THE CHITINOLYTIC BACTERIUM BACILLUS LICHENIFORMIS M3 AND ITS POTENTIAL APPLICATION IN BIOLOGICAL PLANT PROTECTION

Urszula Jankiewicz¹, Monika Kozłowska¹, Maria Świontek-Brzezińska², Jarosław Nowosielski³, Sylwia Stępniewska-Jarosz^4
1 Department of Biochemistry, Warsaw University of Life Sciences – SGGW, Poland
² Faculty Biology and Environment Protection, Department of Environmental Microbiology and Biotechnology, Nicolaus Copernicus University in Toruń, Poland
³ Plant Breeding and Acclimatization Institute – National Research Institute at Radzików, Poland
⁴ Bank of Plant Pathogens and Investigation on Their Biodiversity, Institute of Plant Protection – National Research Institute, Poznań, Poland

ABSTRACT

The bacterium Bacillus licheniformis M3, which is antagonistic towards pathogenic fungi and oomycetes, synthesizes at least two different isoforms of chitinases. The use of a three-stage purification procedure enabled the isolation of two of these isoforms: Chi1 and Chi2. Both of these enzymes have been classified as chitobiosidases of the exochitinase group. Chi1 exhibits a temperature optimum of 60°C and pH 6.0, and has the higher thermal stability. Chi2 exhibits its highest activity at 55°C and pH 7.0. Both of the studied chitinases exhibited highest activity towards colloidal chitin as substrate. Calcium and sodium ions caused a slight stimulation of the activity of both of these enzymes, whereas they were markedly inhibited by Hg²⁺ and Cd²⁺. Chi1 and Chi2 exhibited fungistatic activity towards Fusarium solani.

To summarize, B. licheformis M3 produces chitinases with fungistatic activity which could potentially find use as components of biopreparations for plant protection.

Key words: chitinase, purification, antifungal activity, Bacillus licheniformis, microbial antagonism, biological plant protection.

INTRODUCTION

The interest in bacterial strains exhibiting antagonistic activities related to filamentous fungi and oomycetes harmful to plants stems from the search for alternatives to chemical fungicides. Crop protection chemicals are expensive and toxic to the natural environment. Additionally, due to pathogens and pests gaining immunity to their effects, chemical preparations eventually become ineffective [8, 13, 24]. For this reason, the practical application of natural biocontrol mechanisms appears promising. Biological methods of plant protection involve the use of natural interactions, mainly antagonism and competition between living organisms inhabiting a common environment. An important role in inhibiting the growth of phytopathogenic mould is played by plant growth promoting rhizobacteria (PGPR). The beneficial effects of PGPR on plant health are a result of, for example, secreting antibiotics, siderophores or growth regulators, as well as lytic enzymes: peptidases, chitinases and β-1,3 glucanases [2, 7, 27]. As the literature data shows, chitinases, which are enzymes that break down β-1,4-glycosidic bonds in chitin, are particularly effective for biocontrol [29]. Chitin, as one of the most commonly occurring natural polysaccharides, is an important component of the cell walls of fungi and algae, as well as being a structural component of nematode egg shells and the exoskeletons of arthropods [25, 26]. The chitinases released into the environment by bacteria can therefore degrade the cell walls of phytopathogenic moulds and damage nematode egg shells [6, 29]. PGPR also include Bacillus licheniformis, one of the more common species of bacteria, present both in the soil, where it intensively decomposes plant remains, and in spoiled food [11, 15]. This bacterium is often a cause of food poisoning in humans [5]. At the same time, B. licheniformis is utilised industrially to acquire enzymes, mainly proteases and amylases [16]. In recent years, there have been a number of scientific reports on the important role of these bacteria in limiting the growth of phytopathogenic fungi [10, 28]. Our studies partly focus on the issue of biocontrol, while the object of our studies involve the fungistatic properties of chitinases synthesised by the B. licheniformis M3 rhizospheric bacterium, newly isolated from the soil. The main goal of our studies was to seek rhizospheric bacteria with a high biocontrol potential, and to assess the role of the chitinases of these bacteria in controlling phytopathogenic filamentous fungi.

