A dual-H-NOX signaling system in Saccharophagus degradans

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A dual-H-NOX signaling system in Saccharophagus degradans Yirui Guo, Matthew M. Cooper, Raquel Bromberg, and Michael A Marletta Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01058 • Publication Date (Web): 06 Nov 2018 Downloaded from http://pubs.acs.org on November 6, 2018

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Biochemistry

A dual-H-NOX signaling system in Saccharophagus degradans Yirui Guo1†, Matthew M. Cooper2†, Raquel Bromberg3 and Michael A. Marletta1,2,4,* 1 California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720 2Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720 3Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX 75390 4Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720 †Authors

contributed equally to this work

*Corresponding

author, email: [email protected]

Abstract Nitric oxide (NO) is a critical signaling molecule involved in the regulation of a wide variety of physiological processes across every domain of life. In most aerobic and facultative anaerobic bacteria, heme-nitric oxide/oxygen binding (H-NOX) proteins selectively sense NO and inhibit the activity of a histidine kinase (HK) located on the same operon. This NO-dependent inhibition of the cognate HK alters the phosphorylation of the downstream response regulators. In the marine bacteria Saccharophagus degradans (Sde), in addition to a typical H-NOX (Sde 3804)/HK (Sde 3803) pair, an orphan H-NOX (Sde 3557) with no associated signaling protein has been identified distant from the H-NOX/HK pair in the genome. The characterization reported here elucidates the function of both H-NOX proteins. Sde 3557 exhibits a weaker binding affinity with the kinase, yet both Sde 3804 and Sde 3557 are functional H-NOXs with proper gas-binding properties and kinase inhibition activity. Additionally, Sde 3557 has a significantly slower NO-dissociation rate compared to that of Sde 3804, which may confer prolonged kinase inhibition in vivo. While it is still unclear whether Sde 3557 has another signaling partner or it shares the histidine kinase with Sde 3804, Sde 3557 is the only orphan H-NOX identified to date. S. degradans is likely using a dual-H-NOX system to fine-tune the downstream response of NO signaling.

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Introduction Nitric oxide (NO) is an important signaling molecule that is involved in many physiological functions in all domains of life from bacteria to humans.1-3 This highly reactive gas can diffuse freely across the cell membrane and be sensed by protein receptors to mediate downstream signaling processes. Among all of the NO signaling components in prokaryotes and eukaryotes, the HNOX (Heme-Nitric oxide/OXygen) domain plays a central role in NO sensing, either as a stand-alone protein or as part of a larger multi-domain protein.3-6 The H-NOX protein/domain typically contains ~180 residues and a heme cofactor to bind gas ligands.7 As the name suggests, H-NOXs can also interact with O2 as a physiological ligand in obligate anerobes.7-9 The most intensively studied H-NOX-dependent NO signaling system is centered on the mammalian soluble guanylate cyclase (sGC).5, 10 sGC is a multi-domain protein consisting of an α-subunit and a β-subunit, with a functional H-NOX domain at the N-terminus of the β-subunit.11-13 NO binding to the H-NOX domain is an essential step in activating the guanylate cyclase activity that converts guanosine 5triphosphate (GTP) to guanosine 3,5-cyclic monophosphate (cGMP). cGMP then initiates a signaling cascade that regulates the activity of downstream proteins such cGMP-dependent kinases and cGMP-gated ion channels.5, 12 In contrast to the eukaryotic H-NOX-containing proteins, bacterial homologs in obligate anaerobes are fused to a methyl-accepting chemotaxis protein (MCP) and serve as an O2 sensing domain.9 Alternatively, there are stand-alone NO sensing proteins in aerobes or facultative anaerobes that directly interact with signaling partners such as histidine kinases (HK) in two-component signaling system and diguanylate cyclases in cyclic-di-GMP metabolism.14-19 The key for H-NOX signaling is gas ligand binding to the heme cofactor that induces a conformational change in the protein. In the case of Shewanella oneidensis (So) and Vibrio cholerae (Vc), NO binding to the H-NOX protein induces the Fe-His bond cleavage between the heme ligating histidine and the heme cofactor, and a subsequent displacement of distaland proximal- subdomains.20-22 This conformational change is transduced to the cognate signaling partner, a histidine kinase, and inhibits kinase autophosphorylation that leads to biofilm formation23-25 (Figure 1).


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Biochemistry

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Figure 1. Overview of H-NOX/HK signaling pathway Typical bacterial NO signaling by H-NOX/HK complex using Shewanella oneidensis as an example. In the absence of NO, the H-NOX-associated kinase autophosphorylates a histidine residue. This phosphoryl-group is then transferred to the downstream response regulator HnoB and its inhibitor HnoD. Phosphorylated HnoB stimulates its phosphodiesterase activity and phosphorylated HnoD weakens the inhibition of HnoB. As a result, the conversion of c-di-GMP into pGpG is enhanced. In the presence of NO, the autophosphorylation of the H-NOX-associated kinase is inhibited, resulting a less active HnoB and a more active HnoD, which leads to accumulation of c-di-GMP and subsequent biofilm formation.

