Team:XJTLU-CHINA/Lysis
Lysis
AcmA and the application of the toggle switch in our project
The reason of utilization of the lacI-tetR regulatory switch
AcmA in Lactococcus lactis
Enzymatic function
N-acetylmuramidase, AcmA, is an autolysin protein of Lactococcus lactis, which is responsible for cell wall hydrolysis at the stationary phase and is involved in cell division of this organism（Buist, et al., 1997）. Consisting of two different domains in the structure, a glucosaminidase domain at the N-terminus and three so-called LysM domains at the C-terminus, the enzyme can specifically bind to peptidoglycan of L. lactis and of other Gram-positive bacteria. Peptidoglycan is a major component in the outer plasma membrane of Gram-positive bacteria and forms a mesh-like layer on the exterior of the cell. The known mechanism on the function of AcmA to date is that it can hydrolyze the N-acetylmuramyl-1,4-β-N-acetylglucosamine bonds in the peptidoglycan, whereby the peptidoglycan chain is broken and thus contribute to the cell lysis occurred to stationary L.lactis.
Physiological effects
The physiological functions of AcmA in the bacteria contribute to cell division, separation, motility, cell-wall turnover and other physiological processes (Smith, Blackman & Foster, 2000). However, overproduction of AcmA in L. lactis can cause cell autolysis, and hence its name (Buist, et al., 1997).
Merits and a possible concern of employing AcmA
Merits:
- Since AcmA is an endogenous molecule of Lactococcus lactis, the expression of AcmA in its original host is much more natural and applicable.
- Compared to other secretion systems, the autolysis process makes the secretion of AMPs more thorough because it does not require an energy consuming secretory system.
Concern:
As a broad-range autolysin due to its similar lysis mechanism to the other autolysin family members, i.e. hydrolyzing N-acetylmuramyl-1,4-β-N-acetylglucosamine bonds in the peptidoglycan, it is not clear whether AcmA has any adverse effects on the enteric microbiome. If AcmA lyse other gram-positive bacteria, the homeostasis of intestinal bacterial community may be affected.
Testing the expression of autolysin in L. lactis
Testing Protocol
Nisin-induced lysis of L. lactis
- Inoculate a colony of pNZ8148-acmA transformed L. lactis and a colony of non-transformed L. lactis separately into M17 broth (containing 10 g/ml chloramphenicol) and incubate at 30℃ without shaking.
- Dilute the two overnight cultures 25-fold with M17 broth and transfer the diluted cultures in a 96-well plate, with 8 repeats for the transformant and 1 for the negative control. Put the plate in a plate reader, and read successive OD600 value until bacteria grow to the stationary phase.
- Induce the transformant bacteria with different concentrations (0.1-5 ng/ml) of nisin when the OD600 reaches 3. Continue to measure their optical density for 2 days (can be longer).
References
- Inoculate a colony of pNZ8148-acmA transformed L. lactis and a colony of non-transformed L. lactis separately into M17 broth (containing 10 g/ml chloramphenicol) and incubate at 30℃ without shaking.
- Dilute the two overnight cultures 25-fold with M17 broth and transfer the diluted cultures in a 96-well plate, with 8 repeats for the transformant and 1 for the negative control. Put the plate in a plate reader, and read successive OD600 value until bacteria grow to the stationary phase.
- Induce the transformant bacteria with different concentrations (0.1-5 ng/ml) of nisin when the OD600 reaches 3. Continue to measure their optical density for 2 days (can be longer).
