Journal of Bacteriology, August 2004, p . 5376-5383, Vol . 186, No . 16

Bacillus subtilis YhcR, a High-Molecular-Weight, Nonspecific Endonuclease with a Unique Domain Structure

Irina A . Oussenko,¹ Roberto Sanchez,² and David H . Bechhofer^1*

Departments of Pharmacology and Biological Chemistry,¹ Physiology and Biophysics, Mount Sinai School of Medicine of New York University, New York, New York 10029²

Received 11 February 2004/ Accepted 19 May 2004

 
In a continuing effort to identify ribonucleases that may be involved in mRNA decay in Bacillus subtilis, fractionation of a protein extract from a triple-mutant strain that was missing three previously characterized 3'-to-5' exoribonucleases (polynucleotide phosphorylase [PNPase], RNase R, and YhaM) was undertaken . These experiments revealed the presence of a high-molecular-weight nuclease encoded by the yhcR gene that was active in the presence of Ca²⁺ and Mn²⁺ . YhcR is a sugar-nonspecific nuclease that cleaves endonucleolytically to yield nucleotide 3'-monophosphate products, similar to the well-characterized micrococcal nuclease of Staphylococcus aureus . YhcR appears to be located principally in the cell wall and is likely to be a substrate for a B . subtilis sortase . Zymogram analysis suggests that YhcR is the major Ca²⁺-activated nuclease of B . subtilis . In addition to having a unique overall domain structure, YhcR contains a hitherto unknown structural domain that we have named "NYD," for "new YhcR domain."

 
A general model for mRNA decay in prokaryotes has been developed, based on studies of Escherichia coli . Decay appears to proceed by a combination of an initiating endonucleolytic cleavage, executed by RNase E, followed by degradation in the 3'-to-5' direction by polynucleotide phosphorylase (PNPase) or RNase II (5, 23) . The final turnover of mRNA is accomplished by oligoribonuclease (9) . An E . coli strain lacking PNPase and RNase II is inviable (7) . Thus, it is assumed generally that mRNA turnover is an essential function .

The genome sequence of B . subtilis has revealed that several of the major E . coli ribonucleases have no homologues in B . subtilis, including RNase E, RNase II, and oligoribonuclease . We have been pursuing biochemical experiments in an effort to identify ribonucleases in B . subtilis that might be responsible for mRNA decay . These studies have resulted in the identification of genes encoding several 3'-to-5' exoribonucleases: PNPase (17), RNase R (19), and YhaM (20) . Mutant strains deficient in these exoribonucleases, alone or in combination, show several different phenotypes (16, 19, 20, 28) . The fact that such strains are viable indicates that one or more B . subtilis RNase activities remain to be discovered . In the search for such an RNase, a broad-specificity nuclease encoded by the yhcR gene has been identified and characterized .

 
Bacterial strains and plasmid constructions. The wild-type B . subtilis host was BG1, which is trpC2 thr-5 . The RNase triple mutant, which was pnpA rnr yhaM, was described previously (20) . To delete the yhcR gene, an internal SalI-SacI fragment (nucleotides [nt] 612 to 2336 of the yhcR coding sequence) was replaced with a SalI-SacI fragment from plasmid pBEST501 that contained a neomycin resistance gene cassette (11) . Preparation of B . subtilis growth media and competent B . subtilis cultures was performed as described previously (8). E . coli strain DH5 (10) was the host for plasmid constructions .

To construct His-tagged YhcR, the yhcR coding sequence was amplified by PCR, using genomic DNA from B . subtilis strain BG1 as template, and cloned between the NcoI and BglII sites of plasmid pQE60 (Qiagen) . The full-length version (minus the signal sequence [see Results and Discussion]) contained YhcR amino acids 36 to 1217, and the N-terminal construct contained amino acids 36 to 529 . In both cases, the cloning resulted in a change of phenylalanine to valine at codon 37 . Constructs were verified by sequence analysis, performed by the Mount Sinai Department of Human Genetics DNA sequencing facility . His-tagged constructs were maintained in a DH5 strain that contained the lacI repressor plasmid pREP4 (Qiagen) .

