A New Real-Time Simple Method to Measure the Endogenous Nitrate Reductase Activity (Nar) in Paracoccus denitrificans and Other Denitrifying Bacteria.

The transmembrane nitrate reductase (Nar) is the first enzyme in the dissimilatory alternate anaerobic nitrate respiratory chain in denitrifying bacteria. To date, there has been no real-time method to determine its specific activity embedded in its native membrane; here, we describe such a new method, which is useful with the inside-out membranes of Paracoccus denitrificans and other denitrifying bacteria. This new method takes advantage of the native coupling of the endogenous NADH dehydrogenase or Complex I with the reduction of nitrate by Nar through the quinone pool of the inner membranes of P. denitrificans. This is achieved under previously reached anaerobic conditions. Inner controls confirming the specific Nar activity determined by this new method were made by the total inhibition of the Nar enzyme by sodium azide and cyanide, well-known Nar inhibitors. The estimation of the Michaelis-Menten affinity of Nar for NO3- using this so-called Nar-JJ assay gave a Km of 70.4 µM, similar to previously determined values. This new Nar-JJ assay is a suitable, low-cost, and reproducible method to determine in real-time the endogenous Nar activity not only in P. denitrificans, but in other denitrifying bacteria such as Brucella canis, and potentially in other entero-pathogenic bacteria.

In Rhodotorula mucilaginosa, active oxidative metabolism increases carotenoids to inactivate excess reactive oxygen species.

Carotenoids produced by bacteria, yeasts, algae and plants inactivate Free Radicals (FR). However, FR may inactivate carotenoids and even turn them into free radicals. Oxidative metabolism is a source of the highly motile Reactive Oxygen Species (ROS). To evaluate carotenoid interactions with ROS, the yeast Rhodotorula mucilaginosa was grown in dextrose (YPD), a fermentative substrate where low rates of oxygen consumption and low carotenoid expression were observed, or in lactate (YPLac), a mitochondrial oxidative-phosphorylation (OxPhos) substrate, which supports high respiratory activity and carotenoid production. ROS were high in YPLac-grown cells and these were unmasked by the carotenoid production-inhibitor diphenylamine (DPA). In contrast, in YPD-grown cells ROS were almost absent. It is proposed that YPLac cells are under oxidative stress. In addition, YPLac-grown cells were more sensitive than YPD-grown cells to menadione (MD), a FR-releasing agent. To test whether carotenoids from cells grown in YPLac had been modified by ROS, carotenoids from each, YPD- and YPLac-grown cells were isolated and added back to cells, evaluating protection from MD. Remarkably, carotenoids extracted from cells grown in YPLac medium inhibited growth, while in contrast extracts from YPD-grown cells were innocuous or mildly protective. Results suggest that carotenoid-synthesis in YPLac-cells is a response to OxPhos-produced ROS. However, upon reacting with FR, carotenoids themselves may be inactivated or even become prooxidant themselves.

Tissue-specific differences in Ca2+ sensitivity of the mitochondrial permeability transition pore (PTP). Experiments in male rat liver and heart.

Permeability transition pore (PTP) opening dissipates ion and electron gradients across the internal mitochondrial membrane (IMM), including excess Ca2+ in the mitochondrial matrix. After opening, immediate PTP closure must follow to prevent outer membrane disruption, loss of cytochrome c, and eventual apoptosis. Flickering, defined as the rapid alternative opening/closing of PTP, has been reported in heart, which undergoes frequent, large variations in Ca2+. In contrast, in tissues that undergo depolarization events less often, such as the liver, PTP would not need to be as dynamic and thus these tissues would not be as resistant to stress. To evaluate this idea, it was decided to follow the reversibility of the permeability transition (PT) in isolated murine mitochondria from two different tissues: the very dynamic heart, and the liver, which suffers depolarizations less frequently. It was observed that in heart mitochondria PT remained reversible for longer periods and at higher Ca2+ loads than in liver mitochondria. In all cases, Ca2+ uptake was inhibited by ruthenium red and PT was delayed by Cyclosporine A. Characterization of this phenomenon included measuring the rate of oxygen consumption, organelle swelling and Ca2+ uptake and retention. Results strongly suggest that there are tissue-specific differences in PTP physiology, as it resists many more Ca2+ additions before opening in a highly active organ such as the heart than in an organ that seldom suffers Ca2+ loading, such as the liver.