Most H-NOX-containing bacteria have only one H-NOX in their genome, with a few exceptions where multiple copies of the H-NOX gene are found. Bioinformatic analysis has shown that in both cases, the H-NOX gene is often located on the same operon as other H-NOX-associated signaling partners.26 This gene organization allows the signaling proteins that function together as a complex (e.g. H-NOX/HK pair) to be expressed and regulated simultaneously for a fast and robust response to environmental stimuli. Interestingly, previous computational analysis has shown that Saccharophagus degradans encodes a typical H-NOX (Sde 3804)/HK (Sde 3803) pair as well as an orphan H-NOX (Sde 3557) that has no adjacent signaling partner.3, 26 S. degradans is a Gram-negative, aerobic, free-living marine bacterium with the ability to degrade over 10 polysaccharides from algae and higher plants. This “super-degrader” role of S. degradans makes it an important component in the marine carbon cycle.27, 28 The function of this orphan H-NOX in S. degradans is not obvious especially in relation to the H-NOX/HK pair. In this study, both H-NOXs and the H-NOXassociated HK were purified and characterized. Computational analysis was also


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performed to assess the origin of the orphan H-NOX, leading to a hypothesis that S. degradans may use a dual-H-NOX system to fine-tune the downstream response to NO. Methods Protein expression and purification For expression, full length Sde 3804 (1-180) and Sde 3557 (1-182) were cloned from S. degradans gDNA (ATCC) and inserted into a pET22b vector with a TEV-cleavable His6 tag at the C-terminus. Sde 3804 was expressed in RP523(DE3) cells, grown in Terrific broth (Research Products International) at 37 °C in the presence of 100 μg/mL ampicillin, 0.5% w/v glucose and 30 mg/L hemin. Sde 3557 was expressed from BL21(DE3) cells in Terrific broth supplemented with 500 μM FeCl3, 100 μg/mL ampicillin and 0.5% w/v glucose. The heme precursor 5-aminolevulinic acid hydrochloride (1 mM final concentration, Acros Organics) was added prior to induction.24 Cells were induced with 1 mM IPTG at OD600 of 0.7. After overnight growth at 18 °C, cells were harvested by spinning at 4 °C, 4700 ×g for 25 min. The pellet was re-suspended in buffer (50 mM phosphate, pH 8.0, 300 mM NaCl, 5% v/v glycerol) and stored at –80 °C. Protein was purified by affinity chromatography (His60 Ni superflow resin, Clontech). Tag-free protein was generated by TEV cleavage of the His6 tag overnight at 4 °C and further purified by size-exclusion chromatography (Superdex 75, GE Healthcare) in 50 mM HEPES, pH 8.0, 150 mM NaCl, 2 mM TCEP and 5% v/v glycerol. Purified Sde 3804 and Sde 3557 were transferred to a glove bag (Coy Laboratory Products) under an argon/hydrogen (95:5%) atmosphere, at ~25 °C. Sde H-NOX was fully oxidized (λmax 403 nm) using 2 mM ODQ (Cayman Chemical) followed by buffer exchange through a Bio-Spin 6 column (Biorad). Each protein was then fully reduced to the Fe(II) state (λmax 430 nm) using 25 mM sodium dithionite (final concentration). Residual dithionite was removed by exchanging the protein into fresh pulldown buffer (50 mM HEPES, pH 8.0, 50 mM NaCl, 10 mM MgCl2, 2 mM TCEP and 5% v/v glycerol) or kinase assay buffer (50 mM TEA, pH 8.0, 50 mM NaCl, 10 mM MgCl2, 2 mM TCEP, 5 mM DTT and 5% v/v glycerol). Full length Sde HK (1-308) (Sde 3803) was cloned from S. degradans gDNA (ATCC) into a pHisGST parallel vector with an N-terminal glutathione-S-transferase (GST) tag. The fusion protein was expressed in BL21(DE3) cells in Terrific broth at 37 °C with 100 μg/mL ampicillin. Cells were induced at OD600 ~0.6 with 500 μM IPTG and grown overnight at 18 °C before harvest. The pellet was re-suspended in buffer (50 mM Tris, pH 8.0, 50 mM NaCl, 10 mM MgCl2, 25 mM L-glutamate and 10% v/v glycerol) and stored at –80 °C.23 HisGST Sde HK was purified using affinity chromatography (His60 Ni superflow resin) and size-exclusion chromatography (Superdex 200, GE Healthcare) in 50 mM HEPES, pH 8.0, 50 mM NaCl, 10 mM MgCl2, 25 mM L-glutamate, 2 mM TCEP and 10% v/v glycerol. Pulldown assay Pulldown experiments were carried out in the pulldown buffer described above. In a 300 μL reaction volume, 15 μL Pierce glutathione agarose beads (Thermo Fisher