MATERIALS AND METHODS

Isolation and identification of microorganism and screening of chitinolytic bacteria
Chitinolytic microorganisms were isolated from the rhizosphere of ecologically grown vegetable. Rhizospheric bacteria were isolated following the methodology described by Buyer [4]. The bacterial isolates were transferred onto an agar medium with 0.5% colloidal chitin. The isolates were cultivated at 28°C for 96 hours. After this time, the presence and size of halo zones around the grown bacterial colonies were checked. For further studies, the strongest chitin degraders were selected.

Bacteria identification
The bacterial isolate was identified on the basis of morphological and biochemical traits according to Bergey’s Manual of Determinative Bacteriology. Additionally, identification of the studied strain was confirmed by analysis of the 16S rRNA gene sequence. Amplification of the 16S rRNA gene was performed using 27F and 1492R universal primers [35]. Genomic DNA was isolated from bacterial cells during the late logarithmic growth phase using the Genomic DNA Purification Kit (Fermentas). The obtained nucleotide sequences were compared with sequences deposited in the available GenBank/EMBL/DDBJ databases using the BLAST program.

Optimization of media components for chitinase production
The following composition of growth medium was used (g/L): KH₂PO₄, 3.0; K₂HPO₄, 3.0; MgSO₄, 0.5; NaCl, 2.0; FeCl₃, 0.005; Bacto-peptone 8.0; yeast extract 2.0, enriched with 5 or 10.0 g/L of colloidal chitin or crystalline chitin in flake (Sigma, from shrimp shells), powder form (Roth, from crab shells) or chitosan (Sigma, Aldrich; ≥ 75% deacylated chitin from prawn shell). Control growth media contained all the components except a source of chitin or chitosan. Colloidal chitin was prepared from commercial chitin (Sigma) according to Lee et al. [20].

Enzymatic activity
Chitinase activity was determined with 1% colloidal chitin as substrate in 50 mM Tris – HCl buffer, pH 6.8. The amount of reductive sugars released was measured according to Miller [21]. Calibration curve was prepared for 5 concentrations of N-acetylglucosamine in the range of 10–100 µg/ml. One unit (U) of the chitinase activity was defined as the amount of enzyme which that releases 1 μmol N-acetyl-D-glucosamine (GlcNAc) from colloidal chitin per 60 min.

Purification of the enzymes
B. licheniformis M3 was cultured in 100 mL of growth medium enriched with 1% colloidal chitin at 28°C on a rotary shaker at 120 rpm for 6 days. The bacterial culture was centrifuged 15 min at 12,000 x g, the obtained supernatant was treatment with ammonium sulphate up to 85% of saturation. Subsequently the preparation was centrifuged for 20 min at 16,000 x g, the protein pellet was dissolved in 50 mM Tris-HCl buffer, pH 6.8. The obtained solution was dialysed for 12 h against the same buffer. The enzyme solution was supplemented with (NH₄)₂SO₄ to final concentration in sample 0.8 M and then subjected to hydrophobic chromatography on Phenyl-Sepharose CL-4B (Sigma). Prior to separation the column was equilibrated with 1 M (NH₄)₂SO₄ in 50 mM Tris-HCl buffer, pH 6.8. Protein was eluted with a decreasing gradient of (NH₄)₂SO₄, from 1.0 to 0.0 M. The most active fractions collected during the chromatography were pooled and dialysed against 20 mM Tris-HCl buffer pH 7.2. The obtained preparation was subjected to ion-exchange chromatography on anionite DEAE – Sepharose. Prior to chromatography, the column was equilibrated with 20 mM Tris-HCl buffer, pH 7.2. Protein was eluted with a linear gradient  of KCl, from 0.2 to 0.4 M. Fractions with highest enzymatic activity were dialyzed for 12 hours against the same buffer and used for characterizing these enzymes. Protein concentration was determined using the method of Bradford [3] with BSA as a standard.

