Ubiquinone 8 (coenzyme Q8 or Q8) mediates electron transfer within the aerobic respiratory chain, mitigates oxidative stress, and contributes to gene expression in Escherichia coli. In addition, Q8 was proposed to confer bacterial osmotolerance by accumulating during growth at high osmotic pressure and altering membrane stability. The osmolyte trehalose and membrane lipid cardiolipin accumulate in E. coli cells cultivated at high osmotic pressure. Here, Q8 deficiency impaired E. coli growth at low osmotic pressure and rendered growth osmotically sensitive. The Q8 deficiency impeded cellular O2 uptake and also inhibited the activities of two proton symporters, the osmosensing transporter ProP and the lactose transporter LacY. Q8 supplementation decreased membrane fluidity in liposomes, but did not affect ProP activity in proteoliposomes, which is respiration-independent. Liposomes and proteoliposomes prepared with E. coli lipids were used for these experiments. Similar oxygen uptake rates were observed for bacteria cultivated at low and high osmotic pressures. In contrast, respiration was dramatically inhibited when bacteria grown at the same low osmotic pressure were shifted to high osmotic pressure. Thus, respiration was restored during prolonged growth of E. coli at high osmotic pressure. Of note, bacteria cultivated at low and high osmotic pressures had similar Q8 concentrations. The protection of respiration was neither diminished by cardiolipin deficiency nor conferred by trehalose overproduction during growth at low osmotic pressure, but rather might be achieved by Q8-independent respiratory chain remodeling. We conclude that osmotolerance is conferred through Q8-independent protection of respiration, not by altering physical properties of the membrane. Ubiquinone 8 (coenzyme Q8 or Q8) mediates electron transfer within the aerobic respiratory chain, mitigates oxidative stress, and contributes to gene expression in Escherichia coli. In addition, Q8 was proposed to confer bacterial osmotolerance by accumulating during growth at high osmotic pressure and altering membrane stability. The osmolyte trehalose and membrane lipid cardiolipin accumulate in E. coli cells cultivated at high osmotic pressure. Here, Q8 deficiency impaired E. coli growth at low osmotic pressure and rendered growth osmotically sensitive. The Q8 deficiency impeded cellular O2 uptake and also inhibited the activities of two proton symporters, the osmosensing transporter ProP and the lactose transporter LacY. Q8 supplementation decreased membrane fluidity in liposomes, but did not affect ProP activity in proteoliposomes, which is respiration-independent. Liposomes and proteoliposomes prepared with E. coli lipids were used for these experiments. Similar oxygen uptake rates were observed for bacteria cultivated at low and high osmotic pressures. In contrast, respiration was dramatically inhibited when bacteria grown at the same low osmotic pressure were shifted to high osmotic pressure. Thus, respiration was restored during prolonged growth of E. coli at high osmotic pressure. Of note, bacteria cultivated at low and high osmotic pressures had similar Q8 concentrations. The protection of respiration was neither diminished by cardiolipin deficiency nor conferred by trehalose overproduction during growth at low osmotic pressure, but rather might be achieved by Q8-independent respiratory chain remodeling. We conclude that osmotolerance is conferred through Q8-independent protection of respiration, not by altering physical properties of the membrane. Phospholipid membranes are highly permeable to water but not to polar solutes. Thus, abrupt changes in external solute concentration cause water to rapidly leave or enter cells. In Escherichia coli, osmotically induced dehydration is associated with inhibition of energy-linked functions that include respiration and active transport (1Houssin C. Eynard N. Shechter E. Ghazi A. Effect of osmotic pressure on membrane energy-linked functions in Escherichia coli.Biochim. Biophys. Acta. 1991; 1056 (1984787): 76-8410.1016/S0005-2728(05)80075-1Crossref PubMed Scopus (63) Google Scholar, 2Meury J. Immediate and transient inhibition of the respiration of Escherichia coli under hyperosmotic shock.FEMS Microbiol. Lett. 1994; 121 (7926682): 281-28610.1111/j.1574-6968.1994.tb07113.xCrossref PubMed Scopus (22) Google Scholar, 3Culham D.E. Romantsov T. Wood J.M. Roles of K+, H+, H2O and ΔΨ in solute transport mediated by major facilitator superfamily members ProP and LacY.Biochemistry. 