Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences Bethesda, MD 20814, USA1
Department of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen, Sølvgade 83, 1307 Copenhagen K, Denmark2
Department of Microbiology, Technical University of Denmark, 2800 Lyngby, Denmark3
Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, PA 19140, USA4
Author for correspondence: Per Nygaard. Tel: +45 353 22005. Fax: +45 353 22040. e-mail: Nygaard{at}mermaid.molbio.ku.dk
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ABSTRACT |
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Keywords: purine-nucleoside phosphorylase, phosphopentomutase, ribonucleoside, deoxyribonucleoside, catabolism
The GenBank accession number for the sequence data reported in this paper is U32685.
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INTRODUCTION |
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Previously, we cloned and sequenced two adjacent B. subtilis genes, encoding products similar to phosphopentomutase and purine-nucleoside phosphorylase. These loci were designated drm and pnp (now referred to as pupG), and their sequences were submitted to GenBank in 1995 (accession number U32685). Here, we report the characterization of the products of these loci with respect to the regulation of their expression and their functions. These studies identify distinct differences between the B. subtilis nucleoside-catabolizing pathway and that of E. coli.
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METHODS |
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Sporulation.
Sporulation was induced as described by Piggot & Curtis (1987) . Time is indicated in hours after the end of exponential growth. At t20t24, the extent of heat-resistant-spore formation was determined by plating appropriate diluted aliquots of the cultures on L-broth; the remainder was heated at 90 °C for 20 min, and aliquots were plated on L-broth. Colonies arising from heat-treated and non-heat-treated samples were enumerated to determine the proportion of the starting population of cells that were heat-resistant spores.
Enzyme assays.
ß-Galactosidase was assayed at 30 °C as described by Nicholson & Setlow (1990) . Activities of nucleoside-catabolizing enzymes were determined at 37 °C as described previously (Hammer-Jespersen et al., 1971
; Jensen, 1978
). Enzyme activity is given as nmol product formed min-1 (=1 unit).
DNA preparation and sequencing.
Methods used for transformation and for chromosomal and plasmid DNA isolation have been described previously (Wu et al., 1989 ; Saxild et al., 1995
). DNA sequencing was using a Sequenase kit (US Biochemical). All sequencing analysis was done on double-stranded plasmid DNA templates. Using plasmids pHM2 and pKE5, we determined the nucleotide sequence of the drmpupG operon.
RNA analysis.
RNA preparation, Northern-blot analysis and primer extensions were performed as described previously (Penn et al., 1984 ; Wu et al., 1989
; Nygaard et al., 1996
). For Northern analyses, RNA samples (20 µg per sample) and size markers (Promega) were separated on 1·2% agarose gels, transferred to nylon membranes (Stratagene) and probed as described by Sambrook et al. (1989)
. The pupG-specific probe was the 364 bp EcoRI fragment (nt 24457642446128), and the drmpupG-specific probe was the 823 bp HindIII fragment (nt 24456932446516) (Kunst et al., 1997
).
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RESULTS |
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Complementation of E. coli purine-nucleoside catabolism defects
To confirm the identity of drm and pupG, plasmids pKE5 (expressing drm from its native promoter) and pHM2 (expressing pupG from a vector-encoded promoter) were transformed into E. coli strains HO1077 (deoB) and SØ446 (purE deoD), respectively, and scored for complementation. Strain HO1077 is a phosphopentomutase-deficient mutant and cannot use thymidine as its sole carbon source (Hammer-Jespersen, 1983 ). Introduction of pKE5 into HO1077 restored the ability of this strain to grow on minimal medium containing inosine as a carbon source. Additionally, phosphopentomutase activity increased from <1 unit (mg protein)-1 in HO1077 to 8 units (mg protein)-1 in HO1077/pKE5. Strain SØ446 is purine auxotrophic and is unable to use thymidine as its sole purine source. The transformed strain SØ446/pHM2 did grow with inosine as the sole purine source, indicating that the B. subtilis pupG gene could complement the salvage-pathway defect arising from mutation of the E. coli deoD gene. Inosine phosphorylase activity increased from <1 unit (mg protein)-1 in SØ446 to 85 units (mg protein)-1 in SØ446/pHM2. The B. subtilis drm and pupG loci, therefore, do encode phosphopentomutase and purine-nucleoside phosphorylase activity, respectively.
