Ubiquinone 8 Research Articles

Exploring and characterizing the sensitivity of Bdellovibrio bacteriovorus against different stressors.

Given the increasing concern of antibiotic resistance, scientists have been pushed to look for new and interesting ways to kill bacteria. One area of research focuses on using bacterial predators, such as the Gram-negative predatory bacteria Bdellovibrio bacteriovorus, which has been proposed as a “living antibiotic.” B. bacteriovorus consumes other Gram-negative bacteria, burrowing into the periplasmic space and digesting the prey from the inside before releasing several progeny cells. Using a host-independent mutant, we have exposed B. bacteriovorus to various physiologically relevant stressors, such as temperature and the presence of a model antimicrobial peptide (AMP), used as part of the innate immune response. We first noticed that B. bacteriovorus cultures grown at 37°C become dark yellow after one day of growth, while B. bacteriovorus grown at 30°C remain light yellow even after several days of growth. The dark yellow color is caused by the production of ubiquinone-8 (UQ8), an essential member of the bacterial electron transport chain (ETC).We also found that B. bacteriovorus grown at 37°C has increased sensitivity to the AMP magainin 2 (MAG2). MAG2 kills bacteria by forming pores in bacterial membranes, which will disrupt the bacterial ETC, and lead to the production of reactive oxygen species. We monitored the autofluorescence of B. bacteriovorus during MAG2 treatment and observed an oxidative burst directly after adding MAG2.With the increased production of UQ8, B. bacteriovorus grown at 37°C could have a difference in membrane fluidity, making it easier for MAG2 to insert into the cell membrane, disrupting the ETC and killing the cell. By determining how we can manipulate its stress response, we can engineer B. bacteriovorus to become a true “living antibiotic.”

The Biosynthetic Pathway of Ubiquinone Contributes to Pathogenicity of Francisella novicida.

Francisella tularensis is the causative agent of tularemia. Because of its extreme infectivity and high mortality rate, this pathogen was classified as a biothreat agent. Francisella spp. are strict aerobes, and ubiquinone (UQ) has been previously identified in these bacteria. While the UQ biosynthetic pathways were extensively studied in Escherichia coli, allowing the identification of 15 Ubi proteins to date, little is known about Francisella spp. In this study, and using Francisella novicida as a surrogate organism, we first identified ubiquinone 8 (UQ8) as the major quinone found in the membranes of this bacterium. Next, we characterized the UQ biosynthetic pathway in F. novicida using a combination of bioinformatics, genetics, and biochemical approaches. Our analysis disclosed the presence in Francisella of 10 putative Ubi proteins, and we confirmed 8 of them by heterologous complementation in E. coli. The UQ biosynthetic pathways from F. novicida and E. coli share similar patterns. However, differences were highlighted: the decarboxylase remains unidentified in Francisella spp., and homologs of the Ubi proteins involved in the O2-independent UQ pathway are not present. This is in agreement with the strictly aerobic niche of this bacterium. Next, via two approaches, i.e., the use of an inhibitor (3-amino-4-hydroxybenzoic acid) and a transposon mutant, both of which strongly impair the synthesis of UQ, we demonstrated that UQ is essential for the growth of F. novicida in respiratory medium and contributes to its pathogenicity in Galleria mellonella used as an alternative animal model. IMPORTANCE Francisella tularensis is the causative bacterium of tularemia and is classified as a biothreat agent. Using multidisciplinary approaches, we investigated the ubiquinone (UQ) biosynthetic pathway that operates in F. novicida used as a surrogate. We show that UQ8 is the major quinone identified in the membranes of Francisella novicida. We identified a new competitive inhibitor that strongly decreased the biosynthesis of UQ. Our demonstration of the crucial roles of UQ for the respiratory metabolism of F. novicida and for the involvement in its pathogenicity in the Galleria mellonella model should stimulate the search for selective inhibitors of bacterial UQ biosynthesis.

