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Genetics and Molecular Biology |
Dipartimento di Genetica e Microbiologia, Università degli Studi di Pavia, Via Abbiategrasso, 207, 27100 Pavia, Italy1
Genencor International, 925 Page Mill Road, Palo Alto, CA 94304, USA2
Author for correspondence: Alessandra M. Albertini. Tel: +39 0382 505549. Fax: +39 0382 528496. e-mail: albert{at}pillo.unipv.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Keywords: Bacillus subtilis, subtilase, stationary phase, transcription analysis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The major extracellular proteolytic enzymes are neutral (metallo-) and alkaline serine proteases, such as subtilisin produced by Bacillus subtilis. Subtilisin is one of the most extensively studied of all bacterial proteins, due to its commercial importance (Harwood, 1992 ; Wells & Estell, 1988 ). This protease is produced at the onset of sporulation, and its expression is controlled by the complex network regulating the transition state, including the action of several spo0 genes, abrB, sinR, hpr, degU and other regulators (Ferrari et al., 1993 ). Subtilisin is synthesized as a pre-pro-protein, translocated through the cell membrane via the pre-peptide (or signal peptide) and finally activated by autoproteolytic removal of the pro-peptide (Power et al., 1986 ), which functions as an intramolecular chaperone (Shinde et al., 1997 ). This protease is not essential for either growth or sporulation, but is probably used in nature as a scavenging enzyme.
At present more than 200 proteases have been assigned to the superfamily of subtilases (subtilisin-like serine proteases), with representatives both in micro-organisms (archaea, bacteria, fungi and yeast) and in higher eukaryotes (Siezen & Leunissen, 1997 ). All the enzymes belonging to this superfamily have in common a core structure, the catalytic domain, characterized by the presence of ‘structurally conserved regions’, which correspond to common secondary structure elements. Most of the subtilases characterized so far are extracellular and are subdivided into six families, according to the sequence similarity. The subtilisin family includes true subtilisins as well as minor intracellular proteases (Siezen & Leunissen, 1997 ). Some of the intracellular serine proteases from B. subtilis, such as ISP-I (Koide et al., 1986 ), also called IspA, play an important role in protein turnover or processing during sporulation or may be involved in the heat-shock response (HtrA or serine protease Do; Devine & Noone, 1998 ).
We report here the identification and partial characterization of the aprX gene, whose product reveals high similarity with subtilisins from different Bacillus species. To investigate the physiological role of aprX, we studied the regulation of its transcription.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. Escherichia coli DH5
(supE44 lacU169 
80 lacZ 
M-15 hsdR17 recA1 endA1 gyrA96 thi-1 relA1) was used as a host for plasmid constructions.
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) and pJM115, a derivative of pDH32 (Perego, 1993 ), were used to obtain transcriptional fusions to the lacZ gene. PCR products of different length were amplified from the aprX upstream region using primers with EcoRI–BamHI sites. The primer L378 (nt 378–358 in Fig. 1 and 80640–660 in EMBL sequence no. Z99113), internal to the coding sequence, was used in combination with one of the following upstream primers, U147 (nt 147–167 in Fig. 1 and 80871–851 in EMBL sequence no. Z99113), 5'-GGAATTCGTTTTGCTTTTCTGTCTCA-3' (nt 81095–074 in EMBL sequence no. Z99113) and 5'-GGAATTCTATGAGGTTTAGCCAATAGGT-3' (nt 81453–433 in EMBL sequence no. Z99113), to generate, respectively, 232, 455 and 814 bp PCR products. The purified products were cloned into pJM783 and/or pJM115.
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) to perform aprX inactivation by single crossover insertion. Fragments of 315 bp and 243 bp from nt 80972 to nt 80658 and from nt 79651 to nt 79409, respectively, obtained by PCR, were cloned in plasmid pJM105A (Perego, 1993 ) to perform aprX disruption by double crossover insertion. E. coli DH5 was grown in LB medium. When required, ampicillin was added at a concentration of 100 µg ml-1. B. subtilis strains were grown in NB medium (Difco), supplemented, when required, with 2·5 or 5 µg chloramphenicol ml-1, 2 µg kanamycin ml-1 or 1 µg erythromycin ml-1.
