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From 2010.igem.org

Expression

Introduction

To form a biofilm with hydrophobic surface properties, expression of the hydrophobic proteins(in our case chaplins) should coincide with the late stages of biofilm formation. There are several reasons for this: mainly because not all bacteria participate in biofilm formation, there is significant cell differentiation prior to the formation of a biofilm. Also the expression of chaplins should not interfere with the growth speed and biofilm formation. The latter could be problematic due to the hydrophobic properties of the chaplins which might interfere with the formation of the extracellular matrix(ECM). We found that the ComXPA quorum sensing system and related gene expressions might suitable for this purpose.

ComXPA quorum sensing system

Quorum sensing in bacteria means that bacteria ‘talk’ to each other via signaling molecules. These molecules accumulate extracellularly as cultures grow to high cell densities. The response to these signaling molecules can result in dramatic changes in gene expression. In Bacillus subtilis a quorum sensing pathway named ComX-ComP-ComA is present which regulates the development of genetic competence. The ComX pheromone is a 10-amino-acid peptide which is secreted.

When ComX accumulates extracellularly, it stimulates the activity of a membrane bound receptor histidine kinase called ComP. ComP then autophosphorylates and activates the response regulator ComA by donating a phosphate molecule. The activated ComA functions as a transcriptional activator and activates the expression of 20 genes directly and the expression of another 150 genes is affected indirectly. The affected genes seem to have three different functions: the coordination of physiological changes involved in developmental pathways, the production of extracellular products and the enhancement of survival, growth and colonization. ComA directly activates the srfA operon which encodes enzymes needed for the production of the lipopeptide surfactin. The binding site for the activated ComA is known, the consensus sequence is a 12 bp palingdrome with a 4-5 bp spacer (TTGCGGNNNNCCGCAA). A DNA microarray study determined which genes were mostly activated by the active ComA (Comella et. al., 2005) . For our project we chose to link our chaplins to the promoter of the srfA gene which was most activated, which was srfAA.

Surfactin and biofilm formation

Recently, it has been found that not all bacteria respond to the signaling molecules in the same way. It was shown that biofilm formation in Bacillus subtilis involves paracrine signaling (Lopez et al., 2009). Although most cells produce and secrete ComX, only a subpopulation of the cells is triggered to make surfactin. Surfactin serves as signaling molecule and the cells which are not able to make surfactin, respond to surfactin by making extracellular matrix components for the biofilm.

It is believed that the extracellular matrix interferes with the interaction between ComX and ComP and therefore prevents surfactin production in extracellular matrix producing cells. In a mutant which did not express extracellular matrix proteins, the surfactin expression was more than three times higher.

During our project we would like to explore the possibilities of expressing the chaplins directly as a extracellular matrix protein. This could be done by using the yqxM promotor, responsible for TasA expression, in both a TasA (null mutant) and a wild type strain.

The model

The activation of ComP-P expression by ComX (X) is modelled with a Hill’s equation. The degradation of the formed mRNA is taken into account. The equation describes the formation of ComP-P mRNA (Mp) in time.

After the formation of mRNA, the protein ComP-P (Pp) will be formed which is modelled with the following equation:

The next reaction within the metabolism is the phosphorylation of ComA by ComP-P. The formation of ComA-P (Ap) is described by the following equation:

After phosphorylation, ComA-P is able to activate the expression of the gene that is behind the srfA promoter. This will be one of the chaplins in our case or GFP to test the system. Again, the Hill’s equation is used to model this process combined with the degradation of mRNA and the formation of the protein. The first equation shows the formation of mRNA of chaplins (Mc), the second equation describes the formation of the chaplin (Pc)

Below a table is shown with all the values of the used constants.

The figure shows the formation of ComP-P, ComA-P and chaplins in time.

References

Comella N, Grossman AD., Conservation of genes and processes controlled by the quorum response in bacteria: characterization of genes controlled by the quorum-sensing transcription factor ComA in Bacillus subtilis, Molecular Microbiology (2005) 57 (4), 1159–1174. http://www.ncbi.nlm.nih.gov/pubmed/16091051

Roggiani M, Dubnau D., ComA, a phosphorylated response regulator protein of Bacillus subtilis, binds to the promoter region of srfA, Journal of Bacteriology 1993 175(10):3182-7. http://www.ncbi.nlm.nih.gov/pubmed/8387999

López D, Vlamakis H, Losick R, Kolter R., Paracrine signaling in a bacterium, Genes & Development, 2009 23(14):1631-8. http://www.ncbi.nlm.nih.gov/pubmed

Shah IM, Dworkin J., Microbial interactions: bacteria talk to (some of) their neighbors, Current Biology, 2009 19(16): 689-91. http://www.ncbi.nlm.nih.gov/pubmed/19706277