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National Laboratory of Protein Engineering and Plant Genetic Engineering, Department of Biology, Beijing University, Beiiing 100671, China
Rice leaf blight disease, caused by the bacterium Xanthomonas campestris pv. oryzae, results in great losses in yield in many rice fields in the world. Recent technical advances made it possible to introduce foreign genes and provided promising ways to obtain transgenic rice plants resistant to leaf blight bacterial infection. However, the key point of achieving this goal is to isolate genes encoding anti-bacterial protein.
Recently, an antagonistic bacterium A014 has been screened in our laboratory and classified as Bacillus subtilis A14 (unpublished data). The strain can secrete large amount of anti-bacterial proteins and has strong inhibitory activity against the pathogen of rice leaf blight disease. We report here the purification and characterization of anti-bacterial polypeptide LCI.
1. Purification of the anti-bacterial polypeptide LCI
The screening and isolation of Bacillus strains, from various fields in China, which can secrete anti-rice leaf blight bacterial proteins have been reported. For purification of LCI, crude extracts were prepared from overnight culture of Bacillus subtilis A014, dialyzed against buffer A (50 mM sodium phosphate, pH 7.2) and applied to a carboxymethyl cellulose (Whatman CM52) column. The antibacterial activity was recovered in three peaks (data not shown). In this study, the peak 1, which had a strong activity against Xanthomonas as measured by the plate diffusion method, was collected, dialyzed extensively against buffer B (50 mM sodium acetate, pH 4.8) and concentrated to a suitable volume by PEG.
For further purification, the concentrated active preparation was loaded onto a cation exchange FPLC column of Mono S HR 515 (Pharmacia) and eluted with a linear gradient of 0-1M NaCl in buffer B at a flow rate of 0.7 ml/min. Proteins were detected by measuring absorbance at 280 nm. The protein peak I was found to have anti-bacterial activity, and was subjected to further purification by chromatography on a reverse phase FPLC column of Pep RPC HR 515 (Pharmacia) with a linear gradient of 0-70% (v/v) acetonitrile containing 0.1% TFA (trifluoroacetic acid). A single major peak was detected, as shown in Fig. 1. The critical dilution assay was applied to determine the recovery of ABP-LCI activity (ABP: antibacterial polypeptide). Serial twofold dilutions of the test samples peptide were spotted (10 ul) onto fresh indicator lawns of Xanthonionas campestris pv. oryzae G and the plates were incubated for 24 hr at 28 deg C. The highest dilution showing complete inhibition of the indicator lawn was expressed as the activity units (AU). A purification scheme of ABP-LCI, starting from 2 liter of a culture supernatant, is presented in Table 1. The specific activity increased by 49-fold for the perified ABP-LCI as compared to the crude extract.
Fig. 1. Elution profile of purified antibacterial polypeptide LCI on a Pep
RPC HR 5/5 column.
2. Characterization of the antibacterial polypeptide LCI
When the major peak, shown in the elution profile (Fig. 1) was analyzed by 13.8% SDS-PAGE, only a single band with the molecular weight of 6 kD was observed, in agreement with the result of FPLC Superose 12 column chromatography (data not shown). Isoelectric focusing of the purified ABP-LCI in a 7.5 % polyacrylamide gel containing 0.7% ampholyte (pH 3.5-10) at 24 deg C showed a single band with pH 10.25, indicating that it is a basic protein. Purified ABP-LCI was hydrolysed with 6N HCI in an evacuated seated tube for 24 hr at 1100 deg C and the amino acid composition of the hydrolysate was determined on an automatic amino acid analyzer (Beckman 121BM). The result showed that ABP-LCI consisted of 17 kinds of amino acid residues lacking histidine, cysteine and methionine (Table 2). The sequence of the polypeptide was analyzed using a protein sequencer (Applied Biosystems 470A) and the partial sequence is shown in Fig. 2.
The stability of the purified ABP-LCI against heat and enzyme treatments was studied. First, samples (1 ml; 256 AU/ml) were heated for 20 min at various temperatures (0-100 deg C), and the remaining activities were determined. Second,
Table 1. Purification of Antibacterial polypeptide LCI
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Volume ABP-LCI Total Amt of Specific Fold
Purification stage activity ABP-LCI protein activity purification
(ml) (AU/ml) (AU) (mg/ml)* (AU/mg)
==============================================================================
Crude extract 200 32 6400 1.5 21 1
Chromatography on CM52 60 64 3200 0.5 128 6
Chromatography on Mono S 24 128 3072 6.25 512 24
Chromatography on PepRPC 10 256 2560 0.25 1024 49
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Estimated by the 1.45A/260-0-74A/280 as described by Kalckar and the method of
Bradford with Coomassie brilliant blue R-250
Fig. 2. Partial sequence of the antibacterial polypeptide LCI.
Table 2. The amino acid composition of the antibacterial polypeptide LCI
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Experimental data Calculated data
Amino acid =================================================
n mol % mole/mole
================================================================
Asp* 112.534 10.02 5.11(5)
Thr 22.343 1.78 1.02(1)
Ser 119.041 8.34 5.41(5)
Glu* 52.515 5.15 2.39(2)
Pro 22.004 1.69 1.00(1)
Gly 94.844 4.75 4.31(4)
Ala 72.410 4.30 3.29(3)
Cys 0 0 0 (0)
Val 91.447 7.14 4.16(4)
Met 4.770 0.48 0.22(0)
I le 62.603 5.47 2.85(3)
Leu 47.689 4.17 2.18(2)
Tyr 89.393 10.79 4.06(4)
Phe 69.324 7.69 3.15(3)
Lys 126.670 12.34 5.76(6)
His 2.627 0.27 0.12(0)
Trp 60.135 8.18 2.73(3)
Arg 12.191 1.42 0.55(1)
================================================================
Total 93.92 47
================================================================
* Including the corresponding amide
Table 3. Effect of heat and enzyme treatments on the activity of purified
ABP-LCI preparations
================================================================ Treatment Activity recovery (%)* ================================================================ 40 deg C 100 60 deg C 100 80 deg C 85.3 100 deg C 12.5 Lysozyme 100 trypsin 81.5 pepsin 90.5 pronase E 0 proteinase K 0 ================================================================ Activity of ABP-LCI in buffers was considered as 100%
the purified polypeptide was added to enzyme solutions to yield a final concentration of 256 AU/ml. The following enzymes were employed: trypsin (type 1; Sigma) pepsin, pronase (type VI; Sigma), proteinase K (type XI; Sigma) and lysozyme (grade 1; Sigma). The enzyme-LCI mixtures were incubated for 1 hr at 37 deg C, and the remaining activities were determined. The results of these treatments are shown in Table 3. These data suggest that the purified ABP-LCI is very stable against heat, trypsin and pepsin treatments. The stability is probably due to a specific structure of ABP-LCI.
3. Application of the antibacterial polypeptide LCI
The purified polypeptide LCI exhibited a strong inhibitory activity against the rice leaf blight bacterial pathogens X. campestris pv. oryzae G, X4, X17 and X61. The same activity has also been demonstrated against Pseudomonas solanacearum PEI. However, it could not inhibit the Erwinia carotovora subsp. carotovora and Escherichia coli. These observations altogether indicate that the gene encoding this polypeptide can be used for producing transgenic plants resistant to bacterial infection. Work is in progress to clone the gene encoding this polypeptide and to introduce it into rice.
