My bacterial strain was found to be positive for AHL production using CV026 as a biosensor strain and gave big zones of purple coloration, but no peaks for AHL were detected on LC-MS analysis. I can't understand the reason. Extraction was done with ethyl acetate as well as with dichloromethane, at pH 2 and also without any change in pH.
First did you try to open the lactone cycle with 0.5M ammonia overnight and vaccum evaporation and resolubilization in ethyl acetate? Indeed the lactone ring in its open form is not recognized by CVO.
It will show if the compund detected by CVO26 possess a lactone ring.
Use the NTLR4 bioindicator. It senses the long chain AHL.
If you have the genome of your bacteria, check the presence of AHL biosynthesis gene
Most probably you are not detecting AHL with CV026. Biosensor strains are highly sensitive but sometimes are also not very specific. Maybe you have an unspecific (false positive) result with CV026. Several articles report that biosensors detect compounds from the culture medium not related to AHLs.
May it is just below detection level by LCMS, I will suggest try extraction with a larger volume of culture, to increase yield ....
Yes. I agree with Carlos and Carine.The AHL (in my case: C8-HSL) HPLC detection range is over 1000nM (under the 210nm, C18 column, Gradient). However, i used 100 nM C8-HSL for detecting bioassay (A136 reporter strain, Luminometer or LB plate assay). Also, I tried to detect some type of HSL using LCMS. In this case, the extraction method is very important. Ah~ Did you check the MS detector (negative and positive charge)? What kind of LCMS did you use? If you used quadroful MS, check your detector charge.
CV026 could not recognize long-acyl-chain homoserine lactones such as C10-C14. NTLR4 reporter strain based on the TraI/R system can be used to detect a broad range of AHLs and showed marked sensitivity to AHLs, including the long-acyl-chain HSLs, however, the handling of reporter is complicated especially in comparison with the simpler cross-streak assay on agar plates used for reporter strains such as CV026. There is a new reporter strain VIR24 that was derived from strain ATCC 12472, and could detect long-acyl-chain HSL molecules that could not be detected by CV026.
Therefore, the results from the combination of simple cross-streak assay using both C. violaceum strains can be used to identify your AHL of interest. Be careful with the production of several AHLs by the same strain. Look, in Someya et al., Microbs Environ 2009.
Regards from Spain
I dont know whether you have already considered this but
Instead of checking for m/z 102 characteristic for Lactone in LCMS you can also check whether there is a peak with an m/z of 143/144 (known as a Mc Lafferty fragment ) and a corresponding molecular ion peak for a known AHL
For eg. an m/z of 144 and a molecular ion peak 229 could indicate that your compound is C8HSL
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