Nitrogen Assimilation and Energy Conservation in Nitrosomonas

WAITT, INSTITUTE þ.4-8+ LIBRARY

NITROG EN ASSII{ILATION AND ENERGY CONSERVATION

IN ¡VT?ROSOþIONAS EUROPAEA AND NT?ROBACTER AGILTS

Sharad Kumar, M.Sc.

A thesis submitted in fulfilment of the requirements for the degree of

Doctor of PhiTosoPhY

Department. of AgriculÈural Biochemistry, l,laite Agricultural Research Institute' The University of Adelaide. -x- October, 1983. l_

PREFACE

Part of the work described in this thesis has been presented in scientific meetings (Australian Biochemical Society, 1981' 1983; X.II International Congress of Biochemistry, L982). Some of the results have been published or submitted for publication in the journals listed below:

1). Oxygen-dependent nitrite uptake and nitrate production in cells, spheroplasts and membrane vesicles of iVj¿robacter agiTis. S. Kumar and D.J.D. Nicholas FEMS MicrobioT. Lett. 11: 202-206 (1981).

2). The uptake of nitrite and oxygen and the production of nitrate by cells, spheroplasEs and vesicles of Ni trobacter agiTis. S. Kumar and D.J.D. Nicholas Proc. Aust. Biochen. Soc. I4z 48 (1981).

3). Assimilation of inorganic nitrogen compounds by tVitrobacter agiTis. S. Kumar and D.J.D. Nicholas J. Gen. MicrobioT. 128: 1795-1801 (1982).

4). RespiraÈion-dependent proton transloction in NiÈrosononas europaea and its apparent absence in Nitrobacter agiTis during inorganic oxidations. T.C. Hollocher, S. Kumar and D.J.D. Nicholas J. BacterioT. I49z 1013-1020 (1982).

5). A proton-moÈive force dependent adenosine-5t-triphosphate synthesis in spheroplasts of Nitrosononas europaea. S. Kumar and D.J.D. Nicholas FEMS MicrobioT. Lett. l4z 2I-25 (1982).

6). ATP biosynthesis in the niÈrifying bacterium Nitrosomonas eurapaea; S. Kumar and D.J.D. Nicholas Abstracts of 12th InternaËiona7 Congress of Biochemistry, Perth, p.299 (1982)

7). The proton-elecÈrochemical gradienEs in washed cel1s of IVitrosononas europaea and Nitrobacter agilis. S. Kumar and D.J.D. Nicholas J. BacterioT. I54z 65-71 (f983). 1l_

8). Definitivu 15N-NMR evidence that water serves as a source of tgt durì-ng nitrite oxidation by Nitrobacter agiTis. S. Kumar, D.J.D. Nicholas and E.J. Williams FEBS Lett. l52z 7l-74 (1983).

9). Adenylylation and deadenylylation of gluËamine synthetase in the niÈrifying bacteria Nitrosomonas europaea and Nitrobacter agiTis, S. Kumar and D.J.D. Nicholas Proc. Aust. Biochen. Soc. 15: 56 (1983).

10). Purification, properLies and regulation of glut.amine synt.hetase from Ni:robacter agilis. S. Kumar and D.J.D. Nicholas J. Gen. MicrobioT. Submitted for publication.

l1). NAD+ and NADP+ dependent glutamate dehydrogenases in Nitrobacter àgi7is. S. Kumar and D,J.D. Nicholas J. Gen. MicrobioT. Submitted for publication.

II 12). Na- and K- transport in Nitrosononas europaea and Nitrobacter agiTis. S. Kumar and D.J.D. Nicholas Biochin. Biophys. Acta. SubmiÈted for publication. l_11_

ACKNO\^ILEDGEI"IENTS

I record my deep and sincere Èhanks to my supervisor, professor D.J.D. Nicholas, Chairman of the Department of Agricultural Biochemistry, Llaite Agricultural Research Institute, The University of Adelaide, for his guidance, constant encouragenent and consÈrucLive criticism throughout the progress of the present investigation and the preparaÈion of this manuscript.

I would also like to thank Drs. M.E. Tate and T.C. Hollocher for helpful suggestions, Eechnical advice and critical discussions during the course of present investigation and to Dr. E.H. hlilliams for the help w-ith NMR spectroscopy.

Thanks are due to Mr. R.G; Batt for reading the draft of this manuscript, Mr. B.A. Palk for preparing Èhe photographic prints, Mrs. M. Brock for skillful typing and to all others who helped me from time to tine.

The award of U.R.G. scholarship by The University of Adelaide is gratefully acknowledged- 1V

DECLARATION

I hereby declare that thÏs thesis contains no naterial which has been accepted for the award of any other degree or diplorna in any university. To the best of my knowledge and belief, no naterial described herein has been previously published or written by another person except when due reference is nade in the text.

If accepted for the award of a Ph,D. degree, this thesis will be available for loan and photocopying.

SHARAD KUMAR. NOMENCULATURE AND ABBREVIATI ONS

The major enzymes mentioned in this thesis are listed below with their numbers and systematic names as recommended by Enzyme Commission (Enzyme nomenculature I97B).

Trivial Name E.C. Name and Number Adenosine triphosphatase (ATPase) ATP phosphohydrolase E.C. 3.6.1.3 Glutamate dehydrogenase (GDH) L-gluLamate : NAD(P)+ oxidoreductase ( deaminating ) NAD+-GDH E.C. I.4.r.2 NAD(P)+-cDH E.C. 1.4.1.3 NADPï-GDH E.C. r.4.r.4 Glutamate synthase (GOGAT) L-glutamate : NAD+ oxidoreductase ( transaminating ) E.C. r.4.r.14 Glutamine synthetase (GS) L-glut.amate : ammonia ligase (ADP forming) E.C.6.3.r.2

The abbreviations for chemicals, symbols and units in general follow either the tentaÈive rules of IUPAC-IUB Commission on Biochemical Nomenculature (Biochen. J. (1966) 101: 1-7) or the Instruction to Authors for the Biochinica et Biophysica Acta (BBA (1982) 7l5z l-23).

Chemicals

ADP adenosine 5r-diphosphate | AMP adenosine 5 -monophosphate ATP adenosine 5t-triphosphate BSA bovine serum albumin cccP carbonyl cyanide rn-chlorophenyl hydrazone CDP cytidine 5t-diphosphate CMP cytidine 5r-monophosphate CTAB cetyl trimethyl ammonium bromide CTP cytidine 5t-triphosphate DBP 2.4 dibromophenol t DCCD N, N dicyclohexylcarbodiimide DESB diethylstilbestrol DIECA diethyl dithiocarbamate (sodium salt) 2,4 DNP 2.4-dinitrophenol DBP 2 .4-dibromophenol v1

Chemicals continued/. .

BDTA ethylenediamine tetra acetic acid

Y-GH .¡-glutamyl hydroxamate | GDP guanosine 5 -diphosphate t GMP guanosine 5 -monophosphate GTP guanosine 5t-triphosphaEe

HEPES 4- (Z-hy droxyethyl ) -1 -piperazine-eLhanesul phonic acid H0QN0 2-hept.yl-4-hydroxy quinoline-N-oxide 8-HQ 8-hydroxy quinoline IDP . ionosine 5t-diphosphate I IMP ionosine 5 -monophosphate ITP ionosine 5r-triphosphate

MSX L-methionine-DL-su1 phoximine _J- NAD. nicotinamide-adenine dinucleotide (oxidised)

NADH nicot,inamide-adenine dinucleoti de (reduced)

NADP+ nicotinamide-adenine dinucleotide phosphate (oxidised)

NADPH nicotinamide-adenine dinucleotide phosphate (reduced) NBD-chloride 4-chloro-7-nitrobe nzo-Z-oxa-1, 3-diazole

NEM N-ethyl-maleimide N-serve 2-chloro-6-trichloromethyl pyridine pCl4B p-chloro mercuribenzoaLe

PCP penta chlorophenol

POPOP 1,4 bis (2,(4 methyl-5-phenyl oxazolyl)) benzene PPO 2r5 diphenyloxazole SDS sodium-dodecylsulf ate TCA trichloroacetic acid t- lMP thymidine 5 monophosphate 2-TMP 2-t,richloromethyl pyridine TPB tetra phenyl boron Tris 2-amino-2-hydroxymethyl propane-1' 3-diol TPMP+ t.riphenyl methyl phosphosphonium cation J. TPP. teEra-phenyl phosphonium cation TTP thymidine 5r-triphosphate

UDP uridine 5r-diphosphate t TIMP uridine 5 -monophosphate UTP uridine 5t-triphosphate VI].

Symbols and Units

A absorbance

amu atomic mass unit "c degree centrigrade (celcius) I,I curie

cm centlm6ter

d day(s) Ap or ür+ proton-motive force or proton-electrochemical gradient apH transmembrane pH gradient Aü transmembrane potenLial difference (membrane potential) F free energy or Faradayrs constant

ëo unit of gravitational field èo gram G Gibbfs energy constant h hour(s) *tt+/0 no. of protons translocated per 2e- Eransferred to 0 Hz hertz J joule(s ) k kilo K equilibrium constant kcycle kilo cycle K. inhibitor constanÈ I KJ kilojoules K Michaelis constant m KsoI solubility constanÈ KV kilo volt ì. wave length 1 litre

M molar

m meter mCi nillicurie

mg milligram uci microcurie min rninute(s) m1 millilitre mmol millimole ( s )

mM millimolar ug microgram v11l_

ul microlitre Þrnol miçromole ( s )

Ul"I micronolar

N normal

nm nanometer nmol nanamole ( s )

7" percent Pi ilorganic phosphate pHe extracellular pH pHi intracellular pH

R gas constant

S second ( s )

S substrate concentration t time rà half time T thermodyamic Lemperature (Kelvin) v volume v velociEy of the reaction Vmax rate of enzyme catalysed reaction aÈ infinite concentration of substrate wt weight

0thers

approx. approximately atm. atmosphere conc. concentration cpm counts per minute e. g. for example et a7. et aTia (and others) a GC/MS gas chro'matography linked to mass-sPectrometry a. e. that is max. maximum min. minimum IIt-t p-t 0= neta-r pâra-¡ ortho- Nl"fR nuclear magneËic resonance No. number / per p. (plural pp.) page PAGE Polyacrylamide gel electrophoresis S.D. standard deviation l-x

Others continued/. .

S.E.M. standard error of means soln. solution / temp. temperature Viz. namely Vs. versus v/v volume : volume w/v weight : volume less than less Fhan or equal to greater Lhan greater than or equal to approximately equal TABLE OF CONTENTS x

TABLE OF CONTBNTS

Page No.

PREFACE i ACKNOI,ILEDGEMENTS )-aI DECLARATION iv NOMENCULATURE AND ABBREVIATIONS v TABLE OF CONTENTS x LIST OF TABLES xv LIST OF FIGURES xviii SUMMARY xxii

1. INTRODUCTION 1 1.1 NITRIFYING BACTERIA I

1.1.1 Morphology 1 r.t.2 The concept of chenoTithotrophy 1 1.1.3 Amnonia and hydroxyTanine oxidation by Nitrosomonas 3

1.1.3.1 Ammonia Oxidation 3 1.1.3.2 Hydroxylamine Oxidation 5 1.1 .4 Nitrite oxidation by Nitrobacter agiTis I 1.1 .5 Energy coupTing 10

1.1.5.1 Nitrosononas species 10 L.L.5.2 Nitrobacter species I2 1.1.6 Nitrogen assiniTation' I4

T.2 AIMS OF THE STUDY 15

2. I"ÍATERIALS AND METHODS 17

2.7 MATERIALS

2.T.L ChenicaT and biochenicaTs T7 2.t.2 StabTe isotopes 77 2.r.3 Radioisotopes 18 2.r.4 SoTutions, buffers and solvents 18 2.t.5 Chronatographic naterial s 18 2.r.6 Enzynes and marker proteins 19 2.L.7 BacteriaT cuTtures I9

2.2 METHODS I9 2.2.r Growth and harvesting the bacteria 19 2.2.2 Preparation of spheropTasts and nenbrane vesicles 20 2.2.3 Preparation of cel,l-freet'N extracts 20 2.2.4 Incorporation of 7ub"77ed conpounds into ce71 nitrogen 2O 2.2.5 Enzyne purification 2I xl-

Paee No.

2.2.5.I Glutamine synthetase 2L 2.2.5.2 Glutamate dehydrogenase 22 2.2.6 Enzyne assays 23 2.2.6 .r Adenosine triphosphatase (ATPase) 23 2.2.6.2 Glutamine synthetase 23 2.2.6.3 Glutamate synthase 24 2.2.6 .4 Glutamate dehydrogenase 24 of and K. for gTutanine synthetase 24 2.2.7 Deternination -K, and gTutamate dehY?lrogenase 2.2.8 Deternination of noTecular weight of gTutanine 24 synthetase by ge7 fiTtration 2.2.9 CaTcuTation of cunuTative inhibition for 25 gTutanine synthetase 2.2.tO Native and SDS poTyacrylanide ge7 eTectrophoresis 25 (PAGE and SDS-PAGE) 2.2.rr Measurement of oxygen uptake with oxygen eTectrode 26 2.2.I2 Proton transTocation 26 2.2 .L2.1 Fluorescence quenching method 26 2.2 .r2.2 Oxygen pulse exPeriments 27 2.2 .t2.3 Reductant Pulse exPeriments 28 2.2 .r2.4 Estimation of stoichiometric protons 28 2.2 .t2.5 Permeant ions 29 2.2.I3 Deternination of nembrane potentiaT (Lþ) and 29 transmenbrane pH gradient (LpH) in washed ce77s 29 2 .2.r3.1 EDTA treatment of cells 29 2 .2.r3.2 Intracellular water space 2 .2.r3.3 Uptake of radioactive Probes 30 2 .2.L3.4 Cãlculations of proton motive force (Ap or lr+) 30

2.2.14 Na* and K+ transPort 31

2.2.r4.L K+, depletion of cells 31 2.2.r4.2 Nar and K'determinations by atomic 32 absorption spectroseoPY 15N 780 2.2.I5 Stable isotope experiments with und 7ub"rt"d 32 compounds to studY NO| oxidation by ce77s of Nitrobacter agiTis

2.2 .15.1 GCIMS studies 32 2.2 .15.2 151¡-uun studies 34 2.2.L6 GeneraT techniques 35 2.2.16.r Electron-microscoPY 35 2,2,16.2 High voltage paper electrophoresis (llVPE) 36 2.2.L6.3 Liquid scintillation spectrometry 36 2.2.16.4 Preparation of chromatographic columns 36 x11

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2.2.17 ChenicaT deterninations 37

2.2 .r7 . 1 Nitrite 37 2.2 .r7 . 2 Ammonia 37 2.2 . 17. 3 Protein 37 2,2 .L7. 4 Inorganic phosPhate 38 2.2 .17 5 ATP 38

3. RESULTS 3.1 NITRITE OXIDATION BY WASHED CELLS, SPHEROPLASTS AND 39 MBMBRANB VESICLES OF NITROBACTER AGILIS 3.1.1 39 3,L.2 39 3.1 .3 42 3.1.4 42 3.1.5 Optinum conditions for NO, oxidation by membrane 42 vesicTes 3.1.6 Effects of netaboTic inhibitors onNO, oxidation 42 3.r.7 ATPase activity in nenbrane vesicTes- 54 3.1.8 Effects of inhibitors of ATPase activity in 54 menbrane vesicTes 3.1.9 Effects of phosphoTipase A" on nitrite oxidase and 54 ATPase activities in nenbrãne vesicles

