Discovery of a Nitric Oxide Responsive Quorum Sensing Circuit in

Subscriber access provided by Kaohsiung Medical University

Letter

Discovery of a Nitric Oxide Responsive Quorum Sensing Circuit in Vibrio cholerae Sajjad Hossain, Ilana Heckler, and Elizabeth M Boon ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00360 • Publication Date (Web): 30 Jul 2018 Downloaded from http://pubs.acs.org on August 1, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Biology

Discovery of a Nitric Oxide Responsive Quorum Sensing Circuit in Vibrio cholerae

Sajjad Hossain, Ilana Heckler, and Elizabeth M. Boon*

Department of Chemistry and Institute of Chemical Biology & Drug Discovery, Stony Brook University, Stony Brook, New York 11794, United States

*To whom correspondence should be addressed: Elizabeth M. Boon, Department of Chemistry, Stony Brook University, Stony Brook, NY, USA 11790; Tel.: (631) 632-7945; Fax: (631) 6327960; orchid id: orcid.org/0000-0003-1891-839X; E-mail: [email protected]

ACS Paragon Plus Environment

ACS Chemical Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Group behavior of the human pathogen Vibrio cholerae, including biofilm formation and virulence factor secretion, is mediated by a process known as quorum sensing. Quorum sensing is a way by which bacteria coordinate gene expression in response to population density through the production, secretion, and detection of small molecules called autoinducers. Four autoinducer-mediated receptor histidine kinases have been implicated in quorum sensing through the phosphotransfer protein LuxU: CqsS, LuxP/Q, CqsR, and VpsS (Vc1445). Of these receptor kinases, VpsS is predicted to be cytosolic and its cognate autoinducer is currently unknown. In this study, we demonstrate that the nitric oxide-bound complex of a member of the recently discovered family of nitric oxide-responsive hemoproteins called NosP (VcNosP is encoded by Vc1444; this gene product is also known as VpsV) inhibits the autophosphorylation activity of VpsS and thus phosphate flow to LuxU. Therefore, we propose that VpsS contributes to the regulation of quorum sensing in a nitric oxide-dependent manner through its interaction with NosP.


Page 2 of 26

Page 3 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC GRAPHIC



Nitric oxide (NO) is a bacterial toxin at high concentrations, requiring detection by flavohemoglobins, nitric oxide reductases, and cytochrome c nitrite reductases, which convert NO into less toxic products.1 Interestingly, it appears that bacteria also respond to nanomolar or lower concentrations of NO to regulate physiological processes such as biofilm formation and dispersal.2

The best characterized sensors of low concentrations of NO in bacteria are a family of hemoproteins named H-NOX (heme-nitric oxide/oxygen binding) that bind NO with high affinity and selectivity.3,4 Based on currently available data, NO/H-NOX signaling is primarily implicated in biofilm formation through regulation of cellular cyclic-di-GMP pools.5-8 However, our laboratory has discovered that NO/H-NOX signaling also plays a role in quorum sensing in the bioluminescent bacterium Vibrio harveyi.8,9 Quorum sensing is a way that bacteria communally regulate gene expression in response to population density through the production, secretion, and detection of small molecules called autoinducers.10 We found that NO ligation to H-NOX regulates V. harveyi light production8 and biofilm formation,9 both of which are quorum sensing-mediated behaviors, through NO/H-NOX regulation of the autokinase activity of HqsK (H-NOX-associated quorum sensing kinase) and subsequent phosphate flux through the LuxU/LuxO quorum sensing circuit. These studies suggest that NO can act analogously to an autoinducer in some bacteria.

H-NOX proteins are not encoded in all bacteria that are responsive to low concentrations of NO, however, suggesting that another bacterial NO sensor must exist. Indeed, our laboratory recently discovered an additional NO sensing protein, NosP, which is broadly conserved in bacterial


Page 4 of 26

Page 5 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


genomes.11 Like H-NOX, NosP coordinates NO via ligation to the iron atom of a heme cofactor. We first characterized NosP from the opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa does not encode an hnoX gene) and demonstrated that NosP causes biofilm dispersal in the presence of NO through regulation of an associated histidine kinase signaling pathway.11

