FEBS Letters
Volume 582, Issue 13 , Pages 1840-1846, 11 June 2008

Globin-coupled sensors and protoglobins share a common signaling mechanism

Edited by Peter Brzezinski

  • Jennifer A. Saito

      Affiliations

    • Department of Microbiology, University of Hawaii, Honolulu, HI 96822, United States
    • ,
  • Xuehua Wan

      Affiliations

    • Department of Microbiology, University of Hawaii, Honolulu, HI 96822, United States
    • ,
  • Kit Shan Lee

      Affiliations

    • Department of Microbiology, University of Hawaii, Honolulu, HI 96822, United States
    • ,
  • Shaobin Hou

      Affiliations

    • Advanced Studies in Genomics, Proteomics and Bioinformatics, University of Hawaii, Honolulu, HI 96822, United States
    • ,
  • Maqsudul Alam

      Affiliations

    • Department of Microbiology, University of Hawaii, Honolulu, HI 96822, United States
    • Advanced Studies in Genomics, Proteomics and Bioinformatics, University of Hawaii, Honolulu, HI 96822, United States
    • Corresponding Author InformationCorresponding author. Address: Department of Microbiology, University of Hawaii, Honolulu, HI 96822, United States. Fax: +1 808 956 5339.

Received 17 March 2008; received in revised form 28 April 2008; accepted 6 May 2008. published online 15 May 2008.

Article Outline

Abstract 

The globin-coupled sensors (GCSs) and protoglobins (Pgbs) form one lineage of the globin superfamily. The GCSs are multidomain sensory proteins involved in aerotaxis or gene regulation, while the Pgbs are single-domain globins of yet unknown function. We postulate that the GCSs and Pgbs share a common signaling mechanism to modulate diverse physiological functions. To elucidate the signaling properties of individual globin domains, we constructed and expressed chimeric receptors in Escherichia coli. We demonstrate that all the chimeric receptors reversibly bind oxygen in vitro and trigger aerotactic responses in vivo. Thus, oxygen binding to the globin domains of diverse GCSs and Pgbs form a common signaling state that can trigger aerotactic responses.

Keywords: Chimeric receptor, GCS, Pgb, Oxygen, Signal transduction

Abbreviations: GCS, globin-coupled sensor, Pgb, Protoglobin, MCP, methyl-accepting chemotaxis protein

 

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1. Introduction 

Globins are present in all kingdoms of life and exhibit a wide variety of functions, ranging from oxygen transport and storage to detoxification [1], [2] to signal transduction [3]. Their role as oxygen sensors in Archaea and Bacteria were first described for two aerotactic transducers, HemAT-Hs from the archaeon Halobacterium salinarum and HemAT-Bs from the Gram-positive bacterium Bacillus subtilis [3]. HemAT-Hs and HemAT-Bs consist of an N-terminal globin domain and a C-terminal signaling domain homologous to the bacterial methyl-accepting chemotaxis proteins (MCPs). These receptors form a new family of proteins coined as the globin-coupled sensors (GCSs), including a subgroup of proteins with C-terminal domains predicted to be involved in gene regulation [4], [5]. Single-domain protoglobins (Pgbs) are found in both Archaea and Bacteria [6], but their physiological functions are unknown.

Phylogenetic analysis of the entire globin superfamily indicates that the multidomain GCSs and single-domain Pgbs are related and form one of three distinct globin lineages [7], [8]. The physiological functions of these GCSs and Pgbs have not been experimentally determined, except for HemAT-Hs and HemAT-Bs. We postulate that the globin domains of the GCSs and Pgbs use a common signaling mechanism, which would allow the latter to communicate with heterologous C-terminal transmitter domains to perform signal transduction and gene regulation. To test our hypothesis, we constructed chimeric receptors consisting of the GCS or Pgb globin domains and the C-terminal MCP signaling domain of the E. coli chemotaxis transducer Tsr. Computerized motion analysis coupled to flash photolysis of caged oxygen was used to determine the function of the chimeras in E. coli. We used the globin domains from five HemATs: HemAT-Hs, HemAT-Bs, HemAT-Af (Anoxybacillus flavithermus), McpB and McpM (Caulobacter crescentus); a gene-regulatory GCS: AvGReg (Azotobacter vinelandii); and a Pgb: ApPgb (Aeropyrum pernix). Here, we report that these chimeras reversibly bind oxygen and mediate aerotactic responses in E. coli. We also identify the minimum sensing domain of HemAT-Hs and investigate the role of Arg196 in signaling.

