Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), Abteilung Molekulare Genetik, Corrensstraße 3, 06466 Gatersleben, Germany1
FB Biologie/Chemie, Universität Osnabrück, Barbarastraße 11, 49069 Osnabrück, Germany2
Author for correspondence: Hildgund Schrempf. Tel: +49 541 969 2895. Fax: +49 541 969 2804. e-mail: schrempf{at}biologie.uni-osnabrueck.de
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ABSTRACT |
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Keywords: chitin-binding protein, chbB gene
The GenBank/NCIB accession number for the sequence reported in this paper is AF181997.
a Present address: Institute of Biotechnology, Nghia do, Tu liem, Hanoi, Vietnam.
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INTRODUCTION |
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In contrast to the above polysaccharides, only a few Bacillus species are known to hydrolyse the second most abundant polysaccharide in nature, chitin, and its deacetylated derivative chitosan. B. amyloliquefaciens, Bacillus megaterium and Bacillus subtilis are counted among the degraders of shrimp shell waste (Sabry, 1992 ), although their detailed degradation properties have been rarely investigated (Frädberg & Schnürer, 1998
). Chitinases have been identified within Bacillus cereus (Pleban et al., 1997
; Trachuk et al., 1996
), Bacillus circulans (Watanabe et al., 1992
) and Bacillus thuringiensis (Sampson & Gooday, 1998
). Much is known about B. circulans chitinases and their corresponding genes (Watanabe et al., 1992
; Wiwat et al., 1999
; Zeng et al., 1998
). Chitosanases have been characterized from B. circulans (Mitsutomi et al., 1998
; J. Saito et al., 1999
), B. cereus (Kurakake et al., 2000
) and some unidentified Bacillus species (Izume et al., 1992
).
In contrast to members of the Bacillaceae, most Streptomyces species produce a multitude of chitinases (Robbins et al., 1988 ; Blaak et al., 1993
; Blaak & Schrempf, 1995
; A. Saito et al., 1999
) and use chitin as carbon and nitrogen source (Kutzner, 1981
). Studies by Schrempf (1999)
revealed that many streptomycetes secrete small (1819 kDa) proteins adhering specifically to
-chitin, which is composed of poly-N-acetylglucosamine chains arranged in an anti-parallel fashion. They have thus been named chitin-binding proteins (CHBs). Two of these types, CHB1 and CHB2, and their corresponding genes have been identified in Streptomyces olivaceoviridis (Schnellmann et al., 1994
; Zeltins & Schrempf, 1995
, 1997
) and Streptomyces reticuli. respectively (Kolbe et al., 1998
), and have been characterized in detail.
In this report we reveal for the first time that B. amyloliquefaciens ALKO 2718 secretes a small protein (ChbB) which cross-reacts with anti-CHB1 antibodies. The ChbB protein was purified and its characteristics were analysed. Using reverse genetics, the chbB gene and neighbouring regions were identified and studied. Additional studies showed that ChbB homologues are abundant among different B. amyloliquefaciens strains.
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METHODS |
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Isolation and analyses of DNA.
Chromosomal DNA from B. amyloliquefaciens ALKO 2718 was prepared as described by Cutting & Vander Horn (1990) . Plasmid DNA was isolated from E. coli (Sambrook et al., 1989
). Digestion and ligation of DNA were performed with various restriction enzymes and T4 DNA ligase following the suppliers instructions. Gel electrophoresis was performed in 11·2% agarose gels using either TBE or TAE buffer. After staining in buffer containing ethidium bromide (Sambrook et al., 1989
) DNA fragments were inspected under UV light.
Cloning and transformation.