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Biochemistry

Scientific) and 3 μM purified HisGST Sde HK were incubated with 15 μM Sde 3804 or Sde 3557 with or without NO in sealed tubes at 4 °C. NO was added using 10x DEA NONOate and 5x DETA NONOate (Cayman Chemicals) to the reaction mixture. After overnight incubation, beads were washed twice with buffer before elution. Protein was eluted using 20 μL of 6x SDS loading dye and was resolved by SDS-PAGE. Band intensity was quantified using ImageJ.29 Experiments were performed in two independent replicates. Binding kinetics measurement The binding kinetics between HisGST Sde HK and each Sde H-NOX were measured by Bio-Layer Interferometry (BLI) on a BLITz instrument (ForteBio) as previously described.23 Experiments were performed in advanced kinetics mode. After recording a 30 s initial baseline with kinase assay buffer alone, HisGST Sde HK (1 μM, 4 μL) was loaded to a pre-hydrated anti-GST biosensor (ForteBio) for 120 s followed by another 30 s to establish the baseline. Various concentrations of Sde HNOX (1-5 μM, 4 μL) in both the absence and presence of NO were then added for the kinetics measurement allowing 120 s for both the association and dissociation of the H-NOX. On-rates (k1), off-rates (k2) and the apparent dissociation constant (KD) were calculated using BLItz software in global fitting mode. Kinase assay All kinase assays were performed in the kinase assay buffer described above. Sde HK (5 μM) was incubated anaerobically for 30 min with Sde 3804 or Sde 3557 (5, 10, 25, 50, or 125 μM) treated without or with NO (10x DEA NONOate, 5x DETA NONOate) in a final volume of 20 μL. Assays were performed aerobically by initiating with a mixture of 2.5 μCi [γ-32P] ATP (PerkinElmer) and 1 mM ATP (final concentration). After 60 min incubation, reactions were quenched with SDS loading dye containing 20 mM EDTA (final concentration). Sde HK autophosphorylation was analyzed by SDS-PAGE as described previously.23, 24 Dried gels were exposed overnight on a phosphor imager plate (GE Healthcare) and imaged using a Typhoon Trio (GE Healthcare) set to storage phosphor mode at 100 μm resolution. Phosphorylated Sde HK bands were quantified using ImageJ software.29 Experiments were performed in two independent replicates. Determination of NO off-rates The NO off-rates were measured as previously described.30 NO-treated H-NOX samples were generated by mixing 2.5-5 μM Sde 3804 or Sde 3557 with 10x DEA NONOate and incubated for ~30 min to ensure complete decomposition of the NO donor. The CO/DT trapping solution (60 mM sodium dithionite, 50 mM TEA, pH 8.0, 50 mM NaCl, 5% v/v glycerol) was prepared anaerobically in a Teflon-sealed ReactiVial (Pierce) and saturated with CO (99.99%; Praxair, Inc.) immediately before use. The reaction was initiated by equal volume addition and rapid mixing of the CO/DT solution and the protein-NO complex. The final dithionite concentration in the reaction mixture was 30 mM. The reaction was monitored by electronic absorption spectroscopy using a Stopped-flow spectrometer (TgK Scientific) or a Cary 300 UVVis spectrophotometer (Agilent Technologies) at 20 °C. The dissociation of NO from


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the heme was monitored by the conversion of the Fe(II)–NO complex (Soret maximum at 399 nm) to the Fe(II)–CO complex (Soret maximum at 420 nm). Difference spectra were calculated by subtracting the first spectrum from each subsequent spectrum. The values extracted from the difference spectra were plotted as a function of time and fit to two parallel exponentials (of the form 𝑓(𝑥) = 𝐴 × (1 ― 𝑒 ―𝑘𝑥), where 𝑓(𝑥) is the change of signal amplitude at time 𝑥, 𝐴 is the total change in signal amplitude and 𝑘 is the observed reaction rate constant). Measurements were repeated in triplicate. S. degradans growth conditions Colonies of S. degradans Strain 2-40T were grown at 27 °C in half-strength Marine Agar (Difco Marine Agar 2216) for 48 hours. Liquid cultures were obtained by inoculating colonies from the agar plate into semi-defined medium (23 g/L Instant Ocean sea salt, 2 g/L urea, 2 g/L Tris base, 5 g/L tryptone, 2 g/L yeast extract) supplemented with 0.2% w/v glucose and grown at 27 °C with shaking at 200 rpm. The growth rate was monitored by OD600. For qPCR experiments, S. degradans was first grown in semi-defined medium containing 0.2% w/v glucose to OD600 ~0.35. The cells were harvested and transferred to fresh semi-defined medium supplemented with 0.2% w/v glucose, xylan (beechwood, Sigma-Aldrich), β-glucan (barley, Megazyme) or Avicel (Sigma-Aldrich) as the carbon source and grown for 4 hours. For each carbon source, 15 mL of cells were harvested for RNA extraction. RNA extraction and qPCR analysis For RNA extraction, cells were first incubated with 1 mL RNAprotect Bacteria Reagent (Qiagen) at room temperature for 5 min and then purified using Quick-RNA kit (Zymo Research) following manufacturer’s instructions. DNA contamination was removed using a TURBO DNA-free kit (Life technologies). Purified RNA was reversetranscribed to cDNA using SuperScript III First-Strand Synthesis System (Life technologies) using the random hexamers protocol. Primers for qPCR were designed using PrimerQuest Tool (Integrated DNA Technologies) to amplify ~120 bp regions of the ORF of the targeted gene. Primers for each gene were as follows: Sde 3557, 5’- GAG CAG TTT GCC GGT TAT TG-3’ and 5’-GCT CAA CAG TCT CTC CAT AGT G-3’; Sde 3803/Sde 3804, 5’- GGC AAA GGA ATG GAC CCT AA-3’ and 5’- CCA TGA GAG ATA GAA AGC CCT AAC-3’; guanylate kinase, 5’- CAC GCG CAG GTG TTT ACT A-3’ and 5’- CGC CTT GCC AGT CTA TTT CT-3’. qPCR reactions were performed on a QuantStudio 3 real-time PCR system (Thermo Fisher Scientific) using DyNAmo HS SYBR Green qPCR kit (Thermo Fisher Scientific). The cycling conditions were as follows: initial denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s and extension at 72 °C for 30 s. The melting curve was also measured from 60 °C to 95 °C with a 0.15 °C/s increment. The reaction volume was 10 μL. Primer efficiency for each gene was over 90%. Gene expression level was normalized with primer efficiency to give the final results. Computational analysis Five hundred bacterial proteomes, selected for diversity to span the whole domain, were downloaded from the Refseq ftp website.31 The SlopeTree conservation filter