Cryo-EM structures of Escherichia coli cytochrome bo3 reveal bound phospholipids and ubiquinone-8 in a dynamic substrate binding site

Two independent structures of the proton-pumping, respiratory cytochrome bo3 ubiquinol oxidase (cyt bo3 ) have been determined by cryogenic electron microscopy (cryo-EM) in styrene-maleic acid (SMA) copolymer nanodiscs and in membrane scaffold protein (MSP) nanodiscs to 2.55- and 2.19-Å resolution, respectively. The structures include the metal redox centers (heme b, heme o3 , and CuB), the redox-active cross-linked histidine-tyrosine cofactor, and the internal water molecules in the proton-conducting D channel. Each structure also contains one equivalent of ubiquinone-8 (UQ8) in the substrate binding site as well as several phospholipid molecules. The isoprene side chain of UQ8 is clamped within a hydrophobic groove in subunit I by transmembrane helix TM0, which is only present in quinol oxidases and not in the closely related cytochrome c oxidases. Both structures show carbonyl O1 of the UQ8 headgroup hydrogen bonded to D75I and R71I In both structures, residue H98I occupies two conformations. In conformation 1, H98I forms a hydrogen bond with carbonyl O4 of the UQ8 headgroup, but in conformation 2, the imidazole side chain of H98I has flipped to form a hydrogen bond with E14I at the N-terminal end of TM0. We propose that H98I dynamics facilitate proton transfer from ubiquinol to the periplasmic aqueous phase during oxidation of the substrate. Computational studies show that TM0 creates a channel, allowing access of water to the ubiquinol headgroup and to H98I.

Cysteiniphilum marinum sp. nov., isolated from coastal seawater.

Six aerobic Gram-negative bacteria were isolated from seawater in Guangdong Province, P.R. China. Cells were observed to be Gram-negative, aerobic, non-motile and non-spore forming. Growth of the designated type strain 19X3-30T occurred at a temperature range of 14-37°C (optimum, 28°C), a pH range of 6.0-8.0 (optimum, pH 7) and up to 7.5% NaCl (optimum, 1.5%; w/v), and was enhanced by CO2 and L-cysteine supplementation. The major polar lipids identified in strain 19X3-30T were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The principal cellular fatty acids profile showed the presence of anteiso-C15:0, anteiso-C17:0 and C18:0 (> 8% of total fatty acids), and the respiratory quinone was ubiquinone 8 (UQ-8). According to the analysis of 16S rRNA gene sequences, these strains represented a novel species within the family Fastidiosibacteraceae, sharing maximum similarities with Cysteiniphilum litorale DSM 101832T (96.6%) and Cysteiniphilum halobium DSM 103992T (95.3%). Phylogenetic dendrograms based on 16S rRNA gene and protein marker genes from the genomic sequences both indicated that the strains formed a monophyletic lineage closely linked to the genus Cysteiniphilum, which was also supported by the UPGMA dendrogram based on the MALDI-TOF MS profile. The genomic DNA G + C contents of six strains ranged from 38.0% to 38.1%. Based on different taxonomic genomic metrics, phylogeny and phenotypic features, we propose that the strains warrant the assignment to a novel species, for which the name Cysteiniphilum marinum sp. nov. is proposed. The type strain is 19X3-30T (= KCTC 82154T = CGMCC 1.18585T).

Francisella salimarina sp. nov., isolated from coastal seawater.