Cloning of the orf3 DNA fragment (see Fig. 2) was done using the RAGE (rapid amplification of genomic ends) protocol for PCR cloning, as described previously (Hoang & Hofemeister, 1995
). EcoRI-cleaved chromosomal DNA of B. amyloliquefaciens ALKO 2718 was ligated with pUC18 digested by EcoRI. The initial PCR amplification was performed with primers A and B designed according to the N-terminal amino acids from the mature 20 kDa protein after Edman degradation (see Results). Subsequent steps of RAGE were done using the primer pairs EF or GH and CD or IK, respectively (see Fig. 2b
). PCR was carried out with KlenTaq Polymerase (Sigma) or the Expand Long Template PCR System (Boehringer Mannheim, now Roche). The PCR fragments were purified with a QIAEX gel elution kit (Qiagen) and cloned by using the pGEM-T vector kit (Promega). E. coli was transformed with plasmid DNA using CaCl2 or electroporation (Sambrook et al., 1989
). Using chromosomal DNA of B. amyloliquefaciens as well as primer L (corresponding to the ShineDalgarno sequence AAAGAAGGGAG) and primer M (which replaces the stop codon of orf3 by a BglII site), the complete orf3 (about 650 bp) was amplified and the DNA fragment was cloned into the pQE16 vector, resulting in the plasmid pQEC1. Primers used are listed in Table 1
. Plasmid pHBC1 was constructed using pHB201 (Bacillus Genetic Stock Center, Ohio State University, Columbus, OH, USA) after EcoRI and EcoRV digestion. The chbB DNA fragment was isolated from pQEC1 after HindIII digestion and subsequently, after blunting and MunI digestion, ligated into the prepared vector DNA. Plasmid pHBC1 was transformed into naturally competent cells of B. subtilis 168 using standard conditions (Dubnau & Davidoff-Abelson, 1971
).
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Purification of the His-tag protein.
Plasmid pQEC1 was transformed into E. coli M15(pREP4) and overexpression was achieved following the procedure outlined in the QIAexpressionist handbook (Qiagen). The His-tag fusion protein was accumulated in the periplasm and was thus available by osmotic shock treatment. The washed cells were suspended in 30 mM Tris/HCl, pH 8·0, 20% sucrose, incubated on ice for 10 min, sedimented (8000 g, 15 min, 4 °C), resuspended in 5 mM ice-cold MgSO4 for 10 min and centrifuged for 15 min at 4 °C. The supernatant was collected, equilibrated to 50 mM Na2HPO4, pH 8·0, 300 mM NaCl, 10 mM imidazole, and applied to an Ni-NTA column (Qiagen). After five washes, corresponding to the volume of the column, in the same buffer supplemented with 40 mM imidazole, proteins were released by increasing the imidazole concentration. The fusion protein was found to be released in the presence of 200 mM imidazole. The purity of the protein was analysed by SDS-PAGE and by immunodetection using anti-ChbB antibodies (see Fig. 4a).
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SDS-PAGE and amino acid sequencing.
SDS-PAGE was performed with 12·5% polyacrylamide gels and 0·1% SDS (Laemmli, 1970 ). For N-terminal amino acid sequencing proteins were transferred to a PVDF membrane (Immobilon P; Millipore) and subjected to Edman degradation using a model LF 3400 gas-phase sequencer (Beckman), followed by HPLC of the phenylthiohydantoin amino acids.
Immunological studies.
Antiserum was obtained by immunization of a rabbit with the purified His-tagged ChbB protein isolated from an E. coli M15(pREP4) transformant containing the construct pQEC1. Proteins were separated by SDS-PAGE (Laemmli, 1970 ) and blotted onto a nitrocellulose (Satorius) or a nylon membrane (Fluorotrans; Pall). The membrane was treated with blocking buffer (1% BSA, 150 mM NaCl, 10 mM Tris/HCl, pH 7·5) for 1 h and incubated in the same buffer in the presence of antiserum (dilution 1:500) for 2 h. After two washes with TBS (20 mM Tris/HCl, pH 7·5, 500 mM NaCl containing 0·05%, v/v, Tween 20 and 0·2%, v/v, Triton X-100) and one wash with TBS only, the membrane was treated with alkaline-phosphatase-conjugated goat anti-rabbit secondary antibodies (diluted 1:10000) for 2 h. Colour development was performed in TBS buffer containing 0·33 mg Nitro blue tetrazolium chloride ml-1 and 0·165 mg 5-bromo-4-chloro-3-indolyl phosphate (BCIP) ml-1 (Sigma).
Binding tests.