DNA manipulations.
Standard DNA manipulations were carried out as described by Sambrook et al. (1989) . B. subtilis chromosomal DNA was prepared by phenol extraction (Albertini & Galizzi, 1985 ). DNA sequencing on both strands was conducted by the dideoxy chain-termination method with the T7 (Pharmacia) or Thermo Sequenase (USB) sequencing kits.
RNA extraction, RT-PCR and primer extension analysis.
Samples (0·3–3 ml) of PB1768 parental strain cells growing in 2x NB (Difco) medium were collected at various times. Total RNA was extracted using the Qiagen ‘RNeasy mini’ or ‘midi’ kit, incubating the cell suspension at 37 °C for 20 min during the lysis step. All the RNA samples were treated with DNase I (RNase free) (Boehringer) and then repurified with the ‘RNeasy clean-up protocol’ (Qiagen). The absence of contaminating DNA was tested by performing PCR reactions on RNA with 15 pmol reverse primer L468 (annealing at nt 468–450 in Fig. 1
, corresponding to nt 80550–568 in EMBL sequence no. Z99113) and of forward primer 5'-TATGAGGGTTTAGCCAATAGGT-3' (negative control primer, annealing at nt 81453–433 of EMBL sequence no. Z99113). Reverse transcription (RT) experiments were performed with 2 µg total RNA, according to the Promega Primer Extension System, using the quoted reverse primer. Following the primer extension reaction, we incubated the samples for 2 min at 94 °C. PCR reactions were performed adding to one-third of the samples 1 U Taq polymerase (Pharmacia), PCR buffer and 15 pmol of one of the following alternative forward primers: negative control primer (as above), positive control primer U262, annealing at nt 262–281 in Fig. 1 (nt 80756–737 of EMBL sequence no. Z99113), and discriminating primer U95, annealing at nt 95–115 in Fig. 1 (nt 80923–903 of EMBL sequence no. Z99113).
The primer extension experiment was performed according to the Promega Primer Extension System with total RNA extracted from PB1768 cells grown in 2x NB medium and harvested at t2. Sixteen picomoles of the primer L378 (Fig. 1) was radiolabelled by T4 polynucleotide kinase using 30 µCi (1·11 MBq) [
-32P]ATP (>5000 Ci mmol-1; Amersham). Primer extension was performed using 1·6 pmol labelled L378 and 100 µg total RNA.
ß-Galactosidase assays.
ß-Galactosidase activity in B. subtilis strains harbouring aprX–lacZ transcriptional fusions was measured as described previously (Scotti et al., 1996 ). The cultures, aerated by shaking, were grown at 37 °C in TM (Anagnostopoulos & Spizizen, 1961 ) minimal medium or 2x NB (Difco) medium.
Sporulation assays.
The sporulation frequencies of B. subtilis strains were measured in samples of liquid cultures grown at 37 °C in Schaeffer medium (Schaeffer et al., 1965 ) at t16 and t22 in the late-stationary phase. Viable spores were counted by plating on NB medium suitable dilutions of 0·5 ml samples treated for 10 min at room temperature with 50 µl chloroform; the titre was compared to that of untreated samples.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) we isolated, via plasmid rescue in the genome region around 158·9°, pDA11 and pAA12, derivatives of the integrative vectors pDIA5304 (Glaser et al., 1993 ) and pJM103 (Perego, 1993 ), respectively, bearing two overlapping fragments covering the region from pksS to miaA. Nucleotide sequence analysis of the region, part of the EMBL accession number Z99113, revealed a 1326 bp ORF (aprX) followed by a short ORF of 699 bp (ymaE), both in reverse orientation with respect to the direction of replication (Kunst et al., 1997 ). aprX is preceded by two putative 
A promoter consensus sequences (PL and PS), each consisting of conserved -35 and -10 sequences. Two start codons (ATG and TTG) preceded by a sequence with good consensus to the canonical ribosome-binding site (Fig. 1) are in the same frame, therefore translation could generate two hypothetical gene products, AprXL and AprXS, 442 or 417 amino acids long, respectively. Downstream of aprX there is a potential hairpin terminator (G -105·1 kJ mol-1), followed by a putative A promoter sequence with a weak -35 consensus, upstream of ymaE. Amino acid sequence similarity analyses with the BLASTP (Altschul et al., 1990 ) and FASTA (Pearson, 1990 ) programs strongly indicated that the deduced product AprX is a serine protease of the subtilase superfamily. Dendrogram analysis based on alignment of multiple sequences