TÉCNICAS E INSTRUMENTOS DE RECOLECCIÓN DE DATOS

regulator that binds in its phosphorylated form to the motor switch factor FliM. Upon this event, the rotational bias of the flagellar motor is changed towards a higher counterclockwise to clockwise switching probability. The ability of CheY-phosphate to induce clockwise flagellar rotation is about 100 times the corresponding activity of unphosphorylated CheY (BARAK &

EISENBACH, 1992). In other bacterial species with more than one CheY-like

response regulator it is questionable which of these proteins is the actual switch factor. In this study, it was therefore attempted to examine the differences in CheY and CheV protein behavior beyond the findings that the two proteins are differentially phosphorylated by the histidine kinase. Binding to FliM of either of the proteins would further the understanding of the role of these proteins in chemotaxis.

It was first attempted to reproduce the results from BREN & EISENBACH,

1998, as a control reaction for possible interaction studies with H. pylori derived proteins. The genes coding for E. coli FliM and CheY were cloned into pET28a(+) expression vectors and expressed in E. coli BL21(DE3)Gold cells as N-terminal His6-tagged fusion proteins. CheY was

expressed in the cytosol, whereas FliM was refolded and purified following the protocol given by BREN & EISENBACH, 1998. Fig. 41 shows a typical

Fig. 41. Purification of E.coli FliM and CheY as described in Materials & Methods. A.

Purification of FliM refolded from inclusion bodies. Eluent fractions from a 2.5x10 cm Ni-NTA column. Fraction size: 5 ml, gradient: 0 to 250 mM imidazole in 300 mM sodium chloride, 10 mM Tris HCl pH 8.0 at 4°C, 10 % glycerol in 300 min at a flow rate of 1 ml/min. Lane indicated by an asterisk: flow-through of column during sample application. B. Purification of CheY from E. coli cytosol performed as described under A except that no glycerol was included in the buffer. Fraction numbers are above the respective lanes, and the probe volume was 15 µl of sample per each lane.

The pronounced tendency to precipitate made it difficult to handle the purified FliM. The protein could not be stored either frozen or on ice, and it was necessary to prepare it fresh from inclusion bodies stored at –32°C. Furthermore, the concentration of FliM solutions was below 5 mg/ml, which made it difficult to use FliM in the CheY interaction assays. In spite of the purification of FliM being a difficult task, it was possible to show that E. coli CheY-Pi interacts with the motor switch factor (Fig. 42).

Acetyl-Pi - + + - - +

FliM - - + + + +

CheY + + + + - -

Fig. 42. Interaction of E. coli CheY-Pi with the E. coli motor switch protein FliM. After

crosslinking, the proteins were separated by SDS-PAGE and silver-stained. All reactions contained 5 mM magnesium chloride and 1 mM ortho-phthaldialdehyde as crosslinking agent. Protein concentrations were CheY: 25 µM, FliM: 7.9 µM, and acetyl phosphate was 22 mM. Lane 1: marker protein. The faint bands in lanes 2 and 3 running at around 43 kDa and above are CheY trimers and higher crosslinking products. Similarly, FliM oligomers can be detected in reactions where FliM was present.

Only in the presence of acetyl phosphate (22 mM), CheY and FliM (25 and 7.9 µM, respectively), a protein band running at an appropriate molecular weight corresponding to crosslinked CheY-FliM heterodimers (51.9 kDa) appeared clearly visible before a background of CheY and FliM oligomers as was first shown by BREN & EISENBACH, 1998. In analogy to the

experiments described above, it was attempted to heterologously overexpress H. pylori FliM in E. coli. Whereas the E. coli protein could easily be expressed as inclusion bodies, it was not possible to express H. pylori FliM. In the hope that H. pylori CheY might bind to the E. coli FliM protein that is homologous to the H. pylori ortholog, the crosslinking assay was performed with these two proteins, but no distinct band corresponding to a FliM-CheY dimer appeared (Fig. 43.)

Fig. 43. Interaction assay with H. pylori CheY-Pi with the E. coli motor switch protein

FliM. After crosslinking, the proteins were separated by SDS-PAGE and silver-stained. All reactions contained 5 mM magnesium chloride and 1 mM ortho-phthaldialdehyde as crosslinking agent. Lane 1: CheY, lane 2: FliM, lane 3: CheY and FliM, Lane M: marker proteins. Protein concentrations were CheY: 40 µM, FliM: 5 µM; and acetyl phosphate was 22 mM.

The amino acid sequence alignment of FliM from E. coli, S. enterica serovar Typhimurium and H. pylori reveals that despite the high homology of the three proteins, several residues conserved in the enteric proteins are different in H. pylori FliM. Fig. 44 gives an overview of the N-terminal sequence similarity of the two proteins from two enteric bacteria, E. coli and S. enterica serovar Typhimurium, respectively, and H. pylori FliM.

Fig. 44. Protein sequence alignment of the N-terminal 60 amino acids of FliM from two enteric bacteria and H. pylori. In E. coli and S. enterica serovar Typhimurium, CheY-Pi

binds to amino acids one to 16. The proteins show high sequence homologies but the differences might be sufficiently different to prevent E. coli CheY-Pi to bind to H. pylori

Some of these residues not present in E. coli FliM might be important for H. pylori CheY-phosphate binding to FliM. In the enteric chemotaxis system, the activated response regulator only binds to the 16 N-terminal amino acids of its target, and CheY-phosphate even binds to this region in the absence of the rest of FliM (LEE et al., 2001b). It is conceivable that in

H. pylori, binding to FliM occurs in a similarly defined region of the protein. To examine this, a chimeric FliM protein was constructed where the N- terminal 42 amino acids of H. pylori FliM were fused to the E. coli FliM C- terminal part (see Materials & Methods). This protein was expressed in E. coli and could successfully be purified from the cells following the method for wild type FliM (Fig. 45).

Fig. 45. Purification of chimeric FliM. A. FliM refolded from purified inclusion bodies. Lane 1: 10 µl sample, lane 2: 20 µl sample, lane 3: 30 µl sample. B. Eluent fractions from a 2.5x10 cm Ni-NTA column. Fraction size: 5 ml, gradient: 0 to 250 mM imidazole in 300 mM sodium chloride, 10 mM Tris HCl pH 8.0 at 4°C, 10 % glycerol in 300 min at a flow rate of 1 ml/min. Fraction numbers are indicated above the respective lanes. Probe volume was 15 µl of sample per lane.

When the crosslinking experiments were performed with the chimeric FliM protein, again no band corresponding to response regulator-FliM dimers appeared. The same holds true for experiments with the CheV2 protein

that were performed despite the knowledge that this protein autophosphorylates only weakly in the presence of acetyl phosphate.