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Referee Comments: Referee 1

Posted by PLOS_ONE_Group on 27 May 2008 at 08:55 GMT

Referee 1's review:

N.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication, the manuscript has been revised in light of these comments and to address other editorial requirements.

I have a technical question about the incorporation of proteins into liposomes. As far as I can see, there is no methods of determining the directionality of the protein insertion into the liposome. If this is the case, then it does not seem possible to definitively determine the orientation of the proteins within the membrane using this method. Two protected domains are apparent at 26 and 29kDa, but if there are two populations of protein in the liposome, some SBD and some ATPase domain would be available for cleavage, even if they were on opposite sides of the membrane. The western blot is also confusing: in the text the band in lane 2 is described as 29kDa, in the figure as 26kDa. Also, why does the remaining full length protein visible in lane 2 not show up on the western blot?

In the RT-PCR showing induction of genes by iron, the quantitation does not appear correct. The low iron bands are _much_ more intense than the iron replete, yet are listed in the histogram as '8%' increased: what is this compared to? If time=0 under low iron conditions gave a similar band intensity to iron replete conditions, then the induction looks to be several fold i.e. >>100%

Also, if the experiments have been performed in triplicate, some estimate of errors should be possible. This is most important for Rv2895c, where the difference in calculated Kd values is quite modest and the binding curves look very similar.
The comment about 'biphasic binding curves' is also unclear: the red line in Fig 5A does not appear to be biphasic.

The liposomal transport assays illustrated in Figure 7 suffer from the same uncertainty about protein orientation in the membrane as was highlighted above for the tryptic digest experiments. However, this deficiency is overcome in this assy by the unidirectional nature of the transport measured. The plotting of phosphate generation vs fluorescence is a non-intuitive way of presenting this time-dependent data. It would be better to plot both of these measurements vs time. Also, it would be very helpful to show the control data that has been described but not shown, i.e. the lack of activity without intra-lysosomal ATP, the inability of Rv1349 to import Fe-cMyco without Rv2895c etc.

Is msmeg_6554 the orthologue of Rv1348? Are there any other candidates in the M.smegmatis genome?