Morgan, Hugh W.Janssen, P.H.Cook, Gregory Murray2026-07-072026-07-071992https://hdl.handle.net/10289/18432Five extremely thermophilic organisms representing the Eubacterial and Archaebacterial Kingdoms were studied for controlled expression of β-galactosidase, β-glucosidase and α-glucosidase synthesis. Under the culture conditions used, these organisms exhibited a high constitutive level of enzyme synthesis which was modulated in the presence of added carbohydrate. The lack of observed classical induction in these organisms could either reflect a lack of control or a less elaborate control of enzyme synthesis, which may be advantageous to these bacteria in environments low in nutrients. This absence of highly regulated control is common in thermophilic organisms and may be a reflection of the limited evolutionary divergence of thermophiles as is suggested by phylogenetic studies. Cl. thermohydrosulfuricum Rt8.B1 inhabits thermophilic environments where the natural concentrations of sugars would be expected to be quite low. The results of this study demonstrated that glucose and xylose were used simultaneously i.e. the bacterium exhibited hyperbolic growth when both glucose and xylose were supplied together at nonlimiting concentrations. Under conditions of hyperbolic growth, Cl. thermohydrosulfuricum Rt8.B1 exhibited neither catabolite repression nor inducer exclusion. Although classical control mechanisms were not seen, the utilization of glucose and xylose were tightly controlled at both low and high concentrations of single and multiple substrates. Such regulation to allow controlled hyperbolic growth is consistent with the idea of Cl. thermohydrosulfuricum Rt8.B1 being well adapted as an opportunist. The transport of glucose and xylose across the bacterial cell membrane of Cl. thermohydrosulfuricum Rt8.B1 was governed by permeases which did not catalyze concomitant substrate transport and phosphorylation, and thus was not a phosphoenolpyruvate-dependent carbohydrate: phosphotransferase system. Glucose and xylose transport was not driven by a proton motive force (ΔμH⁺) nor coupled to sodium and potassium ion gradients. An involvement of ATP in the uptake process was indicated by the reduction of glucose and xylose uptake by iodoacetate and sodium fluoride, both inhibitors of ATP synthesis. The phosphorylation of glucose was carried out by a constitutive ATP-dependent glucokinase and that of xylose by an inducible ATP-dependent xylulokinase following isomerization by xylose isomerase. These enzymes not only initiated the metabolism of glucose and xylose in Cl. thermohydrosulfuricum Rt8.B1, but also served to “capture” sugars from the cytoplasm through their phosphorylation. Since glucokinase was regulated primarily by the concentration of ATP, ADP, and AMP, both glucose phosphorylation and uptake appeared to be dependent upon the energy status of the cell. Xylulose inhibited glucokinase activity, indicating that xylose metabolism may regulate the activity of glucokinase in glucose and xylose-grown cells. Xylose utilization by Cl. thermohydrosulfuricum Rt8.B1 was mediated by two systems. Both systems were inducible and dependent on the xylose concentration used for cell growth. Cells grown on 5 mM xylose had a high-affinity, low-capacity system for xylose uptake which was saturable (low Vₘₐₓ). Cells grown on 50 mM xylose had a low-affinity, high-capacity (high Vₘₐₓ) system for xylose uptake and the kinetics were indicative of facilitated diffusion. The xylose isomerase of Cl. thermohydrosulfuricum Rt8.B1 had a high Kₘ for xylose (low-affinity) and xylulokinase had a low Kₘ for xylose (high-affinity). The Kₘ for xylulokinase was lower than the Kₜ for the high-affinity xylose permease; this indicated that the xylulokinase phosphorylating system was fully saturated at external xylose concentrations near or above the Kₜ concentration for uptake and that the affinity of xylulokinase regulated both the rate of xylose uptake and its subsequent utilization. When Cl. thermohydrosulfuricum Rt8.B1 was grown on high concentrations of xylose (low-affinity), xylulose leaked from the cell. This indicated that a futile cycle for xylose may be operative under conditions of high xylose and may explain the facilitated diffusion kinetics observed. A semi-defined minimal medium for the growth of Clostridium thermohydrosulfuricum Rt8.B1 was developed in this study. This medium was able to support the growth of several thermophilic anaerobes. Most strains studied showed morphological changes. In one instance, spores were seen in an apparently nonsporulating strain of Thermoanaerobium brockii DSM 1457 which could not be attributed to a contaminant. This observation has resulted in the species being renamed Thermoanaerobacter brockii comb. nov. and the genus Thermoanaerobium being removed due to the removal of the type strain.enAll items in Research Commons are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.Thesis