2008; 47 (18620422): 8176-818510.1021/bi800794zCrossref PubMed Scopus (17) Google Scholar). 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Recent reports indicate that cultivation in a high-osmotic pressure medium increases the concentrations of two E. coli membrane lipids: cardiolipin (CL) 3The abbreviations used are: CLcardiolipinΠosmotic pressureAthe initial rate of substrate (radiolabeled proline) uptake at a given osmolalityAmaxthe maximum rate of proline uptake via ProP extrapolated to infinite osmolalityBa constant inversely proportional to the slope of the osmolality response curveCEcollision energyDPHdiphenylhexatrieneGBglycine betaineIPTGisopropyl β-d-1-thiogalactopyranosideMRMmultiple-reaction monitoringOD600optical density measured at a wavelength of 600 nmPEphosphatidylethanolaminePGphosphatidylglycerolPRLproteoliposomeQubiquinoneQ8ubiquinone 8 or coenzyme Q8Q10ubiquinone 10 or coenzyme Q10LBLuria bertani. (8Tsatskis Y. Khambati J. Dobson M. Bogdanov M. Dowhan W. Wood J.M. The osmotic activation of transporter ProP is tuned by both its C-terminal coiled-coil and osmotically induced changes in phospholipid composition.J. Biol. Chem. 2005; 280 (16239220): 41387-4139410.1074/jbc.M508362200Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) and ubiquinone 8 (also known as coenzyme Q8 or Q8) (9Sévin D.C. Sauer U. Ubiquinone accumulation improves osmotic-stress tolerance in Escherichia coli.Nat. Chem. Biol. 2014; 10 (24509820): 266-27210.1038/nchembio.1437Crossref PubMed Scopus (98) Google Scholar). CL associates with particular membrane proteins, including respiratory enzymes (10Arias-Cartin R. Grimaldi S. Arnoux P. Guigliarelli B. Magalon A. Cardiolipin binding in bacterial respiratory complexes: structural and functional implications.Biochim. Biophys. Acta. 2012; 1817 (22561115): 1937-194910.1016/j.bbabio.2012.04.005Crossref PubMed Scopus (64) Google Scholar). In addition, the osmotic pressure required to activate osmosensing transporter ProP is a direct function of the proportion of anionic phospholipid in E. coli (CL plus phosphatidylglycerol (PG)) (11Romantsov T. Stalker L. Culham D.E. Wood J.M. Cardiolipin controls the osmotic stress response and the subcellular location of transporter ProP in Escherichia coli.J. Biol. Chem. 2008; 283 (18326496): 12314-1232310.1074/jbc.M709871200Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Q8 is a redox-active lipid that plays three well-established physiological roles in E. coli: it mediates electron transfer from dehydrogenases to terminal oxidases within the respiratory chain, its reduced form (ubiquinol 8) mitigates oxidative stress by serving as an antioxidant, and it is implicated in the regulation of gene expression (12Søballe B. Poole R.K. 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However, in most cases, the physiological significance of these effects is unclear. cardiolipin osmotic pressure the initial rate of substrate (radiolabeled proline) uptake at a given osmolality the maximum rate of proline uptake via ProP extrapolated to infinite osmolality a constant inversely proportional to the slope of the osmolality response curve collision energy diphenylhexatriene glycine betaine isopropyl β-d-1-thiogalactopyranoside multiple-reaction monitoring optical density measured at a wavelength of 600 nm phosphatidylethanolamine phosphatidylglycerol proteoliposome ubiquinone ubiquinone 8 or coenzyme Q8 ubiquinone 10 or coenzyme Q10 Luria bertani. Sévin and Sauer (9Sévin D.C. Sauer U. Ubiquinone accumulation improves osmotic-stress tolerance in Escherichia coli.Nat. Chem. Biol. 2014; 10 (24509820): 266-27210.1038/nchembio.1437Crossref PubMed Scopus (98) Google Scholar) reported that the Q8 content of E. coli increased 110-fold during growth in a high-osmotic pressure medium, that Q8 deficiency impaired the osmotolerance of E. coli although it did not exacerbate oxidative stress, and that exogenous Q10 (the mammalian ubiquinone variant) restored osmotolerance to Q8-deficient E. coli. Reasoning that Q10 could not substitute for Q8 as a respiratory electron carrier in E. coli, they concluded that the elevation of Q8 concentration contributed to the osmotic stress tolerance of E. coli by affecting physical properties of the cytoplasmic membrane. In fact, evidence indicates that Q10 can substitute for Q8 as a respiratory electron carrier in E. coli (19Okada K. Kainou T. Tanaka K. Nakagawa T. Matsuda H. Kawamukai M. Molecular cloning and mutational analysis of the ddsA gene encoding decaprenyl diphosphate synthase from Gluconobacter suboxydans.Eur. J. Biochem. 