YhcR purification. The first steps in purification, up until ammonium sulfate precipitation, were performed as described previously (20) . Briefly, 25 g (wet weight) of B . subtilis cells was disrupted by lysozyme treatment and passage through a French press . The cell homogenate was cleared of cell debris, membranes, and nucleic acid, and proteins were precipitated with ammonium sulfate . The 40 to 60% ammonium sulfate fraction was dialyzed overnight against buffer A (20 mM Tris-HCl [pH 7.8], 100 mM KCl, 0.2 mM EDTA, 1 mM dithiothrietol, 0.1 mM phenylmethylsulfonyl fluoride, 10% glycerol) . Proteins were bound to DEAE-Sepharose CL-6B (Sigma-Aldrich), and the fraction containing activity was eluted in one step with buffer A containing 250 mM KCl . The eluate was passed through Affi-Gel Blue (Bio-Rad) equilibrated with buffer A containing 250 mM KCl . Unbound proteins were collected, concentrated by precipitation with 80% ammonium sulfate, and dissolved in 2 ml of buffer A with 250 mM KCl and no glycerol . Proteins were fractionated by fast protein liquid chromatography (FPLC) (LKB Pharmacia LCC-500 Plus) on a HiLoad Superdex 200 column (Amersham) that was equilibrated with the same buffer . Active fractions were pooled, dialyzed against buffer A without glycerol, and fractionated by FPLC on a Mono Q column that was equilibrated with the same buffer . The column was developed with a two-step linear gradient (0 to 5% in 5 ml and 5 to 25% in 45 ml of 20 mM Tris-HCl [pH 7.8], 1 M KCl) . One-milliliter fractions were collected and assayed for RNase activity . Active fractions were concentrated by precipitation with acetone, and proteins were resolved in a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (7.5% polyacrylamide) gel . Further purification of proteins by transfer to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad) was performed as described previously (20) . His-tagged versions of YhcR were purified on Ni²⁺-nitrilotriacetic acid (NTA) under denaturing conditions, as described in the Qiagen overexpression protocol (Qiagen manual, Qiagen GmbH, Hilden, Germany) . Further purification of the His-tagged proteins was achieved by blotting to a PVDF membrane . Purified YhcR was aliquoted and stored at –80°C in 25 mM Tris-HCl (pH 8.7), 25% glycerol, 5 mM ß-mercaptoethanol, and 0.5% Triton X-100 .

B . subtilis protoplasts were prepared as described previously (4) . Protoplasts were lysed by resuspension in buffer A and sonication for three 20-s bursts (7 W) with a thin probe (Microson XL2000), with cooling on ice for 45 s between bursts . The extract was cleared by centrifugation at 15,000 x g for 20 min, dialyzed overnight at 4°C in buffer A and 1 h the following day against fresh buffer, and aliquoted for storage at –80°C .

Nuclease assay. Uniformly labeled RNA substrates for RNase assay were synthesized by T7 RNA polymerase transcription (MAXIscript T7 kit; Ambion) in the presence of [-³²P]UTP . Templates for the 110- and 187-nt RNAs have been described previously (20) . To assay hydrolysis of single-stranded DNA, oligonucleotides were 5'-end labeled with T4 polynucleotide kinase and [-³²P]ATP . The standard RNase reaction mixture contained 50 mM Tris or 50 mM Tricine (pH 8.0), 100 mM KCl, 1 mM divalent cation, 0.2 to 0.5 pmol (2 x 10⁵ to 4 x 10⁵ cpm/pmol) of labeled substrate, and approximately 0.05 µg of YhcR protein . The reaction mixture was incubated for 20 min at 37°C and then extracted with an equal volume of phenol-chloroform . Ten microliters of the aqueous phase was mixed with gel loading buffer (Ambion) and run on a 20% denaturing polyacrylamide gel to resolve mononucleotides . The amount of mononucleotide product was quantitated on a PhosphorImager instrument (Molecular Dynamics) . For thin-layer chromatography, a polyethyleneimine cellulose sheet (J . T . Baker) was spotted with 2 µl of a reaction mixture and chromatographed in 1 M LiCl at room temperature . For thin-layer chromatography analysis, substrate RNA was also treated with Staphylococcus aureus micrococcal nuclease (Worthington) and S1 nuclease (Invitrogen) . Assay of double-stranded DNA hydrolysis was done with 0.5 µg of an 8.5-kbp plasmid . The reaction buffer was the same as for the RNase assay, incubation was for 60 min at 37°C, and the products were separated on a 0.8% Tris-borate agarose gel .

