关键词: Gac系统, gacS基因, 东湖假单胞菌HYS, 毒性因子

Abstract: Bacterial pathogenic factors are an important part of bacterial toxicity. The Gac system is a global regulatory system of bacteria that regulates the production of various virulence factors in bacteria. In previous studies, a gram-negative bacterium, siderophore high-yielding Pseudomonas donghuensis (HYS), was isolated from the waters of East Lake in Wuhan for the first time. In order to explore the effect of Gac system on HYS toxicity, firstly, the position of GacS protein from Gac system in HYS was located through amino acid sequence BlastP alignment. Through bioinformatics analysis, Gac system was determined to exist in HYS. Then gacS fragments derived from P. donghuensis HYS and P. aeruginosa PAO1 were respectively supplemented into gacS gene knockout HYS strain ΔgacS through gene cloning, and the obtained strains were fed to Caenorhabditis elegans, and the toxicity of the strains were estimate by comparing the survival number and days of the nematodes. The results showed that the toxicity of ΔgacS strain was obviously weakened. However, the toxicity of the two complementary strains to nematodes recovered to different degrees, which indicated that Gac system in HYS participated in toxicity regulation pathway, and gacS fragment of P. aeruginosa PAO1 could also play a certain toxicity regulation role in HYS. In order to further explore whether the attenuated characteristics of ΔgacS strain were related to the loss of virulence factors in HYS, the activity of extracellular protease of the strain was detected by defatted milk plate, and the exercise capacity of the strain was detected by swimming motility plate, swarming motility plate and twitching motility plate. Meanwhile, metabolites such as lipopolysaccharide and hydrocyanic acid were also detected. The results showed that the protease secretion of ΔgacS strain completely disappeared compared with HYS, and the three kinds of exercise abilities of ΔgacS were significantly decreased compared with HYS (P<0.05). In addition, the yield of extracellular lipopolysaccharide of ΔgacS decreased to different degrees compared with that of wild type HYS. However, the phenotypes of these virulence factors could all be recovered to the wild type level in the complementary strains ΔgacS/pBBR2-gacS_HYS, and the complementation of homologous gacS gene from PAO1 could even complement the function of gacS in regulating toxic substances to a certain extent. Studies have shown that the potential animal pathogenic bacteria, P. donghuesis HYS, was positively regulating and producing toxic factors through Gac system, thus exerting lethal effect on Caenorhabditis elegans, which provided theoretical basis for organisms to resist HYS infection in the future.

Key words: Gac system, gacS gene, Pseudomonas donghuensis HYS, virulence factor