Four strains (SYSU SYW-1T, SYW-2, SYW-3 and XLW-1) were isolated from seawater near the shore in Guangdong Province, China. Cells were Gram-stain-negative, aerobic, non-motile and non-spore-forming. Growth was observed at a temperature range of 16-40 °C (optimum, 32 °C), a pH range of 4-8 (optimum, pH 7) and in the presence of up to 10 % (w/v) NaCl. The major polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine and an unidentified phospholipid. The respiratory quinone was ubiquinone 8 (UQ-8), and the predominant fatty acids were C18 : 0 3-OH, C10 : 0, C14 : 0 and C18 : 1ω9c. Comparison of 16S rRNA gene and genome sequences confirmed that these strains represented a novel member of the genus Francisella, with less than 98.8 % 16S rRNA gene sequence similarity and less than 95 % genomic average nucleotide identity to recognized Francisella species. The phylogenetic tree based on 16S rRNA gene sequences and the protein-concatamer tree based on a concatenation of 28 protein marker sequences both indicated that the strains clustered with 'Francisella salina' TX07-7308 and 'Francisella marina' E95-16, but formed a distinct lineage group among the other members of the genus Francisella. The DNA G+C contents of the four strains were determined to be 32.9, 32.7, 32.9 and 32.9 %, respectively (genome). On the basis of phenotypic and genotypic features, the strains are considered to represent a novel species of the genus Francisella, for which the name Francisella salimarina sp. nov. is proposed. The type strain is SYSU SYW-1T (=CGMCC 1.17031T=NBRC 113781T).

Characterization and expression analysis of seven putative JHBPs in the mud crab Scylla paramamosain: Putative relationship with methyl farnesoate.

Juvenile hormone binding proteins (JHBPs) serve as the carriers that transport juvenile hormone (JH) to target tissues to activate JH signals. In this study, seven JHBPs from mud crab Scylla paramamosain were obtained. All Sp-JHBPs contained one JHBP domain, and Sp-JHBP4, 5, 6 also contained one Grp7_allergen domain. The predicted secondary and 3D structures are conserved among the seven Sp-JHBP domains, but the complete 3D structure of Sp-JHBP4, 5, 6 were more similar to lipopolysaccharide binding protein. Additionally, the potential ligands for Sp-JHBP1, 2, 3, 7 were ubiquinone 8 (UQ8) and JH II or JH III, and the predicted binding location and sites for JHs were similar to the structured known BmJHBP, suggesting that MF is a potential ligand for these Sp-JHBPs. Furthermore, the expression analysis showed that Sp-JHBP1 expression is relatively low in the 14 detected tissues, and Sp-JHBP2 has the highest expression in the ovary, while the other five JHBPs are highly expressed in the hepatopancreas. During larval development, Sp-JHBP2 is highly expressed during the Z1, M and C1 stages, which are three post-metamorphosis stages, while Sp-JHBP3 and 4 expression levels were significantly lower during the Z5 and M stages, which are two pre-metamorphosis stages. Moreover, the in vivo and in vitro study revealed that Sp-JHBP2 expression was reduced by MF, while the in vitro studies showed that Sp-JHBP3, 6, 7 are induced by MF in a concentration-independent manner. Taken together, we hypothesized Sp-JHBP1, 2, 3, 7 has the potential to bind MF and Sp-JHBP2, 3, 7 play a more important roles in MF signal, while Sp-JHBP4, 5, 6 were more likely to participate in immune response.

Molecular and eco-physiological characterization of arsenic (As)-transforming Achromobacter sp. KAs 3–5T from As-contaminated groundwater of West Bengal, India

