1. Function of the FtsH metalloprotease during stage 0 sporulation of Bacillus subtilis
Hue Bach Thi Nguyen (PhD student) and Anja Kittel (Diploma student)

When B. subtilis cells are running out of nutrients, different enetic programs are activated including synthesis and secretion of proteases, production of antimicrobial peptides to kill competitors, development of competence and, as an answer of last resort, development of endospores (Piggot and Hilbert, 2004;Hilbert and Piggot, 2004). Under laboratory conditions, production of endospores takes about 7 h, and the whole process has been divided into different morphological stages. The first stage, called stage 0, takes about 2 h and results in the production of activated Spo0A (Spo0A~P). Spo0A~P is a DNA-binding protein which recognizes an operator sequence called Spo0A-box and regulates a total of 121 genes either negatively or positively (Molle et al., 2003). Small amounts of Spo0A are already present in exponentially growing cells. When cells enter the transition phase defined as the phase where cells stop growth and enter the stationary phase, one or more of five different kinases (KinA through KinE) will register so far unknown starvation signals, autophosphorylate and transfer the phosphoryl group to Spo0F with further transfer to Spo0B and finally to Spo0A (Burbulys et al., 1991). Small amounts of Spo0A~P activate an autoregulatory loop resulting in enhanced amounts of Spo0A~P. On the average, about 60% of the cells are able to synthesize a sufficient amount of Spo0A~P allowing them to enter to next stage of sporulation, called stage II, while the remaining 40% fail to do so. Cells being able to produce a high amount of active Spo0A have been designated to be in the Spo0A-ON stage and the others in the Spo0A-OFF stage (Fujita et al., 2005). The reason for this bistability (a population of genetically identical cells consists of two subpopulations) is still unknown.

Seven different phosphatases have been described which either dephosphorylate Spo0F~P and Spo0A~P. Four out of seven Rap phosphatases specifically attack Spo0F~P termed RapA, RapB, RapE and RapH (Perego et al., 1996). Upon removal of the phosphoryl group from Spo0F~P, there is a flow back of phosphate from Spo0A~P via Spo0B to Spo0F thereby converting Spo0A from its active to its inactive state thus preventing cells from leaving phase 0. The four Rap phosphatases are under negative control by appropriate pentapeptides designated PhrA, PhrB, PhrE and PhrH. These pentapeptides are produced as larger precursors, secreted, processed and taken up again by the cells were they interact with their cognate Rap phosphatase to inhibit its activity (Perego et al., 1996). The remaining three phosphatases attack Spo0A~P; while YisI and YnzD are present during vegetative growth and assumed to dephosphorylate Spo0A~P if it becomes phosphorylated by chance during logarithmic growth (Perego, 2001), Spo0E is produced during the transition phase (Perego and Hoch, 1987). No modulators of the phosphatase activity of these three phosphatases have been described so far.

FtsH is a ATP- and Zn2+-dependent metalloprotease anchored in the cytoplasmic membrane using two transmembrane segments (Schumann, 1999). Several years ago, we constructed an ftsH knockout and could show that the mutant cells exhibit a pleiotropic phenotype (Deuerling et al., 1997). This includes filamentous growth, sensitivity towards a heat and an osmotic shock and a significantly reduced sporulation frequency. Recently, we could show that ftsH knockout cells fail to enter stage II due to the absence of Spo0A~P (Le and Schumann, 2009). We hypothesize that FtsH has to degrade and/or control the steady-state of one or more proteins influencing negatively synthesis or/and the phosphorylation state of Spo0A. So far, we have identified one target of FtsH, the Spo0E phosphatase. Furthermore, we could show that the recognition site for FtsH is located within the C-terminal 25 amino acid residues of Spo0E (Le and Schumann, 2009). Experiments are in progress to identify additional substrate proteins of FtsH.


Objectives:
1. To identify additional genes coding for proteins acting as negative regulators of the synthesis or/and phosphorylation of Spo0A using four different strategies
2. To understand the biological function of these genes in reducing the sporulation frequency
3. To identify the amino acid sequence present on the C-terminal end of Spo0E and recognized by the FtsH protease


Literature:
Burbulys,D., Trach,K.A., and Hoch,J.A. (1991) Initiation of sporulation in B. subtilis is controlled by a multicomponent phosphorelay. Cell 64: 545-552.

