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1 微生物学通报 Microbiology China JAN 20, 2011, 38(1): by Institute of Microbiology, CAS 研究报告青枯菌细胞表面的疏水性林娟 * 曾金晶刘树滔饶平凡 ( ) 摘要 : 分别采用 BATH 和 HIC 方法测定青枯菌细胞表面疏水性 (CSH), 并比较菌液与正十二烷比例 (BATH 方法 ) 和菌液上样量 (HIC 方法 ) 对 CSH 测定结果的影响确定在 BATH 方法中菌液 (OD 600 =0.5) 与正十二烷的比例为 2:1, HIC 方法中菌液 (OD 600 =1.0) 上样量为 0.2 ml; 在此条件下, BATH 和 HIC 两种方法之间呈现出良好的线性关系 (r=0.99) 进一步采用 HIC 方法测定青枯菌在生长过程中 CSH 的变化情况, 结果显示随培养时间的延长, CSH 逐渐降低, 24 h 后 CSH 趋于稳定, CSH 与青枯菌细胞表面的 EPSⅠ ( 胞外酸性多糖 ) 含量呈负相关 3 株不同致病强度的青枯菌的试验结果进一步验证了青枯菌细胞表面的 CSH 随 EPSⅠ 含量的增加而降低关键词 : 青枯菌, 细胞表面, 疏水性, EPSⅠ 含量 Cell surface hydrophobicity of Ralstonia solanacearum LIN Juan * ZENG Jin-Jing LIU Shu-Tao RAO Ping-Fan (College of Biological Science and Technology, Fuzhou University, Fuzhou, Fujian , China) Abstract: Bacterial adhesion to hydrocarbons (BATH) and hydrophobic interaction chromatography (HIC) were employed to determine cell surface hydrophobicity (CSH) of Ralstonia solanacearum, respectively. The effects of the ratio of bacterial suspension to n-dodecane in the BATH assays and the injection volume of bacterial suspension in the HIC assays on the measurement of CSH were investigated. The optimal conditions for CSH analysis were as follow: the ratio of bacterial suspension (OD 600 =0.5) to n-dodecane was 2:1 and the injection volume of bacterial suspension (OD 600 =1.0) was 0.2 ml. Under these conditions, a good linear relationships between BATH and HIC methods was obtained (r=0.99). Moreover, HIC method was applied to detect the variation of CSH during growth of Ralstonia solanacearum. The results indicated that the CSH decreased with culture time and approached a plateau after 24 h of incubation, and a negative correlation between CSH and EPSⅠ (extracellular polysaccharide) content of cell surface was observed. The experimental results of three Ralstonia solanacearum stains of different virulence further confirmed that the CSH decreased with increasing EPSⅠ content. 基金项目 : (No. 2008J0066); (No. XSJRC ); * 通讯作者 :Tel: ; Fax: ; : ljuan@fzu.edu.cn 收稿日期 : ; 接受日期 :

2 46 微生物学通报 2011, Vol.38, No.1 Keywords: Ralstonia solanacearum, Cell surface, Hydrophobicity, EPS content ( ) [1 2], [3 5] Mudd, [6 7] (CSH), (BATH) (HIC) (SAT) (CAM) (TPP) [4 5],, , [8 9], (BATH) (HIC) CSH, CSH, CSH 1 材料与方法 1.1 材料菌种 : GMI1000 Institut National dela Recherche Agronomique ; 51# 84# ( ) 培养基 : NA ( 5.0 g, 10.0 g, 3.0 g, 1.0 g, 15.0 g, ml, ph ), SPA ( 20.0 g, 5.0 g, K 2 HPO g, MgSO g, ml, ph ) 缓冲液 : A : 0.02 mol/l -HCl (ph 8.0); B : 0.02 mol/l -HCl (ph 8.0) +1 mol/l NaCl 主要仪器设备 : (Agella, Beckman ); - (UV1700, ); (Varioskan, Thermol Labsystems ); (MILLI-Q, Millipore ); (1285, Thermo Forma ); (PYX-PHS-X, ); (CHA-S, ) 1.2 实验方法青枯菌的培养方法 [10] : NA (30 C, 48 h), 1% (V/V) (OD 600 =0.1) 25 ml SPA, 30 C 200 r/min 24 h 烃类化合物吸附法 (BATH) 测定青枯菌 CSH [6] : [10] B, 600 nm OD 0 ; 8 ml 4 ml, 15 s, 20 min,, 600 nm OD 1 ( 8 ml B 4 ml,, ) : CSH=[(OD 0 OD 1 )/OD 0 ] 100% 疏水相互作用色谱法 (HIC) 测定青枯菌 CSH [5] : [10] B, OD 600 = ml, Octyl Sepharose TM CL-4B (Amersham Pharmacia Biotech AB, Sweden), 1 cm 3 ml A, B, 1 ml/min 0.05 ml, 0.2 ml B, 3 ml B,, 600 nm OD ml B, 600 nm OD 0, : CSH=[(OD 0 OD 1 )/OD 0 ] 100% 青枯菌细胞表面胞外酸性多糖 (EPS Ⅰ ) 含量的测定 [11] : SPA 0.22 μm,,, A1320