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2. Materials and methods 

A full description of the methods is provided in the supplemental information.

2.1. Construction of chimeric receptors 

The gene fragments encoding the N-termini of HemAT-Hs (codons for 1–195 and 1–227 residues) and HemAT-Bs (codons for 1–176 and 1–193 residues) were amplified by PCR using primers (Table S1) which introduced an NdeI restriction site to the 5′ end and EcoRI restriction site to the 3′ end of the genes. Two gene fragments encoding the C-terminal of Tsr (codons for 216–551 and 269–551 residues) were amplified using primers (Table S1) which introduced an EcoRI site to the 5′ end and 6 His codons and a BamHI site to the 3′ end of the genes. The PCR products were purified, digested with EcoRI, and ligated according to Fig. 1. The recombinant chimeric genes were cloned into the pET-3a expression vector (Novagen).

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  • Fig. 1. 

    Construction of the Hs-Tsr chimeric receptors. Analogous receptors were constructed with the N-terminal of HemAT-Bs, using residues 1–176 or 1–193. TM1 and TM2, transmembrane helices 1 and 2; HAMP, linker region found in histidine kinases, adenylyl cyclases, MCPs, and phosphatases.

To clone the chimeric genes into the pTrc99A vector [9], PCR was used to introduce SacI (Hs-Tsr) or EcoRI (Bs-Tsr) restriction sites to the 5′ end of the genes. The 6 His-tag was also eliminated and replaced with a BamHI site at the 3′ end. The final plasmids were transformed into E. coli strain BT3388 Δ(aer tar tsr trg tap) [10] for motion analysis.

The HemAT-Af, McpB, McpM, AvGReg, and ApPgb chimeras were constructed using the strategy described above (with the HAMP domain). Chimeras with SwMb were constructed with and without the Tsr HAMP domain.

2.2. Construction of Hs-Tsr196 and Hs-Tsr227 mutants 

The hs-tsr196/pTrc99a and hs-tsr227/pTrc99A constructions were used as template for mutagenesis of Arg196 using the QuikChange Site-Directed Mutagenesis protocol (Stratagene). Mutations were confirmed by DNA sequencing.

2.3. Expression and purification of His-tagged chimeric proteins 

The His-tagged chimeric proteins were expressed in E. coli BL21(DE3) pLysS cells (Novagen). One liter cultures were grown until OD600=0.5–0.6 at 37°C, induced with 0.6mM isopropyl-β-d-thiogalactopyranoside (IPTG), further incubated for 3h, and harvested by centrifugation. The proteins were purified by Co2+-affinity chromatography according to Piatibratov et al. [11]. Purified recombinant proteins were analyzed by SDS–PAGE. Absorption spectra were measured using a Cary 1E UV–Visible spectrophotometer (Varian).

2.4. Motion analysis 

Transformed BT3388 E. coli cells were selected for motility on tryptone 0.28% agar plates supplemented with 1μM thiamine and then grown at 30°C in LB medium with 100μg/ml ampicillin and 1μM thiamine to OD600=0.2–0.3. Protein expression was induced with 0.5mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 45min. The swimming response of cells to photoreleased oxygen was monitored by computerized motion analysis as previously described [10].

