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Mini Project III - Chemistry

Introduction:

In a process known as quorum sensing, bacteria communicate with one another using chemical signaling molecules called autoinducers. This cell-cell communication allows a population of bacteria to coordinate their gene expression, and therefore the behaviour of the group. Some bacteria, for example, produce visible light in response to autoinducers that accumulate in dense cultures; Pathogenic bacteria can use quorum sensing to coordinate the formation of antibiotics and enzymes. In this project, we used Gram-negative bacteria P. aureofaciens PGS12 which is known to produce N-Hexanoyl-DLhomoserine lactone as a signaling molecule during their growth.

 

Aims:

To detect if PGS12 produce other signaling molecules of similar structures and determine their chemical structure by comparing with eight standard AHLs (acyl-homoserine lactone).

 

Methods:

P. aureofaciens PGS12 was grown in NB medium and signal molecules were monitored and compared with eight standard AHLs: (1) N-Butanoyl-DL-HL, (2) N-Hexanoyl-DL-HL, (3) N-Heptanoyl-DL-HL, (4) N-Octanoyl-DL-HL, (5) N-Decanoyl-DL-HL, (6) N-Dodecanoyl-DL-HL, (7) N-Tetradecanoyl-DL-HL, (8) N-(3-Oxohexanoyl)-DL-HL. Standards (2), (3), (4), (7), and (8) were purchased from Fluka. Compounds (1), (5), and (6) were made by chemical synthesis. The chemical identity of AHLs was ascertained using NMR, IR, and Mass spectrometry. TLC analysis of the crude extracts from PGS12 against the standards above was used to determine if they were present. In bioassays, bioreporter C. violaceum CV026 was coated over the TLC plate to detect signaling molecules produced by PGS12.

 

Results:

We have shown that there may be three signaling molecules in PGS12 which act in the mechanism called quorum sensing. From the result of reversed-phase TLC plates, we could detect that these three compounds fall into between N-Hexanoyl-DL-H(2) and N-Butanoyl-DL-HL(1) (or N-(3-Oxohexanoyl)-DL-HL(8)). NMR spectra showed that PGS12 produced several compounds, not just AHLs. As the quantity of AHLs produced by PGS12 was so small compared to that of other compounds in the mixture. NMR spectra from PGS12 didn�ft show us the characteristic AHL peaks for the comparison with other standard AHLs. To detect and identify the chemical structures of these quorum sensing signal molecules, the use of bioassays seems to be the best way as we can detect AHLs specifically from other compounds. The use of GC-MS of the crude AHL fractions may prove more sensitive to analyse the compounds in the mixture for the future.