Part:BBa_K802000

Lysostaphin generator for B. subtilis

This part associates the Bacillus subtilis Constitutive Promoter (P_veg) with the lysostaphin gene. Lysostaphin is a bacterial biocide isolated from Staphylococcus simulans and which speciﬁcally cleaves the pentaglycine cross bridges found in the staphylococcal peptidoglycan. BBa_K802000 contains the necessary RBS to work in Bacillus. With this part, Bacillus subtilis strains cause the lysis of Staphylococcus aureus cells.

Characterization

The following results show that BBa_K802000 enables B. subtilis 168 strains to kill the S. aureus and in a lesser extent S. epidermidis cells.

In our plasmid collection, this part is named pBK23 in the pSB1C3 backbone (Cm^R) and pBKL28 in the shuttle vector E. coli – B. subtilis. The pBKL28 plasmid was transformed in the NM522 strain to perform tests in E. coli and in the 168 strain to perform tests in Bacillus subtilis. The empty shuttle vector (pBKL25 in our collection) was used as a negative control in both species of bacteria.

If you have any question on the following experiments, don’t forget that all the information relative to our strains, plasmids and protocols are on our wiki notebook.

Confocal Microscopy

Biofilms are formed by the S. aureus fluorescent strain RN4220 pALC2084 expressing GFP. It is a nonmotile laboratory strain, used to form biofilm in 96-well microscopic-grade microtiter plate.

Bacillus subtilis 168 transformed with pBK28 (lysostaphin in the shuttle vector) and by pBKL25 (the shuttle vector without any gene to have a negative control) were grown on LB medium supplemented with erythromycin (15µg/mL).
After 24 hours of culture at 30°C without shaking, biofilms were observed under a time-lapse confocal microscope.
Cells expressing GFP were excited at 488 nm with an argon laser, and fluorescent emission was collected on a detector in the range of 500-600 nm using an oil-immersion objective with a magnification of 63x. The overall three-dimensional structures of the biofilms were scanned from the solid surface to the interface with the growth medium, using a step of 1 µm.
The 3D constructions were obtained with IMARIS software.

Three cases are analysed :

Blank : it is a non treated S. aureus biofilm (just with growth medium).
Negative control : it is a S. aureus biofilm treated by B. subtilis containing the shuttle vector without the Lysostaphin gene.
Strain with our part : it is a S. aureus biofilm treated with B. subtilis containing the part BBa_K802000 in the shuttle vector.

S. aureus Blank : untreated S. aureus

S. aureus biofilm treated with the strain containing the shuttle vector without the lysostaphin gene (Negative control)

S.aureus biofilm treated by the strain with the part

Conclusion :
With these observations, we concluded that the part causes the death of a large part of the S. aureus cells which constitute the biofilm. There is also a good expression of the lysostaphin in the B. subtilis strain, thanks to this part.

Quantitative image analysis :

A quantitative analysis was performed with the MATLAB software. Different parameters^[1] were used to quantify the biofilm, particularly :

Total Biovolume (µm³) : it corresponds to the overall volume of the biofilm and also allows to have an estimation of the biomass in the biofilm.
Mean thickness (µm) : it corresponds to the spatial size of the biofilm.

The same three cases as previously are analysed.

Conclusion :
These statistical results demonstrate that the S. aureus biofilm treated with B. subtilis containing the part is considerably reduced. Indeed, the total biovolume of the biofilm and its thickness strongly decrease after the treatment.
Moreover, the lysostaphin causing lysis of cells, we don't need to wash the biofilm to see the results, unlike the dispersin action (refer to the results for the part BBa_K802001).

[1]Quantification of biofilm structures by the novel computer program COMSTAT. Heydorn A, Nielsen AT, Hentzer M, Sternberg C, Givskov M, Ersbøll BK, Molin S.Molecular Microbial Ecology Group, Department of Microbiology, Technical University of Denmark, DK-2800 Lyngby, Denmark.July 2000.

Quantification of biofilm thickness after lysostaphin treatment

In order to measure and to appreciate the Lysostaphin effect on Staphylococcus epidermidis biofilms, tests on microtiter plates, test tubes and lamella were leaded.

Each well was inoculated with a 5 mL Staphyloccocus epidermids culture grown for 24 hours in TSB and diluted 1:100 with TSB supplemented with 1% glucose. After incubation and crystal violet coloration, it can easily be seen that S. epidermidis biofilms adhere to well's surface and by measuring the D.O. at 600 nm ,the adherence is estimated to be around 75%.