We have been interested in NO signaling in the pathogenic bacterium Vibrio cholerae because although NO/H-NOX signaling has been demonstrated, it cannot fully explain the action of low concentrations of NO in this species. For example, in the presence of NO-bound H-NOX, autophosphorylation of an H-NOX-associated histidine kinase is inhibited.12,13 This results in decreased phosphotransfer to two response regulator proteins: an active EAL-type cyclic-diGMP

phosphodiesterase

(Vc1086),

and

a

degenerate

HD-GYP-type

cyclic-di-GMP

phosphodiesterase (Vc1087).7 Based on this biochemical characterization, NO ought to yield an increase in intracellular cyclic-di-GMP concentrations and increased biofilm. On the contrary, V. cholerae has been shown to disperse from biofilm in response to nanomolar NO.14 In addition, the inhibition of the H-NOX-associated histidine kinase was shown to be independent of NO binding to H-NOX, as the ferric un-ligated form of H-NOX inhibited the kinase comparably.12

Thus, we reasoned that NosP may play a role in the response of V. cholerae to NO. V. cholerae encode a NosP protein (Vc1444; Vc1444 is also known as VpsV) in the same putative operon as the soluble hybrid histidine kinase VpsS (Vc1445).15 Interestingly, VpsS has been implicated in the regulation of biofilm through quorum sensing processes. The current model of quorum sensing in V. cholerae suggests that there are at least four quorum sensing pathways that work in parallel to allow detection of, and response to, multiple quorum sensing inputs.16 Each quorum



sensing pathway centers on a hybrid histidine kinase receptor that specifically binds to its cognate autoinducer. Three of these receptors, CqsS, LuxP/Q, and Vc1831 (CqsR), are membrane associated and the fourth, VpsS, is predicted to be cytosolic (Figure 1). [Figure 1] V. cholerae relies on quorum sensing for the regulation of biofilm-specific genes, such as those involved in the synthesis of the exopolymeric matrix (Vibrio polysaccharide genes, vps), and for virulence factor secretion.16 In contrast to other bacteria, in V. cholerae, low cell density triggers biofilm formation and secretion of virulence factors, and high cell density promotes biofilm dispersal and latency. When autoinducer concentrations are high, typically at high cell density, each receptor kinase binds its cognate autoinducer and acts as a phosphatase, drawing phosphate away from the common phosphotransfer protein LuxU. LuxU, in turn, dephosphorylates LuxO, a transcription factor that regulates the expression of four homologous regulatory small RNAs (Qrrs 1-4). Under these conditions, the transcription factor HapR (a homolog of V. harveyi’s LuxR) is expressed, which inhibits the transcription of genes responsible for biofilm formation as well as the master regulator of virulence factor production, AphA.17 However, when autoinducer concentration is low, the autoinducer receptors act as kinases and increase the phosphate flux towards LuxU and LuxO. Phosphorylation of LuxO, through the Qrrs, results in inhibition of HapR expression, which ultimately results in de-repression of both AphA and the biofilm vps genes, resulting in increased virulence and biofilm formation.18

As mentioned above, V. cholerae encodes a nosP gene in the same operon as the soluble quorum sensing kinase VpsS. Previous studies have shown that a mutant strain of V. cholerae lacking vpsS exhibits a decrease in the expression of vps genes and that overexpression of vpsS results in


Page 6 of 26

Page 7 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


LuxO-mediated activation of vps genes and a hyperbiofilm phenotype; indeed VpsS is named for vibrio polysaccharide biosynthesis sensor.15 It has also been demonstrated VpsS is one of four independent kinases that activate LuxO through LuxU to regulate quorum sensing in V. cholerae and that VpsS activity alone is sufficient for V. cholerae colonization of mouse intestine.19 Therefore, VpsS is involved in LuxU/LuxO quorum sensing in V. cholerae, but at present, no cognate autoinducer has been identified for VpsS. Because VpsS is predicted to be co-cistronic with a NosP protein, we hypothesized that NO-bound VcNosP might serve this role, participating in V. cholerae quorum sensing by regulating the kinase activity of VpsS, perhaps analogously to the NO/H-NOX/HqsK-mediated quorum sensing pathway we reported in V. harveyi.8