Electrophoresis and zymograms
Electrophoresis under native and denaturing conditions was according to the procedure described by Laemmli [18]. SDS – PAGE was performed using a 10% polyacrylamide separating gel. The gel was then stained with silver nitrate to visualize proteins. Isoforms of chitinases synthesized by the tested bacteria were analysed on zymograms following native electrophoretic separations. The separation was conducted on 8% polyacrylamide gel with incorporated 0.05% glycol chitin. The gel was incubated at 40°C in 0.1 M Tris-HCl buffer pH 6.8 for 2 h. Finally, the gel was submerged for 30 min in a 0.1% solution of Congo Red dye, then transferred to 1 M NaCl solution. Glycol chitin was prepared according to Trudel and Asselin [32].

Biochemical characterisation of the purified chitinases
Substrate specificity of the purified chitinases was determined using chromogenic, synthetic substrates: 4-Nitrophenyl N,N′-diacetyl-β-D-chitobioside – for exochitinase activity detection (chitobiosidase activity), 4-Nitrophenyl N-acetyl-β-D-glucosaminide – for exochitinase activity detection (β-N-acetylglucosaminidase activity) and 4-Nitrophenyl β-D-N,N′,N′′-triacetylchitotriose for endochitinase activity detection. One unit of chitinase activity (U) was defined as the amount of enzyme yielding 1 μmol of p-nitrophenol per minute.

The optimum pH and temperature as well as the thermal stability of the purified enzymes were determined in enzymatic reactions using colloidal chitin as substrate.

The optimum pH was determined in a range of 5.0–8.5. The optimal temperature was determined in the range from 35 to 65°C. Thermal stability was determined after 30, 60 and 90 min preincubation of the enzyme at temperatures: 50 and 60ºC.

The effect of metal ions on activity was determined by following preincubation of the enzyme for 30 min at 4°C in the presence of metal ions in final concentration of 5 mM. After that the substrate was added and the residual activity tested. Control sample without metal ions was taken as 100%.

Fungistatic activity of B. licheniformis M3 bacteria and the isolated chitinases
The tested fungal cultures came from the collection of the Phytopathology Unit of the Faculty of Horticulture, Biotechnology and Landscape Architecture at the Warsaw University of Life Sciences. The Oomycetes strain Phytophthora cinnamomi  was obtained from the Bank of Plant Pathogens at the Institute of Plant Protection – National Research Institute in Poznań. In studies on the degree (in %) of the inhibition of mycelium growth by B. licheniformis M3 bacteria the method of two-organism growth on PDA solid medium with 0.4% chitin was employed. The rate of fungal growth inhibition by B. licheniformis M3 bacteria was determined as % of growth inhibition,  following the formula = (K – F/K) x 100; K – culture diameter in the control combination, F – culture diameter in the test combination.

The fungistatic  and anti-oomycetes activity of the studied chitinases was carried in PDA medium by the well diffusion method, as described by Narayana and Vijayalakshmi [22]. Antifungal activity was classified according to Ghasemi et al. [9] as: no inhibition, 0 mm; weak inhibition,  <2 mm; moderate inhibition 2–10 mm; strong inhibition >10 mm of inhibition zone diameter).

All results presented in this paper in the form of numerical values are means from three independent repetitions. The mean error, reflecting maximal deviation of the results of measurements from the mean, did not exceed 5%.

RESULTS

The studied strain was identified as Bacillus licheniformis M3. The nucleotide sequence of the gene coding 16S RNA was deposited in DDBJ/ EMBL/GenBank under the accession number AB699713.