For quantitative assays of YhcR activity, total B . subtilis RNA, which was pulse-labeled with [³H]uridine, was used . Two hundred fifty microliters of [5,6-³H]uridine (specific activity, 1 mCi/ml; Perkin-Elmer Life Sciences) was added to 40 ml of a B . subtilis culture at late logarithmic phase, grown in RNA isolation medium (28) . After a 10-min labeling period, total RNA was isolated by the hot phenol method . Thirty nanomoles of labeled RNA (nucleotides) was used per 100 µl of reaction mixture containing 50 mM Tris-HCl (pH 8.0), 100 mM KCl, and 1 mM CaCl₂ . For assaying over the pH range, the buffer was 50 mM Bis-Tris propane . The reaction mix was preincubated at 37°C for 5 min before addition of 15 ng of purified YhcR-His₆ . After incubation at various times at 37°C, 100-µl aliquots of the reaction mixture were removed into 300 µl of ice-cold 0.5-mg/ml E . coli tRNA, 400 µl of 20% trichloroacetic acid was added, and undigested RNA was precipitated by incubation on ice for 30 min followed by centrifugation at 14,000 x g at 4°C . Four hundred microliters of supernatant was removed into 5 ml of Ecoscint A (National Diagnostics), and acid-soluble radioactivity, representing nucleotides released, was determined .

Zymogram analysis. Whole-cell protein extracts for zymogram analysis were prepared as follows . Twenty milliliters of cell culture, grown in Luria-Bertani medium until the end of exponential phase, was collected, washed in buffer A, resuspended in 1 ml of buffer A containing 0.2-mg/ml lysozyme, and incubated for 20 min at 37°C . Sonication and subsequent steps were performed as described above for the protoplast extract . Purified proteins or 20 µg of a protein extract were electrophoresed on SDS-PAGE gels (10% polyacrylamide) containing 20-µg/ml B . subtilis RNA in the resolving gel and 0.2 mM EDTA in the gel and the running buffer . To reconstitute proteins, the gels were incubated at 4°C for 16 h in 20 mM Tris-HCl (pH 7.8), 100 mM NaCl, 0.2 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and 10% glycerol . Incubation was continued at room temperature for 4 h in fresh buffer and then for 20 h in buffer containing 2 mM Ca²⁺ . The gels were stained for 15 min with ethidium bromide and then destained for 15 min . Dark bands indicate RNase activity .

 
Identification of YhcR RNase activity. In previous work, a strain of B . subtilis deficient in three exoribonucleases (PNPase, RNase R, and YhaM) was constructed (20) . Extracts from the pnpA rnr yhaM strain were prepared, and proteins were fractionated by ammonium sulfate precipitation, DEAE Sepharose, Affi-Gel Blue column chromatography, gel filtration, and Mono Q column chromatography (see Materials and Methods) . Fractions were tested for RNase activity in the presence of Mn²⁺ (1 mM), as this condition gave residual phosphate-independent exonuclease activity in a strain that had a disrupted rnr gene (19) .

Degradation of a uniformly labeled 110-nt RNA (20) was assayed by the presence of labeled mononucleotides in a 20% polyacrylamide-8 M urea gel . In one experiment, partially purified proteins were assayed for RNase activity also in the presence of Ca²⁺ (1 mM), and this condition gave much more activity than in the presence of Mn²⁺ (Fig . 1A) . In subsequent assays, the Mn²⁺- and Ca²⁺-dependent activities always copurified . From the pattern in Fig . 1A, lane 3, it appears that this RNase activity generated more than one species of rapidly migrating nucleotide, suggesting an endonucleolytic mode . The most active fraction was resolved in an SDS-polyacrylamide gel and blotted to a PVDF membrane . Proteins were eluted from segments of the PVDF membrane spanning the whole range of molecular weights and were tested for RNase activity (data not shown) . The Ca²⁺-dependent activity was restricted to a segment containing high-molecular-mass proteins of 120 to 150 kDa .

