Isolation And Characterization Of Quorum-sensing-interfering Bacteria

Quorum sensing is an environmental-signal-sensing system in bacteria, it is believed that interfering of the normal function of quorum-sensing system could be developed as a new strategy for bacterial disease control. The aim of this study was to screen soilborne bacterial strains which could interfere with the quorum-sensing system and to understand primarily their interfering mechanisms.Totally ca. 5,000 bacterial strains were isolated from Beijing city and Yunnan province. The bacterial strains that could interfere with the model quorum-sensing system of Agrobacterium tumefaciens were screened by using an "agar slice" method and then a "reporter plate" method, fifteen isolates were confirmed to be able to inactivate 7V-3-oxohexanoyl-homoserine lactone(3OC6-HSL, 10-2 pmol)-mediated quorum-sensing system. Under our detecting conditions, these fifteen strains showed different levels and characteristics for disturbing quorum-sensing system.With C11 as a model, primary study on mechanism of its cells interfering action was performed. The twice-rinsed cells of Cll can interfere intensely and fast with many kinds of quorum-sensing signals which have various structures. To confirm that the action target of Cll cells was quorum-sensing regulatory system, the possibility of direct inhibition of C11 to P -galactosidase must be excluded. Biparental mating between Escherichia coli S17-1( λ -pir) that carried plasmid pUT/acz and A. tumefaciens NT1 which could not produces acyl-HSLs resulted in NTl::/acZ strains that could express P -galactosidase constitutively. C11 cells did not showed any obvious inhibitory effect on the P -galactosidase activities produced from NT1::/acZ strains, indicating that the functional factor(s) of C11 should target on the bacterial quorum-sensing system instead of P -galactosidase itself. Further analysis of quorum sensing inactive mechanism was performed with Baker Si-C 18F reversed phase thin layer chromatography (TLC). We confirmed primarily that C11 inactivated the biological functions of Af-acyl-L-homoserine lactones (acyl-HSLs) through an intracellular enzymatic mechanism. TLC also showed C11 cells destroyed various acyl-HSLs with different molecular structures and the interfering action was wide.The morphology observation revealed that strain C11 was a short-rod bacterium, which form a round colony with pink colour on LB plate. Cl 1 grew faster in 30℃ than in 37℃. Gram strain showed it was gram-negative. The 16S rDNA of C11 was amplified by PCR, purified, cloned into pGEM-T easy vector and sequenced partly. The sequensing results indicated that C11 shared the highest sequence identity with three species of Shigella (99.9%). Therefore the strain Cll was primarily identified as a bacterium in genus of Shigella. It has been reported that only two species of bacteria, Variovorax paradoxus and Bacillus sp. 240B1, have the ability to interfere with quorum-sensing system. It is obvious that C11 is different from the above two species and is possibly a new quorum-sensing-interfering bacterium discovered.