of the catalytic domain, from residue 112 of AprXL, emphasizes the relationship of AprX with the families of subtilases sharing highest similarity scores in the FASTA search (Fig. 2). The similarity to subtilases starts from residue 117 of AprXL and extends to the C-terminus of the protein. The active site residues Asp-155, His-187 and Ser-384 are clearly conserved, as well as the entire catalytic domain, including the ‘structurally conserved regions’, as defined by Siezen & Leunissen (1997) . Within the catalytic domain the peculiar characteristics of a subtilase are obvious even if, unlike the majority of subtilases, this enzyme lacks a canonical signal sequence for membrane translocation (signal peptide). This finding suggests an intracellular location for AprX. However, the low sequence similarity with the intracellular serine proteases of Bacillus polymyxa (38·5% identity with BpIsp) and B. subtilis (33·3% identity with BsIsp1) does not allow the classification of this subtilase in the sub-group of known intracellular subtilisins. AprXL contains an N-terminal extension (residues 1–116), relative to the catalytic domain, with no similarity to known proteins. It should also be noted that this new serine protease contains nine Cys residues, four of which are in the catalytic portion. Some of these residues, extremely rare in subtilases from Gram-positive bacteria, are nevertheless conserved in subtilases from other organisms (Siezen & Leunissen, 1997 ). In conclusion, AprX, although strongly related to subtilases, appears to be a member of a new family.
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). These results indicated that aprX is transcribed in rich medium (2x NB) from t2 to t6 (t0 represents the time of transition from the exponential to the stationary growth phase; the growth time is measured in hours before or after t0). The use of alternative forward primers gave us some indications about the transcription start site: no product was present in samples obtained using the discriminating primer U95 (3, 6, 9 and 12). These data suggested that, at the times tested, the transcription start site of aprX must be located in the proximity of the A PS promoter of Fig. 1. Moreover these results ruled out the use of PL as a secondary promoter. The primer extension analysis allowed us to map the transcription start site at nt 238 in Fig. 1 (nt 80780 in EMBL sequence no. Z99113). The start site is in fact located in the PS region, but does not match the A consensus sequence previously identified in this region. Only a sequence with a moderate A consensus results in being properly located.
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-amylase gene of the PB1768 strain. The 232 bp fragment contained the proximal PS region (Fig. 1), the 455 bp fragment also included the distal PL sequence, whereas the 814 bp fragment corresponded to the entire intercistronic stretch between ymaC and aprX. None of the corresponding transformed strains (PB1881, PB1882 and PB1894) produced significant levels of ß-galactosidase activity [less than 2·0 Miller units (mg protein)-1 for all the times tested from t-1 to t6, data not shown]. To evaluate if the absence of expression was due to a lack of promoter activity or to the particular ectopic position of the fusion, the same 232 and 455 bp fragments were cloned in pJM783. This integrative plasmid allows the insertion of the lacZ transcriptional fusion by single crossing-over only into the aprX locus. The eventuality of amplification of the constructs was excluded by Southern blot analysis of chromosomal DNA extracted from the transformed colonies, appropriately digested and hybridized to the integrative vector probe pJM783/455EB.
The integration of the two plasmids pJM783/232EB and pJM783/455EB placed the entire aprX promoter region upstream of the lacZ gene, and generated two different constructs downstream of the fusion, in front of the intact aprX gene. In the case of strain PB1883, aprX was preceded by the PS region of 232 bp; in the case of strain PB1884, aprX was preceded by the PL and PS sequences (Fig. 1). Only strain PB1884 gave significant levels of ß-galactosidase expression (Table 2) and enabled the analysis of aprX transcription. As shown in Fig. 5, aprX–lacZ fusion is expressed in the late-stationary phase in both 2x NB rich medium and TM minimal medium.
