Role of Nitric Oxide in Hydroxylamine Oxidation by Ammonia-Oxidizing Bacteria.

An important role of nitric oxide (NO) as either a free intermediate in the NH3 oxidation pathway or a potential oxidant for NH3 or NH2OH has been proposed for ammonia-oxidizing bacteria (AOB) and archaea (AOA), respectively. However, tracing NO metabolism at low concentrations remains notoriously difficult. Here, we use electrochemical sensors and the mild NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) to trace apparent NO concentration and determine production rates at low micromolar concentrations in the model AOB strain Nitrosomonas europaea. In agreement with previous studies, we found that PTIO does not affect NH3 oxidation instantaneously in both Nitrosospira briensis and Nitrosomonas europaea, unlike inhibitors for ammonia oxidation such as allylthiourea and acetylene, although it effectively scavenged NO from the cell suspensions. Quantitative analysis showed that NO production by N. europaea amounted to 3.15% to 6.23% of NO2- production, whereas N. europaea grown under O2 limitation produced NO equivalent to up to 40% of NO2- production at high substrate concentrations. In addition, we found that PTIO addition to N. europaea grown under O2 limitation abolished N2O production. These results indicate different turnover rates of NO during NH3 oxidation under O2-replete and O2-limited growth conditions in AOB. The results suggest that NO may not be a free intermediate or remain tightly bound to iron centers of enzymes during hydroxylamine oxidation and that only NH3 saturation and adaptation to O2 limitation may lead to significant dissociation of NO from hydroxylamine dehydrogenase. IMPORTANCE Ammonia oxidation by chemolithoautotrophic ammonia-oxidizing bacteria (AOB) is thought to contribute significantly to global nitrous oxide (N2O) emissions and leaching of oxidized nitrogen, particularly through their activity in nitrogen (N)-fertilized agricultural production systems. Although substantial efforts have been made to characterize the N metabolism in AOB, recent findings suggest that nitric oxide (NO) may play an important mechanistic role as a free intermediate of hydroxylamine oxidation in AOB, further implying that besides hydroxylamine dehydrogenase (HAO), additional enzymes may be required to complete the ammonia oxidation pathway. However, the NO spin trap PTIO was found to not inhibit ammonia oxidation in AOB. This study provides a combination of physiological and spectroscopic evidence that PTIO indeed scavenges only free NO in AOB and that significant amounts of free NO are produced only during incomplete hydroxylamine oxidation or nitrifier denitrification under O2-limited growth conditions.

Serotonin signals through postsynaptic Gαq, Trio RhoGEF, and diacylglycerol to promote Caenorhabditis elegans egg-laying circuit activity and behavior.

Activated Gαq signals through phospholipase-Cß and Trio, a Rho GTPase exchange factor (RhoGEF), but how these distinct effector pathways promote cellular responses to neurotransmitters like serotonin remains poorly understood. We used the egg-laying behavior circuit of Caenorhabditis elegans to determine whether phospholipase-Cß and Trio mediate serotonin and Gαq signaling through independent or related biochemical pathways. Our genetic rescue experiments suggest that phospholipase-Cß functions in neurons while Trio Rho GTPase exchange factor functions in both neurons and the postsynaptic vulval muscles. While Gαq, phospholipase-Cß, and Trio Rho GTPase exchange factor mutants fail to lay eggs in response to serotonin, optogenetic stimulation of the serotonin-releasing HSN neurons restores egg laying only in phospholipase-Cß mutants. Phospholipase-Cß mutants showed vulval muscle Ca2+ transients while strong Gαq and Trio Rho GTPase exchange factor mutants had little or no vulval muscle Ca2+ activity. Treatment with phorbol 12-myristate 13-acetate that mimics 1,2-diacylglycerol, a product of PIP2 hydrolysis, rescued egg-laying circuit activity and behavior defects of Gαq signaling mutants, suggesting both phospholipase-C and Rho signaling promote synaptic transmission and egg laying via modulation of 1,2-diacylglycerol levels. 1,2-Diacylglycerol activates effectors including UNC-13; however, we find that phorbol esters, but not serotonin, stimulate egg laying in unc-13 and phospholipase-Cß mutants. These results support a model where serotonin signaling through Gαq, phospholipase-Cß, and UNC-13 promotes neurotransmitter release, and that serotonin also signals through Gαq, Trio Rho GTPase exchange factor, and an unidentified, phorbol 12-myristate 13-acetate-responsive effector to promote postsynaptic muscle excitability. Thus, the same neuromodulator serotonin can signal in distinct cells and effector pathways to coordinate activation of a motor behavior circuit.

Presynaptic Gαo (GOA-1) signals to depress command neuron excitability and allow stretch-dependent modulation of egg laying in Caenorhabditis elegans.

Egg laying in the nematode worm Caenorhabditis elegans is a two-state behavior modulated by internal and external sensory input. We have previously shown that homeostatic feedback of embryo accumulation in the uterus regulates bursting activity of the serotonergic HSN command neurons that sustains the egg-laying active state. How sensory feedback of egg release signals to terminate the egg-laying active state is less understood. We find that Gαo, a conserved Pertussis Toxin-sensitive G protein, signals within HSN to inhibit egg-laying circuit activity and prevent entry into the active state. Gαo signaling hyperpolarizes HSN, reducing HSN Ca2+ activity and input onto the postsynaptic vulval muscles. Loss of inhibitory Gαo signaling uncouples presynaptic HSN activity from a postsynaptic, stretch-dependent homeostat, causing precocious entry into the egg-laying active state when only a few eggs are present in the uterus. Feedback of vulval opening and egg release activates the uv1 neuroendocrine cells which release NLP-7 neuropeptides which signal to inhibit egg laying through Gαo-independent mechanisms in the HSNs and Gαo-dependent mechanisms in cells other than the HSNs. Thus, neuropeptide and inhibitory Gαo signaling maintain a bi-stable state of electrical excitability that dynamically controls circuit activity in response to both external and internal sensory input to drive a two-state behavior output.