AcmA and the application of the toggle switch in our project

Figure 1.    Construction of the AMPs-Toggle Switch-Autolysin cassette. The upstream sensing device for S. aureus is omitted, which can be seen at the Sensing Device module.

Considering low efficiency and complexity of conventional secretory pathway, this year, we built a based-on-lysis system for aiming at releasing AMPs (antimicrobial peptides) efficiently. Therefore, we designed the acmA gene to be preceded by a bistable toggle switch so as to make cell lysis more regimented (see the reason in the following section). The switch we used was the lacI-tetR regulated expression system. The tetR linked downstream to plac is constituitively transcribed when free from the repression of the LacI repressor protein. This leads to the expression of TetR protein and subsequently an inhibitive effect on AcmA expression. Hence, under the normal situation in a host bacterial cell (such as L. lactis), the expression of AcmA autolysin is always inhibited. In this case, L. lactis can keep its integrity and maintain the capability of sensing S. aureus. Thereafter, with the initiation of P2 by phosphorylated AgrA (the mechanism can be seen at the Sensing Device module) and the expression of LacI, the switch turns on the ptet promoter and the expression of AcmA starts, as subsequently elicits the autolysis process and the accumulated AMPs burst out, culminating the extermination of the pathogen—S. aureus in the gut.

The reason of utilization of the lacI-tetR regulatory switch

Considering the prompt effect of AcmA on cell number (see Figure 2), after its induction, there might be a relatively early cell lysis (decrease in OD) occurring before AMPs are accumulated to a concentration at which pathogenic bacteria can be effectively killed. In the light of this finding, we adopted a lacI-tetR regulatory switch to postpone the autolysis process.

Figure 2.    

Buist’s demonstration of the effect of overexpression (A, B & C) of AcmA (1997). The optical density of the bacterial cells was measured at OD₆₀₀. After the induction of AcmA with mitomycin at the stationary phase (10 hours in the graph), there was a conspicuous decrease in bacterial optical density during the next 2 days.
In Buist’s work, A, C, D respectively represents the L. lactis that contains different kind of plasmids harboring the acmA gene and B stands （which is not shown in the graph） for the bacteria that does not have the acmA gene.

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:

1. Since AcmA is an endogenous molecule of Lactococcus lactis, the expression of AcmA in its original host is much more natural and applicable.
2. 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

Figure 3.    Construction of the autolysin part (Main elements in pNZ8148 haboring acmA, the detailed result of the assembly of BBa_K2309004 in the pNZ8148 plasmid can be seen at Figure 4)

Before we assembled all of our parts together, we tested each independent functional part. The acmA gene along with an upstream RBS was first joined to pNZ8148 which contains a nisin-inducible promoter and a terminator (Figure 3), and then the whole construct was transformed into the Lactococcus lactis NZ9000 strain. After the bacteria was cultured to the stationary phase, nisin was added to induce the expression of the AcmA protein. We employed an experimental procedure similar to Buist, et al. (1997). In their experiment (see their result at Figure 2, after induction with mitomycin, the bacteria cells at the stationary phase had a prominent decrease in optical density in several hours. (Buist et al., 1997)

Figure 4.    Integrated plasmid construction of the testing part (The detailed illustration of pNZ8148 harboring the acmA gene)

Testing Protocol

Nisin-induced lysis of L. lactis

1. 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.
2. 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.
3. 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