|
|
Establishment and Condition Optimization for Natural Transformation Method of Haemophilus parasuis |
|
|
Abstract Natural transformation can be used to construct deficient mutant of Haemophilus parasuis (HPS), and it has significance on the study of pathogenesis and virulent factors. The experimental conditions of natural transformation are optimized by control variable methods. This research was conducted to obtain the optimal conditions for natural transformation of HPS. The transformation frequency was compared under the differential conditions, which including the bacterial phases (OD600=0.15, 0.40, 0.70, 1.00, 1.30, 1.50, 1.60, 1.90, 2.00), bacterial incubation time (T1=7, 9, 11, 13, 15, 17, 19 h), bacterial concentration (1/100 OD600=0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7), the concentration of exogenous cyclic adenosine monophosphate (cAMP) (terminal concentration C1=0.0, 0.1, 0.2, 0.4, 0.8, 1.0, 2.0, 4.0, 8.0 mmol/L), plasmid (terminal concentration C2=0.000, 0.005, 0.010, 0.020, 0.040, 0.050, 0.100, 0.250, 0.350 μg/μL), and the incubation time after transformed (T2=0, 1, 2, 3, 4, 5, 6, 7 h). The results indicated that the transformation on agar plates of HPS occurred in a high frequency when the spotting bacteria was in the late-log phage(OD600≈1.5~1.7), the incubation time was 13 h, the concentration of exogenous plasmid was more than 0.1 μg/μL, and the incubation time after transformed was 5 h. The optimal conditions of natural transformation are determined, which lay a foundation for the research of virulence factor and pathogenic mechanism associated with Haemophilus parasuis.
|
Received: 01 April 2017
Published: 10 December 2017
|
|
|
|
谨瑾,张禄滑,文心田,等.2016. 副猪嗜血杆菌potD基因缺失株的构建及其部分生物学特性 [J]. 畜牧兽医学报,47(11): 2274-2279.(Jin J, Zhang L H, Wen X T, et al. 2016. Construction and Characterization of an Haemophilus parasuis potD Mutant Strain. [J]. Acta Veterinaria et Zootechnica Sinica, 47(11): 2274-2279.)马艳平. 2010. 副猪嗜血杆菌生物被膜形成相关基因luxS和pilA的克隆和功能鉴定[D]. 硕士学位论文,中国农业科学院,导师:刘永生.pp.27-53(Ma Y P.2010.Cloning and Funcation Identification of the Genes luxS and pilA Related to Biofilm Formation of Haemophilus parasuis. [D]. Thesis for M.Agr. Chiness Academy of Agricultural Sciences, supervisor:Liu Y S, pp.27-53)任梅渗,王印,杨泽晓,等. 2016. 副猪嗜血杆菌Hfq基因缺失株的构建与鉴定 [J]. 中国兽医科学,46(2): 203-209.(Ren M S, Wang Y, Yang Z X, et al. 2016. Construction and Characterization of Hfp deletion strain of Haemophilus parasuis. [J]. Veterinary Science in China, 46(2): 203-209.)曾泽,何欢,岳华,等. 副猪嗜血杆菌lgtF基因缺失株的构建及其部分生物学特性 [J]. 畜牧兽医学报, 2015, 46(11): 2056-2062.(ZENG Z, HE H, YUE H, et al. 2015. Construction and Characterization of a Haemophilus parasuis SC96 ΔlgtF Mutant Stain. [J]. Acta Veterinaria et Zootechnica Sinica, 46(11): 2056-2062.)Bigas A, Garrido ME, Rozas AM, et al. 2005. Development of a genetic manipulation system for Haemophilus parasuis [J]. Veterinary microbiology, 105(3-4): 223-228.Blokesch M. 2012. Chitin colonization, chitin degradation and chitin-induced natural competence of Vibrio cholerae are subject to catabolite repression [J]. Environmental microbiology, 14(8): 1898-1912Bossé JT, Nash JHE, Kroll JS, et al. 2004. Harnessing natural transformation in Actinobacillus pleuropneumoniae : a simple method for allelic replacements [J]. FEMS microbiology letters, 233(2): 277-281.Ding L Q, Wen X T, He L Q, et al. 2016. The arcA gene contributes to the serum resistance and virulence of Haemophilus parasuis serovar 13 clinical strain EP3 [J]. Veterinary microbiology, 196(1):67-71.He L Q, Wen X T, Yan X F, et al. 2016. Effect of cheY deletion on growth and colonization in a Haemophilus parasuis serovar 13 clinical strain EP3 [J]. Gene, 577(1): 96-100.Macfadyen LP, Ma C, Redfield RJ. 1998. A 3′,5′ Cyclic AMP (cAMP) Phosphodiesterase Modulates cAMP Levels and Optimizes Competence in Haemophilus influenzae Rd [J]. Journal of bacteriology, 180(17): 4401-4405.Seitz P, Blokesch M. 2012. Cues and regulatory pathways involved in natural competence and transformation in pathogenic and environmental Gram-negative bacteria. FEMS Microbiol Rev [J]. Fems Microbiology Reviews, 37(2013):336-363.Tarek M. 2005. Membrane Electroporation: A Molecular Dynamics Simulation [J]. Biophysical Journal, 88(6): 4045-4053.Lancashire JF, Terry TD, Blackall PJ, et al. 2005. Plasmid-encoded Tet B tetracycline resistance in Haemophilus parasuis [J]. Antimicrobial agents and chemotherapy, 49(5): 1927-1931.Wu R, Zhao M, Li J, et al. 2015. Direct regulation of the natural competence regulator gene tfoX by cyclic AMP (cAMP) and cAMP receptor protein (CRP) in Vibrios [J]. Scientific Reports, 5(10):14921-14936.Zhang B, He Y, Xu C, et al. 2012. Cytolethal distending toxin (CDT) of the Haemophilus parasuis SC096 strain contributes to serum resistance and adherence to and invasion of PK-15 and PUVEC cells [J]. Veterinary microbiology, 157(12): 237-242.Zhang L H, Li Y, Dai K, et al. 2015. Establishment of a Successive Markerless Mutation System in Haemophilus parasuis through Natural Transformation [J]. PloS one, 10(5):1-12.Zhang L H, Li Y, Wen Y T, et al. 2016. HtrA is important for stress resistance and virulence in Haemophilus parasuis [J]. Infection and immunity, 84(8): 2209-2258. |
|
|
|