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Journal of Bacteriology, April 1999, p. 2118-2123, Vol. 181, No. 7
Mitsubishi-Kasei Institute of Life Sciences,
Received 29 October 1998/Accepted 28 January 1999
Two genes encoding functional RNase H (EC 3.1.26.4) were isolated
from a gram-positive bacterium, Bacillus subtilis 168. Two
DNA clones exhibiting RNase H activities both in vivo and in vitro were
obtained from a B. subtilis DNA library. One (28.2 kDa) revealed high similarity to Escherichia coli RNase
HII, encoded by the rnhB gene. The other (33.9 kDa) was
designated rnhC and encodes B. subtilis
RNase HIII. The B. subtilis genome has an rnhA homologue, the product of which has not yet shown
RNase H activity. Analyses of all three B. subtilis genes revealed that rnhB and
rnhC cannot be simultaneously inactivated. This observation indicated that in B. subtilis both the
rnhB and rnhC products are involved in certain
essential cellular processes that are different from
those suggested by E. coli rnh mutation studies. Sequence
conservation between the rnhB and rnhC genes
implies that both originated from a single ancestral RNase H gene. The roles of bacterial RNase H may be indicated by the single
rnhC homologue in the small genome of
Mycoplasma species.
RNase H (EC 3.1.26.4)
endonucleolytically cleaves RNA in RNA-DNA hybrid molecules
(26). This activity is present in almost all
organisms, from viruses to humans (3). An RNase H gene that
encodes bacterial RNase HI (rnhA) (2) was first
cloned from Escherichia coli K-12 by measurement of
biochemical activity (21, 31). Subsequently, use of
conditional lethal E. coli rnhA mutants, very sensitive to
the residual levels of RNase H activity in vivo
(18), permitted isolation of a second RNase H gene
(rnhB) from E. coli that encodes bacterial RNase
HII (13).
Isolation of rnhA genes from Salmonella
typhimurium LT2 (18), Thermus thermophilus
HB8 (17), Mycobacterium smegmatis
(34), and the yeast Saccharomyces cerevisiae
(18) has been reported. The three-dimensional
structure has been determined by X-ray crystallographic analysis
for E. coli RNase HI (25, 41), the
heat-stable RNase HI from T. thermophilus
(12), and the retroviral homologue RNase H domain of
human immunodeficiency virus (HIV) reverse transcriptase (28). Extensive mutagenesis of E. coli
RNase HI (6, 9, 22-24) has been carried out, and a
detailed mechanism for the enzyme's catalytic reaction has been
proposed (24). Based upon studies of various E. coli rnhA mutants, physiological roles of RNase HI
(rnhA) in DNA replication (1, 4, 16, 27, 35),
repair (15), and transcription (36) have been
proposed. In contrast to the highly active RNase HI
(rnhA), constituting more than 90% of the total RNase H
activity of E. coli (3), RNase HII
(rnhB), which exhibits only 0.4% of the activity of
wild-type RNase HI with poly(rA) · poly(dT) used as a
substrate (13), has not been studied in any detail.
By computer-assisted searches of the complete genomes of
bacteria, a clearly recognizable homologue of RNase HI
(rnhA) cannot be found in the
Archaebacteria or Mycoplasma genomes. In
contrast, ubiquitous RNase HII (rnhB) homologues have
been recognized in Archaebacteria (33, 42). The
lack of obvious bacterial RNase HI (rnhA) or
RNaseHII (rnhB) homologues in the genomes of
Mycoplasma species (8, 11), along with the
viability at low temperature of an E. coli mutant
strain that lacks both RNase HI and RNase HII (12a),
suggests that RNase H is dispensable for cell viability or can be
replaced by another enzyme possessing exonuclease activity (29).
We attempted to isolate a functional RNase H gene(s) from the
gram-positive bacterium Bacillus subtilis by screening a DNA library. Two RNase H genes are present in the B. subtilis genome, including the newly characterized rnhC
gene. Mutational analysis indicated that loss of rnhB and
rnhC renders B. subtilis unable to grow,
suggesting essential roles for these RNase H genes in cell
viability. Discovery of the B. subtilis rnhC gene
allowed computational identification of the rnhC homologue
in the Mycoplasma genomes, where no RNase H homologue
had yet been reported (8, 11).
Bacterial strains and plasmids.
