Monasterio de Santa María de Matallana (Valladolid)

Ferrándiz et al had shown population expansion of E2348/69 changed in response to different temperatures [68]. The same observation was made during this study, but at different temperatures to the Ferrándiz study [68].

Positive population expansion was observed for wild type E2348/69 at both 25°C and 37°C, producing distinct circular areas of ‘diffusion’ on motility agar plates (Figure 13). In all cases 2 biological replicates each consisting of 5 technical replicates were analysed. Representative images of these plates are shown in Figure 13. These images were chosen to illustrate the differences in the density of the motility zone therefore the diameter of the area of motility on the image may not be illustrative of the overall effect of the supplement. At 25°C the area of motility was clearly defined, with distinguishable rings (Figure 13). In comparison at 37°C, the motility zone appeared uniform and more dense throughout. At 25°C the bacteria were able to produce an area of motility with an average diameter of 20.8 ± 1.03mm (diameter ± 1

25°C 37°C

Figure 13 - Effect of temperature on E2348/69 population expansion

Images are representative of the motility zones produced after 18 hours incubation. Each image is representative of 2 independent biological cultures, each with 5 technical replicates.

White bars mark the boundaries of population expansion on each plate. Scale bars are indicated in the left hand corner and are set at 10mm.

N.B. Images are representative, therefore the size of motility ring on the image may not be illustrative of the overall effect of the supplement.

standard deviation). When the incubation temperature was increased to 37°C the area of motility increased to an average of 80.9 ± 3.6mm in diameter. Analysis of all the replicates at both temperatures by ANOVA showed this increase to be statistically significant (F = 1966.996; p = <0.00001).

The role of AHL-dependent quorum sensing and the link to temperature was investigated using a range of both short and long chain modified and non-modified AHLs to assess the impact on population-based motility of E2348/69. To ensure any effect observed was as a direct result of addition of the AHL, inactive D-isomers were used as a negative control in all experiments. As stated in the introduction (Section 1.3.2), there are two possible stereoisomers of AHLs; the L- isomer is able to activate the ligand-binding domain of the LuxR protein while the D-isomers are inactive [93]. In this study, L-isomers are defined as active AHLs, with D-isomers categorised as inactive. AHLs were added to both the motility media prior to pouring, and to the overnight culture in order to stimulate the quorum sensing regulon. AHLs were added twice to the overnight culture, once at time zero and again 1 hour prior to use in the motility experiments. Previous experimentation with bioreporters such as C. violaceum and exogenous AHL during this study had shown that 5nM of signal added to a culture of E. coli strain E2348/69 was sufficient to affect phenotypic behaviour for a limited period of time. As E. coli is unable to produce AHL this was hypothesised to be as a direct result of signal being internalised and used, thereby dropping the external concentration below the required quorum sensing threshold. To counteract this reduction in AHL and thereby the response, the signals were added multiple times to prime the bacteria and maintain a sufficient concentration of AHL to exert an observable effect during the course of experiment. Representative images of E2348/69 population expansion in the presence of different AHLs at 25°C are shown in Figure 14, and the average diameters of the area of motility are summarised in Table 10. In the absence of AHL, E2348/69 had an area of motility of average diameter 20.8 ± 1.03mm with clearly definable rings (Table 10; Figure 15).

No AHL N-(3-oxo-hexanoyl)-D-HSL _(C6) N-(3-oxododecanoyl)-D-HSL _(C12)

N-butanoyl-DL-HSL (C4) N-hexanoyl-DL-HSL (C6) N-(3-oxohexanoyl)-L-HSL (C6)

N-octanoyl-DL-HSL (C8) N-(3-oxo-octanoyl)-DL-HSL _(C8) N-dodecanoyl-L-HSL (C12)

N-(3-oxododecanoyl)-DL-HSL (C12)

Figure 14 - Effect of AHLs on E2348/69 population expansion at 25°C

Images are representative of the motility zones produced after 18 hours incubation. Each image is representative of 2 independent biological cultures, each with 5 technical replicates. Supplementation with AHL was performed to a final concentration of 5nM.

White bars mark the boundaries of population expansion on each plate. Scale bars are indicated in the left hand corner and are set at 10mm.

