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CO2 hydration and bicarbonate dehydration activity of CanB. In (A) the rates of uncatalysed and catalysed CO2 hydration and KHCO3 dehydration are shown, with 40 mM initial concentration of substrate. The enzyme (0.1 μM) was assayed in a reaction buffer of 50 mM HEPES, 50 mM MgSO4, 50 mm Na2SO4, 0.004 % (w/v) phenol red, pH 8.3 with CO2 as a substrate, and pH 6 for KHCO3 substrate at 4°C. In (B) the CO2 concentration was varied at pH 8.3, and the difference between the uncatalysed and catalysed rates determined. The data points represent the mean and SEM of at least six independent assays. The solid curve is the fit to the Michaelis-Menten equation, which gave a Km of 34 ± 10 mM.

CO2 hydration and bicarbonate dehydration activity of CanB. In (A) the rates of uncatalysed and catalysed CO2 hydration and KHCO3 dehydration are shown, with 40 mM initial concentration of substrate. The enzyme (0.1 μM) was assayed in a reaction buffer of 50 mM HEPES, 50 mM MgSO4, 50 mm Na2SO4, 0.004 % (w/v) phenol red, pH 8.3 with CO2 as a substrate, and pH 6 for KHCO3 substrate at 4°C. In (B) the CO2 concentration was varied at pH 8.3, and the difference between the uncatalysed and catalysed rates determined. The data points represent the mean and SEM of at least six independent assays. The solid curve is the fit to the Michaelis-Menten equation, which gave a Km of 34 ± 10 mM.

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Campylobacter jejuni, the leading cause of human bacterial gastroenteritis, requires low environmental oxygen and high carbon dioxide for optimum growth, but the molecular basis for the carbon dioxide requirement is unclear. One factor may be inefficient conversion of gaseous CO2 to bicarbonate, the required substrate of various carboxylases. Two p...

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... Species that need high carbon dioxide partial pressures (capnophiles) often have no detectable CA activity and some have lost CA genes (246,247). In the capnophile Campylobacter jejuni a CA was found only active at high pH, but not active under normal physiological pH (248). ΔCA mutants often can only grow under high carbon dioxide partial pressures (220,249,250) making them functional capnophiles. ...
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... Supporting this, even epsilonproteobacterial genera that associate with terrestrial animals, such as Campylobacter, Helicobacter and Wolinella, retain physiological characteristics reflective of a hydrothermal environment where they most likely evolved. These include a need for high CO 2 (Al-Haideri et al., 2016), intolerance to high levels of O 2 (Kendall et al., 2014), and the ability to use hydrogen as an electron donor (Wolin et al., 1961). Below, the underlying geochemistry of deep-sea vents and its effect on the growth conditions of the microbes inhabiting this habitat are discussed. ...
... Another important and poorly understood phenomenon is the capnophilic (CO 2 -loving) nature of Epsilonproteobacteria. As mentioned above, heterotrophic Campylobacter strains require high levels of CO 2 (≈ 10% v/v in headspace gas), which may be related to the activity of carbonic anhydrases that convert CO 2 to bicarbonate for carboxylation reactions (Al-Haideri et al., 2016). Previous studies have shown that oxygen tolerance may be enhanced when CO 2 above atmospheric levels is provided (Bolton and Coates, 1983). ...
Thesis
Chemoautotrophic ecosystems at deep-sea hydrothermal vents were discovered in 1977, but not until 1995 were free-living autotrophic Epsilonproteobacteria identified as important microbial community members. Because the deep-sea is food-starved, the autotrophic metabolism of hydrothermal vent Epsilonproteobacteria may be very important for deep-sea consumers. However, quantifying their metabolic activities in situ has remained difficult, and biochemical mechanisms underlying their autotrophic physiology are poorly described. To gain insight into environmental processes, an approach was developed for incubations of microbes at in situ pressure and temperature (25 MPa, 24°C) with various combinations of electron donors/acceptors (H₂ , O₂ and NO₃- and ¹³HCO₃-) as a tracer to track carbon fixation. During short (18-24 h) incubations of low-temperature vent fluids from Crab Spa (9°N East Pacific Rise), the concentration of electron donors/acceptors and cell numbers were monitored to quantify microbial processes. Measured rates were generally higher than previous studies, and the stoichiometry of microbially-catalyzed redox reactions revealed new insights into sulfur and nitrogen cycling. Single-cell, taxonomically-resolved tracer incorporation showed Epsilonproteobacteria dominated carbon fixation, and their growth efficiency was calculated based on electron acceptor consumption. Using these data, in situ primary productivity, microbial standing stock, and average biomass residence time of the deep-sea vent subseafloor biosphere were estimated. Finally, the population structures of the most abundant genera Sulfurimonas and Thioreductor were shown to be strongly influenced by pO₂ and temperature respectively, providing a mechanism for niche differentiation in situ. To gain insights into the core biochemical reactions underlying autotrophy in Epsilonprotebacteria, a theoretical metabolic model of Sulfurimonas denitrificans was developed. Validated iteratively by comparing in silico yields with data from chemostat experiments, the model generated hypotheses explaining critical, yet so far unresolved reactions supporting chemoautotrophy in Epsilonproteo bacteria. For example, it provides insight into how energy is conserved during sulfur oxidation coupled to denitrification, how reverse electron transport produces ferredoxin for carbon fixation, and why aerobic growth yields are only slightly higher compared to denitrification. As a whole, this thesis provides important contributions towards understanding core mechanisms of chemoautrophy, as well as the in situ productivity, physiology and ecology of autotrophic Epsilonproteobacteria.