Bacterial strains and
plasmids used in this study are listed in Table
1. Preparation and transformation of
competent E. coli cells were by the method of Mandel
and Higa (32). Preparation and transformation of competent
B. subtilis cells were as previously described
(40). Luria-Bertani (LB) broth was used for growth of
E. coli. Antibiotic medium 3 (Difco Laboratories,
Detroit, Mich.) was used for growth of B. subtilis.
Bacteria were grown at 37°C unless otherwise mentioned. Antibiotic
resistance gene cassettes were prepared from the E. coli plasmids listed in Table 1. All plasmids were purified by
CsCl-ethidium bromide ultracentrifugation.
In vitro DNA manipulations.
Type II restriction enzymes and
T4 DNA ligase were obtained from Toyobo (Tokyo, Japan), except for
NotI (Takara Shuzo, Kyoto, Japan). A Takara exonuclease III
(ExoIII) deletion kit was purchased from Takara. DNA manipulations in
vitro were done according to procedures described in reference
38 or the manufacturers' instructions, unless
specified otherwise. Southern hybridization was carried out with
nylon membranes (Nytran 13N; Schleicher & Schuell, Dassel, Germany), as previously described (14).
Complementation of conditional-lethal E. coli
RNase H mutants.
Three E. coli
mutants RNase H activity in vitro.
A 0.5-ml overnight culture of
E. coli was harvested in a 1.5-ml microcentrifuge tube.
The pellet was suspended in 50 µl of 10 mM Tris-HCl (pH 7.5)-1 mM
EDTA, sonicated, and centrifuged. The supernatant was transferred to a
fresh tube and adjusted to 40 mM Tris-HCl (pH 6.8)-1% sodium dodecyl
sulfate (SDS)-50 mM dithiothreitol-5% (vol/vol) glycerol in 100 µl. Ten microliters was boiled for 3 min immediately before loading
on an SDS-polyacrylamide gel containing
poly([32P]rA) · poly(dT), and the renaturation gel
assay was carried out as described previously (13).
Construction of a B. subtilis DNA library.
Genomic DNA for library construction or analysis by conventional gel
electrophoresis was prepared by a liquid isolation method (37). Agarose (1.0%, wt/vol) in TAE solution (50 mM
Tris-acetate [pH 8.0], 1.0 mM EDTA) was used for conventional gel
electrophoresis at room temperature.
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Isolation of RNase H Genes That Are Essential
for Growth of Bacillus subtilis 168
and
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Bacterial strains and plasmids used in this study
MIC3037, MIC1021, and MIC2067form colonies at 30°C but not
at 42°C. The temperature-dependent growth defect of MIC3037 and
MIC1021 was explained as the adverse effect of recBC and
rnhA double mutations at restrictive temperature (16). This temperature-sensitive phenotype was relieved by
introduction of the recBC or rnhA gene (16,
17). The temperature-sensitive growth of MIC2067 resulted from
the rnhA and rnhB double mutation and was
relieved only by delivering plasmids that carry an RNase H gene
(12a), such as the following: pSK760,
rnhA from E. coli (21); pMIC27,
rnhB from E. coli (13); pMY2051,
RNH1 from S. cerevisiae (18); pRET4,
rnhA from T. thermophilus (17). The versatility of the cloning system using MIC3037 and MIC2067 is demonstrated in this study. MIC1021 was used only when chloramphenicol was needed as a plasmid selection marker.
Determination of the nucleotide sequence. To obtain deletion clones of plasmid pMIB15-3 or pMIB21F at average intervals of 250 bp from the T7 promoter, the ExoIII digestion method (10) was applied. The sequences of the two clones were determined by dideoxy chain termination sequencing with 35S-labeled nucleotides with the T7 promoter-primer as the sequence primer. Alignment of sequences was done with GENETYX, version 7.0.
Overexpression of RNase H enzymes in E. coli.
For B. subtilis RNase HII expression in
E. coli, the 984-bp EcoRI-BamHI
segment from a deletion derivative of pMIB21F (see Fig. 2) was inserted
in the XbaI site 25 bp downstream of the T7 promoter of
pBEST6, yielding pMIB2106. The plasmid was introduced into
E. coli BL21(DE3) (39) and selected by
ampicillin (100 µg/ml). The resultant transformant was grown in LB
medium containing ampicillin at 37°C.
