Because SIB1 RNase HII was also much more active than SIB1 RNase HI, RNases HI and HII rep-resent low- and high-activity type RNases H, respectively, in SIB1.. Because c-proteobacteria,
Trang 1Shewanella sp SIB1 as a high-activity type RNase H
Hyongi Chon1, Takashi Tadokoro1, Naoto Ohtani1, Yuichi Koga1, Kazufumi Takano1,2
and Shigenori Kanaya1
1 Department of Material and Life Science, Graduate School of Engineering, Osaka University, Japan
2 PRESTO, Osaka, Japan
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
replica-tion, repair, and transcription [2–9] RNase H is widely
present in various organisms, including bacteria,
arch-aea, and eukaryotes [10] RNase H is also present in
retroviruses as a C-terminal domain of reverse
tran-scriptase 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 1 and type 2 RNases H, which are evolutionarily unre-lated [10] Bacterial RNases HI, eukaryotic RNases H1, and retroviral RNases H are members of the type 1 RNase H family Bacterial RNases HII, bacterial RNases HIII, archaeal RNases HII, and eukaryotic RNases H2 are members of the type 2 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
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 6 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, Shewa-nellasp SIB1 was cloned, overexpressed in Escherichia coli, and the recom-binant 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 30C 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
30C 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 rep-resent low- and high-activity type RNases H, respectively, in SIB1 In con-trast, 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
Abbreviations
APase, alkaline phosphatase; BSA, bovine serum albumin; IPTG, isopropyl thio-b- D -galactoside; RNase H, ribonuclease H.
Trang 2a helices This folding motif, termed RNase H fold, has
been found in the crystal structures of various
function-ally unrelated proteins, such as integrase [19,20],
trans-posase [21], RuvC Holliday-junction resolvase [22], and
the PIWI domain of argonaute protein [23,24] In
addi-tion, 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
scis-sile 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
phe-notypes of E coli and B subtilis are changed
consider-ably 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,
Bsu-RNase HIII, and Bst-Bsu-RNase HIII, prefers Mg2+ to
Mn2+for activity, whereas other three, such as E coli
RNase HII, Bsu-RNase HII, and Bst-RNase HII,
pre-fer 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 20C [32] This strain
can grow at 0C, but not at temperatures > 30 C
We previously cloned the rnhA gene encoding
RNase HI from this strain and biochemically
charac-terized 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
RNase HI, prefers Mg2+ to Mn2+ for activity Nevertheless, SIB1 RNase HI is considerably less sta-ble 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 com-plete 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 character-ize 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 frag-ment of the SIB1 genome contained the entire Sh-rnhB gene (data not shown) Determination of the nucleo-tide 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 sub-unit 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]
Trang 3Amino 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 1 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
expec-ted 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 mgÆL)1 for the soluble form and
20 mgÆL)1 for the insoluble form, and 10 mg of the purified protein was obtained from 1 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 calcu-lated value These results strongly suggest that, like other RNases HII, SIB1 RNase HII exists in a mono-meric 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 consid-erably different from that of E coli RNase HII (Fig 3B), suggesting that the local conformations
Fig 1 Alignment of the RNase HII sequences Amino acid sequences of RNases HII from Shewanella sp SIB1 (SIB1), E coli (Eco), B subtilis (Bsu), B ste-arothermophilus (Bst), and T maritima (Tma) are shown Accession numbers are P10442 (E coli RNase HII), Z99112 (Bsu-RNase HII), AB073670 (Bst-(Bsu-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 posi-tions 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.
Trang 4around the aromatic residues of SIB1 RNase HII are
considerably different from those of E coli RNase
HII E coli RNase HII contains one tryptophan
resi-due (Trp68), whereas SIB1 RNase HII does not Both
proteins contain five tyrosine residues, but only three
are conserved These differences may be responsible for the difference in their near-UV CD spectra
Enzymatic activity 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, 30C, 110 mm KCl, 1 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 pres-ence 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 spe-cific 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 activ-ities in the presence of 1 mm MnCl2, 5 mm MgCl2, and 0.5 mm CoCl2, respectively (Fig 4) The specific
Fig 2 SDS ⁄ PAGE of SIB1 RNase HII overproduced in E coli cells.
Samples were subjected to 15% SDS ⁄ PAGE and stained with
Coo-massie Brilliant Blue Lane 1, low molecular mass marker kit
(Amer-sham 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
induc-tion; 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.
