Naoto Ohtani

Molecular diversities of RNases H

RNase H is an enzyme that specifically cleaves RNA hybridized to DNA. The enzyme is ubiquitously present in various organisms. Single bacterial and eucaryotic cells often contain two RNases H, whereas single archaeal cells contain only one. To determine whether there is a physiological significance in the ubiquity and multiplicity of the enzyme, and whether all enzymes are evolutionarily diverged from a common ancestor, we carried out phylogenetic analyses of the RNase H sequences. In this report, we demonstrated that RNases H are classified into two major families, Type 1 and Type 2 RNases H, of which only the Type 2 enzymes are present in all living organisms, including bacteria, archaea, and eucaryotes, suggesting that they represent an ancestral form of RNases H. Based on this information, we discuss the evolutionary relationships and possible cellular functions of RNases H.

An Extreme Thermophile, Thermus thermophilus, Is a Polyploid Bacterium

An extremely thermophilic bacterium, Thermus thermophilus HB8, is one of the model organisms for systems biology. Its genome consists of a chromosome (1.85 Mb), a megaplasmid (0.26 Mb) designated pTT27, and a plasmid (9.3 kb) designated pTT8, and the complete sequence is available. We show here that T. thermophilus is a polyploid organism, harboring multiple genomic copies in a cell. In the case of the HB8 strain, the copy number of the chromosome was estimated to be four or five, and the copy number of the pTT27 megaplasmid seemed to be equal to that of the chromosome. It has never been discussed whether T. thermophilus is haploid or polyploid. However, the finding that it is polyploid is not surprising, as Deinococcus radiodurans, an extremely radioresistant bacterium closely related to Thermus, is well known to be a polyploid organism. As is the case for D. radiodurans in the radiation environment, the polyploidy of T. thermophilus might allow for genomic DNA protection, maintenance, and repair at elevated growth temperatures. Polyploidy often complicates the recognition of an essential gene in T. thermophilus as a model organism for systems biology.

Gene cloning, overproduction, and characterization of thermolabile alkaline phosphatase from a psychrotrophic bacterium.

The gene encoding alkaline phosphatase from the psychrotrophic bacterium Shewanella sp. SIB1 was cloned, sequenced, and overexpressed in Escherichia coli. The recombinant protein was purified and its enzymatic properties were compared with those of E. coli alkaline phosphatase (APase), which shows an amino acid sequence identity of 37%. The optimum temperature of SIB1 APase was 50 °C, lower than that of E. coli APase by 30 °C. The specific activity of SIB1 APase at 50 °C was 3.1 fold higher than that of E. coli APase at 80 °C. SIB1 APase lost activity with a half-life of 3.9 min at 70 °C, whereas E. coli APase lost activity with a half-life of >6 h even at 80 °C. Thus SIB1 APase is well adapted to low temperatures. Comparison of the amino acid sequences of SIB1 and E. coli APases suggests that decreases in electrostatic interactions and number of disulfide bonds are responsible for the cold-adaptation of SIB1 APase.

Junction ribonuclease activity specified in RNases HII/2

Junction ribonuclease (JRNase) recognizes the transition from RNA to DNA of an RNA–DNA/DNA hybrid, such as an Okazaki fragment, and cleaves it, leaving a mono‐ribonucleotide at the 5′ terminus of the RNA–DNA junction. Although this JRNase activity was originally reported in calf RNase H2, some other RNases H have recently been suggested to possess it. This paper shows that these enzymes can also cleave an RNA–DNA/RNA heteroduplex in a manner similar to the RNA–DNA/DNA substrate. The cleavage site of the RNA–DNA/RNA substrate corresponds to the RNA/RNA duplex region, indicating that the cleavage activity cannot be categorized as RNase H activity, which specifically cleaves an RNA strand of an RNA/DNA hybrid. Examination of several RNases H with respect to JRNase activity suggested that the activity is only found in RNase HII orthologs. Therefore, RNases HIII, which are RNase HII paralogs, are distinguished from RNases HII by the absence of JRNase activity. Whether a substrate can be targeted by JRNase activity would depend only on whether or not an RNA–DNA junction consisting of one ribonucleotide and one deoxyribonucleotide is included in the duplex. In addition, although the activity has been reported not to occur on completely single‐stranded RNA–DNA, it can recognize a single‐stranded RNA–DNA junction if a double‐stranded region is located adjacent to the junction.

