Introduction Mycoplasma mycoides mycoides 1987 2003 1994 1994 1997 1999 1999 2003 1997 2000 2003 M. mycoides . mycoides 2000 1 2000 2000 M. mycoides . mycoides 2000 M. mycoides . mycoides 2000 Fig. 1 M. mycoides mycoides 28 2000  M. mycoides . mycoides 2002 M. mycoides . mycoides M. mycoides . mycoides 2004 M. mycoides . mycoides 2004 2006 M. mycoides . mycoides 2005 Materials and methods Bioinformatic analysis 1993 http://www.ch.embnet.org/software/TMPRED_form.html 1992 http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html 2004 http://phobius.cgb.ki.se/ http://minnou.cchmc.org/ 2006 http://meme.sdsc.edu/meme/meme.html 1994 M. mycoides mycoides M. mycoides mycoides 2001 1998 M. mycoides mycoides 9 2000 Production of antibodies against recombinant LppQ peptides Escherichia coli 2000 2002 2+ 2 4 2 4 1976 Immunoblot analysis 1999 M. mycoides mycoides 1998 2000 2 3 3 2 M. mycoides mycoides l 2 4 2 4 Scanning electron microscopy 1993 2002 2002 Samples were examined without further metal coating. Secondary electron micrographs and corresponding backscattered images were obtained with a fully digital field emission scanning electron microscope DSM 982 Gemini (Zeiss, Oberkochen, Germany) at an accelerating voltage of 5 kV, a working distance of 6–8 mm and a magnification from 50,000 to 100,000×. Control experiments included omission of primary antibody as well as the use of a rabbit anti-calcitonin antibody (Anawa, Zurich, Switzerland) and of IgGs from rabbit pre-immune sera as irrelevant substitutes for the anti-LppQ primary antibodies. Results Structure of the C-terminal domain of lipoprotein LppQ 1 Monospecificity of IgG preparations M. mycoides mycoides 2 2 Fig. 2 M. mycoides mycoides 1998 2000  Morphology of the mycoplasmas M. mycoides mycoides 3 Fig. 3 M. mycoides mycoides A C E B D F B D F  Immunogold labelling 3 3 1 3 P 1 Table 1 Statistical analysis Statistical data Treatment Anti-LppQ-N’ Anti-LppQ-C’ Anti-calcitonin Pre-immune serum No primary antibody Single cells investigated 27 48 19 20 23 Labelled particles Total 333 17 6 7 5 Min 2 0 0 0 0 Max 31 4 3 3 2 a 12.33 ± 7.58 0.35 ± 0.84 0.32 ± 0.82 0.35 ± 0.81 0.22 ± 0.52 P b  <0.0001 <0.0001 <0.0001 <0.0001 a b Discussion M. mycoides mycoides 1981 2000 2000 2000 1 Bacillus subtilis 1999 2001 Mycoplasma pneumoniae 0 1 1998 High resolution scanning electron microscopy in combination with immunogold labelling revealed extracellular epitopes with antibodies against LppQ-N′ but no signal above the background of the three negative controls was detected with the antibody against LppQ-C′. These results substantiate the predicted localization of the two domains, as they unambiguously corroborate the accessibility of the N-terminal domain at the extracellular side of the plasma membrane. The failure to immunolabel the C-terminal epitopes is an indication that they may be imbedded in the membrane. However, one cannot rule out the possibility that such epitopes (i) are buried in the centre of a globular protein, (ii) are masked by other surface components or (iii) are not recognized in their native forms by rabbit antibodies produced by inoculation of the recombinant peptide. Beyond providing ultrastructural evidence for the location of the hydrophilic and hydrophobic domains of LppQ, these results also demonstrate that immunogold labelling in scanning electron microscopy allows for subtle topographical discrimination and that selective visualization of peptides being exposed at the outer surface of microorganisms can be achieved with high accuracy and reliability. M. mycoides mycoides 2000 M. mycoides . mycoides 2004 Conclusions M. mycoides mycoides M. mycoides mycoides