References
Buist, G., et al. (1997) ‘Autolysis of Lactococcus lactis Caused by Induced Overproduction of Its Major Autolysin, AcmA’, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1997, pp. 2722-2728
Smith, Thomas J., Blackman, Steve A. and Foster, Simon J. (2000) ‘Autolysins of Bacillus subtilis: multiple enzymes with multiple functions’, Microbiology, 146, pp. 249-262, Emerald Insight [Online]. DOI: 10.1099/00221287-146-2-249
Table 1 Definitions of the parameters
| Parameters | Pate constant for | Value | Units | Note |
|---|---|---|---|---|
| αpi | Phosphorylation of AgrA | 10[1] | μmol-1 ml-1 h-1 | |
| αpidi | Dephosphorylation of AgrA | 1[1] | h-1 | |
| μx | Degradation and dilution | 2[1] | h-1 | |
| αcbind | AgrC that anchors to the cell membrane | 10 | μmol-1 ml-1 h-1 | Assume the same as αpi |
| αbind | Binding of AIP to AgrC | 1[1] | μmol-1 ml-1 h-1 | |
| αunbind | Separation of AIP from AgrC | 0.1[1] | h-1 |
| Parameters | Definitions | Value | Units | Note |
|---|---|---|---|---|
| X | Nisin | 1.42×10-7[2] | μmol ml-1 | |
| k2 | The Phosphorylated AgrA concentration required for half-maximal transcription rate of P2 | 1[1] | μmol ml-1 | |
| β1 | Maximum transcription rate of pnisA | 10 | μmol h-1 | Assume the same as β2 |
| β2 | Maximum transcription rate of P2 | 10[1] | μmol h-1 |
Table 2 Definitions of the variables
| Variables | Concentration of | Units |
|---|---|---|
| A | AgrA | μmol ml-1 |
| C | AgrC | μmol ml-1 |
| Cbind | AgrC that anchors to the cell membrane | μmol ml-1 |
| AIP | Free AIP molecules | μmol ml-1 |
| Cp | AIP-bound AgrC | μmol ml-1 |
| Api | The phosphorylated AgrA | μmol ml-1 |
| sfGFP | The product of P2 promoter | μmol ml-1 |
The three Hill equations represent the rates of translation of AgrA, AgrC and sfGFP. Β1 is the highest efficiency for the promoter pnisA to initiate the transcription of the agrC and agrA genes, and β2 is the highest efficiency for the promoter P2 to initiate the transcription of the sfGFP gene. X is the concentration of nisin which is needed to activate the promoter pnisA, to this extent, k1 equals to the concentration of Api when the rate of reaction is up to half of Vmax. K2, which is controlled by another regulatory factor, is the concentration of phosphorylated AgrA when the rate of reaction is up to half of Vmax.
By assuming that 0.25 μM of AIP molecules is present in the intestine, we run the MATLAB script to check whether AIP molecules can successfully activate the promoter P2 by binding to AgrC and phosphorylating AgrA. We set the threshold concentration of sfGFP to be 0.5 μM, and at this point, we consider the promoter P2 is activated. The results are shown below.
Fig 1. State values of AgrA, Cbind, AgrC, Cp, Api and sfGFP.
Fig 2. Individual display of 6 variables
As it is shown in the second graph (values are hard to observe in the first one), concentration of sfGFP reaches 0.5 μM at the third hour. Therefore, we made a conclusion that the amount of AIP molecules can activate the promoter P2 to transcribe the genes downstream.
Modelling on peptide synthesis and cell lysis
Our design uses the tandem repeat strategy to express three copies of each peptide gene, LL-37, GF-17 and Grammistin-Pp1, aiming to producing peptides quickly and at a higher rate. To release the peptides to kill Staphylococcus aureus in the intestine, we choose lysis of the cells instead of secretion. A lysis gene is used to open up the cells, then all the peptides will surely be released into the guts. In addition, we plan to use a toggle switch to provide more time for peptide synthesis before lysis. When the cells are lysed, it will result in the release of intracellular proteins and stop all life activities. Therefore, we use modeling to identify:
- How long does cell lysis take from the point of induction?
- At this time point, how much peptides are produced by the gene circuit?
Results: Inspired by the team TU-Delft (2013), we came up with the idea that the promoters P2, plac and ptet may serve as binary switches between the active and inactive promoter states instead of continuous activities from fully on to fully off. We used the parameter--s, a binary state descriptor, to refer to the situation when a promoter produces one of the two levels of activity: on or off.
