The Aer2 chemoreceptor from Vibrio vulnificus is a tri-PAS-heme oxygen sensor

Abstract

The marine pathogen Vibrio vulnificus senses and responds to environmental stimuli via two chemosensory systems and 42-53 chemoreceptors. Here, we present an analysis of the V. vulnificus Aer2 chemoreceptor, VvAer2, which is the first V. vulnificus chemoreceptor to be characterized. VvAer2 is related to the Aer2 receptors of other gammaproteobacteria, but uncharacteristically contains three PAS domains (PAS1-3), rather than one or two. Using an E. coli chemotaxis hijack assay, we determined that VvAer2, like other Aer2 receptors, senses and responds to O₂ . All three VvAer2 PAS domains bound pentacoordinate b-type heme and exhibited similar O₂ affinities. PAS2 and PAS3 both stabilized O₂ via conserved Iβ-Trp residues, but PAS1, which was easily oxidized in vitro, was unaffected by Iβ-Trp replacement. Our results support a model in which PAS1 is largely dispensable for O₂ -mediated signaling, whereas PAS2 modulates PAS3 signaling, and PAS3 signals to the downstream domains. Each PAS domain appeared to be positionally optimized, because PAS swapping caused altered signaling properties, and neither PAS1 nor PAS2 could replace PAS3. Our findings strengthen previous conclusions that Aer2 receptors are O₂ sensors, but with distinct N-terminal domain arrangements that facilitate, modulate and tune responses based on environmental signals.

Keywords: Vibrio vulnificus; PAS domain; chemoreceptor; oxygen sensing; signal transduction.

FIGURE 1

Vibrio vulnificus chemosensory proteins and gene clusters. (a) A summary of the chemosensory proteins encoded in the V. vulnificus YJ016 genome as determined by analyzing YJ016 in the Microbial Signal Transduction 3 (MiST3) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. (b) V. vulnificus has two chemosensory gene clusters. The che1 cluster on chromosome I is the chemotaxis cluster. It encodes core chemosensory proteins CheA (histidine kinase), CheW and CheW2 (coupling proteins), CheY2 (response regulator), CheZ (phosphatase), CheB (methylesterase), and a chromosome partitioning protein from the ParA family. The che2 cluster on chromosome II encodes a complete chemosensory system including Aer2 and an additional MCP, CheA2 (histidine kinase), CheW3 and CheW4 (coupling proteins), CheY3 (response regulator), CheR3, CheD and CheB3 (adaptation proteins), and the two‐component system RsbU/RsbV.

FIGURE 2

VvAer2 structural models. The structure of a VvAer2 dimer as predicted by Alphafold2 (left; monomers in blue and gold). VvAer2 is predicted to contain three N‐terminal PAS domains followed by a di‐HAMP unit (HAMP1‐2) and a kinase control module with 36 heptad repeats. By analogy to the methylation sites of E. coli Tsr, VvAer2 has three potential methylation sites for adaptational modification at E541, Q548 and E730 (shown as orange spheres). Although not shown, the C‐terminus of VvAer2 has a pentapeptide sequence “EWEEF” for possible adaptation enzyme binding. On the top right, a VvAer2 PAS1 dimer model is shown based on the unliganded dimer structure of the P. aeruginosa Aer2 PAS domain (PDB: H4I4) with heme in dark red spacefill. Residues known to be involved in heme and O₂ binding in other PAS‐heme domains (His, Trp and Tyr) are shown as sticks.

FIGURE 3

VvAer2 directed behavior in E. coli and the impact of sequential N‐terminal truncations. (a) The behavior of E. coli BT3388 cells expressing WT VvAer2, at both the center and edge of an 8 μl bacterial drop spread over 1 cm on a glass slide. A typical time course is shown on the left with measurements every 10 s for 1 min. Air was introduced into the gas perfusion chamber at time zero. On the right, the average percentage of cells tumbling 30 s after switching to air or N₂ is shown. The approximate location of the center and edge measurements are indicated (X) on a cartoon of the gas perfusion chamber (~5 mm and ~ 1.5 mm from the edge of the drop, respectively). (b) Behavioral responses of BT3388 cells expressing full‐length VvAer2_1‐807, N‐terminally‐truncated VvAer2 receptors, and the plasmid alone (pProEX). Relevant Aer2 models are shown to the right of each graph. For all bar graphs, the average percent of cell tumbling and standard deviation are shown; *p < .05.

FIGURE 4

Aer2 PAS sequence alignment and comparisons. (a) An alignment of Aer2 PAS sequences from V. vulnificus YJ016 (VvPAS1, 2 and 3), V. cholerae N16961 (VcPAS1 and 2) and P. aeruginosa PAO1 (PaPAS) as generated by Clustal Omega. Asterisks represent conserved residues, colons represent similar amino acids and periods represent amino acids with weakly similar characteristics. The conserved Eη‐His that coordinates heme is highlighted purple, the Iβ‐Trp and Gβ‐Tyr that stabilize O₂‐binding are teal and green respectively, the Hβ‐Leu that occupies the ligand binding cleft is orange and the Thr of the DxT motif that conformationally links PAS to a C‐terminal domain is light blue. The DxT motif is indicated by bolded text. Secondary structure elements are based on the structure of PaPAS (PDB: H4I4). (b) VvPAS3 monomer model based on the structure of PaPAS (PDB: H4I4) with secondary elements labeled. Relevant residues are shown as sticks with the same colors as the sequence alignment in (a). (c) Sequence identities of the Aer2 PAS sequences from (a). V. vulnificus PAS sequences were compared with each other and with those from V. cholerae and P. aeruginosa.