N.B. Images are representative, therefore the size of motility ring on the image may not be illustrative of the overall

10mm 10mm 10mm 10mm 10mm 10mm 10mm 10mm 10mm 10mm

Table 10 - Statistical analysis of the effect of AHL on E2348/69 population expansion at 25°C

Side chain lengths of the AHLs are in brackets. Significant increases in population expansion are highlighted in blue, those that decrease population expansion are highlighted in pink.

Average diameter of motility ring ± 1 standard deviation

(mm) ANOVA *F-value p-value No AHL 20.8 ± 1.03 N-(3-oxo-hexanoyl)-D-HSL (C6) 20.0 ± 1.41 2.09 0.166 N-(3-oxododecanoyl)-D-HSL (C12) 21.2 ± 1.39 0.53 0.476 N-butanoyl-DL-HSL (C4) 24.4 ± 1.35 44.86 2.80 x 10-6 N-hexanoyl-DL-HSL (C6) 18.3 ± 0.95 31.78 2.40 x 10-5 N-(3-oxohexanoyl)-L-HSL (C6) 13.8 ± 1.87 107.04 5.30 x 10-9 N-octanoyl-DL-HSL (C8) 18.8 ± 0.63 27.27 5.80 x 10-5 N-(3-oxo-octanoyl)-DL-HSL (C8) 14.9 ± 1.52 102.72 7.30 x 10-9 N-dodecanoyl-L-HSL (C12) 25.2 ± 1.39 64.06 2.40 x 10-7 N-(3-oxododecanoyl)-DL-HSL (C12) 23.9 ± 1.19 38.44 7.50 x 10-6 *F- critical value = 4.414

Figure 15 - Variation in the diameter of the motility zones produced by E2348/69 in the presence and absence of different AHLs at 25°C

For each of the 2 independent biological replicates, 5 technical replicates were analysed. Each point on the graph represents one technical replicate. Petri dishes measured 95 mm in diameter.

(Blue) No AHL

(Red) N-(3-oxo-hexanoyl)-L-HSL (Green) N-dodecanoyl-L-HSL (Purple) N-(3-oxo-hexanoyl)-D-HSL (Orange) N-(3-oxo-dodecanoyl)-D-HSL

The grey line represents the average diameter of the motility zones measured for the unsupplemented isolates.

0 20 40 60 80 100 120 Di am e te r o f Ar e a o f M o til ity (m m )

Biological variation between the two E2348/69 cultures was not significant (F = 0.348; p = 0.572) (Figure 15).

To determine which AHLs had the greatest effect on E2348/69 population expansion, each AHL was added individually. Three non-modified short chain AHLs namely N-butanoyl-DL- HSL, N-hexanoyl-DL-HSL and N-octanoyl-DL-HSL were tested. Addition of N-butanoyl-DL-HSL increased the average diameter of the motility zone from 20.8mm diameter to 24.4 ± 1.35mm, a statistically significant increase in bacterial population expansion (F = 44.86; p = <0.0001) (Table 10). In the presence of N-hexanoyl-DL-HSL the average diameter of the motility zone was 18.3 ± 0.95mm, a significant decrease in E2348/69 population expansion (F = 31.78; p = <0.0001) (Table 10). Addition of N-octanoyl-DL-HSL resulted in a significant decrease in the average diameter of the motility zone produced by E2348/69, measuring 18.8 ± 0.63mm (F = 27.27; p = <0.0001) (Table 10). A more significant decrease in population expansion was observed in the presence of the modified form of N-hexanoyl-DL-HSL, N-(3-oxohexanoyl)-L-HSL, which had an average diameter of 13.8 ± 1.87mm (F = 107.04; p = <0.0001) (Table 10; Figure 15). The modified form of N-octanoyl-DL-HSL, N-(3-oxo-octanoyl)-DL-HSL, caused a significant decrease in E2348/69 population expansion with the average diameter of the area of motility measuring 14.9 ± 1.52mm (F = 102.72; p = <0.0001) (Table 10; Figure 15). Furthermore, the areas of motility produced in the presence of the AHLs with an R-group of between 6 and 8 carbons in length were less dense with increasing distance from the point of inoculation compared to motility zones produced in the absence of AHL, suggesting fewer bacteria were able to swim. By comparison, in the presence of N-butanoyl-DL-HSL a dense motility zone was produced, which was more similar in density to the motility zone observed in the absence of AHL (Figure 14).