After a 24-hour incubation of the biofilm with a Bacillus subtilis supernatant containing or not Lysostaphin (negative control) , the biofilm is detached. Indeed, S. epidermidis biofilms are very sensible to medium growth changes and the fact of not using TSB for supernatant tests, does not allow to conclude if the biofilms destruction is the result of the Lysostaphin action.

The same kind of tests were done in test tubes containing a lamella.

The test tube with the lamella in is inoculated with 5 mL culture of S. epidermidis diluted 1:100 with TSB supplemented with glucose 1%. After 24 h the supernatant is replaced with a B. subtilis supernatant containing or not the Lysostaphin (negative control). But, the results are similar to those done on microtiter plates biofilms is destroyed even in presence of the negative control.

In conclusion, S. epidermidis biofilms are not the best model to test the lysostaphine effect on biofilms. Other experiences have demonstrated better results were tests were done on S. aureus biofilms.

SDS-PAGE Protein gel

The SDS-PAGE was run in order to visualize and quantify the production of lysostaphin by Bacillus subtilis.

Bacillus subtilis 168 transformed by pBKL28 (lysostaphin in the shuttle vector) and by pBKL25 (empty shuttle vector as a negative control) were grown on LB medium supplemented with erythromycin (15µg/mL) overnight at 30ºC.

Cultures were centrifuged to separate the cells (pellet) from the supernatant. Cell pellets were resuspended in Cracking Buffer as described in the protocol. Proteins from the supernatants were analyzed on SDS-PAGE gel after addition of cracking buffer, either directly, or after concentration (4X) by using cold acetone (-20ºC).

Image description :

Well 1 : prestained protein ladder;
Well 2: Bacillus subtilis 168 transformed by pBKL28 (pellet);
Well 3: Bacillus subtilis 168 transformed by pBKL25 (pellet).

Result : The gel shows a band migrating around 27 kDa specifically in well 2. This result suggests that Bacillus subtilis 168 cells transformed by pBKL28 produces a low but detectable level of lysostaphin.
However, proteins in the supernatant were not concentrated enough, even after the acetone precipitation, to allow the detection of the lysostaphin by a SDS-PAGE gel without a step of purification (data not shown).

OD₆₀₀ Test

We have conducted OD₆₀₀ measurements to test lysostaphin effect on S. epidermidis. Our results are not detailled here because they did not bring new informations compared to confocal microscopy results.
Our first protocol was not correct because antibiotic doses were too high. Thus, the effect seen was due to antibiotic and not lysostaphin.
Our second protocol was established according to several publications (please refer to the protocol link) was better but any lysostaphin effect was observed even with our positive control.
After more reading we have found a possible explanation : higher concentrations of lysostaphin are required to kill S. epidermidis than for S. aureus. Our samples were maybe not enough concentrated to see the lysostaphin effect and maybe S. epidermidis also was not the most appropriate model to use.

Combined action between the parts BBa_K802000 and BBa_K802001

As we demontrated with the previous tests, the part BBa_K802000 has a real effect on the S. aureus biofilm. For our Biofilm Killer project, two complementary agents (lysostaphin with the part BBa_K802000 and dispersin with the part BBa_K802001) were used to destroy an installed biofilm. Thus, it was interesting for us to combine these two agents.
We have also made new tests on 96-well plates, according to the same protocole than the one used to characterize the lysostaphin part with the confocal microscope. The only difference was that we add 125µL of B. subtilis with the part BBa_K802000 and 125µL of B. subtilis with the part BBa_K802001.
Two cases are analysed :

Negative control : it is a S. aureus biofilm treated with B. subtilis strains containing the shuttle vectors without the Lysostaphin and Dispersin genes.
Strain with our parts : it is a S. aureus biofilm treated with B. subtilis strains containing the part BBa_K802000 and BBa_K802001 in their shuttle vectors.

S.aureus biofilm treated with the shuttle vectors without the lysostaphin and the dispersin genes (Negative control)

S. aureus biofilm treated with the strain containing parts BBa_K802000 and BBa_K802001

................... General conclusion ...................

Our results clearly show the dramatic increase in efficiency of Biofilm Killer, which expresses the lysostaphin and dispersin parts in combination, over the other Bacillus subtilis strains harboring only the lysostaphin or dispersin parts.

Usage and Biology

This part was designed to be used in a motile strain like Bacillus subtilis 168 or Bacillus thuringiensis in order to cause lysis of the Staphylococcus aureus cells. The motility of the strain makes its penetration inside the biofilm easier.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1316
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]