Identification of RNase HII from psychrotrophic bacterium, Shewanella sp SIB1 as a high-activity type RNase H Hyongi Chon1, Takashi Tadokoro1, Naoto Ohtani1, Yuichi Koga1, Kazufumi Takano1,2 and Shigenori Kanaya1 Department of Material and Life Science, Graduate School of Engineering, Osaka University, Japan PRESTO, Osaka, Japan Keywords cold-adaptation; gene cloning; psychrotrophic bacterium; ribonuclease H; Shewanella sp Correspondence S Kanaya, Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2–1, Yamadaoka, Suita, Osaka 565-0871, Japan Tel ⁄ Fax: +81 6879 7938 E-mail: kanaya@mls.eng.osaka-u.ac.jp (Received 21 Feburary 2006, revised 21 March 2006, 22 March 2006) doi:10.1111/j.1742-4658.2006.05241.x 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  °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 lowand high-activity type RNases H, but these types are not correlated with RNase H families Ribonuclease H (RNase H) (EC 3.1.26.4) is an enzyme that degrades the RNA of RNA ⁄ DNA hybrids at the PO-3¢ bond in the presence of divalent metal ions, such as Mg2+ and Mn2+ [1] It is involved in DNA replication, repair, and transcription [2–9] RNase H is widely present in various organisms, including bacteria, archaea, and eukaryotes [10] RNase H is also present in retroviruses as a C-terminal domain of reverse transcriptase This activity is required in the conversion of a single-stranded genomic RNA to a double-stranded DNA and is therefore required for the proliferation of retroviruses [11] Based on differences in the amino acid sequences, RNases H are classified into two major families, type and type RNases H, which are evolutionarily unrelated [10] Bacterial RNases HI, eukaryotic RNases H1, and retroviral RNases H are members of the type RNase H family Bacterial RNases HII, bacterial RNases HIII, archaeal RNases HII, and eukaryotic RNases H2 are members of the type RNase H family According to the crystal structures of bacterial RNases HI [12–14], archaeal RNases HII [15–17], and bacterial RNase HIII [18], these RNases H share a main chain fold consisting of a five-stranded b sheet and two Abbreviations APase, alkaline phosphatase; BSA, bovine serum albumin; IPTG, isopropyl thio-b-D-galactoside; RNase H, ribonuclease H 2264 FEBS Journal 273 (2006) 2264–2275 ª 2006 The Authors Journal compilation ª 2006 FEBS H Chon et al a helices This folding motif, termed RNase H fold, has been found in the crystal structures of various functionally unrelated proteins, such as integrase [19,20], transposase [21], RuvC Holliday-junction resolvase [22], and the PIWI domain of argonaute protein [23,24] In addition, steric configurations of the four acidic active-site residues are similar in these RNases H, suggesting that they share a common catalytic mechanism It has recently been shown that two metal ions bind to the RNase H–substrate complex, such that both metal ions coordinate with acidic active-site residues and the scissile phosphate group of the substrate [25], indicating that RNase H utilizes a two metal ion mechanism According to this mechanism, one metal ion is required for activation of an attacking water molecule and the other is required for stabilization of the penta-covalent intermediate Many organisms contain two different RNases H within a single cell [10,26] For example, Escherichia coli cells contain RNases HI and HII, yeast and human cells contain RNases H1 and H2, and Bacillus subtilis and Bacillus stearothermophilus cells contain RNases HII and HIII The physiological significance of the multiplicity of the RNase H genes in a single genome remains to be understood However, the phenotypes of E coli and B subtilis are changed considerably only when both RNase H genes are absent [27], suggesting that their functions overlap RNases H from E coli [28,29], B subtilis (Bsu-RNases HII and HIII) [26], and B stearothermophilus (Bst-RNases HII and HIII) [30,31] have been overproduced in E coli, purified, and biochemically characterized The two RNases H from each strain differ greatly in metal ion preference and specific activity One of the pair of these RNases H, such as E coli RNase HI, BsuRNase HIII, and Bst-RNase HIII, prefers Mg2+ to Mn2+ for activity, whereas other three, such as E coli RNase HII, Bsu-RNase HII, and Bst-RNase HII, prefer Mn2+ to Mg2+ for activity The specific activities of E coli RNase HI, Bsu-RNase HIII, and Bst-RNase HIII are higher than E coli RNase HII, Bsu-RNase HII, and Bst-RNase HII by 13, 20, and 100 times, respectively These results suggest that bacterial cells usually contain high- and low-activity type RNases H, which differ in metal ion preference Shewanella sp SIB1 is a psychrotrophic bacterium, which grows most rapidly at 20 °C [32] This strain