Multiplexed transcriptional repression identifies a network of bactericidal interactions between mycobacterial respiratory complexes

Mycobacterium tuberculosis remains a leading cause of infectious disease morbidity and mortality for which new drug combination therapies are needed. Combinations of respiratory inhibitors can have synergistic or synthetic lethal interactions suggesting that regimens with multiple bioenergetic inhibitors will drastically shorten treatment times. However, realizing this potential is hampered by a lack of on-target inhibitors and a poor understanding of which inhibitor combinations have the strongest interactions. To overcome these limitations, we have used CRISPR interference (CRISPRi) to characterize the consequences of transcriptionally inhibiting individual respiratory complexes and identify bioenergetic complexes that when simultaneously inhibited result in cell death. In this study, we identified known and novel synthetic lethal interactions and demonstrate how the engineering of CRISPRi-guide sequences can be used to further explore networks of interacting gene pairs. These results provide fundamental insights into the functions of and interactions between bioenergetic complexes and the utility of CRISPRi in designing drug combinations.

Effect of respiratory inhibitors and quinone analogues on the aerobic electron transport system of Eikenella corrodens

The effects of respiratory inhibitors, quinone analogues and artificial substrates on the membrane-bound electron transport system of the fastidious β-proteobacteriumEikenella corrodensgrown under O2-limited conditions were studied. NADH respiration in isolated membrane particles were partially inhibited by rotenone, dicoumarol, quinacrine, flavone, and capsaicin. A similar response was obtained when succinate oxidation was performed in the presence of thenoyltrifluoroacetone and N,N’-dicyclohexylcarbodiimide. NADH respiration was resistant to site II inhibitors and cyanide, indicating that a percentage of the electrons transported can reach O2without thebc1complex. Succinate respiration was sensitive to myxothiazol, antimycin A and 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Juglone, plumbagin and menadione had higher reactivity with NADH dehydrogenase. The membrane particles showed the highest oxidase activities with ascorbate-TCHQ (tetrachlorohydroquinone), TCHQ alone, and NADH-TMPD (N,N,N’,N’-tetramethyl-p-phenylenediamine), and minor activity levels with ascorbate-DCPIP (2,6-dichloro-phenolindophenol) and NADH-DCPIP. The substrates NADH-DCPIP, NADH-TMPD and TCHQ were electron donors to cyanide-sensitivecbb'cytochromecoxidase. The presence of dissimilatory nitrate reductase in the aerobic respiratory system ofE.corrodensATCC 23834 was demonstrated by first time. Our results indicate that complexes I and II have resistance to their classic inhibitors, that the oxidation of NADH is stimulated by juglone, plumbagin and menadione, and that sensitivity to KCN is stimulated by the substrates TCHQ, NADH-DCPIP and NADH-TMPD.

Pathway and driving forces of selenite absorption in wheat leaf blades

Selenium (Se) deficiency in the human diet is a widespread problem. Se biofortification of wheat crop by spraying foliage with selenite could effectively increase Se intake by enhancing the Se concentration in wheat grains. However, pathway and driving forces of selenite absorption in wheat leaf blades are not fully understood. In this study, the effects of selenite-applied concentration, selenite-exposed duration, stomatal inhibitors, respiratory inhibitors, and competitive anions on selenite absorption in wheat leaf blades were investigated. The results indicated that the selenite absorption rate increased linearly with increasing selenite concentrations, but it decreased greatly and reached a low level with treatment times of 4 h and longer. Stomatal inhibitors significantly inhibited selenite absorption. Respiratory inhibitors and inorganic phosphate (P_i) strongly inhibited selenite absorption. Therefore, selenite passively enters wheat leaf blades via cuticle and stomata, and then enters mesophyll cells via P_i transporters. Concentration gradients and selenite uptake by mesophyll cells provide continual driving forces for selenite absorption in leaf blades.

