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[email protected] Current Proteomics, 2014, 11, 23-26
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Synergistic Action of the Antifungal -chitin Binding Protein CBP50 from Bacillus thuringiensis with Bacterial Chitinases Muhammad Aamer Mehmood1*, Khadim Hussain1, Farooq Latif2, Muhammad Rizwan Tabassum1, Munazza Gull3, Saba Shahid Gill1, Anam Saqib1 and Zahid Iqbal4 1
Department of Bioinformatics and Biotechnology, Faculty of Science & Technology, Government College University Faisalabad, Pakistan; 2Industrial Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan; 3Biochemistry Department, Faculty of Science, King Abdul Aziz University, Jeddah, Kingdom of Saudi Arabia; 4Department of Pharmacology, Al-Nafees Medical College, Isra University Islamabad Campus, Pakistan Abstract: Purified chitin binding protein the CBP50 from Bacillus thuringiensis and three bacterial chitinases (Chi74, Chi39 and ChiA) from two different genome sources i.e. Bacillus thuringiensis (Bt) and Serratia proteamaculans, were used to study the crystalline chitin degradation using different combinations of chitinases with and without chitin binding protein CBP50. Overall 2-4 folds increase in enzymatic activity of all three chitinases was observed when used in combination with the CBP50. But the highest increase, 4.64 folds, was observed when combination Chi74+Chi39+CBP50 was used. Gene loci of all proteins involved in Bt-chitin degradation and protein association network were also analyzed. This is the first report of synergistic action of Bt-proteins on chitin degradation. Present study will lead us to develop a technology for environmental friendly conversion of chitin to valuable products.
Keywords: B. thuringiensis, CBP50, chitinases, Non-specific synergistic action. INTRODUCTION Chitin binding proteins are believed to assist the chitinases for chitin degradation. They might bind to the chitin present in the peritrophic membrane, insect shells and fungal cell walls, causing hindrance in the biosynthesis of the chitin, subsequently may be exploited as biological control agent. Chitin binding proteins, along with chitinases and insecticidal system of the Bacillus thuringiensis, may be exploited to improve the resistance in crop plants against insect-pests. So, the present study is not only important for environmental friendly biomass turnover but may also be considered for the biotechnological applications in the plant sciences. Several chitin binding proteins have been exploited in different biotechnological applications. The AFP1 was characterized from nikkomycin-producing Streptomyces tendae Tü901 and was shown to have antifungal activity against Paecilomyces variotii. AFP1 had limited similarity to cellulose-binding domains of microbial plant cell wall hydrolases and bound to crab shell chitin, chitosan, and cell walls of P. variotii but showed no enzyme activity. The AFP1 was the first chitin binding protein from bacteria exhibiting antifungal activity and shown synergistic interaction with the chitin synthetase inhibitor nikkomycin [1]. Similarly, a chitin binding protein CBP50 has shown antifungal activity against plant pathogenic fungi Fusarium oxysporum. It was demonstrated that both the N-terminal and C-terminal domains of *Address correspondence to this author at the Department of Bioinformatics and Biotechnology, Faculty of Science & Technology, Government College University Faisalabad, Pakistan; Tel: +92 300 966 2288; E-mail:
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CBP50 are essential for the efficient binding of chitin [2]. The CBP21 is the most studied chitin binding protein from Serratia marcescens. Binding of CBP21 to chitin have been found synergistic with the chitinases of S. marcescens [3-5]. In a recent study, SpCBP21 (CBP21 from S. proteamaculans) have shown synergistic action with the chitinases of S. proteamaculans [6]. A chitin-binding protein from Moringa oleifera seeds, named Mo-CBP4 was shown to have antinociceptive and anti-inflammatory effect in vivo [7]. In the present study we have shown that the chitin binding protein CBP50 acts synergistically with the chitinases of B. thuringiensis and S. proteamaculans for chitinolytic activity. Previously, the CBP50 was shown to have -chitin binding activity and antifungal activity against the pathogenic fungi such as Fusarium oxysporum and Aspergillus niger [2]. This is the very first report of synergistic