ABSTRACTMolecular and eco-physiological characterization of arsenic (As)-transforming and hydrocarbon-utilizing Achromobacter type strain KAs 3–5T has been investigated in order to gain an insight into As-geomicrobiology in the contaminated groundwater. The bacterium is isolated from As-rich groundwater of West Bengal, India. Comparative 16S rRNA gene sequence phylogenetic analysis confirmed that the strain KAs 3–5T is closely related to Achromobacter mucicolens LMG 26685T (99.17%) and Achromobacter animicus LMG 26690T (99.17%), thus affiliated to the genus Achromobacter. Strain KAs 3–5T is nonflagellated, mesophilic, facultative anaerobe, having a broad metabolic repertoire of using various sugars, sugar-/fatty acids, hydrocarbons as principal carbon substrates, and O2, NO3−, NO2−, and Fe3+ as terminal electron acceptors. Growth with hydrocarbons led to cellular aggregation and adherence of the cells to the hydrocarbon particles confirmed through electron microscopic observations. The strain KAs 3–5T showed high As resistance (MIC of 5 mM for As3+, 25 mM for As5+) and reductive transformation of As5+ under aerobic conditions while utilizing both sugars and hydrocarbons. Molecular taxonomy specified a high genomic GC content (65.5 mol %), ubiquinone 8 (UQ-8) as respiratory quinone, spermidine as predominant polyamine in the bacterium. The differential presence of C12:0, C14:0 2-OH, C18:1 ω7c, and C 14:0 iso 3-OH/ C16:1 iso fatty acids, phosphatidylglycerol (PG), phosphatidylcholine (PC), two unknown phospholipid (PL1, PL2) as polar lipids, low DNA-DNA relatedness (33.0–41.0%) with the Achromobacter members, and unique metabolic capacities clearly indicated the distinct genomic and physiological properties of strain KAs 3–5T among known species of the genus Achromobacter. These findings lead to improve our understanding on metabolic flexibility of bacteria residing in As-contaminated groundwater and As–bacteria interactions within oligotrophic aquifer system.

Mitsuaria noduli sp. nov., isolated from the root nodules of Robinia pseudoacacia in a lead-zinc mine.

A novel endophytic bacterium, designated strain HZ7T, was isolated from the root nodules of Robinia pseudoacacia growing in a lead-zinc mine in Mianxian County, Shaanxi Province, China. Cells were Gram-reaction-negative, aerobic, motile, rod-shaped, methyl-red-negative, catalase-positive, positive for chitosan-degrading activity and did not produce H2S. Strain HZ7T grew at 4-45 °C (optimum 25-30 °C), at pH 5-9 (optimum pH 7-8) and with 0-1 % (w/v) NaCl. The quinone type was ubiquinone 8 (UQ-8). The major fatty acids were identified as C16 : 0, C17 : 0 cyclo and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The G+C content of the genomic DNA was 68.5 mol% by whole genome sequencing. According to 16S rRNA gene sequence analysis, the closest phylogenetic relative was Mitsuaria chitosanitabida 3001T (99.05 % similarity). Genome relatedness was computed using average nucleotide identity and genome-to-genome distance analysis, both of which strongly supported strain HZ7Tas belonging to the genus Mitsuaria as a representative of a novel species. On the basis of phylogenetic analysis, chemotaxonomic data and physiological characteristics, strain HZ7T represents a novel species of the genus Mitsuaria, for which the name Mitsuaria noduli sp. nov. is proposed. The type strain is HZ7T (=JCM 31671T=CCTCC AB 2014353T).

Glaciimonas alpina sp. nov. isolated from alpine glaciers and reclassification of Glaciimonas immobilis Cr9-12 as the type strain of Glaciimonas alpina sp. nov.

Psychrophilic bacterial strains were isolated from alpine glaciers in Switzerland and characterized taxonomically. On the basis of phylogenetic analysis of partial 16S rRNA and rpoB genes, three of those strains, strain 79 ( = CCOS 247), strain 4/58 ( = CCOS 250) and strain 4/56 ( = CCOS 258) clustered together with strain Cr9-12T and separately from the type strains Glaciimonas immobilis Cr9-30T and Glaciimonas singularis LMG 27070T. Strain Cr9-12T has been previously described as a strain of G. immobilis. The three newly isolated strains were compared phenotypically with strain Cr9-12T and with the type strains of the species G. immobilis and G. singularis. Cr9-12T and the three novel strains from an alpine glacier in Switzerland were Gram-stain-negative, non-motile, rod-shaped and psychrophilic and showed good growth throughout a temperature range of 1-20 °C and characteristically oxidized d-mannitol, l-fucose and bromosuccinic acid. The predominant cellular fatty acids of strain Cr9-12T and the three novel strains were summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH), C16 : 0 and C18 : 1ω7c. The respiratory quinone of these strains was ubiquinone 8 (UQ-8). The genomic DNA G+C content of Cr9-12T was 49.2 mol%. The combined data from phenotypic, phylogenetic and DNA-DNA relatedness studies strongly support the reclassification of strain Cr9-12T as representing a novel species. This strain and the isolates 79 ( = CCOS 247), 4/58 ( = CCOS 250) and 4/56 ( = CCOS 258) are representatives of a novel species of the genus Glaciimonas, for which the name Glaciimonas alpina sp. nov. is proposed. The type strain of Glaciimonas alpina is Cr9-12T ( = CCOS 761T = DSM 22814T).

