Signal binding at both modules of its dCache domain enables the McpA chemoreceptor of Bacillus velezensis to sense different ligands.

Bacteria have evolved multiple signal transduction systems that permit an adaptation to changing environmental conditions. Chemoreceptor-based signaling cascades are very abundant in bacteria and are among the most complex signaling systems. Currently, our knowledge on the molecular features that determine signal recognition at chemoreceptors is limited. Chemoreceptor McpA of Bacillus velezensis SQR9 has been shown to mediate chemotaxis to a broad range of different ligands. Here we show that its ligand binding domain binds directly 13 chemoattractants. We provide support that organic acids and amino acids bind to the membrane-distal and membrane-proximal module of the dCache domain, respectively, whereas binding of sugars/sugar alcohols occurred at both modules. Structural biology studies combined with site-directed mutagenesis experiments have permitted to identify 10 amino acid residues that play key roles in the recognition of multiple ligands. Residues in membrane-distal and membrane-proximal regions were central for sensing organic acids and amimo acids, respectively, whereas all residues participated in sugars/sugar alcohol sensing. Most characterized chemoreceptors possess a narrow and well-defined ligand spectrum. We propose here a sensing mechanism involving both dCache modules that allows the integration of very diverse signals by a single chemoreceptor.

Neuromedin B-Expressing Neurons in the Retrotrapezoid Nucleus Regulate Respiratory Homeostasis and Promote Stable Breathing in Adult Mice.

Respiratory chemoreceptor activity encoding arterial Pco2 and Po2 is a critical determinant of ventilation. Currently, the relative importance of several putative chemoreceptor mechanisms for maintaining eupneic breathing and respiratory homeostasis is debated. Transcriptomic and anatomic evidence suggests that bombesin-related peptide Neuromedin-B (Nmb) expression identifies chemoreceptor neurons in the retrotrapezoid nucleus (RTN) that mediate the hypercapnic ventilatory response, but functional support is missing. In this study, we generated a transgenic Nmb-Cre mouse and used Cre-dependent cell ablation and optogenetics to test the hypothesis that RTN Nmb neurons are necessary for the CO2-dependent drive to breathe in adult male and female mice. Selective ablation of ∼95% of RTN Nmb neurons causes compensated respiratory acidosis because of alveolar hypoventilation, as well as profound breathing instability and respiratory-related sleep disruption. Following RTN Nmb lesion, mice were hypoxemic at rest and were prone to severe apneas during hyperoxia, suggesting that oxygen-sensitive mechanisms, presumably the peripheral chemoreceptors, compensate for the loss of RTN Nmb neurons. Interestingly, ventilation following RTN Nmb -lesion was unresponsive to hypercapnia, but behavioral responses to CO2 (freezing and avoidance) and the hypoxia ventilatory response were preserved. Neuroanatomical mapping shows that RTN Nmb neurons are highly collateralized and innervate the respiratory-related centers in the pons and medulla with a strong ipsilateral preference. Together, this evidence suggests that RTN Nmb neurons are dedicated to the respiratory effects of arterial Pco2/pH and maintain respiratory homeostasis in intact conditions and suggest that malfunction of these neurons could underlie the etiology of certain forms of sleep-disordered breathing in humans.SIGNIFICANCE STATEMENT Respiratory chemoreceptors stimulate neural respiratory motor output to regulate arterial Pco2 and Po2, thereby maintaining optimal gas exchange. Neurons in the retrotrapezoid nucleus (RTN) that express the bombesin-related peptide Neuromedin-B are proposed to be important in this process, but functional evidence has not been established. Here, we developed a transgenic mouse model and demonstrated that RTN neurons are fundamental for respiratory homeostasis and mediate the stimulatory effects of CO2 on breathing. Our functional and anatomic data indicate that Nmb-expressing RTN neurons are an integral component of the neural mechanisms that mediate CO2-dependent drive to breathe and maintain alveolar ventilation. This work highlights the importance of the interdependent and dynamic integration of CO2- and O2-sensing mechanisms in respiratory homeostasis of mammals.

Identification of candidate genes associated with host-seeking behavior in the parasitoid wasp Diachasmimorpha longicaudata.

