Liquid Chromatography Mass Spectrometry (LCMS)
Liquid chromatography mass spectroscopy (LCMS) is an analytical technique which combines the separation power of liquid chromatography, along with the detection capability of mass spectroscopy. In our project, we have utilised LCMS to determine the degradation of each of our standards. 4-Hydroxy-5-methyl-3-furanone was used to replicate the presence of AI-2 in the reaction. C4-HSL and N-(beta-Ketocaproyl)-L-homoserine lactone are examples of different AHLs, so we used these as a test for our AHL lactonase. First, we created a calibration curve for each of these standards.
4-hydroxy-5-methyl-3-furanone calibration curve
C4-HSL calibration curve
N-(β-Ketocaproyl)-L-homoserine calibration curve
These initial results confirmed that our standards could be detected on the LCMS and provided us with the necessary equations for calculating the concentration of each standard in the assay. In these equations, y is the area under the curve and x is the calculated concentration in micromolar.
4-hydroxy-5-methyl-3-furanone calibration:
$$ y = 68.727454*x - 70.794631 $$C4-HSL calibration:
$$ y = 636.920486*x - 168.173302 $$N-(β-Ketocaproyl)-L-homoserine calibration:
$$ y = 7852.257148*x - 6124.904398 $$Following detection of LuxS and SP-AHL lactonase on the Western blot following the E. coli transformation we set up a 0-hour reaction, 6-hour reaction and a 24-hour reaction by adding the mix standard a then lysate of the enzyme. After the set duration time, we stopped the reaction by adding in a set volume of ice-cold methanol.
The LuxS was not expected to have any degrading action against any of our standards as it produces 4,5,-dihydroxy-2,3-pentanedione (DPD), a precursor of AI-2 [2]. As there are no other enzymes in the assay, DPD cannot be converted into AI-2 molecules. Therefore, we compared the activity of SP-AHL Lactonase to both the mCherry and the LuxS as controls.
We tested against a mixed sample, containing all three standards. As expected, the degradation of 4-hydroxy-5-methyl-3-furanone over time by AHL lactonase was negligible, shown in Graph A below. This was analysed by assessing the line for being non-zero and the p values, displayed on the Graph A, are greater than 0.05. The data presented shows the assay with AHL lactonase compared to both mCherry and LuxS. With mCherry and LuxS compared on Graph D.
The following graphs display the results obtained from measuring 4-hydroxy-5-methyl-3-furanone concentration:
A
B
C
(A) Scatter plot of 4-Hydroxy-5-methyl-3-furanone concenration over 24 hours for AHL lactonase and mCherry (B) Scatter plot of 4-Hydroxy-5-methyl-3-furanone concenration over 24 hours for LuxS and mCherry (C) Scatter plot of 4-Hydroxy-5-methyl-3-furanone concenration over 24 hours for AHL lactonase and LuxS normalised
The following graphs display the degradation of C4-HSL and N-(beta-Ketocaproyl)-L-homoserine lactone by AHL lactonase compared to mCherry and LuxS:
D
E
F
G
H
I
(D) Scatter plot of C4-HSL concentration over 24 hours for AHL lactonase and mCherry (E) Scatter plot of C4-HSL concentration over 24 hours for AHL lactonase and LuxS (F) Scatter plot of C4-HSL concentration over 24 hours for LuxS and mCherry (G) Scatter plot of N-(beta-Ketocaproyl)-L-homoserine concentration over 24 hours for AHL lactonase and mCherry (H) Scatter plot of N-(beta-Ketocaproyl)-L-homoserine concentration over 24 hours for AHL lactonase and LuxS (I) Scatter plot of N-(beta-Ketocaproyl)-L-homoserine concentration over 24 hours for LuxS and mCherry.
Graph D shows that the degradation was increased by approximately 2.5 times what was observed when cell lysate from the E. coli transformed with mCherry was used. Graph E shows that the degrading action of AHL lactonase compared to LuxS was over 4 times greater. A one-tailed Wilcoxon signed rank test was carried out to validate this and we obtained a p=0.027 for Graph D and p=0.002 for Graph E. As the p value is less than 0.05, we can reject the null hypothesis (that the samples follow the same distribution) and conclude that there is a significant difference in the distributions of the two samples.
From Graph G it can be concluded that the AHL lactonases were not degrading the N-(beta-Ketocaproyl)-L-homoserine lactone. This homoserine-lactone was isolated from V. fischeri [3]. Perhaps indicating that the enzymes we are producing might not be effective against certain bacterial strains.
Graph I shows that the mCherry had greater degradation than LuxS, confirming that the LuxS has no degrading action on N-(beta-Ketocaproyl)-L-homoserine lactone.
From Graph D and Graph E we have shown that there was a significantly increased rate of C4-HSL degradation by AHL lactonase, which is part of the quorum sensing (QS) system in P. aeruginosa [4] - a bacterium which is responsible for catheter-associated biofilm formation [5]. Meaning we have successfully shown that we can produce an enzyme which can target the QS system of a biofilm-forming species.