Adaptive mitochondrial response of the whiteleg shrimp Litopenaeus vannamei to environmental challenges and pathogens.

In most eukaryotic organisms, mitochondrial uncoupling mechanisms control ATP synthesis and reactive oxygen species production. One such mechanism is the permeability transition of the mitochondrial inner membrane. In mammals, ischemia-reperfusion events or viral diseases may induce ionic disturbances, such as calcium overload; this cation enters the mitochondria, thereby triggering the permeability transition. This phenomenon increases inner membrane permeability, affects transmembrane potential, promotes mitochondrial swelling, and induces apoptosis. Previous studies have found that the mitochondria of some crustaceans do not exhibit a calcium-regulated permeability transition. However, in the whiteleg shrimp Litopenaeus vannamei, contradictory evidence has prevented this phenomenon from being confirmed or rejected. Both the ability of L. vannamei mitochondria to take up large quantities of calcium through a putative mitochondrial calcium uniporter with conserved characteristics and permeability transition were investigated in this study by determining mitochondrial responses to cations overload. By measuring mitochondrial swelling and transmembrane potential, we investigated whether shrimp exposure to hypoxia-reoxygenation events or viral diseases may induce mitochondrial permeability transition. The results of this study demonstrate that shrimp mitochondria take up large quantities of calcium through a canonical mitochondrial calcium uniporter. Neither calcium nor other ions were observed to promote permeability transition. This phenomenon does not depend on the life cycle stage of shrimp, and it is not induced during hypoxia/reoxygenation events or in the presence of viral diseases. The absence of the permeability transition phenomenon and its adaptive meaning are discussed as a loss with biological advantages, possibly enabling organisms to survive under harsh environmental conditions.

Functional characterization of the mitochondrial uncoupling proteins from the white shrimp Litopenaeus vannamei.

Mitochondrial uncoupling proteins (UCPs) play an essential role in dissipating the proton gradient and controlling the mitochondrial inner membrane potential. When active, UCPs promote proton leak across the inner membrane, oxidative phosphorylation uncoupling, oxygen uptake increase and decrease the ATP synthesis. Invertebrates possess only isoforms UCP4 and UCP5, however, the role of these proteins is not clear in most species since it may depend on the physiological needs of each animal. This study presents the first functional characterization of crustacean uncoupling proteins from the white shrimp Litopenaeus vannamei LvUCP4 and LvUCP5. Free radicals production in various shrimp organs/tissues was first evaluated, and mitochondria were isolated from shrimp pleopods. The oxygen consumption rate, membrane potential and proton transport of the isolated non-phosphorylating mitochondria were used to determine LvUCPs activation/inhibition. Results indicate that UCPs activity is stimulated in the presence of 4-hydroxyl-2-nonenal (HNE) and myristic acid, and inhibited by the purine nucleotide GDP. A hypoxia/re-oxygenation assay was conducted to determine whether UCPs participate in shrimp mitochondria response to oxidative stress. Isolated mitochondria from shrimp at re-oxygenation produced large quantities of hydrogen peroxide and higher levels of both LvUCPs were immunodetected. Results suggest that, besides the active response of the shrimp antioxidant system, UCP-like activity is activated after hypoxia exposure and during re-oxygenation. LvUCPs may represent a mild uncoupling mechanism, which may be activated before the antioxidant system of cells, to early control reactive oxygen species production and oxidative damage in shrimp.

Metabolism, ATP production and biofilm generation by Staphylococcus epidermidis in either respiratory or fermentative conditions.

