Investigating Production of AHL-Acylase and AHL-Lactonase in Deinococcus radiodurans

 Introduction

    Deinococcus radiodurans is a gram negative extremophilic bacterial species that is frequently recognized for being able to withstand environmental conditions with high levels of radiation. It is also recently found to be one of the few, potentially only, species that produces both AHL-acylase and AHL-lactonase in order to degrade AHLs. AHL, or N-acyl homoserine lactones, are signal molecules produced by cells, typically in a biofilm community, in order to alter gene expression in the recipient cells. The communication between cells is described as quorum sensing, a survival strategy that regulates the behavior of a community of cells. Most cells that have enzymes that degrade AHLs have either AHL-acylase or AHL-lactonase, but it has been found that Deinococcus radiodurans has genes to synthesize both enzymes, which is incredibly unusual. AHL-acylase is a protein that hydrolyzes the amide bond between the lactone ring and the acyl chain of AHLs, while AHL-lactonase breaks the lactone ring. Both enzymes effectively deactivate any AHL signals that the cell receives, preventing any changes in gene expression. The concentration of AHLs in the environment therefore must be much higher for Deinococcus radiodurans to respond in comparison to other cells with only one quorum-quenching enzyme (Koch et al 2014). The procedures done are in preparation for an experiment which will evaluate the quantitative comparison between acylase and lactonase production in D.radiodurans when the cell is under oxidative stress and under no specific stressors. Methodology for detection of AHL production in D,radiodurans will also be explored. The first steps in the process of being able to run qPCR experiments was to evaluate the efficiency of the primers needed for the experiment, which included producing cDNA from a sample D.radiodurans RNA, and running a set of dilutions to determine from graphing data, and the subsequent melt curve, the primer efficiency of lactonase primers and to confirm the optimal dilution factor (Taylor et al 2019). 

Methods

    RNA was originally extracted from Deinococcus radiodurans by Chad Albert, in preparation of the cDNA sample needed to verify the lactonase primers. RNase spray was used to wipe down the surface that would be used to prepare the sample, and an initial nanodrop reading was taken on the Nanodrop 2000.  the RNA tested as 156 ng/ul and a total of  one microgram was needed for the sample. Therefore 6.5 microliters was taken from the RNA sample and combined with 7.5 microliters of RNase free water, for a total of 14 microliters. This tube was combined with 1.5 microliters of DNase buffer and 0.5 microliters of DNase, and mixed by pipetting. The tube was pulse centrifuged and loaded into a PCR machine and the preset protocol cDNA1 was ran, with incubation temperature at 25 degrees Celsius and then 75 degrees Celsius for proper DNase efficiency. Once completed, the sample should only contain RNA molecules for the next step. Four microliters of Supermix is then quickly added to the 16 microliter solution, and mixed with pipetting. Once pulse centrifuged, it went back into the PCR machine for preset protocol cDNA2, in which reverse transcriptase enzymes convert the RNA samples into cDNA. The sample was stored at -80 degrees Celsius until qPCR was run. 

    The lactonase primers were tested at a 1:5 dilution (10 microliters of cDNA sample and 40 microliters of RNase free water), which this solution was then diluted further into 1:10, 1:100, 1:1,000, 1:10,000, and 1:100,000 dilutions labeled A, B, C, D, and E respectively. The tubes were mixed and tapped to homogenize the solution, and then pulse centrifuged. The Mastermix was calculated to need 82.5 ul of  powerup SYBR Green, 8.3 ul of forward primer solution, and 8.3 of reverse primer solution. The NTC tube being used as control was 4.3 ul of RNase free water, 5.5 ul of powerup SYBR Green, and 0.6 ul of forward and 0.6 ul of reverse primer solutions. In following a 60:40 ratio for well samples, 6 ul of Mastermix was loaded into 15 wells, and 4 ul of each dilution was loaded into 3 wells per dilution factor. Sample A was loaded into A1, A2, and A3, sample B was B1, B2, and B3, and so one for each sample. The NTC tube was loaded into F1 on the plate. Each well was mixed with careful pipetting technique five times to try to prevent any air bubbles in the wells. The plate was covered in the appropriate plastic wrap, and sealed on the edges. The plate was then centrifuged at room temperature for approximately ten minutes to get rid of present air bubbles. QPCR was started once the plate was placed into the machine, and ran for a total of 39 cycles with an annealing temperature of 60 degrees Celsius. 

    An identical process was conducted the following day with the original cDNA sample produced that was described above, except with a 1:2 dilution of the initial stock solution rather than a 1:5 dilution. This secondary test was completed by Chad Albert. All other methodology of the process remained identical. 

Results

    The first run of qPCR testing the efficiency of the lactonase primers was unsuccessful due to the fact that a significant amount of detection was not seen until cycle 28 of 39, which indicated that the cDNA solution was too dilute. The samples of identical dilution factors were also not as tight in Figure 1 as they are in Figure 3, which was the second run done by Chad at the 1:2 dilution. The spacing between each dilution factor was also not ideal in Figure 1 like that of Figure 3. The primer efficiency was calculated to be 160% in the first run, and 101.6% in the second run. The melt curve of the first run also showed a stray value within the shelf that could have potentially indicated a primer dimer, but was not evident in the second run as seen in Figure 2. Figure 2 also indicated the appropriate tightness of data which we were looking for. It was also noted that even in the second run, significant data was not collected until cycle 24. 

Figure 1

Figure 2

Figure 3

Conclusion

    

    The variance in data of the first run of qPCR was likely due to the cDNA sample being too dilute, as well as a lack of experience in plate preparation and micropipetting errors. The second run validated the lactonase primers with an efficiency of 101.6% which is well within the margins of 90-100% that is needed to have accurate qPCR runs later on. It also indicates that lactonase may potentially be an enzyme within Deinococcus radiodurans that is not very abundant, since a 1:2 dilution of the cDNA still did not show significant data until the 24th cycle, which is considered late. The next steps of the process will include an RNA extraction to produce more cDNA for the primer validation of acylase. 

References:

1. Gudrun Koch, Pol Nadal-Jimenez, Robbert H. Cool, Wim J. Quax,  Deinococcus radiodurans can interfere with quorum sensing by producing an AHL-acylase and an AHL-lactonase, FEMS Microbiology Letters, Volume 356, Issue 1, July 2014, Pages 62–70, https://doi.org/10.1111/1574-6968.12479

2. Taylor, S. C., Nadeau, K., Abbasi, M., Lachance, C., Nguyen, M., & Fenrich, J. (2019). The ultimate qPCR experiment: producing publication quality, reproducible data the first time. Trends in Biotechnology, 37(7), 761-774.