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

Abstract

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 Ca 2؉ and Mn 2؉ . 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 Ca 2؉ -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 3Ј-to-5Ј 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 3Ј-to-5Ј 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. MATERIALS AND METHODSBacterial 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 codi...

Assembly of Minicellulosomes on the Surface of Bacillus subtilis

“…It is also possible that the protein substrates were missing features not yet identified that are required for enzyme activity. The genome of B. subtilis contains two putative sortase-encoding genes whose functions have not been characterized (46,47). As functional Cel8A protein was not displayed when SrtA was absent, these endogenous enzymes were presumably unable to anchor Cel8A to the cell wall.…”

Functional Characterization and Localization of a Bacillus subtilis Sortase and Its Substrate and Use of This Sortase System To Covalently Anchor a Heterologous Protein to the B. subtilis Cell Wall for Surface Display

“…These data indicate that the ␤-lactamase reporter fused to CWAD YhcRMO2 was biologically active. (45) suggests that YhcR is a nuclease that may anchor to the cell wall covalently. In this study, B. subtilis yhcS and the sequence encoding the cell wall anchoring domain of YhcR were characterized.…”

“…It is hypothesized that anchoring YfkN to the cell wall minimizes the proteolytic cleavage of the membrane-bound form of YfkN by B. subtilis proteases. Since both YhcR and YfkN can be detected in the culture medium (8,22,45), this observation poses an interesting question concerning the purpose of immobilizing these enzymes on the cell surface. Although there is no definite answer to this question, the presence of YhcR and YfkN in both the wall-bound forms and the proteolytic cleaved forms in the culture supernatant can possibly be considered a very elegant strategy employed by B. subtilis.…”

“…Although YhcR and YfkN have been identified as an endonuclease (45) and a trifunctional nucleotidase (8), respectively, conclusive demonstration that these proteins are covalently anchored to the cell wall has proven to be difficult. This is primarily due to the presence of high levels of extracellular proteases produced by B. subtilis that degrade these wall-bound proteins.…”

Ribonucleases J1 and J2: two novel endoribonucleases in B.subtilis with functional homology to E.coli RNase E

“…RNase III and a sugar-nonspecific endoribonuclease have been identified in B.subtilis (16–18), but this organism has no homologues of the two essential E.coli endoribonucleases, RNase E ( rne ) and oligoribonuclease ( orn ). However, an activity similar to the former probably exists in this organism (19).…”