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A promoter, the downstream gene ymaE could be under the control of the aprX promoter. In support of this interpretation, we observed that the induction of ymaE, obtained in strain BFS2601, in which this gene is under the control of the spac promoter of pMUTIN4 (Vagner et al., 1998 ), causes a twofold increase of expression from the aprX promoter (Valbuzzi, 1998 ). In rich medium the presence of 1 mM IPTG increased the expression level from 4·5 to 9·8 Miller units (mg protein)-1 at t2 and from 6·5 to 11·5 at t4. Together, these observations suggest an operon organization for these two genes, and a possible regulatory role for YmaE. Anyhow, even if primer extension analysis (Fig. 4) showed that transcription starts from the PS region, additional upstream sequences seem to be required in combination with the minimal promoter sequence to establish aprX transcription.
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). The presence of the degUhy, degQhy and hpr mutations, which highly increase subtilisin expression (Ferrari et al., 1993 ), did not cause analogous effects on aprX expression (strains PB1886, PB1887 and PB1885, compared to parental strain PB1888). The inactivation of sinR and abrB did not stimulate the expression of aprX (strains PB1889 and PB1891 compared to parental strain PB1884 in Table 2 and Fig. 5). Nevertheless a significant increase of activity was observed in TM minimal medium for the sinR mutant (Fig. 5 and Table 2). It is not clear, however, if the negative role of SinR is exerted directly or indirectly.
An involvement of aprX in the sporulation process was suggested by the oligosporogenous phenotype associated with its inactivation, performed in strain BFS2601 according to the procedure for the systematic analysis of unknown genes (Vagner et al., 1998 ; Valbuzzi, 1998 ). Therefore we decided to analyse in detail this aspect. aprX disruption was obtained in strain PB1906 by means of a more reliable construct, deriving from the insertion of the pJM105A CAT cassette by double crossover. No difference was detected in sporulation assays between parental strain 168 and PB1906 (sporulation frequency around 45% at t22).
Conclusions
We have identified the aprX gene of B. subtilis strain 168, encoding a subtilisin-like serine protease. Interestingly, its primary sequence presents several Cys residues (nine over the entire protein, four in the catalytic domain). This amino acid, usually absent in subtilases from Gram-positive bacteria, may play an important role in contributing to the overall stability of the protein, via disulfide bridge formation. The absence of a canonical signal sequence in the deduced N-terminus suggests an intracellular location; the N-terminal extension, relative to the catalytic domain, with no similarity to known proteins, could play a role as a pro-peptide or as a regulator domain.
The data obtained from RT-PCR and primer extension experiments suggest that PS region sequences are responsible for promoting transcription and, together with the data obtained by means of pJM783 transcriptional fusions, indicate that aprX is expressed in stationary phase. The moderate A consensus displayed by the promoter sequence associated with the transcription start site could be the cause of the low aprX expression level, which was monitored by means of ß-galactosidase assays. This could in turn explain why a long exposure time was necessary to detect the primer extension product.
The sequences upstream of aprX tested in pJM115 transcriptional fusions do not promote transcription of lacZ in the amyE locus (‘in trans’ position). The reason for this can be found in the fact that the putative promoter sequences assayed ‘in trans’ do not comprise the entire regulatory region and/or by the fact that the promoter is not active in the ectopic position.
aprX is expressed during the stationary phase, but according to a scheme which can not be superimposed on that described for aprE (Pero & Sloma, 1993 ; Ferrari et al., 1993 ). Only sinR exerts a negative effect on aprX transcription. The regulation of aprX expression resembles the control of a late-stationary-phase phenomenon. However, aprX is not essential for either growth or sporulation.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 5 February 1999;
revised 3 June 1999;
accepted 21 June 1999.
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, 
, Enzyme activity of strain PB1884 grown, respectively, in 2x NB and TM medium; 
, 
, enzyme activity of strain PB1889 grown, respectively, in 2x NB and TM medium. Plotted values are the mean of values obtained in two different experiments.