Caldalkalibacillus thermarum

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1. Classification

a. Higher order taxa

Domain: Bacteria Phylum: Bacillota (formerly Firmicutes) Class: Bacilli Order: Bacillales Family: Bacillaceae (Schoch et al., 2020)

2. Description and significance

Caldalkalibacillus thermarum is an extremophile isolated from hot springs in China that has potential biotechnological applications, such as bleaching of pulp, stabilization of beverages, and bioremediation of real and synthetic dyes (Xue et al., 2006; Ghatge et al., 2018). C. thermarum has never been identified as a human pathogen, classified as BioSafety Level 1 organism (JCM). Current research focuses on the structure of C. thermarum’s non-proton pumping type II NADH dehydrogenase (NDH-2), which is not well understood, to advance its potential as a drug target for microbial pathogens (Heikal et al., 2014). A novel laccase from C. thermarum (CtLac) has exceptional stability at high temperatures and alkaline pH and holds promise for industrial applications, specifically delignification and detoxification of lignocellulosic biomass where high thermal and alkaline tolerance are critical (Kalamorz et al., 2011; De Jong et al., 2020; Ferguson et al., 2016).

3. Genome structure

The genome of the C. thermarum strain TA2.A1 consists of a a circular chromosome (De Jong et al., 2020) that is approximately 3.34 Mb, with 3,652 coding sequences (CDSs) and a GC content of 47.5% (Kalamorz et al., 2011). Genes encode F1Fo-ATP synthase modified to support efficient proton translocation under high pH (De Jong et al., 2020; Ferguson et al., 2016). The genome also contains genes homologous to plant photorespiration and ROS-detoxification pathways, including catalase, superoxide dismutase, and glutathione peroxidase. Superoxide dismutase is responsible for the survival of C. thermarum in high oxygen conditions (De Jong et al., 2020). The genome of C. thermarum TA2.A1 includes a suite of thirteen ltrA genes, which code for self-splicing group II intron-encoded proteins with reverse transcriptase, maturase, and endonuclease activities (De Jong et al., 2020). The reverse transcriptase activity of these ltrA proteins shows 53% query alignment to the TERT gene of Arabidopsis thaliana, suggesting potential evolutionary links to eukaryotic telomerase. Comparison with other Bacilli shows higher ltrA copy numbers in alkaliphilic taxa, with the C. thermarum ltrA sequence more similar to plant TERT than the neutrophilic B. subtilis version (De Jong et al., 2020).​ For defense against phages, C. thermarum has nine CRISPR-associated genes: three copies each of Cas1 and Cas2, two of Cas3, and one of Cas9 (De Jong et al., 2020). This corresponds to two type I and one type II CRISPR systems, which together form an adaptive immune mechanism that allows bacteria to recognize and destroy viral DNA. The genome also encodes six copies of the chromosome-partitioning gene smc, at least three operons for spore germination (gerABC and yndE), and three complete copies of the pdhABCD operon for pyruvate dehydrogenase[1] , a key entry molecule to the citric acid cycle (De Jong et al., 2020). There are multiple sets of the gsiBCD transporter genes (glutathione import), though only one forms a functional operon, suggesting possible alternative or backup roles. Similar incomplete duplications exist for other transporters; C4-dicarboxylate, lactose, arabinose, and trehalose have multiple import gene sets, as well as for DNA polymerase III, where both unique and triple-copy genes occur (De Jong et al., 2020). While transporter degeneracy is common among Bacilli, the repeated copies for spore germination and chromosomal partitioning are unusual in this lineage.​ Additionally, C. thermarum encodes ccmL and ccmM genes usually associated with CO2 capture in photosynthetic organisms, with unclear function in C. thermarum (De Jong et al., 2020). C. thermarum lacks genes for strictly anaerobic mesaconate pathways and instead likely feeds glutamate directly into the TCA cycle via a glutamate dehydrogenase with deaminase activity.​

4.Cell structure

C. thermarum is a thermophilic, alkaliphilic[1] [JB2] , Gram-positive, catalase-positive, motile, spore-forming bacterium with a distinctive thin, rod cellular shape. Cells are about 0.5 micrometers wide and 3.0 to 6.5 micrometers long, typical for members of the Bacillaceae family (Xue et al., 2006). The bacterium has 158 cell surface proteins, including enzymes Ndh-1, cytochrome complex b6c1, cytochrome c, Sdh, and cytochrome oxidase ba3, allowing for efficient aerobic respiration (De Jong et al., 2023). The cell wall is composed of peptidoglycan with meso-diaminopimelic acid, which increases the cell’s rigidity (Xue et al., 2006). C. thermarum also expresses a surface nitrile hydratase enzyme (NHCTA1) that converts nitrile organic compounds into amide organic compounds (Shen et al., 2021). C. thermarum has voltage-gated sodium channels alternating between two conformational structures (Mol A and Mol B), allowing the bacterium to engage in pH homeostasis and motility maintenance (Tsai et al., 2013).

