Study Iron Metabolism Related Gene Function Of Enterobacter Cloacae In Ningbo Coastal Of Sewage Outfall

Enterobacter cloacae is a gram-negative bacteria and also a mode strain of Enterobacter. Because of superiordrug resistance, it has become one of the most serious pathogens in hospital infection. Iron is an essential nutrientfor bacteria, because it not only play a significant role in the growth and pathogenicity of pathogens, but alsoregulate the expression of virulence genes. Iron regulatory protein2and ferrichrome-iron uptake receptor Aencode outer membrane proteins that intake and receive the iron pigment from bacterial outer membrane. sltA is airon-chelating related gene. sodB is a group of metalloenzymes in Fur that can transform peroxide group into H202+O2. Under oxidative stress, bacteria enhance their cytoplasmic Fe2+binding capacity, inhibit iron transportationand induce the expression of iron storage system by increasing the number of Fur. Therefore, researches onfunctions of irp2, fhuA, sodB, sltA are of much significance in undrstanding iron metabolism and pathogenic andinfectional mechanisms of Enterobacter cloacae, and thus taking precautions and controlling.First of all, a total of98heterotrophic bacterial strains were isolated from the water of terrigenous outlet ofXiangshan Port by micobe automatic identification system and16S rRNA-based technique after a one-yearfollow-up survey. They were then classified into fifteen groups according to their colony morphologicalcharacteristics. Results of RFLP showed that they belonged to98species of five classes, and most of thembelonged to γ-Proteobacteria (67strains,68.4%) and Bacillus (16strains,16.3%). Among them, E.cloacae ofEnterobacteriaceae was the dominat strain. Growth curve and animal experiments indicated that the increase ofiron concentration significantly stimulated the growth and pathogenicity of E.cloacae.Secondly, diffrential expression under different cultural conditions of outer membrane proteins in E.cloacae wereanalysed by using2D-DIGE and mass spectrometry. A total of108protein spots that expressed diffrentially wereobtained，and76of them were then identified, annotated and classified. The annotated proteins were grouped intofour major categories based on their function: transportation (49.4%), recognition (16.3%), cellular structurecontinents (18.6%) and biological regulation (15.7%), and23.5%of such proteins were involved in ironmetabolism. Validation expriments by real-time PCR showed that the expression of fhuA、sodB、irp2、sltA inE.cloacae increased significantly though under different Fe3+concentration. Results demonstrated that all of thefour genes took part in iron metabolic mechanisms in E.cloacae.Finally, With homologous PCR amplification technology, were amplified specific band of sltA, sodB, irp2,fhuA. Application of MEGA4for multiple sequence analysis, topology display with E. coli, Klebsiellapneumoniae, Yersinia pestis homology relatively close. After multiplex PCR technology, can be detected in a shortperiod of time of4kinds of iron metabolism related virulence genes from virus or bacteria.