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Biochemistry

was then applied with a parameter o = 5 to remove less well-conserved proteins.32 The reduced proteomes were then passed to the D2 code to build a phylogenetic tree.33 Separately, a blast database was built for the full, unfiltered 500 proteomes and searched for H-NOX proteins using the default blast parameters (evalue = 10).34 The H-NOX hits were then mapped onto the phylogenetic tree to assess the pattern of presence and absence. In addition, the number of bacteria having H-NOX and the number of copies of H-NOX for each bacterium are counted for each bacterial phylum. Broader search for H-NOX containing bacteria was performed using Vibrio cholerae (Vc) H-NOX as a query sequence in the Refseq database of 8163 total bacteria genomes with default blast parameter. Results and Discussion Bioinformatics analysis of S. degradans H-NOXs Two H-NOXs (Sde 3804 and Sde 3557) were identified in the S. degradans genome.3, 26 Sde 3804 has a gene local context similar to many previously described species where a cognate histidine kinase (Sde 3803, abbreviated Sde HK) is located on the same transcription unit (Figure 2a, top panel, Figure 2b).35-37 On the other hand, Sde 3557 is the only gene in the predicted transcription unit and lacks signaling partner protein in close proximity (Figure 2a, bottom panel).35-37 Sequence analysis revealed that both proteins contain a single H-NOX domain with ~180 residues (Figure 2b). They share 41% sequence identity and have the conserved heme ligating histidine and the YxSxR motif for stabilizing heme cofactor binding8 (Figure S1a). Residues forming the H-bonding network found in O2-sensing H-NOXs are not present in both H-NOX sequences, suggesting that Sde 3804 and Sde 3557 use NO as the physiological ligand.8, 9 Homology models of Sde 3804 and Sde 3557 generated by SWISS-MODEL exhibit a typical H-NOX fold38-42 (Figure S1b).


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Figure 2 a H-NOX H-NOX-associated HK RR other

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Figure 2. Sde H-NOXs gene local context and domain arrangements a) Top panel: gene local context and transcription unit of Sde 3803 (HK)/Sde 3804 (H-NOX). This HK/H-NOX pair is located on the same operon with a HD-GYP containing response regulator (RR) Sde 3805 and a hypothetical protein Sde 3806. Bottom panel: gene local context and transcription unit of Sde 3557. Sde 3557 is an orphan H-NOX that does not have any signaling partner on the same operon. b) Domain arrangement of Sde 3803, Sde 3804 and Sde 3557. Sde 3803 is the histidine kinase with an N-terminal domain (NTD), a dimerization histidine phosphotransfer domain (DHp) and a catalytic ATP-binding domain (CA). Sde 3804 is the kinase-associated H-NOX, with a single H-NOX domain. Sde 3557 is the orphan H-NOX.

Ligand binding properties of Sde 3804 and Sde 3557 and Sde HK interactions UV-Vis absorption spectra of purified Sde 3804 and Sde 3557 were measured to examine the ligand binding properties of both H-NOXs. Spectra of Sde 3804 and Sde 3557 exhibit the typical features of functional H-NOXs21, 22, 30 (Figure 3a). The Fe(II)unliganded state of both proteins exhibited a Soret maximum at 430 nm, representing the 5-coordinate Fe(II)–histidyl resting state. The NO complex has a


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Soret maximum at 399 nm, indicating the formation of the 5-coordinate Fe(II)– nitrosyl complex. CO binding to the heme cofactor forms a 6-coordinate complex with a Soret peak at 420 nm. As predicted from the sequence analysis, neither of the H-NOXs forms a complex with O2. To further characterize both H-NOXs, their ability to interact with the histidine kinase Sde HK was determined using pulldown assays in the absence and presence of NO (Figure 3b). Sde 3804 was observed to interact with Sde HK both with and without NO. This is expected because Sde 3804 is located on the same operon as Sde HK and they are likely to function as a protein complex as observed for other HNOX/HK cognate pairs.14, 24 Additionally, the normalized band intensity of Sde 3804 is higher when NO is present, consistent with what was observed in other HNOX/HK complexes where NO binding increases the affinity of the complex.23, 24 Conversely, only faint bands of Sde 3557 were observed by pulldown with Sde HK, suggesting that the affinity between Sde 3557 and Sde HK is weak and beyond the detection limit of the assay. Figure 2

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Biochemistry

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Figure 3. Ligand binding and kinase interaction of Sde 3804 and Sde 3557 a) Soret peaks of Sde 3804 and Sde 3557 upon NO and CO binding. b) Affinity pulldown assays to test kinase interaction with both H-NOXs in the absence and presence of NO. Error bars represent one standard deviation. Bands of Sde 3804 when HisGST Sde HK is present suggests tight interaction between the two proteins. Faint band of Sde 3557 pulled down by HisGST Sde HK indicates weaker interaction between the two proteins.