can grow at °C, but not at temperatures > 30 °C We previously cloned the rnhA gene encoding RNase HI from this strain and biochemically characterized the recombinant protein (SIB1 RNase HI) [33] SIB1 RNase HI shows an amino acid sequence identity of 63% to E coli RNase HI and, like E coli High-activity type RNase HII from a psychrotroph RNase HI, prefers Mg2+ to Mn2+ for activity Nevertheless, SIB1 RNase HI is considerably less stable and less active than E coli RNase HI These results suggest that, unlike E coli RNase HI, SIB1 RNase HI represents low-activity type RNase H The question therefore arises whether the SIB1 genome contains an additional gene encoding high-activity type RNase H Because c-proteobacteria, for which complete genome sequences are available, always contain RNases HI and HII, and SIB1 belongs to this bacterial group, it is highly likely that the SIB1 genome contains the rnhB gene encoding RNase HII Therefore, it would be informative to clone this gene and characterize the recombinant protein of SIB1 RNase HII In this study, we cloned the gene encoding SIB1 RNase HII, overexpressed it in E coli, purified the recombinant protein, and compared its enzymatic properties with those of the E coli counterpart We showed that, like other bacterial RNases HII, SIB1 RNase HII prefers Mn2+ to Mg2+ for activity, but, unlike them, this RNase H represents a high-activity type RNase H Thus, SIB1 was shown to have a unique combination of high- and low-activity type RNases H Results Gene cloning When the amino acid sequences of various bacterial RNases HII are compared, sequences VAGVDEVG and HRRSFGPVK, which correspond to Val12–Gly19 and His183–Lys191 of E coli RNase HII, respectively, are highly conserved [10] Using primers constructed based on these sequences, part of the gene (Sh-rnhB) encoding SIB1 RNase HII was amplified by PCR from the genomic DNA of Shewanella sp SIB1 Southern blotting and colony hybridization using this DNA fragment as a probe indicated that a 3.0 kb PstI fragment of the SIB1 genome contained the entire Sh-rnhB gene (data not shown) Determination of the nucleotide sequence of the Sh-rnhB gene revealed that SIB1 RNase HII is composed of 212 amino acid residues with a calculated molecular mass of 22 776 Da and an isoelectric point of 6.6 The rnhB gene is arranged in the SIB1 genome such that it is located immediately upstream of the dnaE gene, which encodes the a subunit of DNA polymerase III These genes have the same arrangement in the E coli genome [34] Likewise, the rnhA gene encoding RNase HI and the dnaQ gene encoding the e subunit of DNA polymerase III are arranged in the same way in the SIB1 and E coli genomes, such that they overlap [33] FEBS Journal 273 (2006) 2264–2275 ª 2006 The Authors Journal compilation ª 2006 FEBS 2265 High-activity type RNase HII from a psychrotroph H Chon et al Amino acid sequence The amino acid sequence of SIB1 RNase HII deduced from the nucleotide sequence is compared with those of other bacterial RNases HII in Fig SIB1 RNase HII shows amino acid sequence identities of 61.8% to E coli RNase HII, 43.9% to Bst-RNase HII, 43.4% to Bsu-RNase HII, and 43.4% to RNase HII from Thermotoga maritima (Tma-RNase HII), for which the crystal structure is available (PDB code 2ETJ) The four conserved amino acid residues, which are expected to form the active site of the enzyme, are also fully conserved in the SIB1 RNase HII sequence (Asp28, Glu29, Asp120, and Asp138), suggesting that SIB1 RNase HII structurally and functionally resembles to other RNases HII Biochemical properties of the recombinant protein For overproduction of SIB1 RNase HII, the rnhA ⁄ rnhB double mutant strain E coli MIC2067(DE3), which lacks all functional RNases H, was used as a host strain to avoid contamination of host-derived RNases H Upon induction for overproduction, recombinant protein accumulated in the cells in both soluble and insoluble forms, and the soluble form of the protein was purified to give a single band on SDS ⁄ PAGE (Fig 2) The production level of SIB1 RNase HII was  30 mgỈL)1 for the soluble form and  20 mgỈL)1 for the insoluble form, and  10 mg of the purified protein was obtained from L of culture The molecular mass of the protein was estimated to be 25 kDa by both SDS ⁄ PAGE and gel-filtration column chromatography, which is comparable with the calculated value These results strongly suggest that, like other RNases HII, SIB1 RNase HII exists in a monomeric form The far-UV CD spectrum of SIB1 RNase HII was similar to that of E coli RNase HII (Fig 3A), suggesting that its overall main-chain fold is similar to that of E coli RNase HII In contrast, the near-UV CD spectrum of SIB1 RNase HII was considerably different from that of E coli RNase