Colin W. Jones

Electron transport in Azotobacter vinelandii

Summary 1. The nature and intracellular distribution of the respiratory enzyme system of Azotobacter vinelandii has been investigated. 2. Whole cells of this organism were found to contain high concentrations of ubiquinone (Q-8) but, in contrast to Azotobacter vinelandii strain O, no menaquinone (vitamin K2) was detected. 3. Cell-free extracts prepared by means of the French pressure cell were resolved by differential centrifugation into large particles, small particles and highspeed supernatant. The respiratory chain was almost completely located in the particulate material and comprised flavin, ubiquinone and cytochromes c4 + c5, b1, a1 and α2. Ubiquinone was present in approx. 6-fold molar excess over the individual cytochrome components. 4. The unsupplemented small particles catalysed the rapid oxidation of a variety of substrates including succinate, reduced nicotinamide—adenine dinucleotide, L -malate and DL -lactate. 5. The highly active L -malate and DL -lactate oxidases present in these particles were not linked to pyridine nucleotide, but a low-activity nicotinamide—adenine dinucleotide-linked malate dehydrogenase ( L -malate:NAD+ oxidoreductase, EC 1.1.1.37) was detected in the high-speed supernatant. 6. The oxidation of DL -β-hydroxybutyrate was nicotinamide—adenine dinucleotide-linked and required the presence of both a soluble dehydrogenase ( D -3-hydroxy-butyrate:NAD+ oxidoreductase, EC 1.1.1.30) and a particulate cytochrome system. 7. Antimycin A and 2-alkyl-4-hydroxyquinoline-N-oxide inhibited respiration only at very high concentrations. Inhibition of nicotinamide—adenine dinucleotide oxidase was appreciably higher than succinate oxidase.

The cytochrome system of Azotobacter vinelandii

1. Difference spectra and carbon monoxide action spectra of Azotobacter vinelandii small particles indicate the presence of cytochromes c4 + c5, b1, a1, a2 and o, of which a1, a2 and possibly o may be acting as terminal oxidases. 2. The aerobic steady-state reduction of cytochrome b1 was unexpectedly found to be much lower than that of cytochromes c4 + c5, a result which was incompatible with redox potential and anaerobic state data. 3. 50 μM KCN and 2 mM azide were found to maximally inhibit ascorbate-2,6-dichlorophenolindophenol oxidase whereas NADH oxidase was not significantly inhibited. These results, together with the ability of these inhibitors to increase greatly the aerobic steady-state reduction of cytochromes c4 + c5 but not of b1 suggested the presence of a branched cytochrome system, each branch linked to a functionally separate oxidase system. 4. The concept of a branched cytochrome system was supported by the pattern of light relief of CO inhibition exhibited by the terminal oxidases.

Respiratory protection of nitrogenase in Azotobacter vinelandii

Azofobacter spp. are obligately aerobic bacteria which are capable of reductively assimilating atmospheric nitrogen via a particulate, oxygen-sensitive nitrogenase [ 1,2]. Studies with A. chroococcum under continuous culture [3] suggest that, in the presence of excess oxygen, the nitrogenase is protected by a dual mechanism consisting of: i) Conformational protection, a reversible process, probably manifested via a rapid conformational change within the nitrogenase complex which causes the oxygen-sensitive sites to become inaccessible to oxygen and the nitrogenase to become temporarily inactive, and ii) Respiratory protection, which removes the excess oxygen via enhanced respiration and causes reversal of the conformational protection process (see [4, 51). More recent studies on the complex respiratory system of batch-cultured A. vinelandii [6-81 clearly show that the ambient oxygen concentration during growth has profound effect upon the activity and composition of the respiratory chain of this organism. This present paper describes the changes which occur within the respiratory system of A. vinelandii following the sudden exposure of low aeration cultures to excess oxygen conditions and which, by lowering the energy conservation efficiency, appear to be responsible for the resultant phenomenon of enhanced respiration.

Molecular analysis of the lac operon encoding the binding‐protein‐dependent lactose transport system and β‐galactosidase in Agrobacterium radiobacter

The genes coding for the binding‐protein‐dependent lactose transport system and β‐galactosidase in Agrobacterium radiobacter strain AR50 were cloned and partially sequenced. A novel lac operon was identified which contains genes coding for a lactose‐binding protein (lacE), two integral membrane proteins (lacF and lacG), an ATP‐binding protein (lacK) and β‐galactosidase (lacZ). The operon is transcribed in the order lacEFGZK, The operon is controlled by an upstream regulatory region containing putative ‐35 and ‐10 promoter sites, an operator site, a CRP‐binding site probably mediating catabolite repression by glucose and galactose, and a regulatory gene (lacl) encoding a repressor protein which mediates induction by lactose and other galactosides in wild‐type A. radiobacter (but not in strain AR50, thus allowing constitutive expression of the lac operon). The derived amino acid sequences of the gene products indicate marked similarities with other binding‐protein‐dependent transport systems in bacteria.