5. Metabolic processes

C. thermarum is an obligate aerobe and chemoorganoheterotroph, oxidizing organic compounds for energy and electron source. It can utilize several environmental carbon sources including D-glucose, sucrose, mannose, pyruvate, citrate, and succinate (Xue et al., 2006). C. thermarum has both type-I and type-II NADH hydrogenases in its electron transport system. A key F1F0 ATPase works in conjunction with the NADH hydrogenases to synthesize ATP under aerobic conditions (De Jong et al., 2024). This F1-ATPase creates hydrolysis products like lauryldimethylamine oxide that regulates the amount of ATP hydrolysis the bacterium engages in, allowing it to adjust to aerobic or anaerobic conditions (Ferguson et al., 2016).

C. thermarum produces laccase, an enzyme that carries out the oxidation of phenolic compounds and dimerization with a guaiaglycerol-beta-guaciyl ether (GGGE) to form a tetramer that enhances cell wall rigidity and thermostability for survival at extremely high temperatures (Ghatge et al., 2018). C. thermarum also synthesizes a non-proton pumping type II NADH dehydrogenase (NDH-2) that is essential to its respiration. NDH-2 is associated with the cytoplasmic side of the membrane by two separated C-terminal membrane-anchoring regions that play a central role in FAD binding (Heikal et al., 2014).

6. Ecology

C. thermarum is a thermophile and alkaliphile, as it grows optimally at a temperature of 60°C, salinity level of 1.5% NaCl, and pH of 8.5. C. thermarum inhabits alkaline hotsprings, where nutrient supplies are limited and roles of the microbial community are poorly understood. The geographic distribution of C. thermarum includes the geothermal springs in China (Xue et al., 2006). As a strictly aerobic bacterium, C. thermarum likely contributes to organic carbon oxidation in hot alkaline ecosystems (Xue et al., 2006).

7. Pathology

Unlike some relatives in the family Bacillaceae, C. thermarum has never been identified as a human pathogen in case studies or clinical isolation. C. thermarum cannot grow at 37 °C (Xue et al., 2006), such that it would not survive or multiply inside a warm-blooded host. Research on C. thermarum has focused on its extremophilic metabolism, enzymes, and evolutionary interest rather than disease association (Xue et al., 2006; De Jong et al., 2020). The German Collection of Microorganisms and Cell Cultures assigns C. thermarum to Risk Group 1, meaning it is not associated with disease in healthy adults (LPSN). Consistently, the Japan Collection of Microorganisms lists this strain as Biosafety Level 1 at the lowest risk level (JCM).

8. Current Research

Recent studies on C. thermarum have focused on its membrane proteome to understand how it produces energy and its genomic adaptations to survive in hot alkaline environments (De Jong et al., 2023; De Jong et al., 2020; De Jong et al., 2024). Proteomic analyses have revealed sodium- coupled transporters work in conjunction with proton pumps to maintain cell function in hot environments. The two types of sodium-proton antiporters pump sodium ions and protons in opposite directions across the membrane to maintain the homeostasis of monovalent cations,crucial for any alkaliphile (De Jong et al., 2023). Because free protons are limited in alkaline environments, C. thermarum relies more on sodium gradients to supplement the proton motive force and sustain ATP synthesis. The sodium-proton antiporter is downregulated in response to low oxygen availability and due to its partial involvement as an acetate exporter, as high concentrations of acetate are produced under low oxygen conditions. Oxygen limitation triggers a broader metabolic shift toward pathways that reduce sodium cycling while maintaining energy production. By adjusting which membrane proteins are active, the bacterium can respond to changes in oxygen levels and maintain efficient energy production (De Jong et al., 2024).