action of Btchitin binding proteins with bacterial chitinases. Our findings may be exploited for efficient bio-conversion of chitin waste to valuable by-products and for the biological control of insect-pests and plant pathogenic fungi. MATERIALS AND METHODS The CBP50, Chi74 and Chi39 from B. thuringiensis [2, 8, 9] and ChiA from S. proteamaculans were cloned into pQE30 using E. coli M15 as host strain. The E. coli strains carrying the recombinant vectors were used to induce expression of the recombinant proteins as described previously [10]. Purified elutes were dialyzed against 25 mM sodium phosphate buffer (pH 8.0). After dialysis, purity of the proteins was further confirmed by SDS-PAGE (12.5 %) analysis and concentrations were measured by Bradford method [11]. ©2014 Bentham Science Publishers
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Polysaccharide binding assays were performed as described previously [4]. Protein solution (CBP50) of known concentrations was diluted in 50mM sodium phosphate buffer (pH 7.0). Before adding substrate, the protein concentration was measured and crystalline chitin (0.5mg mL-1) was added, and the reaction volume was brought to 1 mL. The solutions were kept at room temperature for 16 h with continuous shaking at 60 rpm. Subsequently, the sample tubes were centrifuged to pellet the substrate, and the protein concentration in the supernatant was measured. The amount of bound protein was calculated by taking the difference between the initial and final concentrations of protein in the supernatant. All assays were performed in triplicate with blanks (buffer + 0.5 mg mL-1 substrate) and controls (buffer+proteins) to correct for nonspecific binding of the protein. Moreover, genome sequence of the B. thuringiensis was analysed using gene taxonomy genome browser at NCBI (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax. cgi?id=281309) to find the locus of the chitinases and chitin binding proteins. The association of the chitin binding protein CBP50 with the chitinases of the B. thuringiensis was analysed using online tool STRING9.05 (http://stringdb.org). RESULTS AND DISCUSSION The present study was focused to elucidate role of CBP50 protein in the catalytic activity of the chitinases (Chi74, Chi39) of B. thuringiensis. To investigate whether the effects of CBP50 on the efficiency of chitinases from B. thuringiensis was due to specific enzyme-protein interactions, we also included ChiA from S. proteamcualans 18A1 [13]. The binding of CBP50 -chitin occurred rapidly and reached equilibrium within 6-12 h (Fig. 1), that was similar to the chitin binding proteins (SpCBP21 and SpCBP50) of S. proteamaculans [6]. Whereas, SmCBP21 established binding equilibrium after 16 h of incubation [3]. Chitin degradation assays performed using combinations of ChiA, Chi39, Chi74 and CBP50. In agreement with previous experiments [4, 6] it was found that the all the three chitinases act synergistically on -chitin (Fig. 2A). In all cases, CBP50 was shown to increase the chitin degradation efficiency (Table 1). The highest efficiency (4.64 folds increase) was obtained when combining both enzymes (Chi39, Chi74) along with the CBP50. The synergistic action of the CBP50 is higher as compared to the previously reported smCBP21 and spCBP21 [4, 6]. The present results have shown that CBP50 may interfere with the crystalline structure of -chitin which leads to an overall increased enzymatic turnover apart
100
Bound Protein (%)
The enzyme activity was measured using -chitin. The 50 pmol of the enzyme (Chi74, Chi39, ChiA) was incubated with 1.5 mg substrate in 600 L of 100 mM sodium phosphate buffer, pH 6.0, at 55 °C for 1 h along with 50 pmol of the chitin binding protein CBP50. One unit of enzyme activity was defined as the amount of the enzyme required to release 1 mol of reducing sugar in one min. The reducing sugars were measured according to DNS assay [12]. All the assays were performed in triplicates and the data were analyzed using GraphPad Prism (GraphPad Software Inc., San Diego, CA).
Mehmood et al.
80 60 40 20 0 0
6
12
18
24
30
36
Time (Hours) Fig. (1). Time course study for the chitin binding protein CBP50; purified protein was mixed with the substrate and samples were taken at different time intervals. The total protein before and after binding was measured to see the percent bound protein in the specified course of time. Table 1.
Increase in chitinase activity when used in combination with CBP50.
Sr. No.