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3. Results and discussion 

3.1. Globin domains of HemAT-Hs and HemAT-Bs trigger aerotactic responses in E. coli 

To study aerotactic signaling by the archaeal-bacterial chimeric transducers, we constructed chimeric proteins consisting of the N-terminal sensor globin domains of HemAT-Hs or HemAT-Bs and the C-terminal signaling domain of Tsr (with or without the HAMP linker segment) (Fig. 1). SDS–PAGE confirmed that the recombinant receptors were expressed as predicted (Fig. 2A). The absorption spectra (Fig. 2B and C) were similar to that of native HemAT-Hs and HemAT-Bs [3], with peaks at 413nm (Soret), 578nm (α-band), and 542nm (β-band). The proteins were deoxygenated with sodium dithionite and subsequently reoxygenated by exposure to air, demonstrating that they bind oxygen reversibly (data not shown).

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  • Fig. 2. 

    Characterization of the Hs-Tsr and Bs-Tsr receptors. (A–C) Purification of His-tagged Hs-Tsr and Bs-Tsr chimeras. (A) SDS-PAGE. Lanes: 1, molecular weight marker; 2, Hs-Tsr227H; 3, Hs-Tsr227; 4, Hs-Tsr195H; 5, Hs-Tsr195; 6, Bs-Tsr193H; 7, Bs-Tsr193; 8, Bs-Tsr176H; 9, Bs-Tsr176. (B) Absorption spectra of Hs-Tsr chimeras. (C) Absorption spectra of Bs-Tsr chimeras. (D–L) Excitation responses of E. coli strains to photoreleased oxygen. Caged oxygen was released 5s after the initiation of data collection, and an average of 1000 cell paths (30 repetitive assays) were recorded for 30s. Solid and dotted lines indicate the prestimulus rcd and its standard deviation, respectively. The domain structures of the receptors, as described in Fig. 1, are pictured with their corresponding response data.

To determine whether these recombinant chimeric receptors function as aerotaxis receptors, we expressed them in an E. coli strain lacking all five native chemotaxis transducers (Fig. 2D; [10]). We used previously optimized computerized motion analysis coupled to flash photolysis of caged oxygen to analyze the role of the chimeras in vivo [10]. In this assay, the swimming behavior of a population of cells was monitored by a computerized cell-tracking system, and changes in their tumbling frequency resulting from the photorelease of oxygen from a molecular cage was determined by measuring the increase or decrease in their rate of change of direction (rcd). The Hs-Tsr chimeric proteins with the full-length globin domain (residues 1–227) showed aerotactic responses (Fig. 2E and F), while those containing only the minimum heme-binding domain (residues 1–195 [4]) did not show any response (Fig. 2G and H). In contrast, all four Bs-Tsr chimeras mediated repellent responses to oxygen (Fig. 2I–L). Compared with E. coli cells solely expressing its aerotaxis transducer, Aer (Supplementary Fig. 1), signaling by the chimeras was not as effective. This was not surprising, since other chimeric receptors, such as an Aer/Tsr hybrid [12], also exhibited lower signaling efficiency. Our data also showed that the HAMP domain, which plays an important role in signal transduction [13], [14], [15], [16], [17], [18], was not required for these chimeras to signal (Fig. 2F, J, and L). Although the presence of the HAMP domain in several of the chimeras did appear to affect signaling (Fig. 2E and K), we are unable to make a general conclusion about the role of the Tsr HAMP domain in the chimeric constructions at this time. It is interesting to note that HemAT-Hs and HemAT-Bs do not possess a HAMP-like structure. We tested aerotactic signaling by a proximal histidine mutant, Hs-Tsr227 H123A, and this receptor failed to mediate aerotaxis (Supplementary Fig. 2). As a control experiment, we expressed full-length HemAT-Hs and HemAT-Bs in E. coli and found that they could not trigger aerotactic responses (Supplementary Fig. 3). These results establish that the N-terminal globin domain of the HemATs can use the signaling domain of Tsr to transmit aerotactic signals.