Deuerling,E., Mogk,A., Richter,C., Purucker,M., and Schumann,W. (1997) The ftsH gene of Bacillus subtilis is involved in major cellular processes such as sporulation, stress adaptation and secretion. Mol Microbiol 23: 921-933.

Fujita,M., Gonzalez-Pastor,J.E., and Losick,R. (2005) High- and low-threshold genes in the Spo0A regulon of Bacillus subtilis. J Bacteriol 187: 1357-1368.

Hilbert,D.W., and Piggot,P.J. (2004) Compartmentalization of gene expression during Bacillus subtilis spore formation. Microbiol Mol Biol Rev 68: 234-262.

Le,A.T.T., and Schumann,W. (2009) The Spo0E phosphatase of Bacillus subtilis is a substrate of the FtsH metalloprotease. Microbiol.

Molle,V., Fujita,M., Jensen,S.T., Eichenberger,P., Gonzalez-Pastor,J.E., Liu,J.S., and Losick,R. (2003) The Spo0A regulon of Bacillus subtilis. Mol Microbiol 50: 1683-1701.

Perego,M. (2001) A new family of aspartyl phosphate phosphatases targeting the sporulation transcription factor Spo0A of Bacillus subtilis. Mol Microbiol 42: 133-143.

Perego,M., Glaser,P., and Hoch,J.A. (1996) Aspartyl-phosphate phosphatases deactivate the response regulator components of the sporulation signal transduction system in Bacillus subtilis. Mol Microbiol 19: 1151-1157.

Perego,M., and Hoch,J.A. (1987) Isolation and sequence of the spo0E gene: its role in initiation of sporulation in Bacillus subtilis. Mol Microbiol 1: 125-132.

Piggot,P.J., and Hilbert,D.W. (2004) Sporulation of Bacillus subtilis. Curr Opin Microbiol 7: 579-586.

Schumann,W. (1999) FtsH - a single-chain charonin? FEMS Microbiol Rev 23: 1-11.


2. Use of whole cells and endospores of Bacillus subtilis for surface display of peptides and proteins
In collaboration with Prof. June-Hyung Kim from the Dong-A University in Busan, South-Korea
Quynh Anh Nguyen (PhD student) and Nadine Lückemeier (Diploma student)

Surface display is an important tool, to immobilize peptides or proteins on the surface of bacteriophages or cells of bacteria and yeast collectively called bioparticles (Lee et al., 2003). Less known are endospores produced by the genera Bacillus and Clostridium. We have developed an experimental system to anchor proteins covalently on the cell wall of B. subtilis cells (Nguyen and Schumann, 2006). Using an α-amylase as a reporter enzyme, we have been able to immobilize up the about 240,000 molecules per cell. Endospores of B. subtilis consist of different layers. The internal part, the spore core contains all the components of the cytoplasm in a largely dehydrated state. The spore core is surrounded by two membranes which sandwich peptidoglycan material. The outer layer of the spore called spore coat consists of at least 70 different proteins. Three of these proteins, CotB, CotC and CotG, have been shown to be exposed on the outside and have been used to display passenger proteins such as different antigens (Oggioni et al., 2003) and enzymes (Kim et al., 2007).


Objective:
1. To display different cellulases on the outside of B. subtilis cells and spores and to analyze their possibilities to degrade cellulose to glucose using different substrates
2. To isolate novel cellulases using the metagenomic approach (see under 5) and to anchor them on both bioparticles.
The final goal will be to convert cellulose from plant waste material such as straw into glucose which will serve as a starting compound for products such as ethanol and butanol.


Literature:
Kim,J.H., Roh,C., Lee,C.W., Kyung,D., Choi,S.K., Jung,H.C. et al. (2007) Bacterial surface display of GFP(uv) on Bacillus subtilis spores. J Microbiol Biotechnol 17: 677-680.

Lee,S.Y., Choi,J.H., and Xu,Z. (2003) Microbial cell-surface display. Trends Biotechnol 21: 45-52.

Nguyen,D.H., Phan,T.T.P., and Schumann,W. (2007) Expression vectors for the rapid purification of recombinant proteins in Bacillus subtilis. Current Microbiol 55: 89-93.

