Structural insights into thermophilic chaperonin complexes.

Group I chaperonins are dual heptamer protein complexes that play significant roles in protein homeostasis. The structure and function of the Escherichia coli chaperonin are well characterized. However, the dynamic properties of chaperonins, such as large ATPase-dependent conformational changes by binding of lid-like co-chaperonin GroES, have made structural analyses challenging, and our understanding of these changes during the turnover of chaperonin complex formation is limited. In this study, we used single-particle cryogenic electron microscopy to investigate the structures of GroES-bound chaperonin complexes from the thermophilic hydrogen-oxidizing bacteria Hydrogenophilus thermoluteolus and Hydrogenobacter thermophilus in the presence of ATP and AMP-PNP. We captured the structure of an intermediate state chaperonin complex, designated as an asymmetric football-shaped complex, and performed analyses to decipher the dynamic structural variations. Our structural analyses of inter- and intra-subunit communications revealed a unique mechanism of complex formation through the binding of a second GroES to a bullet-shaped complex.

Unlocking microalgal host-exploring dark-growing microalgae transformation for sustainable high-value phytochemical production.

Microalgae have emerged as a promising, next-generation sustainable resource with versatile applications, particularly as expression platforms and green cell factories. They possess the ability to overcome the limitations of terrestrial plants, such non-arable land, water scarcity, time-intensive growth, and seasonal changes. However, the heterologous expression of interested genes in microalgae under heterotrophic cultivation (dark mode) remains a niche area within the field of engineering technologies. In this study, the green microalga, Chlorella sorokiniana AARL G015 was chosen as a potential candidate due to its remarkable capacity for rapid growth in complete darkness, its ability to utilize diverse carbon sources, and its potential for wastewater treatment in a circular bioeconomy model. The aims of this study were to advance microalgal genetic engineering via dark cultivation, thereby positioning the strain as promising dark-host for expressing heterologous genes to produce high-value phytochemicals and ingredients for food and feed. To facilitate comprehensive screening based on resistance, eleven common antibiotics were tested under heterotrophic condition. As the most effective selectable markers for this strain, G418, hygromycin, and streptomycin exhibited growth inhibition rates of 98%, 93%, and 92%, respectively, ensuring robust long-term transgenic growth. Successful transformation was achieved through microalgal cell cocultivation with Agrobacterium under complete darkness verified through the expression of green fluorescence protein and ß-glucuronidase. In summary, this study pioneers an alternative dark-host microalgal platform, using, Chlorella, under dark mode, presenting an easy protocol for heterologous gene transformation for microalgal host, devoid of the need for expensive equipment and light for industrial production. Furthermore, the developed genetic transformation methodology presents a sustainable way for production of high-value nutrients, dietary supplements, nutraceuticals, proteins and pharmaceuticals using heterotrophic microalgae as an innovative host system.

Regulation of the high-specificity Rubisco genes by the third CbbR-type regulator in a hydrogen-oxidizing bacterium Hydrogenovibriomarinus.

The obligate chemolithoautotrophic bacterium, Hydrogenovibrio marinus MH-110, has three ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) isoenzymes, CbbM, CbbLS-1, and CbbLS-2, which differ in CO2/O2 specificity factor values. Expressions of CbbM and CbbLS-1 are regulated differently by transcriptional regulators of the LysR family, CbbRm and CbbR1, respectively. CbbLS-2 has the highest specificity and is induced under low CO2 conditions, but the regulator for the cbbL2S2 genes encoding CbbLS-2 remains unidentified. In this study, the cbbR2 gene encoding the third CbbR-type regulator was identified in the downstream region of the cbbL2S2 and carboxysome gene cluster via transposon mutagenesis. CO2 depletion induced the cbbR2 gene. The cbbR2 knockout mutant could not grow under low CO2 conditions and did not produce CbbLS-2. Recombinant CbbR2 protein was bound to the promoter region of the cbbL2S2 genes. These results indicate that CbbR2 is the specific regulator for CbbLS-2 expression.

Reversible Glutamate Coordination to High-Valent Nickel Protects the Active Site of a [NiFe] Hydrogenase from Oxygen.