As mentioned above, VcNosP (Vc1444) is also known as VpsV, because it is in the same operon as VpsS, and like VpsS, its deletion leads to a decrease in vps gene expression.15 Furthermore, it has previously been predicted that this protein may serve as the signal-sensing partner of VpsS.19 Purified NosP has never been characterized in V. cholerae, however, so we first explored the in vitro gas binding properties of VcNosP (Figure 2A). The spectra of hemoproteins feature a strong absorption band around 400-430 nm, called a Soret band, and two minor peaks (α and β) in the range of 500-650 nm; these spectral characteristics are sensitive to coordination by exogenous ligands as well as the oxidation state of the heme iron.3 Thus, electronic spectroscopy is a convenient method to characterize the ligand biding properties of hemoproteins. [Figure 2] We found that VcNosP (45 kDa), as purified from E. coli, has a Soret band with a maximum at 411 nm, which is indicative of a mixture of ferrous and ferric heme complexes. Treatment of



VcNosP with ferricyanide yielded the ferric complex with an accompanied shift of the Soret band maximum to 409 nm. Upon anaerobic addition of the reducing agent dithionite, the ferrous complex was observed with a Soret maximum of 420 nm and sharp α and β peaks at 554 nm and 524 nm, respectively. When carbon monoxide (CO) was added to ferrous VcNosP, a sharp Soret band with a maximum at 418 nm resulted. The VcNosP FeII-CO spectrum is suggestive of a lowspin, 6-coordinate complex. Incubation of NO with ferrous VcNosP resulted in the formation of a complex with a broad Soret peak at 396 nm, indicative of a high-spin, 5-coordinate FeII-NO complex. These ligand binding properties are similar to what has been observed for NosP from P. aeruginosa,11 as well as other NO-sensing hemoproteins such as H-NOX.3

If NosP is acting as an NO sensor in V. cholerae, we would expect it to bind NO at sub-toxic concentrations. To assess the affinity of NO for VcNosP, the NO dissociation rate constant of VcNosP was determined using a standard CO/dithionite trap, in which dithionite reacts with dissociated NO and CO binds to heme iron upon NO dissociation in order to prevent NOrebinding. NO dissociation is measured by following the formation of the FeII-CO complex.20 Representative spectra for a NO dissociation kinetics experiment are shown in Figure 2B. The data were fit as described previously11 and yield a rate constant of (4.6 ± 0.1) x 10-4 s-1 (Figure 2C). This rate constant is independent of CO concentration as well as dithionite at all concentrations tested (300 µM, 3 mM, 30 mM, and 100 mM dithionite). The VcNosP NO dissociation rate constant is comparable to both the rate constant reported for the dissociation of NO from P. aeruginosa NosP (1.8 x 10-4 s-1),11 and the reported NO dissociation rate constant for sGC (3.6 x 10-4 s-1; sGC is a mammalian member of the H-NOX family).21 Interestingly, the NO dissociation rate constant for VcNosP is much slower than the NO dissociation rate constant


Page 8 of 26

Page 9 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


reported for VcH-NOX (0.3 s-1).22 This difference may indicate that VcH-NOX and VcNosP have differing roles in V. cholerae physiology, or that perhaps they are sensitive to different NO concentration ranges. We anticipate the NO association rate constant for NosP to be similar to that of other histidine-ligated hemoproteins (104-108 M-1 s-1),23 resulting in a NO dissociation constant no worse than nanomolar, consistent with a role for NosP in NO signaling.

To further test our hypothesis that NO/NosP might regulate quorum sensing in V. cholerae, we evaluated the effect of VcNosP on the in vitro autophosphorylation activity of the quorum sensing kinase VpsS. Purified VpsS (65 kDa) is an active kinase, demonstrating stable autophosphorylation over more than 30 minutes upon incubation with ATP (containing trace [γ32

P]-ATP) (Figure 3A). Addition of NO-bound VcNosP inhibits VpsS autophosphorylation in a

concentration dependent manner while ferrous VcNosP shows little effect (Figure 3B, C). These data are consistent with many previous studies demonstrating kinase inhibition by the NO-bound hemoprotein sensors H-NOX and NosP,20,21 including NO/NosP inhibition of NahK in P. aeruginosa,11 and NO/H-NOX inhibition of the quorum sensing kinase HqsK in V. harveyi.8 [Figure 3] If, indeed, NO/NosP is participating in quorum sensing in V. cholerae, we would expect VpsS to transfer phosphate to LuxU. To explore this possibility, we performed a phosphotransfer assay. Phosphotransfer from VpsS to LuxU is evident from the appearance of a radiolabeled band that corresponds to the molecular weight of LuxU (13 kDa) upon incubation of radiolabeled VpsS with LuxU (Figure 4A). This result is consistent with a previous study showing that VpsS is capable of accepting phosphate from phosphorylated LuxU in vitro.15 When a phosphotransferdeficient kinase (VpsSDA), in which the conserved aspartate in its receiver domain was mutated