Glaciimonas singularis sp. nov., isolated from a uranium mine wastewater treatment plant

A bacterial strain, A2-57(T), recovered from a water sample collected in a uranium mine was taxonomically studied in detail. This strain was a Gram-reaction-negative, rod-shaped bacterium that grew optimally at 25 °C and at pH 6.0-7.0 and had a DNA G+C content of 55.0 mol%. Ubiquinone 8 (UQ-8) was the predominant respiratory quinone and the major fatty acids were C16:0, C17:0 cyclo, summed feature 3 (C16:1ω6c and/or ω7c and/or C15:0 iso 2-OH) and C18:1ω7c. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain A2-57(T) belonged to the family Oxalobacteraceae and formed a distinct branch with Glaciimonas immobilis Cr9-30(T). Strain A2-57(T) shared approximately 97.3% 16S rRNA sequence similarity with G. immobilis Cr9-30(T) and also showed high sequence similarity with members of the genera Herbaspirillum (96.3-97.0%) and Collimonas (96.2-97.0%). Although phylogenetically closely related to the type strain of G. immobilis, the low level of DNA-DNA hybridization between the two strains (21.6%) and several physiological and biochemical properties indicated that the novel strain could be clearly distinguished from G. immobilis LMG 25547(T). Therefore, it is concluded that strain A2-57(T) represents a novel species of the genus Glaciimonas, for which the name Glaciimonas singularis sp. nov. is proposed. The type strain is A2-57(T) (=CIP 110539(T)=LMG 27070(T)).

Nitrification performance and microbial community dynamics in a submerged membrane bioreactor with complete sludge retention

A submerged membrane bioreactor (MBR) supplied with inorganic ammonium-bearing wastewater (NH(4)(+)-N, 500 mgl(-1)) was operated for 260 days without sludge purge under decreased hydraulic retention times (HRT) through six steps (from 30 to 5h). Almost complete nitrification was obtained at a volumetric loading rate (VLR)1.2g NH(4)(+)-Nl(-1)day(-1). The sludge nitrification activities were evaluated at each stage. The specific ammonium oxidizing rate (SAOR) decreased from the initial 0.45 to 0.15 kg NH(4)(+)-Nkg(-1)MLSSday(-1) in the last four stages, while the specific nitrate forming rate (SNFR) increased from 0.17 to 0.39 kg NO(3)(-)-Nkg(-1)MLSSday(-1) at the third stage, and then decreased to below 0.1 kg NO(3)(-)-Nkg(-1)MLSSday(-1) from the fourth stage. Microbial population dynamics was investigated by a combination of the MPN method, fluorescence in situ hybridization (FISH) and quinone profiles. During the experiment, although the MLSS increased gradually from 4.5 to 11.5 gl(-1), the number of ammonia-oxidizing bacteria (AOB) decreased from 10(9)l(-1) at the third stage to 10(7)l(-1) in the last two stages, and that of nitrite-oxidizing bacteria (NOB) decreased gradually from 10(8)l(-1) at the second stage (HRT of 20 h) to the final 10(5)l(-1). FISH results showed that the active cells decreased gradually with time from about 60 to 20% in the last two stages, and most of sludge was inert cells. The sum of nitrifiers occupied only about 10% of the total bacteria number in the last stage even though only ammonium-bearing inorganic wastewater was fed in. Nitrosomonas sp. and Nitrospira sp. were confirmed by FISH as the dominant nitrifying genera responsible for ammonia and nitrite oxidation, respectively. In the mean time, a small ratio of Nitrobacter sp. also existed in the system. FISH analysis matched better with the batch activity test results than did the MPN techniques. Quinone profiles revealed that the dominant ubiquinone was ubiquinone-8 (UQ-8), ranging from 84 to 66%, followed by UQ-10 of 7-13%, UQ-7 of 3-5% and UQ-9 of 1.6-2.6%. The dominant menaquinone in the MBR was menaquinone-7 (MK-7) followed by MK-6, MK-8 and MK-8 (H(2)). With the prolongation of operation, the percentage of menaquinones increased from 8 to 14%. The use of the polyphasic approach gave some new insight on variations of microbial community structures.