HII (Fig 3B), suggesting that the local conformations Fig Alignment of the RNase HII sequences Amino acid sequences of RNases HII from Shewanella sp SIB1 (SIB1), E coli (Eco), B subtilis (Bsu), B stearothermophilus (Bst), and T maritima (Tma) are shown Accession numbers are P10442 (E coli RNase HII), Z99112 (BsuRNase HII), AB073670 (Bst-RNase HII), and NP_228723 (Tma-RNase HII) The ranges of the secondary structures of Tma-RNase HII, as well as the disordered regions, are shown below the sequences, based on its crystal structure (PDB code 2ETJ) The positions of the four conserved acidic residues, which form the active site, are indicated by arrows Amino acid residues conserved in at least three different proteins are highlighted in black Gaps are denoted by dashes Numbers represent the positions of the amino acid residues relative to the initiator methionine for each protein 2266 FEBS Journal 273 (2006) 2264–2275 ª 2006 The Authors Journal compilation ª 2006 FEBS H Chon et al High-activity type RNase HII from a psychrotroph are conserved These differences may be responsible for the difference in their near-UV CD spectra Enzymatic activity Fig SDS ⁄ PAGE of SIB1 RNase HII overproduced in E coli cells Samples were subjected to 15% SDS ⁄ PAGE and stained with Coomassie Brilliant Blue Lane 1, low molecular mass marker kit (Amersham Biosciences); lane 2, whole-cell extract (without IPTG induction); lane 3, whole-cell extract (with IPTG induction); lane 4, soluble fraction after sonication lysis of the cells with IPTG induction; lane 5, insoluble fraction after sonication lysis of the cells with IPTG induction; lane 6, purified SIB1 RNase HII Numbers along the gel represent the molecular masses of individual standard proteins around the aromatic residues of SIB1 RNase HII are considerably different from those of E coli RNase HII E coli RNase HII contains one tryptophan residue (Trp68), whereas SIB1 RNase HII does not Both proteins contain five tyrosine residues, but only three The dependencies of the SIB1 RNase HII activity on pH, salt, metal ion, and temperature were analyzed by changing one of these conditions from that used for assay (pH 8.5, 30 °C, 110 mm KCl, mm MnCl2) When enzymatic activity was determined at pH values from 7.1 to 12, SIB1 RNase HII exhibited the highest activity at around pH 10, like E coli RNase HII (data not shown) However, we measured the enzymatic activity at pH 8.5, because solubility of the metal ion decreases as pH increases, and both substrate and enzyme may not be fully stable at a highly alkaline pH When enzymatic activity was determined in the presence of 20, 30, 60, 110, and 220 mm NaCl or KCl, SIB1 RNased HII exhibited the highest activity in the presence of 60 mm NaCl or 110 mm KCl (data not shown) In contrast to the E coli RNase HII activity, which responds equally to NaCl and KCl [29], the specific activity of SIB1 RNased HII determined in the presence of 110 mm KCl was 1.8-fold higher than that determined in the presence of 60 mm NaCl SIB1 RNase HII exhibited enzymatic activity in the presence of MnCl2, MgCl2, and CoCl2, but not CaCl2, ZnCl2, BaCl2, NiCl2, CuCl2, FeCl2, or SrCl2 When enzymatic activity was determined in the presence of various concentrations (from 0.1 to 100 mm) of MnCl2, MgCl2, or CoCl2, SIB1 RNase HII exhibited the highest Mn2+-, Mg2+-, and Co2+-dependent activities in the presence of mm MnCl2, mm MgCl2, and 0.5 mm CoCl2, respectively (Fig 4) The specific Fig CD spectra Far-UV (left) and near-UV (right) CD spectra of SIB1 RNase HII (thick line) and E coli RNase HII (thin line) are shown Spectra were measured as described in Experimental procedures FEBS Journal 273 (2006) 2264–2275 ª 2006 The Authors Journal compilation ª 2006 FEBS 2267 High-activity type RNase HII from a psychrotroph H Chon et al Fig Dependence of SIB1 RNase HII activity on metal ion concentrations The enzymatic activity of SIB1 RNase HII was determined at 30 °C in 10 mM Tris ⁄ HCl (pH 8.5) containing 110 mM KCl, mM 2-mercaptoethanol, 50 lgỈmL)1 BSA, and various concentrations of MnCl2 (n), MgCl2 (s), or CoCl2 (m), using M13 DNA ⁄ RNA hybrid as a substrate The scale for the Mn2+-dependent activity is indicated on the left of the panel (solid line); those for the Mg2+and Co2+-dependent activities are indicated on the right of the panel (broken line) activity of SIB1 RNase HII determined in the presence of mm MnCl2 was 50- and 30-fold higher than those determined in the presence of mm MgCl2 and 0.5 mm CoCl2, respectively, indicating that, like E coli RNase HII, SIB1 RNase HII strongly prefers Mn2+ to Mg2+ or Co2+ for activity In contrast to SIB1 RNase HI, which is less active than E coli RNase HI, SIB1 RNase HII was more active than E coli RNase HII at all