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To complement the pulldown assays, binding kinetics of the Sde H-NOXs and Sde HK were measured by bio-layer interferometry. This method also allows the observation of the interaction between Sde HK and Sde 3557, which was too weak to be detected in the pulldown experiment. Consistent with previous results, Sde 3804 exhibited higher binding affinity with Sde HK than Sde 3557 in both the presence and absence of NO (Table S1, Figure S2). Furthermore, enhanced binding affinity between NO-bound H-NOX and HK was observed for both H-NOXs, suggesting that both could play a role in the kinase regulation. Sde HK autophosphorylation inhibition Kinase autophosphorylation assays were performed with both H-NOXs to determine the effect on kinase activity. To optimize conditions, the autophosphorylation activity of the Sde HK was first examined. The time-dependence of kinase autophosphorylation was determined by incubating HisGST Sde HK with γ-32P ATP for different time periods (Figure S3). An increasing level of phosphorylated kinase was detected between 0 and 90 min, and the signal saturated between 90 min and 120 min. Based on this result, a 60 min incubation time was chosen for the H-NOX inhibition assay. HisGST Sde HK was incubated with Sde 3804 or Sde 3557 in the absence and presence of NO. The kinase activity was then examined by autophosphorylation with γ-32P ATP. As expected, Sde HK activity is inhibited by Sde 3804 in an NOdependent manner, demonstrating that Sde 3804 is a functional H-NOX for its cognate kinase Sde HK (Figure 4a). Surprisingly, Sde 3557 is also capable of inhibiting Sde HK autophosphorylation, despite their distant genomic localization and weak binding affinity (Figure 4b). The kinase inhibition potency of Sde 3804 and Sde 3557 is comparable, indicating that Sde 3557 might also form a functional H-NOX/HK pair with Sde HK in vivo (Figure 4b). It is worth noting that the kinase activity was inhibited with high concentrations of H-NOX in the absence of NO. This has been observed before and is due to the intrinsic flexibility of the H-NOX subdomains, which allows the protein to adopt an inhibitory conformation without NO.24


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Biochemistry

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Figure 4. Sde HK autophosphorylation inhibition assay a) Sde HK autophosphorylation inhibition by Sde 3804. b) Sde HK autophosphorylation inhibition by Sde 3557. Sde HK was incubated with varying concentrations of of Sde H-NOXs in the absence or presence of NO. Kinase activity is quantified in bar graph below. Error bars represents one standard deviation. Both H-NOXs are capable to inhibit kinase activity in NO-dependent manner.

In order to confirm that the NO-dependent kinase inhibition observed above is not due to NO destabilizing or denaturing the kinase, kinase autophosphorylation activity was measured with direct addition of NO (Figure S4a). Kinase activity was also measured with CO and CO-bound H-NOXs (Figure S4a,b). The results showed that the kinase activity was not affected by NO and CO. In addition, CO-bound H-


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NOXs failed to inhibit the kinase activity, suggesting that CO is not a physiologically relevant ligand for S. degradans H-NOXs (Figure S4). NO dissociation rates of Sde 3804 and Sde 3557 In NO-dependent kinase inhibition, the rate at which the NO molecule dissociates from the H-NOX is a key factor determining the timescale of inhibition. Therefore, the NO off-rates of Sde 3804 and Sde 3557 were measured spectroscopically using CO saturated sodium dithionite (NaDT/CO) as an NO trap.6, 43-45 In this experiment, dissociated NO is removed through reaction with dithionite. CO will then bind to the heme cofactor, preventing any NO rebinding. The dissociation of NO with concomitant CO binding will result in a decrease of the Soret peak at 399 nm (5coordinate NO complex) and increase of the Soret peak at 420 nm (6-coordinate CO complex). The NO off-rate of the H-NOX, which is typically relatively slow, is derived from these spectral changes using standard UV-Vis spectroscopy. This method has been frequently used in determining NO dissociation rates for other H-NOXs and sGC.30, 43 The first attempt to measure the NO off-rate of Sde 3804 using a UV-Vis spectrometer was unsuccessful because the reaction was complete during the mixing time (~30 s), indicating a fast NO dissociation rate. As a result, the measurement was performed on a stopped-flow spectrometer. The spectral changes observed for Sde 3804 is shown in Figure 5a. The shoulder at 420 nm at t = 0 indicates that some NO dissociation has occurred during the dead time of the instrument (~1.5 ms). The reaction plateaued after ~375 s; the difference of the absorption at 420 nm and 399 nm was plotted as a function of time (Figure 5c). Similar to other H-NOXs, the data fit to a double-exponential equation with two rate constants (kfast and kslow)30, 43 (Figure 5c,e, Table 1). The kfast is 10x faster than the kslow. However, kslow dominates the fit, contributing ~70% of the total amplitude (ΔA) and therefore is the deciding factor in the NO dissociation rate. When compared to previous data, Sde 3804 has the fastest NO dissociation rate among all bacterial H-NOXs.22, 30