BACKGROUND: Diachasmimorpha longicaudata is a hymenopteran fruit fly endoparasitoid. Females of this species find their hosts for oviposition by using complex sensorial mechanisms in response to physical and chemical stimuli associated with the host and host habitat. Ecological and behavioral aspects related to host-seeking behavior for oviposition have been extensively studied in D. longicaudata, including the identification of volatile organic compounds acting as attractants to females. In this sense, molecular mechanisms of chemoreception have been explored in this species, including a preliminary characterization of odorant-binding proteins (OBPs), chemosensory proteins (CSPs) and odorant receptors (ORs), among other proteins. Functional assays on OBP and CSP have been conducted as a first approach to identify molecular mechanisms associated with the female host-seeking behavior for oviposition. The aims of the present study were to identify the D. longicaudata sensory gene repertoire expressed in the antenna of sexually mature and mated individuals of both sexes, and subsequently, characterize transcripts differentially expressed in the antennae of females to identify candidate genes associated with the female host-seeking behavior for oviposition. RESULTS: A total of 33,745 predicted protein-coding sequences were obtained from a de novo antennal transcriptome assembly. Ten sensory-related gene families were annotated as follows: 222 ORs, 44 ionotropic receptors (IRs), 25 gustatory receptors (GRs), 9 CSPs, 13 OBPs, 2 ammonium transporters (AMTs), 8 pickpocket (PPKs) receptors, 16 transient receptor potential (TRP) channels, 12 CD36/SNMPs and 3 Niemann-Pick type C2 like proteins (NPC2-like). The differential expression analysis revealed 237 and 151 transcripts up- and downregulated, respectively, between the female and male antennae. Ninety-seven differentially expressed transcripts corresponded to sensory-related genes including 88 transcripts being upregulated (87 ORs and one TRP) and nine downregulated (six ORs, two CSPs and one OBP) in females compared to males. CONCLUSIONS: The sensory gene repertoire of D. longicaudata was similar to that of other taxonomically related parasitoid wasps. We identified a high number of ORs upregulated in the female antenna. These results may indicate that this gene family has a central role in the chemoreception of sexually mature females during the search for hosts and host habitats for reproductive purposes.

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

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 Iß-Trp residues, but PAS1, which was easily oxidized in vitro, was unaffected by Iß-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.

Study of NIT domain-containing chemoreceptors from two global phytopathogens and identification of NIT domains in eukaryotes.

Bacterial signal transduction systems are typically activated by the binding of signal molecules to receptor ligand binding domains (LBDs), such as the NIT LBD. We report here the identification of the NIT domain in more than 15,000 receptors that were present in 30 bacterial phyla, but also in 19 eukaryotic phyla, expanding its known phylogenetic distribution. The NIT domain formed part of seven receptor families that either control transcription, mediate chemotaxis or regulate second messenger levels. We have produced the NIT domains from chemoreceptors of the bacterial phytopathogens Pectobacterium atrosepticum (PacN) and Pseudomonas savastanoi (PscN) as individual purified proteins. High-throughput ligand screening using compound libraries revealed a specificity for nitrate and nitrite binding. Isothermal titration calorimetry experiments showed that PacN-LBD bound preferentially nitrate ( K D = 1.9 µM), whereas the affinity of PscN-LBD for nitrite ( K D = 2.1 µM) was 22 times higher than that for nitrate. Analytical ultracentrifugation experiments indicated that PscN-LBD is monomeric in the presence and absence of ligands. The R182A mutant of PscN did not bind nitrate or nitrite. This residue is not conserved in the NIT domain of the Pseudomonas aeruginosa chemoreceptor PA4520, which may be related to its failure to bind nitrate/nitrite. The magnitude of P. atrosepticum chemotaxis towards nitrate was significantly greater than that of nitrite and pacN deletion almost abolished responses to both compounds. This study highlights the important role of nitrate and nitrite as signal molecules in life and advances our knowledge on the NIT domain as universal nitrate/nitrite sensor module.

An Azospirillum brasilense chemoreceptor that mediates nitrate chemotaxis has conditional roles in the colonization of plant roots.

