Phylogenetic Analysis with Prediction of Cofactor or Ligand Binding for Pseudomonas aeruginosa PAS and Cache Domains

ABSTRACT PAS domains are omnipresent building blocks of multidomain proteins in all domains of life. Bacteria possess a variety of PAS domains in intracellular proteins and the related Cache domains in periplasmic or extracellular proteins. PAS and Cache domains are predominant in sensory systems, often carry cofactors or bind ligands, and serve as dimerization domains in protein association. To aid our understanding of the wide distribution of these domains, we analyzed the proteome of the opportunistic human pathogen Pseudomonas aeruginosa PAO1 in silico. The ability of this bacterium to survive under different environmental conditions, to switch between planktonic and sessile/biofilm lifestyle, or to evade stresses, notably involves c-di-GMP regulatory proteins or depends on sensory pathways involving multidomain proteins that possess PAS or Cache domains. Maximum likelihood phylogeny was used to group PAS and Cache domains on the basis of amino acid sequence. Conservation of cofactor- or ligand-coordinating amino acids aided by structure-based comparison was used to inform function. The resulting classification presented here includes PAS domains that are candidate binders of carboxylic acids, amino acids, fatty acids, flavin adenine dinucleotide (FAD), 4-hydroxycinnamic acid, and heme. These predictions are put in context to previously described phenotypic data, often generated from deletion mutants. The analysis predicts novel functions for sensory proteins and sheds light on functional diversification in a large set of proteins with similar architecture. IMPORTANCE To adjust to a variety of life conditions, bacteria typically use multidomain proteins, where the modular structure allows functional differentiation. Proteins responding to environmental cues and regulating physiological responses are found in chemotaxis pathways that respond to a wide range of stimuli to affect movement. Environmental cues also regulate intracellular levels of cyclic-di-GMP, a universal bacterial secondary messenger that is a key determinant of bacterial lifestyle and virulence. We study Pseudomonas aeruginosa, an organism known to colonize a broad range of environments that can switch lifestyle between the sessile biofilm and the planktonic swimming form. We have investigated the PAS and Cache domains, of which we identified 101 in 70 Pseudomonas aeruginosa PAO1 proteins, and have grouped these by phylogeny with domains of known structure. The resulting data set integrates sequence analysis and structure prediction to infer ligand or cofactor binding. With this data set, functional predictions for PAS and Cache domain-containing proteins are made.

The manuscript is well-drafted and describes the methods used and conclusions drawn in sufficient detail. I have only minor comments that the authors might want to address in revision.
I would also encourage the authors to make sequence alignments of the eight colored clusters in Fig. 1, either as supplementary material to this paper.  While the N-terminal helix does cross the membrane, it is most likely the C-terminal helix of this domain that propagates the signal. L. 96. "they are more sensitive" More sensitive than what? L.172. "The similar architecture facilitates a discussion" Where is this discussion? Fig. 4. In the legend, change "The four cascades involve two-component signaling" to "PA5165, PA5512, and PA1336 are sensor histidine kinases, while PA2652 is a chemoreceptor". I am afraid that the use of the same shape for the histidine kinase (HisK/HATPase), methyl-accepting (MA-sensor), and receiver (REC) domains is somewhat misleading.
Reviewer #2 (Comments for the Author): In this paper, Hutchin et al. used protein sequence and phylogenetic analyses to classify and predict ligand/cofactor binding for Pseudomonas aeruginosa PAS and Cache domains. Predictions derived by the authors can be tested by experimentalists working with this model organism. The authors collected and summarized a comprehensive body of information related to ligand and cofactor binding by PAS and Cache domains. This is a very positive aspect of the paper. Computational predictions are technically sound, but without experimental validation of any of them, they remain just "predictions".
Major concerns: 1. As evident from the title and throughout the manuscript, the authors consider Cache domains as a version of PAS domains. This is incorrect and Ref. 20 provides a clear distinction between these two domain superfamilies. Therefore, "PAS and Cache domains" should be used instead of "PAS domains" in the title and throughout the manuscript, as appropriate (e.g. PAS and Cache, not PAS/CACHE). 2. In a nearly similar fashion, "cofactor" and "ligand" are not the same thing. The authors should clearly specify that. For example, in the title and in the heading on line 174, it should be stated "... cofactor and ligand binding..." 3. The authors generated an unusually large bibliography list, and, in many instances, they give full credit to both original discoveries and reviews. Surprisingly, they failed to reference the original discovery of the PAS domain superfamily (by two independent groups, see PMID: 9301332 and 9382818). 4. Tree building methods other than maximum likelihood (e.g., neighbor-joining, Bayesian) should be used for comparison.
Minor concerns: 1. CACHE should not be capitalized, it is "Cache".

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Response to Reviewers
Manuscript Spectrum01026-21 REVIEWER REPORT(S): EDITOR Thank you for submitting your manuscript to Microbiology Spectrum. This is a well-executed project that provides several useful insights for the community. Two reviewers have gone through this manuscript and they have recommended reconsideration of your paper following major revision. Based on their opinions, I invite you to resubmit your manuscript after addressing all reviewer comments. The comments of the reviewers are included at the bottom of this letter.
The first major comment from Reviewer 2 is the most critical one, therefore, please give more emphasis on this comment. Altogether, please address all the comments by both reviewers. Also, pay particular attention to the comments about clear terminology. Please ensure that the added comments are well explained throughout the text and supported by clearly described methods.
Done. In response to comment 1 from reviewer 2, the nomenclature has been updated throughout the manuscript to clarify the difference between PAS and Cache domains.
When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript -we strongly recommend that you submit your paper within the next 60 days or reach out to me. Detailed information on submitting your revised paper is also provided below. The manuscript is well-drafted and describes the methods used and conclusions drawn in sufficient detail. I have only minor comments that the authors might want to address in revision.

Thank you.
I would also encourage the authors to make sequence alignments of the eight colored clusters in Fig. 1, either as supplementary material to this paper.
Done. We have included secondary structure annotated alignments of clades highlighted in Figure 1 and discussed in the text in a new supplement. We reference this in the main text several times and introduce the alignments presented in supplement at the end of the phylogeny section, as follows: "Alignments of individual clades are presented in Supplement, and we give a few examples in the following section." Minor comments