A high chitinolytic activity was detected when using a growth medium with the addition of 1% colloidal chitin (2.1 U/ml) and the addition of 1% powdered prawn shells (1.9 U/ml). A five-fold lower chitinolytic activity was obtained when culturing the bacteria on a growth medium containing flaked crab shells or chitosan. A lack of additional chitin or chitosan in the control growth medium resulted in only trace of chitinolytic activity in these bacteria (Fig. 1).

Fig. 1. Optimization of culture medium composition for the production of chitinases by B. licheniformis M3. The following sources of chitin and chitosan have been used: CC – colloidal chitin, CP – powdered chitin; CF – chitin flakes, CT – chitosan, control – medium not supplemented with chitin or chitosan

Fig. 2. Elution profile of B. licheniformis M3 chitinases in the ion exchange chromatography

An enzymatic preparation acquired from six-day bacteria cultures on a 1.0% colloidal chitin growth medium was subjected to a three-stage purification procedure, involving precipitation with ammonium sulphate, hydrophobic chromatography and ion-exchange chromatography, as per Table 1. After the final purification stage, isoforms of chitinases of B. licheniformis M3 bacteria were separated into two fractions, as shown in Figure 2. Fractions I (Chi1) and II (CHi2) were used for further tests. The molecular weights Chi1 and Chi2 determined by SDS-PAGE were about 66 and 50 kDa, respectively (Fig. 3). Chi1 exhibited an activity in a broad range of pH values, with an optimum at 6.0, while for Chi2 the optimum pH was 7.0 (Fig. 4). Both enzymes had similar values of optimum temperatures: 60°C for Chi1 and 55°C for Chi2 (Fig. 5). However, these chitinases differed in thermal stability. Chi1 maintained a stability of approx. 80% during a 120-minute pre-incubation at 60°C, while Chi2 maintained only 30%.

The effect of metal ions on the activity of the tested chitinases is presented in Figure 6. A minor stimulation by Na⁺ and Ca²⁺ was observed for both enzymes. Chi2 activity was stabilised in the presence of Mg²⁺ and Mn²⁺ whereas Cd²⁺ and Hg²⁺ ions were strong inhibitors for both of these activities.

Table 1. Purification of chitinases produced by B. licheniformis M3

Step		Total protein [mg]	Total activity [U]	Specific activity  [U/mg]	Yield [%]	Purification [fold]
Culture supernatant		169	85	0.5	100	1
(NH4)2SO4 (30–85%), and dialysis		72.7	63	0.9	74.1	1.8
Hydrophobic chromatography		31.3	47	1.5	55.3	3
Ion exchange chromatography	Chi1	2.6	11	4.2	12.9	8.4
	Chi2	1.8	8.7	4.8	10.2	9.6

Fig. 3. SDS-PAGE of the isolated chitinases of B. licheniformis M3: M-markers of molecular weight (Thermo Scientific), lane 1 – isolated Ch1, lane 2 – isolated Chi2, lane 3 – unpurified culture supernatant

Fig. 4. Effect of pH on the purified chitinases activity of B. licheniformis M3

Fig. 5. Effect of temperature on the purified chitinases activity of B. licheniformis M3

Fig. 6. Effect of metal ions on the chitinases activity of B. licheniformis M3

Both of the tested chitinases exhibited the highest activity towards colloidal chitin and a slightly lower one towards powdered crystalline chitin and glycol chitosan. Chi1 exhibited an equally high activity towards glycol chitin. Neither of these enzymes decomposed β-1,3- in laminarin and β-1,4- glycosidic bonds in carboxymethyl cellulose. The preferences of Chi1 and Chi2 chitinases were also tested in terms of the location of the hydrolysed bond in the substrate. The highest activity was observed for 4-nitrophenyl-N,N'-diacetyl-β-D-chitobiose, and trace activity towards 4-nitrophenyl-β-D-N,N',N''-triacetylchitotriose (Tab. 2).