Isopropyl-
-D-thiogalactopyranoside was added at a final
concentration 0.4 mM to the culture when it was at an approximate
A590 of 0.5, with incubation continuing for
4 h. Aliquots were removed at hourly intervals and analyzed by
SDS-polyacrylamide gel electrophoresis.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cloning of two functional RNase H genes from B. subtilis 168. The B. subtilis DNA library was constructed in E. coli MIC3037 (rnhA339::cat rnhB+ recC271) and subdivided into 32 groups designated Bsu clubs (see Materials and Methods). The library was appropriate for direct screening of the RNase H gene(s) by temperature-sensitive complementation assay and activity in the gel assay, because the host strain, MIC3037, did not grow at the restricted temperature (42°C) and lacks the major E. coli RNase HI (rnhA) activity, as shown in lane 2 of Fig. 1. Promoters of B. subtilis genes normally function in E. coli. Therefore, no special sequences were employed for gene expression. By the two independent screenings described below, two clones were positive for both assays.
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Nucleotide and amino acid sequences of the rnhB gene. The nucleotide sequence of the insert (1,685 bp) of pMIB21F was determined as described in Materials and Methods (data not shown). One open reading frame (ORF) was found that encodes a protein of 255 amino acid residues (calculated molecular mass, 28,204 Da; pI, 5.52), consistent with the estimated size of the RNase H activity (approximately 30 kDa) from the gel assay (Fig. 1). Plasmid pMIB2106, described in Materials and Methods, overexpressed the gene product in E. coli BL21(DE3) (data not shown). The expressed protein was subjected to automated sequence analysis in a gas-phase protein sequencer (model 477A; Perkin-Elmer Applied Biosystems), and the N-terminal 15 amino acids, MNTLTVKDIKDRLQE, were identical to those predicted from the nucleotide sequence (Fig. 3). Searches of the B. subtilis genome database (33a) revealed an ORF designated rnh (accession no. BG12666) encoded by nucleotides 1,676,850 to 1,677,614 (30). The rnh gene had a similarity of 63% (amino acid) to that of E. coli RNase HII (rnhB). Consequently, the rnh gene is designated rnhB, encoding B. subtilis RNase HII.
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Nucleotide and amino acid sequences of the rnhC gene. The nucleotide sequence of the insert (1,248 bp) of pMIB156-2 (Fig. 2a) was also determined. One ORF was found that encodes a protein of 313 amino acid residues (calculated molecular mass, 33,915 Da; pI, 10.07), consistent with the estimated size of the RNase H activity (approximately 40 kDa) from the gel assay (Fig. 1). The ORF corresponds to the ysgB gene (accession no. BG12324) encoded by nucleotides 2,925,133 to 2,926,071 of the B. subtilis genome; the function of ysgB is unknown (30). The amino acid sequence is shown in Fig. 3. The ysgB gene product had little similarity to E. coli RNase HI (rnhA). Although the overall similarity between B. subtilis rnhB and the ysgB gene is 20.0%, there are a few well-conserved regions between the two, as indicated in Fig. 3. The ysgB gene, therefore, is designated rnhC, encoding B. subtilis RNase HIII.
Overexpression of the RnhC product in E. coli BL21(DE3) with a pBEST6 vector was unsuccessful. However, a similar expression plasmid constructed with a PCR-amplified DNA fragment overproduced a product whose amino acid sequence was identical to that of the ysgB gene (35a).RNase HI (rnhA) homologue in the B. subtilis genome. On searching the entire B. subtilis genome for proteins related to E. coli RNaseHI (rnhA), a single sequence with 29% homology, ypdQ (accession no. BG11608), encoded by nucleotides 2,309,611 to 2,310,056 of the B. subtilis genome (30) was found. The ypdQ gene was cloned as a 474-bp DNA fragment amplified by using the forward primer 5'-ACCTCGCCATTAGGATGAAC and the reverse primer 5'-TGCAGCCAAAAAAATGATACC from genomic DNA of B. subtilis 1A1. PCR was performed in 20 cycles with a GeneThermoUnit GTU1605 (Taitech, Tokyo, Japan). The PCR fragment was inserted in pGEM4, resulting in pRNHA-1, but this plasmid was unable to suppress the temperature-sensitive growth of any of the E. coli rnh mutants in Table 1. Possibly this protein is not expressed well in E. coli mutants and/or has lower levels of RNase H activity than that needed to give suppression in vivo. This is consistent with the observation that no rnhA homologue was obtained in screening for the Bsu clubs. The ypdQ gene encodes a protein of 132 amino acid residues (calculated molecular mass, 14,529 Da; pI, 5.60). The product expressed in E. coli is being characterized (35a).
