The facultative chemolithoautotroph, Hydrogenophilus thermoluteolus TH-1 has an ability to grow autotrophically or heterotrophically. In this study, the growth under the mixotrophic condition was demonstrated for the first time with carbon dioxide and butyrate as carbon sources. The transcriptome profiles of the cells grown under the autotrophic, heterotrophic, and mixotrophic conditions were investigated. We re-confirmed the function of Calvin-Benson-Bassham (CBB) cycle as a carbon dioxide fixation pathway under the autotrophic condition. The transcriptome profiles also indicated that the CBB cycle is active under the mixotrophic condition. Quite interestingly, we found the concomitant function of phosphoglycolate phosphatase, glycolate oxidase, and malate synthase under the autotrophic or mixotrophic condition. These reactions were thought to be active for the detoxification of glycolate, which originated from the oxygenase reaction of RubisCO. Furthermore, superoxide dismutase/ peroxidase system was considered to be the primary system to detoxify reactive oxygen species in strain TH-1.

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Alkaliphilic actinomycete Nocardiopsis sp. strain F96 has been found to produce a single domain glycoside hydrolase (GH) family 18 chitinase (ChiF1). Recently, genome sequencing of strain F96 was completed and three other homologs of GH18 chitinases were found. One of the chitinase homologs, ChiF3, has a multi-domain architecture. The number of domains in ChiF3 is the most various among the four GH18 chitinase homologs. It contained a signal peptide, a carbohydrate-binding module family 2 chitin-binding domain (ChBD), a Fibronectin type Ⅲ-like domain, and a GH18 catalytic domain including a chitinase insertion domain. C-terminally His-tagged ChiF3 was expressed in Escherichia coli, purified and characterized in comparison with ChiF1. Both ChiF1 and ChiF3 showed bimodal pH profiles. Especially, ChiF3 maintained high activity at pH 7.5-9.4 and showed the highest activity at pH 8.5, which is the highest of all the known chitinases from actinomycetes. ChiF3 having ChBD showed higher binding ability toward insoluble chitin compared to ChiF1, which does not have ChBD. Furthermore, the ratio of specific activity of ChiF3 toward insoluble and soluble substrates [Insoluble/Soluble] is 15.2 times higher than that of ChiF1. This difference of substrate preference of both enzymes might be related to the presence or absence of ChBD.

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Group-3 late embryogenesis abundant (G3LEA) proteins play important roles in the acquisition of desiccation tolerance in some anhydrobiotic organisms such as African sleeping chironomide. From their primary amino acid sequences, the potential functional sites are inferred to be located at segments that include several times repeats of 11-mer motifs, such as AKDGTKEKAGE, characteristic of G3LEA proteins. So far, we have investigated the structural and functional properties of chemically synthesized 22-mer or 44-mer peptides that include one of the consensus 11-mer motifs identified by bioinformatics analysis for various G3LEA proteins. Our findings to be noted are as follows. Such LEA model peptides can reproduce the structural features of the parent G3LEA proteins: they are disordered in water, whereas they adopt -helical coiled coil structure in the dry state. The dried peptides are in the glassy state up to 100 ºC, and have the ability of reinforcing the glassy matrix of co-existing sugar such as trehalose. More importantly, the LEA model peptides have protective activities on various biological molecules against the desiccation stress. For example, they exhibit anti-fusion effect on liposomes in the dry state by covering the membrane surface through the interactions between the side chains of their Lys residues and the polar head groups of phospholipid molecules. In addition, the LEA model peptides are capable not only of suppressing the desiccation-induced aggregation of proteins such as lysozyme and -casein, but also of preserving the catalytic activities of electrostatically different enzymes such as lactate dehydrogenase and -D-galactosidase, whose pI values are 4.6 and 8.2, respectively. On the basis of these results, the LEA model peptides developed by our group are expected to be applicable to dry preservation of a variety of biological molecules.

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In bacteria and eukaryotes, the pentose moiety of nucleosides are degraded through the pentose phosphate pathway. In contrast, since the pentose phosphate pathway is absent in many archaea, it had not been known how nucleosides are degraded in these archaea. On the other hand, the NMP degradation pathway, consisting of AMP phosphorylase, ribose-1,5-bisphosphate isomerase and type III ribulose-1,5-bisphosphate carboxylase/oxygenase, had previously been identified in archaea. In the archaeon Thermococcus kodakarensis, three nucleoside phosphorylases and an ADP-dependent ribose-1- phosphate kinase turned out to convert adenosine, guanosine and uridine to ribose 1,5-bisphosphate, whereas a cytidine kinase was suggested to phosphorylate cytidine to CMP. Since these enzymes link nucleosides to the previously identified NMP degradation pathway, this metabolic network turned out to link nucleosides to central carbon metabolism. In this metabolic network, nucleosides are converted to ribose 1,5-bisphosphate and ribulose 1,5-bisphosphate as intermediates, which is thus designated the pentose bisphosphate pathway.

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The RNA-guided endonuclease Cas9, which is involved in the prokaryotic CRISPR-Cas adoptive immune system, binds to a guide RNA and cleaves double-stranded DNA complementary to the RNA guide. In recent years, Cas9 has been used as a versatile genome-editing tool in a wide range of fields, from fundamental research to clinical applications. However, the molecular mechanism of DNA recognition and cleavage by Cas9 was unknown, and many issues remained to be addressed for its applications to genome editing. We elucidated the crystal structure of S. pyogenes Cas9, which is most widely used for genome editing, in complex with the guide RNA and its target DNA, thus providing the first insights into the Cas9-mediated DNA cleavage mechanism. Furthermore, we solved the crystal structures of Cas9 nucleases from three different bacteria and those of Cas12a (Cpf1) nucleases, which are also harnessed for genome editing. Collectively, these structural studies illuminated the mechanistic convergence and divergence in the CRISPR-Cas nucleases, and paved the way for the engineering of new genome-editing tools with improved functionalities.

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Marine sediments harbor more than half of the total global biomass of the microbes, yet receive less than 1% of total fixed organic carbon in the ocean51) . Therefore, energy sources for bacterial sulfate reduction, one of the most ubiquitous and predominant microbial processes, remain ambiguous, especially in marine sediments with scarce organics and hydrogen. We recently showed the capability of a sulfate-reducing bacteria (SRB) strain isolated from marine sediments, Desulfovibrio ferrophilus IS5, to respire using extracellular solids by direct electron uptake via outer membrane cytochromes, which are widely conserved in various sediment sulfur-species-respiring bacteria. The electron uptake capability of SRB provides a novel explanation for energy acquisition by sediment microbes under energy-limited conditions. Here, we will review the extracellular electron uptake mechanism in sediment SRB and discuss the ubiquity of this mechanism in marine environments, based on the distribution of genes encoding outer membrane cytochromes important for electron uptake in microbes. The results suggest that microbial energy production associated with extracellular electron uptake may be ubiquitous and important in the subsurface environments.

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