Gene Cloning and Biochemical Characterizations of Thermostable Ribonuclease HIII from Bacillus stearothermophilus

The gene encoding RNase HIII from the thermophilic bacterium Bacillus stearothermophilus was cloned and overexpressed in Escherichia coli, and the recombinant protein (Bst–RNase HIII) was purified and biochemically characterized. Bst–RNase HIII is a monomeric protein with 310 amino acid residues, and shows an amino acid sequence identity of 47.1% with B. subtilis RNase HIII (Bsu–RNase HIII). The enzymatic properties of Bst–RNase HIII, such as pH optimum, metal ion requirement, and cleavage mode of the substrates, were similar to those of Bsu–RNase HIII. However, Bst–RNase HIII was more stable than Bsu–RNase HIII, and the temperature (T1⁄2) at which the enzyme loses half of its activity upon incubation for 10 min was 55 °C for Bst–RNase HIII and 35 °C for Bsu–RNase HIII. The optimum temperature for Bst–RNase HIII activity was also shifted upward by roughly 20 °C as compared to that of Bsu–RNase HIII. The availability of such a thermostable enzyme will facilitate structural studies of RNase HIII.

Serial assembly of Thermus megaplasmid DNA in the genome of Bacillus subtilis 168: A BAC-based domino method applied to DNA with a high GC content

Bacillus subtilis is the only bacterium-based host able to clone giant DNA above 1000 kbp. DNA previously handled by this host was limited to that with GC content similar to or lower than that of the B. subtilis genome. To expand the target DNA range to higher GC content, we tried to clone a pTT27 megaplasmid (257 kbp, 69% of G+C) from Thermus thermophilus. To facilitate the reconstruction process, we subcloned pTT27 in a bacterial artificial chromosome (BAC) vector of Escherichia coli. Owing to the ability of BAC to carry around 100 kbp DNA, only 4 clones were needed to cover the pTT27 and conduct step-by-step assembly in the B. subtilis genome. The full length of 257 kbp was reconstructed through 3 intermediary lengths (108, 153, and 226 kbp), despite an unexpected difficulty in the maintenance of DNA >200 kbp. Retrieval of these four pTT27 segments from the B. subtilis genome by genetic transfer to a plasmid pLS20 was attempted. A stable plasmid clone was obtained only for the 108 and 153 kbp intermediates. The B. subtilis genome was demonstrated to accommodate large DNA with a high GC content, but may be restricted to less than 200 kbp by unidentified mechanisms.

Importance of an N-terminal extension in ribonuclease HII from Bacillus stearothermophilus for substrate binding.

The gene encoding ribonuclease HII from Bacillus stearothermophilus was cloned and expressed in Escherichia coli. The overproduced protein, Bst-RNase HII, was purified and biochemically characterized. Bst-RNase HII, which consists of 259 amino acid residues, showed the highest amino acid sequence identity (50.2%) to Bacillus subtilis RNase HII. Like B. subtilis RNase HII, it exhibited Mn2+-dependent RNase H activity. It was, however, more thermostable than B. subtilis RNase HII. When the Bst-RNase HII amino acid sequence is compared with that of Thermococcus kodakaraensis RNase HII, to which it shows 29.8% identity, 30 residues are observed to be truncated from the C-terminus and there is an extension of 71 residues at the N-terminus. The C-terminal truncation results in the loss of the alpha9 helix, which is rich in basic amino acid residues and is therefore important for substrate binding. A truncated protein, Delta59-Bst-RNase HII, in which most of the N-terminal extension was removed, completely lost its RNase H activity. Surface plasmon resonance analysis indicated that this truncated protein did not bind to the substrate. These results suggest that the N-terminal extension of Bst-RNase HII is important for substrate binding. Because B. subtilis RNase HII has an N-terminal extension of the same length and these extensions contain a region in which basic amino acid residues are clustered, the Bacillus enzymes may represent a novel type of RNase H which possesses a substrate-binding domain at the N-terminus.