 
Thin slices of the PVDF membrane in the 120- to 150-kDa range were cut out, and the proteins were eluted and tested for RNase activity (Fig . 1B and C) . Surprisingly, the activity was present in successive slices within the 120- to 150-kDa range . The B . subtilis genome is predicted to have only 18 proteins in this molecular mass range . Of these, only six were of unknown function, one of which was yhcR, a gene that had been predicted to encode an RNase (2, 6) . A yhcR-disrupted mutant strain, generated in the European-Japanese B . subtilis functional analysis project (13), was obtained . Extracts from the wild-type strain and the yhcR-disrupted strain were prepared, resolved in an SDS-PAGE gel, and blotted to a PVDF membrane (Fig . 2A and B) . No RNase activity was detected from the yhcR-disrupted strain in the size range in which the Ca²⁺-dependent activity had been isolated from the wild type (Fig . 2C) . This result suggested strongly that the RNase activity detected in the triple-mutant strain was encoded by yhcR .

 
Domain organization of yhcR. The amino acid sequence of YhcR was analyzed by sequence similarity searches, using PSI-BLAST (1) and the Pfam server (3) . The analysis identified six domains covering most of the sequence, plus an N-terminal signal peptide and a C-terminal gram-positive anchor (Fig . 3A) . The N-terminal end of the sequence (residues 1 to 46) contains a signal peptide that is predicted to direct secretion by the twin-arginine translocation pathway (Fig . 3B) (12, 26, 27) . This is followed by a 110-residue region (residues 51 to 160) that appears to be a domain . This region (labeled "NYD," for new YhcR domain) is not classified in Pfam but is found by PSI-BLAST searches 10 times in six different bacterial proteins, in addition to YhcR (Table 1 and Fig . 3D) . The NYDs vary in sequence identity (between 26 and 40%) with respect to YhcR NYD . Interestingly, three of the proteins with NYDs are classified as nucleases and a fourth contains an OB-fold nucleic acid binding domain as well as a phosphatase and nucleotidase domain . Two of these proteins contain only NYDs and no other identifiable domains, indicating that the NYD itself may have an independent function . The fact that NYD is found in different sequence contexts in various proteins (e.g., next to an OB-fold domain in YhcR and Q9KE43, next to an endonuclease domain in Q8EMI4, next to other NYDs in 23028590, etc.), together with the high conservation of certain positions (Fig . 3D) in spite of the various degrees of overall sequence similarity, supports the idea that this is an independent domain and not merely part of a larger domain .

 
Following the YhcR NYD, there are two OB-fold nucleic acid binding domains (positions 186 to 257 and 290 to 362) . These are relatively divergent OB-folds that are only identifiable by Pfam . The only close matches for these two domains are with a hypothetical protein from Oceanobacillus iheyensis (SP Q8ESW7), a protein that is similar to YhcR over the complete sequence, except for the N-terminal signal peptide, the NYD, and the C-terminal 115 residues (see below) . The two OB-fold domains of YhcR are also divergent from each other, sharing only 22% sequence identity over 71 residues . They share more similarity with their corresponding OB-fold domains in the O . iheyensis protein (45% sequence identity) than with each other .

The two OB-fold domains are followed by a Staphylococcal nuclease homologue (SNase) domain (376 to 517) . This domain shows significant sequence similarity to many other SNase domains, the most similar being the SNase domain of the above-mentioned hypothetical protein from O . iheyensis (SP Q8ESW7, 47% sequence identity) . YhcR SNase shares 31% sequence identity with the S . aureus micrococcal nuclease, and most of the identical residues are around the active site, which is conserved between the two sequences .

The SNase domain is followed by a region of 70 residues that does not show any significant sequence similarity to other domains . The sequence then continues with one metallophosphatase and one 5'-nucleotidase C-terminal domain . These two domains are present together in a large family of 5'-nucleotidases that includes proteins annotated as UDP-sugar hydrolase and 2',3'-cyclic-nucleotide 2'-phosphodiesterase .