FIGURE 5

PAS‐heme spectra and dissociation constants. (a) Purified imidazole‐bound PAS proteins showing typical colors. PAS1 eluted with a unique brown color, whereas PAS2 and PAS3 eluted with a red hue indicative of heme. (b) Absorbance of ~10 μM imidazole‐bound PAS1 immediately after purification with absorbance maxima indicated. The insert shows PAS1 after it had been loaded onto a Ni‐NTA agarose column for purification. (c) Absorption spectra of 10 μM purified PAS1, PAS2 and PAS3 proteins in the reduced (deoxy), oxygen‐bound (oxy), carbon monoxide‐bound (carbonmonoxy) and oxidized (met) states. The wavelengths of absorbance maxima are indicated. Inserts show an expanded view of the peaks between 450 and 700 nm. (d) Dissociation constants for O₂ and CO binding to each WT PAS domain.

FIGURE 6

Heme content, dissociation constants and behavior of PAS Eη‐His, Gβ‐Tyr, and Iβ‐Trp mutants. (a) Heme content of PAS Eη‐His mutants. The graph shows the average heme content of PAS domains (PAS1_38‐157, PAS2_165‐284, and PAS3_292‐409) containing Eη‐His replacements, given as a percentage of WT PAS heme content and corrected for protein concentration. Error bars represent the standard deviation from three independent experiments. (b) The behavior of BT3388 cells expressing full‐length VvAer2 proteins with residue substitutions. >WT behavior is defined as >70% tumbling in air with a WT N₂ response, whereas signal‐on indicates a mutant that tumbles constantly in both air and N₂. Graphs showing the quantitated behavioral data are provided in Figure S3. (c) Heme content of PAS Gβ‐Tyr and Iβ‐Trp mutants. The graph shows the average heme content of PAS domains containing Gβ‐Tyr and/or Iβ‐Trp replacements, given as a percentage of WT PAS heme content and corrected for protein concentration. Error bars represent the standard deviation from three independent experiments. (d) Dissociation constants for O₂ and CO binding to the WT PAS domains (duplicated from Figure 5d) and their respective mutants.

FIGURE 7

The behavior of Vc/VvAer2 chimeras. (a) The behavior of VvAer2 chimeras in BT3388 cells compared with WT VvAer2. All receptors contained the VvAer2 di‐HAMP and kinase control domains: WT VvAer2_1‐807; VvAer2 ∆PAS1 + VcPAS1 (Vv _1‐37/Vc _38‐157/Vv _158‐807); VvAer2 ∆PAS2 + VcPAS1 (Vv _1‐164/Vc _38‐157/Vv _285‐807); and VvAer2 ∆PAS3 + VcPAS2 (Vv _1‐291/Vc _165‐282/Vv _410‐807). The hashtag indicates that tumbling was delayed 15–20 s in air compared to WT Aer2. (b) The behavior of VcAer2 chimeras in BT3388 cells compared with WT VcAer2. All receptors contained the VcAer2 di‐HAMP and kinase control domains: WT VcAer2_1‐678; VcAer2 ∆PAS2 (Vc _1‐157/Vc _283‐678); and VcAer2 ∆PAS2 + VvPAS3 (Vc _1‐164/Vv _292‐409/Vc _283‐678). For all graphs, the average percent of cell tumbling and standard deviation are shown; *p < .05.

FIGURE 8

The behavior of VvAer2 Hβ‐Leu and DxT Thr mutants in BT3388 cells compared with WT VvAer2. See Figure 4b for residue locations in the context of the PAS structure. Under standard inducer conditions (200 μM IPTG), VvAer2‐L259A was signal off. Because the steady‐state level of VvAer2‐L259A was 0.15‐fold that of WT VvAer2, its response was also measured after inducing cells with 1 mM IPTG. For all graphs, the average percent of cell tumbling and standard deviation are shown; *p < .05.

FIGURE 9

Working model of tandem‐PAS signaling in VvAer2 and a summary of relevant VvAer2 mutant behavior. The AlphaFold2 model of VvAer2 PAS1‐3 (minus heme) is shown on the left. All three PAS domains bound b‐type heme and O₂, although PAS1 readily oxidized in vitro. Possible intramolecular dynamics are shown based on O₂‐induced changes known to occur in PaPAS (Orillard et al., 2021). Inward motions at the PAS C‐terminal DxT motifs are shown as red arrows and concomitant outward motions at the PAS N‐terminus (N‐cap) are shown as green arrows. To accommodate opposing motions in tandem PAS domains, a slight compression and downward motion of the DxT loops separating PAS1‐2 and PAS2‐3 may occur (indicated by orange arrows) and the α‐helices connecting the PAS domains may pivot, rotate, and/or alter their crossing angles. The collective data from this study indicate that PAS1 is largely dispensable for VvAer2 function, although some distal PAS1 lesions did influence receptor signaling. In contrast, PAS2 modulates PAS3 signaling, and PAS3 transmits the O₂ response to the downstream HAMP and kinase control domains.

FIGURE 10

Structural models of four different Aer2 receptors highlighting the conserved Aer2 core, different numbers of PAS‐heme domains and distinctive N‐terminal domain arrangements. Alphafold2 dimer models from left to right: P. aeruginosa Aer2 [PaAer2, model from (Anaya et al., 2022)], V. cholerae Aer2 (VcAer2, this study), V. vulnificus Aer2 (VvAer2, this study), and L. interrogans Aer2 [LiAer2, model from (Orillard & Watts, 2022)]. The Aer core comprises an O₂‐sensing PAS‐heme domain connected to C‐terminal di‐HAMP and kinase control domains. Domains N‐terminal to the PAS core include PAS, HAMP and a periplasmic ligand‐binding domain.