Centro Nacional de Biotecnología (CSIC), Campus de la Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain1
Author for correspondence: Rafael P. Mellado. Tel: +34 91 5854547. Fax: +34 91 5854506. e-mail: rpmellado{at}cnb.uam.es
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ABSTRACT |
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Keywords: chitosanase, Bacillus subtilis, gene expression, catabolite repression
Abbreviations: LR-PCR, long-range PCR
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INTRODUCTION |
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An estimated 17% of heterotrophic soil bacteria synthesize chitosanases (Davis & Eveleigh, 1984 ) and chitosanase activities have been reported in a variety of microbial species and plants (rewieved by Somashekar & Joseph, 1996
); genes encoding chitosanases have also been identified in the Chlorella PBCV-1 and CVK2 viruses (Lu et al., 1996
; Yamada et al., 1997
). Some chitosanases have been characterized and their amino acid sequences determined, such as the one from Fusarium solani f. sp. phaseoli SUF368 (Shimosaka et al., 1996
) and a few others of bacterial origin, including those of Bacillus ehimensis EAG1 (Akiyama et al., 1999
), Bacillus circulans MH-K1 (Yabuki et al., 1988
), Streptomyces sp. N174 (N174 chitosanase; Boucher et al., 1992
), Nocardioides sp. N106 (Masson et al., 1995
) and Matsuebacter chitosanotabidus 3001 (Park et al., 1999
). The crystal structures of Streptomyces sp. N174 (Marcotte et al., 1996
) and B. circulans MH-K1 (Saito et al., 1999
) chitosanases are available.
This paper describes the isolation and expression of the csn gene from Bacillus subtilis 168 originally identified in our laboratory (GenBank accession no. X92868; Parro et al., 1997a ). The gene was cloned and propagated in B. subtilis, and the chitosanase was overproduced, partially purified and biochemically characterized.
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METHODS |
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DNA manipulation and PCR amplification.
General recombinant DNA manipulation was carried out as described by Sambrook et al. (1989 ). Restriction endonucleases and DNA-modifying enzymes were from Promega and Boehringer Mannheim. B. subtilis 168 chromosomal DNA was used as a template for PCR amplification and long-range PCR (LR-PCR) amplification (Barnes, 1994
; Cheng et al., 1994
). DNA fragments were purified from low-melting-point agarose gels (LM3; Hispanagar) using Streptomyces coelicolor agarase, which was overproduced and purified in our laboratory (Parro et al., 1997b
). Chromosomal DNA was obtained as described by Harwood & Cutting (1990
). PCR and LR-PCR amplifications were carried out in an automated thermocycler (PTC-100; MJ Research). PCR amplification included a denaturation step at 95 °C for 3 min, followed by 30 cycles of incubation at 95 °C for 1 min, 55 °C for 1 min and 72 °C for 2 min; the reaction was ended by 10 min incubation at 72 °C. DNA fragments were amplified by using chromosomal DNA (500 ng) from B. subtilis 168 with 1 U EcoTaq polymerase (Ecogen) in the presence of 2 mM MgCl2 and 40 pmol of each primer in a final reaction volume of 100 µl. To obtain DNA fragments longer than 2 kb, LR-PCR amplification was carried out using the GeneAmpXL kit (Perkin Elmer) and following the manufacturers instructions. The reaction included a denaturation step at 94 °C for 5 min, followed by 15 cycles of incubation at 94 °C for 30 s and 66 °C for 10 min, and 11 cycles of incubation at 94 °C for 30 s and 66 °C for 10 min with an increment of 15 s per cycle; the amplification was ended by 10 min incubation at 72 °C. DNA fragments were amplified from B. subtilis 168 chromosomal DNA (500 ng) with 2·0 U Tth DNA Polymerase (Promega) containing 1 mM Mg(CH3COO)2 and 40 pmol of each primer in a final reaction volume of 100 µl. For automatic DNA sequencing, a 373 DNA sequencer from Applied Biosystems and an Edit-View 1.0 DNA sequencer viewer (Applied Biosystems) were used.
Transcriptional analysis and RNA manipulations.