HemAT-Bs and HemAT-Hs mediate aerophilic and aerophobic responses, respectively, in their native hosts [3]. Attractants activate CheA autophosphorylation in B. subtilis [19], causing an increase in smooth swimming [20]. In contrast, repellents activate CheA autophosphorylation in H. salinarum, resulting in increased reversal frequency [21]. Based on this, one would expect HemAT-Bs and HemAT-Hs to respond in a similar manner to oxygen by activating CheA. In E. coli, as for H. salinarum, CheA is activated by repellents. Therefore, if a common CheA-activating receptor conformation were used in B. subtilis, H. salinarum, and E. coli, we would expect the chimeras to activate CheA upon binding oxygen, resulting in increased tumbling frequency. Indeed, for both the Bs-Tsr and Hs-Tsr chimeras, the tumbling frequency of the cells increased when oxygen was released. Our data is consistent with the conclusions made by Jung et al. [22], who demonstrated that a chimera between sensory rhodopsin II, its transducer, HtrII, and Tsr (SRII/HtrII/Tsr) could mediate repellent phototaxis responses in E. coli, similar to the response observed in halobacteria. They further showed that SRII/HtrII/Tsr could activate CheA autophosphorylation in vitro [23]. Zhang and Phillips [24] also suggested that the connection between receptor symmetry and CheA activation is conserved in E. coli and B. subtilis (i.e., symmetrical, rather than asymmetrical, receptor structure activates CheA), based on the crystal structure of the HemAT-Bs sensing domain.

3.2. Arginine 196 and its surrounding residues of HemAT-Hs are involved in signaling 

Since the Hs-Tsr chimeric receptors with the full-length globin domain (residues 1-227) mediated aerotactic responses, whereas the mini globin domain (residues 1–195) did not, this indicated that residues 196–227 were involved in signaling (Fig. 2). To determine whether all, or only a subset, of these 32 residues were needed for signal transmission, we generated a series of chimeras using various lengths of HemAT-Hs and tested their function in E. coli. All of the chimeras with 196 or more residues of HemAT-Hs were capable of generating repellent aerotactic responses (Fig. 3, Fig. 4A). We also tested chimeras containing 189 and 193 residues of HemAT-Hs and like those with 195 residues, they failed to respond to oxygen (Fig. 3). Thus, we have identified residues 1–196 of HemAT-Hs as being the smallest functional sensory module.

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  • Fig. 3. 

    Identification of the minimum sensing domain of HemAT-Hs. Chimeric receptors consisting of the indicated HemAT-Hs fragments with the signaling domain of Tsr (without the HAMP domain) were analyzed in E. coli as described in the text. −, no aerotactic response was observed; +, an aerotactic response was observed.

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  • Fig. 4. 

    Aerotactic responses of Hs-Tsr196 and Arg196 mutants. (A) Hs-Tsr196. (B–E) Arg196 mutations in Hs-Tsr196. (F–I) Arg196 mutations in Hs-Tsr227. Responses were measured as described in Fig. 2.

We next investigated the specific role of residue 196 by probing whether it was merely the length that rendered the domain capable of signaling or whether the chemical nature of this residue was important. We used site-directed mutagenesis to change the arginine at position 196 to alanine, phenylalanine, aspartate, or lysine in Hs-Tsr196 and tested their effect on the function of the receptor. Signaling was abolished when arginine was replaced with neutral alanine, aromatic phenylalanine, or negatively charged aspartate (Fig. 4B–D). On the other hand, signaling was preserved when arginine was replaced with another positively charged amino acid, lysine (Fig. 4E). We tested the same set of mutations in Hs-Tsr227 to determine whether residues 197–227 of HemAT-Hs could compensate for the changes at residue 196. Consistent with the previous results, the R196K mutation preserved signaling (Fig. 4I), while the R196F (Fig. 4H) and R196D (Fig. 4G) mutants failed to respond to oxygen. However, in this case the R196A mutant was able to trigger an aerotactic response (Fig. 4F). This data shows that the chemical nature of residue 196, including its surrounding residues, plays a role in signaling.