Staphylococcus epidermidis is a Gram-positive saprophytic bacterium found in the microaerobic/anaerobic layers of the skin that becomes a health hazard when it is carried across the skin through punctures or wounds. Pathogenicity is enhanced by the ability of S. epidermidis to associate into biofilms, where it avoids attacks by the host and antibiotics. To test the effect of oxygen on metabolism and biofilm generation, cells were cultured at different oxygen concentrations ([O2]). As [O2] decreased, S. epidermidis metabolism went from respiratory to fermentative. Remarkably, the rate of growth decreased at low [O2] while a high concentration of ATP ([ATP]) was kept. Under hypoxic conditions bacteria associated into biofilms. Aerobic activity sensitized the cell to hydrogen peroxide-mediated damage. In the presence of metabolic inhibitors, biofilm formation decreased. It is suggested that at low [O2] S. epidermidis limits its growth and develops the ability to form biofilms.

Mitochondrial uncoupling proteins UCP4 and UCP5 from the Pacific white shrimp Litopenaeus vannamei.

Mitochondrial uncoupling proteins (UCP) transport protons from the intermembrane space to the mitochondrial matrix uncoupling oxidative phosphorylation. In mammals, these proteins have been implicated in several cellular functions ranging from thermoregulation to antioxidant defense. In contrast, their invertebrate homologs have been much less studied despite the great diversity of species. In this study, two transcripts encoding mitochondrial uncoupling proteins were, for the first time, characterized in crustaceans. The white shrimp Litopenaeus vannamei transcript LvUCP4 is expressed in all tested shrimp tissues/organs, and its cDNA includes a coding region of 954 bp long which encodes a deduced protein 318 residues long and a predicted molecular weight of 35.3 kDa. The coding region of LvUCP5 transcript is 906 bp long, encodes a protein of 302 residues with a calculated molecular weight of 33.17 kDa. Both proteins share homology with insect UCPs, their predicted structures show the conserved motifs of the mitochondrial carrier proteins and were confirmed to be located in the mitochondria through a Western blot analysis. The genic expression of LvUCP4 and LvUCP5 was evaluated in shrimp at oxidative stress conditions and results were compared to some antioxidant enzymes to infer about their antioxidant role. LvUCP4 and LvUCP5 genes expression did not change during hypoxia/re-oxygenation, and no coordinated responses were detected with antioxidant enzymes at the transcriptional level. Results confirmed UCPs as the first uncoupling mechanism reported in this species, but their role in the oxidative stress response remains to be confirmed.

The shrimp mitochondrial FoF1-ATPase inhibitory factor 1 (IF1).

The whiteleg shrimp species Litopenaeus vannamei is exposed to cyclic changes of the dissolved oxygen concentration of seawater and must neutralize the adverse effects of hypoxia by using ATP as energy source. In crustaceans, the mitochondrial FOF1-ATP synthase is pivotal to the homeostasis of ATP and function prevalently as a FOF1-ATPase. Hitherto, it is unknown whether these marine invertebrates are equipped with molecules able to control the FOF1-ATPase inhibiting the ATP consumption. In this study, we report two variants of the mitochondrial FOF1-ATPase Inhibitory Factor 1 (IF1) ubiquitously expressed across tissues of the Litopenaeus vannamei transcriptome: the IF1_Lv1 and the IF1_Lv2. The IF1_Lv1, with a full-length sequence of 550 bp, encodes a 104 aa long protein and its mRNA amounts are significantly affected by hypoxia and re-oxygenation. The IF1_Lv2, with a sequence of 654 bp, encodes instead for a protein of 85 aa. Both proteins share a 69 % homology and contain a conserved minimal inhibitory sequence (IATP domain) along with a G-rich region on their N-terminus typical of the invertebrate. In light of this characterization IF1 is here discussed as an adaptive mechanism evolved by this marine species to inhibit the FOF1-ATPase activity and avoid ATP dissipation to thrive in spite of the changes in oxygen tension.