University of Zürich, Institute of Medical Microbiology, Gloriastr. 32, CH-8028 Zürich, Switzerland
Correspondence
Brigitte Berger-Bächi
bberger{at}immv.unizh.ch
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ABSTRACT |
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INTRODUCTION |
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With the development of bacterial two-hybrid (BTH) systems (Dove et al., 1997; Karimova et al., 1998
), bacterial proteins can now be assayed for interaction under conditions that match their native environment more closely. In the system developed by Karimova et al. (1998)
(Fig. 1
), the interaction of two proteins fused to Bordetella pertussis adenylate cyclase domains leads to its functional reconstitution and activation of subordinate metabolic pathways, allowing growth on minimal media or assaying for colour formation on MacConkey agar. This BTH system circumvents some of the limitations of the conventional YTH system, in which proteins localized in membranes and transcription factors, for example, are not amenable for analysis. In addition, the spatial separation of the interaction event and the signal readout reduces problems arising from non-specific interactions (Karimova et al., 1998
).
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We show here that homodimerization occurs for FemA and FemB, but not for FmhB, and that an interaction also occurs between FemA and FemB. The findings of the BTH screen were confirmed using two independent methods, namely, analytical gel filtration and the glutathione S-transferase (GST)-pulldown assay.
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METHODS |
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Production of recombinant protein.
His6-tagged protein was expressed in E. coli BL21(DE3) cells at 30 °C induced with 0·5 mM IPTG. Cells were lysed in 1/25 of the original culture volume of lysis buffer (50 mM sodium phosphate pH 8·0/150 mM NaCl/10 mM imidazole/1 mg lysozyme ml-1/0·1 mg DNase ml-1/0·01 mg RNase ml-1/0·1 mM PMSF) on ice for 30 min. The lysates were then sonicated and centrifuged at 20 000 g for 20 min.
Cleared lysates were incubated with 1/20 volume of Ni-NTAagarose (Qiagen) for 1 h, on an overhead shaker, at 4 °C. The resin was washed with 1015 volumes of wash buffer (50 mM sodium phosphate pH 8·0/150 mM NaCl/10 mM imidazole). Protein was eluted with 34 column volumes of elution buffer (50 mM sodium phosphate pH 8·0/100 mM NaCl/200 mM imidazole). The eluates were centrifuged in Centricon centrifugal filter devices (Millipore) with a cut-off of 10 kDa to exchange the buffer for PBS (8·5 g NaCl l-1, 10·7 g Na2HPO4.2H2O l-1, 0·9 g KH2PO4 l-1, pH 7·4). The recombinant protein was more than 95 % pure on SDS-PAGE and Coomassie staining (not shown).
GST fusions of FemA and FemB were purified similarly. The fusion proteins were expressed in E. coli BL21(DE3) cells with 0·05 mM IPTG at 17 °C for 1 h. Cells were lysed in 1/25 of the original culture volume of lysis buffer as above, with addition of 1 % (v/v) Triton X-100, omitting imidazole. Cleared lysates were bound to 1/40 to 1/20 volume of glutathioneagarose (Sigma) at 4 °C for 1 h, on an overhead shaker, washed with 20 volumes of PBS and eluted with 50 mM Tris/HCl pH 8/150 mM NaCl/5 mM GSH. The protein was more than 95 % pure on SDS-PAGE and Coomassie staining (not shown).
Analytical gel filtration.
Protein oligomerization was observed by analytical gel filtration using FPLC on a Superdex 200 HR column on an Äkta FPLC system (Pharmacia). Proteins were run in PBS. In addition to recombinant His6-tagged proteins, a set of protein standards were run (HMW and LMW protein standards; Pharmacia). Peak data of the standards were used to perform linear regression analysis, in order to obtain a standard curve for molecular mass determination of the analysed proteins.
GST-pulldown assay.
Cleared lysates of GSTFemA or GSTFemB fusions, or GST alone, were incubated with equal volumes of GSHagarose beads for 1 h at 4 °C. The bead material was washed with 20 volumes of PBS. Non-specific protein binding was blocked by incubating the bead material for 1 h with BSA (1 mg ml-1) in PBS at 4 °C. The material was again washed with 20 volumes of PBS. His6-tagged FemA or FemB protein was added in excess and incubated with the bead-bound proteins for 2 h at 4 °C. Unbound protein was removed by washing three times with 10 volumes of PBS, and the bead samples with bound protein were suspended in equal volumes of SDS sample buffer.
Western blotting.
SDS-PAGE and Western blotting were done following standard procedures (Ausubel et al., 1997). Western blots were incubated with monoclonal anti-His antibody (Sigma) at 1 : 1000 dilution in PBS containing 0·03 % (w/v) Top Block (Juro) overnight at 4 °C, followed by secondary antibody (Sheep anti-mouse FabHRP; Jackson Immuno Research) at 1 : 10 000 dilution for 2 h at room temperature (22 °C). Detection was done with SuperSignal West Femto reagent (Pierce).
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RESULTS |
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Analytical gel filtration
His6-tagged protein was run on a Superdex 200 HR size exclusion column to determine protein oligomerization. A standard curve was derived by running protein standards under the same conditions and performing linear regression analysis on the peak data. From this standard curve, the peak sizes of the recombinant proteins could be determined. For the monomers of FmhB, FemA and FemB, the measured molecular masses correlated well with the theoretical values derived from the amino acid sequences, and it was determined that FemA and FemB formed dimers (Fig. 3a, b; Table 3
).
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DISCUSSION |
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Additional BTH experiments were done using other members of the FemABX family. The lysostaphin immunity factor Lif protects Staphylococcus simulans biovar staphylolyticus from the action of lysostaphin and has been shown to substitute serine in positions 3 and 5 of the pentaglycine side chain in S. aureus, in cooperation with FemA and FemB (Ehlert et al., 2000; Thumm & Götz, 1997
). We observed interactions between Lif and both FemA and FemB, but not with FmhB, nor Lif homodimerization (data not shown). Streptococcus pneumoniae FibB (MurN) adds alanine or serine to the stem peptide in cooperation with FibA (MurM) to form an Ala2 or AlaSer side chain (Filipe & Tomasz, 2000
; Weber et al., 2000
). FibB proved to be able to form homodimers, but not heterodimers with FibA, and FibA homodimerization was also not observed (data not shown). To bring these additional findings into perspective, the sequences of known FemABX family proteins were compared (Fig. 4
). It appears that all proteins able to form dimers group in one of two main clades of the family. The non-interactors are grouped in the other, evolutionarily older, branch comprising those FemABX family members that add the first amino acid in an interpeptide to the peptidoglycan, and/or are the only family member in the respective species. This may indicate that a gain of function occurred during the diversification of the FemABX protein family. However, this hypothesis would require further experimental testing. Analysis of proteinprotein interactions within the FemABX protein family may yield evidence concerning their function. While there must be a mechanism that determines whether one or two amino acids are attached by a FemABX protein, the data presented here do not imply that interaction or non-interaction contribute to this mechanism.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 21 February 2003;
revised 2 May 2003;
accepted 5 May 2003.