temperatures examined (Fig 5) When enzymatic activity was determined at various temperatures from 15 to 60 °C, SIB1 RNase HII and E coli RNase HII apparently exhibited the highest activity at 40 and 45 °C, respectively However, the amount of digestion product did not increase linearly with incubation time at 35 °C and above for SIB1 RNase HII, and 40 °C and above for E coli RNase HII (data not shown) These results indicate that SIB1 RNase HII and E coli RNase HII are not fully stable at these temperatures Therefore, we measured enzymatic activity at 30 °C and below The specific activities of SIB1 RNase HII, which were determined at 15 and 30 °C and a substrate concentration of 0.4 lm, were 25- and 20-fold higher than those of E coli RNase HII (Table 1) The kinetic parameters of SIB1 RNase HII were determined at 15 and 30 °C and compared with those of E coli RNase HII (Table 1) Vmax values for SIB1 2268 Fig Temperature dependence of the activities of SIB1 and E coli RNases H The M13 DNA ⁄ RNA hybrid (10 pmol) was hydrolyzed by pg of SIB1 RNase HII (d) or E coli RNase HII (s) at the temperatures indicated in 10 lL of the reaction mixture for 15 min, and the amount of acid-soluble digestion products accumulated upon enzymatic reaction was plotted against the temperature The composition of the reaction mixture for assay is 10 mM Tris ⁄ HCl (pH 8.5) containing mM MnCl2, 110 mM KCl, mM 2-mercatoethanol, and 50 lgỈmL)1 BSA for SIB1 RNase HII or 10 mM Tris ⁄ HCl (pH 8.5) containing mM MnCl2, 50 mM KCl, mM 2-mercaptoethanol, and 50 lgỈmL)1 BSA for E coli RNase HII as described in Experimental procedures Temperature dependencies of the activities of SIB1 RNase HI (thick broken line) and E coli RNase HI (thin broken line) are modified from Ohtani et al [33], such that pg of the enzyme was used for hydrolytic reaction The composition of the reaction mixture for assay is 10 mM Tris ⁄ HCl (pH 7.5) containing mM MgCl2, 30 mM KCl, mM 2-mercaptoethanol, and 50 lgỈmL)1 BSA for SIB1 RNase HI or 10 mM Tris ⁄ HCl (pH 8.0) containing 10 mM MgCl2, 50 mM NaCl, mM 2-mercaptoethanol, and 50 lgỈmL)1 BSA for E coli RNase HI Table Specific activities and kinetic parameters of SIB1 and E coli RNases HII Hydrolysis of the M13 DNA ⁄ RNA hybrid by the enzyme was carried out at the temperatures indicated under the conditions described in Experimental procedures Errors, which represent the 67% confidence limits, are all at or below ± 20% of the values reported Protein SIB1 RNase HII E coli RNase HII Temperature (°C) Specific activity (unitsỈmg)1) Km (lM) Vmax (unitsỈmg)1) 15 30 15 30 7.6 22 0.31 1.1 0.071 0.075 0.26 0.26 9.0 26 0.51 1.8 FEBS Journal 273 (2006) 2264–2275 ª 2006 The Authors Journal compilation ª 2006 FEBS H Chon et al High-activity type RNase HII from a psychrotroph RNase HII at 15 and 30 °C were 18- and 14-fold higher than those of E coli RNase HII, respectively Km values for SIB1 RNase HII at these temperatures were both 3.5-fold lower than those of E coli RNase HII These results indicate that SIB1 RNase HII exhibits a higher hydrolysis rate and substrate-binding affinity than E coli RNase HII at both low and moderate temperatures A Substrate specificity and cleavage-site specificity To examine whether SIB1 RNase HII specifically cleaves the RNA strand of RNA ⁄ DNA hybrids, the 12 b RNA, 12 b DNA, 12 b RNA ⁄ RNA duplex, 12 b DNA ⁄ DNA duplex, and 12 b RNA ⁄ DNA hybrid were used as substrates for the enzymatic reaction The enzymatic reaction was performed under the same conditions as used for hydrolysis of the M13 DNA ⁄ RNA hybrid SIB1 RNase HII did not cleave these substrates except for the 12 b RNA ⁄ DNA hybrid (data not shown), indicating that SIB1 RNase HII does not exhibit nuclease activity other than the RNase H activity SIB1 RNase HII cleaved this substrate at multiple sites, but most preferentially at a6–u7 (Fig 6) E coli RNase HII has been reported to cleave this substrate in a similar manner [29] These results suggest that the cleavage-site specificity of SIB1 RNase HII is similar to that of E coli RNase HII The reason why these RNases H preferentially cleave the substrate at a unique site remains to be fully understood B Stability The stabilities of SIB1 and E coli RNases HII against heat inactivation were analyzed by incubating the protein in 20 mm Tris ⁄ HCl (pH 7.5) containing 0.1 m KCl, mm EDTA, 10% glycerol, and 0.1 mgỈmL)1 bovine serum albumin (BSA) at 30 °C, and measuring residual activity at the same temperature with appropriate intervals Half-lives were determined to be  30 for SIB1 RNase HII and  45 for E coli RNase HII (data not shown), indicating that SIB1 RNase HII is less stable than E coli RNase HII, although the difference is marginal It is noted that these proteins are