3.2 ASSIMILATION OF INORGANIC NITROGEN COMPOUNDS BY NTTROBACTER 58 AGILIS 3.2.L Growth studies 58 3.2.2 Inhibition of NOf, qaidation by Unl in washed ce77s 58 3.2.3 Incorporation ofL IJN 7abe77ed coåpounds into ce77 6L nitrogen 3.2.4 Enzvnes of NH+, assiniTation 6I 3.2.5 EffLcts of L-frethionine-DL-suTphoxinine (IíSX) and 66 azaserine on the activities of GS, GOGAT and GDH 3.2.6 Effects of MSX and azaserine pretreatnent of ce77s on 66 the incorporation of 15NO) an'd l5Un)into ce77 nitrogen 70 3.3 PURIFICATfON, PROPERTIES AND REGULATION OF GLUTAÌ4INE SYNTHETASE (cS) FRoM NTTROBACTER AGILIS AND 1VTTROSOIIONAS EUROPAEA 70 3.3. 1 Purification of GS 3.3.2 Properties of GS 70 3.3.2.r Molecular weight 70 3.3.2.2 Substrate requirements for enzyme acEivity 75 3.3.2.3 Effects of metal ions 75 3.3.2.4 K for substrates of transferase and 75 bÏosynthetic reactions 3.3.2.5 Heat stability and effecEs of denaturing agents 82 3.3.2.6 Inhibition of transferase activi-ty by NHOC1 87 xl-l_ r_

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3.3.3 ReguTation of GS 87

3.3.3.1 Feed back inhibition 87 3.3.3.2 Adenylylation and deadenylylation of GS 92

3.4 GLUTAMATE DEHYDROGENASES OF JVTTROBACTER AGILIS ro2

3.4. I Evidence for two isolynes of GDH in Nitrobacter agiTis ro2 3,4.2 Purification of NADP' -GDH ro2 3.4.3 Anination and deanination reactions in S,,ntuþili" fraction 106 3.4.4 Properties of NADP+ -GOtt from Nitrobactert 106

3.5 ENERGY CONSERVATION IN NITROSOI,IONAS EUROPAE,4 AND NITROBACTER 11i AGILIS

3.5.1 Proton transTocation and oxygen puTse experinents 111

3.5.1.1 Kinetic parameters of respiration in Nitrosononas 111 europaea and Nitrobacter agiTis 3.5,r.2 Permeant ion requì-rement 116 3.s.1.3 Stoichiometric proton production 118 3.s.1.4 Oxygen pulse experiments with Nitrosononas europaea T2T 3.5. 1 .5 Oxygen pulse experiments with Nitrobacter agilis r24 3.s.I.6 Duration of respiraÈion after an oxygen pulse r29 3.5.2 Proton eTectrochemicaT-gradients in washed ce77s r29 of Nitrosomonas europaea and Nitrobacter agiTis 3.s.2.L Uptake of radioactive probes I29 3.5.2.2 MeasuremenÈ of ApH as a function of external pH (pHe) r29 3.5.2.3 MeasuremenÈ of Âù as a function of pHe r32 3.5.2.¿+ Total proton-motive force (Ap) r32 3.5,2.5 Proton--motive force in cells of .Mi trosomonas euîopaea harvested at various stages of growth 134 3.5.2,6 Effects of some inhibit.ors on the components of Ap 134 3.5.2.7 Effects of NHI and NH,OH on Ar! ánd ÂpH 138

3.5.3 ATP biosynthesis driven by artìficia77y induced 140 proton notive force

3.5.3.1 ATP biosynthesis in Nitrosononas europaea 140 3.5.3.2 ATP biosynthesis in iVi trobacter agilis L45 3.5.4 Nat and K+ transport L45 3.5.4.1 r46 3.5.4.2 r46 3.5.4.3 1s0

3.s.4.4 22¡¡u+ loadins of K+ deoleted cells 150 3.5.4.5 22¡¡u+ extruslon fror 22Nu* loaded cells r52 15¡'¡ lB0 3. s.5 StabTe isotope experiments witn und 7ub"17"d 155 compounds to study NO" oxidation by ce77s of Nitrobacter agiTis

3.5.5.1 GC/MS studies of N0" oxidation L56 3.5,5.2 r)N-N¡,IR studíes of Ñ02 oxiclation 1 60 xrv

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4. DISCUSSION 163

4.T NITRTTE OXIDATION BY hIASHED CELLS, SPHEROPLASTS AND 163 I"IEMBRANE VESICLES OF NTTROBACTER AGILIS

4.2 ASSIMILATION OF INORGANIC NITROGEN COMPOUNDS IN 164 NITROBACTER AGILIS AND NTTROSOMONAS EUROPAEA

4 2 .1 Pathways of nitrogen assiniTation in Nitrobacter agilis 164 4 2 .2 Purificationt properties and regulation of gltttanine 166 synthetase and glutamate dehydrogenase from Nitrobacter agiTis and Nitrosononas euroPaea

4.3 ENERGY CONSERVATION IN NITROSOMONAS EUROPAEA AND 77r NITROBACTER AGILIS

4 3 .1 Respiration driven proton transTocation T7I 4 3 .2 Prqton-nolive fcrce and ATP biosynthesis t74 4 3 .3 NA' 44d K' transport-studies 177 4 3 .4 15N,180 isotope of No) oxidation by 180 Nitrobacter agiTis

5 BIBLIOGRAPHY 183 XV

LIST OF TABLES

Table

I EffecLs of various metalli-c ions.on oz dependent Noz uptake and NO3 production by membra ne vesicles.

2 Effects of metal inhibitors on the uptake of N02 and 02 and extrusion of NOj by washed cells. 48

3 Effects of metal inhibition on the uptake of NOf and 02 and extrusion of N0! by spheroplasts' 49

4 Effects of metal inhibitors on the uptake of NOJ and 02 and the extrusion of NOJ by membrane vesicles. 50

5 EffecÈs of inhibiLors of electron transport, oxidative phosphorylation and ATPase on uptake of N02 and 02 and the production of NOj by washed cells. 51

6 Effects of inhibitors of electron transport, oxidative phosphorylat.ion and ATPase on uPtake of NOj and 02 and the productiort of NOJ by spheroplasts. 52

7 Effects of inhibitors of electron transporÈ oxidaLive phosphorylation and ATPase on uptake of NOj' and 02 and the production of NOj by membrane vesicles. 53

I Effects of NEM and pCMB on the uptake of N02 and 02 and the production of NOJ by spheroplasEs and membrane vesicles. 55

9 Effects of sorne inhibitors on ATPase activity in nembrane vesicles. 57 15N-1ubu11ed 10 Incorporation of compounds into washed cells. 63 l5N-t.b"tled 11 Incorporation of compounds into protein of cell extracts (Sr). 65 l2 Specific activities of glutamine synthetase (GS), q1ut9- mãte synt.hase (GOGAT) and glutamate dehydrogenase (GDH) in cell extracts (Sf+a). 67 tt*o2 15llu| 13 rncorporat.ion of una into cells pretreated l/ith MSX and azaserine. 69

T4 Purification of GS from Nitrobacter agiTis 7I

15 Purification of GS from Nitrosononas europaea 72

16 Substrate requirements for the y-glutamyl transferase reaction of purified GS from Nittobacter agiTis. 78

T7 Substrate requirements for the biosynthetic activity of purified GS from lVi trobacter agiTis. 79 xva

Table Page No.

18 Effects of various divalenE cations on the y-glutamyl transferase activity of purified GS from Nitrobacter agiTis. 80 Effects of various divalent caLions on the biosynthetic 1', activity of purified GS from Nitrobacter agiTis' B1

20 Propertíes of purified GS,from Nitrobacter agiTis. 85 + 2L Effect of NH[ on K¡¡ of the substrates of y-glutamyl transferase activity of purified GS from Nitrobacter agiTis. 89

22 Effects of various amino acids on '¡-glutamyl transferase and biosynthetic activities of purified GS fror¡ Nitrobacter agiTis. 90

23 Combined effects of amino acids on y-glutamyl transferase activity of purified GS from Nitrobacter agiTis. 93

24 Effects of nucleotides on y-gluEamy1 Lransferase activity of purified GS from lVi trobacter agilis 94

25 Effects of CTAB treatment on y-glutamyl transferase acLivity in cell extracts from Njtrobacter agiTis and Nitrosononas euroPaea. 96

26 Effects of some amino acids and 5t-AMP on adenylylated and deadenylylated forms of purified GS from Nitrobacter agiTis. 101 104 27 Purification of NADP+-GDH from Ni¿robacter agiTis.

28 purification of NADP+-GDH from iVitrosononas europaea. 105

29 Amination and deamination reaction raLes in exÈracts (S110) of Nitrobacter agiTis. 107

30 Effects of organic acids on the amination activity of partially putlfi-"d NADP+-GDH f¡e¡¡ Nitrobacter agiTis' 110

31 Effects of nucleotides on the amination activity of partially purified NADP+-GDH from lVitrobacter agiTis. II2.

32 Kinet.ic parameters of respiration in washed cells of Nitrosononas europaea. Lt4

33 Kinetic parameters of respiration in washed cellsri: spheroplasts and membrane vesicles oL Nitrobacter agiTis. 115

34 Bffects of salts of three permeant anions on aerobic oxidation of substrates by washed cells of Nitrosononas europaea and Nitrobacter agiTis. LL7

35 Effects of lipophilic cation TPMP+ on aerobic oxidation of substrates by washed cells of Nj trosomonas europaea and Nitrobacter agiTis. 119 xv11

Table Pase No. 36 Production of stoichiometric protons by Ni trosomonas europaea and Nitrobacter agiTis. r20

I 37 Apparent ->H' /O ratios in rvashed cells of Ni trosononas europaea and ilitrobaçter agiTis during substraLe oxidation using TPMP' as permeant ion. 125 38 Probes for determining Aü and ApH in washed cells of Nitrosononas europaea and Nitrobacter agiTis. 130 39 Effects of some inhibitors on respiration and proton- motive force in Nitrosomonas europaea. r36 40 Effects of some inhibit.ors on respiration and proton- motive force in Nitrobacter agiTis. r37

4T lntracellular concentration of Na* and Kf in washed cells of NiÈrosomonas europaea and ffitrobacter agilis. r47 42 Effects of Kl depletion and K* extrusion on respirat.ion in lVitrosononas europaea and ilitrobacter agilis. r49 43 Stable isotope experiments with washed cells of Nitrobacter agiTis. 158 xvl_l_l-

LIST OF FIGURES

Fie. Pase No. I Blectrode assembly for measuring N03 production and 02 uptake. 40

2 Uptake of. 02 and extrusion of N03 by washed ce11s- 41

3 Stoichiometry of NOl and d2 uptake and NOj exLrusion by washed cells. 4t

4 Electron micrographs of lysozyme-EDTA cells of Nitrobacter agiTis. 43

5 Electron micrographs of Nitrobactet agiTis membrane vesicles. 44

6 Stoíchiometry of N0) and 02 uptake and NOa extrusion by spheroplasts and membrane vesicles. 45

7 ATPase activity in membrane vesicles. s6

8 Effects of phospholipase-A2 treatment on ATPase and N0) oxidase acÈivity in membrane vesicles. s6

9 Growth of. Nítrobacter agiTis in NH4C1 containing mediurn. s9

10 Cell yields from culÈures of Nitrobactet agiTis grown with various concentrations of NH¿*CI. s9

11 Inhibition of NOf oxidation in washed cells by NH4C1 ' 60 62 12 Competitive inhibition of NO! oxidation by NH4CI'

13 Double reciprocal plot of inhibiÈion of NOi oxidation at various concentrations of NH4C1. 62 15llo7, ttto5, 15t'l02oH 14 Time cour se for incorporation of and lsunt respectively into washed cel1s. 64

l5 Effects of MSX and azaserine on glutamine synÈhetase, glutamate synthase and glutarnate dehydrogenase. 68

16 Elution profile for GS of Nitrobacter agilis from Blue Sepharose CL-68 column. 73

T7 Elution profile for GS of Nitrobacter agiTis from Sepharose-48 column. 73

1B Polyacrylamide gel electrophoresis of purified GS from Nitrobacter agiTis. 74

19 Polyacrylamide gel electrophoresis of purified GS from Nitrosononas europaea. 74 76 20 SDS-PAGE of purified GS from Nitrobactet agiTis' xrx

Fie. Paee No.

2T Determination of subunit molecular weight of purified GS from Nitrobacter agiTis. 76

22 Determination of native molecular weight of purified GS from Nitrobacter agiTis. 77

23 The effects of various concentrations of L-glutamine on y-glutamyl transferase activity of purified GS from Nitrobacter agiTis. 83

24 The effects of various concentrations of NH20H on J-glutamyl transferase activity of purified GS from Nitrobacter agiTis. 83

25 The effects of various concentrations of L-glutamate on the biosynthetic activity of purified GS from Nitrobacter agilis. 84

26 The effects of various concentrations of NH4C1 on the biosynthetic activity of purified GS from Nitrobacter agiTis. 84

27 Tj-rnecourse for inactivation by NH20H of y-g1utamy1 transferase and biosynthetic acÈivities of purified GS from Nitrobacter agiTis, 86

28 NHI intribition of Y-glutamyl transferase activity of purified GS from Nitrobacter agiTis. 86

29 Competitive inhibition by NH4CI of '¡-glutamyl transferase activity of purified GS from lVitrobacter agiTis. 88

30 Mixed type ínhibition by NH4C1 of y-glutamyl transferase activity of purified GS from Nitrobacter agiTis. 88

31 Inhibition of y-glutamyl transferase activity of purified GS from Ni trobacter agiTis by various concentrations of alanine, glycine and serine. 91

32 Effects of NHI concentration in culture mediurn on ]-glutamyl transferase activit.y of GS in crude exLracts of Nitrosononas europaea at various stages of growth. 97

33 Effects of phosphodiesterase treatment on purified GS from Nitrobacter agiTis and NjtÍosomonas euroPaea. 99

34 DeLermination of isoactivity pH for purified GS from Nitrobacter agiTis. 100

35 Detection of NAD+ and NADP+ dependent GDH in Nitrobacter agiTis. 103

36 Determination of pH optima for amination and deaminatj-on activities of partialiy purified NADP+-GDH from Nitrobacter agiTis. 108 XX

Fie. Page No.

37 The effects of various concentrations of a-ketoglutarate on the anination reacLion of partially purified NADP+-GDH from /Vitrobacter agiTis. 108

3B The effects of various concentrations of NH4CI on the aminaLion reacLion of partially purified NADP+- GDH from Nitrobacter agilis. 109

39 The effects of various concentrations of NADPH on the amination reaction of Lhe partially purified NADP+-GDH frorn ilitrobacter agiTis. 109

40 Pi'oton extrusion in washed cells of Nitrosononas europaea and Ni trobacter agiTis determined by fluorescence met.hod. 113

4L Oxygen pulse responses in washed cel1s of NitrosoÍnonas europaea. L22

42 Dependence of +Ht/O ratio on the sj-ze of the oxygen pulse for oxidation of NH20H by rVitrosomonas europaea. r23

43 Dependence of +H*/O ratio on the substrate concentrati-on for oxidation of amine-like substrate by Nitrosononas europaea. 723

4l+ Oxygen pulse responses in spheroplasts oL Nitrobacter agiTis. r27

45 Oxygen pulse responses in membrane vesicles of Nitrobacter agiTis. 128

46 Uptake or []aCl benzoic acld, tl4Cl acetylsalicylic acid and [tuC] rnethylarnine as a function of pHe by EDTA treated cells of Ni trosomonas europaea and Nitrobacter agiTis. 131

47 Effects of pHe on pHi, ApH, Arþ and Ap in.EDTA-treated cel1s of ilitrosononas europaea and iVitrobnacter agiTis. 133

48 Intracellular pH and Arþ in cel1s of Nitrosononas europaea at various stages of growth, determined at pHe values of 6 and 8. 135

49 Effects of NHf and NH20H on Aü and ApH in washed cells of Nitrosononàs europaea. 139

50 ATP synthesis in washed cel-ls of Nitrosononas europaea. I4L

51 Valinomycin induced ATP synthesis in spheroplasts of T4T N itrosononas europaea.

52 Bffect of DCCD on valinomycin induced ATP synthesis in spheroplasts of Nitrosononas europaea. r42

53 Effect of K+ on valinomycin induced ATP synthesis in spheroplasts of Nitrosononas europaea. r42 xxr_

Fie ' Paee No. 54 Nigericin mediated f+/U+ exchange in spheroplasts of Nitrosomonas euroPaea. r44

55 Schematic representation of nigericin rnediated K+/H+ exchange across the spheroplast membrane. It+4

s6 Time course for K* extrusion from uhe cells of Nitrosononas europaea and Nitrobacter agil-is during diethanolamine treatment. t48

57 Bffccts of external pH (pHe) on ApH, Aü and Ap in K+ depleted cel1s of Nitrosononas europaea and Nitrobacter agitis and the effects of added K*. 151 22N.+ 58 Uptake of by diethanolamine loaded cel1s of Nitrosononas europaea and Nitrobacter agiTis. 153 22Na+ 22Nu* s9 exLrusion ftorn loaded cel1s of Nitros ononas el,ropaea and Nltrobacter agiTis. 154

60 Mass spectra of Lrimethyl phosphate. r57

61 Mass spectra of NrO gas. 159 180/160 62 NMR spectra of derivatives of nitrate. 16r

xxl_a

SUI"IMARY

1. This thesis embodies results of an investígation on some aspects of nitrogen assinilation and energy conservation in nit.rifying bacteria, Nitrosononas europaea and Nitrobacter agilis'

2. Electrode techniques have been developed to measure N0, producLion and 0, uptake simultaneously and continuously during N0, utilization by Nitrobacter agiTis. The stoichiometry of NO, oxidation by washed cells was lNOl : 0.502 : 0.75 N0, compared to lNOl : 0.502 : 1N0l for spheroplasts and membrane vesicles of Nitrobacter agiTis. The effects of several merabolic inhibitors on N0] and 0, utilization ancl N0! lroduction were invesLigated. Nitrite oxidation was found to be particularly sensitive to the inhibitors of cyÈochrome oxidase (eg. azide), ATPase (eg. DCCD), -SH group (eg. pCMB) and uncouplers of oxidaÈive phosphorylation (ee. CCCP).

