Cloning And Characterization Of LsrK Gene In Salmonella Enterica SM1

Quorum sensing (QS) is a physiological phenomenon which microorganisms regulate gene expression according to cell density. Many bacteria can secret signal molecules called autoinducers, with which bacteria moniter the numbers of microorganism in the vicinal community and regulate gene expression. Resent researches have proved that AI-2, a kind of autoinducers who transmit messages between different species, is universal in pathogenic microorganisms, and play an important role in virulence expression or biofilm formation. In all AI-2-producing bacteria, the core part of AI-2 molecular is 4,5-dihydroxy-2,3-pentanedione (DPD), synthesized through methionine cycle. However, the mechanisms of AI-2 degradation and signal transduction are not clear. In some kinds of salmonella, LsrK phosphorylates AI-2 and DPD, which is the first step of degradation. So characterizing the AI-2 degradation genes is important to understanding QS system and provide the possibility of therapy based on QS system.Salmonella enterica SM1 was isolated from clinical patients and identified to be Salmonella Cholereasuis. However it was different from any reported strains. SM1 can spontaneously synthesize and utilize AI-2. To search the AI-2 degradation mechanism in SM1, we firstly synthesize DPD by 2-(tert-butyldimethylsilyloxy)ethanal and 3-buten-2-one based on Baylis–Hillman reaction. To make sure exact configuration, DPD was identified by TLC and HPLC-MS.To search the gene related to AI-2 degradation, we cloned the conserved part of lsrK gene from SM1 by PCR, and identifying its non-conserved part by Two-Step Gene Walking method. According to sequence analysis, the lsrK gene in SM1 had over 99% similarity to the homologous sequence from E.colli K12. Then, we constructed a lsrK-over-expression strain (SM1-pGEX-lsrK) and a chromosomal lsrK disrupted mutant strain (SM1-lsrK-Gm). Comparison of the three strains in growth and extracellular AI-2 actiity was carried out, we found that there were no difference between the three strains in degrading AI-2. Then we collected the cultures during logarithmic phase, transferred them into inorganic salt medium with DPD as sole carbon source. After a period of culturing, we found that SM1-pGEX-lsrK degrade DPD more quickly than the wild strain while the SM1-lsrK-Gm.strain was the same as the wild strain. The results indicated that LsrK kinase is not the only way to phosphorylate AI-2 in Salmonella enterica SM1.