to an alanine, is incubated with LuxU, no phosphotransfer is observed. The increased phosphorylation of VpsSDA relative to WT VpsS is presumably due to the higher stability of the phosphoramidate formed when phosphate is trapped on histidine in VpsSDA, in comparison to the phosphoanhydride formed when phosphate is transferred to aspartate in the WT enzyme.25 Further, in the presence of NO-bound NosP, phosphotransfer from VpsS to LuxU is inhibited, as evident by a decrease in phosphorylated LuxU (Figure 4B). [Figure 4] Taken together, these experiments suggest that V. cholerae may detect NO to modulate gene expression through regulation of VpsS in a LuxU/LuxO-dependent manner. This proposed NO/NosP-mediated quorum sensing pathway is very similar to the NO/H-NOX-mediated quorum sensing pathway we reported in V. harveyi.8 In V. harveyi, NO/H-NOX regulates the autophosphorylation activity of the soluble hybrid histidine kinase HqsK, which in turn contributes to phosphate flux through LuxU/LuxO to ultimately regulate bioluminescence and other high cell density quorum sensing-mediated group behaviors. As in V. harveyi, it appears that in V. cholerae, NO acts analogously to an autoinducer to mimic a high cell density state (Figure 1B) and mediates phosphate flux through LuxU through regulation of a quorum sensing kinase (VpsS) in complex with a hemoprotein (NosP). Interestingly, this pathway would explain the biofilm dispersal phenotype of V. cholerae in the presence of NO,14 as inhibition of VpsS autophosphorylation by NO-bound VcNosP should repress vps biofilm genes by decreasing phosphate flow through the LuxU-based quorum sensing pathway in V. cholerae.

The existence of an NO responsive QS circuit in V. cholerae may be relevant to host-microbe interactions during infection. V. cholerae may sense NO as an environmental cue to regulate


Page 10 of 26

Page 11 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


biofilm and virulence during infection of the human intestine. In eukaryotes, NO is produced enzymatically by NO synthases (NOS) from arginine, however there are no predicted NOS genes found in the V. cholerae genome.26 NO produced by the eukaryotic host may therefore act as an interkingdom signaling molecule. Acute cholera infection has been previously shown to elicit NO production in humans.27 The link between V. cholerae infection and host NO production raises the possibility that the bacterium may use endogenously produced NO to monitor stages of pathogenesis and accordingly regulate biofilm modes.

What has yet to be elucidated is the potential advantage to bacteria of encoding more than one NO sensor (such as H-NOX and NosP). A possible explanation for why V. cholerae encodes both H-NOX and NosP could be drawn from consideration of a recent proposal suggesting that V. cholerae H-NOX may be involved in heme-independent redox sensing.12 This finding suggests that VcH-NOX may act as a dual NO/redox sensor, depending on the environment. Therefore, under certain circumstances NosP may act as a primary NO sensor. It would also be worth considering possible crosstalk between the H-NOX and NosP pathways.

METHODS Cloning. Genes were amplified using PCR from Vibrio cholerae genomic DNA and cloned into a pET20b vector appending His6 at the C terminus by the use of NdeI and XhoI restrictions enzymes. Primers used to clone and strains of E. coli used to clone and express proteins are listed in Table1 in the Supporting Information.



Expression and purification. All proteins were expressed and purified as described previously.11 The desalted protein was incubated with 300 µM hemin overnight at 4 ºC. The following day, unbound heme was separated from the heme-protein complex using size exclusion chromatography on a Superdex 200 column. Colored fractions were collected and stored at -80 ºC in 10% glycerol.

Electronic spectroscopy. All electronic spectra were recorded on a Cary 100 spectrophotometer equipped with a constant temperature bath. NosP complexes were prepared in an oxygen-free environment using a COY anaerobic glove bag as described previously.28

NO dissociation kinetics. The formation of a Soret band at 418 nm corresponding to the FeII-CO protein complex was monitored over time upon addition of a CO-saturated dithionite solution to the FeII-NO complex of Vc1444, as previously described.29 Reactions were kept at 22 °C using a temperature bath. Controls reactions were run with varying dithionite concentration (300 µM, 3 mM, 30 mM, and 100 mM dithionite) and in the absence of CO to ensure that the measured rate constants were independent of dithionite and CO.