Oggioni,M.R., Ciabattini,A., Cuppone,A.M., and Pozzi,G. (2003) Bacillus spores for vaccine delivery. Vaccine 21 Suppl 2: S96-101.


3. High level intra- and extracellular production of recombinant proteins in Bacillus subtilis
Kelly Leite (PhD student) and Johannes Martini (Diploma student)

Three different pathways have been described involved in the translocation of proteins through the cytoplasmic membrane of bacteria: The Sec pathway, the Tat pathway and the SRP pathway (Brüser, 2007;Rapoport, 2007;Rapoport et al., 1996;Lee et al., 2006). All polypeptide chains translocated by these three different pathways carry a signal sequence at their N-terminus which is cleaved off by a signal peptidase after their translocation through the cytoplasmic membrane. The Sec pathway recognizes “normal” signal peptides, and the polypeptide chains have to be in an unfolded, translocation-competent state and are translocated post-translationally (are fully synthesized in the cytoplasm) (Rusch and Kendall, 2007). The Tat pathway recognizes signal peptides containing two consecutive arginine residues (twin arginines) and the proteins are also post-translationally translocated in their correctly folded form (Lee et al., 2006). The SRP pathway recognizes signal peptides enriched in hydrophobic amino acid residues when they emerge from the exit tunnel of the ribosomes, targets them to the SecYEG translocon where translocation occurs co-translationally (Shan and Walter, 2005).

We have created a strong promoter by optimizing the Pgrac promoter (Nguyen et al., 2007;Phan et al., 2006) leading to the production of about 30% recombinant protein intracellularly (unpublished). Furthermore, we created a 5' stabilizing element resulting in half-life of mRNAs to more than 60 min (unpublished). The next step will be to convert this strong intra- into an extracellular expression system based on the Sec- and the SRP pathway.


Objectives:
1. To identify a signal-sequence recognized specifically by the SRP
2. To proof translocation of a fast-folding model protein by the SRP pathway
3. To develop an SRP-dependent secretion system which translocates any fast-folding recombinant protein


Literature:
Brüser,T. (2007) The twin-arginine translocation system and its capability for protein secretion in biotechnological protein production. Appl Microbiol Biotechnol 76: 35-45.

Lee,P.A., Tullman-Ercek,D., and Georgiou,G. (2006) The bacterial twin-arginine translocation pathway. Annu Rev Microbiol 60: 373-395.

Nguyen,H.D., and Schumann,W. (2006) Establishment of an experimental system allowing immobilization of proteins on the surface of Bacillus subtilis cells. J Biotechnol 122: 473-482.

Phan,T.T.P., Nguyen,H.D., and Schumann,W. (2006) Novel plasmid-based expression vectors for intra- and extracellular production of recombinant proteins in Bacillus subtilis. Protein Expr Purif 46: 189-195.

Rapoport,T.A. (2007) Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes. Nature 450: 663-669.

Rapoport,T.A., Jungnickel,B., and Kutay,U. (1996) Protein transport across the eukaryotic endoplasmic reticulum and bacterial inner membranes. Annu Rev Biochem 65: 271-303.

Rusch,S.L., and Kendall,D.A. (2007) Interactions that drive Sec-dependent bacterial protein transport. Biochemistry 46: 9665-9673.

Shan,S.O., and Walter,P. (2005) Co-translational protein targeting by the signal recognition particle. FEBS Lett 579: 921-926.


4. Development of vaccines based on spores of Bacillus subtilis
In collaboration with Prof. Luis Carlos Ferreira from the University of São Paulo, Brazil
Ines Brandherm (Diploma student)

Spores of B. subtilis are extremely resistant against irradiation, high temperature and a wide variety of chemicals (Driks, 2002). This behaviour makes spores an ideal vaccine for animals since no cooling chain is needed and spores can be stored like a powder at room temperature for decades. If nutrients are added to spores, they germinate and form vegetative cells. Germination occurs after parenteral or oral application to animals. Our vaccination concept is based on the application of spores to animals and their germination after uptake by cells, e.g., by macrophages where they will be directed to phagosomes. The appropriate antigen will be produced by the vegetative cells under the control of a stress-inducible promoter. Stress factors are the acidic pH, the absence of oxygen and of glucose. Using the mouse model, we have shown that the animals acquire immunity against the model antigen LTB of E. coli when spores were given parenterally or orally (Paccez et al., 2006;Paccez et al., 2007). The final goal of this project is to develop spores which induce an immune response after a single dose when applied orally. Spores can be added to the drinking water or the food.


