Shewanella oneidensis MR-1 H-NOX regulation of a histidine kinase by nitric oxide

Abstract

Nitric oxide (NO) signaling in animals controls processes such as smooth muscle relaxation and neurotransmission by activation of soluble guanylate cyclase (sGC). Prokaryotic homologues of the sGC heme domain, called H-NOX domains, have been identified and are generally found in a predicted operon in conjunction with a histidine kinase. Here, we show that an H-NOX protein (SO2144) from Shewanella oneidensis directly interacts with the sensor histidine kinase (SO2145), binds NO in a 5-coordinate complex similar to mammalian sGC, and in that form inhibits the activity of a histidine kinase (SO2145). We also describe the first account of NO formation by S. oneidensis under anaerobic growth conditions derived from nitrate and nitrite. These observations suggest that the S. oneidensis H-NOX and histidine kinase pair function as part of a novel two-component signaling pathway that is responsive to NO formation from higher nitrogen oxides used as electron acceptors when oxygen is low and thereby functioning as an environmental sensor.

Figure 1

Genomic organization of H-NOX proteins. H-NOX operon organization is shown for Shewanella oneidensis (SO2144, SO2145), Vibrio cholerae El Tor (VCA0720, VCA0719), Legionella pneumophilia Philadelphia (LPG 2459, LPG2458, LPG2457), Caulobacter crescentus (CC2992, CC2993), and Vibrio fischeri (VFA0071, VFA0072). The H-NOX gene can be followed by a predicted cytoplasmic sensor histidine kinase, sometimes with a response regulator (LPG genes). Legionella pneumophilia Philadelphia also contains and additional H-NOX operon (LPG1056, LPG1057) with the H-NOX upstream of a predicted a diguanylate cyclase gene. CC2993 contains a receiver domain. VFA0071/VFA0072 is unique in that the hybrid kinase that follows the H-NOX gene contains an HPT domain as well as a receiver domain. TTE0680 illustrates the gene from Thermoanaerobacter tengcongensis, and is a fusion of an H-NOX domain to a methyl accepting chemotaxis protein. Overlap in arrows indicates overlapping reading frames. Operon predictions were accessed at MicrobesOnline (www.microbesonline.org) from the VIMSS database (23).

Figure 2. UV/Vis Spectra of H-NOX SO2144

A: The spectrum of the 6-coordinate, low-spin Fe²⁺-CO complex (solid line), with the Soret maximum at 424 nm; B: The spectrum of the 5-coordinate Fe²⁺-NO complex (dotted line), with the Soret maximum at 398 nm; C: The spectrum of the 5-coordinate, high-spin Fe²⁺ unligated species (dashed line), with the Soret maximum at 427 nm.

Figure 3. Autophosphorylation of SO2145

Kinase autophosphorylation assays were carried out with radiolabeled ATP as described in the Materials and Methods, and aliquots were taken at 0, 0.16, 0.5, 1, 3, 5, 10, 20 and 60 min and analyzed by SDS-PAGE and autoradiography. Panel A: Autoradiograph of a kinase assay with a plot of the relative signal intensity ± standard error from two experiments. Panel B: Anti-Histag Western blot of WT SO2145 kinase (lane 1) and the mutant H72A (lane 2) at the predicted site of autophosphorylation accompanied by an autoradiograph of WT (lane 1) and the mutant kinase (lane 2).

Figure 4. Effect of the SO2144 H-NOX on the kinase activity of SO2145

Kinase assays containing 1 μM SO2145 were carried out for 30 min with radiolabeled ATP in the presence of different H-NOX heme complexes as described in the Materials and Methods, and samples were analyzed by SDS-PAGE and autoradiography. (a) The H-NOX Fe²⁺-NO complex inhibits kinase activity. Top: Autoradiograph of a kinase assay in the presence of increasing amounts of H-NOX Fe²⁺-NO complex. B is a blank lane; C is an assay carried out with 100 μM H-NOX-Fe²⁺. Bottom: Plot of relative signal intensity ± standard error from three experiments. (b) The H-NOX Fe²⁺-CO complex has little effect on kinase activity. Top: Autoradiograph of a kinase assay in the presence of increasing amounts of H-NOX Fe²⁺-CO complex. Bottom: Plot of relative signal intensity ± standard error from three experiments. (c) The H-NOX-Fe³⁺ species inhibits kinase activity. Top: Autoradiograph of a kinase assay in the presence of increasing amounts of H-NOX-Fe³⁺. Bottom: Plot of relative signal intensity ± standard error from three experiments. The background in the scanned gels is different and this was left unadjusted.

Figure 5. SO2144 H-NOX interacts with SO2145 Kinase

Pull-down experiments were carried out using magnetic NiNTA beads, His-tagged kinase, and E. coli lysate containing overexpressed untagged HNOX as described in the Materials and Methods. Lane 1: + His-tagged SO2145 kinase – SO2144 H-NOX; Lane 2: + SO2144 H-NOX loading control; Lane 3: + S02144 H-NOX – His-tagged SO2145 kinase; Lane 4: + SO2144 H-NOX + His-tagged SO2145 kinase.

Figure 6. NO production by S. oneidensis

Bacteria were grown anaerobically in the presence of 5 mM nitrite (solid line) or 5 mM nitrate (dashed line) and headspace NO was measured with a chemiluminescence NO analyzer. S. oneidensis is capable of producing NO in the presence of either alternate electron acceptor.