The YhcR sequence continues with another unidentified region of 120 residues and ends with a putative gram-positive anchor (Fig. 3C) . Proteins with such anchors are cleaved after the threonine residue in a conserved LPXTG motif and are bound to the cell wall via the action of sortase (18) . The existence of a sortase substrate in B . subtilis has been the subject of some interest, as B . subtilis appears to have two sortase homologues, yet only one identifiable sortase substrate—YhcR (21) . Interestingly, one of the B . subtilis sortase homologues is encoded by yhcS, which is located immediately downstream of yhcR; in fact, the yhcR stop codon and the yhcS start codon overlap . Although the consensus gram-positive sortase cleavage signal is LPXTG and YhcR has an LPDTS sequence, at least one other protein thought to be a sortase substrate, the FNZ protein of Streptococcus equi (15), has an LPXTS sequence, like YhcR . Experiments to determine YhcR localization are described below .

It is interesting to note that there is only one other known protein sequence with a domain structure similar to that described here for YhcR . The sequence of the hypothetical protein from O . iheyensis (SP Q8ESW7) is strikingly similar over 930 residues (positions 168 to 1102), with a sequence identity of 50% . This region includes the two OB-fold domains, the SNase, metallophosphatase, and 5'-nucleotidase C-terminal domains, but it does not include the NYD . As shown in Table 1, another protein from O . iheyensis annotated as an extracellular RNase (SP Q8EMI4) contains the closest homologue of the YhcR NYD domain, with 40% identity, but the rest of this O . iheyensis sequence (a predicted endonuclease I domain) does not share any sequence similarity with YhcR .

Cloning of yhcR and characterization of the protein product. To confirm the predicted enzymatic activities, we attempted to clone the yhcR coding sequence in a His-tag vector in E . coli, but it appeared that even low-level expression of the complete YhcR was toxic to E . coli . Such toxicity is often associated with hydrophobic signal peptide sequences (Qiagen manual, Qiagen GmbH, Hilden, Germany) . Therefore, the coding sequence was cloned without the N-terminal signal peptide sequence, and this construct proved to be stable . His-tagged YhcR was purified from an isopropyl-ß-D-thiogalactopyranoside (IPTG)-induced E . coli strain (Fig . 4A, lanes 1 and 2) . The major induced bands seen in lane 2 are YhcR specific, as demonstrated by zymogram analysis of whole-cell extracts . A control E . coli strain containing empty plasmid vector had no detectable Ca²⁺-specific RNase activity upon zymogram analysis (data not shown) . As will be discussed later, YhcR appears to be subject to proteolysis in both E . coli and B . subtilis . After purification through an Ni²⁺-NTA column, the fraction containing YhcR-His₆ (Fig . 4A, lane 3) was electrophoresed on a preparative SDS-PAGE gel (7.5% polyacrylamide) and blotted to a PVDF membrane, and the full-length YhcR-His₆ was cut out of the PVDF membrane and eluted (see Materials and Methods) . Despite these precautions, bands of lower molecular weight were always detected in the most purified YhcR preparations (Fig . 4A, lane 4) . These lower-molecular-weight species had Ca²⁺-activated RNase activity, as shown by zymogram analysis (data not shown) . We assume that these species are degradation fragments of full-length YhcR-His₆ (see below) .

 
YhcR activity was assayed by using a uniformly labeled 187-nt RNA (20) . The purified protein showed nuclease activity in the presence of Mn²⁺ and even more activity in the presence of Ca²⁺, but no activity in the presence of Mg²⁺ (Fig . 4B) . The presence of fast-migrating nucleotide products other than mononucleotides suggested that the RNase activity was endonucleolytic .

The products of complete YhcR digestion were determined, using the 5'-[-³²P]UTP uniformly labeled 187-nt RNA as a substrate and isolated His-tagged YhcR protein that was purified further from a PVDF membrane . The products were separated by thin-layer chromatography and were run in parallel with the products of S . aureus micrococcal nuclease and S1 nuclease digestions . Micrococcal nuclease cleaves RNA endonucleolytically, generating 3'-monophosphate nucleosides (reviewed in reference 22) . S1 nuclease cleaves RNA endonucleolytically to generate 5'-monophosphate nucleosides, and the only detectable S1 nuclease product from 5'-[-³²P]UTP-labeled RNA should be 5'-UMP . The results in Fig . 4C show identical products for YhcR and micrococcal nuclease, indicating strongly that YhcR cleaves RNA in the same manner as micrococcal nuclease, as could be predicted from the SNase domain homology . Quantitation of radioactivity in the spots was consistent with the nucleotide composition of the labeled substrate (data not shown) . The N-terminal portion of the yhcR coding sequence (codons 36 to 529), specifying two OB-folds and the SNase domain, was also cloned into the His-tag vector . Purified N-terminal YhcR possessed the same RNase activity as micrococcal nuclease, although it was less active than the full-length YhcR (Fig . 4C, lanes 3 and 4) .

