Mitochondrial DNA and Electron Transport Chain Protein Levels Are Altered in Peripheral Nerve Tissues from Donors with HIV Sensory Neuropathy: A Pilot Study.

Distal sensory polyneuropathy (DSP) and distal neuropathic pain (DNP) remain significant challenges for older people with HIV (PWH), necessitating enhanced clinical attention. HIV and certain antiretroviral therapies (ARTs) can compromise mitochondrial function and impact mitochondrial DNA (mtDNA) replication, which is linked to DSP in ART-treated PWH. This study investigated mtDNA, mitochondrial fission and fusion proteins, and mitochondrial electron transport chain protein changes in the dorsal root ganglions (DRGs) and sural nerves (SuNs) of 11 autopsied PWH. In antemortem standardized assessments, six had no or one sign of DSP, while five exhibited two or more DSP signs. Digital droplet polymerase chain reaction was used to measure mtDNA quantity and the common deletions in isolated DNA. We found lower mtDNA copy numbers in DSP+ donors. SuNs exhibited a higher proportion of mtDNA common deletion than DRGs in both groups. Mitochondrial electron transport chain (ETC) proteins were altered in the DRGs of DSP+ compared to DSP- donors, particularly Complex I. These findings suggest that reduced mtDNA quantity and increased common deletion abundance may contribute to DSP in PWH, indicating diminished mitochondrial activity in the sensory neurons. Accumulated ETC proteins in the DRG imply impaired mitochondrial transport to the sensory neuron's distal portion. Identifying molecules to safeguard mitochondrial integrity could aid in treating or preventing HIV-associated peripheral neuropathy.

Membrane-Bound Redox Enzyme Cytochrome bd-I Promotes Carbon Monoxide-Resistant Escherichia coli Growth and Respiration.

The terminal oxidases of bacterial aerobic respiratory chains are redox-active electrogenic enzymes that catalyze the four-electron reduction of O2 to 2H2O taking out electrons from quinol or cytochrome c. Living bacteria often deal with carbon monoxide (CO) which can act as both a signaling molecule and a poison. Bacterial terminal oxidases contain hemes; therefore, they are potential targets for CO. However, our knowledge of this issue is limited and contradictory. Here, we investigated the effect of CO on the cell growth and aerobic respiration of three different Escherichia coli mutants, each expressing only one terminal quinol oxidase: cytochrome bd-I, cytochrome bd-II, or cytochrome bo3. We found that following the addition of CO to bd-I-only cells, a minimal effect on growth was observed, whereas the growth of both bd-II-only and bo3-only strains was severely impaired. Consistently, the degree of resistance of aerobic respiration of bd-I-only cells to CO is high, as opposed to high CO sensitivity displayed by bd-II-only and bo3-only cells consuming O2. Such a difference between the oxidases in sensitivity to CO was also observed with isolated membranes of the mutants. Accordingly, O2 consumption of wild-type cells showed relatively low CO sensitivity under conditions favoring the expression of a bd-type oxidase.

LncRNA FGD5-AS1 Alleviates Inflammation in Allergic Rhinitis through the miR-223-3p/COX11 Axis.