Five micrograms of the His-tag ChbB protein purified from E. coli or the purified ChbB protein secreted by B. amyloliquefaciens was mixed with 2 mg substrate in 50 µl of the indicated buffer (see below), containing 1% BSA, and shaken for 16 h at 4 °C or for 3 h at room temperature. The samples were centrifuged at 10000 g for 5 min, then each supernatant was collected. The pellet was washed twice in the same buffer and resuspended with 50 µl buffer. Each sample (supernatant or resuspended pellet) was mixed with loading buffer, heated for 10 min at 100 °C and analysed using 12% SDS-PAGE. The relative quantities of the protein were estimated after Coomassie staining or immunodetection (Zeltins & Schrempf, 1995 ) by scanning of corresponding bands and subsequent analysis using Scion Image software (Scion, MD, USA). To study the effect of pH, crab shell powder was mixed with ChbB protein in aliquots of various buffers adjusted to different pH values. Citrate/phosphate buffer (10 mM) was used for tests at pH 37, Tris/HCl buffer (10 mM) for pH 710. The effect of salt was tested after the addition of NaCl (0·5 or 1 M) to citrate/phosphate buffer (pH 7, 10 mM, 1% BSA) containing the chitinous sample. Various substrates, i.e.
-chitin (crab shells), ß-chitin from Sepia and from Siboglinum fjordicum, chitosan, ß-glucan (yeast), ß-glucan (barley), cellulose from cotton linters and xylan (oat spells), were used to study the binding specificity of the ChbB protein in citrate/phosphate buffer (pH 7, 10 mM, 1% BSA).
Analysis by immunofluorescence light microscopy.
A suspension (0·1 ml) of chitin (1%) (10 mM citrate/phosphate buffer and 1% BSA, pH 7·0) was centrifuged and washed three times with the buffer plus 1% BSA. The chitinous sample was then covered with 100 µl of a solution containing 0·5 µg ChbB protein and left at room temperature for 1 h. After three washes in the same buffer, a 1:100 dilution of the antiserum [anti-His-tag ChbB antibodies (rabbit)] was added for 1 h, followed by another three washes in buffer. A fluorescein-labelled secondary antibody (rabbit) was added for 1 h. After three washes in buffer, the remaining layer was directly visualized using UV light, Kodak Ektachrome professional film and an Axiovert microscope.
Chemicals and materials.
Chitin from crab shells (practical grade; Sigma) was used after grinding and colloidal chitin was prepared as described by Jeuniaux (1966) . For kinetic studies, highly purified chitin powder from crab shells and ß-glucan from barley (Sigma) were utilized. ß-Glucan (from yeast), chitosan, Avicel and xylan were from Sigma and cotton powder from Fluka. The ß-chitin samples were a gift from H. Chanzy (Grenoble, France). Ni-NTA agarose was supplied by Qiagen. DEAE, MonoS and phenyl Superose (HR5/5) columns were from Pharmacia. Restriction enzymes were either from Boehringer Mannheim (now Roche) or Amersham. T4 DNA ligase, T4 DNA polynucleotide kinase, Klenow enzyme and the Expand Long Template PCR system were purchased from Boehringer Mannheim (now Roche). KlenTaq polymerase was from Sigma. Serratia marcescens chitinase was from Sigma. All other chemicals were obtained from Merck, Sigma or Serva.
Test for chitinolytic activity.
Cultures were grown in Spizizen minimal medium with 0·5% colloidal chitin and 0·2% yeast extract at 37 °C for 24 h. The cell-free supernatant was used for enzyme testing. The test was performed using carboxymethyl chitin/Remazol brilliant violet (no. 04106; Loewe Biochimica) by mixing 0·1 ml citrate/phosphate buffer (0·2 M, pH 6·0) and 0·1 ml substrate (0·2% in water) with 0·2 ml of the respective culture supernatant or enzyme sample. The incubation was performed for 12 h at 37 °C. The reaction was stopped by adding 0·1 ml 1 M HCl and absorption was estimated at 600 nm. The plate assay was performed by streaking cells on TBY agar containing 2% colloidal chitin. The plates were incubated for 48 h at 37 °C and rinsed with KJ/J2 reagent. Chitin hydrolysis was recorded by haloes in zones of colony growth.