1998; 255 (9692900): 52-5910.1046/j.1432-1327.1998.2550052.xCrossref PubMed Scopus (83) Google Scholar). We further examined the impact of Q8 on respiration, osmotolerance, and membrane properties in E. coli. Proton-solute symporters ProP and LacY served as indicators of those impacts: ProP activates as lactose transporter LacY inactivates in response to osmotic upshifts (3Culham D.E. Romantsov T. Wood J.M. Roles of K+, H+, H2O and ΔΨ in solute transport mediated by major facilitator superfamily members ProP and LacY.Biochemistry. 2008; 47 (18620422): 8176-818510.1021/bi800794zCrossref PubMed Scopus (17) Google Scholar). Here we confirm that a Q8 biosynthetic lesion impairs the osmotolerance of E. coli during growth in minimal salts media without exogenous osmolytes. We further show that Q8 deficiency impairs respiration and the activities of ProP and LacY in E. coli cells. We report that Q8 supplementation decreases the membrane fluidity of liposomes prepared from an E. coli polar lipid extract but does not alter the osmotic activation of ProP in proteoliposomes (ProP-supplemented liposomes in which a protonmotive force can be imposed without respiration). Thus, Q8 influences the osmoregulatory action of ProP by supporting respiration, not by altering physical properties of the membrane. During these studies, we observed similar oxygen uptake rates for E. coli cells cultivated in low- and high-osmotic pressure media. This was surprising, because comparable osmotic upshifts dramatically inhibit oxygen uptake (2Meury J. Immediate and transient inhibition of the respiration of Escherichia coli under hyperosmotic shock.FEMS Microbiol. Lett. 1994; 121 (7926682): 281-28610.1111/j.1574-6968.1994.tb07113.xCrossref PubMed Scopus (22) Google Scholar). Such data suggest that respiration is protected by cellular changes that occur during growth at high osmotic pressure. Remarkably, our data show no difference in Q8 content between E. coli cells cultivated at low and high osmotic pressure. Thus, respiration is not protected by elevating Q8. Here, we show that respiration is also not protected by elevating CL or trehalose. Thus, other changes, such as Q8-independent remodeling of the respiratory chain, may protect respiration in E. coli cells grown at high osmotic pressure. Sévin and Sauer (9Sévin D.C. Sauer U. Ubiquinone accumulation improves osmotic-stress tolerance in Escherichia coli.Nat. Chem. Biol. 2014; 10 (24509820): 266-27210.1038/nchembio.1437Crossref PubMed Scopus (98) Google Scholar) reported that the salinity tolerance of E. coli BW25113 was impaired by mutation ΔubiG785::kan, which blocks Q8 synthesis (20Hsu A.Y. Poon W.W. Shepherd J.A. Myles D.C. Clarke C. Complementation of coq3 mutant yeast by mitochondrial targeting of the Escherichia coli UbiG polypeptide: evidence that UbiG catalyzes both O-methylation steps in ubiquinone biosynthesis.Biochemistry. 1996; 35 (8703953): 9797-980610.1021/bi9602932Crossref PubMed Scopus (97) Google Scholar), in Keio Collection strain JW2226. Key characteristics of Keio Collection strains such as growth rate, ribosomal content, and protein expression may be affected by other mutations present in BW25113 (21Cardinale S. Joachimiak M.P. Arkin A.P. Effects of genetic variation on the E. coli host-circuit interface.Cell Rep. 2013; 4 (23871664): 231-23710.1016/j.celrep.2013.06.023Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). However, the ΔubiG785::kan mutation also impaired salinity tolerance in the background of WT E. coli (MG1655) during cultivation in the M9 minimal salts medium used by Sévin and Sauer (9Sévin D.C. Sauer U. Ubiquinone accumulation improves osmotic-stress tolerance in Escherichia coli.Nat. Chem. Biol. 2014; 10 (24509820): 266-27210.1038/nchembio.1437Crossref PubMed Scopus (98) Google Scholar) (Fig. S1A) or in MOPS, a standard minimal medium for physiological studies of E. coli (22Neidhardt F.C. Bloch P.L. Smith D.F. Culture medium for enterobacteria.J. Bacteriol. 