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Gene cloning and biochemical characterization of chitinase CH from Bacillus cereus 28-9



Bacillus cereus 28-9 is a chitinolytic bacterium showing antagonistic activity against several fungi. One chitinase of 37 kDa, named chitinase CH (ChiCH), was purified by ammonium sulphate fractionation and anion exchange chromatography. The N-termi-nal sequence of purified ChiCH was determined as ANNLGSKLLVGYWHNFD. The chiCH (1,083 bp), cloned from the genomic DNA of B. cereus 28-9, encodes a polypep-tide of 360 amino acids containing the N-terminal signal peptide and a catalytic domain. ChiCH, partially purified from an Escherichia coli transformant harbouring chiCH, exhibit-ed chitinase activity with an optimal pH of 6.0 and an optimal temperature of 40 °C. This ChiCH was slightly inhibitory to conidial germination of Botrytis elliptica. It was suggested that ChiCH is one of the factor involved in the antagonism of B. cereus 28-9 toward fungi.
Annals of Microbiology, 54 (3), 289-297 (2004)
Gene cloning and biochemical characterization
of chitinase CH from Bacillus cereus 28-9
Department of Plant Pathology and Microbiology, National Taiwan University, No. 1,
Sec. 4, Roosevelt Rd., Taipei, Taiwan 106, Republic of China
Abstract - Bacillus cereus 28-9 is a chitinolytic bacterium showing antagonistic activity
against several fungi. One chitinase of 37 kDa, named chitinase CH (ChiCH), was purified
by ammonium sulphate fractionation and anion exchange chromatography. The N-termi-
nal sequence of purified ChiCH was determined as ANNLGSKLLVGYWHNFD. The
chiCH (1,083 bp), cloned from the genomic DNA of B. cereus 28-9, encodes a polypep-
tide of 360 amino acids containing the N-terminal signal peptide and a catalytic domain.
ChiCH, partially purified from an Escherichia coli transformant harbouring chiCH, exhibit-
ed chitinase activity with an optimal pH of 6.0 and an optimal temperature of 40 °C. This
ChiCH was slightly inhibitory to conidial germination of Botrytis elliptica. It was suggested
that ChiCH is one of the factor involved in the antagonism of B. cereus 28-9 toward fungi.
Key words: chitinase, ChiCH, glycosyl hydrolase family 18, gene cloning.
Bacillus cereus is a large, Gram-positive, endospore-forming bacterium that is
very common in soils and plants (Brunel et al., 1994; Martinez et al., 2002). For
plant disease control, B. cereus UW85 has been proven as a reliable biocon-
trol agent of Phytophthora damping off and root rot of soybean (Emmert and
Handelsman, 1999), and capable of producing two antibiotics responsible for
disease suppression (Silo-Suh et al., 1994). In addition, an endophytic B.
cereus strain 65 producing a chitobiosidase is effective against Rhizoctonia
solani in cotton (Pleban et al., 1997). However, the role of chitobiosidase in the
antagonism of B. cereus strain 65 toward fungal plant pathogens is not clearly
In this study, we analysed the chitinases produced by a chitinolytic strain of
B. cereus and found that this B. cereus strain excreted two chitinases. One of
them was partially purified and its encoding gene was cloned. In addition, this
chitinase was characterized and investigated on its antifungal activity toward
Botrytis elliptica, a fungal pathogen of lily leaf and blossom blight.
* Corresponding Author. E-mail:
Achitinolytic strain 28-9 was classified as Bacillus cereus / Bacillus thuringien-
sis according to carbon source utilization ability by using BIOLOG plate (Bacte-
ria & Yeast Identification System, Biolog, Inc., Hayward, CA) and identified as a
strain of B. cereus by PCR analysis of a gyrase gene (Yamada et al., 1999).
Chitinase activity was determined using a fluorometric substrate, 4-methy-
lumbelliferyl β-D-N, N’-diacetylchitobioside (Sigma, St. Louis, MO, USA), fol-
lowing the method of Morimoto et al. (1997). One unit of chitinase activity was
defined as the amount of enzyme required to release 1 µmol of 4-methylum-
belliferone per min. In addition, protein concentration was measured using
Bradford’s method (1976) and bovine serum albumin was used as a standard.