3. A Mg2+ dependent ATPase was detected in membrane vesicles of Nittobacter agiTis which was inhibited by classical ATPase inhibitors (eg. DCCD) but was unaffected by uncouplers (eg. 2,4.DNP,CCCP). The ATPase activity of membrane vesicles was progressively lost by delipidation of membranes by phospholipase Ar.

4 The growth yields of Nitrobacter agilis were increased about 2 fold by growing the bacterium in a N0, medium supplemented with 2ml"f-NHOCl. Higher concentrations of NHOCI however competitively inhibited N0, oxidation and the growth of the bacterium.

It*tl l5lln 0H' 5. trlashed cells of Nitrobacter agiTis readily íncorporated 2 15n0, ttto; una respectively (in decreasing order) into cell nitrogen Inhibitors of glutamine synÈhetase (L-met.hionine Dl-sulphoximine) and glutamate synthase (azaserine) did not affect the incorporaLion of either tt*tl or 15llO, into cell nitrogen, indicating that glutamate dehydrogenase pathway is the main route for the assimilation of uHf in Nitrobacter agilis.

6. Glutamine synthetase was purifi-ecl 430 fold from IVitrobacter agilis and iLs properties and regulation studied. The eîzyme (molecular weight 700,000) which contained 12 subunits of 58,000 each was regulated by feed back inhibition involving amino acids and nucleotides, substrate xxtl].

inhibirion by NHf as well as by an adenylylation/deadenylylation mechanism. An isoactivity pl{ of 7.4 was recorded 'for the purified enzyme.

7. Glutamine synthetase fromrilitrosomonas euroPaea \Ias purified 710 fold' In crude exLracts, the Mg'- effecL on the 'y-glutamyl Lransferase to NHI concentration in growth medium. This activity was related ;he enzyme activity was stimulated two fold by Mg'* in crude extracts from cells of culture from whÍch nHf naa been depleted. Unlike the Njtrobacter enzyme, the J-glutamyl transferasè activity of eiEher crude extracts or the purified enzyme from Nitrosononas europaea was unaffected by snake venom phosphodiesterase treatmenL. NAD* and NADP* re- B Two isozynes of glutamate dehydrogenase sPecific for specgively \,/ere detected in Lhe cytosol fraction of Nitrobacter agiTis. The NAD+ enzyme functioned in both directions ie. amination and deamination whereas the NADP+ enzyme was primarily for the amination of o-ketoglutarate to glutamate. The NADP+ enzyme was purified 52 fold (free of and NAD+ enzyme) by affinity chromatography on 2'-5rADP Sepharose-4B some of its properties studied. Subs¡rate activation of the NADP+ enzyme was observed with NHI and NADPH. A comparison r¡/as made of the properties of the purified NADP+ enzyme from Nitrobacter agilis with that from Nitrosononas euroqaea.

9. Oxygen pulse experirnents were carried out with washed cells of Nitro- soínonas europaea and iVi trobacter agiTis and with spheroplasts and everÈed membrane vesicles prepared from Nj trobacter agiTis. In addition to thiocyanate, the saltingin anions perchlorate and trichloroacetate and lipophilic cation triphenyl methyl phosphonium (TP!ÍP+) proved to be perneant and effective in allowing respiration-dependent proton translocation in ceIIs of Ni trosomonas europaea' The observed *H+/O ratio for NHI oxidation by Nitrosomonas europaea was 3.4 and that for both NHrOH and NrHr+ oxidation was 4.4. These values when corrected for the production of stoichiometric protons and for the fact. that the firsE step *H+/0 in NUf oxidation is mediated by a mono-oxygenase, gave effective ratsios of about 4 f.or these Ehree substraÈes. No convincing evidence was obtained for the operation of a respiratory Proton pump in Nítrobacter agiTis during NOI oxidation' (Aü) 10. The components of proton-motive force (Àp), namely mernbrane potential of and Eransmembrane pH gradient (ApH) were determined in washed cells both Nitrosononas europaea and Nitrobacter agiTis' In Ehese bacter1a, 8 ce1ls of Aü and ÂpH were dependenE on external pH (pHe)' Thus at PHe XXAV

Nitrosononas europaea and Nitrobacter agiTis had AÜ of l73mV and 125mV (inside negative) respectively as deterrnined by the distribution of t3g] tetraphenyl phosphonium cation (TPP+). Intracellular pH was deternined by rhe distribution of tlaC] benzoic and IlaC] acetyl salicylic acids and tlaC] methylamine. At pHe of.7 for Nitrosomonas europaea and 7.5 for Nitrobacter agilis there was no detectable ApH so that only Atþ conLributed to Ap. Intracellular pH (pHi) in cel1s of Ni¿rosomonas europaea varied from 6.3 at pHe 6 to 7.8 at pHe 8.5. In /Vitrobacter agiTis however pHi was relatively consLant (7.3 to 7.8) over the pH range of 6 to 8.5. The components of Ap (Arþ and ApH) remained constant at various stages of gror'rth of Ni trosononas europaea so that the metabolic state of the ce1ls did not. affect Ap. Such àn experimenE was not possible with Nitrobacter agiTis because of low cell yields.

11. Spheroplasts of Nitrosononas europaea synthesized ATP in response to an artificially created Ar!. This ATP synthesis was inhibired by DCCD indi- caLing that it was mediated by an A'l'Pase.

12. Cation (Nr*, K+ and NHO+ trarìsport systems in Nj trosononas europaea and Nitrobacter agiTis were investigated. In K+ deplet.ed cel1s it was shown that K+ is transported by an êlectrogenic mechanism in both bacteria and its uptake resulted inpartial conversion of Arl.r into ApH. NHI was Èrans- ported essentially as a neutral^sPecies (NH3) by a simple diffusion 22Na* loaded ãe1ls indicated that anti- mechanisrn. Experiments "l-th porters for Na+/H+, Nu+/f+ and K+/H+ were present in both bacteria At least one of these antiporters (Na+/K+) .equired an electrochemical gradienE for its operati n.

15N 15 180 18or.r,d 13. Using stable isotopes of (t5l¡oi und NO!) and {ttrl8o, l5N-NMR ttto?-> it was shown by GC/MS und techniques that the third '0' in NOJ produced bV ll0, oxidation by cells of ili trobacter agiTis originated from HrO and not from 0, or POi

WAITE 1 LIBRÂRY'NSTITUTE

1 INTRODUCTION

1.1 NITRIFYING BACTERIA

1.1.1 Mor 7o øv

NiLrifying bacteria, which belong to family Nítrobacteriaceae, have been classified into seven genera (Breed et a7., 1957)z Nitrobacter, Nitrosonofiâs¡ Nitrosococ.cus t Nitrosospira, . Nitrosocystis r Nitrosogloea- and Nitrocystis . htatson (1971) and lr/atson and Waterbury (197f) confirmed the first four genera and added three new ones, namely Nitrospina, Nitrococcus and ilitrosoTobus. These bacteria fa11 into two main grou[.s; (i) ammoni,a oxidisers and (ii) nit.rite oxidisers. The Èwo predominant genera of the family are the ammonia oxidising genus NiËrosononas and the nitrite oxidiser Nitrobacter.

Nitrosononas and Nitrosocystis are ovoid and approximately 2 and 2.5V in lengLh respectively (Murr:ay and hlatson, 1965). Nitrobacter, which is snraller (1.5U) than Nitrosononas is enlarged at one endrtapering towards the other end (Murray and hlatson, 1965). lvi trosomonas europaea contains a few con- ..centric- mernbranes at the periphery of the cell enclosing the cytoplasn while Nitrobacter possess a unique lamellar a-Iray of membranes located peripherally at the swollen end of the cell (Murray and l,iatson, 1965). Polyhedral bodies have been reported in lVi trosomonas europaea (h/ullenweber et a7., 1977) and Nitrobacter agiTis (Pope et a7.t f968).

Polar flagella have been reported in species of Ni trobacter (Breed et a7. ¡ 1957). Nitrosomonas cells are usually motile and possess two sub-terminal flagella (Engel, 1961). Non-f1agellar species of Nitrosononas have been observed by Engel (1961), but it is likely that the flagella were lost duri-ng preparative centrifugation of the cells for electron-microscop!;

I.L.2 The conceDt of chenoT ithotroohv

Chemolithotrophic bacteria can obtain all the energy needed for grorvLh and carbon assimilation from the oxidation of inorganic compounds eg. inorganic sulfur compounds (Hutchinson eË a7., t969; Kelly, L967), iron metal or ferrous 2.

ion (peck, 1968), hydrogen (Peck, t96B; Kelly, l-97L) ammonia and nitrite (Peck, 1968; Wallace and Nicholas, 1969b; Aleem, I97O; Suzuki, L97t+; Aleem, L977; Hooper, Ig78; Nicholas, 1978; Suzuki et aI., 1981b; Hooper, I9B2). The two imporÈant genera of t.he nitrifying bacteria mainly responsible for the bio- Iogical oxidation of ammonia to nitrate in soil are Nitrosononas which converts ammonia to nitrite (Meyerhoff, 19L71, Lees and Quastel , L945; Quastel and Scholefield, 1951; Zavarzín, 1960; Alexander, 1965) and Nitrobacter, which then oxidises nitrite to nitrate (Lees, 1955). trlinogradsky (1890) established Ehat these bacteria are chemolithotrophic tyPes because they derive their energy from the oxidation of reduced inorganic nitrogen compounds and carbon from COr. Heterotrophic growth of both Nitrosononas europaea and Nitrobacter agiTis has been reported (Smith and Hoare, 1968; Pan and Umbreit, L972; Bock, 1976, L978i Marin, 1978). The fixation of. CO, by nitrifiers is well establj-shed i¡ Nitrosononas (Rao and Nicholas , 1966), Nitrobacter (Aleem, 1965) and Nitrocystís (Campbell eË al., 1966). It seems thaE C0, fixation is common to all chemoautorrophs (Kelly , 1967 , L97L). Delwiche eÊ a-l. (1963) have shown 14C-1ubul1ed that ili trosononas actively fixed CO, into cornpounds that were precipitaÈed by trichloroacetic acid. Carboxydismutase, enzymes of the re- phosphoenol pyruvate carboxylase have been ductive penËose pathway and i described in Nitrosononas (Nicholas and Rao, Lg64; Rao and Nicholas' 1966)' The triose-phosphate dehydrogenase in both Nitrosononas and Nitrobacts¡ wâs found ro be NAD-specific (Aleem, 1965; Rao and Nicholas, L966). Malic enzyme which carboyxlates pyruvate, was not deEecÈed in Nitrosononas euroPaea (Rao and Nicholas, 1966). There was no evidence for glycollic or phosphoglycollic acids as intermediates in the fixation of CO, and the enzymes that utilize glucose, fructose, ribose and aceLate were not detected (Rao, 1966).

Based on the work with the sulfur oxidising bacterium ThiobaciTTus, íE has been proposed that chemolithotrophic bacteria have a defective tricarboxylic acid cycle, since they do not appear to have q,-ketoglutarate dehydrogenase and Kiesow NADH oxidase enzymes (Smit.h et aI . , 1967). In Nitrobacter however, (1968) (1964) found an active NADH oxidase which was linked to 0r. Aleem keto- also studied NADH oxidase in /VlËrobacter' but was unable Eo detects- glutarate dehydrogenase in either this or other chemolithotrophic bacterla cycle (lrlallace et a7. , 1970). Thus there is some evidence now thaL the Krebs 3

in chemolithotrophs is irnpaired at the level of q-ketoglutarate.

1.1.3 Anmonia and hvdroxvTanine oxidation bv Nitrosomonas

1 .1 .3.1 Ammonia Oxidation

The oxidation of ammonia to nitrite involves a net transfer of 6 electrons . Hofman and Lees (L952, 1953) confirmed the postulate of Kluyer and Donker (L926) that the oxidation of ammonia to nitrite occurs in two stepst ,o_ (i) NH, --å NH2OH ¿t-, (ii) NH2OH nOã

Engel and Alexander (1958a,b) reported that washed cells of NiËrosononas europaea progressively lost their ammonia oxidising ability upon ageing. Oxidation of ammonia to hydroxylamine is inhibited by a variety of ínhibitors (Lees, I952a,b, 1955; Campbell and A1eem, T965; Hooper and Terry, Lg73; Ashworth er af., 1977; Bhandari and Nicholas, L979a,b). Lees (1946, I952a, 1962) postulated that a metal enzyme is involved in ammonia oxidation and this metal could be Cu (Hofman and Lees' 1953; Lees, 1955; Nicholas ët a7., 1962; Aleem, 1965; Hooper and Terry, 1973; Hooper, 1978; Nicholas, 1978; Bhandari and Nicholas, 1979a,b). It was then suggested by Anderson (1964a, 1965b) thaL ammonia oxidation requires a form of tthigh energytt oxygen generated by the oxidation of cuprous copper. Based on inhibitor studies Hooper and Terry (1973) indicated that ammonia oxidation requires, in addition to Cu, a C0- binding factor namely P-460 as well as an intact membrane.

Hooper (1969Þ) reported that an initial lag phase of ammonia oxidation was eliminated on adding a sma1l quantity of NH'OH to washed cells of ilitrosomonas. Suzuki et a7. Q97l+) established that Kn' values for ammonia oxidation in washed cells were pH dependent, ranging from 4 to 0.3ml,l over the pH range 7.0 to 8.5. Sirnilar K* values were also reported by Laudeloul et a7. (L976) and Drozd (1976). 4

Suzuki and Kwok (f969) prepared spheroplasts from the cells of Nitrosononas europaea which oxidised amrnonia only in the presence of Mg2+ or NHTOH. Rees and Nason (1966) found a small (7%) incorporation of 180 troí 1BO, into N0, (uncorrected for possible loss by exchange reactions) duriíg ammonia oxidation by cel1s of Nj trosononas. Suzuki and Kwok (1969) then suggested that a mixed funct.ion oxygenase caLalysed the oxidation of ammonia to hydroxylamine, which is coupled to the oxidation of NH,OH to NOr. Dua et a7. (1979) reported an incorpora- 180 18õ, rion of fro* {o.g uio* % excess) into NH2oH produced by the oxidatiol of ammonia by cel1s in the presence of hydrazine, but not from nrttO. This exp.eriment was reptated with the yields of. >92 atom % en- 180 l5N-NMR, richment (tlollocher et a7.,1981). Using i"otope shift in Andersgon et a7. (T982) reported that NOl produced by cells from NH, or NH2OH has t.he isotopic oxygen composition of water. Cells were found to catalyse rapid exchange of both the oxygen atoms of NOl with water. Thus the origin of second oxygen atom in NOJ was uncertain. All this evidence indicates that NH, oxidation to NHrOH involves an oxygen insertion reaction via a monooxygenase. The origin of second oxygen in NOJ nroduced in rhe second srep of NH, oxidation (NH20H+NOr) is still unclear.

