563rd MEETING, LONDON

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Electron Transport in the AIkaIopbiIe Bacilluspasteurii(N.C.I.B. 8841) BRUCE A. HADDOCK and JOHN G. COBLEY Department of Biochemistry, Medical Sciences Institute, Unimrsity of Dundee, Dundee DD1 4HN, Scotland, U.K. Bacillus pasteurii requires unusual conditions for growth in that it develops only in alkaline medium (pH9 or higher) and in the presence of relatively high concentrations of NH4+salts; urea can substitute for NH4+salts since the organism is highly ureolytic. The organism is an obligate aerobe and the requirements for growth were needed also for the oxidative metabolism of whole cells (Wiley & Stokes, 1962). However, substrate oxidation by disrupted cells was NH4+-independent and optimal at pH7.5, and, in addition, indirect experimental evidence indicated that the intracellular pH was near neutrality (Wiley & Stokes, 1963). This implies that the alkaline pH and NH4+requirements affect whole cells externally and not internally. As the external alkaline pH would favour the conversion of NH4+into NH3, it was suggested that NH3 was required by the cells for the transport of low concentrations of substrate across the cell membrane (Wiley & Stokes, 1963). However, these proposals do not explain how the bacterium is able to maintain a pH gradient across its cytoplasmic membrane of some 1.5 units inside acid, during growth, nor do they take into account the possible consequences of this transmembrane pH gradient on the ability of the organism to generate ATP by electron transport. We have begun an investigation of the mechanism of oxidative phosphorylation in this bacterium with the aim of functionally characterizing these phenomena. This report presents a preliminary description of the electron-transport chain of B. pasteurii grown under different conditions. Cells were grown aerobically at 37°C in 2-litre conical flasks, each with 6251111 of growth medium, agitated in a rotary shaker operating at about 200rev./min. Two different growth media were used containing either yeast extract (2 %, w/v), (NH4),S04 (1 %, w/v) and Tris/KOH (0.1 3 M),pH9.0, to yield NH,+-grown cells or yeast extract (1 %, w/v), urea ( 5 % , w/v) and Tris/KOH (0.13~),pH9.0, to yield urea-grown cells. Cells were harvested in the lateexponential phase of growth and washed as a routine with MgCI2 (5m)/(NH4),S04 (lSmM)/Tris/KOH (1OOm)buffer, pH9.0. Membrane particles were prepared from cells, after ultrasonic disruption, by differential centrifugation as described previously for Escherichia coli (Haddock, 1973) in MgCI, (5m)/Tris (100 mM) buffer adjusted to either pH7.5 or pH9.0 as appropriate. Oxidase activities (at 30°C) and cytochrome measurements were performed as described previously (Haddock & Garland, 1971).

NH4+-growncells Cells grown under these conditions were reddish-brown in colour. Polarographic measurements of whole cells, suspended in MgClz (Sm)/Tris/KOH (IOOmM), pH9.0, buffer indicated that, although the cells possessed a significant oxidase activity due to endogenous substrates, there was no appreciable oxidation of added L-glutamate, L-isoleucine, L-threonine or fumarate (each at 3 m ) in the presence or absence of either (NH&SO, (3 m)or urea (3mM). Interestingly, the addition of the proton-conducting agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone (lOpg/mg of protein) stimulated the oxidation of endogenous substrates by about 50% (from 120 to 185ngatom of O/min per mg of protein in a typical experiment). The inability to oxidize these added substrates is at variance with previous results (Wiley &Stokes, 1962) and remains unexplained. However, it precluded measurements of the stoicheiometry of respirationdriven proton translocation coupled to the oxidation of these added substrates and therefore attempts to assess the potential number of energy-conservation sites available to this bacterium (see Lawford &Haddock, 1973). Pulses of air-saturated KCI ( 1 5 0 m ) to an anaerobic suspension of cells, in K,S04 (lOOmM)/glycylglycine (1 m),pH8.8, buffer containing valinomycin (4,ug/ml), did, however, result in transient acidification

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Wavelength (nrn) diference spectra recorded at 77 K obtained Fig. 1. Na2S204-reduced-minus-oxidized with B. pasteurii

NH4+-grown cells (trace a) or urea-grown cells (trace 6) of B. pasteurii were grown and harvested as indicated in the text. Low-temperature difference spectra were recorded as described previously (Haddock & Garland, 1971). The final protein concentrations in the cuvettes were lS.Omg/ml (trace a) and 3.5mg/ml (trace 6).