Indirect identification of isoprenoid quinones in Escherichia coli by LC‐MS with atmospheric pressure chemical ionization in negative mode

A novel analytical method was applied for identification of isoprenoid quinones in Escherichia coli by liquid chromatography atmospheric press chemical ionization mass spectrometry in negative mode (LC-NI-APCI-MS). Extraction and clean-up of sample were carried out on Sep-Pak Plus Silica solid-phase extraction cartridges. Ubiquinone-7 (UQ-7), Ubiquinone-8 (UQ-8) and Mequinone-8 (MK-8) were determined directly using combined information on retention time, molecular ion mass, fragment ion masses and UV characteristic spectrometry without any standard reagent. It was found that UQ-8 was the major component of isoprenoid quinones in Escherichia coli under aerobic condition. Compared with UQ-8, the relative abundance of UQ-7 and MK-8 is only 15% and 14%, respectively. The average recoveries of UQ-6, UQ-10 and vitamin K(1) in Escherichia coli were investigated by standard spiking experiment. The recoveries were achieved in the range from 94 to 106%, and the relative standard deviations (RSD) of the triplicate analysis of the spiked samples (UQ-6, UQ-10 and vitamin K(1)) ranged from 3 to 8%. The detection limits of LC-NI-APCI-MS were estimated to be 5, 40 and 0.8 microg/g dry cell for UQ-6, UQ-10 and vitamin K(1), respectively.

Comparison between a submerged membrane bioreactor and a conventional activated sludge system on treating ammonia-bearing inorganic wastewater

A submerged membrane bioreactor (SMBR) and a conventional activated sludge system (CAS) were compared in parallel over a period of 210 days on treating synthetic ammonia-bearing inorganic wastewater under similar conditions. Except for a short period of pH control failure, almost complete conversion of NH 4 +N to NO 3 −N was constantly achieved over an NH 4 +N concentration range from 180 to 1300 mg l −1 at a hydraulic retention time (HRT) of 24 h in the SMBR, compared to an average conversion ratio of 95.0% in the CAS. Scanning electron micrographs (SEMs) demonstrated the accumulation of extracellular polymeric substances (EPSs) in the SMBR. Ubiquinone-8 (UQ-8), followed by UQ-10, UQ-7 and UQ-9, was the dominant ubiquinone in both the systems. The dominant menaquinone in the SMBR was menaquinone-6 (MK-6), while that in the CAS was MK-7, indicating that some differences existed between the two systems in terms of microbial community structure. Soluble microbial products (SMPs) tended to accumulate, and then biodegrade in SMBR.