For the purification of ChiCH, all steps were carried out at 4 °C. Bacillus
cereus 28-9 was cultured in 500 mL of M9 broth that contained 0.4% GlcNAc at
37 °C on a rotary shaker at 175 rpm for three days. The culture supernatant
was collected by centrifugation at 10,000 ×gfor 15 min, and proteins in the su-
pernatant were precipitated with ammonium sulphate at 40-70% saturation. The
precipitate was dissolved in 0.1 M of Tris-HCl buffer (pH 8.0) and dialyzed
overnight in the same buffer. The dialysate was loaded onto a Hyper-D anion
exchange column (Sigma) and proteins were eluted with 0.1-0.5 M NaCl gradi-
ent in 0.1 M of Tris-HCl buffer (pH 8.0). ChiCH was eluted with 0.1 M NaCl and
fractions that exhibited chitinase activity were pooled, concentrated by ultrafil-
tration through a Centriplus YM-10 membrane (10 kDa MW cut-off, Millipore,
Bedford, MA, USA), and finally stored at –20 °C.
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)
was performed following the method of Laemmli (1970) using Mini-Protein II ap-
paratus (Bio-Rad, Herculus, CA, USA). A separating gel (10%) containing
0.01% of glycol chitin was used for detection of chitinase activity. After elec-
trophoresis, separated proteins were renatured by soaking the gel in 0.1 M ac-
etate buffer (pH 5.0) containing 1% Triton X-100 at 37 °C with gentle shaking
for 2 h. The gel was stained with 0.01% Calcofluor White M2R (Sigma) in 0.5 M
Tris-HCl (pH 8.9). Protein bands exhibiting chitinolytic activities were visualized
under a UV transilluminator (Trudel and Asselin, 1989). Proteins in the poly-
acrylamide gel were stained with Coomassie Brilliant Blue G-250.
The protein was electroblotted onto a polyvinylidene difluoride (PVDF)
membrane (Millipore), using a Mini-Electroblot apparatus (Bio-Rad). Proteins
on the membrane were stained with 0.1% amido black. The protein band cor-
responding to that exhibiting chitinase activity was cut out from the membrane
and subjected to N-terminal amino acid sequencing by automated Edman
degradation using the Applied Biosystems model 477A protein sequencer (Ap-
plied Biosystems, Perkin Elmer, Foster City, Calif., USA).
The N-terminal amino acid sequence of ChiCH and the conserved se-
quence of family 18 chitinases were used to design degenerated primers.
Primer dchf (5’-TAITGGCAIAACTTTG-3’) corresponding to the amino acid se-
quence YWHNF and primer dchr (5’-TTCITCITCIATITCTATTCC-3’) correspon-
ding to the amino acid sequence G(L/I)D(L/I)DXE were used in polymerase
chain reaction (PCR). “I” refers to inosine. For DNA amplification, PCR was
done with melting at 94 °C for 10 min, followed by 30 cycles of 94 °C 1 min, 54
°C 1.5 min, and 72 °C 1 min, with final extension at 72 °C for 10 min after the
290 C.-J. HUANG and C.-Y. CHEN
last cycle. Amplified DNA fragments were cloned into pGEMT-easy vector and
sequenced. The insert of recombinant plasmid, encoding amino acid sequence
of chitinase was used as a probe in Southern blot analysis and subsequent
colony hybridisation. Probe was prepared using a PCR DIG Probe Synthesis
Kit (Roche Molecular Biochemicals, Mannheim, Germany) following the method
described by the manufacturer. A subgenomic library of B. cereus 28-9 was
constructed in pBluescript II KS(-) and transformed into E. coli TOP10F’. After
colony hybridisation, the insert DNA from a selected clone was sequenced
using the ABI-310 autosequencer (Applied Biosystems).
The DNA fragment carrying the chiCH gene and 17-bp upstream region was
amplified by PCR with primer chf, 5’-GTATAGGAGTGTTGATAATGTTAAA
CAAG-3’, and primer chr, 5’-GTTATTTTTCGAAGGAAAGACCATC-3’. The am-
plified chiCH-containing fragment was cloned into pGEMT-easy vector to cre-
ate recombinant plasmid, pGH51, and transformed into E. coli DH5α. The re-
sulting E. coli DH5α(pGH51) was cultured in LB broth containing 50 µg/ml
ampicillin under constant shaking at 37 °C for 20 h. The periplasmic protein of
E. coli DH5α(pGH51) was extracted following the method of Manoil and Beck-
with (1986). Purification of ChiCH from the periplasmic fraction of B. cereus 28-
9 was performed by the same procedures used for purification of ChiCH from
culture supernatant of B. cereus 28-9.