3.3. Globin domains of diverse GCSs trigger aerotactic responses 

Since the physiological functions of the GCSs, besides HemAT-Hs and HemAT-Bs, have not been experimentally determined, we chose to test the signaling properties of three other HemAT globin domains from HemAT-Af, McpB, and McpM. Af-Tsr204H (Fig. 5A), McpB-Tsr177H (Fig. 5B), and McpM-Tsr214H (Fig. 5C) all mediated aerotactic responses similar to the responses seen for the Hs-Tsr and Bs-Tsr chimeras. These results indicate that the globin domain of MCPs from evolutionarily diverse microorganisms use common signaling mechanisms. The nature of the response (attractant or repellent) in the native hosts, however, cannot be inferred without knowledge of each individual chemotaxis pathway, as in the case for HemAT-Bs.

It has been shown that functional chimeras between MCPs and gene-regulatory sensor kinases use common mechanisms for signaling [25], [26], [27]. To test whether this applied to the GCSs, we constructed a chimera using the globin domain from AvGReg, a putative gene-regulatory GCS with a GGDEF domain (cyclic di-GMP synthesis; [28], [29]). AvGReg-Tsr178H was also able to elicit an aerotactic response in E. coli (Fig. 5D). The ability of the AvGReg-Tsr chimera to trigger an aerotactic response demonstrates the flexibility of the globin domain to interact with various regulatory domains, suggesting that they utilize a common signaling mechanism. The globin ancestor of the GCSs may have used a similar mechanism, which became fine-tuned for functioning with specific domains in the course of evolution.

3.4. Single-domain protoglobin also triggers aerotactic responses 

To further test our hypothesis that single-domain Pgbs can also form a signaling state and trigger aerotactic responses, we constructed ApPgb-Tsr chimeras containing full-length (residues 1–195) and truncated (residues 1–182) ApPgb. These chimeric transducers indeed had the ability to transmit a physiological aerotactic signal (Fig. 6), and surprisingly, the truncated version produced a better response. This result led us to ask whether distantly related single-domain globins, such as myoglobin, retained the capability for signal transduction. We analyzed chimeras between sperm whale myoglobin (SwMb) and Tsr, but these receptors failed to trigger any aerotactic responses (Supplementary Fig. 4).

It is intriguing that single-domain ApPgb is functionally compatible with the MCP signaling domain for aerotactic signaling. This suggests that, besides sequence and structural similarities, a functional relationship exists between the Pgbs and GCSs. Although we have not yet determined the physiological function of ApPgb in its native host or whether it interacts with a cognate signaling partner, our data demonstrates that it has the inherent capacity for signal transduction. We previously proposed that Pgbs may be used for protection against oxidative or nitrosative stress [6], so existing as a single-domain globin could allow for the flexibility to perform multiple functions. A similar scenario is proposed for the single-domain Vitreoscilla hemoglobin (VHb). Evidence suggests that VHb can either form a homodimer to facilitate oxygen delivery or pair with a flavoreductase domain to form a flavohemoglobin-like structure for NO detoxification [30].

In summary, we have demonstrated that reversible oxygen binding to the globin domains of diverse GCSs and Pgbs can form a common excitatory signal and transmit this signal to a transmitter domain of another receptor to trigger aerotactic responses. We propose that the GCSs and Pgbs share a common signaling mechanism to modulate diverse physiological functions.

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Acknowledgements 

We thank Jimmy Saw and Alex Yu for technical assistance, Dr. John S. Parkinson for providing the anti-Tsr antibody, and Dr. Luc Moens, Dr. Sylvia Dewilde, and Tracey Freitas for helpful comments on the manuscript. We also thank several unknown reviewers for their constructive comments of the manuscript. This investigation was supported by National Science Foundation Grant MCB0446431 and partially by US Army Award TATRC #W81XWH-05-2-0013.

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Appendix A. Supplementary data 

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Fig. S1.

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Fig. S2.

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Fig. S3.

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Fig. S4.

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Supplementary data 1.

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Supplementary data 2.

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Supplementary Table.

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PII: S0014-5793(08)00401-8

doi:10.1016/j.febslet.2008.05.004

FEBS Letters
Volume 582, Issue 13 , Pages 1840-1846, 11 June 2008

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