NAD+-reducing [NiFe] hydrogenases are valuable biocatalysts for H2-based energy conversion and the regeneration of nucleotide cofactors. While most hydrogenases are sensitive toward O2 and elevated temperatures, the soluble NAD+-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus (HtSH) is O2-tolerant and thermostable. Thus, it represents a promising candidate for biotechnological applications. Here, we have investigated the catalytic activity and active-site structure of native HtSH and variants in which a glutamate residue in the active-site cavity was replaced by glutamine, alanine, and aspartate. Our biochemical, spectroscopic, and theoretical studies reveal that at least two active-site states of oxidized HtSH feature an unusual architecture in which the glutamate acts as a terminal ligand of the active-site nickel. This observation demonstrates that crystallographically observed glutamate coordination represents a native feature of the enzyme. One of these states is diamagnetic and characterized by a very high stretching frequency of an iron-bound active-site CO ligand. Supported by density-functional-theory calculations, we identify this state as a high-valent species with a biologically unprecedented formal Ni(IV) ground state. Detailed insights into its structure and dynamics were obtained by ultrafast and two-dimensional infrared spectroscopy, demonstrating that it represents a conformationally strained state with unusual bond properties. Our data further show that this state is selectively and reversibly formed under oxic conditions, especially upon rapid exposure to high O2 levels. We conclude that the kinetically controlled formation of this six-coordinate high-valent state represents a specific and precisely orchestrated stereoelectronic response toward O2 that could protect the enzyme from oxidative damage.

Complete Genome Sequence of the Acetic Acid Bacterium Acetobacter aceti NBRC 14818.

We report here the complete genome sequence of the acetic acid bacterium Acetobacter aceti type strain NBRC 14818. The genome comprises a chromosome of 3,596,270 bp with 57.1% GC content and four plasmids/phages of 63,279 bp, 25,755 bp, 4,858 bp, and 2,964 bp, with an average GC content of 57.0%.

Complete Genome Sequence of a Mesophilic Obligately Chemolithoautotrophic Hydrogen-Oxidizing Bacterium, Hydrogenovibrio marinus MH-110.

Hydrogenovibrio marinus is a mesophilic, obligately chemolithoautotrophic, and hydrogen-oxidizing bacterium that uses three different RubisCOs at different carbon dioxide tensions. Here, we report its complete genome sequence, which is 2,491,293 bp long, with an average GC content of 44.1%.

Draft Genome Sequence of Comamonas testosteroni TA441, a Bacterium That Has a Cryptic Phenol Degradation Gene Cluster.

Comamonas testosteroni TA441 has a complete phenol degradation gene cluster but does not degrade phenol because the cluster is tightly repressed. However, mutant strains that can degrade phenol arise by spontaneous mutations of a repressor gene during incubation with phenol. Here, we report the draft genome sequence of strain TA441.

Physiological characterization of poly-ß-hydroxybutyrate accumulation in the moderately thermophilic hydrogen-oxidizing bacterium Hydrogenophilus thermoluteolus TH-1.

Hydrogenophilus thermoluteolus strain TH-1 is a thermophilic hydrogen-oxidizing microorganism that has the highest growth rate among autotrophs. Genomic analysis revealed that this strain comprises the complete gene set for poly-ß-hydroxybutyrate (PHB) synthesis, i.e., three copies of acetyl-CoA acetyltransferase and polyhydroxyalkanoate synthase and one copy of acetoacetyl-CoA reductase and 3-hydroxyacyl-CoA dehydrogenase/3-hydroxybutyryl-CoA epimerase. An investigation on PHB accumulation in strain TH-1 demonstrated that PHB accumulation was induced by nitrogen limitation under autotrophic as well as heterotrophic conditions. This strain accumulated up to 430.4 ± 14.3 mg L-1 PHB during a 3-h incubation under nitrogen-limited heterotrophic conditions. The highest PHB accumulation rates under autotrophic and heterotrophic conditions were 38.6% (w/w) of the dry cells after a 6-h induction and 53.8% after 3 h, respectively. Although PHB granules started to accumulate after 15 min of nitrogen limitation under heterotrophic conditions, a drastic decrease of PHB was observed after 9 h of induction.