fully stable at 30 °C for at least 15 under the assay conditions, probably because they are stabilized in the presence of metal cofactor and substrate To compare the conformational stability of SIB1 RNase HII with that of E coli RNase HII, ureainduced unfolding of the protein was analyzed using CD Neither protein was fully reversible in urea-induced unfolding under the conditions examined Comparison of the urea denaturation curves of these Fig Cleavage of 12 b RNA ⁄ DNA hybrid by SIB1 RNase HII (A) Autoradiograph of cleavage reactions Hydrolyses of the 5¢-endlabeled 12 b RNA hybridized to the 12 b DNA with SIB1 RNase HII were carried out at 30 °C for 15 Hydrolysates were separated on a 20% polyacrylamide gel containing M urea as described in Experimental procedures The substrate concentration was 1.0 lM Lane 1, partial digest of the 5¢-end-labeled 12 b RNA with snake venom phosphodiesterase; lane 2, untreated substrate; lane 3, hydrolysate with 2.9 pg of the enzyme; lane 4, hydrolysate with 29 pg of the enzyme; lane 5, hydrolysate with 290 pg of the enzyme; lane 6, hydrolysate with 2.9 ng of the enzyme; lane 7, hydrolysate with 29 ng of the enzyme (B) Sites and extents of cleavage by SIB1 RNase HII Cleavage sites of the 12 b RNA ⁄ DNA hybrid by the enzyme are shown by arrows The differences in the lengths of the arrows reflect relative cleavage intensities at positions indicated Deoxyribonucleotides are shown in upper case and ribonucleotides are shown in lower case FEBS Journal 273 (2006) 2264–2275 ª 2006 The Authors Journal compilation ª 2006 FEBS 2269 High-activity type RNase HII from a psychrotroph H Chon et al Fig Urea-induced unfolding of proteins The apparent fraction of unfolded protein, determined by CD measurement, is shown as a function of urea concentration for SIB1 (d) and E coli (s) RNases HII The fraction unfolded was calculated with an equation given by Pace [52] in which a least-squares analysis of the pre- and post-transition base lines is applied proteins indicated that SIB1 RNase HII is slightly less stable than E coli RNase HII (Fig 7) The midpoint of the urea denaturation curve, [D]1 ⁄ 2, was determined as  1.6 m for SIB1 RNase HII and 1.8 m for E coli RNase HII, indicating that SIB1 RNase HII is less stable than E coli RNase HII by  0.2 m in [D]1 ⁄ Complementation assay E coli MIC2067 [27] and E coli MIC2067(DE3) [18] show a RNase H-dependent temperature-sensitive (ts) growth phenotype This ts phenotype can be complemented by the functional RNase H genes To examine whether the Sh-rnhB gene complements this ts phenotype, a strain of E coli MIC2067(DE3) that can overproduce SIB1 RNase HII was grown in the absence of isopropyl thio-b-d-galactosidase (IPTG) at permissive (30 °C) and nonpermissive (42 °C) temperatures This strain was able to grow at 42 °C (data not shown), indicating that the Sh-rnhB gene complements the ts growth phenotype of MIC2067(DE3) This result suggests that SIB1 RNase HII exhibits the enzymatic activity in vivo Discussion Multiple RNases H in the SIB1 cells We have shown that the SIB1 genome contains the rnhB gene encoding RNase HII We have previously shown that this genome also contains the rnhA gene encoding 2270 RNase HI [33] Thus, the SIB1 genome contains the rnhA and rnhB genes, like the E coli genome SIB1 RNases HI and HII are similar to E coli RNases HI and HII, respectively, in terms of optimum pH, metal ion preference, and cleavage-site specificity These SIB1 proteins are less stable than their E coli counterparts as expected However, SIB1 RNase HII is more stable and more active than SIB1 RNase HI, whereas E coli RNase HII is less stable and less active than E coli RNase HI It has also been reported that B subtilis [26] and B stearothermophilus [30,31] contains two RNases H (RNases HII and HIII), which differ greatly in activity The specific activities of Bsu-RNase HIII (10 unitsỈmg)1) and Bst-RNase HIII (1.9 unitsỈmg)1) are 20- and 95-fold higher than those of Bsu-RNase HII (0.5 unitsỈmg)1) and Bst-RNase HII (0.02 unitsỈmg)1), respectively, at 30 °C, indicating that RNase HIII is more active than RNase HII in these Bacillus strains These results suggest that the bacterial cells usually contain low- and high-activity type RNases H, but these types are not correlated with the RNase H families Gene-disruption studies [27] suggest that the functions of two different RNases H within the single cells overlap Phylogenetic analyses suggest that type RNases H have diverged from a common ancestor by neutral drift, whereas type RNases H have been transferred horizontally among different organisms [10] An RNase H transferred horizontally may provide a selective advantage to recipients However, once a cell that already has an RNase H