Fig 3 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.
Trang 5activity of SIB1 RNase HII determined in the presence
of 1 mm MnCl2was 50- and 30-fold higher than those
determined in the presence of 5 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
deter-mined at various temperatures from 15 to 60C, SIB1
RNase HII and E coli RNase HII apparently
exhib-ited the highest activity at 40 and 45C, respectively
However, the amount of digestion product did not
increase linearly with incubation time at 35C and
above for SIB1 RNase HII, and 40C 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 30C and below
The specific activities of SIB1 RNase HII, which were
determined at 15 and 30C and a substrate
concentra-tion 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 30C and compared with those
of E coli RNase HII (Table 1) Vmax values for SIB1
Fig 4 Dependence of SIB1 RNase HII activity on metal ion
con-centrations The enzymatic activity of SIB1 RNase HII was
deter-mined at 30 C in 10 m M Tris ⁄ HCl (pH 8.5) containing 110 m M KCl,
1 m M 2-mercaptoethanol, 50 lgÆmL)1BSA, and various
concentra-tions of MnCl2(n), MgCl2(s), or CoCl2(m), using M13 DNA ⁄ RNA
hybrid as a substrate The scale for the Mn 2+ -dependent activity is
indicated on the left of the panel (solid line); those for the Mg2+
-and Co 2+ -dependent activities are indicated on the right of the
panel (broken line).
Fig 5 Temperature dependence of the activities of SIB1 and
E coli RNases H The M13 DNA ⁄ RNA hybrid (10 pmol) was hydro-lyzed by 4 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 m M Tris ⁄ HCl (pH 8.5) containing 1 m M MnCl2, 110 m M KCl, 1 m M 2-mercatoeth-anol, and 50 lgÆmL)1BSA for SIB1 RNase HII or 10 m M Tris ⁄ HCl (pH 8.5) containing 5 m M MnCl 2 , 50 m M KCl, 1 m M 2-mercaptoeth-anol, and 50 lgÆmL)1 BSA for E coli RNase HII as described in Experimental procedures Temperature dependencies of the activit-ies of SIB1 RNase HI (thick broken line) and E coli RNase HI (thin broken line) are modified from Ohtani et al [33], such that 4 pg of the enzyme was used for hydrolytic reaction The composition of the reaction mixture for assay is 10 m M Tris ⁄ HCl (pH 7.5) contain-ing 5 m M MgCl 2 , 30 m M KCl, 1 m M 2-mercaptoethanol, and
50 lgÆmL)1 BSA for SIB1 RNase HI or 10 m M Tris ⁄ HCl (pH 8.0) containing 10 m M MgCl2, 50 m M NaCl, 1 m M 2-mercaptoethanol, and 50 lgÆmL)1BSA for E coli RNase HI.
Table 1 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 rep-resent the 67% confidence limits, are all at or below ± 20% of the values reported.
Protein
Temperature (C)
Specific activity (unitsÆmg)1)
Km (l M )
Vmax (unitsÆmg)1)
Trang 6RNase HII at 15 and 30C 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
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
con-ditions as used for hydrolysis of the M13 DNA⁄ RNA
hybrid SIB1 RNase HII did not cleave these
sub-strates 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
activ-ity 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
Stability
The stabilities of SIB1 and E coli RNases HII against
heat inactivation were analyzed by incubating the
pro-tein in 20 mm Tris⁄ HCl (pH 7.5) containing 0.1 m KCl,
1 mm EDTA, 10% glycerol, and 0.1 mgÆmL)1 bovine
serum albumin (BSA) at 30C, and measuring residual
activity at the same temperature with appropriate
inter-vals Half-lives were determined to be 30 min for
SIB1 RNase HII and 45 min for E coli RNase HII
(data not shown), indicating that SIB1 RNase HII is
less stable than E coli RNase HII, although the
differ-ence is marginal It is noted that these proteins are fully
stable at 30C for at least 15 min under the assay
con-ditions, 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,
urea-induced 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
A
B
Fig 6 Cleavage of 12 b RNA ⁄ DNA hybrid by SIB1 RNase HII (A) Autoradiograph of cleavage reactions Hydrolyses of the 5¢-end-labeled 12 b RNA hybridized to the 12 b DNA with SIB1 RNase HII were carried out at 30 C for 15 min Hydrolysates were separated
on a 20% polyacrylamide gel containing 7 M urea as described in Experimental procedures The substrate concentration was 1.0 l M 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 posi-tions indicated Deoxyribonucleotides are shown in upper case and ribonucleotides are shown in lower case.