Kinase and phosphotransfer assays. Kinase assays were performed in triplicate as described previously,11 with the following exceptions. Reactions were started by the addition of an ATP solution so that the final ATP concentration was 2 mM with 10 µCi [γ-32P]-ATP per reaction. Reactions were incubated for 15 minutes at room temperature before quenching with 5x SDS loading dye. Reactions were separated on 10% Bis-Tris gels. All kinase assay reactions were performed at 25°C under anaerobic conditions using a COY anaerobic glove bag. ImageJ


Page 12 of 26

Page 13 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


software was used to quantify band intensity of autoradiographs and Coomassie stained SDSPAGE gels. Values of kinase autophosphorylation were normalized to protein loading band intensities. Relative levels of autophosphorylation were further normalized to kinase only autophosphorylation. Phosphotransfer assays between WT or DA VpsS with LuxU were conducted as previously described.11 Phosphotransfer assays which included NO-bound NosP were started by incubating a solution of VcFeII-NO and VpsS with ATP so that the final ATP concentration was 2.5 µM with 10 µCi [γ-32P]-ATP per reaction. Incubation of VcFeII-NO and VpsS with ATP was allowed to proceed for 15 minutes, after which LuxU was added. Phosphotransfer reactions were quenched after 15 minutes with 5x SDS loading dye.



SUPPORTING INFORMATION Supporting information is available: Supplemental Table 1, strains, plasmids, and primers used in this study. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION To whom correspondence should be addressed: Elizabeth M. Boon, Department of Chemistry, Stony Brook University, Stony Brook, NY, USA 11790; Tel.: (631) 632-7945; Fax: (631) 6327960; orchid id: orcid.org/0000-0003-1891-839X; E-mail: [email protected]

AUTHOR CONTRIBUTIONS E.M.B. and S.H. designed the experiments, I.H. and S.H. performed the experiments, and E.M.B., I.H. and S.H. wrote the manuscript.

ACKNOWLEDGEMENTS This work was supported by the Stony Wold-Herbert Fund, the National Science Foundation, (grant CHE-1607532 to E.M.B.), and the National Institutes of Health (grant GM118894-01A1 to E.M.B.). We thank R. Johnson and the Boon Group for helpful discussions.


Page 14 of 26

Page 15 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


REFERENCES (1) Poole, R. K., and Hughes, M. N. (2000) New functions for the ancient globin family: bacterial responses to nitric oxide and nitrosative stress. Mol. Microbiol. 36, 775–783. (2) Arora, D. P., Hossain, S., Xu, Y., and Boon, E. M. (2015) Nitric Oxide Regulation of Bacterial Biofilms. Biochemistry 54, 3717–3728. (3) 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. (4) Plate, L., and Marletta, M. A. (2013) Nitric oxide-sensing H-NOX proteins govern bacterial communal behavior. Trends Biochem. Sci. 38, 566–575. (5) Carlson, H. K., Vance, R. E., and Marletta, M. A. (2010) H-NOX regulation of c-di-GMP metabolism and biofilm formation in Legionella pneumophila. Mol. Microbiol. 77, 930–942. (6) Liu, N., Xu, Y., Hossain, S., Huang, N., Coursolle, D., Gralnick, J. A., and Boon, E. M. (2012) Nitric oxide regulation of cyclic di-GMP synthesis and hydrolysis in Shewanella woodyi. Biochemistry 51, 2087–2099. (7) Plate, L., and Marletta, M. A. (2012) Nitric Oxide Modulates Bacterial Biofilm Formation through a Multicomponent Cyclic-di-GMP Signaling Network. Molecular Cell 46, 449–460. (8) Henares, B. M., Higgins, K. E., and Boon, E. M. (2012) Discovery of a nitric oxide responsive quorum sensing circuit in Vibrio harveyi. ACS chemical biology. (9) Henares, B. M., Xu, Y., and Boon, E. M. (2013) A nitric oxide-responsive quorum sensing circuit in Vibrio harveyi regulates flagella production and biofilm formation. Int J Mol Sci 14, 16473–16484. (10) Bassler, B. L., and Losick, R. (2006) Bacterially speaking. Cell 125, 237–246.