Micrococcal nuclease is a sugar-nonspecific nuclease (22) . To test whether YhcR could cleave single-stranded DNA, an 18-nt, 5'-end-labeled oligodeoxyribonucleotide was incubated with purified YhcR . The results (Fig . 4D) show that the DNA oligonucleotide substrate was degraded completely in the presence of Ca²⁺ and partially in the presence of Mn²⁺ . No activity was detected in the presence of Mg²⁺ . Nuclease activity on double-stranded DNA was also tested, using plasmid DNA as a substrate (Fig . 4E) . Again, the DNA substrate was degraded completely in the presence of Ca²⁺ . On the other hand, addition of Mn²⁺ resulted in nicked and linearized plasmid forms but not complete degradation . This was observed as well in the presence of Mg²⁺ . No YhcR activity was detected in the absence of free divalent cation (data not shown) .

The C-terminal half of YhcR is annotated in the databases as a "5'-nucleotidase." The isolated full-length protein was tested for 5'-nucleotidase activity, using 5'-AMP as the substrate and assaying for release of phosphate (14) . No 5'-nucleotidase activity was detected in the presence of a variety of divalent metals (Mg²⁺, Mn²⁺, Ca²⁺, Co²⁺, and Zn²⁺) at pH 6.0, 8.0, and 9.0 . (The control 5'-nucleotidase, from Crotalus atrox venom [Sigma-Aldrich], was positive for phosphate release.)

Biochemical parameters of YhcR activity were determined by using ³H-labeled total cellular RNA as substrate (see Materials and Methods) . YhcR did not require monovalent cation for activity, was most active with KCl between 50 and 200 mM, and was inhibited by the presence of NaCl at concentrations of 100 mM and higher (Fig . 5A) . YhcR required a minimum of 0.1 mM Ca²⁺ for significant activity . Maximal activity was observed in the range of 1 to 5 mM Ca²⁺ (Fig . 5B) . YhcR was 10-fold less active in the corresponding concentrations of Mn²⁺ . pH dependence of YhcR activity was tested in the pH range of 6.0 to 9.5 . YhcR was active at pH 6.5 and greater, with maximal activity at pH 9.0 (Fig . 5C) . This was similar to the activity of micrococcal nuclease, which has a pH optimum of 9.0 to 10.0 (22) .

 
Localization of YhcR activity. Although the initial isolation of YhcR was from cell extracts, suggesting a cytosolic protein, the YhcR amino acid sequence contained an N-proximal signal sequence that was predicted to allow secretion of YhcR by the twin-arginine secretion pathway (Fig . 3B) . Indeed, YhcR protein was found previously to be part of the B . subtilis secretome (12) . On the other hand, as discussed above, the C-terminal domain of YhcR is predicted to contain an anchor sequence, which would bind YhcR to the cell wall (and thus explain the presence of YhcR in cell extracts) . A mutant strain was constructed in which the yhcR sequence encoding amino acids 205 to 777 was replaced with a neomycin resistance cassette . (We found that the YhcR-disrupted strain used in the experiment shown in Fig . 2 still specified a YhcR-encoded RNase activity present in the culture medium.) YhcR activities in cell extracts from wild-type and yhcR deletion strains and from an equivalent volume of culture medium were assayed by zymogram analysis (Fig . 6A and B) . As a control for Ca²⁺-dependent nuclease activity, cell extracts from a strain that is missing two low-molecular-weight nucleases, yncB and yokF (24), were included in the zymogram analysis . The Ca²⁺-dependent yncB and yokF gene products are thought to be membrane bound . In the wild-type whole-cell extract (Fig . 6A), prominent bands of a high-molecular-mass (>115 kDa), Ca²⁺-dependent RNase activity were observed, in addition to multiple bands of activity migrating between 70 and 115 kDa (Fig . 6A, lane 1) . These bands were also present in the yncB yokF strain (lane 3) . Disruption of yhcR resulted in a loss of all activity in the >70-kDa range (Fig . 6A, lanes 2 and 4) . Of note is that YhcR appears to be the dominant Ca²⁺-dependent nuclease . Except for a faint band at about 65 kDa (present in all four lanes), all of the Ca²⁺-dependent nuclease activity is attributable to either YhcR (in the high-molecular-weight range) or yncB/yokF (in the low-molecular-weight range) . Also of interest were the multiple forms in which YhcR activity was present, as was observed in the initial identification of YhcR (Fig. 2B and C) . These may be due to posttranslational processing, protein degradation, or YhcR bound to cell wall fragments digested by lysozyme .

