The thermoalkaliphile Caldalkalibacillus thermarum possesses a highly branched respiratory chain. These primarily facilitate growth at a wide range of dissolved oxygen levels. The aim of this study was to investigate the regulation of C. thermarum respiratory chain. C. thermarum was cultivated in chemostat bioreactors with a range of oxygen levels (0.25% O2–4.2% O2). Proteomic analysis unexpectedly showed that both the type I and the type II NADH dehydrogenase present are constitutive. The two terminal oxidases detected were the cytochrome c:oxygen aa3 oxidase, whose abundance was highest at 4.2% O2. The cytochrome c:oxygen ba3 oxidase was more abundant at most other O2 levels, but its abundance started to decline below 0.42% O2. We expected this would result in the emergence of the cytochrome c:oxygen bb3 complex or the menaquinol:oxygen bd complex, the other two terminal oxidases of C. thermarum; but neither was detected. Furthermore, the sodium-proton antiporter complex Mrp was downregulated under the lower oxygen levels. Normally, in alkaliphiles, this enzyme is considered crucial for sodium homeostasis. We propose that the existence of a sodium:acetate exporter decreases the requirement for Mrp under strong oxygen limitation.@en

Alkaliphiles thrive in environments with a pH of 8.5 or above, while maintaining an internal pH closer to neutral. Thus, alkaliphilic microorganisms have a proton gradient inverted with respect to the normal orientation. Intuitively, this would nullify the potential to generate energy via respiration with regularly oriented respiratory chains that rely on proton-coupled ATP synthases. Yet, alkaliphilic respiratory chains are oriented traditionally and are actively used. The question therefore is how they are able to create conditions conducive to such behaviour. In addition, attempts to answer that question will hopefully also clarify how alkaliphiles acidify their cytoplasm with respect to the exterior milieu in the first place. This thesis details methods required to study these questions and provides some answers regarding alkaliphilic life. This thesis focuses on a single category of alkaliphiles: the low-salt gram positive alkaliphiles. These microbes have just a single membrane, the proteins therein, and a cell wall to generate conditions suitable for energy generation and other transport mechanisms. In short, it can be regarded as the most basic system to study an alkaline, or basic, problem....@en

Proteomics has greatly advanced the understanding of the cellular biochemistry of microorganisms. The thermoalkaliphile Caldalkalibacillus thermarum TA2.A1 is an organism of interest for studies into how alkaliphiles adapt to their extreme lifestyles, as it can grow from pH 7.5 to pH 11. Within most classes of microbes, the membrane-bound electron transport chain (ETC) enables a great degree of adaptability and is a key part of metabolic adaptation. Knowing what membrane proteins are generally expressed is crucial as a benchmark for further studies. Unfortunately, membrane proteins are the category of proteins hardest to detect using conventional cellular proteomics protocols. In part, this is due to the hydrophobicity of membrane proteins as well as their general lower absolute abundance, which hinders detection. Here, we performed a combination of whole cell lysate proteomics and proteomics of membrane extracts solubilised with either SDS or FOS-choline-12 at various temperatures. The combined methods led to the detection of 158 membrane proteins containing at least a single transmembrane helix (TMH). Within this data set we revealed a full oxidative phosphorylation pathway as well as an alternative NADH dehydrogenase type II (Ndh-2) and a microaerophilic cytochrome oxidase ba₃. We also observed C. thermarum TA2.A1 expressing transporters for ectoine and glycine betaine, compounds that are known osmolytes that may assist in maintaining a near neutral internal pH when the external pH is highly alkaline.

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It is a conjecture that the ε subunit regulates ATP hydrolytic function of the F1Fo ATP synthase in bacteria. This has been proposed by the ε subunit taking an extended conformation, with a terminal helix probing into the central architecture of the hexameric catalytic domain, preventing ATP hydrolysis. The ε subunit takes a contracted conformation when bound to ATP, thus would not interfere with catalysis. A recent crystallographic study has disputed this; the Caldalkalibacillus thermarum TA2.A1 F1Fo ATP synthase cannot natively hydrolyse ATP, yet studies have demonstrated that the loss of the ε subunit terminal helix results in an ATP synthase capable of ATP hydrolysis, supporting ε subunit function. Analysis of sequence and crystallographic data of the C. thermarum F1Fo ATP synthase revealed two unique histidine residues. Molecular dynamics simulations suggested that the protonation state of these residues may influence ATP binding site stability. Yet these residues lie outside
the ATP/Mg2+ binding site of the ε subunit. We then probed the effect of pH on the ATP binding affinity of the ε subunit from the C. thermarum F1Fo ATP synthase at various physiologically relevant pH values. We show that binding affinity changes 5.9 fold between pH 7.0, where binding is weakest, to pH 8.5 where it is strongest. Since the C. thermarum cytoplasm is pH 8.0 when it grows optimally, this correlates to the ε subunit being down due to ATP/Mg2+ affinity, and not being involved in blocking ATP hydrolysis. Here, we have experimentally correlated that the pH of the bacterial cytoplasm is of critical importance for ε subunit ATP affinity regulated by second shell residues thus the function of the ε subunit changes with growth conditions.@en

The aerobic thermoalkaliphile Caldalkalibacillus thermarum strain TA2.A1 is a member of a separate order of alkaliphilic bacteria closely related to the Bacillales order. Efforts to relate the genomic information of this evolutionary ancient organism to environmental adaptation have been thwarted by the inability to construct a complete genome. The existing draft genome is highly fragmented due to repetitive regions, and gaps between and over repetitive regions were unbridgeable. To address this, Oxford Nanopore Technology’s MinION allowed us to span these repeats through long reads, with over 6000-fold coverage. This resulted in a single 3.34 Mb circular chromosome. The profile of transporters and central metabolism gives insight into why the organism prefers glutamate over sucrose as carbon source. We propose that the deamination of glutamate allows alkalization of the immediate environment, an excellent example of how an extremophile modulates environmental conditions to suit its own requirements. Curiously, plant-like hallmark electron transfer enzymes and transporters are found throughout the genome, such as a cytochrome b₆c₁ complex and a CO₂-concentrating transporter. In addition, multiple self-splicing group II intron-encoded proteins closely aligning to those of a telomerase reverse transcriptase in Arabidopsis thaliana were revealed. Collectively, these features suggest an evolutionary relationship to plant life.

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