Sulfide oxidation by gene expressions of sulfide-quinone oxidoreductase and ubiquinone-8 biosynthase in Escherichia coli

Sulfides (S 2−,SH −) such as hydrogen sulfide belong to a class of sulfur compounds with unpleasant odors. In order to confer sulfide-oxidizing ability on the intestine-inhabiting bacteria, the sulfide-quinone oxidoreductase gene (sqr) in Rhodobacter capsulatus DSM-155 and genes for quinone biosynthesis ( ubiC, ubiA and ispB) in Escherichia coli XL1 Blue-MRF' were transduced into E. coli BL21(DE3). Plasmids pT7-7 and pSTV were used as vectors of sqr, and ubiCA and ispB, respectively. The recombinants sqr-BL21(DE3) and ubiCA,ispB-sqr-BL21(DE3) were successfully constructed. The maximal sulfide-removing activities of the whole cells and membrane fractions of sqr-BL21(DE3) attained at pH 8.0 and 7.8, were 267 nmol/mg cells (dry weight)/min and 1250 nmol/mg membrane fraction (protein)/min, respectively. The molecular ratio of sulfide (S 2−) oxidized and oxygen (O 2) consumed was 2:1. SQR activity in the recombinant cells was positively restricted under anaerobic conditions and also by the addition of electron transfer inhibitors. Ubiquinone-8 (UQ-8) biosynthesis in the cells of ubiCA,ispB-sqr-BL21(DE3) increased as much as 2.2-fold compared with that of (pSTV)- sqr-BL21(DE3) during the 12–16 h incubation period. The maximal sulfide removal in the quinone-raised E. coli was attained slightly earlier, however, SQR activities thereafter were lower than those in (pSTV)- sqr-BL21(DE3).

Mechanism of Oxidation of Dimethyl Disulphide by Thiobacillus thioparus Strain E6

Summary: A recently isolated organism, capable of chemolithoautotrophic growth on dimethyl disulphide, was characterized as a strain of Thiobacillus thioparus. It had DNA with a base composition of 60.5 ± 1.0 mol% G + C, and ubiquinone-8 (UQ-8) as its only respiratory quinone. Its growth in chemostat culture (at a growth rate of 0.07-0.08 h−1) showed yields of 14.4, 11.8 and 2.45 g cell-carbon per mol of dimethyl disulphide (DMDS), dimethyl sulphide (DMS) and thiosulphate, respectively. This is consistent with energy generation from the oxidation of the methyl and the sulphide moieties of DMDS, with oxidation of sulphide to sulphate contributing a yield of about 2.8 g cell-carbon mol−1. From whole organism and cell-free extract studies, DMDS oxidation was shown to proceed by its (NADH-stimulated) reduction to methanethiol (MT), which was oxidized via sulphide, formaldehyde and formate to CO2 and sulphate by MT oxidase, formaldehyde and formate dehydrogenases, and an uncharacterized sulphide-oxidizing system. The MT oxidase had a K m of 9.7 μM and showed substrate inhibition with a K i of about 8 μM. The essential role of catalase during growth on DMDS was shown by the sensitivity of growth on DMDS (but not on thiosulphate) to 3-amino-1,2,4-triazole. Catalase is believed to destroy the peroxide produced by the MT oxidase reaction. DMDS-grown organisms oxidized sulphide, thiosulphate and tetrathionate (with the latter indicated to be an intermediate in thiosulphate oxidation), suggesting the pathway of sulphide oxidation to be similar to that in some other thiobacilli. Carbon assimilation was by the Calvin cycle, with ribulose bisphosphate carboxylase being present in cell-free extracts at a specific activity of 80 nmol CO2 fixed min−1 (mg protein)−1. Hydroxypyruvate reductase (HPR) was not detected at levels sufficient to indicate any role in primary carbon assimilation.

Hydroxylation of 2-octaprenylphenol in [formula omitted] K 12

A membrane-bound pathway for the biosynthesis of ubiquinone 8 (UQ8) in Escherichia coli has been identified from the analysis of the precursors accumulated by mutants blocked in the pathway. Ubiquinone 8 (UQ8) deficient mutant which accumulate 2 octaprenylphenol (2-OPP) allowed to show that two components are involved in the hydroxylating system of this compound: one membranous, is cytochrome o and the second cytoplasmic, is an NADPH cytochrome c reductase.