Stay Warm, Stay Close

Our initial idea was to let the bacteria produce the silk in a bath and when the implant is put into the bath, the implant will be coated with silk. This is a wasteful and inelegant method. A low production yield can be expected and a solution is needed to overcome this problem. Therefore the heat motility was developed. With the help of heat motility the silk will be produced on site, this will also save energy in the form of nutrition and energy of heating the bath. An overview of our 'Coating GEM' is shown in figure 1.

Native Motility

The Motility in most bacteria is governed by the Che proteins [1], they control if the bacteria is swimming straight or tumbling (changing directions). They do this by controlling the flagella. If the flagella spin counter-clockwise (CCW) they will group together in one pole of the bacterium, causing straight swimming. On the other hand if the flagella are spinning clockwise (CW) then the flagella will disperse over the membrane and cause tumbling.

CheY

The Che proteins are present in many motile bacteria, however they can have different effects depending on the species [1]. The CheY protein in Bacillus subtilis, for example, has the complete opposite effect as in Escherichia coli. CheY is an important protein for controlling the spinning of the flagella. When the concentration of phosphorylated CheY (CheY-p) is sufficiently high the flagella turn CCW (straight swimming), but when the concentration of CheY decreases the chance of tumbling also increases, and the bacterium will reorient themselves more often.

Attractant Receptor

The chemotaxis process is initiated at the receptor, which can sense the concentration of an attractant (or repellent) [2]. Increasing concentrations of attractant correspond to an increased chance of swimming. If the concentration decreases, the bacteria will start to tumble more frequently, and will reorient its swimming direction, hopefully to more desirable regions.

cheA & CheC-CheD

Binding of the attractant receptor causes straight swimming via a small cascade [2]. When the receptor is bound, the CheA protein (which is attached to it) gets phosphorylated. The CheA-p phophorylates cheY, which then causes straight swimming. The protein complex CheC-CheD causes dephophorylation of cheY-p (when receptor is bound) resulting in a negative feedback. Two more negative feedback systems are also activated following the binding of attractant, which also result in decreased values of CheY-p. In such a way, CheY-p adaption occurs, and B. subtilis is ready to sense new changes in its environment. For more information about how this pathway works please visit the heat motility section.

Controllable motility

cheY Knockout

To make our coating mechanism a success we need to have control over the motility. This is achieved by knocking out cheY. This might sound strange since CheY-p is controlling straight swimming. However since we know that a CheY null mutant is immobile due to excessive tumbling. This makes it possible to insert cheY with a promoter of our choosing, and make it the sole producer of CheY. Also a cheC knockout is made in order to prevent negative feedback. How this is effecting the cell is explained in more detail with our model.

DesK pathway

We envision a bacterium that moves towards a heat source, in order to do this it needs a temperature sensor. B. subtilis natively has a protein that fits this requirement nicely. It is called DesK, it is a membrane protein that senses cold [3][4] (25°C). When the environment is cold, DesK autophophorylates, after which it phosphorylates DesR. DesR in turn activates the promoter of the des gene (P_des), which would be the promoter we are looking for. The des gene expresses a protein that provides negative feedback to the system, so we need to knockout this des gene to keep our promoter active.

Thermal control of fatty acid synthesis.

In order to maintain the fluidity of the cell membrane when the environmental temperature is decreasing, B. subtilis (among other bacteria) adapts the membrane by increasing the fraction of unsaturated phospholipids acyl chains.

The desaturation of the membrane starts with the membrane protein, DesK. DesK senses temperature of its environment and when the temperature is 30°C, DesK autophosphorylates its conserved histidine. Sequentially the phosphoryl group is transferred to the aspartate residue in desR that activates the promoter of des. The gene des is translated into a fatty acid desaturase (Δ5-Des), that changes the fluidity of the membrane by introducing double bonds into pre-existing saturated fatty acyl chains.

The promoter activity of des

Strain	Description
JH642	trpC2 pheA1
AKP3	JH642 amyE::[Pdes(-2269 to +31)lacZ]
AKP4	AKP3 des::kan
AKP21	AKP3 desKR::kan

Bredeston et al. 2011

The coating

The silk proteins are secreted with a Strep tag. The implant will be covered with biotin (vitamine B8). The Strep tag will automatically form a Van der Waals binding with the biotin and thus, if the silk is secreted closely the implant will be coated automatically. B.subtilis has a tendency to form a biofilm on a structure, or in our case an implant (insert proof that biofilm can grow in plastic/titanium) . To coat the implant we have thought of to option 1 via secretion and 2 via a biofilm formation. The secretion process is the most elegant and best option, but secretion of large proteins is proven to be difficult and has never been done before with silk. So a ‘backup’ plan was needed.

Animation

Motility of the knockout strains

To observe whether or not the mutant strains are less motile than the wild type strain different tests are done. One of the tests is performed using microscopy.

Microscope movies

Microscope movies (4x real time) are made for the wildtype, the ΔcheY and the ΔcheYΔdes strain. These movies show that the wildtype strain (Movie 1) is more motile than both mutant strains. The comparison between the movie of the ΔcheY (Movie 2) and ΔcheYΔdes (Movie 3) strain shows just as seen in the motility assay, that the ΔcheYΔdes strain is less motile than the ΔcheY strain.


Movie 1: Motility of the wild type strain


Movie 2: Motility of ΔcheY


Movie 3: Motility of ΔcheYΔdes

References

[1] Liam F. Garrity and George W. Ordal, Chemotaxis in Bacillus subtilis: How bacteria monitor environmental signals, Pharmacology and Therepeutics (1995), Vol. 68 No.1, pp. 87-104.
[2] Christopher V. Rao, George D. Glekas and George W. Ordal, The three adaptation systems of Bacillus subtilis chemotaxis, Trends in Biology (2008), Vol. 16 No 10, pp. 480-487.
[3] Mariana Martin and Diego de Mendoza, Regulation of Bacillus subtilis DesK thermosensor by lipids, Biochemical Journal (2013), Vol 451 No 2, pp. 269–275.
[4] Thermal Regulation of Membrane Lipid Fluidity by a Two-Component System in Bacillus subtilis, January 31 2011, BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Vol. 39, No. 5, pp. 362–366, 2011.