Transcription of the drmpupG locus
Transcription of drm and pupG during both late-exponential and early-stationary-phase L-broth cultures of strain SL4 was examined by Northern-blot analysis. Using a pupG-specific probe, we detected a predominant 2·1 kb transcript (Fig. 2a, lanes 1 and 2). With a probe containing both drm and pupG sequences, a similar 2·1 kb message was visualized (Fig. 2b
, lanes 1 and 2). The transcript size observed in each case strongly suggests that drm (a 1·2 kb gene) and pupG (a 0·8 kb gene) are cotranscribed as an operon. A minor 0·9 kb band was also noted. This band may reflect a low level of expression of a specific pupG transcript or a processing/breakdown product of the 2·1 kb transcript. Expression of the drmpupG transcript was barely detectable in mRNA samples isolated at t2 (Fig. 2
, lanes 3), suggesting that it may be repressed in later stationary phase. To confirm the operon structure of drmpupG, we also analysed expression of single-copy drmpupG transcriptional fusions to lacZ integrated at the chromosomal amyE locus. Fusion to lacZ of several distinct DNA fragments extending from within pupG to at least 300 bp upstream of it yielded background levels of ß-galactosidase activity (data not shown), indicating that pupG is not likely to be transcribed from its own promoter. In contrast, a 1·4 kb PvuIIBglII fragment containing the ripXdrm intergenic region and the 5' end of drm fused to lacZ in SL6479 does support high-level ß-galactosidase activity in L-broth cultures: 135 units (mg protein)-1 at t-1, 176 at t0 and 141 at t2. pupG is, therefore, probably cotranscribed with drm from a promoter in the ripXdrm intergenic region. The lower enzyme level observed at t2 reflects the fact that the drmpupG operon is no longer transcribed at this stage of growth (Fig. 2
).
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Identification of the low-molecular-mass effector of induction
Preliminary analysis had indicated that the levels of Drm and PupG, but not that of PupA, were increased by the addition of ribonucleosides and deoxyribonucleosides to the growth medium. The level of PupA was uninducible and remained between 27 and 34 units (mg protein)-1. The activities of Drm and PupG in wild-type cells grown in the presence of thymidine, inosine and adenosine are shown in Table 2. No induction was observed in the presence of any of the naturally occurring purine and pyrimidine bases (data not shown). To investigate whether a nucleoside must be degraded to induce, experiments were conducted with mutants defective in nucleoside catabolism. In a pupG mutant defective in inosine degradation, inosine no longer induced Drm synthesis, while increased Drm activity was seen when adenosine was added (Table 2
). This indicates that a nucleoside must be phosphorylized to act as an inducer. To determine whether the first products of nucleoside degradation, ribose 1-phosphate or deoxyribose 1-phosphate, were the low-molecular-mass effector molecules, induction experiments were performed in a drm mutant strain. This mutant is unable to convert ribose 1-phosphate and deoxyribose 1-phosphate, formed from inosine and thymidine, respectively, to ribose 5-phosphate and deoxyribose 5-phosphate. The level of PupG and ß-galactosidase activity from a drmlacZ transcriptional fusion was measured in the drm knock-out mutant HH245 grown in the presence of either inosine or thymidine. The PupG level was low due to the interruption of the drm gene and this level was not affected by addition of nucleosides to the growth medium. However, the ß-galactosidase activity, driven by the drm promoter, was also unaffected. This indicates that ribose 1-phosphate and deoxyribose 1-phosphate must be further metabolized to affect the enzyme levels. In the dra mutant HH234, which can degrade deoxyribose 1-phosphate one step more to deoxyribose 5-phosphate and no further, addition of thymidine results in increased levels of Drm and PupG, indicating that the deoxyribose 5-phosphate formed may act as a low-molecular-mass effector molecule. Since no induction is observed in the drm mutant unable to convert ribose 1-phosphate to ribose 5-phosphate, ribose 5-phosphate is a likely low-molecular-mass effector molecule too. An alternative way of forming ribose 5-phosphate and deoxyribose 5-phosphate is through phosphorylation of ribose and deoxyribose (Fig. 1
). Induction experiments with ribose and deoxyribose were performed with cultures grown on succinate as the carbon source, because neither ribokinase (OReilly et al., 1994
) nor deoxyribokinase (X. Zeng, personal communication) is synthesized when glucose serves as carbon source. When added to cultures growing on succinate both ribose and deoxyribose caused slightly increased enzyme levels in both wild-type and the drm mutant strain, supporting the suggestion that both ribose 5-phosphate and deoxyribose 5-phosphate are low-molecular-mass effector molecules. Addition of thymidine or inosine to a deoR mutant (HH232) resulted in the same level of induction as in wild-type cells (Table 2
). This indicates that the B. subtilis deoxynucleoside regulator protein DeoR, which negatively regulates the expression of the dranupCpdp operon, does not appear to be involved in the regulation of expression of the drmpupG operon.
