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
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. ). In subsequent assays, the Mn2+
- and Ca2+
-dependent activities always copurified. From the pattern in Fig. , 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.
FIG. 1. Partial purification of a high-molecular-weight RNase. (A) Products of RNase activity using a 110-nt, uniformly labeled RNA substrate were separated on a 20% denaturing polyacrylamide gel. Substrate RNA was incubated with no protein (lane 1), with protein (more ...)
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. ). 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
). 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. ). 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. ). This result suggested strongly that the RNase activity detected in the triple-mutant strain was encoded by yhcR
FIG. 2. Identification of yhcR as the gene encoding the high-molecular-weight RNase. (A) Coomassie-stained SDS-PAGE gel (7.5% polyacrylamide) of whole-cell extracts from wild-type (wt) and yhcR-disrupted strains. (B) PVDF membrane containing proteins blotted (more ...) 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. ). 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. ) (12
). 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 and Fig. ). 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. ) 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.
FIG. 3. (A) Domain structure of YhcR. Two regions that do not show significant sequence similarity to other domains are indicated by question marks. (B) Twin-arginine signal sequence. The numbering is from the beginning of the YhcR sequence, with the amino acid (more ...)
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. ). 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
), 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 , 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-β-d-thiogalactopyranoside (IPTG)-induced E. coli strain (Fig. , 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. , 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. , 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).
FIG. 4. Purification and characterization of YhcR nuclease activity. (A) Expression of His-tagged YhcR was induced by addition of 1 mM IPTG. Shown in lanes 1 and 2 are whole-cell extracts before and after induction with 1 mM IPTG, respectively. Migration of major (more ...)
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. ). 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′-[α-32
P]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′-[α-32
P]UTP-labeled RNA should be 5′-UMP. The results in Fig. 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. , 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. ) 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. ). 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+
, 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 3
H-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. ). 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. ). 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. ). This was similar to the activity of micrococcal nuclease, which has a pH optimum of 9.0 to 10.0 (22
FIG. 5. Biochemical parameters of YhcR RNase activity. (A) Monovalent cation dependence of YhcR activity. Purified YhcR (15 ng) was assayed with 30 nmol of [3H]-labeled total RNA and the indicated concentrations of KCl () or NaCl (). Specific (more ...) 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. ). Indeed, YhcR protein was found previously to be part of the B. subtilis
). 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. 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. ). As a control for Ca2+
-dependent nuclease activity, cell extracts from a strain that is missing two low-molecular-weight nucleases, yncB
), were included in the zymogram analysis. The Ca2+
gene products are thought to be membrane bound. In the wild-type whole-cell extract (Fig. ), 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. , 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. , 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
(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. ). These may be due to posttranslational processing, protein degradation, or YhcR bound to cell wall fragments digested by lysozyme.
FIG. 6. Zymogram analyses. Purified proteins (lanes FL and NT, for full-length and N-terminal half) or total protein from whole-cell extracts (A) or culture medium (B) were separated on SDS-PAGE gels (10% polyacrylamide) containing 20-μg/ml B. subtilis (more ...)
When protein isolated from equivalent amounts of culture medium was analyzed (Fig. ), high-molecular-mass bands of RNase activity were seen, as well as a medium-molecular-mass (approximately 50 kDa) RNase activity (Fig. , 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. , 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. ), 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. ). A comparison of a whole-cell extract versus a protoplast extract in wild-type and yhcR deletion strains (lanes 1 in Fig. ) 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. ) 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.