RNase H genes are conserved in related B. subtilis species. Southern hybridization to SfiI and NotI fragments by three clones, pRNHA-1, pMIB21F, and pMIB15-3 (data not shown), placed these clones in the B. subtilis physical map shown in Fig. 2b. The same DNA probes gave clear signals to genomic DNAs from closely related B. subtilis species, B. subtilis W23 and Bacillus natto IFO1212 (data not shown). The results indicated that all three genes are conserved in these two strains.
RNase H-deficient mutant of B. subtilis. Mutations of each of the three B. subtilis genes were constructed in E. coli plasmids. The ypdQ gene (rnhA homologue) was disrupted by insertion of a spectinomycin resistance gene cassette (spc) in the unique BsmI site of pUC2.2, resulting in pRNHA-4. The spc cassette was prepared by SmaI digestion of pBEST517A (Table 1). Similarly, the rnhB gene in pMIB21F was disrupted by insertion of a neomycin resistance gene cassette (neo) in the internal HindIII site, resulting in pMIB21Lneo (Fig. 2a). The neo cassette gene was prepared by HindIII digestion from pBEST512 (14). The rnhC gene in pMIB156-2 was disrupted by insertion of a chloramphenicol resistance gene cassette (cat) in the unique NotI site after being blunt ended, resulting in pMIB15-N1 (Fig. 2a). The cat gene was prepared by SmaI digestion of pBEST4F (20).
Mutations carried in these plasmids were introduced into the B. subtilis genome by gene replacement (40). Single mutants were selected by appropriate antibiotic resistance: spectinomycin for the ypdQ mutant, neomycin for the rnhB mutant, and chloramphenicol for the rnhC mutant at 30°C (Table 1). The genomic structures of these mutants were verified by Southern analysis by using the parental plasmids as probes (data not shown). The rnhC mutant BEST220 (rnhC151::cat) gave colonies slightly smaller than the other two single mutants. Construction of double mutants by genetic crosses of the single mutants was successful except for rnhB and rnhC. Attempts to introduce the rnhB21::neo mutation of BEST218 into BEST220 (rnhC151::cat) resulted in no neomycin-resistant transformants. Other markers unrelated to RNase H, proB or leuB, could be introduced into BEST220, giving approximately 103 transformants per microgram of donor DNA. In contrast, introduction of the rnhC151::cat mutation of BEST220 into BEST218 (rnhB21::neo) resulted in strains that formed very tiny colonies on selection plates containing chloramphenicol at frequencies similar to those of proB or leuB transfer. However, these tiny colonies did not produce viable colonies when restreaked on fresh plates, even in the absence of antibiotics. Introduction of the ypdQ mutation gave no phenotype, regardless of what other mutations were present in the recipient strain. All of the viable mutants were able to grow in the temperature range of 25 to 50°C, as does the wild-type strain. The results are interpreted to indicate that loss of both RNase HII (rnhB) and RNase HIII (rnhC) renders B. subtilis inviable. From the formation of colonies of inviable cells in the BEST220 × BEST218 cross, it can be interpreted that low levels of RnhC allow for consecutive cell divisions. The number of cell divisions may be enough to form visible (tiny) colonies. In the other cross, BEST218 × BEST220, RnhB was rapidly degraded or diluted out before producing sufficient numbers of cells.rnhC homologues in the database. On searching the current database for sequences related to B. subtilis rnhC, homologues were detected in various bacterial and eukaryotic genomes. However, most had been already designated rnh or rnhB in the database due to the slight similarity between rnhB and rnhC, as indicated in Fig. 3, with the exception of three genes. Two of these were from Mycoplasma species, the putative gene from Mycoplasma genitalium (accession no. MG199) (8, 33a) and the putative gene from Mycoplasma pneumoniae (accession no. C09_orf143b) (11, 33a), and the third was from the hyperthemophilic bacterium Aquifex aeolicus (accession no. aq_1768) (5, 33a). Their alignment with B. subtilis rnhC is shown in Fig. 4. As the rnhB gene in A. aeolicus was already reported (accession no. aq_1955) (5), it seems likely that this hyperthermophilic bacterium has an rnhC homologue and an rnhB homologue, although no obvious rnhA homologue was detected. It should be mentioned that an rnhC homologue was not found in the E. coli genome (33a).