Trang 7proteins 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
sta-ble than E coli RNase HII by 0.2 m in [D]1 ⁄ 2
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
comple-mented by the functional RNase H genes To examine
whether the Sh-rnhB gene complements this ts
pheno-type, a strain of E coli MIC2067(DE3) that can
over-produce SIB1 RNase HII was grown in the absence of
isopropyl thio-b-d-galactosidase (IPTG) at permissive
(30C) and nonpermissive (42 C) temperatures This
strain was able to grow at 42C (data not shown),
indi-cating 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
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 unitsÆmg)1) and Bst-RNase HIII (1.9 unitsÆmg)1) are 20- and 95-fold higher than those of Bsu-RNase HII (0.5 unitsÆmg)1) and Bst-RNase HII (0.02 unitsÆmg)1), respectively, at 30C, 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 func-tions of two different RNases H within the single cells overlap Phylogenetic analyses suggest that type 2 RNases H have diverged from a common ancestor by neutral drift, whereas type 1 RNases H have been transferred horizontally among different organisms [10] An RNase H transferred horizontally may pro-vide 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 substi-tute for RNase HI in SIB1 cells The SIB1 genome contains one additional gene encoding another type 1 RNase H, which complements the RNase H-dependent
ts growth phenotype of MIC2067 (H Chon, unpub-lished 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-Fig 7 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.
Trang 8binding domain (dsRBD) at the N-terminus, like
var-ious eukaryotic type 1 RNases H, including human
RNase H1 [35,36] The Shewanella oneidensis and
E coli genomes do not contain this gene, indicating
that SIB1 is unique in that it has both type 1
RNases H with and without dsRBD
Cold adaptation
Psychrophiles and psychrotrophs adapt to low
temper-atures 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
iso-merase activity [44,45] These proteins are highly
ther-molabile compared with their mesophilic counterparts
For example, SIB1 APase is rapidly inactivated at
tem-peratures 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⁄ 2 [33] Tm is the
midpoint of the thermal denaturation curve and T1 ⁄ 2
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
counter-parts by 30, 20, and at least 15C, respectively
SIB1 RNase HII, however, does not show typical
features of cold-adapted enzymes, as long as its
activ-ity and stabilactiv-ity 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
men-tioned above, RNase HII is probably functionally
degenerated in E coli due to the lack of selective
pres-sure 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-activ-ity type RNase H in E coli, SIB1 RNase HII shows
typical features of cold-adapted enzymes The
opti-mum temperature of SIB1 RNase HII for activity is
shifted downward by 10C 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 < 45C and > 45 C, respectively
(Fig 5) In addition, SIB1 RNase HII is considerably
less stable than E coli RNase HI by 3.4 m in [urea]1⁄ 2 [46] Therefore, like other cold-adapted enzymes, SIB1 RNase HII may acquire a conformational flexibility at low temperatures at the cost of stability
Experimental procedures
Cells and plasmids The psychrotrophic bacterium Shewanella sp SIB1 was pre-viously isolated from Japanese oil reservoir water in our laboratory [32] E coli MIC2067(DE3) [F– k IN(rrnD-rrnE)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 con-structed previously [47] E coli MIC2067(DE3)
containing 50 mgÆL)1 ampicillin and 0.1% (w⁄ v) glucose Other E coli transformants were grown in Luria–Bertani medium containing 50 mgÆL)1ampicillin
Materials [32P]ATP[cP] (> 5000 CiÆmmol)1) was obtained from Amersham Biosciences (Piscataway, NJ, USA) Snake venom phosphodiesterase from Crotalus durissus was from Boehringer-Mannheim (Tokyo, Japan) Recombinant
E coliRNase HII was purified as described previously [29] All DNA oligomers for PCR were synthesized by
Hokkai-do System Science (Sapporo, Japan) Restriction and modi-fying 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
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 sup-plier 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 (Perkin-Elmer) Nucleotide and amino acid sequence analyses,
Trang 9including the localization of open reading flames and
deter-mination of molecular mass were performed using dnasis
software (Hitachi Software) The nucleotide sequence of the