receives a second RNase H by lateral gene transfer, the responsibilities can be shared in ways that would not necessarily be repeated following other occurrences of transfer In some instances, the incoming RNase H may retain the selective traits, whereas in others, the resident and incoming RNase H may swap some or all of their properties Because RNases HI and HII represent high-activity type RNases H in E coli and SIB1, respectively, these RNases H may retain the selective traits However, it remains to be determined whether the RNase HI and RNase HII activities represent the minor and major RNase H activities in the SIB1 cells, respectively, because the production levels of these RNases H in the SIB1 cells have yet to be analyzed In addition, the third RNase H may function as a substitute for RNase HI in SIB1 cells The SIB1 genome contains one additional gene encoding another type RNase H, which complements the RNase H-dependent ts growth phenotype of MIC2067 (H Chon, unpublished data) This protein consists of 262 amino acids and shows amino acid sequence identity of 26% with SIB1 RNase HI and 17% with E coli RNase HI Interestingly, this protein has a double-stranded RNA- FEBS Journal 273 (2006) 2264–2275 ª 2006 The Authors Journal compilation ª 2006 FEBS H Chon et al High-activity type RNase HII from a psychrotroph binding domain (dsRBD) at the N-terminus, like various eukaryotic type RNases H, including human RNase H1 [35,36] The Shewanella oneidensis and E coli genomes not contain this gene, indicating that SIB1 is unique in that it has both type RNases H with and without dsRBD less stable than E coli RNase HI by 3.4 m in [urea]1 ⁄ [46] Therefore, like other cold-adapted enzymes, SIB1 RNase HII may acquire a conformational flexibility at low temperatures at the cost of stability Cold adaptation Cells and plasmids Psychrophiles and psychrotrophs adapt to low temperatures by producing cold-adapted enzymes, which are characterized by increased activity at low temperatures and decreased stability at any temperature compared with their mesophilic and thermophilic counterparts [37–42] SIB1 cells also produce cold-adapted enzymes, such as alkaline phosphatase (APase) [43], RNase HI [33], and FKBP22 with peptidyl prolyl cis–trans isomerase activity [44,45] These proteins are highly thermolabile compared with their mesophilic counterparts For example, SIB1 APase is rapidly inactivated at temperatures at which E coli APase is stable [43], SIB1 FKBP22 is less stable than E coli FKBP22 by  30 °C in Tm [45], and SIB1 RNase HI is less stable than E coli RNase HI by  35 °C in T1 ⁄ [33] Tm is the midpoint of the thermal denaturation curve and T1 ⁄ is the temperature at which the enzyme loses half of its activity In addition, the optimum temperatures of SIB1 APase, SIB1 RNase HI, and SIB1 FKBP22 for activity are lower than those of their E coli counterparts by 30, 20, and at least 15 °C, respectively SIB1 RNase HII, however, does not show typical features of cold-adapted enzymes, as long as its activity and stability are compared with those of E coli RNase HII SIB1 RNase HII is more active than E coli RNase HII over the entire temperature range examined SIB1 RNase HII is less stable than E coli RNase HII, but only slightly This small difference in stability is probably caused by decreased stability of mesophilic E coli RNase HII, rather than increased stability of psychrotrophic SIB1 RNase HII As mentioned above, RNase HII is probably functionally degenerated in E coli due to the lack of selective pressure against stability and activity If the stability and activity of RNase HII, however, are compared with those of E coli RNase HI, which represents high-activity type RNase H in E coli, SIB1 RNase HII shows typical features of cold-adapted enzymes The optimum temperature of SIB1 RNase HII for activity is shifted downward by 10 °C compared with that of E coli RNase HI, and the enzymatic activity of SIB1 RNase HII is higher and lower than that of E coli RNase HI at < 45 °C and > 45 °C, respectively (Fig 5) In addition, SIB1 RNase HII is considerably The psychrotrophic bacterium Shewanella sp SIB1 was previously isolated from Japanese oil reservoir water in our laboratory [32] E coli MIC2067(DE3) [F– k IN(rrnDrrnE)1 rnhA339::cat rnhB716::kam kDE3] was constructed previously [29] Plasmids pBR322 and pUC18 were obtained from Takara Shuzo (Otsu, Japan) and pET-3a was from Novagen (Madison, WI, USA) Plasmid pBR860 containing the E coli rnhA gene and its promoter was constructed previously [47] E coli MIC2067(DE3) transformants were grown in NZCYM medium (Novagen) containing 50 mgỈL)1 ampicillin and 0.1% (w ⁄ v) glucose Other E coli transformants were grown in Luria–Bertani medium containing 50 mgỈL)1 