(11) Hossain, S., and Boon, E. M. (2017) Discovery of a Novel Nitric Oxide Binding Protein and Nitric-Oxide-Responsive Signaling Pathway in Pseudomonas aeruginosa. ACS Infect Dis acsinfecdis.7b00027. (12) Mukhopadyay, R., Sudasinghe, N., Schaub, T., and Yukl, E. T. (2016) Heme-independent Redox Sensing by the Heme-Nitric Oxide/Oxygen-binding Protein (H-NOX) from Vibrio cholerae. J. Biol. Chem. 291, 17547–17556. (13) Guo, Y., Iavarone, A. T., Cooper, M. M., and Marletta, M. A. (2018) Mapping the HNOX/HK Binding Interface in Vibrio cholerae by Hydrogen/Deuterium Exchange Mass Spectrometry. Biochemistry 57, 1779–1789. (14) Barraud, N., Storey, M. V., Moore, Z. P., Webb, J. S., Rice, S. A., and Kjelleberg, S. (2009) Nitric oxide‐mediated dispersal in single‐ and multi‐species biofilms of clinically and industrially relevant microorganisms. Microbial Biotechnology 2, 370–378. (15) Shikuma, N. J., Fong, J. C. N., Odell, L. S., Perchuk, B. S., Laub, M. T., and Yildiz, F. H. (2009) Overexpression of VpsS, a hybrid sensor kinase, enhances biofilm formation in Vibrio cholerae. J. Bacteriol. 191, 5147–5158. (16) Jung, S. A., Hawver, L. A., and Ng, W.-L. (2016) Parallel quorum sensing signaling pathways in Vibrio cholerae. Curr Genet 62, 255–260. (17) Rutherford, S. T., van Kessel, J. C., Shao, Y., and Bassler, B. L. (2011) AphA and LuxR/HapR reciprocally control quorum sensing in vibrios. Genes Dev. 25, 397–408. (18) Shao, Y., and Bassler, B. L. (2012) Quorum-sensing non-coding small RNAs use unique pairing regions to differentially control mRNA targets. Mol. Microbiol. 83, 599–611. (19) Jung, S. A., Chapman, C. A., and Ng, W.-L. (2015) Quadruple Quorum-Sensing Inputs Control Vibrio cholerae Virulence and Maintain System Robustness. PLoS Pathog. (Weiss, D.,


Page 16 of 26

Page 17 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Ed.) 11, e1004837. (20) Boon, E. M., Huang, S. H., and Marletta, M. A. (2005) A molecular basis for NO selectivity in soluble guanylate cyclase. Nat. Chem. Biol. 1, 53–59. (21) Stone, J. R., and Marletta, M. A. (1994) Soluble guanylate cyclase from bovine lung: activation with nitric oxide and carbon monoxide and spectral characterization of the ferrous and ferric states. Biochemistry 33, 5636–5640. (22) Wu, G., Liu, W., Berka, V., and Tsai, A.-L. (2013) The selectivity of Vibrio cholerae HNOX for gaseous ligands follows the “sliding scale rule” hypothesis. Ligand interactions with both ferrous and ferric Vc H-NOX. Biochemistry 52, 9432–9446. (23) Tsai, A.-L., Martin, E., Berka, V., and Olson, J. S. (2012) How do heme-protein sensors exclude oxygen? Lessons learned from cytochrome c', Nostoc puntiforme heme nitric oxide/oxygen-binding domain, and soluble guanylyl cyclase. Antioxid. Redox Signal. 17, 1246– 1263. (24) Arora, D. P., and Boon, E. M. (2012) Nitric oxide regulated two-component signaling in Pseudoalteromonas atlantica. Biochemical and Biophysical Research Communications 421, 521–526. (25) Uhl, M. A., and Miller, J. F. (1996) Central role of the BvgS receiver as a phosphorylated intermediate in a complex two-component phosphorelay. J. Biol. Chem. 271, 33176–33180. (26) Crane, B. R., Sudhamsu, J., and Patel, B. A. (2010) Bacterial nitric oxide synthases. Annu. Rev. Biochem. 79, 445–470. (27) Janoff, E. N., Hayakawa, H., Taylor, D. N., Fasching, C. E., Kenner, J. R., Jaimes, E., and Raij, L. (1997) Nitric oxide production during Vibrio cholerae infection. Am. J. Physiol. 273, G1160–7.



(28) 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. (29) Boon, E. M., and Marletta, M. A. (2005) Ligand specificity of H-NOX domains: from sGC to bacterial NO sensors. J. Inorg. Biochem. 99, 892–902.