INTRODUCTION: Long noncoding RNAs (lncRNAs) have been implicated in the pathogenesis of allergic rhinitis (AR). The current investigation is focused on elucidating the functional impact of a specific lncRNA, FGD5 antisense RNA 1 (FGD5-AS1), on the development and progression of AR through its interaction with miR-223-3p. METHODS: An experimental framework for AR was constructed in both cellular and animal models. Quantitative assessment of FGD5-AS1, miR-223-3p, and COX11 mRNA expression was conducted using real-time quantitative reverse transcription PCR. The expression of inflammatory factors, immunoglobulin E, LTC4, and ECP, was examined using ELISA. Apoptosis in human nasal epithelial cells was assessed by the flow cytometry method. The protein expression of COX11 was examined using Western blotting. Nasal mucosal function was further evaluated by hematoxylin and eosin staining. Furthermore, bioinformatics evaluations, dual-luciferase reporter assays, and a series of experimental procedures unveiled a putative competitive endogenous RNA regulatory mechanism. RESULTS: We found the expression of lncRNA FGD5-AS1 was decreased in AR. In vitro lncRNA FGD5-AS1 attenuated the production of inflammatory cytokines in nasal epithelial cells. Furthermore, elevated FGD5-AS1 expression significantly alleviated AR symptoms by reducing nasal epithelial apoptosis and inflammation. MiR-223-3p was identified as a direct target of FGD5-AS1. Moreover, miRNA-223-3p directly downregulated the expression of COX11 mRNA. Subsequent experiments confirmed that FGD5-AS1 regulated AR through the miR-223-3p/COX11 axis, thereby inhibiting inflammation. CONCLUSION: The FGD5-AS1/miR-223-3p/COX11 axis plays a pivotal role in the pathogenesis of AR, suggesting that FGD5-AS1 could serve as a potential diagnostic biomarker and therapeutic target for AR.

[Cytochrome bd as Antioxidant Redox Enzyme].

One of the main functions of enzyme complexes that constitute electron transport (respiratory) chains of organisms is to maintain cellular redox homeostasis by oxidizing reducing equivalents, NADH and quinol. Cytochrome bd is a unique terminal oxidase of the chains of many bacteria including pathogenic species. This redox enzyme couples the oxidation of ubiquinol or menaquinol by molecular oxygen to the generation of proton motive force, a universal energy currency. The latter is used by the organism to produce ATP, another cellular energy currency, via oxidative phosphorylation. Escherichia coli contains two bd-type oxidases, bd-I and bd-II, encoded by the cydAB and appCB operons, respectively. Surprisingly, both bd enzymes make a further contribution to molecular mechanisms of maintaining the appropriate redox balance in the bacterial cell by means of elimination of reactive oxygen species, such as hydrogen peroxide. This review summarizes recent data on the redox-modulated H2O2-scavenging activities of cytochromes bd-I and bd-II from E. coli. The possibility of such antioxidant properties in cytochromes bd from other bacteria is also discussed.

Mutating the environment of heme b595 of E. coli cytochrome bd-I oxidase shifts its redox potential by 200 mV without inactivating the enzyme.

Cytochrome bd-I catalyzes the reduction of oxygen to water with the aid of hemes b558, b595 and d. Here, effects of a mutation of E445, a ligand of heme b595 and of R448, hydrogen bonded to E445 are studied electrochemically in the E. coli enzyme. The equilibrium potential of the three hemes are shifted by up to 200 mV in these mutants. Strikingly the E445D and the R448N mutants show a turnover of 41 ± 2 % and 20 ± 4 %, respectively. Electrocatalytic studies confirm that the mutants react with oxygen and bind and release NO. These results point towards the ability of cytochrome bd to react even if the electron transfer is less favorable.

Cytochrome bd-I from Escherichia coli is catalytically active in the absence of the CydH subunit.

Cytochrome bd-I from Escherichia coli is a terminal oxidase in the respiratory chain that plays an important role under stress conditions. Cytochrome bd-I was thought to consist of the major subunits CydA and CydB plus the small CydX subunit. Recent high-resolution structures of cytochrome bd-I demonstrated the presence of an additional subunit, CydH/CydY (called CydH here), the function of which is unclear. In this report, we show that in the absence of CydH, cytochrome bd-I is catalytically active, can sustain bacterial growth and displays haem spectra and susceptibility for haem-binding inhibitors comparable to the wild-type enzyme. Removal of CydH did not elicit catalase activity of cytochrome bd-I in our experimental system. Taken together, in the absence of the CydH subunit cytochrome bd-I retained key enzymatic properties.

Mitochondrial respiratory chain dysfunction alters ER sterol sensing and mevalonate pathway activity.

