Construction And Preliminary Evaluation Of Biological Characteristics Of RfbN Gene Mutant Of Salmonella Enterica Serovar Typhimurium

Rhamnose,a important component of O-antigen, is transferred by rhamnose transferase which was synthesized by Salmonella enterica serovar Typhimurium rfbN gene.Here,we constructed a rfbN gene deletion mutant (ArfbN) and an arabinose regulated rfbN gene mutant.Then, biological characteristics of the mutants were systematically anaiyzed.1 construction of Salmonella enterica serovar Typhimurium ArfbN mutant strainThe Salmonella enterica serovar T yphimurium ArfbN mutant strain were constructed by the method of suicide plasmid-mediated homologous recombination.LPS map showed that ArfbN had an incomplete LPS structure composed by lipid A, core polysaccharide and a galactose.The covered plasmid 15a-rfbN was electroporated into ArfbN mutant, resulting in the expression of the complete LPS.It indicating that Salmonella enterica serovar Typhimurium ArfbN mutant strain was successfully constructed.2 construction of Salmonella enterica serovar Typhimuriu marabinose regulated rfbN genemutant strainSuicide plasmid pYA4286-ΔpagL-TT araC PBADrfbN was constructed by changing the SD sequence and the start codon of rfbN gene. We constructed two arabinose regulated rfbN gene mutants, ΔrfbNΔpagL::TT araC PBADrfbN(start codon,ATG) and ΔrfbNΔpagL::TT araC PBADrfbN(start codon,GTG).In the presence of arabinose,ΔrfbNΔpagL::TT araC PBADrfbN (start codon,ATG), the gene rfbN expressed, bacteria capable to synthesize a complete LPS; However, In the presence of arabinose, the gene rfbN of ΔrfbNΔpagL::TT araC PBADrfbN(start codon,GTG) little expressed, which incapable to synthesize a complete LPS.3 Some studies on biological characteristics of those mutantsIn order to compare the role of rfbN in biological functions of salmonella enterica serovar Typhimurium, the bacteria growth, swimming and swarming motility, polyxin B resistance, attachment and invasion and virulence between rfbN mutant strain and parent strain were systemically analyzed. Our results showed that there were no significant difference in bacteria growth, attachment and invasion in ArfbN strain and ΔrfbNΔpagL::TT araC PBADrfbN(start codon,ATG)strain compared with parent strain in vitro. However, the swimming and swarming motility and virulence of ΔrfbN strain were significant reduce, and more sensitive than parent strain. Also, the swimming and swarming motility and polyxin B resistance of ΔrfbNApagL::TT araC PBADrfbN(start codon,ATG)strain were fully recover supplement with arabinose in growth medium. In summary, rfbN deletion mutant of Salmonella enterica serovar Typhimurium could not influence the bacteria growth, attachment and invasion in vitro, but it plays a important role in bacteria virulence and positive antibacterial peptide resistance.