The first attempts to prepare cel1 free-ext,racts of Nitrosononas were made by Imshenetskii and Ruban (L952, 1954a,b, 1956, 1957). Very slow rate of ammonia oxidation was observed when the autolysates of Nitrosononas were incubated f.or 24h at 40oC. Subsequently Engel and Alexander (f959) attempted preparaEions of cell free extracts, but these were relatively inactive in oxidising NH, to NOJ. The cell-free preparations by Nicholas and Jones (f960), however oxidised NH2OH to N0, provided an electron carrier such as cytochrome c or phenazine metho- sulphate was added. Attempts were also made to demonstrate ammonia oxidation in cell free systems of Nitrosononas europaea (Suzuki and Kwok I97O,1981; Suzuki et a7., L974, 1976,1981a,b; Tokuyama and Asano, l97}a,b,ci Bhan

Hooper (f982) suggested thaE ammonia oxidation requires 5 possible components in an appropriat,e aggregate : amnlonia oxidase protein(s), hydroxylamine oxidoreductase, cytochromes c554 and c552 and a terminal oxidase. Suzuki and Kwok (1981) attempted the separation and reconsti- tuti-on of the ammonia oxidising system. Crude fractions wère separat,ed by gel filtration on a Sepharose-68 column. A membranous fraction 1, which contained hydroxylamine oxidoreductase, cytochrome a and cyt.ochrome oxidase activity catalysed the oxidation of NH20H. Two of the fractions contained a hydroxylamine oxidoreductase and a cytochrome c552 respectively. Maximum rates for ammonia oxidation were recorded when the three fracLions h/ere reconsEituted or when fraction one u¡as added to purified cytochrome c554. The latter cytochrome (c554) (K^ 3.3 UM) was suggested Eo be an electron donor for ammonia hydroxylation. Using reconstiLuted membrane fraction with reduced cytochrome c554 Tsang and Suzuki (1982) reported that the reoxidaEion of cytochrome c554 and 0, utilization takes place when either CO or NH, is added to Èhe membrane fraction.

I.I.3.2 Hydroxylamine Oxidation

The NH'OH produced from NHf, by ammonia mono-oxygenase (Suzuki et a7.¡ 1974; Hollocher et a7.,1981) is further oxidised to NOJ by hydroxylamine oxidoreductase (HAO). Hofman and Lees (1952, f953) found that the oxidation of NH.,OH¿- by whole cells was inhibited by hydrazine and under these conditions NH'OH was accumulat.ed. Three two-elecÈron steps for the oxidation process vrere then postulated (Lees, L952b; Hofman and Lees, 1953): lluf 2e , NH2oHg (lron¡$lto,

Chromatographic methods were used to idenEify NH,OH as an inËermediate during t.he oxidation of NHI to NO, (Yostrida and Alexander, 1964). Hy'droxylamine oxidaEion in cell-free extracts was first achieved by Nicholas and Jones (1960). Cell-free extracts oxidised NH,OH only in the presence of air and an electron carrier such as cytochrome' c, phenazine methosulfate, methylene blue, benzyl viologen or ferricyanide (Nicholas and Jones, 1960; Aleem and Lees, L963; Falcone et a7., 1963; Anderson , L964a; Aleem, 1970). Nicholas and Jones (1960) demonsErated 6

that the added cytochr:ome c was reduced by NH20H as well as by hydrazine but N, gas instead of NOl was produced in the presence of hydrazine. They demonstrated that hydrazine competitively inhibited NH20H oxidation to NOl. Based on these and other investigations, Nicholas and Jones (1960) and Falcone eÉ af. (1963) further suggested that the oxidation of NH20H involves the participation of the respiratory chain involving flavoproteins and cytochrornes.

The hydroxylamine oxidase was located in the membranes and it r+as resolved into hydroxylamine cytochrome c reductase and cytochrome oxidase by Falcone et at. (L962, 1963). The role of the particulate hydroxylamine cytochrome c reductase enzyme system was subsequently confirmed by Aleern and Lees (1963) and Hooper and Nason (1965).

Hydroxylamine oxidoreductase (HAO) was purified and its properties determined (Hooper and Nason, L965; Hooper et a7 ., 1978; Tokuyama s¿ a7., LgTgi Yamanaka et a7., I979bi Yamanaka and Sakano, 1980; Miller and l.Jood, 1983b). Various elecEron transporÈ components of HAO were then purified (Payne, 1978) . Hydroxylami-ne oxidoreductase constitutes 5% of the soluble proteins and 40% of the c haem of Nitrosononas (Hooper et a7., 1978). Rees and Nason (1965) reported an unusual absorption maximum at 465nm from the dithionite reduction of extracts of Nitrosononas. The addition of C0 resulted j-n a shift of absorbancy to approximately 450nm suggesting a cytochrome P-450 like pigment. The 465nm haem protein was found to be soluble (Hooper et a7. , f972) and was located in fraction containing HAO (R:-tchie and Nicholas, I974) but was considered of uncertain significance since it was not reduced by either NH2OH or NHrNHr. Erickson and Hooper (1972) purified a small fraction of the total cellular pigment with absorption maxima aL 435 (oxidised), 460 (dirhionire reduced) and 450 (dithionite reduced C0 complex). Based on its ligand binding properties it was ident.ified as a haem and named P46O. Hooper et a7. (1978) subsequently reported that essentially all the cellular haem P460 was associaLed with HAO. Selective destruction of haem P46O of HAO with HrO, resulted in a loss of both hydroxylamine dehydrogenase activity and the hydroxylamine reduction of c type haerns indicating that haem P460 is a part of or near to the substrate binding site (Hooper and Terry , L977; Hooper and Balny, L9B2). Miller and tfreet l,/ood (1983c) recently reported that about 57" of total P460 was in form. The free trimeric cytochrome P46O had a native molecular weight of 52,000. 7

The purified HAO contained )20 moles Fe per mole enzyme (molecular weight 200,000) (Hooper et a7., 1978; Terry and Hooper, fg8l). The ratio of protein per heme was reported as 10,000 (Hooper et al',1978) or 17,500 (Yamanaka et a7.,1979b). Vickery and Hooper (198I) detected heme c centres in HAO by electron paramagnetic resonance (EPR). EstimaLes of the ratio of P460: c heme range from 6 to 9 (Hooper et a1., I97B; Hooper, Lg82),8 (Lipscomb and Hooper, 1982) and 7 (Lipscomb et a7.¡ 1982).

Some cellular cytochromes not associated with HAO have been purified and characterized (Rees , 1968; Tronson et a7., 1973', Yamanaka and Shinra, Lg74; l"lillar and lJood, 1982, 1983a,d). Cytochrome a r^/as ex- tracted from KCl washed membranes with Triton X-100 by Erickson et aI. (Ig72) and purj-f ied r^¡ith ammonium sulf ate. This cytochrome contained non-covalently bound haem a identified as cytochrome a1 which oxidised cytochrome c-55t+ from Njtrosononas (Yamanaka and Shinra, 1974) ' Hooper et a7. (Lg72) identified the elecÈron transfer comPonents of the membrane envelope fractions, namely ubiquinone as well as cytochromes b, c and a1.

Aleem et a7. (f962) suggested that nit.rohydroxylamine is the intermediate formed by initial oxidaEive condensation between NH20H and NOJ as follows:

NH20H 4 (NOH) + 2(H) +2H+ 2(H)+ 2cyt "F"3* ------+2cyl."F"2* (NOH) + HN02 ------r N02.NH0H NO2.NHOH + à Oz 2HN02 --à It was then proposed that dehydrogenation of the nitroxyl to N0 and NrQ occurs (Falcone et a7., L962, 1963; Anderson, I964a, 1965a). It was observed that under anaerobic conditions when NH20H was added to extrâcts of iljtrosomonas equivalent amounts of both Nro and N0 were formed (Anderson, L964a, !965c; Hooper, 1968; Yoshida and Alexander' Lg|O, I97l; Ritchie and Nicholas, L}TZ). Ritchie and Nicholas (L974) reported a nitrite reCuctase closely associated with HAO. Subsequently the ammonia oxidising chemolithotrophic bacteria have been shown to account for significant. amounts of NrO production (Bremner and Blackmer' L97B; Blackmer et a7. t 1980) or Nro and NO (Goreau et a7., 1980) ' I

Recently Miller and hrood (1983b) purified a soluble cytochrome oxidase (molecular r+eight 120,000). These authors reported that the oxidase v/as a copper protein devoid of haem, and not a cytochrome o as was previously assumed (Rees and Nason, 1965). Soluble cyLochrome oxidase activity co-purified with nitrite reductase activity and appeared to be associated with the sarne protein (Miller and l,Jood, 1983b).

The similarities in the mechanism of oxidation of ammonia by Nitrosononas and methane by methanotrophic bacteria have been discussed (Higg j¡s et af ., 1981; Hooper , LgBz). Recently, cells of Ni'Ë¡*osononas eu.opaea have been shown to oxidise methane (Jones and Morita, 1983; Hyman and hiood, 1983).

1.1.4 Nitrite oxi dation b Nitrobacter asiTis

The reduction of cytochromes 551 and 559 during nitrite oxidation by whole cel1s of Nitrobacter was first observed by lees and Simpson (1957). Subsequentty But.t and Lees (1958) proposed the following scheme for nitrite oxidation:

Cyt 551. Fu3+ + NO; -----+Cyr 551. F"2+ + "NO?" Cyt 551. F"2+ + "*õr" * à oz4yt 551.F"31 + NO;

According to the scheme proposed by Lees and Simpson (1957) and BuÈt and lees (1958), NO; mediated by cytochrome c and NOl is formed as a result oxidation is J of an 0 insertion from molecular 0r. Aleem and Nason (1959) further characterized N0, oxidase that catalysed the followíng reaction:

NOr+ cytochrome c reductase + cytochrome c+cytochrome uL* cytochrome oxidase * O2

As this scheme involves the reduction of 0, to HrO by the elecÈrons supplied Uy NOJ, it was proposed that molecular oxygen could not possibly supply an 0 atom to NOj for the formation of NOl. Subsequently Aleem et a7. (1965) provided evidence fror 180 isotope stuãies that HrO but not 0, contribuEed 0 during N0] oxidation to NOJ. They proposed the following mechanism to account for their resulLs: 9

(i) uol + rrtto' i'roã.nrtuo + 2 cvt + 2H+ (íi ) uo;.Hrl8o + 2 cyL 1.u"3* --**tuoi 1.u.'* t3.F"3+ 2 cyL + 2 cy-_ t3.Ft2+ (iii) 2 cyt 1..F"2* + 2 cyr --> 1.t"3* (iu) 2 cyl uu,F"z+ + 2u++àor-----'+ 2 cyt t3.F"3+ + Hrg

sum: llO, + åOZ -----* UOã

The four steps in the scheme are .as follows (Aleem, 1977): (i) hydration of the NO, molecule prior to removal of electrons from NOJ and protons from HrO; (ii) electrons are transferred from hydrated N0, molecule to cytochrome a, and tOt NO, is formed in turn as a result of the incorporation of from HrO ; (iii) a proton current is generated; (iv) finally electrons and protons are transferred to molecular 0 via a cyt aa3 type oxidase.

Aleem (1968) and Sewell and Aleem (1969) established that there is a second NO. oxidising system in Nitrobacter which is coupled to the generaÈion ¿ of reduced pyridine nucleotides and is highly endergonic:

J- NO * + NAD(P) ' ----> NO^ + NAD(P)H + H+ 2 HzO _1 = *35 kcal.mol -1 ^G"

Sewell et a7. G972) reported that there are five different types of cyto- chromesPresentj:nNitrobacter:cytochromec(E7=274nY)rcytochromea (fu7 = 240mV), cytochrome a3 (h7 = 400mV) and two al type cyt.ochromes (Emt = 352mV and Em, = 100mV). Similar results were subsequently reported by Cobley (1973). The oxidation-reduction potentials of cytochromes c, a and a, showed no pH dependence but a high potential component of cytochrome a, had a gradient of approximately 25nY/pH unit and Èhe lower potential component had a gradient of SgmV/pH unit (Ingledew et a7., L974). Similar results $/ere reported by Aleem (1977) with mid point potential of N0, / t'tO; couple in extracts of Nitrobacter. The Em became more positive from pH 9.0 (Em = 328mV) to pH 6'8 (E* = 414mV). Because the Em for NO, and of the high potential component of cytochrome a, at pH 8.0 (optimum for N0, oxidation) are at 360mV and 327mV respectively, the reduction of high potential a1 component by NOJ Poses no thermodynamic barrier. Moreover since the Em of cytochrome c is not affected by pH changes, its reduction Uy nOJ and cytochrome a, would be endergonic.

It is well established now that the site of entry of NO, in the electron 10

transport chain is cytochrome aI and the reduction of cytochrome c by N0, i.s energy dependent (Kiesow, 1967; Aleern, L967, 1968; Sewell and Aleem, I969i cobley, lg73; Ingledew et a7., 1974; Cobley, 1976a,b). In recent years components of nitrite oxidase have been purified and sLudied in detail. yamanaka et a7. (1979a)purified a cytochrome a type terminal oxidase from Nitrobacter agiTis which consisted of two heterologous subunits of molecular weighr 40,000 and 27,000 respectively. Chaudhry et a7. (1980) purified the aa3 type terminal oxidase by hydrophobic interaction chromatography and reported that the enzyme had three subunits of molecular weight 37'000, 25,000 and 13,000 respectively. The absorption maxima were at 42O and 600nm in the oxidised form and aL 443 and 606nm when reduced. Yamanaka et a7. (1981) reported that this terminal oxidase contained I mole of haem a¡ 1.6 g-atom of Cu per 4lr0OOg. The enzyme rapidly oxidised ferrocytochrome of several eukaryotes as well as Nitrobacter cytochrom¿ u552.

Chaudhry et ai. (1980) found a b type cytochrome in Nitrobacter agiTis which reacted with NO, and CO. Three c type cytochromes have been purified (Chaudhry et a7.¡ 1981) : c-553, c-550, c-559, 554 and their amino acid compositions studied. Yamanaka et a7. (1982) independently isolated cytochrome c-550. 'The e1zyme v,ras composed of 108 amino acid residues, sixteen of which were lysine. The cytochrome rapidly reacted with NiËrobacter cytochrome c oxidase. The complete amino acid sequence of cytochrome .c-552 has been determined (Tanaka et a1., L982) and was found to be homologous with eu,karyotic cytochrome c and with cytochrome c, from photosynthetic bacteria.

Fukumori and Yamanaka (1982) studied the effects of cardiolipin on the reaction rates of cytochrome c oxidase at various concentrations of phosphate buffer. In contrast to the oxidations of horse and ferrocytochrome c which were stimulated by cardiolipin, the cytochrome c-550 oxidation by cytochrome oxidase was unaffected. They suggesÈed that cardiolipin was not necessary for the reaction of cytochrome c oxidase with cytochrome c-550 in Nitrobacter.

1.1.5 Energv coupTing

1.1.5.f Nitrosomonas sPecies

Energy coupling in Nj trosononas has been discussed by Peck (f968)t t{allace and Nicholas (1969b), Aleem (1970, 1977), Suzuki (1974), and Hooper (1982). The oxidation of NH2oH Lo NOt is an energy yielding 11

step. The free energy changes during MIf, oxidation are as follows: LGi' ^ci' (i) unþåor-----+NHroH + H+ -o.7 + 3.85 .L (ii) NH,OH + O¡--NO, + Ht0 + H' -83.3 -68.98 t Sum: NHI * t.5 02- NOr+HrO+2H' -84.0 -6s .04 Where AG!' and AG"'2 denote the free energy values according to Gibbs and Schiff (196f) and Aleem (1970) respectively.