Mitochondrial dysfunction induces a strong adaptive retrograde signaling response; however, many of the downstream effectors of this response remain to be discovered. Here, we studied the shared transcriptional responses to three different mitochondrial respiratory chain inhibitors in human primary skin fibroblasts using QuantSeq 3'-RNA-sequencing. We found that genes involved in the mevalonate pathway were concurrently downregulated, irrespective of the respiratory chain complex affected. Targeted metabolomics demonstrated that impaired mitochondrial respiration at any of the three affected complexes also had functional consequences on the mevalonate pathway, reducing levels of cholesterol precursor metabolites. A deeper study of complex I inhibition showed a reduced activity of endoplasmic reticulum-bound sterol-sensing enzymes through impaired processing of the transcription factor Sterol Regulatory Element-Binding Protein 2 and accelerated degradation of the endoplasmic reticulum cholesterol-sensors squalene epoxidase and HMG-CoA reductase. These adaptations of mevalonate pathway activity affected neither total intracellular cholesterol levels nor the cellular free (nonesterified) cholesterol pool. Finally, measurement of intracellular cholesterol using the fluorescent cholesterol binding dye filipin revealed that complex I inhibition elevated cholesterol on intracellular compartments. Taken together, our study shows that mitochondrial respiratory chain dysfunction elevates intracellular free cholesterol levels and therefore attenuates the expression of mevalonate pathway enzymes, which lowers endogenous cholesterol biosynthesis, disrupting the metabolic output of the mevalonate pathway. We conclude that intracellular disturbances in cholesterol homeostasis may alter systemic cholesterol management in diseases associated with declining mitochondrial function.

Alternative oxidase (AOX) 1a and 1d limit proline-induced oxidative stress and aid salinity recovery in Arabidopsis.

Proline (Pro) catabolism and reactive oxygen species production have been linked in mammals and Caenorhabditis elegans, while increases in leaf respiration rate follow Pro exposure in plants. Here, we investigated how alternative oxidases (AOXs) of the mitochondrial electron transport chain accommodate the large, atypical flux resulting from Pro catabolism and limit oxidative stress during Pro breakdown in mature Arabidopsis (Arabidopsis thaliana) leaves. Following Pro treatment, AOX1a and AOX1d accumulate at transcript and protein levels, with AOX1d approaching the level of the typically dominant AOX1a isoform. We therefore sought to determine the function of both AOX isoforms under Pro respiring conditions. Oxygen consumption rate measurements in aox1a and aox1d leaves suggested these AOXs can functionally compensate for each other to establish enhanced AOX catalytic capacity in response to Pro. Generation of aox1a.aox1d lines showed complete loss of AOX proteins and activity upon Pro treatment, yet full respiratory induction in response to Pro remained possible via the cytochrome pathway. However, aox1a.aox1d leaves displayed symptoms of elevated oxidative stress and suffered increased oxidative damage during Pro metabolism compared to the wild-type (WT) or the single mutants. During recovery from salt stress, when relatively high rates of Pro catabolism occur naturally, photosynthetic rates in aox1a.aox1d recovered slower than in the WT or the single aox lines, showing that both AOX1a and AOX1d are beneficial for cellular metabolism during Pro drawdown following osmotic stress. This work provides physiological evidence of a beneficial role for AOX1a but also the less studied AOX1d isoform in allowing safe catabolism of alternative respiratory substrates like Pro.

Mechanistic and structural diversity between cytochrome bd isoforms of Escherichia coli.

