Using Mini-Tn10 Transposon System to Research the Genes Involved in Biofilm Formation in Bacillus

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1 Microbiology MAR 20, 2009, 36(3): 345~ by Institute of Microbiology, CAS mini-tn10 1, ,2* ( ) ( ) :, mini-tn10 NK10.BAhjaWT 400, 90% 4, citbcitggpsa yvfb citb citg gpsa, yvfb mini-tn10, :,,mini-tn10 Using Mini-Tn10 Transposon System to Research the Genes Involved in Biofilm Formation in Bacillus GAO Wei-Hua 1,2 HAO Jian-An 1 XIA Si-Yuan 1 XU Hai-Jin 1 BAI Yan-Ling 1 ZHANG Xiu-Ming 1 QIAO Ming-Qiang 1,2* (1. Institute for Molecular Biology, College of Life Science, NanKai University, Tianjin , China) (2. Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Tianjin , China) Abstract: Biofilm has great influence for bacteria to resist the harm from outer environment. Its forming and development are controlled by many different genes. In this research, an insertional mutagenesis library of a wide type Bacillus amyloliquefaciens NK10.BAhjaWT was built via mini-tn10 transposon system to find out genes involved in biofilm formation. Four such genes have been screened, in which, citb, citg, gpsa are all related to the energy metabolism. The function of another gene of yvfb is unknown. In a word, mini-tn10 transposon system was proved to be efficient and stable in Bacillus. Keywords: Biofilm, Bacillus amyloliquefaciens, Mini-Tn10 transposon system (bacterial biofilm), (exopo1ymeric matrix) (microcolony) [1,2] (No ) * Tel: ; : mingqiangqiao@yahoo.com.cn ;

2 , Vol.36, No.3,,, [3],,,,, [4,5] pic333, mini-tn10 [6], ColE1, 35 C ColE1, DNA, ColE1,,,, ( 1),, mini- Tn10,,, (Bacillus amyloliquefaciens)nk10.bahjawt DH5α pic T4 DNA DNA Marker () ; ;, Sigma ;, EP (0.625 mol/l, 1 mmol/l MgCl 2 ) M9-YE (Oxoid ) 3 g/l, (Sigma )10 g/l, l0 M9 10%, 200 g/l 1 Fig. 1 pic333 DNA Insertion of pic333 into chromosome 1%, 1 mo1/l MgSO 4 0.2%, 1 mol/l CaCl %LB (Oxoid ) 5 g/l, 10 g/l, NaCl 10 g/l LB 1 mmol/l MgSO 4 0.1%, LB (Ery)1 μg/ml, (Spc)100 μg/ml, 30 C, (Spc)100 μg/ml 100 μg/ml, 37 C 1.1.4, P15 -GCCGCGTTGGCCGATTC-3 P25 -GATATTCACGGTTTAC

3 : mini-tn ml 5 g/l M9-YE, 30 C200 r/min ; l:l00 25 ml 5 g/l M9-YE, 37 C220 r/min OD ; 4 C6000 r/min 10 min ; 1/3 EP, 4 C 6000 r/min 5 min ; 1/50 EP, l h 100 μl l μl pic333 ( 10 ng)(0.2 cm ), Micro pulser (Bio-Rad ) 2 kv, 1 ml 30 C M9-YE, 30 C120 r/min 1 h 100 μl LB, 30 C, 1.2.2, pic333, 70 C 5 ml LB 30 C 1:100 5 ml LB, 3 h, 42 C 4 h, 10 3, LB, 30 C 48 h,, [7], NK10.BAhjaWT, 1.2.3,, [8], OD , 1 μl 99 μl LB 96, 30 C 24 h 48 h,, 2, 150 μl 1%, 25 min 2, 150 μl DMSO, 570 nm LB 30 C, DNA Hind III DNA ( ), DNA, DH5α, LB, ml 100 μl, 156, pic333 = 156 CFU 10/10 ng= CFU/μg NK10.BAhjaWT, 4 1~4#, 2 1# 2#, 3# 4#,, 2 Fig. 2 Colony morphology of the mutants 2.2 NK10.BAhjaWT, 24 h 48 h, 3 NK10.BAhjaWT,, NK10.BAhjaWT 50%, 48 h 24 h, 2.3,,,

4 , Vol.36, No.3 1 Table 1 Transposon integration sites 3 Fig. 3 Comparison of biofilm formation ability between the wide type strain and the mutants 1 NCBI 95%, citb citg gpsa (EC ), (EC ) 3- (EC ),,,, ;, IRP-1(iron regulatory protein 1),,, RNA, (iron response elements, IREs), 2, 1#,, [9 12] 3-3-, [13] 3# yvfb,, EpsK, EpsK epsa-o epsk,, 2006 Branda eps, [14], 3# Strain Gene 1# citb 2# citg 3# yvfb 4# gpsa Function 3- Insertion site ACGTA-Tn10-TACTTTA TGAAG-Tn10-ATGCGTG CAAAG-Tn10-CATGCAA AAAGA-Tn10-GGCTGTT,,,,,, Mu Tn917 mini-tn10 3 Mu,, 10 2 CFU/μg, Tn917 mini-tn10, 5 kb [15], Tn917, pltv kb [16], Tn917,,, mini-tn10 pic333 7 kb, 10 5 mini-tn10,, [17], mini-tn10 pic333,,, 4

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