Burge et a7. (1963) were unable to detecL ATP synthesis during 32P NH?OH oxidation, based on experiments. Ramaiah and Nicholas (1964) however demonstrated phosphorylation in particulate fractions r4liÈh P/0 raLio of around O.2O. Drozd (fg76), reported *H+/0 raiio of about 2 for either llHf or NH,OH oxidation and suggested that substraLe oxidation is closely associated with the reduction of cyLochrome c so that Lhere is only one active proton translocating loop (loop 3) composed of at least hydroxylamine cyLochrome c reductase, cytochrome c and cytochrome oxidase of the a andfor 0 type (Aleem and Lees, 1963; Aleem, 1970).

The requirements for NADH and ATP for endergonic reducÈion reactions assocíaLed with C0, fixation in chemoliLhotrophic bacteria are well established (t/allace and Nicholas, 1969b). The generation of reduced nicotinamide adenine nucleotides coupled to oxidation of NHf or NH20H would be thermodynamically unfavourable due Eo positive values of AGo' for these coupled reactions (Gibbs and Schiff, 1961). The reducEion of Nl,O+ Uy energy dependent electron flow was proposed by Chance and Hollunger (1961) in animal mitochondria. Energy dependent reduction of UnD+ Uy reversed electron transfer has been demonstrated in extracts of Nitrosononas and Nitrobacter as well as in other chemolithoUrophic bacteria (Aleem et a7., 1963). This was subsequently confirmed by Aleem (1965 , 1966).

The following scheme of NADH product.ion v¡as then proposed: t2

NH 0H --> Cyt b cyt.c cyt oxidase ----+ 02 2

ATP TP

Flavins

Pyridine nuc leotides

I.1.5.2 Nitrobacter sPecies

The oxidat.ion of NOt by Nitrobacter is linked to the generation of ATP. (Aleem and Nason, 1960; Malavolta et a7., 1960; Fischer and Laudelout, 1965; Kiesow, 1964; Aleem, 1968; 0rKe11y et a7., I97O; Cobley and Chappel\L974; Cobley, 1976a,b; Aleem, L977). Aleem and Nason (1960) reported P/0 ratios of 0.2, 0.1 and 0.03 in Nitrobacter particles for the oxidation of NOr, succinate and NADH, and succinate respectively. Kiesow (1964) reported that NADH oxidation by Nitrobacter particles could yield P/O ratios of 3.0 and P/2e- ratios of 2 with 0, and N0, as respectj-ve electron acceptors. Further ' Kiesow (L964) suggested that N0, oxidation is an energy consuming process than energy generating one. However observations by Aleem (1968, L977) ruled out the possibitity that NAD* involvement is obligatory duri ne lto, oxidation. Aleem (1968) reported P/0 ratios of abouÈ I in the presence of an NADH trap or rotenone. Similar results were obtained for ascorbate as an electron donor indicating that ATP generation takes place in the terminal segment of electron transport chain involving coupling site 3. The NADH oxidation was also reported to be coupled to ATP biosynthesis with P/0 ratios of about 2 (Aleem, 1968, 1977). It was found that rotenone, antimycin A and H0QN0 all inhibited NADH oxidation as well as coupled phosphorylation. Sewe1l and Aleem (L979) observed P/0 ratios of abour 1.1 and P/NOã ratios of about 0.7 for NADH oxidation wit.h 0, or NO, as respective electron acceptors. They suggested that aerobic NADH oxidation and associat.ed ATP formation involves all the three coupling sites and NADII-NO] reductase sites I and 2.

Cobley (1976a,b) proposed a chemiosmotic mechanism for ATP generation in Nitrobacter, Using membranes of Nitrobacter winogrdskyi 13

Cobley (I976a,b) found that uncouplers eg. CCCP inhibited N0, oxidation but stimulated aerobic NADII oxidation. He also reporued Lhat ghe compounds known to collapse membrane potential (AÜ) inhibited N0] oxidation and proposed that the flow of elecLrons from cYtochrome a, Lo cyt. c is facilítated by Arþ, Very 1ow H+/0 ratios (=O.t) were reported for NO, oxidation by membrane particles. The following mechanism of proton translocaLion was put forward by Cobley (f976b):

OUT IN *o; + H2o + -1. IH cyt c cytat + NO +H 3 2e

I Lro + 2H' 2 e cyt oxidase n20

An alternative mechanism proposed by Aleem (L977) predicts the movemenÈ of two protons and two net positive charges out of the cell for the flow of t.wo electrons OUT rN

NO + nzo 2 I 2H, cyt c 2H cyt a1 NO 3 2e T + 2H, \o2

cyt oxidase Hzo

Aleemts scheme (Aleem, Lg77 ) did not account for the stimulation of N0, oxidation by ArÞ whereas Cobleyrs scheme (Cobley' L976b) predicted an unusual H- (hydride) transfer. Ferguson (1982) proposed that cytochrome oxidase (aar) can alternatively act as a proton pump in Nitrobacter to generaLe proton-motive force: T4

OUT IN

NO + 2 "2o C cyt + NO ; +2H 2e LO + zd 2 2e+ oxidase H^O cyt +2 2t* 2 H

Interestingly, a recent report (Sone et a7., 1983) indicates that unlike the cytochrome c oxidase of other bacteria (Solioz et a7. , l-9BZ) and rnitochondria (hlikstrom and Krab, l9B0) the cytochrome c oxldase from Nitrobacter agilis lacked the proton-pump activiEy'

1.1.6 Nitropen assiniTation I,Jallace and Nicholas (1968) reported that assimilatory nitrite and hydroxylamine reductases \^/ere present in both Nitrosononas and Nitrobacter. In addition jVjtrobacter also contained a nitrate reductase (Straat and Nason, 1965; ir/allace and Nicholas, 1968; Faull et a7.t 19691 Herrera and Nicholas Ig74). The product of N0, and NHrOH reduction by either bacteriurn was found'to be ammonia (I^/allace and Nicholas, 1968). Both Nitrobacter and ttttT, l5NUrOH 15WO; Nitrosononas incorporated una into cell,nitro8en and in t'*Oã addition Nitrobacter but not lVitro somonas also incorporated (VJallace and Nicholas, 1968) .

Glutamate dehydrogenase has been characterized and purified from Nitrosomonas (Hooper et a7.t 1967). The purified gluEamata dehydrogenase from Nitrosomonas was specific for NADP+ and exhibiEed both amination and deamination reactions (Hooper et a7. t 1967). The specific activity of the enzyÍLe was over BO fold greater than the rate required to synthesize all the organic nitrogen by the bacterium. Wallace and Nicholas (1968) reporËed thaL Ehe specific activity of glutamat.e dehydrogenase i-n Nj trobacter was about 80 Uimes less than that of Nitrosononas. They also showed that glutamate dehydrogenase from 15

Nitrosomonas is competitively inhibited by NH'OH and oxime of o-ketoglutarate (hlallace and Nicholas, 1969a).

Glutamate was found to. be major amino acid in the water soluble fraction of iVitrobacter, contributing about 257" (w/w) of total amino acid poo1, while in /Vitrosomonas alanine was the main amino acid constituting about 17.57" (w/r+) of total pool (l,rlallace et a7.,1970).

Bhandari and Nicholas (1981) purifíed glutamine synthetase from Nitrosononas europaea (molecular wL.440,000) and studied some of its properEies. GlutamaLe synthase was.not detected in lVitrosononas europaea (Bhandari and Nicholas ' 1981 ) . It was suggested that glutamine synthetase in Ni trosomonas is required to produce glutamine needed for the biosynthesis of various metabolic compounds. The purified enzyme was inhibited by a variety of amino acids and nucleotides.

T.2 AIMS OF THE STUDY

This thesis is concerned with the biochemical studies with two nitrifying bacÈeria; ivitrosomonas europaea and Nitrobacter agiTis. The following lines of enquiry h,ere pursued:

(i) Determination of the stoichiometry of N0, oxidation by washed cells, spheroplasts and membrane vesicles of Nitrobacter agiTis using electrode techniques to measure NOJ production and O, uptake simultaneously and continuously. In addition the effects of various metabolic inhibitors on NO, oxidation were also investigated. (ii) Investigation of possible pathways of nitrogen assimilatiqq in l5NHrOH Nitrobacter agiTis using srable isoropes of 15N (i5Not, ttro;, lsttU4*) "nd and specific inhibitors of glutamine synthetase and glutamate synthase. (iii) Purification, properties and regulation of gluÈamine synthetase frorn Nitrobacter agiTis and /Vi trosononas europaea and the role of glutamate dehydrogenase in Nitrobacter- agiTis. (iv) Studies on proton translocation during substrate oxidation by Nitrosononas eur-opaea and Nitrobacter agiTis using oxygen pulse technique to determine +H*/0 ratio for specific substrate. 16

(") Determination of proLon-motive force (Ap) in Nitrosononas eutopaea and Nitrobacter agiTis and the role of Ap in mediating ATP biosynthesis. ("i) Studies on Na* and K* transport in Nitrosomonas europaea and Nitrobacter agiTis and their relationship to Áp. (vii) Investigation of the source of oxygen in nitrate, produced from the oxidatiôn of nitrite by Nitrobacter agiTisr using stable iso- 15uoJ) tto ropes of 15N u.a and (ttor, trtto una rl8of-) l5N-NMRflt*o, in GC/MS urrd studies. 2. MATERIALS AND METHODS T7

2. I'IATERIALS AND I'{ETHODS

2.I MATERIALS

2.I.I Chenicals and biochemical-s

All nucleotides and amino acids r' imidazole, Y glutarnyl hydroxamate, valinomycin, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), sodium tetraphenyl boron (NaB(C6H5)4), triphenyl methyl phosphonium bromide (TPMP+Sr-) dimethyl sulfate, trimethyl phosphaLe, N-methyl-N-nitroso-p-toluene sulfonamide (for the preparation of diazomethane), c-ketoglutarate, hydroxylamine hydrochloride, azaserine, L-methionine-Dl-sulphoximine (l'lSX), N-N'-dicyclohexyl carbodiimide (DCCD) , antimycin-A, 2-heptyl-4-hydroxyquinoline- N-oxide (H0QN0) , quinacrine-hydrochloride, rotenone, pyruvic acid, succinic acid and Tris(hydroxymechyl)-aminoethane were from Sigma Chemical Co., St. Louis, U.S.A. 2-trichloromethyl pyridine (2-TllP) was synthesized by Miss Tina Chambers and Dr. S.F. Lincoln of the Botany and Physical and Inorganic Chemistry Departments respectively, University of Adelaide. Nigericin was purchased from Eli Lil1y & Co., Indiana, U.S.A. 2.5 diphenyloxazole (PPO) and 1,4 bis (2,(4 methyl-5-phenyl oxazolyl)) benzene(fOeOf¡ were from Packard Instrument Co. Chicago, U.S.A. Phase combining system (PCS@) liquid scintillation fluid was from Amersham, Bucks, England.

All other chemicals, the best purity available, were obtained from the following sources: Ajax Chemical Co. (Alburn, Australia), B.D.H. Chemicals Ltd. (Poole, England), ICN Pharmaceuticals (Cleveland, U.S.A.), May and Baker, (Dagenham, England), Aldrich Chemical Co. (l"li1waukee, U.S.A.), Dow Chemial Co. (Midland, U.S.A.) and Drughouse Ltd., (Adelaide, Australia).

2.r.2 Stable isoto lst'tuocr (30 arom % excess), KI5NO, u'd N.15NO, (boch 32.5 atom % excess) h'ere purchased from Lroffice NaLional Industriel de'1' Azote (ONIA), l4arseille, tt*tr0H France. (=97 atom % excess) and trtuo (97 atom % excess) were from Merck, Sharpe and Dohme, Montreal, CanaJa. nrttO (98.3 auom7. excess) was 1BO, from Prochem, London, England and {Ol aton % excess) from Yeda Research and Development, Israel. H15t'lO3 (99 atom % excess) was obtained from Isomet 18

Corp. N.J., U.S.A. f15l¡03 (99 aLom 7" excess) was prepared by neutralizing 99 atom % excess) was piepared through reduction of y lead (Pb) at 42OoC in silica crucible (Jolly , Lg61+). dissolved in distilled water, filtered and dri-ed. O, contained 10 and 30% rlsl'to, and rest K15NO, in tr,/o separate preparations used for GC/MS and NlfR studies respectively. UrflBOO vras prepared by the reaction of excess nrt*O (97 atom excess) (0.5m1) wirh PCl, (1.3g) : PCl, + 4Ur18O +5HCl. Hydrochloric acid and --IH3P180O !ùater were evaporated from the reaction mixture in a 1O0oC oven and this step also served to hydrolyse traces of pyr:ophosphate present initially in the preparation. Pyrophosphate was detected by means of high voltage paper electro- 15N 180-tabel1ed phoresis (Section 2.2.16 .2) . , nitrate standards (f or Nl'fR study) were prepared by the method of Bunton et a7. (1953).

2.L3 Radioisotopes

The radj-oisoropes [3U] r"rraphenyl phosphonium bromide (TPP+gr-) T4 (23,7 Ci.rnmol-l), aceryl Icarboxyl-toa] salicylic acid (2OmCi.mmol-l), and cl tnrO 1 inulin (5.6 Ci.r*o1-1) were from Amersham, Bucks, England. (lCi.mol- ) ll-I4C), benzoic acrd (22.6 mCi.*ro1-1), tlaC] merhylamine hydrochloride 22Nuc1 (51.8 mci.mmol -1), tlacl sucrose (r mci.*rol-1) and (293.4 mci.rg-1 ) were purchased from New England Nuclear (NEN), Boston' U.S.A.

2.r.4 SoTutions, buffers and soTvents

Unless stated otherwise, the aquous solutions, buffers and reagents used in Ëhis study were prepared in double glass distilled water. All the organic solvents used were redistilled from glass before use.

2.L.5 Chrona toeraphic nateriaTs

Affinity chromatographic media (B1ue sepharose cL-68 and 2"5f-ADP Sepharose-4B) and gel filtration media (Sepharose-48 and Sepharose-6B) were purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. Dowex-50 H+ form, was from Sigma Chemical Co., St. Luois, U.S.A. 19

2.r,6 Enzvnes and marker oroteins

Phospholipase A' firefly lanÈerns, carbonic anhydrase, catalase and snake venom phosphodiesterase r,rere purchased from Sigma Chemical Co. , St. Louis, U.S.A. Molecular weight sLandards were from Pharmacia Fine Chemicals, Uppsala, Sweden.

2.r.7 BacteriaT cuTtures

Nitrobacter agiTis was kindly suppl.ied by Prof. M.I.H. Aleem of University of Kentucky, U.S.A. and one strain (ATCC-I4123) was also purchased from the American Typ".Culture Colleci.ion, U.S.A. The strain of Njtrosononas europaea used was obtained from Dr. Jane Meiklejohn of Rothamsted Experimental Station, U.K.

2.2 METHOÐS

2.2.I Growth and harves tinø the bacteri a

Cultures of Nitrosononas europaea r{ere gror^¡n in 401 batches at 28oC with vigorous aeration in the following growth medium (g.f-l), (NHO)rSOO,4; KH2P04,0.5; CaCLr.2H2O,0.004; MgSOa.7H2O,0.05; chelated iron (methyldiamine bis-orthohydroxy-ptrenylacetic acid), 0.0001 and CuSOO, 0.00002. The pH of the cultures were continuously adjusted to 7.8 with sterile 20% (w/v) K2C03 using an automatic pH stat unit (Radiometer, Copenhagen).

Nìtrobacter agiTis \^ras groh/n in batch cultures (B or 401) in a growth medium based on those of Aleem (1968) and t/allace and'Nicholas (f968). The medium conrained (g.r-1): NaNOr,l; KHTPOO,l; NaCl,O.3; MgSO, .llzo,O.5; MnS00,0.002 and Fer(SO4):,0.005. After sterilization the pH of the medium was adjusted to 7.5 with sterile 20% (w/v) K2C03.

Unless stated otherwi-se, the media were inoculated with 102 (v/v) of an exponantially growing culture of Nitrosononas europaea or Nitrobacter agiTis respectively.

