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Our Project

The aim of our project is to genetically engineer Bacillus subtilis to be able to detect and sense cadmium which has been taken up from the soil environment and sequester them into a metallothionein. This metallothionein will then become incorporated into a Bacillus spore; the resilience of which means that the cadmium ions can become isolated from the environment (and made bio-unavailable) for many years.

This project involves a number of steps, each of which can be considered as sub projects:

1. Cadmium Intake
2. Cadmium Sensing
3. Tuning of Bacillus subtilis normal stochastic switch
4. Cadmium sequestration by metallothionein
5. Second Stochastic Switch
6. Synthesizing a Promoter Library for Bacillus subtilis

Cadmium Intake

For this project we want to be able to process cadmium and cadmium only. Therefore it would be logical to find a way in which we can increase the intake of cadmium without increasing the intake of other metals too.

It is known that a Bacillus subtilis cell (from the 168 strain) takes up cadmium naturally through the manganese transport system (Laddaga. R.A., Bessen. R., Silver. S.; 1985). Additionally it has been demonstrated that mutations to the manganese transport system can affect its ability to uptake cadmium without affecting its ability to transport manganese (Zeigler. D.R., et al; 1987).

The manganese ion channel that we intend to either upregulate or control is the mntH ion channel (also goes under the name ydaR). mntH is part of the Nramp family of proton-coupled, metal ion transporters (Que. Q., Helmann. J.D.; 2000). It is also classified as a secondary transporter (Membrane Transport website).

It is also known that mntH is regulated negatively by increasing manganese ion concentrations. This is allowed to happen through the promoter called mntR (Que. Q., Helmann. J.D.; 2000). This means that B.subtilis has the ability to limit the manganese metal intake system when the intracellular concentrations of Mn+ starts to near cytotoxic levels.

Cadmium Sensing

If our project is to process cadmium and not other metals, we need to genetically engineer Bacillus subtilis to carry out a set of cellular processes based on the action of metal sensors. These metal sensors will detect cadmium through a system known as AND Gating.

There are two metal sensing repressors, which are known to respond to cadmium: arsR and czrA (also known as yozA).

i) arsR

arsR (also known as yqcJ) is a protein which is part of the ArsR-SmtB family of transcriptional regulators. It is a regulating protein for the arsenic resistance operon in Bacillus subtilis (NCBI website – arsR family transcription regulator profile) (Moore. C.M and Helmann. J.D; 2005).

The arsR protein acts as a repressor until it is conformationally changed by the presence of arsenic ions (Harvie. D.R, et al; 2006). However, arsenic is not the only metal which can cause this action to happen; it has been noted that silver (Ag(I)), cadmium (Cd) and copper (Cu) (Moore. C.M and Helmann. J.D; 2005) can cause this action to happen.

Metal Sensor	Metals Sensed
arsR	As(III)	Ag(I)	Cu	Cd

ii) czrA (yozA)

czrA (also known as yozA) is a member of the ArsR-SmtB family of transcriptional regulators (Moore. C.M and Helmann. J.D; 2005)(Harvie. D.R, et al; 2006). Like arsR, it is a regulator protein which can be relieved from binding to the DNA by being bound to by metal ions – these include zinc (Zn), cobalt (Co), nickel (Ni) and cadmium (Cd) (Moore. C.M and Helmann. J.D; 2005). This can be summarised in the table below:

Metal Sensor	Metals Sensed
czrA	Zn	Co	Ni	Cd

AND Gating

If the two ion selectivity tables are put together, it can be seen that the metal common to both sensors is cadmium:

Metal Sensor	Metals Sensed
arsR	As(III)	Ag(I)	Cu	Cd
czrA	Zn	Co	Ni	Cd

This means that if the two repressors can act on a single promoter or binding site by AND gating, cadmium can be detected and a biological response can be triggered.

The presence of cadmium ions releases both arsR and czrA binding proteins from the DNA allowing transcription to occur.

Tuning of Bacillus subtilis normal stochastic switch

Cadmium sequestration by metallothionein

So the cadmium has made its way into the cell; and the cadmium has been detected by the metal sensors arsR and czrA. The question is: what happens to the cadmium now?

In our project, we hope to soak up the intracellular cadmium with a metallothionein known as smtA. Metallothioneins are proteins which have great tendencies to bind to cationic metal ions; examples of which include copper (Cu), zinc (Zn), lead (Pb) and cadmium (Cd) (Creti. P., et al; 2009). This property is due to the richness of cysteine residues in its structure (Creti. P., et al; 2009).

In the cell, metallothioneins generally have two important roles: to remove non-essential metals via sequestration and to control levels of essential metals (Creti. P., et al; 2009). It is the first role with which we are concentrating on.

smtA encodes a metallothionein of the same name – this protein, which is described as a class II metallothionein seems to be synthesized in response to metals such as zinc and cadmium (Morby. A.P., et al; 1993). This suggests it has a role in cadmium sequestration.

We intend to coat our Bacillus spores in the metallothionein by making a fusion protein with cotC a major spore coat protein, our metal sponge should locate to the spore making the cadmium bio-unavailable.

Second stochastic switch

Our second stochastic switch will use an invertible promoter sequence to regulate expression of either germination genes or genes required for our spores to be metal containers, such as smtA the metallothionein. We will carry out stochastic modelling of the system in order to fine tune it to produce a 'biased heads or tails' switch.

Promoter Library

Some of the promoters we intend to use for our BioBricks will require different strengths. Part of our project will involve making a library of Sigma A promoters for Bacillus subtilis and measuring their varying strengths; with this data we will use a sigma A promoter variant for a BioBrick according to the desired strength. As stated in the instructors meeting on the 22/04/09, we could measure the expression of fluorescent protein.

References

Membrane Transport website (http://www.membranetransport.org/all_type_btab.php?oOID=bsub1 )

Charles, M. M., G. Ahmed, et al. (2005). "Genetic and physiological responses of Bacillus subtilis to metal ion stress." Molecular Microbiology 57(1): 27-40.

Cretì, P., F. Trinchella, et al. "Heavy metal bioaccumulation and metallothionein content in tissues of the sea bream Sparus aurata from three different fish farming systems." Environmental Monitoring and Assessment.

Duncan, R. H., A. Claudia, et al. (2006). "Predicting metals sensed by ArsR-SmtB repressors: allosteric interference by a non-effector metal." Molecular Microbiology 59(4): 1341-1356.

Laddaga, R. A., R. Bessen, et al. (1985). "Cadmium-resistant mutant of Bacillus subtilis 168 with reduced cadmium transport." J. Bacteriol. 162(3): 1106-1110.

Moore, C. M. and J. D. Helmann (2005). "Metal ion homeostasis in Bacillus subtilis." Current Opinion in Microbiology 8(2): 188-195.

Morby, A. P., J. S. Turner, et al. (1993). SmtB is a metal-dependent repressor of the cyanobacterial metallothionein gene smtA: identification of a Zn inhibited DNA-protein complex. 21: 921-925.

Qiang, Q. and D. H. John (2000). "Manganese homeostasis in Bacillus subtilis is regulated by MntR, a bifunctional regulator related to the diphtheria toxin repressor family of proteins." Molecular Microbiology 35(6): 1454-1468.

Zeigler, D. R., B. E. Burke, et al. (1987). "Genetic mapping of cadmium resistance mutations inBacillus subtilis." Current Microbiology 16(3): 163-165.