3 : 47 EPSⅠ, ; (ELISA) EPSⅠ (OD 595 ) 2 结果与分析 2.1 采用 BATH 方法测定青枯菌 CSH 烃 - 水两相体系的确定 :,, -,, -,, 3, -,,,, ;, BATH - CSH 菌液与正十二烷的体积比对疏水性测定的影响 : SPA 24 h,, -HCl, OD 600 =0.5; 8 ml ml, ( 1) 1, CSH, 4 ml, CSH,,,, 4 ml, 2:1 ( OD ) 2.2 采用 HIC 方法测定青枯菌 CSH HIC 1978 Smyth, [5,12] SPA 24 h,, -HCl, OD 600 =1.0, ml, ( 2) 图 2 菌液上样量对 CSH 测定结果的影响 Fig. 2 Effect of injection volume of bacterial suspension on measurement of CSH 图 1 正十二烷的添加量对 CSH 测定结果的影响 Fig. 1 Effect of n-dodecane volume on measurement of CSH,, OD 600 ;,,,, 0.2 ml ( OD ) 2.3 BATH 和 HIC 方法的相关性研究 5, BATH HIC CSH, ( 3) 3, BATH HIC

4 48 微生物学通报 2011, Vol.38, No.1 图 3 Fig. 3 采用 BATH 和 HIC 方法测定 CSH 的相关性曲线 Correlation between BATH and HIC methods, r=0.99, BATH,, HIC CSH 2.4 青枯菌在生长过程中 CSH 的变化 NA 48 h, SPA, 30 C 200 r/min,, ph, HIC CSH ELISA EPSⅠ (OD 595 ),, ph CSH OD 595, ph CSH EPSⅠ ( 4 5) Fig. 4 图 4 青枯菌菌数和培养液 ph 值随培养时间的变化曲线 Curves of cell number and ph of fermentation broth against culture time 图 5 青枯菌 CSH 和 EPSⅠ 含量随培养时间的变化曲线 Fig. 5 Curves of CSH and EPS Ⅰ content of cell surface against culture time

5 : 49, EPSⅠ LysR PhcA, phca phcb 3- (3-OH PAME) 10 7 CFU/mL, 3-OH PAME; 10 8 CFU/mL, 3-OH PAME 5 nmol/l, PhcA, EPSⅠ [13 14] EPSⅠ 85%, 15% [15], EPSⅠ [16] 4 5, 0 8 h, ; 12 h ( CFU/mL), EPS Ⅰ, CSH ph ; 24 h ph CSH EPSⅠ CSH EPSⅠ, 2.5 不同致病强度青枯菌 CSH 分析 EPSⅠ,,, EPSⅠ 3 : GMI1000 ( 6 d 100% ) 51# ( 6 d 86.7% ) 84# [17], SPA 24 h, CSH EPSⅠ ( 1) 1,, EPSⅠ CSH, CSH EPSⅠ 表 1 不同致病强度青枯菌 CSH 与 EPSⅠ 含量的比较 Table 1 Comparison of CSH and EPS Ⅰ content of Ralstonia solanacearum of different virulence Strain EPS Ⅰ content (OD 595 ) GMI # 84# 0.99± ± ±0.02 CSH (%) 12.74± ± ± 讨论, [1 5], [6 7] CSH, CSH EPSⅠ, EPSⅠ, CSH, EPSⅠ [16] EPSⅠ, LysR phca phca phcbsrq phca EPSⅠ, ; phca, EPSⅠ, [14], EPSⅠ, EPSⅠ ELISA,, EPSⅠ CSH, CSH, EPSⅠ,, 参考文献 [1] Dickson JS, Koohmaraie M. Cell surface charge characteristics and their relationship to bacterial attachment to meat surface[j]. Appl Environ Microbiol, 1989, 55(4): [2] Gusils C, Cuozzo S, Sesma F, et al. Examination of adhesive determinants in three species of Lactobacillus isolated from chicken[j]. Can J Microbiol, 2002, 48(1): [3] Devasia P, Natarajan KA, Sathyanarayana DN, et al. Surface chemistry of Thiobacillus ferrooxidous relevant to adhension on mineral surfaces[j]. Appl Environ Microbiol, 1993, 59(12): [4] Hamada T, Sameshima Y, Honda K, et al. A comparison of various methods to predict bacterial predilection for organic solvents used as reaction media[j]. J Biosci Bioeng,