Cells were harvested in a Sorvall RC-28 centrifuge fitted with a continuous flow rotor (Ivan Sorva11, Inc., Connecticut, U.S.A.), at îoC at 30,0009 with a 20

f low rate of. I2!,.h -1 Cells were washed several times with 50mM Tris-HCl buffer (pH 7.5 to 7.8) and either used j.mmediately or stored at 4oC.

2.2.2 Preparation of sohetooTasts and menbrane vesicTes

The spheroplasts and membrane vesicles of Nitrobacter agì7is were prepared as follows: 19 wet weight of cells suspended in 50ml of 50m1"1 Tris-HCl buffer (pH 8.0) containing 0.2M sucrose, 1-mM NaTEDTA and 80rng lysozyme in a lOOml Erlenmeyer flask were incubated at 3OoC in a rvaterbath shaker (60 rev. 'l min-') for 2h. Then 2 Ug DNAse and 2ml of 2mM ÙIg-acetate v/ere added and incubation continued for th. A pelleL of spheroplasts was then obtained by centrifuging t.he suspension at 80009 for 15 min at 4oC. The pellet, washed twice with cold Tris-HCl-sucrose buffer was finally suspended in 5ml of the same buffer. The spheroplasts thus prepared remained active for 5 days.

Membrane vesicles h'ere prepared by incubating spheroplats (19 weÈ weight) for 15 min in 25ml 5OmM Tris-HCl buffer containing 10mM NaTEDTA, 10mM MgSOO and 100ug DNAse. They v¡ere then subjected to ultrasonic treatment with an MSE probe uniÈ at maximum output with short bursts of 1 min over a period of 20 min at 4oC in an icebath. After centrifuging at 50009 f.or 20 min to re- nove intact spheroplasts the supernatant fraction was futher centrifuged at 100,0009 for 2h. The pellet containing menbrane vesicles was washed several times rvith cold 100mM Tris-HCl buffer (pH 7.5) containing 10mM NaTEDTA and finally suspended in Èhe same buffer. Both spheroplasts and membrane vesicles were stored at 2oC.

2.2.3 Preparati on of ce77-free extracts

Cell extracts of both Ni trosornonas europaea and Nitrobacter agiTis v¡s¡¿ prepared by sonication of cell suspensions (approx.O.lg wet weight t1:1) with probe (20 kilocycles 1-2 min bursts over a period of an ultrasonic "-1¡ "iah 20-30 min at 4oC. Unless stated otherwise, thb sonicated suspensions k¡ere centrifuged at 3O,00Og lor 20 min aE 4oC and the supernatant (SrO) used for study.

2.2.4 Incorporatio, of 75't¡ lah e77edc Õmnott nds i nto ce77 ni troaen

washed suspension (25ng protein) The incubation mixture contained: cell ' l5NHroH.HCl lst'tHocr, 15N-1uh"11ed substrate (eithu. N.15NO, r15llor, or lmg 2t

ttt) equivalen, and KHCO, (1.5mM fi.nal concentration) in a final volume of 6m1 of lOOmlf-sodium phosphate buffer (pH 7.8). Unless stated otherwise, the incubation was for 2h in a lOOml Erlenmeyer flask in a waterbath shaker at 30oC. The reaction was terminated by adding 30m1 chilled water and the contents were immediately centrifuged at 30,0009 for 10 min at 4oC. The pellet. thus obtained was washed several times with distilled water and finally suspended in a small volume (3-5m1) of water. The cell suspension was then transferred to a 100url Kjeldalil flask containing 4ml 36-N .H2S0O and 29 of the digestion mixture (7gHgO + 93g NarSOr) and digested by heat.ing. The ammonia produced by NaOH treatment of digested samples was distilled into boric acid and concentrated to 2ml after adding 0.lml of lN H2S04. The samples were then transferred into one limb of a Rittenberg tube and alkaline (L% w/v) hypobromite v¡as added to the other (Sims and Cocking, 1958). The Rittenberg tube was evacuated to 133.22 x 10-7 Pa and the contents mixed to generate N, gas from ammonia. The 1511, {* Nr) was then introduced via an evacuated expansion flask into an 15N AE-I-MS2 nass spectrometer (Nicholas and Fisher, f96O) fot enrichment analysis.

For experiments with cell extracts (Sr) the reaction mixture was as for washed cells, buÈ the reaction was stopped by adding IO% (w/v) trichloroacetic acid (TCA) follorved by centrifugation at 2O,000g for 15 min. The pellet was 15N washed several Limes in cold TCA and samples fot analysis were prePared as described above.

2.2.5 Enzvne purification

2.2.5.I Glutamine synthetase

Cell extracts (SrO) were prepared in 10mM Tris-HCl, lrnM lfnCl, buffer (pH 7.2). In Nitrobacter agilis, the SrO fraction was heat- treated at 5OoC for 15 min with constant stirring, then chilled in ice for 15 min and centrifuged at 3O,0O0g for 15 min. The supernatant contained all the glutamine synthetase activity. Longer heat treatment or higher temperatures resulted in a loss of enzyme act.ivity. The supernatant was loaded onto a Blue-Sepharose CL-68 column (1.5 x 9cm) preequilibrated vrith 10mM Tris-HCl, lmM MnCl, buffer (pH 7 .2) . The column was then 22

1 \,/ashed rvith buffer (florv rate 5Oml lt-') until the absorbance (Orr') was close to zero. Glutamine synthetase was elut.ed frorn the col-umn with 2mM ADP in the same buf f er. Act.ive fractions \4/ere pooled, dialysed against the buffer overnight, concentrated on an Amicon PM-10 membrane and loaded onto a gepharose-4B column (2 x 70cm) p..- equilibrated with the buffer. The enzyme was eluted with the same buffer (flow rate 12ml.n-1) ar,¿ ttru active fractions pooled and concentrated as before.

In Ni trosononas europaea, the SrO was heat-treated at 65oC for 15 min with constant stirring, chilled on ice for 15 min and centrifuged as for Nitrobacter agiTis. The pH precipiLation step was carried out essentially as described by Bhandari and Nicholas (198f). The pH of the supernatant obtained after heat treatment adjusted to 5.2 with 1I4 acetic acid, r¿as allorved to stand on ice for 30 min and then centrifuged at 30,0009 for 30 min. The pe11et was discarded and the pH of the supernatant fraction was adjusted Ëo 4.2 with lM acetic acid. After standing on ice for 20 min, it was centrifuged aL 30,0009 for 30 min and the pellet homogenised in 10m1 of 10mM Tris-HCl, lmM MnC1, buffer (pH 7.2). To this suspension was added half volume of 20% (w/v) polyethylene glycol (PEG) in 50mM Tris-HCl buffer (pH 7.5), drop by drop with constant stirring and after standing on ice for 15 min it was centrifuged at 30,0009 for 15 min. The pellet resuspended in 4M NaCl in 50mM Tris-HCl buffer (pH 7.5) was added drop by drop with constant stirring to an equal volume of. 20% (w/v) PEG in 50mM Tris-HCl buffer (pH 7.5) and after sÈanding on ice lor 20 min it h¡as centrifuged at 30,0009 for 20 min. Most of the gluÈamine synthetase was recovered in the supernatant fraction which was dialysed overnight against 10mM Tris-HCl, lmll MnC1, buffer (pH 7.2). To remove PEG, the second pH precipitation step was repeated Eo precipitate the enzyme which was then washed with buffer and resuspended in a small volume of the same buffer.

2.2.5.2 Glutamate dehydrogenase

A similar procedure was used to purify glutamate dehydrogenase from both /Vi trobacter agiTis and Ni trosomonas europaea. Crude extract (in 50mM Tris-HCl., lmM ß mercaptoethanol, pH 7.S) S:O' hras centrifuged at 23

at 110,000 x g fr:r th and the supernaLanL SrrO was loaded onto a 2r5l ADP gepharose-4B column (0.8 x 9cm) preequilibrated with buffer ,(50mM Tris-HCl, lmM ß mercaptoethanol pII 7.5). The column was washed with buffer (f1ow rate 40m1.fr-I) until the absorbance (AZSO) was close to zero. Enzyme was then eluted v/ith 2mM NADPH in buffer and the fractions containing enzyme activity were pooled and dialysed overnight against the same buffer. All purified enzymes were stored at -15oC.

2.2,6 Enzvme assays

2.2.6.1 Adenosine triphosphatase (ATPase)

ATPase activity in membrane fractions of Nitrobacter agiTis was determined by measuring the release of inorganic phosphate (Pi) from ATP. The reaction mixture in a total volume of lml contained membrane proteins (1.0 to 2.Omg), Tris-HCl buffer (pH 7.3), 1.8 pmol of ATP (pH 7.3) and 3 pmol of ÙfgSOO. Incubation was at 30oC in a water bath shaker. After appropriate incubation period, the reaction was terminated by adding 0.5m1 LO% (w/v) Erichloracetic acid (TCA) and then centrifuging at 10,0009 for 15 min. The Pi release from ATP was Lhen determined by the method of Fiske and SubbaRow (1925).

2.2.6.2 Glutamine synthetase

Both the y glutamyl transferase and biosyntheLic activities of glutamine synthetase were determined by the method of Shapiro and Stadtman (1970à). For transferase assay, the reaction mixture in a final volume of lml contained (mM): imidazole-HCl (pH 7.2),401 glutamine,30; hydroxylamine hydrochloride (neutralized with 2M-NaOH),30; Mnclr.4H2O' 0.5; sodium arsenate,20; ADP,0.4; and an appropriate aliquot of enzyme. For the in vivo assay in whole cells, the assay mixture also contained _1 20 Vg ml-' cetyl trimethyl ammonium bromide (CTAB). Control tubes without gluLamine and hydroxylamine respectively were always included. For biosynthetic activity the assay mixture in a final volume of 0.2m1 contained (mM) : imidazole-HCl (pH 7.0)'50; glutamate,100; of enzyme NHOC1 ,50; ATP, 10; I'fgClt ,5; ¿nd an appropriate aliquot ' Glutamate was omitted from control tubes and a correction h¡as also made for non-enzymaLic production of P1 from ATP. All incubations were at 37"C, usually for 15-30 min' 24

2.2.6.3 Glutamate sYnthase

Glutarnate synthase activity was deLermined spectrophotometrically as described by lfeers et al.. (1970) from the rate of oxidation of NADH at 34Onm in a 1cm qttarEz cuvette, following the addition of aliquots of enzyme preparation to a solution containing (mÞf): o-ketoglutarate,5; NADH,0.35; glutamine 5 and 50mM Tris-HCl buffer (pH 7.6) in a final volume of 3m1.

2.2.6.4 Glutamate dehYdrogenase Activity of glutamate deh'.'drogenase was determined as described by Hooper et a7. (1967) either from the rate of oxidatic.¡n of NAD(P)H (amination reaction) or rate of reduction of NAD(P) (deamination reaction) at 34Onm. For amination reaction (NAD(P)H+¡iAD(P)) the assay mixture in a total volume of 3m1 contained (mM): 0-ketoglutaraUe 20; NH,C1 NAD(P)H,0.33; Tris-HCl buffer (pH 7'8-B'0),50; and an .+ ,24O; appropriate aliquot of Uhe enzyme preparation. For the deamination reaction (Nnn(p)+t'¡AD(P)H) the assay mixture in a final volume of 3m1 conrained (mì4): sodium glutamate,17; NAD(P),0.33; Tris-HCl buffer (pH 9.0),50; and an approPriate aliquot of the enzyme preparation. The amination and deamination reactions were started by adding C-ketoglutarate and glutarnaLe respectively. The reaction rates v/ere correcLed for endogenous oxidation/reduction of NAD(P)H/NAD(P).

2.2.7 Deternination of K and K.for Tutanine s thetase m pTutamate dehvdrop enase

The K values were determined as described by Lineweaver and Burk (1934) m from double reciprocal plots of rate of the reaction against the initial substrate concentration. The K. values were determined by a double reciprocal plot of the reaction rate against the substrate concentration in the presence of an inhibiUor as described by Lineweaver and Burk (1934).

2.2,8 Deternination of noTecuTar wei pht of p1 utamine svnthetase bv pe7 fiTtration The molecular weight of glutamlne synthetase k¡as determined by the method of Andrews (f970) using Sepharose 6-B column. The column (1.6 x 100cm) prepared as described in Secrion 2.2.16.4 was equilibrated with 50mM Tris-HCl 25

buffer (pH 7.5) and calibrated rvith aldolase (158,000), catalase (232,000), ferririn (440,000) and rhyroglobulin (669,000). Blue dextran (=2,000,000) was used to determj-ne the void volume. The distribution coefficienL (Kd) was calculated by the formula,

\¡^ Kd=Ë where Ve and Vo are elution and void volume respectively

2.2.9 CaTcuTation of cunuTative inhibition for BTutamine svnthetase

Cumulative inhibition was calculaied by the proceclure of Stadtrnan et a7. (1968): ^B A * lö (100-A) where the percent inhibition due to each inhibitor is represented byAandBrespectively.

2.2.rO Native and SDS poTvacrvTanide pe7 eiectroDhoresis( PAGE and SDS-PAGE)

Discontinuous, nondenaturing PAGE was carried out in 5 and 77" (w/v) polyacrylamide tube gels (Davis , 1964), The stacking gel was 37" ("/u) polyacrylamide in 125mM Tris-HCl buffer (pH 6.8) and the running gel 5 or 7Z (w/v) polyacrylamide in 375mM Tris-HCl buffer (pH 8.8). The electrode buffer contained 12.5mM Tris, 96mM glycine (pH 8.4). Electrophoresis was carried out aL 2mA per gel at constant current. Gels were stained for protein either with coomassie brilliant blue R-250 (Chrambach et a1., L967) or by silver staining method of hlray et a7. (1981). Glut.amine synthetase activiEy was detected in the gels, washed once in cold 50mM Tris-HCl (pH 7.2) and then incubated at 37oC wi-th transferase assay mixture (Section 2.2.6.2) f.or 15 to 20 min in a water bath shaker. Activity band for the enzyme was detected by adding FeClt reagent (0.4g FeCl' O.24g TCA and 0.25m1, I2N-HC1 in a final volume of 10m1). The gels were immedÍately photographed. In another procedure the gels, after electrophoresis were cut into 2mm slices. Each slice was then individually checked for transferase activity using the standard assay mixture (Section 2.2.6.2) . Specific staining for glutamate dehydrogenase in the polyacrylamide gels k'as done following the deamination reaction according to Tally et a7. G9l2). 26

After electrophoresis the gels were incubated for 20 to 30 min in the dark at 37oC in a reaction mixture containing: 4Om1 0.1M Tris-HCl buffer (pH 8.25), 3m0ll"t sodium gluLamate, lml 0.0065M phenzine rnethosulfate, 2ml O.OO57M nitro- blue tetrazoliurn and 1.3m1 O,O22M NAD* or NADP+ or both. After the bands had appeared the gels were rinsed in distilled \,/ater and stored ín 77. (v/v) acetic acid until photographed.

The subunit molecular weight of glutamine synthetase r¡/as determined by discontinuous gel electrophoresis(10 to 12% w/v polyacrylamide) in the presence of 0.17" (w/v) sodium dodecyl sulfate (SDS) using Tris-glycine buffer (pH 8.3) according to the methods of Laemmli (l 970) and l^Jeber and Osborn (1975). The gels were calibrated with the following SDS treated protein standards: phosphorylase b (94,000), albumin (67,000): ovalbumin (43,000), carbonic anhydrase (30,000) and trypsin inhibitor (20,100). Gels were stained by coomassie blue method of Chranbach et a7. (1967).