ampicillin Experimental procedures Materials [32P]ATP[cP] (> 5000 CiỈmmol)1) was obtained from Amersham Biosciences (Piscataway, NJ, USA) Snake venom phosphodiesterase from Crotalus durissus was from Boehringer-Mannheim (Tokyo, Japan) Recombinant E coli RNase HII was purified as described previously [29] All DNA oligomers for PCR were synthesized by Hokkaido System Science (Sapporo, Japan) Restriction and modifying enzymes were from Takara Shuzo Gene cloning The genomic DNA of Shewanella sp SIB1 was prepared as described previously [48] and used as a template to amplify a part of the rnhB gene (Sh-rnhB) by PCR The sequences of the PCR primers are 5¢-ATTGCAGGTGTTGAT GAAGTWGG-3¢ for the 5¢-primer and 5¢-TTTAACTG GACCAAAACTTTTACGRTG-3¢ for the 3¢-primer, where R represents A + G and W represents A + T PCR was performed with GeneAmp PCR system 2400 (Perkin-Elmer, Tokyo, Japan) using a KOD polymerase (Toyobo, Kyoto, Japan) according to procedures recommended by the supplier The amplified DNA fragment (540 bp) was used as a probe for Southern blotting and colony hybridization to clone the entire Sh-rnhB gene Southern blotting and colony hybridization were carried out using the AlkPhos Direct system (Amersham Biosciences) according to procedures recommended by the supplier The DNA sequence was determined with a Prism 310 DNA sequencer (PerkinElmer) Nucleotide and amino acid sequence analyses, FEBS Journal 273 (2006) 2264–2275 ª 2006 The Authors Journal compilation ª 2006 FEBS 2271 High-activity type RNase HII from a psychrotroph H Chon et al including the localization of open reading flames and determination of molecular mass were performed using dnasis software (Hitachi Software) The nucleotide sequence of the Sh-rnhB gene is deposited in DDBJ under accession number AB245507 Construction of plasmids Plasmid pBR1100eS for complementation assay was constructed by performing PCR twice The sequences of the PCR primers are 5¢-TTCAAGAATTCTCATGTTTTGAC -3¢ for the 5¢-primer, 5¢-CGCGTCGACACTAACAG GGCTGATTGACGAGTC-3¢ for the 3¢-primer, 5¢TCTACCAGAG ATG TCGACATTATCGGTTGTG-3¢ for the 5¢-fusion primer and 5¢-TAATGTCGA CAT CTCT GGTAGACTTCCTGTAA-3¢ for the 3¢-fusion primer In these sequences, underlined bases show the positions of the EcoRI (5¢-primer) and SalI (3¢-primer) sites, boxed bases show the position of the codon for the initial methionine residue, and italic bases represent those of the Sh-rnhB gene In the first PCR, the 400 bp DNA fragment containing the promoter and ribosome binding site of the E coli rnhA gene was amplified with 5¢-primer and 3¢-fusion primer using pBR860 as a template Likewise, the 700 bp DNA fragment containing the entire Sh-rnhB gene was amplified with 5¢-fusion primer and 3¢-primer using the cloned Sh-rnhB gene as a template These two DNA fragments were mixed and amplified with 5¢- and 3¢-primers The resultant 1100 bp DNA fragment was ligated to the EcoRI–SalI site of pBR322 In pBR1100eS, transcription and translation of the Sh-rnhB gene are controlled by the promoter and the SD sequence of the E coli rnhA gene Plasmid pET680S for overproduction of SIB1 RNase HII was constructed by ligating the DNA fragment, which was amplified by PCR using the cloned Sh-rnhB gene as a template, to the NdeI–SalI site of pET-3a The PCR primer sequences are 5¢-CTAGGATAAGCTTCATATGTCGACA TTATCGGTT-3¢ for the 5¢-primer and 5¢-CGCGCGGA TCCAACGATAAACTCGCTTA-3¢ for the 3¢-primer, underlined bases show the position of the NdeI (5¢-primer) and BamHI (3¢-primer) sites Overproduction and purification E coli MIC2067(DE3) was transformed with pET680S and grown at 30 °C When the optical density at 660 nm of the culture reached around 0.6, mm of IPTG was added to the culture medium and cultivation was continued at 30 °C for 30 Then, the temperature of the growth medium was shifted to 15 °C and cultivation was continued at 15 °C for an additional 15 h Cells were harvested by centrifugation at 6000 g for 10 min, suspended in 20 mm Tris ⁄ HCl (pH 8.0) containing mm EDTA and mm dithiothreitol (buffer A), disrupted by sonication lysis, and centrifuged at 30 000 g for 30 The supernatant was collected, dia- 2272 lyzed against buffer A, and loaded onto a DE52 column equilibrated with the same buffer The flow-through fraction was collected and loaded onto a Hitrap Heparin HP column (Amersham Biosciences) equilibrated with buffer A The protein was eluted from the column by linearly increasing the NaCl concentration from to 0.5 m The fractions containing SIB1 RNase HII with high purity were combined and used for further analyses The purity of the protein was confirmed by SDS ⁄ PAGE [49], followed by staining with Coomassie Brilliant Blue Protein concentration The protein concentration was determined from