Sh-rnhBgene is deposited in DDBJ under accession number
AB245507
Construction of plasmids
Plasmid pBR1100eS for complementation assay was
con-structed by performing PCR twice The sequences of the
PCR primers are 5¢-TTCAAGAATTCTCATGTTTTGAC
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
contain-ing the promoter and ribosome bindcontain-ing site of the E coli
rnhA gene was amplified with 5¢-primer and 3¢-fusion
pri-mer 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
frag-ments 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
tem-plate, to the NdeI–SalI site of pET-3a The PCR primer
sequences are 5¢-CTAGGATAAGCTTCATATGTCGACA
TTATCGGTT-3¢ for the 5¢-primer and 5¢-CGCGCGGA
underlined bases show the position of the NdeI (5¢-primer)
and BamHI (3¢-primer) sites
Overproduction and purification
E coliMIC2067(DE3) was transformed with pET680S and
grown at 30C When the optical density at 660 nm of the
culture reached around 0.6, 1 mm of IPTG was added to
the culture medium and cultivation was continued at 30C
for 30 min Then, the temperature of the growth medium
was shifted to 15C and cultivation was continued at 15 C
for an additional 15 h Cells were harvested by
centrifuga-tion at 6000 g for 10 min, suspended in 20 mm Tris⁄ HCl
(pH 8.0) containing 1 mm EDTA and 1 mm dithiothreitol
(buffer A), disrupted by sonication lysis, and centrifuged at
30 000 g for 30 min The supernatant was collected,
dia-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 0 to 0.5 m The fractions con-taining 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 coliRNase HII These values were calculated by using e
of 1576 m)1cm)1 for Tyr and 5225 m)1cm)1 for Trp at
280 nm [50]
Biochemical characterizations The molecular mass of the protein was estimated by gel-fil-tration column chromatography using a Superdex 200
16⁄ 60 gel filtration column (Amersham Biosciences) equili-brated with buffer A containing 0.15 m NaCl Elution was performed at a flow rate of 0.5 mLÆmin)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 4C The far-UV CD spectra were obtained using solutions containing protein at 0.25– 0.3 mgÆmL)1 in buffer A containing 0.15 m NaCl in a cell with an optical path length of 2 mm For near-UV CD spectra, the protein concentration and optical path length were increased to 0.9–1.2 mgÆmL)1 and 10 mm, respect-ively The mean residue ellipticity, h, which has the units of deg cm2Ædmol)1, was calculated by using an average amino acid relative molecular mass of 110
Enzymatic activity The RNase H activity was determined at 30C 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 1 mm MnCl2,
110 mm KCl, 1 mm 2-mercaptoethanol, and 50 lgÆmL)1 BSA for SIB1 RNase HII or 10 mm Tris⁄ HCl (pH 8.5) con-taining 5 mm MnCl2, 50 mm KCl, 1 mm 2-mercaptoethanol, and 50 lgÆmL)1 BSA for E coli RNase HII One unit is defined as the amount of enzyme producing 1 lmol of acid-soluble material per min at 30C The specific activity was defined as the enzymatic activity per milligram of protein
Trang 10To determine the kinetic parameters, substrate
concentra-tion 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
en-zymatic 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
(1 lm) were prepared by hybridizing the 5¢-end-labeled
5¢-CGG-AGA(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 30C for 15 min and
separation of the hydrolysates on a 20% polyacrylamide
gel containing 7 m urea were carried out as described
previ-ously [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
phosphodi-esterase
Urea denaturation
Urea denaturation curves were obtained at 10C by
monit-oring the CD values at 220 nm with variation of the urea
concentration Proteins were dissolved in 20 mm Tris⁄ HCl
(pH 8.0) containing 5 mm MnCl2, 1 mm dithiothreitol,
0.15 m NaCl and an appropriate concentration of urea and
incubated for at least 30 min prior to the measurement
The protein concentration was 0.1 mgÆmL)1, and the
optical path length was 2 mm
Acknowledgements
We thank Drs M Morikawa and M Haruki for
help-ful discussions This work was supported in part by a
Grant-in-Aid for National Project on Protein
Struc-tural and Functional Analyses and by a Grant-in-Aid
for Scientific Research (No 16041229) from the
Minis-try of Education, Culture, Sports, Science, and
Tech-nology of Japan, and by an Industrial TechTech-nology
Research Grant Program from the New Energy and
Industrial Technology Development Organization
(NEDO) of Japan
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