Aliquots from the different cultures were lysed (Mellado et al., 1981 ) and total RNA was extracted as described by Kedzierski & Porter (1991
). High-resolution S1 nuclease protection experiments were as described by Barthelemy et al. (1986
), Sambrook et al. (1989
) and Parro et al. (1998
) using 50 µg total RNA. The DNA molecular size ladders were chemically derived (Maxam & Gilbert, 1980
) from the same DNA fragment used as a probe in the experiments. Total RNA was transferred to nylon membranes (Hybond N+; Amersham) and used for Northern analysis as described by Sambrook et al. (1989
). Nylon membranes were incubated overnight at 65 °C in 0·5 M sodium phosphate pH 7·2, 10 mM EDTA, 7% (w/v) SDS. A PCR internal fragment of the csn gene was amplified from genomic DNA with the oligonucleotides csn2 (5'-GGCGAGGCTATACATGCGGACGGG-3') and csn1 (5'-GGCATTATCCGATCGTTTCATGG-3') as primers. The amplified DNA fragment (5 ng) was used as template to extend 10 pmol primer csn1 with 5 U sequencing grade Taq DNA polymerase (fmol DNA Cycle Sequencing System; Promega) in the presence of 1xfmol DNA Sequencing Buffer (Promega). The labelled DNA was used as a probe for Northern analysis.
Pulsechase and Western blot experiments.
One millilitre aliquots from different phases of cell cultures growing in defined medium were labelled with 100 µCi (3·7 MBq) [35S]methionine (Redivue Pro-mix L-[35S] in vitro cell labelling mix; Amersham) in a 0·5 min pulse, following a procedure described previously (Parro & Mellado, 1994 ). A 1000-fold molar excess of non-radioactive methionine and cysteine were then added and the incubation continued; 100 µl aliquots were removed from the labelled cultures at 0, 0·5, 1, 2, 5 and 10 min after the pulse and the extracellular and intracellular labelled proteins were subjected to immunoprecipitation and analysis by SDS-PAGE (Laemmli, 1970
). Proteins were immunoprecipitated as described previously (Parro & Mellado, 1994
) with polyclonal antibodies raised against mature Csn extracted from acrylamide gels (Dunbar & Schwoebel, 1990
). Samples treated with non-immune serum were always run in parallel as a negative control. Pulsechase labelling experiments were repeated at least twice. Gels were exposed to Molecular Dynamics Storage Phospho Screens. Screens were scanned with a Molecular Imager FX (Bio-Rad) and relative amounts of radioactivity were determined with Quantity One version 4 software (Bio-Rad). 14C-methylated molecular mass reference markers were obtained from Amersham.
For Western blot analysis, intracellular and extracellular proteins were fractionated by SDS-PAGE (Laemmli, 1970 ) and transferred to Immobilon PVDF membranes (Millipore) as described by Timmons & Dunbar (1990
). Half of the transferred material was stained with 1% (w/v) Coomassie brilliant blue R-250 in 50% (v/v) methanol, 20% (v/v) acetic acid for 15 min. The other half of the transferred material was incubated with antibodies raised against mature Csn and peptides reacting with the antibodies were revealed by further incubation with 0·1 µCi (3·7 kBq) ml-1 125I-labelled protein A from Staphyloccocus aureus (Amersham), as described by Timmons & Dunbar (1990
). Membranes were exposed to Agfa Curix RP2 film at -70 °C. Protein concentration in the different samples was determined as described by Bradford (1976
), using standard I bovine gamma globulin (Bio-Rad).
Chitosanase assay.
Chitosanase activity was assayed as described by Boucher et al. (1992 ) using the neocuproine reagent (Dygert et al., 1965
) for reducing sugar determination and 0·2% (w/v) chitosan flakes (practical grade; Sigma) dissolved in 50 mM sodium acetate buffer pH 5·7 as substrate. Activity was measured after 15 min incubation at 37 °C. One unit (U) of enzyme is defined as the amount of enzyme that liberated 1 µmol D-glucosamine equivalents min-1 under the assay conditions. For chitosanase substrate specificity studies, the substrates were prepared as 2 mg ml-1 solutions or suspensions in 50 mM sodium acetate buffer pH 5·5 and assayed as described above.
Chitosanase purification and analysis.