Motile plant-associated bacteria use chemotaxis and dedicated chemoreceptors to navigate gradients in their surroundings and to colonize host plant surfaces. Here, we characterize a chemoreceptor that we named Tlp2 in the soil alphaproteobacterium Azospirillum brasilense. We show that the Tlp2 ligand-binding domain is related to the 4-helix bundle family and is conserved in chemoreceptors found in the genomes of many soil- and sediment-dwelling alphaproteobacteria. The promoter of tlp2 is regulated in an NtrC- and RpoN-dependent manner and is most upregulated under conditions of nitrogen fixation or in the presence of nitrate. Using fluorescently tagged Tlp2 (Tlp2-YFP), we show that this chemoreceptor is present in low abundance in chemotaxis-signaling clusters and is prone to degradation. We also obtained evidence that the presence of ammonium rapidly disrupts Tlp2-YFP localization. Behavioral experiments using a strain lacking Tlp2 and variants of Tlp2 lacking conserved arginine residues suggest that Tlp2 mediates chemotaxis in gradients of nitrate and nitrite, with the R159 residue being essential for Tlp2 function. We also provide evidence that Tlp2 is essential for root surface colonization of some plants (teff, red clover, and cowpea) but not others (wheat, sorghum, alfalfa, and pea). These results highlight the selective role of nitrate sensing and chemotaxis in plant root surface colonization and illustrate the relative contribution of chemoreceptors to chemotaxis and root surface colonization.IMPORTANCEBacterial chemotaxis mediates host-microbe associations, including the association of beneficial bacteria with the roots of host plants. Dedicated chemoreceptors specify sensory preferences during chemotaxis. Here, we show that a chemoreceptor mediating chemotaxis to nitrate is important in the beneficial soil bacterium colonization of some but not all plant hosts tested. Nitrate is the preferred nitrogen source for plant nutrition, and plants sense and tightly control nitrate transport, resulting in varying nitrate uptake rates depending on the plant and its physiological state. Nitrate is thus a limiting nutrient in the rhizosphere. Chemotaxis and dedicated chemoreceptors for nitrate likely provide motile bacteria with a competitive advantage to access this nutrient in the rhizosphere.

Blood oxygen regulation via P2Y12R expressed in the carotid body.

BACKGROUND: Peripheral blood oxygen monitoring via chemoreceptors in the carotid body (CB) is an integral function of the autonomic cardiorespiratory regulation. The presence of the purinergic P2Y12 receptor (P2Y12R) has been implicated in CB; however, the exact role of the receptor in O2 sensing and signal transduction is unknown. METHODS: The presence of P2Y12R was established by immunoblotting, RT qPCR and immunohistochemistry. Primary glomus cells were used to assess P2Y12R function during hypoxia and hypercapnia, where monoamines were measured by HPLC; calcium signal was recorded utilizing OGB-1 and N-STORM Super-Resolution System. Ingravescent hypoxia model was tested in anaesthetized mice of mixed gender and cardiorespiratory parameters were recorded in control and receptor-deficient or drug-treated experimental animals. RESULTS: Initially, the expression of P2Y12R in adult murine CB was confirmed. Hypoxia induced a P2Y12R-dependent release of monoamine transmitters from isolated CB cells. Receptor activation with the endogenous ligand ADP promoted release of neurotransmitters under normoxic conditions, while blockade disrupted the amplitude and duration of the intracellular calcium concentration. In anaesthetised mice, blockade of P2Y12R expressed in the CB abrogated the initiation of compensatory cardiorespiratory changes in hypoxic environment, while centrally inhibited receptors (i.e. microglial receptors) or receptor-deficiency induced by platelet depletion had limited influence on the physiological adjustment to hypoxia. CONCLUSIONS: Peripheral P2Y12R inhibition interfere with the complex mechanisms of acute oxygen sensing by influencing the calcium signalling and the release of neurotransmitter molecules to evoke compensatory response to hypoxia. Prospectively, the irreversible blockade of glomic receptors by anti-platelet drugs targeting P2Y12Rs, propose a potential, formerly unrecognized side-effect to anti-platelet medications in patients with pulmonary morbidities.

Evolution of chemosensory genes in Colorado potato beetle, Leptinotarsa decemlineata.