ATP compartmentation in plastids and cytosol ofArabidopsis thalianarevealed by fluorescent protein sensing

Matching ATP:NADPH provision and consumption in the chloroplast is a prerequisite for efficient photosynthesis. In terms of ATP:NADPH ratio, the amount of ATP generated from the linear electron flow does not meet the demand of the Calvin–Benson–Bassham (CBB) cycle. Several different mechanisms to increase ATP availability have evolved, including cyclic electron flow in higher plants and the direct import of mitochondrial-derived ATP in diatoms. By imaging a fluorescent ATP sensor protein expressed in livingArabidopsis thalianaseedlings, we found that MgATP2−concentrations were lower in the stroma of mature chloroplasts than in the cytosol, and exogenous ATP was able to enter chloroplasts isolated from 4- and 5-day-old seedlings, but not chloroplasts isolated from 10- or 20-day-old photosynthetic tissues. This observation is in line with the previous finding that the expression of chloroplast nucleotide transporters (NTTs) inArabidopsismesophyll is limited to very young seedlings. Employing a combination of photosynthetic and respiratory inhibitors with compartment-specific imaging of ATP, we corroborate the dependency of stromal ATP production on mitochondrial dissipation of photosynthetic reductant. Our data suggest that, during illumination, the provision and consumption of ATP:NADPH in chloroplasts can be balanced by exporting excess reductants rather than importing ATP from the cytosol.

Mitochondria are physiologically maintained at close to 50 °C

In endothermic species, heat released as a product of metabolism ensures stable internal temperature throughout the organism, despite varying environmental conditions. Mitochondria are major actors in this thermogenic process. Part of the energy released by the oxidation of respiratory substrates drives ATP synthesis and metabolite transport, while a noticeable proportion is released as heat. Using a temperature-sensitive fluorescent probe targeted to mitochondria, we measured mitochondrial temperature in situ under different physiological conditions. At a constant external temperature of 38 °C, mitochondria were more than 10 °C warmer when the respiratory chain was fully functional, both in HEK293cells and primary skin fibroblasts. This differential was abolished in cells lacking mitochondrial DNA or by respiratory inhibitors, but preserved or enhanced by expressing thermogenic enzymes such as the alternative oxidase or the uncoupling protein 1. The activity of various RC enzymes was maximal at, or slightly above, 50 °C. Our study prompts a re-examination of the literature on mitochondria, taking account of the inferred high temperature.

Expression of multiplecbb3cytochromecoxidase isoforms by combinations of multiple isosubunits inPseudomonas aeruginosa

The ubiquitous opportunistic human pathogenPseudomonas aeruginosahas five terminal oxidases for aerobic respiration and uses them under different growth conditions. Two of them arecbb3-type cytochromecoxidases encoded by the gene clustersccoN1O1Q1P1andccoN2O2Q2P2, which are the main terminal oxidases under high- and low-oxygen conditions, respectively.P. aeruginosaalso has two orphan gene clusters,ccoN3Q3andccoN4Q4, encoding the core catalytic CcoN isosubunits, but the roles of these genes have not been clarified. We found that 16 activecbb3isoforms could be produced by combinations of four CcoN, two CcoO, and two CcoP isosubunits. The CcoN3- or CcoN4-containing isoforms were produced in the WT cell membrane in response to nitrite and cyanide, respectively. The strains carrying these isoforms were more resistant to nitrite or cyanide under low-oxygen conditions. These results indicate thatP. aeruginosagains resistance to respiratory inhibitors using multiplecbb3isoforms with different features, which are produced through exchanges of multiple core catalytic isosubunits.

Influence of hypoxia on protoplast structure in the plant cell

An influence of hypoxia on the protoplast’s structure in the root tips meristematic cells of onion (<i>Allium cepa</i> L.) and of <i>Tradescantia bracteata</i> Small has been investigated. Hypoxia was caused either by respiratory inhibitors (sodium azide, 2,4-dinitrophenol), phosfon-D or by anaeroibic conditions. In both cases characteristic membranization of cytoplasm was observed. It appeared as spherical and parallel structures of rough endoplasmic reticulum. The observed hypertrophy was not connected with the increase of nucleic acids and proteins synthesis. In the examined cells the membranization was accompanied by an increase of the lipids content.

Effect of cyclophosphamide on meristematic plant cells

The action of cyclophosphamide on meristematic plant cells was checked. A mitostatic influence of this preparation was observed, by way of DNA synthesis inhibition. The disturbance in the course of mitosis is described and the characteristic changes in the structure of the ergastoplasm, analogous to those elicited by respiratory inhibitors.