6 50 微生物学通报 2011, Vol.38, No , 106(4): [5] Ahimou F, Paquot M, Jacques P, et al. Influence of electrical properties on the evaluation of the surface hydrophobicity of Bacillus subtilis[j]. J Microbiol Methods, 2001, 45(2): [6],,,. [J]., 2003, 23(2): [7],,. [J]., 1997(1): [8] Denny TP. Ralstonia solanacearum a plant pathogen in touch with its host[j]. Trends Microbiol, 2000, 8(11): [9] Genin S, Boucher C. Ralstonia solanacearum: secrets of a major pathogen unveiled by analysis of its genome[j]. Mol Plant Pathol, 2002, 3(3): [10],,,. HPLC [J]., 2006, 26(5): [11],,,. [J]., 2007, 47(1): [12] Peng JS, Tsai WC, Chou CC. Surface characteristics of Bacillius cereus and its adhesion to stainless steel[j]. Int J Food Microbiol, 2001, 65(1/2): [13] Clough SJ, Flavier AB, Schell MA, et al. Differential expression of virulence genes and motility in Ralstonia (Pseudomonas) solanacearum during exponential growth[j]. Appl Environ Microbiol, 1997, 63(3): [14] Clough SJ, Lee KE, Schell MA, et al. A two-component system in Ralstonia (Pseudomonas) solanacearum modulates production of PhcA-regulated virulence factors in response to 3-hydroxypalmitic acid methyl ester[j]. J Bacteriol, 1997, 179(11): [15] Orgambide G, Montrozier H, Servin P, et al. High heterogeneity of the exopolysaccharides of Pseudomonas solanacearum strain GMI1000 and the complete structure of the major polysaccharide[j]. J Biol Chem, 1991, 266(13): [16] Iwabuchi N, Sunairi M, Anzai H, et al. Relationships among colony morphotypes, cell-surface properties and bacterial adhesion to substrata in Rhodococcus[J]. Colloids Surf B: Biointerfaces, 2003, 30(1/2): [17],,,. [J]., 2009, 15(5): 征订启事欢迎订阅微生物学通报 1974,,, : ; ; ; ; ; ; ; ; ; ;,,, 2000, ,,,, , 576,,,, ( ) : (100101) 1 3 Tel: ; tongbao@im.ac.cn, bjb@im.ac.cn; : : 2-817; : BM413

: 307, [], [2],,,, [3] (Response Surface Methodology, RSA),,, [4,5] Design-Expert 6.0,,,, [6] VPJ33 ph 3,, ph, OD, Design-Expert 6.0 Box-Behnken, VPJ3

: 307, [], [2],,,, [3] (Response Surface Methodology, RSA),,, [4,5] Design-Expert 6.0,,,, [6] VPJ33 ph 3,, ph, OD, Design-Expert 6.0 Box-Behnken, VPJ3 微生物学通报 FEB 20, 2008, 35(2) 306~30 Microbiology 2008 by Institute of Microbiology, CAS tongbao@im.ac.cn 生物实验室响应面分析法优化副溶血性弧菌生长条件刘代新宁喜斌 * 张继伦 2 (. 200090) (2. 20202) 摘要