Associating with plant hosts is thought to have elevated the diversification of insect herbivores, which comprise the majority of global species diversity. In particular, there is considerable interest in understanding the genetic changes that allow host-plant shifts to occur in pest insects and in determining what aspects of functional genomic diversity impact host-plant breadth. Insect chemoreceptors play a central role in mediating insect-plant interactions, as they directly influence plant detection and sensory stimuli during feeding. Although chemosensory genes evolve rapidly, it is unclear how they evolve in response to host shifts and host specialization. We investigate whether selection at chemosensory genes is linked to host-plant expansion from the buffalo burr, Solanum rostratum, to potato, Solanum tuberosum, in the super-pest Colorado potato beetle (CPB), Leptinotarsa decemlineata (Coleoptera: Chrysomelidae). First, to refine our knowledge of CPB chemosensory genes, we developed novel gene expression data for the antennae and maxillary-labial palps. We then examine patterns of selection at these loci within CPB, as well as compare whether rates of selection vary with respect to 9 closely related, non-pest Leptinotarsa species that vary in diet breadth. We find that rates of positive selection on olfactory receptors are higher in host-plant generalists, and this signal is particularly strong in CPB. These results provide strong candidates for further research on the genetic basis of variation in insect chemosensory performance and novel targets for pest control of a notorious super-pest.

A time course transcriptomic analysis of host and injected oncogenic cells reveals new aspects of Drosophila immune defenses.

Oncogenic RasV12 cells [A. Simcox et al., PLoS Genet 4, e1000142 (2008)] injected into adult males proliferated massively after a lag period of several days, and led to the demise of the flies after 2 to 3 wk. The injection induced an early massive transcriptomic response that, unexpectedly, included more than 100 genes encoding chemoreceptors of various families. The kinetics of induction and the identities of the induced genes differed markedly from the responses generated by injections of microbes. Subsequently, hundreds of genes were up-regulated, attesting to intense catabolic activities in the flies, active tracheogenesis, and cuticulogenesis, as well as stress and inflammation-type responses. At 11 d after the injections, GFP-positive oncogenic cells isolated from the host flies exhibited a markedly different transcriptomic profile from that of the host and distinct from that at the time of their injection, including in particular up-regulated expression of genes typical for cells engaged in the classical antimicrobial response of Drosophila.

Azospirillum brasilense AerC and Tlp4b Cytoplasmic Chemoreceptors Are Promiscuous and Interact with the Two Membrane-Bound Chemotaxis Signaling Clusters Mediating Chemotaxis Responses.

Chemotaxis in Bacteria and Archaea depends on the presence of hexagonal polar arrays composed of membrane-bound chemoreceptors that interact with rings of baseplate signaling proteins. In the alphaproteobacterium Azospirillum brasilense, chemotaxis is controlled by two chemotaxis signaling systems (Che1 and Che4) that mix at the baseplates of two spatially distinct membrane-bound chemoreceptor arrays. The subcellular localization and organization of transmembrane chemoreceptors in chemotaxis signaling clusters have been well characterized but those of soluble chemoreceptors remain relatively underexplored. By combining mutagenesis, microscopy, and biochemical assays, we show that the cytoplasmic chemoreceptors AerC and Tlp4b function in chemotaxis and localize to and interact with membrane-bound chemoreceptors and chemotaxis signaling proteins from both polar arrays, indicating that soluble chemoreceptors are promiscuous. The interactions of AerC and Tlp4b with polar chemotaxis signaling clusters are not equivalent and suggest distinct functions. Tlp4b, but not AerC, modulates the abundance of chemoreceptors within the signaling clusters through an unknown mechanism. The AerC chemoreceptor, but not Tlp4b, is able to traffic in and out of chemotaxis signaling clusters depending on its level of expression. We also identify a role of the chemoreceptor composition of chemotaxis signaling clusters in regulating their polar subcellular organization. The organization of chemotaxis signaling proteins as large membrane-bound arrays underlies chemotaxis sensitivity. Our findings suggest that the composition of chemoreceptors may fine-tune chemotaxis signaling not only through their chemosensory specificity but also through their role in the organization of polar chemotaxis signaling clusters. IMPORTANCE Cytoplasmic chemoreceptors represent about 14% of all chemoreceptors encoded in bacterial and archaeal genomes, but little is known about how they interact with and function in large polar assemblies of membrane-bound chemotaxis signaling clusters. Here, we show that two soluble chemoreceptors with a role in chemotaxis are promiscuous and interact with two distinct membrane-bound chemotaxis signaling clusters that control all chemotaxis responses in Azospirillum brasilense. We also found that any change in the chemoreceptor composition of chemotaxis signaling clusters alters their polar organization, suggesting a dynamic interplay between the sensory specificity of chemotaxis signaling clusters and their polar membrane organization.

