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I am satisfied that you have addressed all of the reviewer comments and I think the manuscript is improved as a result. Your paper will be a valuable addition to the literature in this important area of research.
Dear Dr. Gazdik.
Thanks for submitting your manuscript to Peer J. It has been reviewed by two experts in the field who recommended only minor changes to the manuscript.
The methodology of this manuscript are sound.
The findings and conclusions by Casey et al. are scientifically sound.
In this manuscript, Casey et al. report an expression analysis Mycobacterium smegmatis adenylate cyclase genes in response to changing environmental stimuli. They show that all of the cyclase genes are transcribed under the conditions tested and that only a few of them show modulated transcription in response to changing stimuli. They extended their findings to the human pathogen M. tuberculosis using a GFP promoter probe approach and saw similar patterns of expression. These findings are of interest to the field and should be published in PeerJ. I have many minor comments as to how the readability of the manuscript could be improved. In addition, I have included some comments and questions for the authors which may help better frame the manuscript and increase its impact.
Line 20: As M. smegmatis is spelled out each time it is used, I would refrain from jargon like “Mtb” when referring to M. tuberculosis, particularly in figure and table legends. Mtb is used in a few other places in the manuscript other than line 20.
Line 25 (and 14): Since the term prokaryotic includes both bacteria and archaea, would it be more accurate to simply use the term bacteria here, as the knowledge presented sounds specific to this group of organisms and does not include archaea?
Lines 26-28: A few introductory statements about what the major differences between Class I and Class III cAMP cyclases is needed. Presumably there is a Class II that is encoded by organisms other than E. coli and Mycobacteria spp. as well?
Lines 32-42: Though it is clear the authors are referring to M. tuberculosis, I think the language should be tightened up a bit. For instance, the use of mycobacterial should be changed to M. tuberculosis….particularly because there are many other sequenced Mycobacterium spp.
Line 36-39: I would consider re-wording this statement to something along the lines of, “The abundance and diversity of adenylate cyclases in Mycobacterium spp. suggests a more robust and complex role for cAMP signalling in bacteria than currently understood.”
Line 42: Prokaryote vs. bacteria.
Line 43: Consider changing one of the two uses of ‘high’ to ‘large’ … or possibly end the sentence by saying ‘significant redundancy in cAMP production in Mycobacterium spp.”
Line 48: Change to, “Instead, it is more likely that the expression of individual cyclases or groups of cyclases are controlled at the level of transcription and their expression is dependent on specific environmental or lifestyle conditions.”
Line 53: Mtb
Line 59: Space after the degree symbol, space before cm
Line 62: Spell out what DETA stands for
Line 65 / 70: Hyphenate RNase-free
Line 71: Manufacturer’s specifications
Line 72: Hyphenate Semi-quantitative.
Line 73: “…of RNA using the iScript cDNA synthesis kit according to the manufacturer’s instructions.”
Line 79: “16S rDNA products”
Line 81: You attempted to PCR amplify the 16S rDNA not the 16S RNA.
Line 84: These seem to be transcriptional rather than translational fusions, so it should be promoter::gfp rather than promoter::GFP.
Line 91: Presumably normalized to 106 bacteria haboring the pGFPoriM vector only?
Line 102: Eight not 8. Spell out numbers below 10.
Line 104: Although this is clarified later in the manuscript, use of the phrase “greatest level of regulation” confuses the reader here. Please change the wording to be more clear. Perhaps, “change in transcription” could be used instead?
Line 106: Either show the data for the nitrosative and CO2 stress or report as data not shown.
Line 108: Comma after additionally.
Line 108: Change to…”after starvation and exposure to low pH.”
Line 111: Comma after interestingly.
Line 111-113: Consider changing to, “Interestingly, the expression of only four of the eight adenylate cyclases were influenced by the environmental stimuli tested. Although the observed changes in gene expression were not dramatic, they were statistically significantly different.”
Line 118: Change to whose expression was altered by environmental stimuli …. rather than demonstrate transcriptional regulation.
Table 1: Italics for sigA and tuf. What is the story with the N’s in the primer sequences?
Figure 1: (A) Hyphenate PCR-amplified. (B) Reword. Quantification of PCR products depicted in (A) using ImageJ. Italics and uppercase of P
Figure 2: Subscript 2’s for the second mention of H2O2. Italics and uppercase for P
Line 125: Change to “…upstream of the putative ATG start codon.”
Line 131: gfp vs. GFP as mentioned above.
Other comments / questions:
Are the M. smegmatis orthologs analysed here present in other pathogenic Mycobacterium species? What about M. marinum? Can the findings here be applied to this pathogenic system as well?
Is MSMEG_0545 also predicted to be biochemically inactive like Rv1359? What is the bioinformatics basis for this?
MSMEG_0545 (and Rv1359) is both upregulated during starvation and downregulated during oxidative stress. This interesting pattern of expression and the fact that it (or Rv1359 at least) is predicted to not be functional suggests there is something interesting going on with this cyclase. Is there any reasonable speculation the authors can provide here?
Although few changes in expression of the cyclases was seen, can the authors relate what changes were observed back to the host? A small discussion of how starvation and oxidative stress relates to the M. tubercolosis interaction with its host and why specifically cAMP signalling may be important in this context seems warranted.
This manuscript by Casey, Ford & Gazdik describes a transcriptional comparison of the putative adenylate cyclase genes of Mycobacterium smegmatis, relating these expression profiles to the homogous genes in M. tuberculosis, where adenylate cyclases have been shown to be related to virulence.
The manuscript appears to be in required format for PeerJ, containing relevant figures and is a self contained unit of publication. There are however a few amendments that could be included by the authors to improve the manuscript in my opinion.
The article describes original work, in terms of it being a comprehensive survey of expression of adenylate cyclases from M. smegmatis. The experimental design appears to be rigorous, with appropriate statistical methods applied to the data obtained. There are no ethical issues relating to this work.
The data are robust, statistically sound and the appropriate controls have been used by the authors.
There are a few things that could be added to the manuscript to improve its completeness.
Results and discussion - line 94 - the authors state the examination of the M. smegmatis orthologs of M. tuberculosis. Were these orthologs determined simply by identifying the 'best blast hit' or, as generally accepted for ortholog identification 'reciprocal best blast hitting' between the two organisms? it is not entirely clear to me.
Moreover an alignment of the adenylate cyclases and an appropriately constructed phylogenetic tree would be really useful to this manuscript. I guess the authors already have the data available?
line 52 - 'Dass et al focused' should be 'Dass et al., (2008) focused...'
line 112 - I assume 'significant' should be added to this sentence?
I think the authors should mention in the manuscript that there is a cAMP burst upon the onset of infection in M. tuberculosis (eg reference http://www.ncbi.nlm.nih.gov/pubmed/20028978) and also it maybe worth mentioning the existence if cyclic-di-AMP that has recently arisen as an important second messenger, which has yet to be shown to occur in mycobacteria, but maybe related to the diversity in cAMP related regulons.
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