Directed evolution of amidase in Methylophilus methylotrophus; purification and properties of amidases from wild-type and mutant strains

Summary: The obligately methylotrophic bacterium Methylophilusmethylotrophus hydrolyses acetamide and acrylamide using a cytoplasmic amidase. In previous work, continuous culture was used to isolate spontaneous mutants which overexpressed either the wild-type amidase (strain MM6) or a mutant amidase with an apparently higher K cat (strain MM8). We now report that NTG mutagenesis of strain MM8 followed by acrylamide-limited growth at low dilution rate (D 0·025 h-1; 37 °C) led to the selection of a strain which continued to overexpress the amidase, but which exhibited an unexpectedly low amidase activity and a greatly decreased K m for acrylamide (strain MM15). Amidases from the wild-type and mutant strains were purified and shown to be homotetramers (subunit M r 38000, pI 4·1). The N-terminal amino acid sequence of the wild-type enzyme was 90% homologous with the aliphatic amidase from Pseudomonas aeruginosa, and Southern blotting using an oligonucleotide probe for this region showed that overexpression of the enzyme in the mutant strains was not due to gene amplification. Compared with the wild-type and MM6 enzymes, the MM8 enzyme exhibited a threefold higher K m and a slightly lower K m for acrylamide, whereas the MM15 enzyme exhibited a similar K cat and an eightfold lower K m for acrylamide. The MM15 enzyme also reacted more extensively with the thiol group reagent DTNB, had a significantly lower sedimentation coefficient and exhibited a more relaxed substrate specificity, all of which were compatible with a looser tetrameric structure. It was also much more susceptible than the other three enzymes to inactivation by high temperature or by freezing and thawing (MM15“MM8>MM6/wild-type), both of which variably dissociated the enzyme into inactive dimers and monomers. The amidase activity of strain MM15 was almost 15-fold higher following growth at 25 °C than at 37 °C, since at this lower temperature the enzyme exhibited a similar K cat to the MM8 enzyme and was not significantly dissociated. However, as strain MM15 readily outgrew the organism from which it was derived (strain MM8) during acrylamide-limited continuous culture at 37 °C, it is clear that under these conditions a low K m was a greater selective advantage than a high K cat.

Energy conservation in the extreme thermophile Thermus thermophilus HB8

Whole cells of the extreme thermophile Thermus thermophilus HB8 contained a membrane-bound respiratory chain (comprised of nicotinamide nucleotide transhydrogenase, NADH dehydrogenase, menaquinone, and cytochromes b, c, aa3, o), which exhibited a maximum→H+/O quotient of approximately 8 g-ion H+·g-atom O-1 for the oxidation of endogenous substrates. Whole cell respiration at 70° at the expense of endogenous substrates or ascorbate-TMPD generated a transmembrane protonmotive force (Δp) of up to 197 mV and an intracellular phosphorylation poteintial (ΔGp), measured under similar conditions, of approximately 43.9 kJ·mol-1.The measured ΔGp/Δp ratio thus indicated an→H+/ATP quotient of approximately 2.3 g-ion H+·mole ATP-1. Glucose-limited continuous cultures of T. thermophilus at 60°, 70° and 78.5° exhibited extremely low moler growth yields (YO2max≤27.6 g cells·mol O2-1; Yglucosemax≤64.4 g cells ·mol glucose-1) compared with mesophilic bacteria of similar respiratory chain composition and proton translocation efficiency. These low yields are probably at least partly explained by the extremely high permeability of the cytoplasmic membrane to H+, which thus causes the cells to respire rapidly in order to maintain the protonmotive force at a level commensurate with cell growth.

The effect of respiratory chain composition on the growth efficiencies of aerobic bacteria

AbstractThe relationship between respiratory chain redox carrier composition and the efficiency of aerobic growth in continuous culture under carbon-limited conditions has been investigated for nine species of bacteria.True molar growth yields with respect to molecular oxygen (

Respiration-linked proton translocation in Azotobacter vinelandii.

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