Central and peripheral chemoreflexes in humans with treated hypertension.

Increased peripheral chemoreflex sensitivity is a pathogenic feature of human hypertension (HTN), while both central and peripheral chemoreflex sensitivities are reportedly augmented in animal models of HTN. Herein, we tested the hypothesis that both central and combined central and peripheral chemoreflex sensitivities are augmented in HTN. Fifteen HTN participants (68 ± 5 years; mean ± SD) and 13 normotensives (NT; 65 ± 6 years) performed two modified rebreathing protocols in which the partial pressure of end-tidal carbon dioxide ( P ETC O 2 ${P_{{\rm{ETC}}{{\rm{O}}_2}}}$ ) progressively increased while the partial pressure of end-tidal oxygen was clamped at either 150 mmHg (isoxic hyperoxia; central chemoreflex activation) or 50 mmHg (isoxic hypoxia; combined central and peripheral chemoreflex activation). Ventilation ( V ̇ E ${\dot{V}}_{\rm{E}}$ ; pneumotachometer) and muscle sympathetic nerve activity (MSNA; microneurography) were recorded, and ventilatory ( V ̇ E ${\dot{V}}_{\rm{E}}$ vs. P ETC O 2 ${P_{{\rm{ETC}}{{\rm{O}}_2}}}$  slope) and sympathetic (MSNA vs. P ETC O 2 ${P_{{\rm{ETC}}{{\rm{O}}_2}}}$ slope) chemoreflex sensitivities and recruitment thresholds (breakpoint) were calculated. Global cerebral blood flow (gCBF; duplex Doppler) was measured, and the association with chemoreflex responses was examined. Central ventilatory and sympathetic chemoreflex sensitivities were greater in HTN than NT (2.48 ± 1.33 vs. 1.58 ± 0.42 L min-1  mmHg-1 , P = 0.030: 3.32 ± 1.90 vs. 1.77 ± 0.62 a.u. mmHg-1 , P = 0.034, respectively), while recruitment thresholds were not different between groups. HTN and NT had similar combined central and peripheral ventilatory and sympathetic chemoreflex sensitivities and recruitment thresholds. A lower gCBF was associated with an earlier recruitment threshold for V ̇ E ${\dot{V}}_{\rm{E}}$ (R2  = 0.666, P < 0.0001) and MSNA (R2  = 0.698, P = 0.004) during isoxic hyperoxic rebreathing. These findings indicate that central ventilatory and sympathetic chemoreflex sensitivities are augmented in human HTN and perhaps suggest that targeting the central chemoreflex may help some forms of HTN. KEY POINTS: In human hypertension (HTN) increased peripheral chemoreflex sensitivity has been identified as a pathogenic feature, and in animal models of HTN, both central and peripheral chemoreflex sensitivities are reportedly augmented. In this study, the hypothesis was tested that both central and combined central and peripheral chemoreflex sensitivities are augmented in human HTN. We observed that both central ventilatory and sympathetic chemoreflex sensitivities were augmented in HTN compared to age-matched normotensive controls, but no difference was found in the combined central and peripheral ventilatory and sympathetic chemoreflex sensitivities. During central chemoreflex activation, the ventilatory and sympathetic recruitment thresholds were lower in those with lower total cerebral blood flow. These results indicate a potential contributory role of the central chemoreceptors in the pathogenesis of human HTN and support the possibility that therapeutic targeting of the central chemoreflex may help some forms of HTN.

Rethinking O2 , CO2 and breathing during wakefulness and sleep.

We have examined the importance of three long-standing questions concerning chemoreceptor influences on cardiorespiratory function which are currently experiencing a resurgence of study among physiologists and clinical investigators. Firstly, while carotid chemoreceptors (CB) are required for hypoxic stimulation of breathing, use of an isolated, extracorporeally perfused CB preparation in unanaesthetized animals with maintained tonic input from the CB, reveals that extra-CB hypoxaemia also provides dose-dependent ventilatory stimulation sufficient to account for 40-50% of the total ventilatory response to steady-state hypoxaemia. Extra-CB hyperoxia also provides a dose- and time-dependent hyperventilation. Extra-CB sites of O2 -driven ventilatory stimulation identified to date include the medulla, kidney and spinal cord. Secondly, using the isolated or denervated CB preparation in awake animals and humans has demonstrated a hyperadditive effect of CB sensory input on central CO2 sensitivity, so that tonic CB activity accounts for as much as 35-40% of the normal, air-breathing eupnoeic drive to breathe. Thirdly, we argue for a key role for CO2 chemoreception and the neural drive to breathe in the pathogenesis of upper airway obstruction during sleep (OSA), based on the following evidence: (1) removal of the wakefulness drive to breathe enhances the effects of transient CO2 changes on breathing instability; (2) oscillations in respiratory motor output precipitate pharyngeal obstruction in sleeping subjects with compliant, collapsible airways; and (3) in the majority of patients in a large OSA cohort, a reduced neural drive to breathe accompanied reductions in both airflow and pharyngeal airway muscle dilator activity, precipitating airway obstruction.

