Báo cáo khoa học: Identification of RNase HII from psychrotrophic bacterium, Shewanella sp. SIB1 as a high-activity type RNase H pot

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
1 Department of Material and Life Science, Graduate School of Engineering, Osaka University, Japan
2 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 6 6879 7938
E-mail:
(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  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 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 1
and type 2 RNases H, which are evolutionarily unrelated [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

Abbreviations
APase, alkaline phosphatase; BSA, bovine serum albumin; IPTG, isopropyl thio-b-D-galactoside; RNase H, ribonuclease H.

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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 0 °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].

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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. 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 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 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 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. 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. 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.

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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. 2. 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, 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 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 1 mm MnCl2, 5 mm MgCl2,
and 0.5 mm CoCl2, respectively (Fig. 4). The specific

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.

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H. Chon et al.

Fig. 4. 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,
1 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 1 mm MnCl2 was 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 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
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Fig. 5. Temperature dependence of the activities of SIB1 and
E. coli RNases H. The M13 DNA ⁄ RNA hybrid (10 pmol) was hydrolyzed 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 mM Tris ⁄ HCl
(pH 8.5) containing 1 mM MnCl2, 110 mM KCl, 1 mM 2-mercatoethanol, and 50 lgỈmL)1 BSA for SIB1 RNase HII or 10 mM Tris ⁄ HCl
(pH 8.5) containing 5 mM MnCl2, 50 mM KCl, 1 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 4 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 5 mM MgCl2, 30 mM KCl, 1 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, 1 mM 2-mercaptoethanol,
and 50 lgỈmL)1 BSA 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 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

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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,
1 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 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 difference is marginal. It is noted that these proteins are fully
stable at 30 °C for at least 15 min 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. 6. 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 min. Hydrolysates were separated
on a 20% polyacrylamide gel containing 7 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.

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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.

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 ⁄ 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 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
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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 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 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 1
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-

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binding domain (dsRBD) at the N-terminus, like various 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.

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.

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 ⁄ 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 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,

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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, 1 mm of IPTG was added to
the culture medium and cultivation was continued at 30 °C
for 30 min. 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 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-

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 0 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 4 °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 2 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 1 mm MnCl2,
110 mm KCl, 1 mm 2-mercaptoethanol, and 50 lgỈmL)1
BSA for SIB1 RNase HII or 10 mm Tris ⁄ HCl (pH 8.5) containing 5 mm MnCl2, 50 mm KCl, 1 mm 2-mercaptoethanol,
and 50 lgỈmL)1 BSA for E. coli RNase HII. One unit is
defined as the amount of enzyme producing 1 lmol of acidsoluble material per min 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 min and
separation of the hydrolysates on a 20% polyacrylamide
gel containing 7 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

2

3

4

5

6

7

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 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 mgỈmL)1, and the
optical path length was 2 mm.

8

9

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.

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