Complete Genome Sequence of a Moderately Thermophilic Facultative Chemolithoautotrophic Hydrogen-Oxidizing Bacterium, Hydrogenophilus thermoluteolus TH-1.

Hydrogenophilus spp., which are moderately thermophilic aerobic betaproteobacteria, are widely distributed in geothermal environments. They fix carbon dioxide via the Calvin-Benson-Bassham cycle and exhibit rapid autotrophic growth using hydrogen as an energy source. Here, we report the complete genome sequence of Hydrogenophilus thermoluteolus strain TH-1.

Function of three RuBisCO enzymes under different CO2 conditions in Hydrogenovibrio marinus.

The obligate chemolithoautotrophic bacterium, Hydrogenovibrio marinus MH-110 has three ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) isoenzymes, designated CbbLS-1, CbbLS-2, and CbbM, which are encoded by the cbbL1S1, cbbL2S2, and cbbM genes, respectively. Functions of these isoenzymes at different CO2 concentrations were investigated using deletion mutants of their genes. Deletion of cbbL1 had no effect on cell growth under any of the test growth conditions. The cbbL2 mutant was unable to grow under lower (≤0.15%) CO2 conditions, though it grew normally under higher (≥2%) CO2 conditions. Growth of the cbbM mutant was retarded under higher CO2 conditions but was not affected by lower CO2 conditions. These results indicate that CbbLS-2 and CbbM specifically function under lower and higher CO2 conditions, respectively. The growth retardation of the cbbL2 and cbbM mutants was not restored by complementation with plasmids carrying the cbbL2S2 and cbbM genes, respectively. The cbbL2S2 and cbbM genes are followed by the carboxysome genes and the cbbQmOm genes, respectively. Co-expression of these downstream genes was probably necessary for the in vivo function of CbbLS-2 and CbbM. CbbLS-1 was upregulated in the cbbL2 and cbbM mutants under the lower and higher CO2 conditions, respectively, indicating that the expression of cbbL1S1 was controlled to compensate the deficiency of the other RuBisCO isoenzymes.

The Arnon-Buchanan cycle: a retrospective, 1966-2016.

For the first decade following its description in 1954, the Calvin-Benson cycle was considered the sole pathway of autotrophic CO2 assimilation. In the early 1960s, experiments with fermentative bacteria uncovered reactions that challenged this concept. Ferredoxin was found to donate electrons directly for the reductive fixation of CO2 into alpha-keto acids via reactions considered irreversible. Thus, pyruvate and alpha-ketoglutarate could be synthesized from CO2, reduced ferredoxin and acetyl-CoA or succinyl-CoA, respectively. This work opened the door to the discovery that reduced ferredoxin could drive the Krebs citric acid cycle in reverse, converting the pathway from its historical role in carbohydrate breakdown to one fixing CO2. Originally uncovered in photosynthetic green sulfur bacteria, the Arnon-Buchanan cycle has since been divorced from light and shown to function in a variety of anaerobic chemoautotrophs. In this retrospective, colleagues who worked on the cycle at its inception in 1966 and those presently working in the field trace its development from a controversial reception to its present-day inclusion in textbooks. This pathway is now well established in major groups of chemoautotrophic bacteria, instead of the Calvin-Benson cycle, and is increasingly referred to as the Arnon-Buchanan cycle. In this retrospective, separate sections have been written by the authors indicated. Bob Buchanan wrote the abstract and the concluding comments.

Phosphoserine Phosphatase Is Required for Serine and One-Carbon Unit Synthesis in Hydrogenobacter thermophilus.