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Biochemistry

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Figure 5. NO off-rate measurement for Sde 3804 and Sde 3557 a,b) Time courses for NO dissociation of Sde 3804 (left) and Sde 3557 (right) at 20 °C determined using a CO/dithionite trap for NO. NO dissociation is shown as a decrease in the Soret peak at 399 nm and an increase in the Soret peak at 420 nm. Red line indicates spectrum at t = 0. c,d) NO off-rates extracted from double exponential fitting of the difference spectra at 399 nm and 420 nm over time. e,f) Residual plots of the fittings in c) and d). Table 1. NO off-rates for Sde 3804 and Sde 3557 Sde 3804 Sde 3557 -3 -1 kfast (×10 s ) 85.4 ± 1.8 4.2 ± 0.3 -4 -1 kslow (×10 s ) 97.0 ± 1.8 3.3 ± 0.6 ΔAfast (%) 28.2 ± 1.0 32.8 ± 3.5 ΔAslow (%) 71.8 67.2


Biochemistry

Conversely, the NO off-rate for Sde 3557 is similar to that of other bacterial H-NOXs, where the NO dissociation is complete in hours rather than seconds 30 (Figure 5b,d). The 420 nm and 399 nm absorption difference was also fit to a double exponential equation (Figure 5d,f, Table 1). The dominant rate kslow (ΔAs ~70%) for Sde 3557 is similar to that of So H-NOX and is 30x slower than that of Sde 3804. This result indicates that Sde 3557 could inhibit kinase activity for significantly longer than Sde 3804. The slower NO off-rate for Sde 3557 may play an important role in vivo. In vivo expression levels of Sde 3804 and Sde 3557 In vivo expression level of Sde 3804 and Sde 3557 was first measured from S. degradans in standard growth media using glucose as carbon source (Figure S5). Sde 3695, which encodes a guanylate kinase, was chosen as the reference.27, 46 Although both genes are weakly expressed comparing to Sde 3695, the expression level of Sde Figure 3557 is5~4x higher than that of Sde 3804 (Figure 6). Sde 3804 Sde 3557 1.6 1.4 relative expression level

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Figure 6. Expression level of Sde 3804 and Sde 3557 with different carbon sources for growth mRNA levels of Sde 3804 and Sde 3557 are measured from S. degradans cultured in different carbon sources. Expression level is normalized to the reference gene coding for guanylate kinase. Error bars represent one standard deviation. Both genes are expressed in the four conditions tested. The expression level of Sde 3557 is 3-5x higher than Sde 3804.

Since S. degradans is capable of degrading a variety of polysaccharides, the mRNA levels of Sde 3804 and Sde 3557 were also measured from growth on other carbon sources (xylan, β-glucan and Avicel).27 While the absolute expression levels vary depending on the carbon source, the relative expression levels of the two H-NOX proteins is similar (Figure 6). In all cases, Sde 3557 mRNA level remains 3-5x higher than that of Sde 3804. This synchronized up/down-regulation of the H-NOXs may suggest that they are involved in the same signaling pathway. While Sde 3557 has a lower affinity with Sde HK, the bacteria may use a higher expression level of Sde 3557 to compensate for the low affinity in order to form a functional signaling


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Biochemistry

complex between Sde 3557 and Sde HK. However, the in vivo roles of Sde 3557 still require further investigation. Computational analysis Normally when two copies of a gene are present in a genome, it could either be from horizontal gene transfer, in which genetic material was acquired in some way from a separate organism, or from gene duplication, in which the original gene is copied to a second location in the same genome and then often develops a new function separate from the function of the original gene.47 Since both Sde 3804 and Sde 3557 are expressed, this is a good indication that they are functional in vivo. This leads to the following questions: why does the organism have two distinct H-NOXs and what is the origin of these two H-NOXs (horizontal gene transfer vs. gene duplication). This is difficult to address because S. degradans is the only identified species in its genus and it cannot readily be manipulated genetically. Therefore, computational methods were used to potentially provide a hypothesis. A preliminary blast search of H-NOX proteins revealed that they are occasionally present in multiple copy number, with S. degradans having two copies and Pontibacter korlensis, a member of the Bacteroidetes, having three. Multiple copy number, particularly when combined with an uneven distribution of genes (e.g. a gene occurring only once in a phylogenetic group of related species or homologs being frequently absent in such a group) is a signature of horizontal transfer. A phylogenetic tree containing 500 diverse bacterial species was constructed and the prevalence of H-NOX proteins was mapped to the tree. It was found that the tree exhibits an idiosyncratic signature of H-NOX prevalence (Figure 7a). For instance, only one Chloroflexi out of five has a copy of H-NOX (Table S2). Based on known principles of bacterial evolution and horizontal gene transfer, we hypothesize that both H-NOXs from S. degradans were acquired through horizontal gene transfer. Conversely, the hypothesis that a common ancient bacterial had a copy of H-NOX that was then unevenly dropped and then duplicated, giving rise to the scattered presence of H-NOX observed in the tree, is far less plausible.