Combination of Proteins
Increase in Activity
01
ChiA+CBP50
2.16 ± 0.38 fold
02
Chi39+CBP50
2.95 ± 0.30 fold
03
Chi74+CBP50
2.37 ± 0.22 fold
04
Chi39+Chi74+CBP50
4.64 ± 0.11 fold
from of which chitinase is used in combination with the CBP50. The results also show that this effect of CBP50 is not due to formation of specific CBP50-chitinase complexes but rather to a more general effect of CBP50 on substrate availability. These results are in accordance with the earlier report on SmChiB, SpChiA [4, 6]. In conformity to this finding, the non-catalytic chitin binding protein smCBP21 [4] and spCBP21 [6] were found to be synergistic with the chitinolytic activity of chitinases. But, this is the first report of synergistic activity of bacterial chitin binding protein that belongs to a genus other than Serratia. Later, it was found that CBP21 is the only chitin binding protein which has shown to contain the catalytic activity [5], but at present the CBP50 protein has not shown any catalytic activity. Interestingly, in both the organisms (S. marcescens and S. proteamaculans), the cbp21 gene is located 1.5 kb downstream to the chiB gene [5, 6]. Whereas, chi74 and chi39 genes are located 2204 kb upstream and 917kb downstream to cbp50 gene in B. thuringiensis genome, respectively. So, it seems that closeness in gene locus is not critical for the synergistic action among the proteins on a particular substrate. Despite of distant positions of the genes encoding Chi74, Chi39 and CBP50 proteins on the genome, they have shown very close association in their function as revealed by STRING analysis (Fig. 3) and their synergistic action. It was even reported that Sm CBP21 was produced along with three chitinases, SmChiA, SmChiB and SmChiC [14] which is not in accordance in the case of Bt proteins. However, the protein association network analyses [15] have revealed that the
Reducing Sugars(PMoles min-1)
Synergistic Action of the Antifungal -chitin Binding Protein
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600
Chi74
500
ChiA Chi39+CBP50 Chi74+CBP50 Chi39+Chi74
400 300 200 100 0
25
0
4
8
12
16
20
24
28
32
36
CBP50 Chi39 ChiA+CBP50 Chi39+Chi74+CBP50
Time (hours)
6 5 4 3 2 1
C
hi A
+C
BP 50 C C hi hi 39 39 +C BP 50 C C h hi i7 74 4 +C BP C C hi 50 hi 39 39 +C +C h hi i7 74 4 +C BP 50
0 C hi A
Fold Increase in Enzyme activity
A
B Fig. (2). Synergistic assay the CBP50 protein in combination with three different chitinases. (A): Each curve present a different combination and each point present average value of experiment in triplicates. (B) Consecutive bars show that how much fold increase in activity was observed for different chitinase in combination with CBP50 protein.
A
B
Fig. (3). Position of all genes encoding chitinases and chitin binding proteins involved in chitin degradation pathway of B. thuringiensis. Diagram shows that genes are located at distant positions on the genome. Association network of CBP50 protein (A): It is evidence based view; multiple lines show multiple associations among the proteins in network, (B): it is a confidence based view; strength of association is presented by thickness of lines. Both these diagrams are showing that CBP50 (BT9727_2556) protein is strongly associated with Chi39 (BT9727_3469), where; BT9727_0277 is a hypothetical protein with unknown function, BT9727_363 is a ribosomal large subunit pseudouridine synthase D, BT9727_1686 is a fibronectin binding protein, and trmB is a tRNA guanine-N(7)-methyltransferase which Catalyzes the formation of N(7)-methylguanine at position 46 (m7G46) in tRNA.
CBP50 protein may have role in metabolic pathways other than chitinolytic system of the B. thuringiensis (Fig. 3), that may be due to the presence of Fn-III and CBM-49 [2] domains which are present in addition to N-terminal chitin binding domain CBD-N.
CONCLUSION CBP-mediated augmentation of substrate availability increased the efficiency of the bacterial chitinase. It is concluded that there are no specific substrate-protein interactions rather CBP50 exerts an overall general effect for the
26 Current Proteomics, 2014, Vol. 11, No. 1
Mehmood et al.
accessibility of the substrate. Moreover, proximity in the loci of the genes is not critical for the encoded proteins to be the part of a synergistic mechanism of action. Being a modular protein CBP50 may have role in more than one (chitin degradation) pathways. This system of the B. thuringiensis may be exploited to convert the chitin biomass into production of chitooligosaccharides, which are useful in food, agriculture, pharmaceutical and food industries. CONFLICT OF INTEREST
[5]
[6]
[7]
[8]
The authors confirm that this article content has no conflict of interest. [9]
ACKNOWLEDGEMENTS Thanks to Higher Education Commission Pakistan for financial support.
[10]
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Received: July 29, 2013
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Revised: January 13, 2014
Accepted: February 18, 2014