Table 3 Definitions of parameters
| Parameters | Definitions | Value | Units | Note |
|---|---|---|---|---|
| a | translation rate per amino acid | 1020[3] | Min-1 amino acids residues-1 | |
| cp2 | maximum transcription rate of P2 | 0.17[1] | μmol min-1 | |
| ctetR | maximum transcription rate of ptet | 2.79[3] | μmol-1 min-1 | |
| cplac | maximum transcription rate of plac | 2.79 | μmol-1 min-1 | Assume the same as ctetR |
| dmRNA | degradation rate of mRNA | 0.288[4] | min-1 | |
| dLacl | degradation rate of Lacl | 0.1386[4] | min-1 | |
| dtetR | degradation rate of tetR | 0.1386[4] | min-1 | |
| dAcmA | degradation rate of AcmA | 0.0063 | min-1 | Assume the same as GFP |
| dGFn | degradation rate of GFn | 0.0021 | min-1 | Assume the one-third of GFP |
| dGram | degradation rate of Gran | 0.0021 | min-1 | Assume the one-third of GFP |
| dLLn | degradation rate of LLn | 0.0021 | min-1 | Assume the one-third of GFP |
| lp2 | Leakage factor of P2 | 0.002 | - | Assume the same as ltetR |
| ltetR | Leakage factor of tetR | 0.002[3] | - | |
| lplac | Leakage factor of plac | 0.002 | - | Assume the same as ltetR |
| SLacl | length of Lacl | 371 | Amino Acid residues | |
| StetR | length of tetR | 226 | Amino Acid residues | |
| SAcmA | length of AcmA | 438 | Amino Acid residues | |
| S | Activation/Inactivation | 0/1[3] | - | |
| kLacl | dissociation constant of plac | 6 | μmol | Assume the same as ktetR |
| ktetR | dissociation constant of ptet | 6[3] | μmol | |
| ntetR | Hills coefficient | 3[3] | - | |
| nLacl | Hills coefficient | 3 | - | Assume the same as ntetR |
| Variables | Concentration of |
|---|---|
| LacIm | Transcribed LacI |
| tetRm | Transcribed TetR |
| AcmAm | Transcribed AcmA |
| GFnm | Transcribed GF-17 (n=1,2,3) |
| Granm | Transcribed Grammistin-Pp1 (n=1,2,3) |
| LLnm | Transcribed LL-37 (n=1,2,3) |
| LacI | Translated Lacl |
| tetR | Translated tetR |
| GFn | Translated GF-17 (n=1,2,3) |
| Gran | Translated Grammistin-Pp1 (n=1,2,3) |
| LLn | Translated LL-37 (n=1,2,3) |
By running the Matlab script, we obtained the results shown below.
Fig 3. State values of LacIm, GFnm, Granm, LLnm, tetRm, AcmAm, LacI, tetR, AcmA, GFn, Gran, LLn.
Fig 4. Individual display of transcribed LacIm, GFnm, Granm, LLnm, tetRm and AcmAm
Fig 5. Individual display of translated LacI, tetR, AcmA, GFn, Gran and LLn.
From these graphs, we can make a general conclusion that the shift between the two states controlled by LacI and TetR takes at least 20 minutes. By the time the promoter P2 initiates the transcription and later efficiently translation of the mRNA of the tandem repeat genes (ll-37, gf-17, and grammistin-Pp1), the antimicrobial peptides are capable of being synthesized at high rates. When the repression of the promoter ptet (tetR) is relieved and the lysis gene acmA (AcmAm) starts to be transcribed, the antimicrobial peptides can be accumulated to high concentrations. Thereafter, enough amounts of antimicrobial peptides will be released to eradicate Staphylococcus aureus through the cell lysis.
References
[1] Z. Cai, et al. “A simulation of Synthetic agr System in E. coli,”in Bioinformatics Research and Applications. Charlotte, NC: Springer, 2013, pp76-86.
[2] NICE Expression System for Lactococcus lactis. MoBITec GmbH, Germany, 2010.
[3] Team: TU-Delft (2013). Timer Plus Sumo [Online]. Available: https://2013.igem.org/Team:TU-Delft/Timer_Plus_Sumo
[4] C. Wu, H. Lee, and B. Chen, "Robust synthetic gene network design via library-based search method," Bioinformatics, vol. 27, pp. 2700-2706, Oct. 2011.
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