ChiCH purified from the periplasmic fraction of E. coli DH5α(pGH51) was
used to determine the effects of pH and temperature on chitinase activity of
ChiCH. Glycol chitin was used as a substrate. Chitinase activity was analysed
by a procedure described by Imoto and Yogishita (1971). Hydrolysis reaction of
ChiCH was performed at 37 °C for 25 min in the following buffers of 0.1 M: sodi-
um citrate (pH 3-5), potassium phosphate (pH 6-7), Tris-HCl (pH 8), and
glycine-NaOH (pH 9-11) buffers. Temperature effect on chitinase activity of
ChiCH was measured in 0.1 M potassium phosphate buffer (pH 6.0) from 20 °C
to 80 °C. In addition, substrate specificity of ChiCH was examined on soluble
substrates, namely glycol chitin (Sigma), glycol chitosan (Sigma), car-
boxymethylcellulose (Hayashi, Osaka, Japan), laminarin (Sigma), and soluble
starch (Hayashi).
For antifungal assay, conidial suspension of Botrytis elliptica, at a final con-
centration of 4 × 105conidia/ml, was mixed with purified ChiCH and incubated
at room temperature for 12 h. After incubation, the percentage of germinated
spores of Botrytis elliptica was calculated and the inhibition rate was used as
an indication of antifungal activity. The experiment was repeated for three times
and analysed statistically by Duncan’s Multiple Range Test.
The nucleotide sequence data of chiCH have been submitted to Gen-
Bank/EMBL DNA Databases under accession numbers AF510723.
Three chitinolytic B. cereus strains isolated from Israel (Pleban et al., 1997),
United States (Wang et al., 2001), and Japan (Mabuchi et al., 2000), have been
reported. These B. cereus strains produce chitinases, such as chitobiosidase
of strain 65 (Pleban et al., 1997), Chi36 of strain 6E1 (Wang et al., 2001), and
ChiA of strain CH (Mabuchi and Araki, 2001). In this report, we found that
Ann. Microbiol., 54 (3), 289-297 (2004) 291
ChiCH produced by the chitinolytic B. cereus 28-9 from Taiwan was similar to
these chitinases. However, the biological function of this kind of chitinases from
different B. cereus strains has not yet been studied. Therefore, we cloned the
ChiCH-encoding gene and investigated the biological function of ChiCH of B.
cereus 28-9 herein.
B. cereus 28-9 produced at least two chitinases and secreted both enzymes
into culture medium. Fig. 1 shows the zymogram of partially purified chitinases
produced by B. cereus 28-9. One chitinase with estimated molecular mass of
37 kDa was named ChiCH and its N-terminal amino acid sequence was deter-
The N-terminal amino acid sequence of ChiCH and the conserved amino
acid sequence of catalytic domains of family 18 chitinases were used to design
degenerate primers, dchf and dchr. A DNA fragment of about 300 bp was am-
plified and determined as the partial sequence of a chitinase gene based on se-
quence analysis data. This fragment was subsequently used as a probe in
Southern blot analysis and colony hybridisation.
Figure 2 shows the result of Southern blot analysis using the 300-bp DNA
probe. EcoRI-digested genomic DNA of B. cereus 28-9 yielded a single band at
the 1.7 kb-position. Single bands were also detected in the EcoRV- and PvuII-
digested B. cereus 28-9 genomic DNA at the positions of about 4.0 kb and 8.0
kb, respectively. Therefore, EcoRI fragments (1.5-3 kb) of B. cereus 28-9 were
used to construct a subgenomic library. One clone, screened from this subge-
nomic library, harboured a recombinant plasmid carrying a 1.7-kb EcoRI insert.