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RESULTS |
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Identification and characterization of the chbB gene and neighbouring genes
Using total DNA of B. amyloliquefaciens ALKO 2718, the initial PCR amplification was performed with the primers A and B designed according to the determined N-terminal amino acid sequence from the mature 20 kDa protein (Fig. 2). Subsequent steps of the RAGE (rapid amplification of genomic ends) procedure were performed using the primer pairs EF or GH and CD or IK, respectively (Fig. 2
, Table 1
). Restriction fragments of total B. amyloliquefaciens DNA (cut with HindII, HindIII or EcoRI) hybridizing with the labelled PCR fragment corresponded to those predicted from the restriction map of the amplified PCR product. Using chromosomal DNA of B. amyloliquefaciens, a fragment of about 4·5 kb was generated after PCR cloning and was sequenced. Three complete and two incomplete ORFs were found (Fig. 2a
). The experimentally determined N-terminal amino acid sequence of the secreted 20 kDa protein (see first paragraph) corresponded to amino acid residues 2843 of the protein deduced from orf3 (with a predicted signal peptide, 127) (Fig. 3
). The predicted mature 19·8 kDa protein deduced from orf3 shares 39, 37 and 45% identical amino acids with the previously identified chitin-binding protein CHB1 from Streptomyces olivaceoviridis (Schnellmann et al., 1994
), CHB2 from Streptomyces reticuli (Kolbe et al., 1998
) and CBP21 from Serratia marcescens (Suzuki et al., 1998
), respectively. The B. amyloliquefaciens gene was named chbB (Fig. 3
), as it encodes a chitin-binding protein (see above and below) from a Bacillus species.
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The proteins deduced from the additional sequenced ORFs (Fig. 2a) share a high degree of similarity with proteins of unknown function, i.e. part of YnaE (incomplete Orf1), YvgO (Orf4) or part of YobB (incomplete Orf5), the genes of which are scattered in the B. subtilis 168 chromosome (Kunst et al., 1997
). The part of the protein deduced from the sequenced orf2 (Fig. 2a
) is similar to the deduced N5,N10-methylenetetrahydromethanopterin reductase from Staphylococcus aureus (accession No. U96107). It remains to be shown whether the B. amyloliquefaciens Orf2 protein corresponds to a functional reductase catalysing the reversible reduction of N5,N10-methylenetetrahydromethanopterin with the reduced F420 as electron donor, a characteristic feature of methanogenic archaea (Vaupel & Thauer, 1995
) or whether it corresponds to another type of F420-dependent reductase.
Purification of the ChbB protein and the His-tag ChbB fusion protein
B. amyloliquefaciens was pregrown (see Methods) and after washing transferred to minimal medium supplemented with ground crab shell chitin (1%). Since the predominant part of the desired protein was found in the supernatant of the culture, the latter served as isolation source. Like the previously described CHB1 and CHB2 proteins (Schnellmann et al., 1994 ; Kolbe et al., 1998
), the Bacillus protein does not bind to DEAE at a range of various pH values, including pH 9. The concentrated run-through of the DEAE column contained two dominant proteins of 20 and 24 kDa. The 20 kDa protein cross-reacted with anti-CHB1 antibodies and could be purified to near homogeneity by chromatography using MonoS material and citrate/phosphate at pH 5·2 (Fig. 4b
). About 1 mg ChbB protein was gained per 1 l culture.
To gain larger amounts of the protein, the chbB gene was fused in-frame with six histidine codons, resulting in the construct pQEC1 (for details see Methods). After induction with ITPG, an E. coli transformant carrying pQEC1 produced larger quantities (2 mg per 500 ml culture) of the His-tag ChbB fusion protein (Fig. 4a). The latter was used to raise anti-ChbB antibodies, as well as for binding studies.
Binding specificities of the ChbB protein
The binding properties of the purified mature B. amyloliquefaciens ChbB protein were studied (Fig. 5). The quantities of the proteins, bound and/or unbound, were analysed by SDS-PAGE and, if necessary, the proteins were immunodetected. For initial studies anti-CHB1 antibodies were used; however, as their affinity was comparatively low, they were substituted by antibodies newly raised against the His-tag ChbB protein (from E. coli, see above). For binding tests, ChbB (5 µg) was mixed with 2 mg of each substrate. The maximally bound ChbB was set as 100%. The pH optimum was established as 7 (Fig. 5a
), and salt reduced binding to about 30% (0·5 M NaCl) or 50% (1 M NaCl) (Fig. 5b
). ChbB showed a preference for ß-chitin, less for
-chitin; however, weak binding of ß-glucans from yeast and barley, and of crystalline cellulose was detected (Fig. 5c
). For visualization (detection of fluorescence)
- and ß-chitin were treated with ChbB. Binding was detected with primary anti-ChbB antibodies, followed by secondary fluorescein-labelled antibodies. Fluorescence was most intense for ß-chitin and reduced for
-chitin. No fluorescence was scored on the control chitin sample which had not been treated with ChbB (Fig. 6
). After purification, the His-tag fusion protein ChbB obtained from the heterologous E. coli host was found to have identical binding characteristics (data not shown).