1974; 119 (4604283): 736-747Crossref PubMed Google Scholar) (Fig. 1A). Lesion ΔubiG785::kan affected the growth and osmotolerance of E. coli strains MG1655 (Fig. 1 and Fig. S1), BW25113 (Fig. S2), and WG350 (data not shown) similarly. Complementation with plasmid-borne ubiG restored growth to the ΔubiG785::kan derivative of strain MG1655 (ΔubiG pLT1 in Fig. 1A), confirming that the ubiG lesion was directly responsible for the decreased salinity tolerance. Observing similar effects of the ubiG mutation and anaerobiosis, Sévin and Sauer (9Sévin D.C. Sauer U. Ubiquinone accumulation improves osmotic-stress tolerance in Escherichia coli.Nat. Chem. Biol. 2014; 10 (24509820): 266-27210.1038/nchembio.1437Crossref PubMed Scopus (98) Google Scholar) attributed the impact of the ubiG defect on growth at low salinity to impaired respiration. This effect can be masked and the impact of Q8 deficiency on salinity tolerance highlighted by normalizing each growth rate with that obtained at the lowest salinity tested (e.g. Fig. 1 (compare A and B) and Fig. S2 (compare A and B)). The growth of the ΔubiG785::kan mutant appeared to be inhibited more than that of WT E. coli as the medium osmolality increased (Fig. 1). This was true when the osmolality was adjusted with NaCl (Fig. 1, A and B) or with sorbitol, which is not metabolized by E. coli K-12 (Fig. 1, C and D). Thus, the effects of these agents were osmotic, justifying presentation of the data in terms of measured medium osmolalities, where the osmolality (Π/RT, units of mol/kg) is the osmotic pressure (Π) at a particular temperature (T) and R is the gas constant. Redundant osmolyte accumulation mechanisms promote the growth of E. coli K-12 in high-osmolality media (4Wood J.M. Bacterial Osmoregulation: a paradigm for the study of cellular homeostasis.Annu. Rev. Microbiol. 2011; 65 (21663439): 215-23810.1146/annurev-micro-090110-102815Crossref PubMed Scopus (197) Google Scholar). They include the trehalose biosynthetic system (OtsAB) as well as broad specificity osmolyte transporters ProP and ProU. In addition, transporter BetT mediates choline uptake, whereas BetB and BetA mediate the oxidation of choline to glycine betaine. Respiration generates the protonmotive force that powers proton-osmolyte symport via transporter ProP (23Milner J.L. Grothe S. Wood J.M. Proline porter II is activated by a hyperosmotic shift in both whole cells and membrane vesicles of Escherichia coli K12.J. Biol. Chem. 1988; 263 (3049595): 14900-14905Abstract Full Text PDF PubMed Google Scholar) and lactose transport via its paralogue, LacY (24Kaback H.R. Guan L. It takes two to tango: the dance of the permease.J. Gen. Physiol. 2019; 151 (31147449): 878-88610.1085/jgp.201912377Crossref PubMed Scopus (25) Google Scholar), in aerobic E. coli. LacY served as a control in these experiments as it is not osmotically activated, nor does it contribute to osmotolerance. The impact of mutation ΔubiG785::kan on ProP function was first explored by determining the growth rates of E. coli strains WG1230 (proP+ ubiG+) and WG1535 (proP+ ubiG−) as a function of the osmolality in the presence and absence of osmolyte glycine betaine (GB) (Fig. 2). Growth stimulation by glycine betaine indicates ProP activity in these MG1655 derivatives, which lack all other osmolyte accumulation mechanisms (Table 1). GB stimulated the growth of both strains, but it appeared to be more effective in the ubiG+ strain (WG1230) than in its ΔubiG785::kan derivative (WG1535).Table 1E. coli strains and plasmidsStrain or plasmidGenotypeSource or referenceDH5αF− ϕ80 dlacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rk− mk−) supE44 λ− thi-1 gyrA relA1Ref. 59Hanahan D. Studies on transformation of Escherichia coli with plasmids.J. Mol. Biol. 1983; 166 (6345791): 557-58010.1016/S0022-2836(83)80284-8Crossref PubMed Scopus (8170) Google ScholarBKT12W3110 ΔclsA856::FRT ΔclsB861::FRT ΔclsC788::kanRef. 39Tan B.K. Bogdanov M. Zhao J. Dowhan W. Raetz C.R.H. Guan Z. Discovery of a cardiolipin synthase utilizing phosphatidylethanolamine and phosphatidylglycerol as substrates.Proc. Natl. Acad. Sci. 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Recognit. 2000; 13 (10992293): 309-32210.1002/1099-1352(200009/10)13:5%3C309::AID-JMR505%3E3.0.CO;2-RCrossref PubMed Scopus (49) Google ScholarpDC347Encodes full-length clsA under the control of pBAD and AraC in pBAD24Ref. 77Romantsov T. Gonzalez K. Sahtout N. Culham D.E. Coumoundouros C. Garner J. Kerr C.H. Chang L. Turner R.J. Wood J.M. Cardiolipin synthase A colocalizes with cardiolipin and osmosensing transporter ProP at the poles of Escherichia coli cells.Mol. Microbiol. 