Page 18 of 26

Page 19 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


FIGURE LEGENDS Figure 1. Schematic of quorum sensing in V. cholerae. (A) V. cholerae quorum sensing signal transduction at low cell density and in the absence of NO. When cell density is low and autoinducer concentration is small, the hybrid histidine kinase receptors CqsS, LuxPQ, and CqsR act as kinases to transfer phosphate to LuxU. Similarly, our data indicate that the absence of NO promotes kinase activity of VpsS, which increases phosphotransfer to LuxU. Phosphorylated LuxU promotes biofilm formation and virulence factor secretion through downstream signaling. (B) Quorum sensing signal transduction at high cell density and in the presence of NO. When cell density is high, autoinducer concentrations rises. At high autoinducer concentration, CqsS, LuxPQ, and CqsR act as phosphatases, reversing the flow of phosphate from LuxU, effectively dephosphorylating it. Likewise, in the presence of NO, VcNosP FeII-NO inhibits the autophosphorylation activity of VpsS, and contributes to the decrease of phosphate flux through LuxU.

Figure 2. NosP has the ligand binding properties of a NO sensor. (A) UV/vis absorption spectra of ferric NosP (dashed line) with a λmax at 409 nm, ferrous NosP (solid black line) with a λmax at 420 nm, CO-ligated NosP (dash-dot line) with a λmax at 418 nm, and NO-ligated NosP (dotted line) with a λmax at 396 nm. (B, C) Example of a UV/vis spectroscopy scanning kinetics experiment to measure the dissociation rate of NO from NosP at 22 °C using saturated CO and 3 mM dithionite as a trap for released NO. The data were fit as described previously.11 The measured rate of 4.6 x 10-4 is independent of CO and dithionite at all concentrations tested (300 µM, 3 mM, 30 mM, and 100 mM dithionite). (B) Absorbance difference spectrum where the spectrum at time = 0 min is subtracted from all subsequent spectra collected over time as the



Page 20 of 26

FeII-CO complex is formed. (C) A plot of the change in absorbance at 418 nm minus 387 nm (the maximum and the minimum in the difference spectrum) versus time along with the exponential fit of those data.

Figure 3. NO-bound VcNosP inhibits the autophosphorylation of VpsS. Radiolabeled phosphoproteins were detected by SDS-PAGE (A, B, C bottom) and autoradiography (A, B, C top). Experiments were performed in triplicate and representative data are shown. (A) In vitro autophosphorylation of VpsS over time. (B) VcNosP-FeII-NO inhibits VpsS autophosphorylation in a concentration-dependent manner. VpsS (1 µM) was incubated with

32

P-labeled ATP and

varying amounts of VcNosP-FeII-NO (0, 1.4, 2.0, 3.3, 4.5, and 10.4 µM), resulting in the following respective factional values of VpsS phosphorylation with respect to no added VcNosPFeII-NO complex: 1.00, 0.52, 0.36, 0.29, 0.28, 0.19. (C) VcNosP-FeII does not influence the autophosphorylation of VpsS in a concentration dependent manner. VpsS was incubated with 32

P-labeled ATP and varying amounts of VcNosP-FeII (0, 1.0, 1.7, 3.8, 5.0, 8.7 µM), resulting in

the following respective factional values of VpsS phosphorylation with respect to no added VcNosP-FeII complex: 1.0, 1.2, 1.4, 1.2, 1.7, 1.2.

Figure 4. NO-bound VcNosP inhibits the phosphotransfer of VpsS to LuxU. Radiolabeled phosphoproteins were detected by SDS-PAGE (A, B right) and autoradiography (A, B left). (A) VpsS transfers phosphate to LuxU. The following proteins were incubated with 32P-labeled ATP for 15 minutes: WT VpsS only (lane 1); WT VpsS + LuxU (lane 2); VpsSDA + LuxU (lane 3). (B) In the presence of VcNosP-FeII-NO, phosphotransfer from VpsS to LuxU is inhibited. The following proteins were incubated with 32P-labeled ATP for 15 minutes, prior to the addition of


Page 21 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


LuxU: WT VpsS only (lane 1); WT VpsS + VcNosP-FeII-NO (lane 2). The addition of NObound VcNosP decreases the amount of phosphorylated LuxU (lane 2).



Figure 1


Page 22 of 26

Page 23 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2



Figure 3


Page 24 of 26

Page 25 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4


ACS Chemical Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25


Page 26 of 26

Discovery of a Nitric Oxide Responsive Quorum Sensing Circuit in

Recommend Documents