The jugular venous-to-arterial P C O 2 ${P_{{\mathrm{C}}{{\mathrm{O}}_{\mathrm{2}}}}}$ difference during rebreathing and end-tidal forcing: Relationship with cerebral perfusion.

We examined two assumptions of the modified rebreathing technique for the assessment of the ventilatory central chemoreflex (CCR) and cerebrovascular CO2 reactivity (CVR), hypothesizing: (1) that rebreathing abolishes the gradient between the partial pressures of arterial and brain tissue CO2 [measured via the surrogate jugular venous P C O 2 ${P_{{\mathrm{C}}{{\mathrm{O}}_{\mathrm{2}}}}}$ and arterial P C O 2 ${P_{{\mathrm{C}}{{\mathrm{O}}_{\mathrm{2}}}}}$ difference (Pjv-a CO2 )] and (2) rebreathing eliminates the capacity of CVR to influence the Pjv-a CO2 difference, and thus affect CCR sensitivity. We also evaluated these variables during two separate dynamic end-tidal forcing (ETF) protocols (termed: ETF-1 and ETF-2), another method of assessing CCR sensitivity and CVR. Healthy participants were included in the rebreathing (n = 9), ETF-1 (n = 11) and ETF-2 (n = 10) protocols and underwent radial artery and internal jugular vein (advanced to jugular bulb) catheterization to collect blood samples. Transcranial Doppler ultrasound was used to measure middle cerebral artery blood velocity (MCAv). The Pjv-a CO2 difference was not abolished during rebreathing (6.2 ± 2.6 mmHg; P < 0.001), ETF-1 (9.3 ± 1.5 mmHg; P < 0.001) or ETF-2 (8.6 ± 1.4 mmHg; P < 0.001). The Pjv-a CO2 difference did not change during the rebreathing protocol (-0.1 ± 1.2 mmHg; P = 0.83), but was reduced during the ETF-1 (-3.9 ± 1.1 mmHg; P < 0.001) and ETF-2 (-3.4 ± 1.2 mmHg; P = 0.001) protocols. Overall, increases in MCAv were associated with reductions in the Pjv-a CO2 difference during ETF (-0.095 ± 0.089 mmHg cm-1  s-1 ; P = 0.001) but not during rebreathing (-0.028 ± 0.045 mmHg · cm-1  · s-1 ; P = 0.067). These findings suggest that, although the Pjv-a CO2 is not abolished during any chemoreflex assessment technique, hyperoxic hypercapnic rebreathing is probably more appropriate to assess CCR sensitivity independent of cerebrovascular reactivity to CO2 . KEY POINTS: Modified rebreathing is a technique used to assess the ventilatory central chemoreflex and is based on the premise that the rebreathing method eliminates the difference between arterial and brain tissue P C O 2 ${P_{{\mathrm{C}}{{\mathrm{O}}_{\mathrm{2}}}}}$ . Therefore, rebreathing is assumed to isolate the ventilatory response to central chemoreflex stimulation from the influence of cerebral blood flow. We assessed these assumptions by measuring arterial and jugular venous bulb P C O 2 ${P_{{\mathrm{C}}{{\mathrm{O}}_{\mathrm{2}}}}}$ and middle cerebral artery blood velocity during modified rebreathing and compared these data against data from another test of the ventilatory central chemoreflex using hypercapnic dynamic end-tidal forcing. The difference between arterial and jugular venous bulb P C O 2 ${P_{{\mathrm{C}}{{\mathrm{O}}_{\mathrm{2}}}}}$ remained present during both rebreathing and end-tidal forcing tests, whereas middle cerebral artery blood velocity was associated with the P C O 2 ${P_{{\mathrm{C}}{{\mathrm{O}}_{\mathrm{2}}}}}$ difference during end-tidal forcing but not rebreathing. These findings offer substantiating evidence that clarifies and refines the assumptions of modified rebreathing tests, enhancing interpretation of future findings.