Total protein from 100 ml culture medium of B. subtilis 168(pQC10) in LB was precipitated at 80% saturation of ammonium sulfate at 4 °C. The precipitate was collected by centrifugation at 12000 g for 20 min, dissolved in 50 mM Tris/maleate buffer pH 7·3 (buffer A) and applied to a 45x1 cm Sephadex G-100 column (Pharmacia) previously equilibrated with the same buffer. The flow rate of the column was 15 ml h-1. Fractions showing chitosanase activity were pooled (17·5 ml) and applied to an SP-Fast Flow Sepharose 6x2·5 cm column (Pharmacia) previously equilibrated with the same buffer. The flow rate of the column was 15 ml h-1. Unbound protein was washed from the column with buffer A containing 75 mM NaCl. Elution of chitosanase was carried out at the same flow rate in a step-wise manner with 30 ml buffer A containing 100, 150, 200, 250 and 300 mM NaCl. All purification steps were carried out at 4 °C. Fractions showing chitosanase activity were pooled and the purified enzyme was stored at -20 °C in 50% (v/v) glycerol. The enzyme remained active without loss of activity for approximately 6 months. The N-terminal amino acid sequence of the purified mature chitosanase was determined by Edman degradation in a Procise 494 protein sequencer (Applied Biosystems).
Chitosanase sequences were retrieved from the NCBI GenBank database. The B. circulans MH-K1 chitosanase sequence was the version determined by Saito et al. (1999 ). Protein sequence comparison and analysis were carried out using the CLUSTAL W multiple sequence alignment program from the UWGCG package (version 1.7; Thompson et al., 1994
). Sequence alignments were adjusted manually taking into account the structural relationships of chitosanases revealed by Saito et al. (1999
). Phylogenetic analysis of the aligned sequences was performed using the maximum-parsimony analysis of the Phylogeny Analysis Using Parsimony (PAUP) program version 4.0 (Swofford, 1988
) from the UWGCG package.
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RESULTS AND DISCUSSION |
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The csn transcription initiation site was determined by high-resolution S1 nuclease protection experiments, using total RNA from B. subtilis 168 growing in minimal medium supplemented with 0·4% glucose. A 1355 bp PCR fragment containing part of the csn sequence was amplified by PCR from the B. subtilis genome using oligonucleotides 366I (5'-GACATGTACTTGTTCGGGATGGC-3'), derived from the yraK gene immediately preceding csn in the chromosome, and 406I (5'-CAAAGATACTTGTCAGCTGTTCCG-3') and digested with HindIII. A 355 bp fragment thought to contain the csn promoter region was radioactively labelled at its unique 5' blunt end and used as a probe. A 192 nt protected fragment was detected corresponding to a transcript starting at 33 bp upstream of the csn translation start codon (Fig. 2b). The deduced -35 and -10 regions of the csn promoter are separated by 17 bp and showed homology to those of the consensus B. subtilis
A promoters (Parro et al., 1997a
)
The transcription termination site of the chitosanase gene was also determined by S1 nuclease mapping. A 384 bp DNA fragment was amplified by PCR from the B. subtilis genome using oligonucleotides csnt (5'-GAACAACTATAATCTAAACGGACC-3'), derived from the csn coding sequence, and 145d (5'-GACGGAACAGTTTATACGCATGG-3'), derived from the yraM gene immediately after csn in the chromosome, and digested with HinfI. The resulting 359 bp fragment was radioactively labelled at its unique HinfI 3' end and used as a probe. A 109112 nt protected fragment was detected (Fig. 2c), locating the transcription termination site 6568 nt downstream of the csn translation stop codon; the presence of more than one protected band could be due to the S1 nuclease nibbling effect previously described (Christie & Calendar, 1983
; Mellado et al., 1986
). The stemloop structure predicted around the transcription termination site is depicted in Fig. 2(d)
.
To determine the secretion pattern of the chitosanase precursor, total intracellular polypeptides from mid-exponential, transition-to-stationary and stationary phases of growth from B. subtilis 168 cultures grown in minimal medium in the presence of 0·4% glucose were pulse-labelled with [35S]methionine and chased with a 1000-fold molar excess of non-radioactive methionine and cysteine as described in Methods. The labelled proteins were incubated with antibody raised against the extracellular chitosanase and immunoprecipitated polypeptides analysed by SDS-PAGE. Chitosanase was only detected during the transition-to-stationary phase of growth (Fig. 3a), coinciding with csn transcription being more abundant (Fig. 2a
). Pre-Csn was rapidly processed and secreted; within 2 min after the pulse-labelled pre-Csn was chased extracellular mature enzyme was detected (Fig. 3a
). The relative amounts of precursor and mature forms were determined by densitometer scanning of the autoradiographs. The 15% of mature chitosanase that remained cell-associated after the 0·5 min pulse illustrates that the passage through the cell wall of B. subtilis is an active step during secretion as identified by Leloup et al. (1997
) and Bolhuis et al. (1999
).