Hydrogenobacter thermophilus is an obligate chemolithoautotrophic bacterium of the phylum Aquificae and is capable of fixing carbon dioxide through the reductive tricarboxylic acid (TCA) cycle. The recent discovery of two novel-type phosphoserine phosphatases (PSPs) in H. thermophilus suggests the presence of a phosphorylated serine biosynthesis pathway; however, the physiological role of these novel-type metal-independent PSPs (iPSPs) in H. thermophilus has not been confirmed. In the present study, a mutant strain with a deletion of pspA, the catalytic subunit of iPSPs, was constructed and characterized. The generated mutant was a serine auxotroph, suggesting that the novel-type PSPs and phosphorylated serine synthesis pathway are essential for serine anabolism in H. thermophilus. As an autotrophic medium supplemented with glycine did not support the growth of the mutant, the reversible enzyme serine hydroxymethyltransferase does not appear to synthesize serine from glycine and may therefore generate glycine and 5,10-CH2-tetrahydrofolate (5,10-CH2-THF) from serine. This speculation is supported by the lack of glycine cleavage activity, which is needed to generate 5,10-CH2-THF, in H. thermophilus Determining the mechanism of 5,10-CH2-THF synthesis is important for understanding the fundamental anabolic pathways of organisms, because 5,10-CH2-THF is a major one-carbon donor that is used for the synthesis of various essential compounds, including nucleic and amino acids. The findings from the present experiments using a pspA deletion mutant have confirmed the physiological role of iPSPs as serine producers and show that serine is a major donor of one-carbon units in H. thermophilusIMPORTANCE Serine biosynthesis and catabolism pathways are intimately related to the metabolism of 5,10-CH2-THF, a one-carbon donor that is utilized for the biosynthesis of various essential compounds. For this reason, determining the mechanism of serine synthesis is important for understanding the fundamental anabolic pathways of microorganisms. In the present study, we experimentally confirmed that a novel phosphoserine phosphatase in the obligate chemolithoautotrophic bacterium Hydrogenobacter thermophilus is essential for serine biosynthesis. This finding indicates that serine is synthesized from an intermediate of gluconeogenesis in H. thermophilus In addition, because glycine cleavage system activity and genes encoding an enzyme capable of producing 5,10-CH2-THF were not detected, serine appears to be the major one-carbon donor to tetrahydrofolate (THF) in H. thermophilus.

Nitrate reductases in Hydrogenobacter thermophilus with evolutionarily ancient features: distinctive localization and electron transfer.

Dissimilatory nitrate reductase (NAR) and assimilatory nitrate reductase (NAS) serve as key enzymes for nitrogen catabolism and anabolism in many organisms. We purified NAR and NAS from H. thermophilus, a hydrogen-oxidizing chemolithoautotroph belonging to the phylogenetically deepest branch in the Bacteria domain. Physiological contribution of these enzymes to nitrate respiration and assimilation was clarified by transcriptomic analysis and gene disruption experiments. These enzymes showed several features unreported in bacteria, such as the periplasmic orientation of NAR anchored with a putative transmembrane subunit and the specific electron transfer from a [4Fe-4S]-type ferredoxin to NAS. While some of their enzymatic properties are shared with NARs from archaea and with NASs from phototrophs, phylogenetic analysis indicated that H. thermophilus NAR and NAS have deep evolutionary origins that cannot be explained by a recent horizontal gene transfer event from archaea and phototrophs. These findings revealed the diversity of NAR and NAS in nonphotosynthetic bacteria, and they also implied that the outward orientation of NAR and the ferredoxin-dependent electron transfer of NAS are evolutionarily ancient features preserved in H. thermophilus.

Specific expression and function of the A-type cytochrome c oxidase under starvation conditions in Pseudomonas aeruginosa.