2,2.IT Measurenent of oxvøen uDtake with eTec trode

The aerobic oxidation of NH4+, NH2OH and NrHr+ by lVi trosononas europaea and N0, by Nitrobacter agiTis was measured by means of oxygen electrodes (Rank Bros., U.K. and Department of Biochemistry., University of Bristol, U.K.) which take 4.5 and 5.5m1, respectively, of reaction mixture. The concenLra- tions of dissolved 0, in equilibrium with air were determined by the method of Chappell (1964). The relevant reactions at pH 7 to I for Nitrosononas europaea are (Lees, 1952; Nicholas, 1963):

(i) NtlO+ + 30 ----+ nO, + HrO + 2H+

(ii) NH2OH + 20 ------> llo, + HrO + H+

(iii) t2nr* + 20 '-----+ N, + 2Hr0 + H+

and for Nitrobacter agiTis is (Nicholas, L963; A1eem, 1965): (iu) nOJ+0 'N0ã Unless staLed otherwise, for electrode measurements, the buffer was 50mM Tris- HCl buffer (pH 7.5).

2.2.L2 Proton transTocation

2.2.I2,I Fluorescence quenching method

The fluoresence emission was followed as described by Tuovinen 27

et a7. (L977). Measurements were made in a 1cm quartz cell in a Fluorispec (Model SF-l) fluorimeter (Baird Atomic, Cambridge, Massachusetts, U.S.A.) at 42)nn excitation and 485 emission. A potentiometrj-c unit (Sewoscribe 1S) was used to record the data. Fluorescence of the reaction mixture was measured in arbitrary units.

2,2.I2.2 Oxygen pulse experiments

These experimenLs measured the change in proton concenLration in the exLracellular medium of a dense anaerobic suspension of cells, spheroplasts or vesicles caused by a short (1-4s) burst of respiration ir:rtiated by injection of a sma1l amount of 0, (10 to 120ng atom 0). The method used v/as a modification (Kristjansson et a7., 1978) of that described by Scholes and l"litchell (1970a,b). The apparatus was the same as thaL used by hlalter et a7. (1978) excePt that the pH electrode employed was a fast responding combination electrode with a flat pH sensing tip (Activon Scientj-fic Products, N.S.I^I., Australia, Model 92IO). The response of the apparaLus r+as limited by the injection and fluid mixing time which was about 1.5s. Typically, II2ng wet weight of cells in 1.5m1 of 0.151"f KCI was supplemented with carbonic anhydrase (80 Ug 1 m1-') and the sal-t of a permeant ion at sufficient concentration to collapse the membrane potential and allow proton ejection to be observed. This mixture was placed in the electrode chamber and allowed to equili- brate under N, for 10 min at 25oC, at which time the pH was adjusted as required by use of anaerobic 0.1M HC1 or KOH. Once t.he pH had stabiLized, a smal1 volume of 0.151"f KCl saturated under pure 0, was injected to initiate respiratlon and Lhe subsequent proton response was recorded. The response was calibrated by injecting an appropriate amount of anaerobic 5.00mM HC1 (in 0.15M KC1), the concentraLion of which was determined titrimetrically with reference to accurately weighed Tris-base.

A positive response to 0, by bacteria involved a rapid acidification of the medium, followed immediately thereafter by a slow (tå = approximately 1 min) passive diffusion of proLons back across the cell membrane. The latter process tended to diminish the amplitude of t.he initial rapid pH transient by perhaps 5 Lo 2O7", depending on respiration rate and the permeability of the membrane to protons. To correct for 2B

this effect the decay curve for passive proton diffusion v,Ias extrapolated back to a time, approximately I to 2s after 0, inject.ion, at which the initial transienL had reached half its final amplitude, as suggested by Scholes and Mitchell (1970b).

The solubility of O, in 0.15M KCI was taken as 2.32ng atom 0.1-1 at latm. and 25oC (Chappell, Lg64).

Internal and external buffering capacities were estimated as described by Scholes and Milchell (1970a) from the initial pH response of the systern to acid pulses relative to the final equilibrium value after relaxation of protons across the membrane. t\iith bolh Ni trosononas europaea and Ni trobacter agilis about 2/3 of the total buffering capacity v/as external t.o the membrane and about 1/3 internal, under the conditions of experì-ment. A similar distribution applied in the case of spheroplasts and vesicles from Nitrobacter agiTis.

2.2.I2.3 Reductant pulse experiments

These experiments used the same apparatus as described above and are analogous to oxygen pulse experiments, except that small amounts (10 ro 150 nmol) of ruHf , NH2OH or NrHr+ in the case of Nitrosomonas europaea were injected j-nto a system under pure 0r. Because the initial reductant was typically well below its K- value (eg. concentration of m 30 nmol NHO+ in 1.5m1 = 2OuM; K, for NH*+ at pH 7.4 is approximately 1.OmM) respiration rates were relatively slow. The response, especially in the absence of a permeant ion, hras typically a progressive acidification from an initial value to a final value rather than one showing the sharp maxima observed for oxygen pulses.

2.2.I2.4 Estimation of stoichiometric protons

The reactions considered in Equationsi-iii (Section 2,2.LI) involve the stoichiometric production of protons. In order to calculate the true * U+/O ratio in oxygen pulse studies for the protons translocated by the proton purnp it was necessary to determine the yield s¡ stoichiometric protons and substract Èhe value from the overall yield 29

of protons. Stoichiometric protons were estimated by two techniques: (i) reductant pulse experiments in the absence of a permeant ion, and (ii) oxygen pulse experiments in which 3-5UM CCCP was used to bring about the rapid equilibration of protons between internal and external buffer compartments. CCCP is known to be an effective inhibitor of respiration in Nj trosononas europaea (Bhandari and Nicholas , I979a) and NiËrobacter (Cobley, I976a), but the extent of inhibitj-on at 3 to 5UM was in- sufficient Lo interfere seriously with the oxygen pulse experiments.

2.2.I2.5 Permeant ions In oxygen pulse studies, it is necessary to collapse me:,rbrane potential so that optimum proton ejection can be observes (Scholes and Mitchell, 1970b). The salts of all the permeant ions usedin this study (Secrion 3.5.1.2) were dissolved in double distilted water. Itlhere ClO[ was used, it was added as NaC1OO at'a final concentration of 0.3þl to a solution already 0.15M in K*. Inasmuch as KC104 is relatively in- (K^^, ar 25oC) much of the K* and some of the C10O were soluble . SOI =IO-2 precipitated to give actual concentrations of K+, N"* and C10[ of about 0.05,0.3 and 0.2M resPectively.

2,2.r3 Deternination of nenbrane potentiaT (LY) and transnembrane oH pradient (LpH) in washed ce77s

2.2.I3.I EDTA t.reatment of cel1s Cel1s of both Nitrosononas europaea and Nitrobacter agiTis wete suspended in 100mM Tris-HCl buffer, pH 8.0 (20mg . *1-1) and Ereated with 5ml"t EDTA/KOH, pH 7.0 (for Nitrosomonas europaea) and 10mÙi (for.Nitro- bacter agiTis) for 10 min at. 37oC. The ce1ls collected by centrifugation were washed once in the buffer and suspended in the appropriate buffer. The EDTA-treated cells were used within 2h.

2.2.I3.2 Intracellular water space 3H20 I4 This was determined by using (for total pellet water), t cl sucrose (for total pe1let r.trater - intracellular water, which does not include periplasmic space) and ttaC] inulin (for extra cellular water) according to the methods of Maloney et a7. (1975) and Stock et a7' (1977)' 30

Thus for Nitrosononas europaea and Nitrobacter agiTis the intracellul-ar water spaces were I ,7 + O.2 and I.2 t 0.2 U1(mg dry weight)-l r""p"ctive1y.

2.2.13.3 Uptake of radioactive probes

Untreated or EDTA-treated cel1s were incubated at 25oC in either -LI Na'or K'phosphate (100mtf) or Tris-HCl (50mM) buffer at the appropriare pH. The ce1l suspensions krere either vigorously oxygenated for 10 min with pure oxygen or mixed with catalase (0.05mg m1-1) and Hro, (1u1 rl-1). For Nitrosononas, 5mM NH4cl and for Nitrobacter 5mM NaNO, was the substrate. Then the radioactive compound was added and incubation continued for a further 5-15 min. Aliquots (1 rnl) were then centrifuged in Eppendorf microfuge at 13,0009 for 1 min. Aliquots of the supernatant (100il1) and the cell pellet respecrively were added to I ml, 3M perchloric acid in 15ml scintillation glass via1s. After 30 min, when ce11 proteins were completely dissolved, 5m1 of a scintillation counting fluid (PCS) (Amersham, England) was added to each vial, the contents mixed thoroughly and radioassayed in a Packard Tri-Carb 460 CD liquid scintillation specLrometer. fn the standard protocol two consecutive experiments \./ere carried ouL in which At! and ApH were measured. For A$ determination [3u1 frf* Br-(20-50 nCj.ml-l) was added ro a cell suspension (1-1.5mg dry weight. *1-1). For ApH dererminarion [14C1 b"n"oi. acid (2 uCi.ml-l), tlaCl aceryl saricyric acid (2 pCi.m1-1) or tlaCl methylamine-hydrochloride (1 pCi.rl-1) 3Hro was added. "u" used to determine total pellet t'/ater.

2.2.I3.4 Calculations of proton motive force (Ap or frr+)

The calculations of A$ and ApH were made by using the Nerrist equation as described by Mitchell (1966) after correcting the uptake data for non specifically bound ¡3U1 fff+ and extracellular counrs of tlaC] benzoic acid, tlaC] acetyl salicylic acid and tlAC] merhylamine respectively. Membrane potential ( ¿{,) was calculated from the uptake of ¡3H1 rer+ as follows: 31

o* =BËr,ffi]fi, or aü = -2.ros þo*.[ff"* 'At 27"C (T = 300oK) 2.303 RT/F = 60mV Aü=60x1og

is rhe ratio of intracellular [3u] rpp+ to where +iËi+l*"* exrracellular [3u] fPP+.

distribution of 14C Intracellular pH (pHi) was calculated from the "uuk acids (benzoic and acetyl salicylic); pui = pK + log [Ain/Aouu 11g(lHe-pr)+.1)-t] l4C-'n"thylamine; or by the distribution of weak base. pHi = pK - log [Bin/Bout (to(pK-pge)+ 1)-1] ApH was obtained from the difference of pHi and pHe (ApH = pHi - pHe)' Proton-motive force (^p) was calculated as: Ap = A$-2.303 Ë ApH or at 27"C Ap = Atll-60 ÂpH

2.2.14- Na* and Kr transport' 2.2.I4.I K+ dePletion of cells and . The nethod used for K+ depletion of both Nj trosonortas e\ropaea Nitrobacter agiTis was essentially the same as described by Nakamura et a7. (1982). For rourine use, freshly harves¡ed cells (=500m9 wet weighr) washed twice with either lOOmM K-phosphate (pH 7.5) or 50mM Tris- HCl (pH 7.5) were suspended in about 50m1, 50mM dÍethanolamine-HCl ' sus- 150mM NaCl (pH 9.2) and incubated at 30oc for 30 min. The cell pension was then centrifuged at 15,0009 for 10 min. The pellet was resuspended in 50mM diethanolamine-HCl, 150mM NaCl (pH 9.2) and treated similarly as described above but for 15 min instead of 30 min ' The ce1l 32

suspension was then recentrifuged at 15r000g for 10 min and the pellet resuspended in the appropriate buffer. Cells prepared thus rvere washed once in an appropriate buffer and contained .(5nM K*. Unless stated otherwise, these diethanolamine treated cells are referred as t'K+ depleted cellstt in the text.

2.2.I4.2 Na* and K* deterininations by atomic absorption -spectros copy For cellular Na* and K* determination, cell suspensions were filtered through Millipore O.22V or 0.45U filters (Type GS or HI^IAP)' washed twice with at. least 2m1 volume of either 50mll Tris -HCl (pH 7.5) or buffered choline chloride (0.2I1 choline chloride in 10ml'f Tris -HCl pH 7.25). The filters were then immersed in 5ml 5% (w/v) trichloroacetic acid (TCA) in acid washed plasLic centrifuge tubes (10m1 volume) and left overnight in a waterbath shaker at 30"C. The Na* and K* contents of the TCA extracts were deterrnined in a Varian atomic absorption spectrometer af ter ad justing the volume to 10m1 \"/ith deionized distilled v¡ater. The machine was calibrated with standard solutions of KC1 and NaCl before and after each set of 6 determinations. Appropriate controls (including rJ- for K- and Na- contents in filters, plastic tubes, TCA and deionized distilled water) were always included. All solutions used in atomic absorption studies v/ere Prepared in deionized distilled l¡/ater.

15N 180 2.2.15 StabTe iso inents with und 7ub"77"d ds to stud oxidation b s of Nitrobacter iTis t The aim of the experiments was to determi-ne Lhe 0 contents of N0, produced during the aerobic oxidaËion of N0, at pH 7.8 by Nitrobacter agiTis 180, in the presence of Hrl8o o, tISo] pholphate. Two techniques were used for isotopic analysis namely gas chromatography combined with mass specLro- l5N-nuclear metry (GC/MS) .nd magnetic resonance (NMR) spectroscopy. The two sets of studies were carried out independently.

2.2.15 J GCIMS studies 180, For experiments involving o. nrtto washed ce11s Lrere suspended in either 0.1M Tris-HCl (pH 7.8) or 0.1M K-phosphate (pH 7.8). For experiments involving tlBO] phosphate, cells were incubated in O.lM t18O] K-phosphate (pH 7.8). The final volume of the reaction rnixture 33

lBo was 20m1 except for experiments involving H, in which case the final volume was 2ml and contained O.5ml of Hrl8ol All experiments were carried out in 100m1 Erlenmeyer flasks each closed with a Subaseal 180, septum. For ttr" experiment, flask \4/as evacuatecl and back filled 180r. to latm wittr Eacf, complete reaction nixture contained lOOmg (rvet weight) of cells and the reaction \{as started by the addition of 50 Umol of either NaNO, or K15NOr. Nitrite consumption was monitored colorimetrically (Nicholas and Nason, L957 ) and upon i-ts exhaustion another 50 pmol of nitrite was added. This r,/as repeated until the cells l5llOr;. oxidised a total of.25O Umol of NO" All incubations were at ¿12lor 2BoC in a waterbpth shaker (120 rev.*n-t). After utilízation of the final addition of nitrite, each reaction mixture r¡/as chilled to OoC and centrifuged (15r0009 for 10 min) to remove cel1s. Each supernatant was divided into two parts. The first part, if it contained phosphate was treated with stoichiometric amounts of NHOOH and MgC1, in order to precipitate MgNHOPOO, which was removed by centrifugation. This step was omitted if Tris-HCl was the buffer. The phosphate depleted super- naLant was then passed through a Dowex-5O H* form column (10m1 bed volume). The column was eluted with distilled water until the effluent reached a pH of about 6.5. The pooled effluenu (pH 1.5 to 2) was neutralized to pH 7.0 with NH40H and lyophilized to a powder. The powder was pyrolysed in a 10ml Pyrex tube, closed with a Subaseal septum, evacuated and backfilled with Helium gas. The bottom ofthe tube was carefully heated on a burner flame and then 10-100U1 sample of the resulting NrO gas mixture was analysed by GC/MS.

The second part hras passed through a Dowex-5O column as above, but with omission of the precipitation step. In this case the acidic pooled effluent was lyophilized directly without neutralization to yield a small volume of liquid which contained mainly phosphoric and nitric acids. This liquid was methylated using diazomethane (prepared by the method of Fieser and Fieser, 1967). Excess of CH,N2 (in ether) vras added to the sample at room ternperature until the yellow colour of the distillate failed to fade away. A similar method was employed to methylate standard nal?O and HTPIBOO to yield trimethylphosphate (CH3)3Pt"04). Methylation of llt{O, was in the .same nanner. ((CII3)3PO 4 ot 34

but it rvas found necessary to add an approximately equal volume of concentrated H2S04 to ttre HN03. This procedure generated a mixture of dimethyl sulfate and methyl nitrate which coul-d be separated by GC/l'fS. Ether and excess CH'N, were removed from the methylation mixtures by evaporation at 45oC. The methylated products were dissolved in CHrCl, for GC/MS analysis.

The yields of methyl nitrate produced by CH2N, methylation of lyophilized liquid were low presumably because of the unstable nature of met.hyl-nitrate.