of the incubation medium with a value for the +H+/O ratio of about 2.7 due to the oxidation of endogenous substrates. Low-temperature reduced-minus-oxidized difference spectra indicated the presence of a variety of cytochromes in whole cells grown under these conditions (Fig. 1, trace a). Five cytochromes with absorption maxima at 548,552,556,562 and 600nm were clearly resolved, but the presence of additional cytochromes could not be discounted. The cytochrome content ofcells varied from batch to batch particularly in the relative amount of species absorbing at 556 and 562nm. Both a 6-type and an a-type cytochrome were capable of reacting with CO, but their kinetic competence to serve as terminal oxidases, must be determined. The specific activity of both NADH ( 0 . 7 m ) and L-ascorbate ( 0 . 7 m ~ with ) NNN'N'tetramethyl-p-phenylenediamine( 7 0 ~ oxidation ~) by membrane particles was twofold higher in particles prepared and assayed at pH7.5 than in particles prepared and assayed at pH9.0. The oxidation of NADH was insensitive to piericidin A (0.25nmol/mg of protein) and antimycin A (20pg/mg of protein), but completely inhibited by KCN (3 mM). Membrane particles did not oxidize added succinate, D-lactate or formate (each at 3 m) to any significant extent. Urea-growncells

Growth in urea-containing medium was much slower than in the NH.++-containing medium, and the cells obtained were conspicuously white in colour. The functional characterization of the electron-transport chain of cells grown under these conditions has proved difficult. Whole cells failed to oxidize any of the substrates tested and indeed 1976

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showed no respiration due to endogenous substrates. Only L-ascorbate with NNN‘N’tetramethyl-p-phenylenediaminewas oxidized by membrane particles and the specific activity obtained was lower than in corresponding particles prepared from NH4+-grown cells. Low-temperature reduced-minus-oxidized difference spectra (Fig. 1, trace b) suggested that this lack of electron-transport activity may be due, in part, to a marked decrease in synthesis of several cytochromes. Discussion

The cytochrome components of the electron-transport chain of B. pasteurii appear similar to those found in other bacilli, including Bacillus subfilis (Chaix & Petit, 1956; Tochikubo, 1971), Bacillus megaterium KM (Broberg & Smith, 1967; Wilkinson & Ellar, 1975), Bacillus brevis (Seddon & Fynn, 1971) and Bacillus cereus (McFeters et al., 1970), and no novel components or unusual properties essential for growth in alkaline media have yet been determined. However, Downs & Jones (1975) have shown that two strains of B. megaterium (D440 and M) possess superficially similar redox components that are functionally organized into two different electron-transport chains. In addition the content of individual redox carriers in all the bacilli so far studied appears to vary widely depending on the growth conditions used. This work was supported in part by the Science Research Council through grant B/RG/62041. Broberg, P. L. & Smith, L. (1967) Biochim. Biophys. Acta 131,479-489 Chaix, P. & Petit, J. F. (1956) Biochim. Biophys. Acta 22,66-71 Downs, A. J. & Jones, C. W. (1975) Arch. Microbiol. 105,159-167 Haddock, B. A. (1973) Biochem.J. 136,877-884 Haddock, B. A. &Garland, P. B. (1971) Biochern.J. 124,155-170 Lawford, H. G. & Haddock, B. A. (1973) Biochem.J. 136,217-220 McFeters, G. A., Wilson, D. F. & Strobel, G. A. (1970) Can. J. Microbiol. 16, 1221-1226 Seddon, B. & Fynn, G. H. (1971) Arch. Mikrobiol. 77,252-261 Tochikubo, K. (1971) J. Bucteriol. 108,652-661 Wiley, W. R. & Stokes, J. L. (1962) J. Bacteriol. 84,730-734 Wiley, W. R. &Stokes, J. L. (1963)J. Bucteriol. 86, 1152-1156 Wilkinson, B. J. & Ellar, D. J. (1975) Eur. J. Biochem. 55,131-139

Electron Transport in Mutants of Escherichiu coli Deficient in their Ability to Synthesize Adenosine 3’ :5’-Cyclic Monophosphate and the Catabolite-Gene Activator Protein MUNAF S. DAOUD and BRUCE A. HADDOCK Department of Biochemistry, Medical Sciences Institute, University of Dundee, Dundee DD1 4HN, Scotland, U.K. Escherichia coli can synthesize a variety of redox carriers depending on the growth phase, the terminal electron acceptor, the carbon source for growth, the growth-limiting nutrient and the strain used. Although the mechanism of electron transport and oxidative phosphorylation in E . coli and the functional characterization of the various redox carriers involved, has been the centre of much attention recently, considerably less effort has been directed towards assessing the intracellular control mechanisms that serve to regulate the synthesis of these redox carriers and their incorporation into the cytoplasmic membrane. In particular, there are several reports in the literature to suggest that the biosynthesis of certain redox carriers and the efficiency of oxidative phosphorylation in E. coliis subject to catabolite repression, and, although the mechanism of control has not been elucidated, a role for cyclic AMP has been implied.

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Electron transport in the alkalophile Bacillus pasteurii (N.C.I.B. 8841).

563rd MEETING, LONDON 709 Electron Transport in the AIkaIopbiIe Bacilluspasteurii(N.C.I.B. 8841) BRUCE A. HADDOCK and JOHN G. COBLEY Department of B...
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