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Figure 7. Computational analysis of H-NOX in bacteria genomes a) Subtree of bacterial species tree over conserved genes. Subtree containing S. degradans and closely related species from a larger phylogenetic tree that includes over 500 diverse species of bacteria. b) Histogram of H-NOX-containing bacteria. In 8163 bacterial genomes searched, 7499 has 0 copies of H-NOX, 632 has 1 copy of H-NOX, 30 has 2 copies of H-NOX and 2 has 3 copies of H-NOX.

Of the 500 bacterial species analyzed, Alteromonadales bacterium BS08 is most closely related to S. degradans. Although A. bacterium and S. degradans belong to different families, Sde 3804 shares over 60% sequence identity with the H-NOX from A. bacterium BS08, which is significantly higher than the 41% identity with the orphan H-NOX Sde 3557. A synteny analysis between S. degradans and A. bacterium BS08 revealed additional information about the history of the S. degradans H-NOX genes, suggesting very different evolutionary paths for the two genes. The genes


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Biochemistry

near Sde 3804 have high synteny with those of A. bacterium BS08 (Figure S6a). Given the size of the syntenic block, it is likely that the common ancestor for these two bacteria already had this region in its genome, although this shared region could also be the result of large horizontal transfer. Conversely, Sde 3557 does not share high sequence similarity with any other known H-NOX. Moreover, it is located in a gap between two high synteny regions (Figure S6b). Although it is possible that Sde 3557 is a duplication of Sde 3804 with its sequence diverging to develop a new function, Sde 3557 was likely obtained via a more recent horizontal gene transfer event from a species that has not yet been identified and/or sequenced. Search for other orphan H-NOXs in bacteria genomes A broader search in 8163 bacteria genomes was performed to identify whether orphan H-NOXs exist in other species. The search resulted 664 H-NOX-containing bacteria, with 632 species have one copy of H-NOX, 30 species have 2 copies of HNOX and 2 species have 3 copies of H-NOX (Figure 7b, Table S3). Analysis of H-NOXassociated protein in bacteria containing multiple copies of H-NOX showed that in most cases, each H-NOX is associated with a typical signaling partner. However, besides S. degradans, orphan H-NOXs were found in other bacteria species (Table S4). For example, in Fulvivirga imtechensis, only one of the two copies of H-NOX is associated with histidine kinase. There are also a few cases where none of the HNOXs have signaling partner nearby, such as Adhaeribacter aquaticus, Alcanivorax jadensis and Pontibacter korlensis. Without in vivo data, it is difficult to assess whether the orphan H-NOXs in those bacteria are functional. Nevertheless, the search result here suggest that orphan H-NOX is not unique to S. degradans. Other bacteria may also benefit from this strategy in adaptation to the environment. Concluding remarks NO-dependent signaling is critical for numerous physiological processes. Archetypal proteins involved in sensing NO have been well characterized, including H-NOX proteins. Until recently, H-NOX proteins were found either fused to or cotranscribed with signaling partners such as chemotaxis proteins or histidine kinases. Moreover, most H-NOX-containing bacteria have only one copy of an H-NOX gene. However, blast search revealed that a small number of bacteria have multiple copies. Among them, S. degradans is the only identified species that encodes a typical H-NOX (Sde 3804)/HK (Sde 3803) pair as well as an orphan H-NOX (Sde 3557) that has no adjacent signaling partner. As such, it was an interesting target to better understand the role of orphan H-NOXs in bacteria. In this study, two H-NOXs from S. degradans were characterized in terms of ligand binding, kinase binding affinity, NO-dependent kinase inhibition and in vivo expression. A few key conclusions emerged. First, both H-NOXs showed the ability to bind gas ligands like other H-NOXs, and NO is likely to be the physiological ligand. Second, although the orphan H-NOX Sde 3557 exhibits lower affinity with Sde HK than the HK-associated H-NOX Sde 3804, the kinase inhibition assay showed both HNOXs are functional H-NOXs with the ability to adopt a kinase inhibitory conformation in an NO-dependent manner. Third, the NO dissociation rate of