Pseudomonas aeruginosa has one A-type (caa3) and multiple C-type (cbb3) cytochrome c oxidases as well as two quinol oxidases for aerobic respiration. The caa3 oxidase is highly efficient in creating a proton gradient across the cell membrane, but it is not expressed under normal growth conditions and its physiological role has not been investigated. In the present study, a mutant strain deficient in the coxBA-PA0107-coxC genes encoding caa3 exhibited normal growth under any test conditions, but it had low relative fitness under carbon starvation conditions, indicating that the expression of caa3 is advantageous under starvation conditions. A mutant that lacked four terminal oxidase gene clusters except for the cox genes was unable to grow aerobically because of low expression level of caa3. However, suppressor mutants that grew aerobically using caa3 as the only terminal oxidase emerged after aerobic subculturing. Analyses of the suppressor mutants revealed that a mutation of roxS encoding a sensor kinase of a two-component regulator RoxSR was necessary for the aerobic growth in synthetic medium. Two additional mutations in the 5'-flanking region of coxB were necessary for the aerobic growth in LB medium. Although the expression level of caa3 was higher in the suppressor mutants, their growth rates were lower than when the other terminal oxidases were utilized, suggesting that caa3 was not suited for utilization as the only terminal oxidase. Overexpression of the cox genes also inhibited the aerobic growth of the wild-type strain. These results indicate that caa3 is tightly regulated to be expressed only under starvation conditions at low level and it functions in cooperation with other terminal oxidases to facilitate survival in nutrient starvation conditions.

Expression of multiple cbb3 cytochrome c oxidase isoforms by combinations of multiple isosubunits in Pseudomonas aeruginosa.

The ubiquitous opportunistic human pathogen Pseudomonas aeruginosa has five terminal oxidases for aerobic respiration and uses them under different growth conditions. Two of them are cbb3-type cytochrome c oxidases encoded by the gene clusters ccoN1O1Q1P1 and ccoN2O2Q2P2, which are the main terminal oxidases under high- and low-oxygen conditions, respectively. P. aeruginosa also has two orphan gene clusters, ccoN3Q3 and ccoN4Q4, encoding the core catalytic CcoN isosubunits, but the roles of these genes have not been clarified. We found that 16 active cbb3 isoforms could be produced by combinations of four CcoN, two CcoO, and two CcoP isosubunits. The CcoN3- or CcoN4-containing isoforms were produced in the WT cell membrane in response to nitrite and cyanide, respectively. The strains carrying these isoforms were more resistant to nitrite or cyanide under low-oxygen conditions. These results indicate that P. aeruginosa gains resistance to respiratory inhibitors using multiple cbb3 isoforms with different features, which are produced through exchanges of multiple core catalytic isosubunits.

Discovery of an intermolecular disulfide bond required for the thermostability of a heterodimeric protein from the thermophile Hydrogenobacter thermophilus.

Factors that increase protein thermostability are of considerable interest in both scientific and industrial fields. Disulfide bonds are one of such factors that increase thermostability, but are rarely found in intracellular proteins because of the reducing environment of the cytosol. Here, we report the first example of an intermolecular disulfide bond between heteromeric subunits of a novel-type phosphoserine phosphatase from a thermophilic bacterium Hydrogenobacter thermophilus, which contributes to the protein thermostability at the physiological temperature. Comparison of remaining soluble proteins between wild-type and cysteine-deleted mutant using SDS-PAGE revealed that the disulfide bond increases the thermostability of the whole protein by tightly connecting a subunit with low solubility to the partner with higher solubility. Furthermore, it was strongly suggested that the disulfide bond is formed and contributes to the stability in vivo. This finding will open new avenues for the design of proteins with increased thermostability.

Crystallization and preliminary X-ray analysis of the NAD+-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus TH-1.

NAD+-reducing [NiFe] hydrogenases catalyze the oxidoreduction of dihydrogen concomitant with the interconversion of NAD+ and NADH. Here, the isolation, purification and crystallization of the NAD+-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus TH-1 are reported. Crystals of the NAD+-reducing [NiFe] hydrogenase were obtained within one week from a solution containing polyethylene glycol using the sitting-drop vapour-diffusion method and micro-seeding. The crystal diffracted to 2.58 Šresolution and belonged to space group C2, with unit-cell parameters a=131.43, b=189.71, c=124.59 Å, ß=109.42°. Assuming the presence of two NAD+-reducing [NiFe] hydrogenase molecules in the asymmetric unit, VM was calculated to be 2.2 Å3 Da(-1), which corresponds to a solvent content of 43%. Initial phases were determined by the single-wavelength anomalous dispersion method using the anomalous signal from the Fe atoms.