One modified and one non-modified long chain AHL were tested. Addition of N- dodecanoyl-L-HSL and N-(3-oxododecanoyl)-DL-HSL to E2348/69 resulted in a significant increase in the diameter of the motility zone to an average of 25.2 ± 1.39mm (F = 64.06;

p = <0.0001) and 23.9 ± 1.19mm (F = 38.33; p = <0.0001) respectively (Table 10; Figure 15). The motility zones produced in the presence of N-dodecanoyl-L-HSL and N-(3-oxododecanoyl)- DL-HSL had clear boundaries (Figure 14).

It was observed that at 25°C addition of N-(3-oxohexanoyl)-L-HSL resulted in the largest decrease and N-dodecanoyl-L-HSL the largest increase in the population expansion of E2348/69 (Table 10; Figure 15).

The two D-isomers N-(3-oxo-hexanoyl)-D-HSL and N-(3-oxododecanoyl)-D-HSL were selected as negative controls for AHL-dependent activity. The average areas of motility produced by E2348/69 measured 20 ± 1.41mm in diameter (F = 2.09; p = 0.166) in the presence of N-(3- oxo-hexanoyl)-D-HSL, and 21.2 ± 1.39mm (F = 0.53; p = 0.476) after addition of N-(3- oxododecanoyl)-D-HSL (Table 10). Therefore, neither of these AHLs had a significant effect on the population expansion of E2348/69 when incubated at 25°C (Table 10; Figure 15).

Based on the observations of the AHL effect on E2348/69 population expansion, N-(3- oxohexanoyl)-L-HSL and N-dodecanoyl-L-HSL, in addition to the two D-isomers were used in the following experiments to investigate the role of quorum sensing on E2348/69 motility at 37°C. In the absence of AHL E2348/69 produced an area of motility measuring 80.9 ± 3.6mm on average (Table 11). The variation in the diameter of the motility zones produced at this higher temperature was much greater than that observed at 25°C, indicated by higher standard deviations and the spread of diameter measurements (Figure 16). Addition of both N-(3- oxohexanoyl)-L-HSL and N-dodecanoyl-L-HSL had no significant effect the size of the motility zone produced by E2348/69 at 37°C (Table 11; Figure 16). Furthermore, neither of the D-isomer controls had any significant effect on E2348/69 population expansion (Table 11; Figure 16).

Table 11 - Statistical analysis of the effect of AHL on E2348/69 population expansion at 37°C

Side chain lengths of the AHLs are in brackets.

Average diameter of motility ring ± 1 standard deviation

(mm) ANOVA *F-value p-value No AHL 80.9 ± 3.60 N-(3-oxo-hexanoyl)-D-HSL (C6) 78.3 ± 1.89 4.083 0.0584 N-(3-oxododecanoyl)-D-HSL (C12) 81.0 ± 3.97 0.0035 0.953 N-(3-oxohexanoyl)-L-HSL (C6) 83.5 ± 4.72 1.916 0.183 N-dodecanoyl-L-HSL (C12) 81.5 ± 2.95 0.166 0.688 *F- critical value = 4.414

Figure 16 - Variation in the diameter of the motility zones produced by E2348/69 in the presence and absence of different AHLs at 37°C

For each of the 2 independent biological replicates, 5 technical replicates were analysed. Each point on the graph represents one technical replicate. Petri dishes measured 95 mm in diameter.

(Blue) No AHL

(Red) N-(3-oxo-hexanoyl)-L-HSL (Green) N-dodecanoyl-L-HSL (Purple) N-(3-oxo-hexanoyl)-D-HSL (Orange) N-(3-oxo-dodecanoyl)-D-HSL

The grey line represents the average diameter of the motility zones measured for the unsupplemented isolates.

0 20 40 60 80 100 120 D iam e te r o f A re s o f M o til ity (m m )