Analyses of strains ISS4060 and Lilo2 gave similar results (data not shown). Figure 4 Ultrastructural analysis

of the cell surface of C. diphtheriae strains. (A) ISS3319, (B) Lilo1; red boxes in the low magnification images on the left hand side mark three areas shown with a higher magnification on the right hand side (upper row: topography/height, lower row: phase). Colour scale bars at the right hand side give height and phase magnitudes. Discussion In this study, the function of the surface-associated protein DIP1281, a member of the NlpC/P60 family was investigated, which was annotated as hypothetical invasion-associated protein. By fluorescence staining and atomic force microscopy, we could show that DIP1281 mutations cause formation

of chains of bacteria, rearrangements of cell surface structures, Target Selective Inhibitor Library and dramatic changes in protein patterns. Our data indicate that DIP1281 is not crucial for the separation of the peptidoglycan layer of dividing bacteria, since disruption of chains did not decrease the viability of bacteria. Consequently, DIP1281 function seems to be limited to the outer protein layer of C. diphtheriae, which is not uniformly organized in a surface layer lattice, but comprises more than 50 different proteins [16]. If the other NlpC/P60 family members in C. diphtheriae besides DIP1281, namely DIP0640, DIP1621, and DIP1622 [18] have similar functions in cell surface layer organization is unknown and has to be investigated in future projects. Tsuge and co-workers reported cell separation defects in Corynebacterium glutamicum R, when the DIP1281 homolog cgR_1596 and another member of the NlpC/P60 Trichostatin A protein family cgR_2070 were mutated [22]. Also in this study, cell separation was not impaired in respect to separation of peptidoglycan and mycolic

acid layers of daughter cells, but mainly restricted to the surface protein layer of the bacteria. However, using transmission electron microscopy of thin sections of cells, in this study also formation of multiple septa within single bacteria was observed in response to cgR_1596 mutations. Furthermore, growth of mutant strains was examined. In contrast to the situation in C. diphtheriae, where we found an unaltered growth rate 4��8C and a strongly increased biomass formation caused by lack of DIP1281, in C. glutamicum R mutation of cgR_1596 led to a slightly decreased growth rate and unaltered final optical density of the culture. The exact function of the NlpC/P60 protein family members in C. glutamicum was also not unravelled until now. In respect to adhesion and internalization of C. diphtheriae to epithelial cells, the results obtained in this study suggest that DIP1281 is crucial for localization and function of adhesion and invasion factors and consequently, structural alterations caused by lack DIP1281 prevent adhesion of corresponding mutants to host cells and invasion into these cells.