 
When protein isolated from equivalent amounts of culture medium was analyzed (Fig . 6B), high-molecular-mass bands of RNase activity were seen, as well as a medium-molecular-mass (approximately 50 kDa) RNase activity (Fig . 6B, lanes 1 and 3) . We could assign all of these activities to YhcR, based on the absence of such activities in the single- and triple-deletion mutants (Fig . 6B, lanes 2 and 4) . Thus, YhcR protein seems to be processed (or degraded) extracellularly, giving rise to one or more additional polypeptides with RNase activity .

Since the YhcR C-terminal region contains a putative anchor sequence (Fig . 3C), it was likely that the cell-associated activity we observed was due to release of YhcR from the cell wall during processing of cell pellets . To confirm this, we examined whether YhcR was present in protoplasts (Fig . 6C) . A comparison of a whole-cell extract versus a protoplast extract in wild-type and yhcR deletion strains (lanes 1 in Fig. 6C) showed that no YhcR activity was detected in protoplasts from the wild-type strain . This is consistent with the prediction of a putative cell wall anchor sequence . The observation of YhcR activity in the culture medium (Fig . 6B) likely stems from secreted protein that fails to be cell wall anchored .

In summary, biochemical and genetic experiments demonstrate that the B . subtilis yhcR gene encodes a high-molecular-weight, sugar-nonspecific nuclease . Based on the zymogram analyses, it appears that YhcR is the major Ca²⁺-activated nuclease in this organism . (It is possible, however, that other Ca²⁺-activated nucleases are present in the cell extract but their activity does not survive the denaturing/renaturing process inherent in the zymogram analysis.) Earlier reports of Ca²⁺-activated nucleases of B . subtilis are described in a recent review by Condon (6) . None of the nucleases studied previously has the same combined size, divalent cation dependence, and mode of action as YhcR . Thus, it is unlikely that any of these were YhcR .

The bulk of YhcR activity appears to be associated with the cell wall and so could function in acquisition of extracellular nucleic acid . However, in other experiments, the yhcR deletion strain did not show any clear growth phenotype in rich or defined media, was as competent as a wild-type strain, and did not grow slower than the wild type in a minimal medium that contained RNA or DNA as the only phosphate source (data not shown) . The latter finding is unlike the case of YokF, which is required for utilization of RNA or DNA as a phosphate source (24) . Strains in which the yhcR deletion was combined with deletions in any of the three characterized B . subtilis 3'-to-5' exoribonucleases (PNPase, RNase R, or YhaM) also did not show obvious changes in growth phenotype .

The distinctive domain structure of YhcR and its occurrence in only one other prokaryote (O . iheyensis) suggest that YhcR may be responsible for a function that is required in particular environmental niches . That these niches are so diverse (soil for B . subtilis and deep sea sediment for O . iheyensis) is remarkable . The absence of YhcR in protoplasts and its likely extracellular location appear to exclude this nuclease from an mRNA turnover function . It was interesting that our RNase assay system, using a variety of ionic conditions, did not reveal other major exonucleases or nonspecific endonucleases . Perhaps our in vitro assay conditions do not support the activity of other B . subtilis ribonucleases, which could be involved in mRNA decay . The search for such an activity continues .

 
We thank S . Tamasdan for help on the use of FPLC .

This work was supported by Public Health Service grant GM-48804 from the National Institutes of Health .

 
* Corresponding author . Mailing address: Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine of New York University, New York, NY 10029 . Phone: (212) 241-5628 . Fax: (212) 996-7214 . E-mail: david.bechhofer@mssm.edu.

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