2018; 107 (29280215): 623-63810.1111/mmi.13904Crossref PubMed Scopus (17) Google ScholarpLT1Encodes full-length ubiG under the control of pBAD and AraC in pBAD24This work Open table in a new tab ProP activity is a sigmoid function of the assay medium osmolality in cells cultivated at low osmolality (25Culham D.E. Henderson J. Crane R.A. Wood J.M. Osmosensor ProP of Escherichia coli responds to the concentration, chemistry and molecular size of osmolytes in the proteoliposome lumen.Biochemistry. 2003; 42 (12525168): 410-42010.1021/bi0264364Crossref PubMed Scopus (77) Google Scholar). Such direct measurement revealed that mutation ΔubiG785::kan decreased ProP activity ∼10-fold (Fig. 3A). Proline was the ProP substrate for these measurements, as proline and glycine betaine are essentially equivalent as ProP substrates (26MacMillan S.V. Alexander D.A. Culham D.E. Kunte H.J. Marshall E.V. Rochon D. Wood J.M. The ion coupling and organic substrate specificities of osmoregulatory transporter ProP in Escherichia coli.Biochim. Biophys. Acta. 1999; 1420 (10446288): 30-4410.1016/s0005-2736(99)00085-1Crossref PubMed Scopus (86) Google Scholar), and proline is more readily available in radiolabeled form than glycine betaine. The osmoregulation of transporter activity was retained, as there was no significant change in the osmolality at which transporter activity was half-maximal (Fig. 3A (inset) and Table S1 (parameters Π½/RT and B)). The ubiG defect could affect ProP activity by impairing generation of the protonmotive force and by altering membrane properties. A similar, dramatic impairment of LacY activity (Fig. 3B and Table S1) suggested that respiration, and hence generation of the protonmotive force, was impaired in the Q8-deficient bacteria. Osmotic inhibition of LacY activity was not observed, because these experiments involved a narrower osmolality range than was employed for previous work (1Houssin C. Eynard N. Shechter E. Ghazi A. Effect of osmotic pressure on membrane energy-linked functions in Escherichia coli.Biochim. Biophys. Acta. 1991; 1056 (1984787): 76-8410.1016/S0005-2728(05)80075-1Crossref PubMed Scopus (63) Google Scholar, 3Culham D.E. Romantsov T. Wood J.M. Roles of K+, H+, H2O and ΔΨ in solute transport mediated by major facilitator superfamily members ProP and LacY.Biochemistry. 2008; 47 (18620422): 8176-818510.1021/bi800794zCrossref PubMed Scopus (17) Google Scholar). In addition, this work was done with intact cells, whereas an earlier report was based on cytoplasmic membrane vesicles (3Culham D.E. Romantsov T. Wood J.M. Roles of K+, H+, H2O and ΔΨ in solute transport mediated by major facilitator superfamily members ProP and LacY.Biochemistry. 2008; 47 (18620422): 8176-818510.1021/bi800794zCrossref PubMed Scopus (17) Google Scholar). Oxygen uptake measurements directly demonstrated the inhibition of respiration by the ubiG lesion and an osmotic upshift (Fig. 4A). As expected, mutation ΔubiG785::kan dramatically decreased the rate of oxygen uptake by bacteria cultivated and incubated at low osmolality (compare circles and squares, Low - Low). That effect was reversed when the ubiG defect was complemented with plasmid pLT1 (compare circles, squares, and triangles, Low - Low). As reported previously (1Houssin C. Eynard N. Shechter E. Ghazi A. Effect of osmotic pressure on membrane energy-linked functions in Escherichia coli.Biochim. Biophys. Acta. 1991; 1056 (1984787): 76-8410.1016/S0005-2728(05)80075-1Crossref PubMed Scopus (63) Google Scholar), respiration was also dramatically inhibited when bacteria cultivated at low osmolality were introduced to a high-osmolality medium (Fig. 4A, Low - Low versus Low - High). Respiration at high osmolality could be restored to ubiG+ bacteria by long-term cultivation in a high-osmolality medium (Fig. 4A, High - High). Similar patterns were seen when the osmolalities of the media were adjusted with NaCl (Fig. 4A) or sorbitol (Fig. 4B). The above results show that respiration (Fig. 4) and the activities of protonmotive force–dependent transporters ProP and LacY (Figure 2, Figure 3 and Table S1) are inhibited by a ubiG lesion (i.e. Q8 deficiency) or osmotic upshifts in E. coli cells. Also, the restoration of respiration afforded by cultivation of E. coli in a high-osmolality medium