Sequence analysis revealed an open reading frame (ORF) in this region. The
N-terminal amino acid sequence, ANNLGSKLLVGYWHNFD, of excreted
ChiCH coincided with that deduced from the nucleotide sequence of the pre-
dicted ORF as shown in Fig. 3.
292 C.-J. HUANG and C.-Y. CHEN
FIG. 1 – SDS-PAGE and zymogram analysis. (A) Gel was stained with Coomassie
Brilliant Blue G-250. (B) Chitinase activity was detected by staining the gel
containing 0.01% glycol chitin with 0.01% Calcofluor. Lanes: 1, proteins par-
tially purified from the culture supernatant of B. cereus 28-9; 2, ChiCH par-
tially purified from E. coli DH5α(pGH51). ChiCH with estimated molecular
mass of 37 kDa is indicated by an arrow.
Ann. Microbiol., 54 (3), 289-297 (2004) 293
FIG. 2 – Southern blot analysis of the genomic DNA of B. cereus 28-9. The genomic
DNA was digested with EcoRI (lane 1), EcoRV (lane 2) and PvuII (lane 3).
The Southern blot showed signals of hybridization with the 300-bp DNA probe
corresponding to chiCH sequence. The estimated DNA fragment size of each
signal is indicated beside the arrow.
FIG. 3 – Nucleotide and the deduced amino acid sequences of chiCH. Amino acid
residues corresponding to those determined by N-terminal sequencing are in
bold type. The putative active site of ChiCH is underlined. The stop codon of
chiCH is indicated by an asterisk. The putative Shine-Dalgarno sequence,
AGGAG, is italicized.
Sequence analysis indicated that chiCH gene is 1,083 bp in length with an
ATG start codon and a TAA stop codon (Fig. 3). The putative Shine-Dalgarno
sequence, AGGAG, was located 8 nucleotides upstream of the start codon. The
deduced protein (ChiCH precursor) consisted of 360 amino acid residues with a
calculated molecular weight of 39,372 and isoelectric point of 6.21.
Alignment of the deduced amino acid sequence of ChiCH precursor with the
N-terminal amino acid sequence of excreted ChiCH of B. cereus 28-9 showed
that the deduced amino acid sequence of ChiCH precursor contained a signal
peptide which was cleaved off between Ala-27 and Ala-28 by the signal pepti-
dase. The signal peptide of ChiCH produced by B. cereus 28-9 had 27 amino
acid residues with the common characteristics of a signal peptide, including a
positive-charged region, a hydrophobic central core and a signal peptidase
recognition site, Ala-X-Ala (Perlman and Halvorson, 1983).
In addition to the N-terminal signal peptide, the deduced ChiCH contained a
catalytic domain as that shown in the conserved domain database of National
Center for Biotechnology (
The conserved amino acid sequence of the catalytic domain, from Gly-139 to
Leu-150, was homologous to a number of family 18 chitinases (Henrissat and
Bairoch, 1993) (Fig. 3). This region included two aspartate residues, Asp-141
and Asp-143, and one glutamate residue, Glu-145. These residues have also
been found in ChiA1 of Bacillus circulans WL-12 (Watanabe et al., 1993) and
ChiA of Serratia marcescens (Perrakis et al., 1994). Furthermore, Glu-145 in
the deduced ChiCH seemed to correspond to Glu-315 of S. marcescens ChiA,
which has been reported to be involved in the catalysis of chitinase (Perrakis et
al., 1994).
The amino acid sequence of the deduced ChiCH showed 97.5% homology
to that of the ChiA of B. cereus CH (Mabuchi and Araki, 2001) and 94.7% ho-
mology to that of the Chi36 of B. cereus 6E1 (Wang et al., 2001) as analysed by
the comparison program in the GCG package (Fig. 4). In addition, Wang et al.
(2001) have speculated that the chitobiosidase of B. cereus strain 65 is similar
to Chi36 of strain 6E1 (Pleban et al., 1997; Wang et al., 2001). Although ho-
mologous chitinases are distributed in different B. cereus strains from different
countries, it is unclear whether this kind of family 18 chitinase gene is species-
specific in B. cereus. However, a species-specific family 19 chitinase gene has
been found in Burkholderia gladioli (Kong et al., 2001). Therefore, the distribu-
tion of chiCH-like genes in B. cereus strains and other Bacillus species be-
comes a subject to study.
