigated using right side out and inside out membrane vesicles prepared from P. denitrificans. The uptake mechanism involves an uncoupler-sensitive transport mechanism driven either by respiration, or by a transmembrane pH gradient (alkaline inside). The active transport of sulphate was shown to be carrier-mediated, by its sensitivity to sulphydryl-group reagents. It is proposed that the sulphate carrier operates by a mechanism of electroneutral proton symport, and is capable of transporting sulphate in either direction across the plasma membrane (Chapter 5). ATP Sulphurylase, the initial enzyme involved in the activation of sulphate, was purified, and its kinetic and regulatory properties investigated (Chapter 6). ATP Sulphurylase activity was repressed by sulphite, sulphide and cysteine, and inhibited by sulphide. Accumulation of APS, the end-product of the ATP sulphurylase-catalysed reaction, could only be detected in the presence of inorganic pyrophosphatase, an enzyme which removed pyrophosphate, another end-product of sulphate activation. Inorganic pyrophosphatase was purified and its substrate specificity, kinetics and regulatory properties examined, in relation to its part in sulphate metabolism (Chapter 7). Inorganic pyrophosphatase is a constitutive enzyme which functions equally well with either Mg²⁺ or Co²⁺ as the cofactor. APS Kinase activity was detected in crude extracts of P. denitrificans. A new assay is described for measuring APS kinase activity (Chapter 8). APS Kinase was purified. Coupled enzyme assays, with purified ATP sulphurylase, inorganic pyrophosphatase and APS kinase, indicated that all three enzymes were necessary for the synthesis, and accumulation of PAPS (Chapter 8). No 3andprime;-nucleotidase or enzyme "A" activity was detected. Serine transacetylase and O-acetyl serine sulphydrylase were purified and the kinetics and regulation of these two enzymes, investigated (Chapter 9). O-Acetyl serine lyase activity was detected in crude extracts of P. denitrificans, representing the first report of this enzyme in bacteria (Chapter 9). β-Cystathionase was purified and its kinetic and regulatory properties investigated; the unidirectionality of the cysteine to methionine pathway was confirmed (Chapter 10). Cysteinyl- and methionyl-tRNA synthetases were purified and the kinetics and regulation of these enzymes studied (Chapters 11 and 12 respectively). Both these enzymes possess different substrate specificities to the aminoacyl-tRNA synthetases from other organisms. Both enzymes appear to be constitutive. During this investigation of the sulphur metabolism of P. denitrificans, the substrate specificity of the different enzymes, to the selenium analogues of the respective sulphur-containing substrates, were investigated. Selenate competitively inhibits sulphate uptake and ATP sulphurylase, with respect to sulphate, but no APSe or PAPSe synthesis could be detected in the coupled enzyme assays (Chapters 7 and 8). Purified O-acetyl serine sulphydrylase catalysed the synthesis of selenocysteine from selenide and O-acetyl serine (Chapter 9). Both selenocysteine and selenomethionine are activated by the respective aminoacyl-tRNA synthetase (Chapters 11 and 12, respectively).

Thesis (Ph. D.)--Leland Stanford Junior University, 1916.

