Coordinated regulation of chemotaxis and resistance to copper by CsoR in Pseudomonas putida

Reviewer #1 (Public Review):

This report contains two parts. In the first part, several experiments were carried out to show that CsoR binds to CheA, inhibits CheA phosphorylation, and impairs P. putida chemotaxis. The second part provides some evidence that CsoR is a copper-binding protein, binds to CheA in a copper-dependent manner, and regulates P. putida response to copper, a chemorepellent. Based on these results, a working model is proposed to describe how CsoR coordinates chemotaxis and resistance to copper in P. putida. While the second part of the study is relatively solid, there are some major concerns about the first part.

Critiques:

(1) The rigor from prior research is not clear. In addition to talking about other bacterial chemotaxis, the Introduction should briefly summarize previous work on P. putida chemotaxis and copper resistance.

(2) The rationale for identifying those CheA-binding proteins is vague. CheA has been extensively studied and its functional domains (P1 to P5) have been well characterized. Compared to its counterparts from other bacteria, does P. putida CheA contain a unique motif or domain? Does CsoR bind to other bacterial CheAs or only to P. putida CheA?

(3) Line 133-136, "Collectively, using pull-down, BTH, and BiFC assays, we identified 16 new CheA-interacting proteins in P. putida." It is surprising that so many proteins were identified but none of them were chemotaxis proteins, in particular those known to interact with CheA, such as CheW, CheY and CheZ, which raises a concern about the specificity of these methods. BTH and BiFC often give false-positive results and thus should be substantiated by other approaches such as co-IP, surface plasmon resonance (SPR), or isothermal titration calorimetry (ITC) along with mutagenesis studies.

(4) Line 147-149, "Fig. 2a, five strains (WT+pcsoR, WT+pispG, WT+pnfuA, WT+pphaD, and WT+pPP_1644) displayed smaller colony than the control strain (WT+pVec), indicating a weaker chemotaxis ability in these five strains." If copper is a chemorepellent, these strains should swim away from high concentrations of copper; thus, the sizes of colonies couldn't be used to measure this response. In the cited reference (reference 29), bacterial response to phenol was measured using a response index (RI).

(5) Figures 2 and 3 show both CsoR and PhaD bind to CheA and inhibit CheA autophosphorylation. Do these two proteins share any sequence or structural similarity? Does PhaD also bind to copper? Otherwise, it is difficult to understand these results.

(6) Line 195-196, "CsoR/PhaD had no apparent influence on the phosphate transfer between CheA and CheY". CheA controls bacterial chemotaxis through CheY phosphorylation. If this is true, how do CsoR and PhaD affect chemotaxis?

(7) Figure 3 shows that CsoR/PhaD bind to CheA through P1, P3, and P4. This result is intriguing. All CheA proteins contain these three domains. If this is true, CsoR/PhaD should bind to other bacterial CheAs too. That said, this experiment is premature and needs to be confirmed by other approaches.

(8) Figure 5, does PhaD contain these three residues (C40, H65, and C69)? If not, how does PhaD inhibit CheA autophosphorylation and chemotactic response to copper?

(9) Does deletion of cosR or cheA have any impact on P. putida resistance to high concentrations of copper?

Reviewer #2 (Public Review):

Summary:

This manuscript focuses on the apparent involvement of a proposed copper-responsive regulator in the chemotactic response of Pseudomonas putida to Cu(II), a chemorepellent. Broadly, this area is of interest because it could provide insight into how soil microbes mitigate metal stress. Additionally, copper has some historical agricultural use as an antimicrobial, thus can accumulate in soil. The manuscript bases its conclusions on an in vitro screen to identify interacting partners of CheA, an essential kinase in the P. putida chemotaxis-signaling pathway. Much of the subsequent analysis focuses on a regulator of the CsoR/RcnR family (PP_2969).

Weaknesses:

The data presented in this work does not support the model (Figure 8). In particular, PP_2969 is linked to Ni/Co resistance, not Cu resistance. Further, it is not clear how the putative new interactions with CheA would be integrated into diverse responses to various chemoattract/repellents. These two comments are justified below.

PP_2969

(1) The authors present a sequence alignment (Figure S5) that is the sole basis for their initial assignment of this ORF as a CsoR protein. There is a conservation of the primary coordinating ligands (highlighted with asterisks) known to be involved in Cu(I) binding to CsoR (ref 31). There are some key differences, though, in residues immediately adjacent to the conserved Cys (the preceding Ala, which is Tyr in the other sequences). The effect of these changes may be significant in a physiological context.

(2) The gene immediately downstream of PP_2969 is homologous to E. coli RcnA, a demonstrated Ni/Co efflux protein, suggesting that P2969 may be Ni or Co responsive. Indeed PP_2970 has previously been reported as Ni/Co responsive (J. Bact 2009 doi:10.1128/JB.00465-09). The host cytosol plays a critical role in determining metal response, in addition to the protein, which can explain the divergence from the metal response expected from the alignment.

(3) The previous JBact study also explains the lack of an effect (Figure 5b) of deleting PP_2969 on copper-efflux gene expression (copA-I, copA-II, and copB-II) as these are regulated by CueR not PP_2969 consistent with the previous report. Deletion of CsoR/RcnR family regulator will result in constitutive expression of the relevant efflux/detoxification gene, at a level generally equivalent to the de-repression observed in the presence of the signal.

(4) Further, CsoR proteins are Cu(I) responsive so measuring Cu(II) binding affinity is not physiologically relevant (Figures 5a and S5b). The affinities of demonstrated CsoR proteins are 10-18 M and these values are determined by competition assay. The MTS assay and resulting affinities are not physiologically relevant.

(5) The DNA-binding assays are carried out at protein concentrations well above physiological ranges (Figures 5c and d, and S5c, d). The weak binding will in part result from using DNA sequences upstream of the copA genes and not from from PP_2970.

CheA interactions

(1) There is no consideration given to the likely physiological relevance of the new interacting partners for CheA.

(2) How much CheA is present in the cell (copies) and how many copies of other proteins are present? How would specific responses involving individual interacting partners be possible with such a heterogenous pool of putative CheA-complexes in a cell? For PP_2969, the affinity reported (Figure 5A) may lay at the upper end of the CsoR concentration range (for example, CueR in Salmonella is present at ~40 nM).

(3) The two-hybrid system experiment uses a long growth time (60 h) before analysis. Even low LacZ activity levels will generate a blue colour, depending upon growth medium (see doi: 10.1016/0076-6879(91)04011-c). It is also not clear how Miller units can be accurately or precisely determined from a solid plate assay (the reference cited describes a protocol for liquid culture).