ChiCH was expressed and purified from the periplasmic fraction of E. coli
DH5α(pGH51) as shown by SDS-PAGE and in-gel activity assay (Fig. 1, lane
2). A 33-kDa protein without chitinase activity as shown by in-gel activity assay
was consistently co-purified with ChiCH in our procedure. Therefore, the par-
tially purified ChiCH was used for further characterization. The results indicated
that ChiCH had an optimal pH of 6 (Fig. 5) and an optimal temperature of 40 °C
(Fig. 6). It retained over 75% of optimal activity between pH 5.0–7.0. Further-
more, pH and temperature stabilities of ChiCH could be maintained in the
ranges of pH 3-8 (Fig. 5) and 40-50 °C (Fig. 6), respectively.
The ability of ChiCH to hydrolyse various carbohydrates was examined. The
result showed that glycol chitin was efficiently hydrolysed among five soluble
substrates. In addition, when glycol chitosan was used as a substrate, chitinase
294 C.-J. HUANG and C.-Y. CHEN
Ann. Microbiol., 54 (3), 289-297 (2004) 295
FIG. 4 – Sequence comparison of three chitinases from B. cereus strains. ChiCH (this
study); BcchiA, chitinase A of B. cereus CH (accession number AB041931);
Bcexo, exochitinase (Chi36) of B. cereus 6E1 (accession number AF275724).
The asterisks indicate conserved amino acid residues, two Asp and one Glu,
which have been identified as essential amino acid residues.
ChiCH exhibited 10% of relative enzyme activity. However, null effect on other
substrates, including laminarin (β-1,3-glucan), carboxymethyl cellulose (β-1,4-
glucan), and soluble starch (β-1,4/1,6-glucan), was observed.
ChiCH of 17 µunit inhibited about 10% of the conidial germination of Botry-
tis elliptica and the inhibition level was not augmented by increasing chitinase
activity from 17 to 66 µunit. On the other hand, inhibition of the conidial germi-
nation of Botrytis elliptica was much stronger (55.2% of inhibition) by the cul-
ture supernatant of B. cereus 28-9 at 20 µunits than by the partially purified
ChiCH from E. coli DH5α(pGH51).
According to the study of Pleban et al. (1997), a chitobiosidase is present in
the endophytic B. cereus strain 65. They have suggested that chitobiosidase
activity is important for antifungal activity of strain 65. Our present work indicat-
ed that ChiCH possibly has antifungal activity of mild strength. Since this anti-
fungal level was much lower than that exhibited by the culture supernatant of
B. cereus 28-9, we presume that not only ChiCH but also other antifungal fac-
tors were produced by B. cereus 28-9 and exhibited synergistic or combined ef-
fect against target fungi.
This work was financially supported by National Science Council, Taiwan, Re-
public of China.
296 C.-J. HUANG and C.-Y. CHEN
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... In addition, the ability of rhizobacterial isolates to induce systemic resistance to pathogens is also one of the characteristics of rhizobacteria that can act as candidates for biocontrol agents. The results showed that rhizobacteria, which act as biocontrol agents, have the ability to produce enzymes such as chitinase, 1,3-glucanase, 1,4 glucanase, cellulases, lipases, proteases, and inducyl-aminocyclo-propane-carbocylate (ACC) deaminase [18,19,20,21]. ...
Rhizobacteria play a positive role as biocontrol agents as well as Plant Growth Promoting Rhizobacteria (PGPR) agents. The research objective was to obtain indigenous rhizobacteria isolates on cocoa plants that have the potential to inhibit the attack of P. palmivora fungal pathogens, and act as PGPR in vitro and in vivo. The results of the study concluded that isolates TRI 7/1, TRI 8/8, GM 7/9 and GM 7/10 had the highest ability to inhibit the growth of pathogen. The lowest disease severity (20%) was obtained in the seedlings treated using isolates TRI 7/1 and TRI 8/8. Rhizobacterial isolates GM 3/6, GM 5/6, GM 7/9 and GM 8/8 produce high amounts of IAA. Rhizobacteria isolates GM 5/6, GM 7/9 and GM 8/8 has very high peroxidase enzyme activity. High production of HCN compounds was obtained in rhizobacteria isolates TRI 3/3, TRI 4/10 and TRI GM 8/11. All rhizobacterial isolates gave an increase in the value of maximum growth potential, germination and vigor values for growth strength compared with the control. The rizobacteria treatments using isolates TRI 7/1, TRI 8/8, GM 7/9 and GM 7/10 were able to increase plant height, stem diameter and number of leaves at 30, 40, 50, 60, and 70 DAP compared to control treatment.