The treatment of infectious diseases caused by multidrug-resistant pathogens is a major clinical challenge of the 21st century. The membrane-embedded respiratory cytochrome bd-type oxygen reductase is a critical survival factor utilized by pathogenic bacteria during infection, proliferation and the transition from acute to chronic states. Escherichia coli encodes for two cytochrome bd isoforms that are both involved in respiration under oxygen limited conditions. Mechanistic and structural differences between cydABX (Ecbd-I) and appCBX (Ecbd-II) operon encoded cytochrome bd variants have remained elusive in the past. Here, we demonstrate that cytochrome bd-II catalyzes oxidation of benzoquinols while possessing additional specificity for naphthoquinones. Our data show that although menaquinol-1 (MK1) is not able to directly transfer electrons onto cytochrome bd-II from E. coli, it has a stimulatory effect on its oxygen reduction rate in the presence of ubiquinol-1. We further determined cryo-EM structures of cytochrome bd-II to high resolution of 2.1 Å. Our structural insights confirm that the general architecture and substrate accessible pathways are conserved between the two bd oxidase isoforms, but two notable differences are apparent upon inspection: (i) Ecbd-II does not contain a CydH-like subunit, thereby exposing heme b595 to the membrane environment and (ii) the AppB subunit harbors a structural demethylmenaquinone-8 molecule instead of ubiquinone-8 as found in CydB of Ecbd-I Our work completes the structural landscape of terminal respiratory oxygen reductases of E. coli and suggests that structural and functional properties of the respective oxidases are linked to quinol-pool dependent metabolic adaptations in E. coli.

p107 mediated mitochondrial function controls muscle stem cell proliferative fates.

Muscle diseases and aging are associated with impaired myogenic stem cell self-renewal and fewer proliferating progenitors (MPs). Importantly, distinct metabolic states induced by glycolysis or oxidative phosphorylation have been connected to MP proliferation and differentiation. However, how these energy-provisioning mechanisms cooperate remain obscure. Herein, we describe a mechanism by which mitochondrial-localized transcriptional co-repressor p107 regulates MP proliferation. We show p107 directly interacts with the mitochondrial DNA, repressing mitochondrial-encoded gene transcription. This reduces ATP production by limiting electron transport chain complex formation. ATP output, controlled by the mitochondrial function of p107, is directly associated with the cell cycle rate. Sirt1 activity, dependent on the cytoplasmic glycolysis product NAD+, directly interacts with p107, impeding its mitochondrial localization. The metabolic control of MP proliferation, driven by p107 mitochondrial function, establishes a cell cycle paradigm that might extend to other dividing cell types.

Translational activators and mitoribosomal isoforms cooperate to mediate mRNA-specific translation in Schizosaccharomyces pombe mitochondria.

Mitochondrial mRNAs encode key subunits of the oxidative phosphorylation complexes that produce energy for the cell. In Saccharomyces cerevisiae, mitochondrial translation is under the control of translational activators, specific to each mRNA. In Schizosaccharomyces pombe, which more closely resembles the human system by its mitochondrial DNA structure and physiology, most translational activators appear to be either lacking, or recruited for post-translational functions. By combining bioinformatics, genetic and biochemical approaches we identified two interacting factors, Cbp7 and Cbp8, controlling Cytb production in S. pombe. We show that their absence affects cytb mRNA stability and impairs the detection of the Cytb protein. We further identified two classes of Cbp7/Cbp8 partners and showed that they modulated Cytb or Cox1 synthesis. First, two isoforms of bS1m, a protein of the small mitoribosomal subunit, that appear mutually exclusive and confer translational specificity. Second, a complex of four proteins dedicated to Cox1 synthesis, which includes an RNA helicase that interacts with the mitochondrial ribosome. Our results suggest that S. pombe contains, in addition to complexes of translational activators, a heterogeneous population of mitochondrial ribosomes that could specifically modulate translation depending on the mRNA translated, in order to optimally balance the production of different respiratory complex subunits.

OGT Regulates Mitochondrial Biogenesis and Function via Diabetes Susceptibility Gene Pdx1.

O-GlcNAc transferase (OGT), a nutrient sensor sensitive to glucose flux, is highly expressed in the pancreas. However, the role of OGT in the mitochondria of ß-cells is unexplored. In this study, we identified the role of OGT in mitochondrial function in ß-cells. Constitutive deletion of OGT (ßOGTKO) or inducible ablation in mature ß-cells (ißOGTKO) causes distinct effects on mitochondrial morphology and function. Islets from ßOGTKO, but not ißOGTKO, mice display swollen mitochondria, reduced glucose-stimulated oxygen consumption rate, ATP production, and glycolysis. Alleviating endoplasmic reticulum stress by genetic deletion of Chop did not rescue the mitochondrial dysfunction in ßOGTKO mice. We identified altered islet proteome between ßOGTKO and ißOGTKO mice. Pancreatic and duodenal homeobox 1 (Pdx1) was reduced in in ßOGTKO islets. Pdx1 overexpression increased insulin content and improved mitochondrial morphology and function in ßOGTKO islets. These data underscore the essential role of OGT in regulating ß-cell mitochondrial morphology and bioenergetics. In conclusion, OGT couples nutrient signal and mitochondrial function to promote normal ß-cell physiology.

