Bacterial type III secretion systems serve to translocate proteins into eukaryotic cells, requiring a secreton and a translocator for proteins to pass the bacterial and host membranes. We used the contact hemolytic activity of Shigella flexneri to investigate its putative translocator. Hemolysis was caused by formation of a 25-Å pore within the red blood cell (RBC) membrane. Of the five proteins secreted by Shigella upon activation of its type III secretion system, only the hydrophobic IpaB and IpaC were tightly associated with RBC membranes isolated after hemolysis. Ipa protein secretion and hemolysis were kinetically coupled processes. However, Ipa protein secretion in the immediate vicinity of RBCs was not sufficient to cause hemolysis in the absence of centrifugation. Centrifugation reduced the distance between bacterial and RBC membranes beyond a critical threshold. Electron microscopy analysis indicated that secretons were constitutively assembled at 37°C before any host contact. They were composed of three parts: (a) an external needle, (b) a neck domain, and (c) a large proximal bulb. Secreton morphology did not change upon activation of secretion. In mutants of some genes encoding the secretion machinery the organelle was absent, whereas ipaB and ipaC mutants displayed normal secretons.

Attenuated total reflection Fourier-transform infrared spectroscopy of thin hydrated films of soluble and membrane protein included in a phospholipid bilayer is shown to provide useful information as to the secondary structure of the protein. The analysis of the amide I band of deuterated samples by Fourier self-deconvolution followed by a curve fitting was performed by a new procedure in which all the input parameters are generated by the computer rather than by the investigator. The results of this analysis provide a correct estimation of the a-helix and P-sheet structure content with a standard deviation of 8.6% when X-ray structures are taken as a reference. We also show that the orientation of the different secondary structures resolved by the Fourier self-deconvolution/ curve-fitting procedure and of the phospholipid acyl chains can be simultaneously evaluated for membrane proteins reconstituted in a lipid bilayer. Of special interest for reconstitution of membrane proteins, the lipid/ protein ratio can be accurately and quickly determined from the infrared spectrum.Among the spectroscopic techniques currently used for the determination on secondary structures of proteins and peptides (circular dichroism, NMR, X-ray diffraction analysis, etc.) infrared spectroscopy has recently become a prominent one. Although its potential usefulness has been recognized for some time [l, 21, the recognition of the different components of conformation-sensitive amides bands (particularly amide I band) was difficult due to the resulting overlap of bands originating from the different secondary structures such as a-helix, P-sheet, p-turns and random. Pointing out the different components of amide I has been made possible by the advent of numerical analysis of the spectra. The computed second and fourth derivatives [3, 41 for instance replace a broad featureless band by peaks of well-resolved components. More recently, the use of Fourier self-deconvolution has provided the same type of result. The advantage of Fourier selfdeconvolution is that it does not change the integrated areas of the different components of the amide envelope [5]. It must be noted that the Fourier self-deconvolution does not achieve an actual resolution enhancement but rather enhances selected frequencies of the spectrum. The actual resolution of the spectrum is defined by the spectrophotometer settings. Once the number and the approximate frequency of the different components are known, curve-fitting procedures can be applied to quantify the area of the different components of the amide envelope (much care must be taken then to prevent the appearance of artefacts due to the numerical treatment). ItCorrespondence to E. Goormaghtigh, UniversitC Libre de Bruxelles, Campus Plaine CP 206/2, B-1050 Bruxelles, BelgiumAbbreviations. ATR, attenuated total reflection; FTIR, Fouriertransform infrared spectroscopy; Myr2GroPCho, dimyristoylphosphatidylcholine; OcGlc, octyl-glucopyranoside; FWHH, full width at half height.was only in 1986 that the first comprehensi...

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