Full-text available
Authors' Contribution FD performed the experiments. HAS and MK worked on arranging, interpreting and organizing the data. MI helped in statistical analysis. JIQ supervised and facilitated the whole project. Chitinase is most promising natural enzyme present in all life forms. It has various environmental, food, medical industrial and biotechnological applications. Twenty strains were isolated on chitinase producing medium (CPM) from soil samples collected from local termites' influenced areas. Of all these, three isolates gave positive test for chitinase screened on the bases of clear zone on CPM following chitinase assay. The best chitinase producer was selected and identified as Bacillus subtilis employing 16S rRNA gene sequence identification technique. B. subtilis yielded highest chitinase on CPM at pH 7 with 3% inoculum size after incubating at 37 °C for 3 days. Plackett-Burman design was used for screening of medium components. The optimization of concentration of significantly impacted medium components for chitinase production was carried out using central composite design of response surface methodology. The maximum chitinase production was achieved employing 5% chitin, 0.5% rice straw, 0.05% peptone, 0.02% CaCl 2 , and 0.05% yeast extract. The utilization of agro-industrial waste (rice straw) not only decrease the production costs of microbial chitinase but can also providing positive way out for solving environmental pollution problem related to the waste.
Root and rust rot are common fungal diseases that decrease both the yield and quality of ginseng, resulting in severe economic losses for the ginseng industry. In this study, we explored the bacteriostatic and greenhouse control effects that the actinomycetes strain Frankia F1, extracted from the ginseng-rhizosphere soil, has on the pathogenic fungi that cause ginseng root rot (Fusarium solani) and rust rot (Cylindrocarpon destructans). We found that F1 fermentation broth inhibited the rate of mycelia growth by 80.6% and 71.1%, as well as the rate of spore germination by 87.0% and 78.6%, in F. solani. and C. destructans, respectively. Moreover, the antagonistic mechanism of F1 crude extract damaged the cell membranes of pathogens and led to a decreased ability to reduce sugar and soluble protein. The immobilised microorganisms of germ bran and germ bran charcoal improved the physical and chemical properties and soil fertility, improved the microbial community structure, increased the relative abundance of Proteobacteria, and reduced the relative abundance of Acidobacteria. An F1 biocontrol strategy could reduce economic and agricultural losses within the ginseng industry if implemented.
The amino acid sequences of 301 glycosyl hydrolases and related enzymes have been compared. A total of 291 sequences corresponding to 39 EC entries could be classified into 35 families. Only ten sequences (less than 5% of the sample) could not be assigned to any family. With the sequences available for this analysis, 18 families were found to be monospecific (containing only one EC number) and 17 were found to be polyspecific (containing at least two EC numbers). Implications on the folding characteristics and mechanism of action of these enzymes and on the evolution of carbohydrate metabolism are discussed. With the steady increase in sequence and structural data, it is suggested that the enzyme classification system should perhaps be revised.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Thirty-five spore-forming bacilli were isolated from evergreen oak leaves at four stages (one-year-old and two-year-old leaves, upper layer and underlayer little leaves) and over the four seasons within one year. These isolates, plus five reference strains, were characterized morphologically and physiologically by a total of 100 tests and genetically by DNA/DNA hybridization. Phenotypic similarities of all strains were determined by numerical taxonomy, with the unweighted average linkage (UPGMA) algorithm and simple matching (SSM) and Jaccard (SJ) coefficients used as measures for similarity. Three groups (A to C) were defined at a similarity level of 71% (SSM) or 57% (SJ). They contained leaf isolates phenotypically related to Bacillus cereus, B. pumilus and B. circulans species respectively. The majority of the leaf isolates were asigned to B. cereus (34%) and B. pumilus (63%). DNA/DNA hybridization also discriminated three groups (genomic groups 1, 2 and unclassified strains) which presented a good correlation with the numerical analysis. Yet, DNA/DNA hybridization grouping revealed a higher degree of discrimination by defining four subgroups (1a, 1b, 2a, and 2b). Genomic subgroup 1a contained leaf isolates belonging to the B. cereus species; isolates from genomic subgroup 1b belonged to B. mycoides species and isolates from genomic subgroup 2a belonged to B. pumilus species. Subgroup 2b consisted of a new genomic subspecies of B. pumilus which exhibited a degree of homology ranging from 53 to 64% with the B. pumilus type strain and a coefficient of divergence (ΔTm) ranging from 5.5 to 7°C. The different genomic groups presented different substrate metabolism capacities and a different spatial distribution on evergreen oak leaves. B. cereus strains (group 1) were predominantly located on litter leaves whereas B. pumilus strain (group 2) were found on the phyllosphere. In contrast with group 1, group 2 was able to metabolize some sugars and pectin, while group 1 isolates were able to hydrolyze starch and glycogen. Thus, our hypothesis is that group 1 succeeds group 2 when the leaves are littered.