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DISCUSSION |
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The very efficient nucleoside transport and catabolism found in E. coli and the great number of genes encoding regulatory proteins, transport proteins and enzymes involved in nucleoside catabolism do not have counterparts in B. subtilis. In E. coli, the genes encoding most of the proteins involved in purine- and pyrimidine-nucleoside catabolism are organized in a regulon composed of several single genes: nupC, nupG, tsx, cytX (all encoding transport proteins), udp, (encoding uridine phosphorylase), cdd (encoding cytidine deaminase) and the deo operon deoABCD (Valentin-Hansen et al., 1996 ). This regulon is negatively regulated by the DeoR and CytR proteins. A second operon, xapAB (encoding a purine-nucleoside phosphorylase and a nucleoside transport protein), is not part of the regulon, but is positively regulated by the XapR protein (Seeger et al., 1995
). The E. coli deoB and deoD genes (equivalent to the B. subtilis drm and pupG genes) are encoded in the deo operon, together with deoA (encoding thymidine phosphorylase) and deoC (encoding deoxyriboaldolase). The low-molecular-mass effector that reacts with the regulatory DeoR protein of E. coli is deoxyribose 5-phosphate, while ribose 5-phosphate is not active (Hammer-Jespersen, 1983
). CytR, on the other hand, reacts with cytidine. Additionally, the CytR-controlled promoters are activated by the cAMP receptor protein (Valentin-Hansen et al., 1996
). In B. subtilis, the genes identified corresponding to those of the E. coli regulon are located in two operons: the drmpupG operon and the dranupCpdp operon, in addition to the single genes cdd, encoding cytidine deaminase, and pupA. Expression of pupA and cdd has been found not to respond to nucleosides in the growth medium (Nygaard, 1993
). The dranupCpdp operon is regulated by the DeoR protein (Saxild et al., 1996
; Zeng & Saxild, 1999
) and the drmpupG operon is not. However, the expression of both operons is increased by deoxyribonucleosides. Additionally, only the expression of drmpupG is affected by the presence of ribonucleosides. Our finding that the transcription of the drmpupG operon started at two different sites could suggest distinct ribose 5-phosphate- and deoxyribose 5-phosphate-controlled start sites. This turned out not to be the case, as similar increases in transcription from both the drmP1 and drmP2 promoter were detected in the presence of either thymidine or inosine (Fig. 3
). Most likely there is a protein that negatively regulates the initiation of transcription, and which recognizes both ribose 5-phosphate and deoxyribose 5-phosphate. We observed a major difference between growth on inosine or thymidine as the carbon source, compared with growth on free ribose and deoxyribose. Significant growth was only observed when thymidine or ribose served as the sole carbon source. While the level of the nucleoside-catabolizing enzymes is comparable to that of E. coli, the nucleoside-transport activity is lower in B. subtilis. We therefore suggest that what limits the catabolism of nucleosides in this organism is the transport of nucleosides. In agreement with this is our finding that an E. coli strain carrying a plasmid (pHM2) with the cloned drm gene grew well on inosine as the carbon source although the Drm level was only 30% of that in wild-type B. subtilis, which cannot grow on inosine as the sole carbon source.
Synthesis of the nucleoside-catabolic enzymes in B. subtilis is subject to catabolite repression (Saxild et al., 1996 ). Catabolite repression can occur via a common regulatory mechanism that involves the cis-acting cre element, 5'-TGWAANCGNTNWCA-3', which is active whether located in the promoter region or within a gene (Miwa et al., 1997
). Two such putative elements were found in the drmpupG operon. The first putative cre site (nucleotide 24477392447726) has 12 out of 14 matches with the common sequence, and overlaps with the putative -10 region of the drmP1 promoter and extends to within 2 nt of the putative -35 region of the drmP2 promoter. The second putative cre site (nucleotide 24461682446155) lies in the pupG coding region and matches 11 out of 14 positions. For both cre sites the mismatches are located at the 3' end. The presence of these sites offers an explanation for the observed induction pattern (Table 3
): that the extent of induction of Drm and PupG in particular was lower in glucose-grown cultures than in succinate-grown cultures.
A few studies dealing with nucleoside catabolism in other bacilli have shown that the levels of phosphopentomutase and purine-nucleoside phosphorylase in Bacillus cereus and Bacillus thuringiensis are increased by nucleosides and nucleotides in the growth medium (Gardner & Kornberg, 1967 ; Ipata et al., 1983
; Grigorieva & Sukhodolets, 1979
). Another aspect of purine-nucleoside metabolism found in E. coli and incidentally also in B. cereus is the involvement of purine-nucleoside phosphorylases and adenosine deaminase in the interconversion of adenine compounds to guanine compounds. Such a pathway is not present in B. subtilis (Nygaard et al., 1996
).
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ACKNOWLEDGEMENTS |
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Received 22 March 1999;
revised 14 May 1999;
accepted 28 May 1999.