TAT-Conjugated NDUFS8 Can Be Transduced into Mitochondria in a Membrane-Potential-Independent Manner and Rescue Complex I Deficiency.

NADH dehydrogenase (ubiquinone) Fe-S protein 8 (NDUFS8) is a nuclear-encoded core subunit of human mitochondrial complex I. Defects in NDUFS8 are associated with Leigh syndrome and encephalomyopathy. Cell-penetrating peptide derived from the HIV-1 transactivator of transcription protein (TAT) has been successfully applied as a carrier to bring fusion proteins into cells without compromising the biological function of the cargoes. In this study, we developed a TAT-mediated protein transduction system to rescue complex I deficiency caused by NDUFS8 defects. Two fusion proteins (TAT-NDUFS8 and NDUFS8-TAT) were exogenously expressed and purified from Escherichia coli for transduction of human cells. In addition, similar constructs were generated and used in transfection studies for comparison. The results showed that both exogenous TAT-NDUFS8 and NDUFS8-TAT were delivered into mitochondria and correctly processed. Interestingly, the mitochondrial import of TAT-containing NDUFS8 was independent of mitochondrial membrane potential. Treatment with TAT-NDUFS8 not only significantly improved the assembly of complex I in an NDUFS8-deficient cell line, but also partially rescued complex I functions both in the in-gel activity assay and the oxygen consumption assay. Our current findings suggest the considerable potential of applying the TAT-mediated protein transduction system for treatment of complex I deficiency.

Loss of mitochondrial transcription factor A in neural stem cells leads to immature brain development and triggers the activation of the integral stress response in vivo.

Mitochondrial dysfunction is significantly associated with neurological deficits and age-related neurological diseases. While mitochondria are dynamically regulated and properly maintained during neurogenesis, the manner in which mitochondrial activities are controlled and contribute to these processes is not fully understood. Mitochondrial transcription factor A (TFAM) contributes to mitochondrial function by maintaining mitochondrial DNA (mtDNA). To clarify how mitochondrial dysfunction affects neurogenesis, we induced mitochondrial dysfunction specifically in murine neural stem cells (NSCs) by inactivating Tfam. Tfam inactivation in NSCs resulted in mitochondrial dysfunction by reducing respiratory chain activities and causing a severe deficit in neural differentiation and maturation both in vivo and in vitro. Brain tissue from Tfam-deficient mice exhibited neuronal cell death primarily at layer V and microglia were activated prior to cell death. Cultured Tfam-deficient NSCs showed a reduction in reactive oxygen species produced by the mitochondria. Tfam inactivation during neurogenesis resulted in the accumulation of ATF4 and activation of target gene expression. Therefore, we propose that the integrated stress response (ISR) induced by mitochondrial dysfunction in neurogenesis is activated to protect the progression of neurodegenerative diseases.

Evolution of the cytochrome bd oxygen reductase superfamily and the function of CydAA' in Archaea.

Cytochrome bd-type oxygen reductases (cytbd) belong to one of three enzyme superfamilies that catalyze oxygen reduction to water. They are widely distributed in Bacteria and Archaea, but the full extent of their biochemical diversity is unknown. Here we used phylogenomics to identify three families and several subfamilies within the cytbd superfamily. The core architecture shared by all members of the superfamily consists of four transmembrane helices that bind two active site hemes, which are responsible for oxygen reduction. While previously characterized cytochrome bd-type oxygen reductases use quinol as an electron donor to reduce oxygen, sequence analysis shows that only one of the identified families has a conserved quinol binding site. The other families are missing this feature, suggesting that they use an alternative electron donor. Multiple gene duplication events were identified within the superfamily, resulting in significant evolutionary and structural diversity. The CydAA' cytbd, found exclusively in Archaea, is formed by the co-association of two superfamily paralogs. We heterologously expressed CydAA' from Caldivirga maquilingensis and demonstrated that it performs oxygen reduction with quinol as an electron donor. Strikingly, CydAA' is the first isoform of cytbd containing only b-type hemes shown to be active when isolated from membranes, demonstrating that oxygen reductase activity in this superfamily is not dependent on heme d.