Objectives:
1. To enhance the uptake of spores by the M cells in the small intestine
2. To find conditions which either induce preferentially an antigen-specific cytotoxic T lymphocyte (CTL) response or the humoral (antibody) response in animals


Literature:
Driks,A. (2002) Proteins of the spore coat. In Bacillus subtilis and its closest relatives. Sonenshein,A.L., Hoch,J.A., and Losick,R. (eds). Washington, DC: American Society for Microbiolgy, pp. 527-536.

Paccez,J.D., Luiz,W.B., Sbrogio-Almeida,M.E., Ferreira,R.C., Schumann,W., and Ferreira,L.C. (2006) Stable episomal expression system under control of a stress inducible promoter enhances the immunogenicity of Bacillus subtilis as a vector for antigen delivery. Vaccine 24: 2935-2943.

Paccez,J.D., Nguyen,H.D., Luiz,W.B., Ferreira,R.C., Sbrogio-Almeida,M.E., Schumann,W., and Ferreira,L.C. (2007) Evaluation of different promoter sequences and antigen sorting signals on the immunogenicity of Bacillus subtilis vaccine vehicles. Vaccine 25: 4671-4680.


5. Identification of novel cellulases using the metagenomic approach
In collaboration with Prof. Spartaco Filho from the Federal University of Amazonas at Manaus/Brazil

The metagenome is defined as all the bacterial species present in a given habitat, e.g., in a soil sample, in a water sample or in our mouth (Handelsman, 2004). Analysis of all genes present in such a sample is termed metagenomics. The primary goal of metagenomics is to identify and to isolate genes of interest from the bacterial genomes present in such a sample. Metagenomics has become an important technique since about 99% of the bacterial species present in their natural habitat cannot be cultivated in the laboratory (Staley and Konopka, 1985); they have been called the unculturable species. Two different experimental approaches have been developed to identify and to isolate genes of interest: (1) the sequence-driven approach and (2) the function-driven approach. In the sequence-driven approach, new members of a group of known genes coding for, e.g., cellulases, lipases and α-amylases are being identified using either primer pairs derived from conserved regions of these genes or hybridization probes. In the function-driven approach, the genes have to be expressed and methods have to be available to identify their proteins by their activity. For example, cellulases can be identified by plating bacteria on LB plates containing soluble cellulose. Bacterial colonies producing cellulases are surrounded by a halo.

Our objective is to identify novel cellulases using soil samples from Vietnam and the Amazon region using the function-driven approach. We make use of the BAC vector pMBD14 (Martinez et al., 2004). This vector plasmid can be transferred by conjugation from E. coli to Streptomyces lividans and Pseudomonas putida where they will integrate into their chromosome. We have extended the host range of this BAC vector by insertion of the internal part of the amyE gene of B. subtilis to allow its transfer to this species as well with subsequent integration at the lacA locus. In the next step, we will establish metagenomic libraries in E. coli and check them for cellulase activity in all four species.


Objectives:
1. To establish an efficient conjugation system to transfer recombinant BAC plasmids from E. coli to other eubacterial species
2. To establish the methodology to extract bacterial DNA from soil samples
3. To identify and characterize novel cellulases from soil samples


Literature:
Handelsman,J. (2004) Metagenomics: application of genomics to uncultured microorganisms. Microbiol Mol Biol Rev 68: 669-685.

Martinez,A., Kolvek,S.J., Yip,C.L.T., Hopke,J., Brown,K.A., MacNeil,I.A., and Osburne,M.S. (2004) Genetically modified bacterial strains and novel bacterial artificial chromosome shuttle vectors for constructing environmental libraries and detecting heterologous natural products in multiple expression hosts. Appl Environ Microbiol 70: 2452-2463.

Staley,J.T., and Konopka,A. (1985) Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu Rev Microbiol 39: 321-346.