Table 2. Substrate specificity of purified chitinases from Bacillus licheniformis M3

Substrat	Activity [U/ml]
	Chi1	Chi2
4-Nitrophenyl β-D-N,N′,N′′-triacetylchitotriose	1.8	3.5
Nitrophenyl N,N′-diacetyl-β-D-chitobioside	44	31.6
4-Nitrophenyl N-acetyl-β-D-glucosaminide	0	0
Colloidal chitin	2.1	1.8
Glycol chitin	2.0	0.2
Powdered crab shells	1.5	0.9.
Chitosan	0.4	0.2
Glycol chitosan	1.9	0
Laminarin	0	0
CMC	0	0
The mean error, reflecting maximal deviation of the results of measurements from the mean, did not exceed 5%. The experiment was performed in three independent repetitions.

The conducted tests indicate that the most susceptible to the antagonism of B. licheniformis M3 bacteria were: Fusarium solani and Fusarium oxysporum, Phytophthora cinnamomi, Rhizoctonia solani and Chaetomium globosum. The least vulnerable to the presence of the bacteria was Cladosporium sp.and Alternaria alternata (Tab. 3).

Table 3. Anti-phytopathogene activity of B. licheniformis M3 and their isolated chitinases

Phytopatogens	Anti-phytopathogen activity
	Growth inhibition [%] in two-organism cultures:  B. licheniformis M3 + phytopathogen	Diameter of growth inhibition [mm] in agar-well diffusion method
		Chi1	Chi2
Fusarium solani	55	4	5
Fusarium oxysporum	68	3	4
Rhizoctonia solani	45	0	0
Phytophthora cinnamomi	38	0	0
Chaetomium globosum	15	0	0
Cladosporium sp	0	0	0
Alternaria alternata	0	0	0

The fungistatic activity of isolated chitinases, tested using the agar-well diffusion method, was observed towards F. solani and F. oxysporum.The specific activity of the Chi1 and Chi2 it was 4.9 and 4.2 U/mg, respectively (Tab. 3).

DISCUSSION

Damage to agricultural crops caused by pathogens is a serious economic problem not only in ecological farms but in traditional ones as well. The production of healthy food forces the elaboration of new, more effective biological plant protection measures and for this reason studies on soil microorganisms that are antagonistic towards plant pathogens are of prime importance. In the context of limiting diseases caused by phytopathogenic moulds a very important role is played by lytic enzymes, such as chitinases, glucanases or proteases. With future application in mind, studies on the properties of these enzymes, mainly on their stability and optimal conditions for activity, are an absolute necessity.

The results presented in this study concern the isolation and characterisation of chitinases synthesised by B. licheniformis M3 bacteria isolated from the rhizosphere of ecologically grown vegetable plants. The initial stage of the study involved the optimisation of the growth medium composition in order to acquire high levels of chitinolytic activity. This was an important stage of the experiments, as most chitinases are inductive enzymes. Through the selection of an appropriate growth medium composition for the culturing of bacteria, the level of chitinolytic activity can be adjusted. Similarly, in the case of the B. licheniformis M3 bacterium, the results indicated that chitinase secretion by this strain was an inductive characteristic, and colloidal chitin was an effective inducer. The effective induction of chitinase synthesis in the presence of colloidal chitin was also observed in strain Bacillus licheniformis S213 as well as in Paenibacillus thermoaerophilus TC22-2b [28, 33]. However, in some bacteria species, chitinase synthesis is constitutive [1].