Reduced mitochondrial DNA and OXPHOS protein content in skeletal muscle of children with cerebral palsy.

AIM: To provide a detailed gene and protein expression analysis related to mitochondrial biogenesis and assess mitochondrial content in skeletal muscle of children with cerebral palsy (CP). METHOD: Biceps brachii muscle samples were collected from 19 children with CP (mean [SD] age 15y 4mo [2y 6mo], range 9-18y, 16 males, three females) and 10 typically developing comparison children (mean [SD] age 15y [4y], range 7-21y, eight males, two females). Gene expression (quantitative reverse transcription polymerase chain reaction [PCR]), mitochondrial DNA (mtDNA) to genomic DNA ratio (quantitative PCR), and protein abundance (western blotting) were analyzed. Microarray data sets (CP/aging/bed rest) were analyzed with a focused query investigating metabolism- and mitochondria-related gene networks. RESULTS: The mtDNA to genomic DNA ratio was lower in the children with CP compared to the typically developing group (-23%, p=0.002). Out of five investigated complexes in the mitochondrial respiratory chain, we observed lower protein levels of all complexes (I, III, IV, V, -20% to -37%; p<0.05) except complex II. Total peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) messenger RNA (p<0.004), isoforms PGC1α1 (p=0.05), and PGC1α4 (p<0.001) were reduced in CP. Transcriptional similarities were observed between CP, aging, and 90 days' bed rest. INTERPRETATION: Mitochondrial biogenesis, mtDNA, and oxidative phosphorylation protein content are reduced in CP muscle compared with typically developing muscle. Transcriptional pathways shared between aging and long-term unloading suggests metabolic dysregulation in CP, which may guide therapeutic strategies for combatting CP muscle pathology. What this paper adds Cerebral palsy (CP) muscle contains fewer energy-generating organelles than typically developing muscle. Gene expression in CP muscle is similar to aging and long-term bed rest.

TMT-based proteomic and bioinformatic analyses of human granulosa cells from obese and normal-weight female subjects.

BACKGROUND: Increasing evidence supports a relationship between obesity and either infertility or subfertility in women. Most previous omics studies were focused on determining if the serum and follicular fluid expression profiles of subjects afflicted with both obesity-related infertility and polycystic ovary syndrome (PCOS) are different than those in normal healthy controls. As granulosa cells (GCs) are essential for oocyte development and fertility, we determined here if the protein expression profiles in the GCs from obese subjects are different than those in their normal-weight counterpart. METHODS: GC samples were collected from obese female subjects (n = 14) and normal-weight female subjects (n = 12) who were infertile and underwent in vitro fertilization (IVF) treatment due to tubal pathology. A quantitative approach including tandem mass tag labeling and liquid chromatography tandem mass spectrometry (TMT) was employed to identify differentially expressed proteins. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were then conducted to interrogate the functions and pathways of identified proteins. Clinical, hormonal, and biochemical parameters were also analyzed in both groups. RESULTS: A total of 228 differentially expressed proteins were noted, including 138 that were upregulated whereas 90 others were downregulated. Significant pathways and GO terms associated with protein expression changes were also identified, especially within the mitochondrial electron transport chain. The levels of free fatty acids in both the serum and follicular fluid of obese subjects were significantly higher than those in matched normal-weight subjects. CONCLUSIONS: In GCs obtained from obese subjects, their mitochondria were damaged and the endoplasmic reticulum stress response was accompanied by dysregulated hormonal synthesis whereas none of these changes occurred in normal-weight subjects. These alterations may be related to the high FFA and TG levels detected in human follicular fluid.