Graduation date: 1998

The goal of this research was to mathematically simulate the ability of bioaugmented microorganisms to aerobically cometabolize a mixture of chlorinated aliphatic hydrocarbon (CAH) compounds during in-situ treatment. Parameter values measured from laboratory experiments were applied to the transport model with biotransformation processes included. In laboratory microcosm studies, a butane-grown, enriched culture was inoculated in soil and groundwater microcosms and exposed to butane and several repeated additions of 1,1,1-trichloroethane (TCA), 1,1-dichioroethylene (DCE), and 1,1-dichloroethane (DCA) at aqueous concentrations of 200 ��g/L, 100 ��g/L, and 200 ��g/L, respectively. Microcosms containing the bioaugmented culture showed 1,1-DCE to be rapidly transformed, followed by slower transformation of 1,1-DCA and 1,1,1-TCA. After most of the butane had been consumed, transformation of these latter CAHs increased, indicating strong inhibition by butane. With repeat biostimulations, butane utilization and CAH transformation accelerated, showing the increase in cell mass. These trends occurred in two sets of microcosm triplicates. No stimulation was observed in controls containing only the microorganisms indigenous to Moffett Field, confirming that activity seen in the bioaugmented microcosms was a result of the introduced culture's activity. Batch reactor results were simulated using differential equations accounting for Michaelis-Menten kinetics, transformation product toxicity, substrate inhibition, butane utilization, and CAH transformation. The equations were solved simultaneously by Runge-Kutta numerical integration with parameter values adjusted to match the microcosm data. Having defined the parameter values from laboratory studies, the biotransformation model was combined with 1-D advective-dispersive transport to simulate behavior of the culture and the substrates within an aquifer. The model was used to simulate the results of field studies where the butane-utilizing culture was injected into a 7 m subsurface test site and exposed to alternating pulses of oxygen and butane, along with the contaminant mixture studied in the microcosms. Monitoring wells spaced at 1 m, 2.2 m, and 4 m from the injection well allowed temporal and spatial changes in substrate concentrations to be determined. Model simulations of the field demonstration were performed to determine how well the biotransformation/solute transport model predicted actual field observations. To model the influences of solute transport, simulations were run and compared to breakthrough test data (prior to bioaugmentation) to determine the values for advection, dispersion, and sorption. The simulations showed that flow ranged from 1.0 to 1.5 m��/day (average linear velocity of 2.0 m/day). Dispersion was estimated as 0.31 m��/day. Sediment sorption partitioning coefficients for 1,1-DCE, 1,1-DCA, and 1,1,1-TCA were determined to be approximately 0.69, 0.50, and 0.50 L/kg, respectively. It was more difficult to determine an appropriate value of the mass transfer rate coefficient for non-equilibrium sorption, so simulations were run to compare equilibrium and non-equilibrium cases. Results indicated that non-equilibrium (with mass transfer rate coefficient of approximately 0.2 day�����) better simulated the field data. Using these transport parameters and the biotransformation values determined from the laboratory experiments, simulations of the field data showed that the model was capable of simulating the effects of transformation rates, butane inhibition, and 1,1-DCE product toxicity. Simulations for varying pulsing cycles and durations provided possible improvements for future field demonstrations. Overall, this work proved that there is good potential in extrapolating laboratory based kinetics to simulate biotransformation at a field scale. Although the complexity of such systems makes modeling difficult, such simulations are useful in understanding and interpreting field data. / Graduation date: 2003

Graduation date: 1996

Five strains (9L, 642A, 714, 5688T and 3384Y) of Histophilus ovis were investigated with respect to iron acquisition. All strains used ovine, bovine and goat, but not porcine or human, transferrins (Tfs) as iron sources for growth. In solid phase binding assays, total membranes from only two (9L and 642A) of the five strains, grown under iron-restricted conditions, were able to bind Tfs (ovine, bovine and goat, but not porcine or human). However, when the organisms were grown under iron-restricted conditions in the presence of bovine Tf, total membranes from all strains exhibited Tf binding (as above); competition experiments demonstrated that all three Tfs (ovine, bovine and goat) were bound by the same receptor(s). An affinity isolation procedure allowed the isolation of two putative Tf-binding polypeptides (78 and 66 kDa) from total membranes of strains 9L and 642A grown under iron-restricted conditions, and from membranes of all strains if the growth medium also contained Tf. A gene encoding a Pasteurella multocida TbpA homologue was shown to be present in each of two representative strains (9L and 3384Y); these genes were sequenced and determined to be the structural genes encoding the 78-kDa Tf-binding polypeptides. The identification of a fur homologue and a Fur box within the promoter region of tbpA in both strains indicated that Fur (and iron) is responsible for the iron-repressible nature of Tf-binding activity. Although tbpA transcripts were detected by reverse transcription (RT)-PCR with RNA isolated from strains 9L and 3384Y grown under iron-restricted conditions, with strain 3384Y, and depending on the primer pair, tbpA transcripts were detected by RT-PCR predominantly when the RNA was isolated from cells grown under conditions of iron-restriction in the presence of Tf. The presence of an additional G in the tbpA gene of strain 3384Y grown under iron-replete conditions, compared to organisms grown under iron-restricted conditions plus bovine Tf, is

No description available.

Graduation date: 1998

Ovine (strains 9L and 3384Y) and bovine (strains 649, 2336 and 8025) isolates of Histophilus somni were investigated for their ability to acquire iron from haemoglobin (Hb). Bovine isolates were capable of utilizing bovine, but not ovine, porcine or human Hb as a source of iron. Ovine isolates could not obtain iron from Hb. Bovine isolates bound bovine, ovine, and human Hbs by means of the same iron-repressible receptor(s) and produced a ~120-kDa iron-repressible, outer membrane protein. Using PCR approaches, an iron-regulated operon containing hugX and hugZ homologues and a gene (hgbA) that encodes a TonB-dependent, Hb-binding proteins were identified in strains 649, 9L and 3384Y. In strains 9L and 3384Y, HgbA is truncated offering a possible explanation for their lack of utilization of Hb as an iron source. In strains 2336 and 8025, expression of HgbA was also subject to a form of phase variation.

No description available.