Evolution of Olfactory Receptors Tuned to Mustard Oils in Herbivorous Drosophilidae.

The diversity of herbivorous insects is attributed to their propensity to specialize on toxic plants. In an evolutionary twist, toxins betray the identity of their bearers when herbivores coopt them as cues for host-plant finding, but the evolutionary mechanisms underlying this phenomenon are poorly understood. We focused on Scaptomyza flava, an herbivorous drosophilid specialized on isothiocyanate (ITC)-producing (Brassicales) plants, and identified Or67b paralogs that were triplicated as mustard-specific herbivory evolved. Using in vivo heterologous systems for the expression of olfactory receptors, we found that S. flava Or67bs, but not the homologs from microbe-feeding relatives, responded selectively to ITCs, each paralog detecting different ITC subsets. Consistent with this, S. flava was attracted to ITCs, as was Drosophila melanogaster expressing S. flava Or67b3 in the homologous Or67b olfactory circuit. ITCs were likely coopted as olfactory attractants through gene duplication and functional specialization (neofunctionalization and subfunctionalization) in S. flava, a recently derived herbivore.

History and Recent Progress in Carotid Body Studies.

This chapter describes the history of the carotid body (CB) and the subsequent research on its structure and function. The chronological development of ideas about its anatomical structure as a ganglion, the first descriptions of its glandular nature as a ball of highly vascular tissue (glomus), the discovery of its neural crest origin and relevant embryological views as a true paraganglion toward a more conclusive understanding of its sensory nature as a chemoreceptor for chemical changes in blood have been consistently demonstrated. The knowledge of the CB neurochemistry, physiology and pathophysiology has progressed immensely in the past century and a large and compelling body of evidence for the presence of a neurogenic niche in the CB has accumulated over the last two decades, thus underlying its function and possibility for the development of cell replacement therapies.

A Nitrate-Sensing Domain-Containing Chemoreceptor Is Required for Successful Entry and Virulence of Dickeya dadantii 3937 in Potato Plants.

Nitrate metabolism plays an important role in bacterial physiology. During the interaction of plant-pathogenic bacteria with their hosts, bacteria face variable conditions with respect to nitrate availability. Perception mechanisms through the chemosensory pathway drive the entry and control the colonization of the plant host in phytopathogenic bacteria. In this work, the identification and characterization of the nitrate- and nitrite-sensing (NIT) domain-containing chemoreceptor of Dickeya dadantii 3937 (Dd3937) allowed us to unveil the key role of nitrate sensing not only for the entry into the plant apoplast through wounds but also for infection success. We determined the specificity of this chemoreceptor to bind nitrate and nitrite, with a slight ligand preference for nitrate. Gene expression analysis showed that nitrate perception controls not only the expression of nitrate reductase genes involved in respiratory and assimilatory metabolic processes but also the expression of gyrA, hrpN, and bgxA, three well-known virulence determinants in Dd3937.

The Carotid Body "Tripartite Synapse": Role of Gliotransmission.

In mammals, cardiorespiratory reflexes originating in the carotid body (CB) help maintain homeostasis by matching oxygen supply to oxygen demand. CB output to the brainstem is shaped by synaptic interactions at a "tripartite synapse" consisting of chemosensory (type I) cells, abutting glial-like (type II) cells, and sensory (petrosal) nerve terminals. Type I cells are stimulated by several blood-borne metabolic stimuli, including the novel chemoexcitant lactate. During chemotransduction, type I cells depolarize and release a multitude of excitatory and inhibitory neurotransmitters/neuromodulators including ATP, dopamine (DA), histamine, and angiotensin II (ANG II). However, there is a growing appreciation that the type II cells may not be silent partners. Thus, similar to astrocytes at "tripartite synapses" in the CNS, type II cells may contribute to the afferent output by releasing "gliotransmitters" such as ATP. Here, we first consider whether type II cells can also sense lactate. Next, we review and update the evidence supporting the roles of ATP, DA, histamine, and ANG II in cross talk among the three main CB cellular elements. Importantly, we consider how conventional excitatory and inhibitory pathways, together with gliotransmission, help to coordinate activity within this network and thereby modulate afferent firing frequency during chemotransduction.