The SCO2299 gene from Streptomyces coelicolor A3(2) encodes a bifunctional enzyme consisting of an RNase H domain and an acid phosphatase domain

The SCO2299gene from Streptomyces coelicolor encodes a single peptide consisting of 497 amino acid residues. Its N‐terminal region shows high amino acid sequence similarity to RNase HI, whereas its C‐terminal region bears similarity to the CobC protein, which is involved in the synthesis of cobalamin. The SCO2299 gene suppressed a temperature‐sensitive growth defect of an Escherichia coli RNase H‐deficient strain, and the recombinant SCO2299 protein cleaved an RNA strand of RNA·DNA hybrid in vitro. The N‐terminal domain of the SCO2299 protein, when overproduced independently, exhibited RNase H activity at a similar level to the full length protein. On the other hand, the C‐terminal domain showed no CobC‐like activity but an acid phosphatase activity. The full length protein also exhibited acid phosphatase activity at almost the same level as the C‐terminal domain alone. These results indicate that RNase H and acid phosphatase activities of the full length SCO2299 protein depend on its N‐terminal and C‐terminal domains, respectively. The physiological functions of the SCO2299 gene and the relation between RNase H and acid phosphatase remain to be determined. However, the bifunctional enzyme examined here is a novel style in the Type 1 RNase H family. Additionally, S. coelicolor is the first example of an organism whose genome contains three active RNase H genes.

Identification of RNase HII from psychrotrophic bacterium, Shewanella sp. SIB1 as a high-activity type RNase H

The gene encoding RNase HII from the psychrotrophic bacterium, Shewanella sp. SIB1 was cloned, overexpressed in Escherichia coli, and the recombinant protein was purified and biochemically characterized. SIB1 RNase HII is a monomeric protein with 212 amino acid residues and shows an amino acid sequence identity of 64% to E. coli RNase HII. The enzymatic properties of SIB1 RNase HII, such as metal ion preference, pH optimum, and cleavage mode of substrate, were similar to those of E. coli RNase HII. SIB1 RNase HII was less stable than E. coli RNase HII, but the difference was marginal. The half‐lives of SIB1 and E. coli RNases HII at 30 °C were ∼ 30 and 45 min, respectively. The midpoint of the urea denaturation curve and optimum temperature of SIB1 RNase HII were lower than those of E. coli RNase HII by ∼ 0.2 m and ∼ 5 °C, respectively. However, SIB1 RNase HII was much more active than E. coli RNase HII at all temperatures studied. The specific activity of SIB1 RNase HII at 30 °C was 20 times that of E. coli RNase HII. Because SIB1 RNase HII was also much more active than SIB1 RNase HI, RNases HI and HII represent low‐ and high‐activity type RNases H, respectively, in SIB1. In contrast, RNases HI and HII represent high‐ and low‐activity type RNases H, respectively, in E. coli. We propose that bacterial cells usually contain low‐ and high‐activity type RNases H, but these types are not correlated with RNase H families.

Restriction on Conjugational Transfer of pLS20 in Bacillus subtilis 168

Conjugational transfer of pLS20 in Bacillus subtilis Marburg 168 is restricted by the BsuM restriction-modification system. Restriction efficiency was measured using pLS20 derivatives possessing various numbers of XhoI sites, which are known to be recognized by BsuM. An increase in XhoI sites clearly reduced the conjugational efficiency of pLS20 as compared with that of pUB110 plasmid lacking XhoI.