Conductive iron oxides accelerate thermophilic methanogenesis from acetate and propionate.

Anaerobic digester is one of the attractive technologies for treatment of organic wastes and wastewater, while continuous development and improvements on their stable operation with efficient organic removal are required. Particles of conductive iron oxides (e.g., magnetite) are known to facilitate microbial interspecies electron transfer (termed as electric syntrophy). Electric syntrophy has been reported to enhance methanogenic degradation of organic acids by mesophilic communities in soil and anaerobic digester. Here we investigated the effects of supplementation of conductive iron oxides (magnetite) on thermophilic methanogenic microbial communities derived from a thermophilic anaerobic digester. Supplementation of magnetite accelerated methanogenesis from acetate and propionate under thermophilic conditions, while supplementation of ferrihydrite also accelerated methanogenesis from propionate. Microbial community analysis revealed that supplementation of magnetite drastically changed bacterial populations in the methanogenic acetate-degrading cultures, in which Tepidoanaerobacter sp. and Coprothermobacter sp. dominated. These results suggest that supplementation of magnetite induce electric syntrophy between organic acid-oxidizing bacteria and methanogenic archaea and accelerate methanogenesis even under thermophilic conditions. Findings from this study would provide a possibility for the achievement of stably operating thermophilic anaerobic digestion systems with high efficiency for removal of organics and generation of CH4.

Reorganization of the bacterial and archaeal populations associated with organic loading conditions in a thermophilic anaerobic digester.

Organic loading conditions are an important factor influencing reactor performances in methanogenic bioreactors. Yet the underlying microbiological basis of the process stability, deterioration, and recovery remains to be understood. Here, structural responses of the bacterial and archaeal populations to the change of organic loading conditions in a thermophilic anaerobic digester were investigated by process analyses and 16S rRNA gene-based molecular approaches. The biogas was produced stably without the accumulation of volatile fatty acids (VFAs) at low organic loading rates (OLRs) in the beginning of reactor operation. Increasing OLR in stages disrupted the stable reactor performance, and high OLR conditions continued the deteriorated performance with slight biogas production and high accumulation of VFAs. Thereafter, the gradual decrease of OLR resulted in the recovery from the deterioration, giving rise to the stable performance again. The stable performances before and after the high OLR conditions conducted were associated with compositionally similar but not identical methanogenic consortia. The bacterial and archaeal populations were synchronously changed at both the transient phases toward the deteriorated performance and in recovery process, during which the dynamic shift of aceticlastic and hydrogenotrophic methanogens including the recently identified Methanomassiliicoccus might contribute to the maintenance of the methanogenic activity. The distinctive bacterial population with a high predominance of Methanobacterium formicicum as archaeal member was found for the deteriorated performance. The results in this study indicate the coordinated reorganization of the bacterial and archaeal populations in response to functional states induced by the change of organic loading conditions in the anaerobic digester.

Involvement of a novel fermentative bacterium in acidification in a thermophilic anaerobic digester.

Acidification results from the excessive accumulation of volatile fatty acids and the breakthrough of buffering capacity in anaerobic digesters. However, little is known about the identity of the acidogenic bacteria involved. Here, we identified an active fermentative bacterium during acidification in a thermophilic anaerobic digester by sequencing and phylogenetic analysis of isotopically labeled rRNA. The digestion sludge retrieved from the beginning of pH drop in the laboratory-scale anaerobic digester was incubated anaerobically at 55 °C for 4 h during which 13C-labeled glucose was supplemented repeatedly. 13CH4 and 13CO2 were produced after substrate addition. RNA extracts from the incubated sludge was density-separated by ultracentrifugation, and then bacterial communities in the density fractions were screened by terminal restriction fragment length polymorphism and clone library analyses based on 16S rRNA transcripts. Remarkably, a novel lineage within the genus Thermoanaerobacterium became abundant with increasing the buoyant density and predominated in the heaviest fraction of RNA. The results in this study indicate that a thermoacidophilic bacterium exclusively fermented the simple carbohydrate glucose, thereby playing key roles in acidification in the thermophilic anaerobic digester.