the UV absorption on the basis that the absorbance at 280 nm of a 0.1% solution is 0.34 for SIB1 RNase HII and 0.61 for E coli RNase HII These values were calculated by using e of 1576 m)1 cm)1 for Tyr and 5225 m)1 cm)1 for Trp at 280 nm [50] Biochemical characterizations The molecular mass of the protein was estimated by gel-filtration column chromatography using a Superdex 200 16 ⁄ 60 gel filtration column (Amersham Biosciences) equilibrated with buffer A containing 0.15 m NaCl Elution was performed at a flow rate of 0.5 mLỈmin)1 BSA (67 kDa), ovalbumin (44 kDa), chymotrypsinogen A (25 kDa), and RNase A (14 kDa) were used as standard proteins CD spectra were measured on a J-725 spectropolarimeter (Japan Spectroscopic) at °C The far-UV CD spectra were obtained using solutions containing protein at 0.25– 0.3 mgỈmL)1 in buffer A containing 0.15 m NaCl in a cell with an optical path length of mm For near-UV CD spectra, the protein concentration and optical path length were increased to 0.9–1.2 mgỈmL)1 and 10 mm, respectively The mean residue ellipticity, h, which has the units of deg cm2Ỉdmol)1, was calculated by using an average amino acid relative molecular mass of 110 Enzymatic activity The RNase H activity was determined at 30 °C by measuring the amount of radioactivity of the acid-soluble digestion product from the substrate, the [3H]-labeled M13 DNA ⁄ RNA hybrid, as described previously [51] The buffer was 10 mm Tris ⁄ HCl (pH 8.5) containing mm MnCl2, 110 mm KCl, mm 2-mercaptoethanol, and 50 lgỈmL)1 BSA for SIB1 RNase HII or 10 mm Tris ⁄ HCl (pH 8.5) containing mm MnCl2, 50 mm KCl, mm 2-mercaptoethanol, and 50 lgỈmL)1 BSA for E coli RNase HII One unit is defined as the amount of enzyme producing lmol of acidsoluble material per at 30 °C The specific activity was defined as the enzymatic activity per milligram of protein FEBS Journal 273 (2006) 2264–2275 ª 2006 The Authors Journal compilation ª 2006 FEBS H Chon et al To determine the kinetic parameters, substrate concentration was varied from 0.04 to 0.4 lm The hydrolysis of the M13 DNA ⁄ RNA hybrid by the enzyme followed Michaelis– Menten kinetics and the kinetic parameters were determined from the Lineweaver–Burk plot To analyze pH dependence, 10 mm Tris ⁄ HCl (pH 7.1–8.8), glycine ⁄ NaOH (pH 8.3–9.8), or CAPS ⁄ NaOH (pH 9.0–12.0) was used as a buffer for assay To analyze divalent cation or salt dependence, the enzymatic activity was determined in the presence of various concentrations of MgCl2, MnCl2 CoCl2, NaCl, or KCl For cleavage of the oligomeric substrates, the 12 b RNA–DNA, RNA–RNA, and DNA–DNA duplexes (1 lm) were prepared by hybridizing the 5¢-end-labeled 12 b RNA or DNA with a sequence of 5¢-CGGAGA(U ⁄ T)GACGG-3¢ with 1.5 molar equivalent of the complementary 12 b DNA or RNA, as described previously [26] Hydrolyses of the substrates at 30 °C for 15 and separation of the hydrolysates on a 20% polyacrylamide gel containing m urea were carried out as described previously [26] The reaction buffer was the same as that for the hydrolysis of M13 DNA ⁄ RNA hybrid The products were identified by comparing their migration on the gel with those of the oligonucleotides generated by partial digestion of 5¢-end-labeled 12 b RNA with snake venom phosphodiesterase High-activity type RNase HII from a psychrotroph Urea denaturation Urea denaturation curves were obtained at 10 °C by monitoring the CD values at 220 nm with variation of the urea concentration Proteins were dissolved in 20 mm Tris ⁄ HCl (pH 8.0) containing mm MnCl2, mm dithiothreitol, 0.15 m NaCl and an appropriate concentration of urea and incubated for at least 30 prior to the measurement The protein concentration was  0.1 mgỈmL)1, and the optical path length was mm 10 Acknowledgements We thank Drs M Morikawa and M Haruki for helpful discussions This work was supported in part by a Grant-in-Aid for National Project on Protein Structural and Functional Analyses and by a Grant-in-Aid for Scientific Research (No 16041229) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by an Industrial Technology Research Grant Program from the New Energy and Industrial Technology Development Organization (NEDO) of Japan References Crouch RJ & Dirksen M-L (1982) Ribonuclease H In Nuclease 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Bsu -RNase HII, and Bst -RNase HII, ... Escherichia coli cells contain RNases HI and HII, yeast and human cells contain RNases H1 and H2 , and Bacillus subtilis and Bacillus stearothermophilus cells contain RNases HII and HIII The physiological... Mn2+ to Mg2+ for activity The specific activities of E coli RNase HI, Bsu -RNase HIII, and Bst -RNase HIII are higher than E coli RNase HII, Bsu -RNase HII, and Bst -RNase HII by 13, 20, and 100 times,