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A 2881 bp DNA fragment comprising csn and flanking regions was amplified by LR-PCR from the B. subtilis 168 chromosome using primers 366I and yraMR (5'-GCCTACTGGAAATAGTTCGGAG-3'), derived from the yraM gene. The amplified DNA fragment was purified and digested with DraI to obtain a 1634 bp DNA fragment comprising the csn coding region plus the 625 bp preceding it and the 179 bp located behind it in the B. subtilis chromosome. The purified DraI fragment was inserted into pNR2 through its unique SmaI site to generate the high-copy-number plasmid pQC10 that carried csn in the same relative orientation as the cat gene, as confirmed by DNA sequencing. B. subtilis 168(pQC10) produced chitosanase in considerably larger amounts than B. subtilis 168, as determined by Western blot assays (Fig. 3b), and propagation of pQC10 in the csn mutant B. subtilis BSCAT40 restored its ability to produce the enzyme at equivalent levels (Fig. 3b
). B. subtilis 168(pQC10) was able to grow in minimal medium containing chitosan as the sole carbon source, whereas B. subtilis BSCAT40 did not (results not shown), as expected according to their relative levels of chitosanase production.
Purification and characterization of chitosanase
Mature chitosanase was purified from stationary-phase supernatants of B. subtilis 168(pQC10) cultures grown in LB. In that phase of growth, B. subtilis 168(pQC10) accumulates aproximately 60-fold more chitosanase activity than B. subtilis 168(pNR2) in the same culture conditions (results not shown). Mature Csn was purified as described in Methods. The purified enzyme, eluting from the SP-Fast Flow Sepharose column at 250 mM NaCl, was protease-free and almost 95% pure, as determined by SDS-PAGE (Fig. 4). N-terminal sequencing of the purified protein confirmed the predicted length (35 aa) of the leader peptide (not shown). The relative degree of purification of the B. subtilis Csn [specific activity 56·9 U (mg protein)-1, yield 33%, and purification factor 1·83] was comparable to that of the N174 chitosanase, which was purified following a very similar procedure (Boucher et al., 1992
).
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Purified B. subtilis chitosanase can hydrolyse glycol-chitosan at a similar rate (23% relative to 100% activity on solubilized Sigma chitosan) to that of the N174 chitosanase (35% of the maximal rate; Boucher et al., 1992 ). Although it has been reported that chitosanases from several sources (reviewed by Somashekar & Joseph, 1996
) hydrolyse CM-cellulose and/or chitin to a different extent, Csn cannot do this, as also reported for chitosanases of Streptomyces sp. N174 (Boucher et al., 1992
), Nocardioides sp. N106 (Boucher et al., 1992
), B. circulans MH-K1 (Yabuki et al., 1988
) and B. ehimensis (Akiyama et al., 1999
). Chitosan concentrations higher than 1 mg ml-1 inhibited Csn, as also happens with N174 chitosanase (Boucher et al., 1992
). The apparent Km for the B. subtilis 168 chitosanase, determined from a double reciprocal plot (not shown), was 0·110 mg ml-1 and its Vmax was 66·3 U mg-1. These parameter values are similar to those of the N174 enzyme (Km 0·088 mg ml-1, Vmax 96·5 U mg-1; Boucher et al., 1992
) but differ from those of B. circulans MH-K1 (Km 0·63 mg ml-1; Yabuki et al., 1988
) and B. megaterium (Km 0·82 mg ml-1; Pelletier & Sygush, 1990
).
From the results obtained it can be concluded that the csn gene of B. subtilis 168 encodes a chitosanase whose amino acid composition and functional characteristics are close to those of the Gram-positive bacterium Streptomyces sp. N174, despite the phylogenetic distance of their respective genera.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 14 April 2000;
revised 20 July 2000;
accepted 26 July 2000.
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