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Two distinctive RNase H genes of B. subtilis 168. Two clones encoding functional RNase H genes were isolated from a strain of the gram-positive bacterium B. subtilis. The two genes, rnhB and rnhC, were obtained based on the abilities to complement E. coli RNase H mutants in vivo (Fig. 2a) and to exhibit RNase H activity in vitro (Fig. 1). The temperature-sensitive E. coli strain MIC2067 newly adopted in this study was more specific for in vivo RNase H activity than MIC2067 newly adopted in this study was more specific for in vivo RNase H activity than MIC3037 (12a), and the MIC strains in Table 1 can discriminate the rnh genes in vivo if the genes are correctly expressed. In addition to these functional B. subtilis RNase H genes, a gene, ypdQ, that has some similarity to rnhA in the B. subtilis genome was also identified. However, the ypdQ clone was negative for all RNase H criteria. It remains to be demonstrated whether the failure of the ypdQ gene to suppress the temperature-sensitive phenotypes of MIC strains is due to either incorrect expression or low activity in E. coli. Alternatively, as YpdQ lacks the basic protruding region characteristic of E. coli RNase HI (25, 41), it may require an additional polypeptide for RNase H activity, similar to that observed for RNase H of HIV reverse transcriptase (28). It may be worth testing the ypdQ homologues observed in related B. subtilis species for enzyme activity.
Are RNase H proteins essential for B. subtilis? Because the E. coli K-12 genome seems not to have an rnhC homologue, rnhA and rnhB double mutants such as MIC2067 completely abolish RNase H. Although MIC2067 shows a temperature-sensitive growth phenotype, double mutants of a certain E. coli genetic background grow normally (12a). In contrast, the rnhB and rnhC double mutant of B. subtilis 168 is unable to form viable colonies. These observations suggest that RNase H-requiring biological processes in B. subtilis are different from those suggested for E. coli (1, 4, 15, 16, 27, 35, 36). Distinctive pI values of the two B. subtilis RNase H genes (5.5 for rnhB and 10.1 for rnhC), compared with those of E. coli (9.7 for rnhA and 6.9 for rnhB), may imply different roles or substrate recognition properties in vivo. Alternative explanations include the acquisition of a suppressor mutation in laboratory strains of E. coli resulting in complete loss of RNase H proteins. Isolation of a B. subtilis rnhB and rnhC double mutant that has a suppressor mutation(s) is underway.
The requirement for RNase H in B. subtilis raises the question of whether RNase H is dispensable for bacteria. Complete DNA sequences are known for two Mycoplasma genomes, in which no homologue of rnhA and rnhB was reported, indicating that RNase H is dispensable (29). However, the presence of homologues to the newly characterized rnhC in these Mycoplasma species (Fig. 4) may imply that the single bacterial RNase H of the smallest genomes performs a function similar to the two RNase H proteins of B. subtilis. The genome of A. aeolicus, a hyperthermophilic hydrogen-oxidizing bacterium, has both rnhB and rnhC but lacks an rnhA homologue. The bacterium grows at 95°C, similar to primordial forms of life (5). This suggests that the two rnh genes of A. aeolicus represent an ancestral structure of the two present B. subtilis RNase H genes.| |
ACKNOWLEDGMENTS |
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We thank A. Ogura, K. Matsui, K. Fujita, and M. Murayama for technical help. We especially thank N. Ohtani for kindly communicating data prior to publication.
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FOOTNOTES |
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* Corresponding author. Mailing address: Mitsubishi-Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194-8511, Japan. Phone: 81-427-24-6254. Fax: 81-427-24-6316. E-mail: ita{at}libra.ls.m-kagaku.co.jp.
Present address: School of Marine Science and Technology, Tokai
University, Shimizu, Shizuoka 424-8610, Japan.
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Top
Abstract
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Materials and Methods
Results
Discussion
References
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Journal of Bacteriology, April 1999, p. 2118-2123, Vol. 181, No. 7
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