The Cellular Abundance of Chemoreceptors, Chemosensory Signaling Proteins, Sensor Histidine Kinases, and Solute Binding Proteins of Pseudomonas aeruginosa Provides Insight into Sensory Preferences and Signaling Mechanisms.

Chemosensory pathways and two-component systems are important bacterial signal transduction systems. In the human pathogen Pseudomonas aeruginosa, these systems control many virulence traits. Previous studies showed that inorganic phosphate (Pi) deficiency induces virulence. We report here the abundance of chemosensory and two-component signaling proteins of P. aeruginosa grown in Pi deficient and sufficient media. The cellular abundance of chemoreceptors differed greatly, since a 2400-fold difference between the most and least abundant receptors was observed. For many chemoreceptors, their amount varied with the growth condition. The amount of chemoreceptors did not correlate with the magnitude of chemotaxis to their cognate chemoeffectors. Of the four chemosensory pathways, proteins of the Che chemotaxis pathway were most abundant and showed little variation in different growth conditions. The abundance of chemoreceptors and solute binding proteins indicates a sensing preference for amino acids and polyamines. There was an excess of response regulators over sensor histidine kinases in two-component systems. In contrast, ratios of the response regulators CheY and CheB to the histidine kinase CheA of the Che pathway were all below 1, indicative of different signaling mechanisms. This study will serve as a reference for exploring sensing preferences and signaling mechanisms of other bacteria.

Noncanonical Sensing Mechanisms for Bacillus subtilis Chemoreceptors.

Bodhankar et al. reported a noncanonical sensing mechanism that involves signal interaction with the McpA chemoreceptor signaling domain resulting in a chemorepellence response of Bacillus subtilis. The identified repellent binding site is analogous to that for attractant binding in McpB, another B. subtilis chemoreceptor.

Leptospira interrogans Aer2: an Unusual Membrane-Bound PAS-Heme Oxygen Sensor.

In this study, we provide the first characterization of a chemoreceptor from Leptospira interrogans, the cause of leptospirosis. This receptor is related to the Aer2 receptors that have been studied in other bacteria. In those organisms, Aer2 is a soluble receptor with one or two PAS-heme domains and signals in response to O2 binding. In contrast, L. interrogans Aer2 (LiAer2) is an unusual membrane-bound Aer2 with a periplasmic domain and three cytoplasmic PAS-heme domains. Each of the three PAS domains bound b-type heme via conserved Eη-His residues. They also bound O2 and CO with similar affinities to each other and other PAS-heme domains. However, all three PAS domains were uniquely hexacoordinate in the deoxy-heme state, whereas other Aer2-PAS domains are pentacoordinate. Similar to other Aer2 receptors, LiAer2 could hijack the E. coli chemotaxis pathway but only when it was expressed with an E. coli high-abundance chemoreceptor. Unexpectedly, the response was inverted relative to classic Aer2 receptors. That is, LiAer2 caused E. coli to tumble (it was signal-on) in the absence of O2 and to stop tumbling in its presence. Thus, an endogenous ligand in the deoxy-heme state was correlated with signal-on LiAer2, and its displacement for gas-binding turned signaling off. This response also occurred in a soluble version of LiAer2 lacking the periplasmic domain, transmembrane (TM) region, and first two PAS domains, meaning that PAS3 alone was sufficient for O2-mediated control. Future studies are needed to understand the unique signaling mechanisms of this unusual Aer2 receptor. IMPORTANCE Leptospira interrogans, the cause of the zoonotic infection leptospirosis, is found in soil and water contaminated with animal urine. L. interrogans survives in complex environments with the aid of 12 chemoreceptors, none of which has been explicitly studied. In this study, we characterized the first L. interrogans chemoreceptor, LiAer2, and reported its unique characteristics. LiAer2 is membrane-bound, has three cytoplasmic PAS-heme domains that each bound hexacoordinate b-type heme and O2 turned LiAer2 signaling off. An endogenous ligand in the deoxy-heme state was correlated with signal-on LiAer2 and its displacement for O2-binding turned signaling off. Our study corroborated previous findings that Aer2 receptors are O2 sensors, but also demonstrated that they do not all function the same way.