Silver scurf, caused by the fungus Helminthosporium solani, is an important disease affecting potato tubers. Control of the disease has been hampered by the development of H. solani strains resistant to thiabendazole. Currently, there is no alternative to thiabendazole for the efficient control of the disease. In this study, 45 selected soils from the province of Québec were tested in greenhouse assays for their effect on silver scurf development. The results showed that soil influenced significantly silver scurf development and that specific soils displayed an interesting level of suppressiveness against potato silver scurf. Investigations into the cause(s) of soil suppressiveness revealed on the one hand, significant negative correlations between silver scurf severity and soil N-NO3 and available Fe contents, and on the other hand, absence of significant correlations between silver scurf severity and the different soil microbial populations. Investigations also revealed the presence of microorganisms antagonistic to the pathogen in the most suppressive soils. The antagonist microorganisms isolated in those soils were Bacillus cereus, Cellulomonas fimi, Kocuria varians, Pseudomonas putida, Rhodococcus erythropolis and Rhodococcus globerulus. Considering that few microorganisms were previously reported to display antagonism against H. solani, these results open the way to new avenues of investigation towards achieving biocontrol of silver scurf.
Commercial Streptomyces griseus and Serratia marcescens chitinases and purified wheat germ W1A and hen egg white lysozymes were subjected to polyacrylamide gel electrophoresis under native conditions at pH 4.3. After electrophoresis, an overlay gel containing 0.01% (W/V) glycol chitin as substrate was incubated in contact with the separation gel. Lytic zones were revealed by uv illumination with a transilluminator after staining for 5 min with 0.01% (W/V) Calcofluor white M2R. As low as 500 ng of purified hen egg lysozyme could be detected after 1 h incubation at 37 degrees C. One band was observed with W1A lysozyme and several bands with the commercial microbial chitinases. The same system was also used with native polyacrylamide gel electrophoresis at pH 8.9. Several bands were detected with the microbial chitinases. The same enzymes were also subjected to denaturing polyacrylamide gel electrophoresis in gradient gels containing 0.01% (W/V) glycol chitin. After electrophoresis, enzymes were renatured in buffered 1% (V/V) purified Triton X-100. Lytic zones were revealed by uv after staining with Calcofluor white M2R as for native gels. The molecular weights of chitinolytic enzymes could thus be directly estimated. In denaturing gels, as low as 10 ng of purified hen egg white lysozyme could be detected after 2 h incubation at 37 degrees C. Estimated molecular weights of St. griseus and Se. marcescens were between 24,000 and 72,000 and between 40,500 and 73,000, respectively. Some microbial chitinases were only resistant to denaturation with sodium dodecyl sulfate while others were resistant to sodium dodecyl sulfate and beta-mercaptoethanol.
Fusions of the secreted protein alkaline phosphatase to an integral cytoplasmic membrane protein of Escherichia coli showed different activities depending on where in the membrane protein the alkaline phosphatase was fused. Fusions to positions in or near the periplasmic domain led to high alkaline phosphatase activity, whereas those to positions in the cytoplasmic domain gave low activity. Analysis of alkaline phosphatase fusions to membrane proteins of unknown structure may thus be generally useful in determining their membrane topologies.
Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.