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orphan H-NOX Sde 3557 is much slower than that of Sde 3804, suggesting that Sde 3557 could persist in the NO-bound state for a longer period of time and lengthen the lifetime of an NO-induced signal in vivo. Lastly, the expression level of Sde 3557 is always 3-5x higher than that of Sde 3804 and Sde HK, which could potentially help Sde 3557 to compete with Sde 3804 for Sde HK binding in vivo, notwithstanding its lower kinase binding affinity. The genome of S. degradans does not contain a nitric oxide synthase (NOS). Therefore, Sde 3804 and Sde 3557 are likely involved in sensing environmental NO as a host defense or perhaps from denitrification.27 Having two copies of H-NOX with different NO binding properties controlling the same downstream histidine kinase may give the bacteria some advantage in NO signaling, though this is difficult to directly test.18, 48 For example, when a prolonged kinase inhibition is required under certain circumstances, Sde 3557, which has a slower NO off-rate, could serve as a compensation for Sde 3804 to extend the lifetime of the NO signaling. It is also interesting that the expression of both H-NOXs change over different carbon sources, with Avicel giving the highest expression level and glucose giving the lowest expression level. While there is no clear connection between polysaccharides consumption and NO signaling, it is worth investigating the potential signaling pathways that might interlink the carbon metabolism and H-NOX function in S. degradans. Taken together, Sde 3557 is the only identified orphan H-NOX in all bacteria genomes to date. Likely acquired through horizontal gene transfer from a species that has not been sequenced, Sde 3557, the first identified orphan H-NOX, is able to regulate kinase activity from a H-NOX/HK pair in S. degradans. Although the data presented here does not rule out the possibility for Sde 3557 to have its own signaling partner other than Sde HK, this study serves as a unique example of a bacteria exploiting a dual-H-NOX system to fine-tune the downstream response in NO signaling. Supporting information Sequence analysis of Sde 3804 and Sde 3557 (Figure S1); BLItz traces for HisGST Sde HK interaction with Sde 3804 and Sde 3557 in the absence and presence of NO (Figure S2); Time dependence of Sde HK autophosphorylation (Figure S3); Effects of NO and CO to Sde HK activity (Figure S4); Growth curve of S. degradans (Figure S5); Synteny analysis between Saccharophagus degradans and Alteromonadales bacterium BS08 (Figure S6); H-NOX and HK binding kinetics from BLItz (Table S1); Pattern of presence and absence of H-NOX per each bacterial phylum (Table S2); List of H-NOX-containing bacteria (Table S3); H-NOX-associated protein in multicopy H-NOX-containing bacteria (Table S4). Abbreviations CA, catalytic domain; cGMP, guanosine 3,5-cyclic monophosphate; DEA, diethylamine; DETA, diethylenetriamine; DHp, dimerization and histidine


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Biochemistry

phosphotransfer domain; GST, glutathione-S-transferase; GTP, guanosine 5triphosphate; H-NOX, Heme-Nitric oxide/Oxygen protein; HEPES, 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid; HK, histidine kinase; MCP, methylaccepting chemotaxis protein; NO, nitric oxide; pGpG, diguanylic acid; NTD, Nterminal domain; RR, response regulator; sGC, soluble guanylate cyclase; TCEP, tris(2-carboxyethyl)phosphine; TEV, Tobacco Etch Virus protease Author Contributions Y.G. and M.A.M. designed the research; Y.G. and M.M.C. performed the cloning, protein expression/purification, pulldown assays, kinase assays and NO off-rates measurement. Y.G. performed the cell culture and qPCR experiments. R.B. performed computational analysis. Y.G., M.M.C., R.B. and M.A.M. wrote the paper. Acknowledgements We would like to thank current and former members of the laboratory of Michael A. Marletta for frequent helpful discussions and invaluable insight, particularly Benjamin G. Horst, Drs. Charles W. Hespen, Marco Agostoni, Christopher M. Lemon, John Hangasky and Elizabeth C. Wittenborn. We would also like to thank Ko-Chuan Lee and Dr. Chun-Hao Huang (Jennifer Doudna lab) for help with the qPCR experiment. The Saccharophagus degradans Strain 2-40T was kindly provided by Dr. Steven W. Hutcheson at University of Maryland. References [1] Domingos, P., Prado, A. M., Wong, A., Gehring, C., and Feijo, J. A. (2015) Nitric oxide: a multitasked signaling gas in plants, Mol Plant 8, 506-520. [2] Farah, C., Michel, L. Y. M., and Balligand, J. L. (2018) Nitric oxide signalling in cardiovascular health and disease, Nat Rev Cardiol 15, 292-316. [3] Plate, L., and Marletta, M. A. (2013) Nitric oxide-sensing H-NOX proteins govern bacterial communal behavior, Trends Biochem Sci 38, 566-575. [4] Iyer, L. M., Anantharaman, V., and Aravind, L. (2003) Ancient conserved domains shared by animal soluble guanylyl cyclases and bacterial signaling proteins, BMC Genomics 4, 5. [5] Derbyshire, E. R., and Marletta, M. A. (2012) Structure and regulation of soluble guanylate cyclase, Annu Rev Biochem 81, 533-559. [6] Boon, E. M., Davis, J. H., Tran, R., Karow, D. S., Huang, S. H., Pan, D., Miazgowicz, M. M., Mathies, R. A., and Marletta, M. A. (2006) Nitric oxide binding to prokaryotic homologs of the soluble guanylate cyclase beta1 H-NOX domain, J Biol Chem 281, 21892-21902. [7] Karow, D. S., Pan, D., Tran, R., Pellicena, P., Presley, A., Mathies, R. A., and Marletta, M. A. (2004) Spectroscopic characterization of the soluble guanylate cyclase-like heme domains from Vibrio cholerae and Thermoanaerobacter tengcongensis, Biochemistry 43, 10203-10211. [8] Pellicena, P., Karow, D. S., Boon, E. M., Marletta, M. A., and Kuriyan, J. (2004) Crystal structure of an oxygen-binding heme domain related to soluble guanylate cyclases, Proc Natl Acad Sci U S A 101, 12854-12859.


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