Further genomic investigations of C. thermarum reveal its genome includes a cytochrome b 6 c 1 complex and a CO 2 -concentrating transporter, similar to those found in plants. Comparative genomic analysis suggests an evolutionary link between alkaline adaptation in C. thermarum and distantly related organisms that have developed comparable energy generating systems. In alkaline conditions, the scarcity of free protons results in cells primarily utilizing the potential energy stored in their membrane potential to drive oxidative phosphorylation (De Jong et al., 2020). The genes for ATP synthase are modified to work efficiently in proton-poor alkaline environments (De Jong et al., 2020; Kalamorz et al., 2011).

References

De Jong, S. I., Sorokin, D. Y., van Loosdrecht, M. C. M., Pabst, M., and McMillan, D. G. G. 2023. Membrane proteome of the thermoalkaliphile Caldalkalibacillus thermarum TA2.A1. Frontiers in Microbiology 14:1228266.

De Jong, S. I., Van den Broek, M. A., Merkel, A. Y., de la Torre Cortes, P., Kalamorz, F., Cook,

G. M., van Loosdrecht, M. C. M., and McMillan, D. G. G. 2020. Genomic analysis of Caldalkalibacillus thermarum TA2.A1 reveals aerobic alkaliphilic metabolism and evolutionary hallmarks linking alkaliphilic bacteria and plant life. Extremophiles 24: 923–935.

De Jong, S. I., Wissink, M., Yildirim, K., Pabst, M., van Loosdrecht, M. C. M., and McMillan, D. G. G. 2024. Quantitative proteomics reveals oxygen-induced adaptations in Caldalkalibacillus thermarum TA2.A1 microaerobic chemostat cultures. Frontiers in Microbiology 15:1468929.

Ferguson, S. A., Cook, G. M., Montgomery, M. G., Leslie, A. G. W., and Walker, J. E. 2016. Regulation of the thermoalkaliphilic F1-ATPase from Caldalkalibacillus thermarum. PNAS 113:10860–10865.

Ghatge, S., Yang, Y., Song, W. Y., Kim, T. Y., and Hur, H. G. 2018. A novel laccase from thermoalkaliphilic bacterium Caldalkalibacillus thermarum strain TA2.A1 able to catalyze dimerization of a lignin model compound. Applied Microbiology and Biotechnology 102:4075–4086.

Heikal, A., Nakatani, Y., Dunn, E., Weimar, M. R., Day, C. L., Baker, E. N., Lott, J. S., Sazanov, L. A., and Cook, G. M. 2014. Structure of the bacterial type II NADH dehydrogenase: a monotopic membrane protein with an essential role in energy generation. Molecular Microbiology 91:950–964.

Japan Collection of Microorganisms (JCM). Caldalkalibacillus thermarum. RIKEN BioResource Research Center.

Kalamorz, F., Keis, S., McMillan, D. G. G., Olsson, K., Stanton, J.-A., Stockwell, P., Black, M. A., and Cook, G. M. 2011. Draft genome sequence of the thermoalkaliphilic Caldalkalibacillus thermarum strain TA2.A1. Journal of Bacteriology 193:4290–4291.

List of Prokaryotic names with Standing in Nomenclature (LPSN). Species: Caldalkalibacillus thermarum. Leibniz Institute DSMZ–German Collection of Microorganisms and Cell Cultures.

Schoch CL, et al. NCBI Taxonomy: a comprehensive update on curation, resources and tools. Database (Oxford). 2020: baaa062.

Shen, J. D., Cai, X., Ni, Y. W., Jin, L. Q., Liu, Z. Q., and Zheng, Y. G. 2021. Structural insights into the thermostability mechanism of a nitrile hydratase from Caldalkalibacillus thermarum by comparative molecular dynamics simulation. Proteins 89:978–987.

Tsai, C.-J., Tani, K., Irie, K., Hiroaki, Y., Shimomura, T., McMillan, D. G. G., Cook, G. M., Schertler, G. F. X., Fujiyoshi, Y., and Li, X.-D. 2013. Two alternative conformations of a voltage-gated sodium channel. Journal of Molecular Biology 425:4074–4088.

Xue, Y., Zhang, X., Zhou, C., Zhao, Y., Cowan, D. A., Heaphy, S., Grant, W. D., Jones, B. E., Ventosa, A., and Ma, Y. 2006. Caldalkalibacillus thermarum gen. nov., sp. nov., a novel alkalithermophilic bacterium from a hot spring in China. International Journal of Systematic and Evolutionary Microbiology 56:1217–1221.

Edited by Jaeho Kim, Joshua Park, Elijah Porras, Beamlak Mideksa, Tran Phan, student of Jennifer Bhatnagar for BI 311 General Microbiology, 2024, Boston University.