Reviewer 1's Review

“The investigation presented by Kiselev et al. is on the polymorphic membrane protein D (PmpD) from Chlamydia trachomatis Serovar L2, which was reported before to belong to the family of autotransporter proteins. These proteins are produced as precursor molecules consisting of a typical signal peptide at the very N terminus for translocation across the cytoplasmic membrane and a so-called beta-barrel at the C terminus for translocation across the outer membrane. By this way a passenger domain located in between is transported to the cell surface. Kivelev et al. investigated the expression, the processing and the localization of PmpD in particular with respect to the two distinct morphological forms of Chlamydia, the infectious and metabolically inactive form called elementary body (EB) and the metabolically active form, the reticulate body (RB).
In the first part of the paper the authors quantified the expression of PmpD by a real time RT-PCR approach over a period of 48 hours p.i. using 16S rRNA for standardization. They found the highest rate of expression to appear between 16 and 24 hours p.i. being in agreement with the development cycle of Chlamydia, which covers a conversion of the EB form to the RB form and back again to the EB form. This part is quite clear and easy to follow.
In the second part of the paper they expressed four different non-overlapping fragments of PmpD in E. coli, purified the fragments by the use of a His-tag and used the four purified proteins to raise polyclonal antisera. These sera were used in different approaches to analyse expression, localization, and the processing of PmpD with respect to the distinct forms in the Chlamydia development cycle. This part appears to be confusing and speculative.
It would be helpful for the reader, in order to follow the cases of the authors, if the edges of the four different fragments produces could be given as amino acid sequences in Fig. 1.
The authors report on the appearance of a 160 kDa protein, which they call the full size protein. But it remains obscure, whether it still contains the signal peptide or not. If yes, the authors need to comment the observation that the 160 kDa protein appears in the periplasm and in different insoluble fractions (pointing on a membrane-embedding), as transport across the inner membrane is strongly correlated to signal peptide cleavage and a subsequent loss of the corresponding MW. This answer could be given easily by the results of the MS experiments that has been performed in order to verify the identity of PmpD. In this case, it needs to be discussed, why the authors were not able to detect by any means the processed form of the precursor without signal peptide. This was reported by in the investigation of another group, which detected a so-called 155 kDa protein. Or is the 155 kDa protein of that investigation identical with the author's reported 160 kDa form? If the 160 kDa protein does not contain the signal peptide, than the question arises, what happened after processing to result in a localization of this form within the cytoplasm.
Beside the 160 kDa protein, two additional protein bands were detected by the different antibodies, a 120 kDa protein band and a 65 kDa protein band. The authors speculate that the 120 kDa protein band could contain the full size protein with a removed beta-barrel, whereas nothing is said on the origin of the 65 kDa protein. Again the question arises whether the 120 kDa protein still contains the signal peptide or not. Here it would be very helpful, if the identity and the primary structure of both proteins, the 120 kDa and the 65 kDa protein, could be elucidated by a similar approach, as has been performed for the full size protein.
As long as these questions cannot be answered, the presented investigation appears to be somewhat preliminary and it seems to raise more questions than it answers.”

N.B. These are the general comments made by the reviewer when reviewing this paper in light of which the manuscript was revised. Specific points addressed during revision of the paper are not shown.