Loss of LUC7L2 and U1 snRNP subunits shifts energy metabolism from glycolysis to OXPHOS.

Oxidative phosphorylation (OXPHOS) and glycolysis are the two major pathways for ATP production. The reliance on each varies across tissues and cell states, and can influence susceptibility to disease. At present, the full set of molecular mechanisms governing the relative expression and balance of these two pathways is unknown. Here, we focus on genes whose loss leads to an increase in OXPHOS activity. Unexpectedly, this class of genes is enriched for components of the pre-mRNA splicing machinery, in particular for subunits of the U1 snRNP. Among them, we show that LUC7L2 represses OXPHOS and promotes glycolysis by multiple mechanisms, including (1) splicing of the glycolytic enzyme PFKM to suppress glycogen synthesis, (2) splicing of the cystine/glutamate antiporter SLC7A11 (xCT) to suppress glutamate oxidation, and (3) secondary repression of mitochondrial respiratory supercomplex formation. Our results connect LUC7L2 expression and, more generally, the U1 snRNP to cellular energy metabolism.

Cytochrome bd promotes Escherichia coli biofilm antibiotic tolerance by regulating accumulation of noxious chemicals.

Nutrient gradients in biofilms cause bacteria to organize into metabolically versatile communities capable of withstanding threats from external agents including bacteriophages, phagocytes, and antibiotics. We previously determined that oxygen availability spatially organizes respiration in uropathogenic Escherichia coli biofilms, and that the high-affinity respiratory quinol oxidase cytochrome bd is necessary for extracellular matrix production and biofilm development. In this study we investigate the physiologic consequences of cytochrome bd deficiency in biofilms and determine that loss of cytochrome bd induces a biofilm-specific increase in expression of general diffusion porins, leading to elevated outer membrane permeability. In addition, loss of cytochrome bd impedes the proton mediated efflux of noxious chemicals by diminishing respiratory flux. As a result, loss of cytochrome bd enhances cellular accumulation of noxious chemicals and increases biofilm susceptibility to antibiotics. These results identify an undescribed link between E. coli biofilm respiration and stress tolerance, while suggesting the possibility of inhibiting cytochrome bd as an antibiofilm therapeutic approach.

The flexibility of metabolic interactions between chloroplasts and mitochondria in Nicotiana tabacum leaf.

To examine the effect of mitochondrial function on photosynthesis, wild-type and transgenic Nicotiana tabacum with varying amounts of alternative oxidase (AOX) were treated with different respiratory inhibitors. Initially, each inhibitor increased the reduction state of the chloroplast electron transport chain, most severely in AOX knockdowns and least severely in AOX overexpressors. This indicated that the mitochondrion was a necessary sink for photo-generated reductant, contributing to the 'P700 oxidation capacity' of photosystem I. Initially, the Complex III inhibitor myxothiazol and the mitochondrial ATP synthase inhibitor oligomycin caused an increase in photosystem II regulated non-photochemical quenching not evident with the Complex III inhibitor antimycin A (AA). This indicated that the increased quenching depended upon AA-sensitive cyclic electron transport (CET). Following 12 h with oligomycin, the reduction state of the chloroplast electron transport chain recovered in all plant lines. Recovery was associated with large increases in the protein amount of chloroplast ATP synthase and mitochondrial uncoupling protein. This increased the capacity for photophosphorylation in the absence of oxidative phosphorylation and enabled the mitochondrion to act again as a sink for photo-generated reductant. Comparing the AA and myxothiazol treatments at 12 h showed that CET optimized photosystem I quantum yield, depending upon the P700 oxidation capacity. When this capacity was too high, CET drew electrons away from other sinks, moderating the P700+ amount. When P700 oxidation capacity was too low, CET acted as an electron overflow, moderating the amount of reduced P700. This study reveals flexible chloroplast-mitochondrion interactions able to overcome lesions in energy metabolism.