The bacteria B. licheniformis M3 secrete at least three isoforms of chitinases with various electrophoretic mobilities. The presence of numerous chitinases was also observed by Trachuk et al. [31]. They demonstrated the presence of five chitinolytic enzymes, with molecular masses of 66, 62, 53, 49 and 42 kDa, in the post-culture supernatant of B. licheniformis B 6839 bacteria. These results partly overlap with those obtained in the present study, in which the molecular masses of  Chi1 and Chi2 were 66 and 50 kDa respectively. In general, as indicated by the literature, bacteria of the Bacillus genus secrete several isoforms of chitinases with molecular masses in the 35–70 kDa range [36, 37]. The use of synthetic substrates for the activity determinations enabled confirmation of the substrate specificity for both of the tested chitinases. Both Chi1 and Chi2 are exochitinases, so-called chitobiosidases, severing dimeric N-acetylglucosamine molecules from the chitin polymer chain. Similar properties of chitinases were described for the chitinase from S213 bacteria [28]. However, different results are presented by Kudan [17], who describes the properties of endochitinases secreted by B. licheniformis SK-1 in his study.

Both Chi1 and Chi2 exhibited changes of activity under the effects of metal ions. Similarly, the chitinase acquired from the Bacillus licheniformis LHH100 bacteria was slightly activated by calcium and magnesium ions [18]. The activity of both of the tested chitinases was inhibited under the effects of cadmium and mercury ions, which are frequently indicated as bacterial chitinase inhibitors [13, 16].

The chitinases of Bacillus genusdescribed so far exhibited an activity in a broad range of pH, with an optimum at 6.0 [30], although several scientific reports have appeared on alkaline chitinases [34].

As indicated by numerous scientific papers, chitinases synthesised by B. licheniformis bacteria are characterised by a relatively high temperature optimum and thermal stability. The stability of chitinase 1 was similar to that of the chitinase from B. licheniformis Mb-2 whichexhibited stability at 60°C, after a 120-minute incubation of this enzyme [35].

The last goal of the study, and at the same time one of the more important ones, was to demonstrate the fungistatic activity of the tested B. licheniformis M3 strain and the chitinases it releases towards the fungal phytopathogens and oomycetes of arable crops. The presented results indicate that the bacteria effectively inhibit the growth of fungi in two-organism cultures on solid growth media. The isolated chitinases exhibited fungistatic activity, but only towards a limited spectrum of pathogenic species. The limited amount of literature data confirms the capability of inhibiting the growth of fungal phytopathogens of arable crops by B. licheniformis, which reinforces their role as PGPRs. Similarly as in our studies, a chitinase synthesized by B. licheniformis NM 120-17 inhibited the growth of phytopathogens from the genus Fusarium [17]. The antagonism of this bacterial species towards phytopathogenic Fusarium strains was recently described by Slimene et al. [28]. Literature data indicate that certain strains of the genus Bacillus have antagonistic properties towards oomycota [37].

CONCLUSION

To summarize, it can be said that the chitinases of  B. licheniformis M3 that we have characterized possess  valuable biochemical properties, which suggests the possibility of their industrial use. On the other hand, from the viewpoint of plant protection, they have very important fungistatic properties.

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Accepted for print: 3.04.2016

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Urszula Jankiewicz
Department of Biochemistry, Warsaw University of Life Sciences – SGGW, Poland
Nowoursynowska 159
02-776 Warsaw
Poland
email: urszula_jankiewicz@sggw.pl

Monika Kozłowska
Department of Biochemistry, Warsaw University of Life Sciences – SGGW, Poland
Nowoursynowska 159
02-776 Warsaw
Poland

Maria Świontek-Brzezińska
Faculty Biology and Environment Protection, Department of Environmental Microbiology and Biotechnology, Nicolaus Copernicus University in Toruń, Poland
Lwowska 1
87-100 Toruń
Poland

Jarosław Nowosielski
Plant Breeding and Acclimatization Institute – National Research Institute at Radzików, Poland
Radzików
Poland

Sylwia Stępniewska-Jarosz
Bank of Plant Pathogens and Investigation on Their Biodiversity, Institute of Plant Protection – National Research Institute, Poznań, Poland
Poznań
Poland

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Responses to this article, comments are invited and should be submitted within three months of the publication of the article. If accepted for publication, they will be published in the chapter headed 'Discussions' and hyperlinked to the article.

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