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DISCUSSION |
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The Bacillus ChbB carries a tyrosine residue (Y) (see Fig. 3) corresponding in its location to the W57 residue in Streptomyces CHBs. The latter has been shown to be directly involved in the interaction with
-chitin (Zeltins & Schrempf, 1997
). Its replacement by a leucine or a tyrosine residue also leads to nearly complete cessation of binding to
-chitin (Zeltins & Schrempf, 1997
). The two Serratia proteins (CBP21 and Chi) also carry a tyrosine residue in this position, corresponding to W57 in CHB1 (Fig. 3
) (Shin et al., 1996
). It is interesting that the four additional W residues within the CHBs correspond in their relative positions to those present in ChbB and the Serratia CBP21 protein (W99, W114, W134 and W184; see Fig. 3
). Strikingly, ChbB lacks all the cysteine residues which are found in the CHBs, CBP21 and Chi. We have shown (Svergun et al., 2000
) that within CHB1 SS bridges are formed. Since ChbB lacks cysteine residues, SS bridges stabilizing the topology cannot be formed and it is expected that the shape of ChbB is more flexible than that of CHB1.
ChbB does not display relevant amino acid identities with various types of accessory chitin-binding domains within chitinases from different organisms, including those from streptomycetes (Blaak & Schrempf, 1995 ; Saito et al., 1999
) and B. circulans (Watanabe et al., 1992
). Neither does ChbB share relevant common motifs with a recently discovered Streptomyces tendae protein (9·8 kDa) targeting chitin within various fungi (Bormann et al., 1999
), with Vibrio parahaemolyticus chitovibrin (134 kDa) (Montgomery & Kirchman, 1994
) assumed to mediate adhesion to chitin-containing organisms, nor with a small chitin-binding polypeptide (73 residues) from the haemocyte of horseshoe crab (Suetake et al., 2000
). In nature chitin is very diverse in its organization [i.e. parallel (ß) or anti-parallel (
) arrangement of N-acetylglucosamine chains, variable length, different degrees of crystallization] and its associated compounds (i.e. protein, inorganic substances or glucan). It is therefore not surprising that a number of proteins have evolved with subtle differences in recognition.
The effect of glucose on ChbB secretion (tested for B. amyloliquefaciens ALKO 2718) suggests that the expression of the chbB gene is under catabolite control. This assumption is supported by the identification of cre boxes in the vicinity of a putative promoter (A) of the chbB gene, thus indicating transcriptional repression (Hueck et al., 1994
; Gösseringer et al., 1997
; Stülke & Hillen, 2000
).
In contrast to B. subtilis strains 168 and GB 72, all investigated B. amyloliquefaciens strains display varying levels of low and moderate chitinolytic activity and also secreted a protein of about similar size, cross-reacting with anti-ChbB antibodies. All B. amyloliquefaciens strains secreting a ChbB homologue share a DNA region which hybridizes with the chbB gene. The different sizes of the hybridizing DNA fragments as well as the varying efficiency of PCR amplification (using only one set of primers and the same conditions) reflect some evolutionary divergence of homologues of the chbB gene. Our sequence data showed that the B. amyloliquefaciens chbB gene is situated next to genes of so far unknown function, which have counterparts in the B. subtilis 168 genome. There they are found scattered (Kunst et al., 1997 ), while a chbB homologue could not be identified. It thus appears likely that acquisition of the chbB gene by B. amyloliquefaciens leads to ChbB-mediated interaction with chitin-containing substrates (i.e. certain fungi and a number of chitin-containing organisms) which are subsequently degraded by their chitinolytic activity. Therefore, B. amyloliquefaciens strains are, contrary to B. subtilis, expected to have a selective advantage in colonizing and hydrolysing chitin-comprising substrates in their natural habitats (i.e. soil and marine environments) (Gooday, 1990
).
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ACKNOWLEDGEMENTS |
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Received 29 December 2000;
revised 2 March 2001;
accepted 9 March 2001.