Isotopic analyses l^rere carried out by use of a Hewlett-Packard GC/MS model 5gg2-B fitted with a glass column (lm x 2mm i.d.) packed with Tenax GC (60-80 mesh). The helium flow was 25m1.min-1 and the electron multiplier^ aL 22OO volts. Data were obtained by Peakfinder and Selected Ion Monitoring programrnes. The latter programme allowed the monitoring of six selected amu values simultaneously. Trimethyl phosphate, dimethyl sulphate, methyl nitrate and NrO had the following retention times respectively: 3.6 min at l6OoC, 1.8 min at 16OoC, 4.8 min at 50oC and 0.3 rnin at 25oC. The degree of ionj.zation-induced fragmentation of NrO+ into N0+ and Nr+ *.s similar to those reported previously, (Cady and Bartholomew, 1960; St. John and Hollocher, 1977)' Trimet.hyl phosphate and dimethyl sulfate were dissolved tn CHTCI, (1:1000) and usually lp1 was injected. Methyl nitrate was usually injected from the vapour phase without solvent (10-100U1) because it separated only poorly from common solvents such as CHZCI.Z on the column' The Pl+ peak for CH3NO3 (77 amu) v/as generally not observed; identifi- cation was based on the base (int.egral = 100) peak (NOrt-), NO* and a weak (M-1)+ peak at 76 amu.

2.2.r5,2 15t'¡-NYn studies

The incubation mixtures were similar to those described in Section 2.2.15.1 with minor changes. irlashed cells of Nitrobacter agiTis vüere suspended in 10OmM K phosphate (pH 7.8) at a concentration of about lml 5OO rng wet weight.ml-l. The following experiments \"/ere done: (i) ce11 suspension rvas diluted to lOml in 100mM phosphate, 5nM carbonate 35

buffer (pH 7.8); (ii) lmf cell suspension was diluted to 10ml in the same buffer, and the flask closed with a Subaseal septum. The flask 180, was evacuated and backfilled with ao 1atm.; (iii) lml cell sus- pensi-on was added with lml each of 200mlf phosphate, lOmlul carbonate (pH 7.8) and nrt80; (iv) I ml cel1 suspension r{as centrifuged in an Eppendorf tube (1.5m1) at 13,0009 for 5 min and the pellet resuspended 180 in 10ml of phosphater 5mM carbonate buffer (pH 7.8). To all the cell suspensions in 50ml Erlenmeyer fl-asks was added, catalase (lmg) and 40% (v/v) HrO, (5U1) (ecept for experiment ii) followed by incubation ar 2BoC in u ,rt"r barh shaker. Then 50 umol r15llo, (97 atom %) was added to each flask to start the reaction. Aliquots (5-10u1) were withdrawn from the reaction mixtures to check N0, concentration by the method of Nicholas and Nason (1957). As soon as the nitriEe was uti- 15lt0, lized completely another 50 umol of was added and the reaction continued until at least 200 Unol of total nitrite had been oxidised to niÈraLe. At the end of reaction, ceIl suspensions were centrifuged at 20r0009 for 10 min at 4oC and the supernatant fractions hrere carefully dispensed with a Pasteur pipette. The volume of each supernatant fracÈion was made to 10m1 with phosphate-carbonate buffer, the pH adjusted to 8.0 if needed and then immediateLy írozen in liquid N, until used in NMR st.udies.

l5N-NMR 30.42 Utl, spectra vrere obtained on a Bruker CXP 300 NMR spectrometer operating at a field strength of 7.05T. Spectra h¡ere from 2 d*-3 in 10mm NMR tubes as Èhe result of approxi- acquired ".*ples mately 200 scans into an 8K data tab1e. A 15o (10rs) pulse was used lH with a 4.1s recycle time and no du.oupling. After acquisitionr ê line broadening of 0.1H2 was applied, together with apodisation. The data were zero filled to 16K before Fourier transformation.

2.2.16 GeneraT a UES

2,2.\6.I Electron-microscopy

Samples of spheroplasts and membrane vesicles were negatively srained wi:h 27. (w/v) phosphotungstic acid (pH 6.6) on carbon coated 36

copper grids, dried in air prior Lo examination in an electron microscope JEOL (Model JEùf.100 cx) at an acceleration voltage of 60kV.

2.2.16.2 High voltage paper elecgrophoresis (HVPE)

HVPE of phosphate and pyrophosphate was done according to the method of Tate (1968). Standard solutions or aliquots of reaction mixtures were spoLted in the middle of a Whatman 3MM chromatographic paper (15 cm x 60cm) near the cathode. The paper was moistened with 100mM Na- tetraoxalate (pH 4.2) and then lightly blotted to remove surface moisture. It was then laid out on a polythene frame and placed in a ceramic tank filled with Perclene. The ends of the paper l¡/ere connecLed by wicks to the buffer charnbers containíng tOOml"l Na-tetraoxalate (pH a.2). The perclean was cooled by passing cold water through copper coils placed in the centre of t.he tank. A stabilized power pack (Paton Industries Ltd. Australia) was used to supply the current to the buffer chambers, (100m4 and 1500 volts for 30 min).

After electrophoresis the paper was dried by hot air and spoËs were detected either by silver nitraÈe or by rnolybdate staining method (Tate, 1968).

2.2.16.3 Liquid scÍntillation spectrometry 3H¡ Radioactivity in aquous samples (14C ot ru" measured by couriting aliquots in IPCS' scintj-llation fluid in Packard glass vials. The ratio of sample volume Èo scintillation fluid volume was 1:5 according to the recommendations of the manufacturer (Amersham, England).

Radioactivity on dried filters (221¡u) was measured in toluene based scinrillation fluor (O.3% (w/v) PPO and O.O37" (w/v) P0P0P in toluene) in Packard glass via1s. The vials h¡ere assayed in a Packard Tri-Carb liquid scintillation spectrometer (Model 460 CD).

2.2.16.4 Preparation of chromatographic columns

Affinity chromatography columns (Blue Sepharose CL-68 and 2'5'-ADP 37

Sepharose-4B) and gel filtration columns (Sepharose-4B and Sepharose-68) were prepared according to the instructions given by the manufacturers (Pharrnacia Fine Chemicals, Uppsala, Sweden). The columns were equili- þ¡ation with appropriaÈe buffers and when not in use they were stored at 2oC in the appropriate buffer in the presence of 0.I7" (w/v) sodium azide.

2.2.17 ChemicaT deterninations

2 ,2.17 .l Nitrite

Nitrite was determj-ned by the method of Nj-cholas and Nason (f957) and Hewitt and Nicholas G964). An aliquot. containing 30 to 500 nmol of nitrite was diluted to lml with double distilled water. The red azodye was developed by adding lml of L% (w/v) sulphanilamide in lN-HC1' followed by lml of 0.01% (w/v) N-(l-naphthyf) ethylene diamine hydrochloride. After 15 min, the absorbance was read at 540nm in lcm glass cuvettes, employing a Shimadzu (QV-50) spectrophoLometer. The concen- traEion of nitrite was determined from a standard curve.

2.2.I7.2 Ammonia

Ammonía was determirted by a modified Nesslerrs method (Ballentine, 1957). An aliquoL containing O.2 to 2 pmol NHI was diluted Èo 0.5rn1 with distilled water. Color was developed by adding lm1 of reagent A (IO%',¡/v, sodium potassium tartarate) and Zn1- of. reagenÈ B (2.2729 mercuric iodide, L.826g KI and 49 NaOH in 100m1 distilled water). After 20 min the absorbance was read at. 435nm in 1cm glass cuvettes employing a Shimadzu (QV-50) spectrophotometer. The concentration of NHf was determined from a standard curve.

2.2.I7.3 Protein

Protein was determined either by a microbiuret method (Itzhaki and Gill, fg64) or by the dye binding method of Bradford (L976), using bovine serum albumin as a standard. The absorbance was recorded in lcm qttar:-z cuvettes in Shinradzu (QV-50) spectrophotometer. 3B

2.2.I7.4 Inorganic phosphat.e

Inorganic phosphate (Pi) was determined by the method of Fiske and Subba Row (1925). Samples containing Pi were diluted to 2m1 with distilled water and 0.5m1 of acid molybdate (2.57. w/v ammonium molybdate ín 2.5M H2S04) was then added followed by 0.1m1 of colour reagent. The colour reagent was prepared by mixing 1g l-amino 2-naphthol 4-sulphonic acid, 39 anhydrous sodium sulphite and 69 sodium metabi- sulphite and stored at 4oC in the dark. The colour reagent. was prepared fresh before use by dissolving 0.259 of the mixture in 10ml double distilled rttater. The colour was allowed to develop for 20 min and the absorbance read at 750nm.

2.2.I7.5 ATP

ATP was determined by the firefly method of Stanley and Williams (1969). Aliquot (0.4m1) were dispensed from the reaction mixture into 0.1m1 3M perchloric acid in a test tube (1 x 5cm) kept in ice. After 15 min, 0.3m1 lM KOH was added and then 20U1 of this neutralized extract r'/as assayed for ATP. The reaction mixture in a scintillation vial (15 x 45mm) conÈained lml 10mM sodium phosphate (pH 7.5),0.9rn1 distilled water, 0.1m1 5mM MgC1, and 50Ul firefly extract. The vial was then placed in a liquid scint.illation spectrometer (Packard Tri-Carb model 3375) seË at maximum sensiÈivity with the two photomultipliers swítched out of coincidence and assay continued for 6s. Standard solution of ATP (10-50 pmol) or the sarnples (20u1) were then added and after 30s assayed for 6s. The ATP concentration was calculated from a calibration of freshly prepared ATP standards.

3. RESULTS.

3.1 NITRITE OXIDAT]ON BY I.{ASIIED CELLS, SPHEROPLASTS AND MEI'IBRANE VESICLES OF NTTROBACTER AGILIS

3.1.1 Elec trode neasurenent of NO oduction and 0 U take

A technique has been developed to measure 0, uptake and N0, ProducLion simultaneously and conLinuously during NOI oxidatì-on by Nitrobacter agiTis. The apparatus (Fig.-1) consists of a double-walled perspex vessel (5ml volume) closed with a perspex .lid. The port in the lid of the vessel accommodated an 0, electrode (DepartmenÈ of Biochemistry, University of Bristol, U.K.), a N0, sensitive electrode (0rion model 93-07-01) and a reference electrode (Orion model 90-02). The NOJ and reference electrods were connected to a Beckman expanded scale pH meter (model 76). The elecÈrode responses were recorded simultaneously and continuously using a two-channel Rikadenki (model B 181-H) potentiometric recorder connected to the pH meter through a Unicam SP45 concentration readout unit to converE the log response of the NOJ electrode to a linear scale. All the additions were made via a Hamilton microsyringe through a port in the lid of the vessel. The NOJ electrode was calibrated before each experiment using a standard NO; solution, in 5OmÙf Tris-HC1 buffer (pH 8.0) for experiments with washed cells and in 5OmM Tris-HCl buffer containing 0.21"1 sucrose for spheroplasts and membrane vesicles. Corrections vJere made for the response of electrode to NO' after N0, additions.

3.1.2 stoichionetr fNo tion b washed ce77s

The addition of NO, to a reaction mixture containing washed cells of Nitrobacter agiTis in 50mM Tris-HCl buffer (pH 8.0) resulted in an immediate uptake of Orand extrusion of NO; (Fig.z). Nitrite was determined in aliquots of the reaction mixture by the method of Nicholas and Nason (1957). The stoichiometry of NOJ oxidation by washed cells was 1NOJ : 0.50, : 0'75N01 maximum rates of N0, and 0r,uptake and Nor \.iere (Fig.3). The rProduction approximately 0.3, 0.15 and 0.26 Umol min-t. (*g protein)-' respectively and varied from one baLch of cells to another. The uptake of NOi and product- was consurned' ion of NOJ were dependent on O, and when all the O, from medium 40

FIG.I: ELECTRODE ASSEI'{BLY FOR MEASURING N03 PRODUCTION AND 02 UPTAKE

A. NOJ electrode (Orion model 93-07-01) B. Reference electrode (Orion model 9O-O2) C. Oxygen electrode (University of Bristol, U.K.) D. Perspex reaction vessel (5 ml volume)

E. Port for addiuions (2 rnm diameter) FIG.l

& 4T

FTG.2 : UPTAKE OF 02 AND PRODUCTION OF NO3 BY I,JASHED CELLS

100p1 of washed cel1 suspension (40 mg wet weight) waç added to a perspex vessel containing 5p1 catalase (2mg. m1-') and 0.25 m mol Tris-HCl buffer (pH 8.0) in a final volume of 5m1. The reaction mixture was maintained at 25oC. Reaction was started by adding 10pmo1 of N0, via a Hamilton microsyringe through a port in t.he 1id of tñe vessel. 0" was regenerated by injecting =19U1, 2% (v/v) HZOZ into Ehe réaction mixture. The reaction mj-xture v/as continuously stirred with a magnetic flee, . The maximum rates alongside the traces are in pmol. min ' for N0, and 0, Broken line, NO, nroduction; continuous line, 0, uptake.

FIG.3: STOICHIOMETRY 0F N0 AND 02 UPTAKE AND NOz PRODUCTION BY I^JASHED 2 CELLS

The reaction mixture used as given in Fig.2. Nitrite was determined in aliquot.s withdrawn from the react,ion mixture at 1 min int,ervals as described in Section 2.2.I7.I. NOJ utilization (o); NO, lroducrion (O); 0, uprake (t). CELLS FIG. 2 {_

pMoL r 0.9 Nos I

o.r pMoL 02 I

FIG. 3 to

or! I oL lrO zo 6 olC\J z. 4 c, 1E ?

o 2 4 6 I lO mrn 42

both NO, uptake and N0, production ceased. Thus oxygen \,/as regenerated in these experiments by means of catalase and HZOZ.

3.1.3 Preoaration of soheropTas ts and membrane vesic-les

Spheroplasts of Nitrobacter agiTis r{ere prepared by lysozyme-EDTA treatment of washed cells (Section 2.2.2). Under the electron microscope they appeared almost completely devoid of cell walls (Fig.4). The spheroplasts thus obtained were osmotically fragile and rapid lysis occurred when they were suspended in hypotonic solutions or distilled k/ater, resulting in the release of DNA and a decrease in the absorbance of the spheroplast suspension.

Ultrasonic treatment of spheroplasts result.ed in vasicularization of membranes producing inside-out membrane vesicles. Electron microscopy of thc membrane vesicles showed that t.hey were bounded by a single membrane (Fig.5).

3.1.4 S r NO oxidation b ero ts and nembrane

Spheroplasts and membrane vesicles prepared as described in Section 2.2.2 were tested for N0, oxidising activity by the electrode method. BoÈh preparations oxidised NO, aE about 1/8tn of the rate of washed cells. Stoichiometry of lNO, : 0.502 : fNOJ was recorded for bolh spheroplasts and membrane vesicles (Fig.6a and 6b respecEively).

3.1.5 tinum ons for NO oxidation b membrane ves

The optimum pH for N0, oxidation by membrane vesicles was 7.5 and the oxidation rate decreased rapidly above this pH. The optimum temperature was 23oC. The K* val'ues of 0.8mM and 20pM were obtained for N0, and 0, respec¡ively. Additions of any of the metal salts listed in Table 1, except 2+ for Ni2+ and Cu2*, did nor affecr NOt oxidarion while Ni. and Cu2+ inhibited NO, uptake by about 25 and 2O% respectively at lmM final concentration.

3. 1.6 Effects of netabolic inhibitors on NQ oxidation

It is known that N0; oxidation by Ni trobacter is sensitive to a variety of metabolic inhibitors (O'Ke1ly et a7., L97O; A1eem, 1977). The effects of 43

FIG.4: ELECTRON MICROGRAPHS 0F LYSOZYME-EDTA TREATED CELLS 0F NITROBACTER AGILIS

Lysozyme-EDTA treatment of cells was carried out as described in Section 2.2.2. Samples q¡ere negatively stained wíLh 27" (w/v) phosphotungstic acid (pH 6.6) on carbon coated copper grids and examined in an electron microscope JEOL (Model JEM-100cx) at an acceleraÈing voltage of 60 kV (Section 2.2.16.I). From top:

A Cel1s after 30 min lysozyme-EDTA treatment (x 20,000)

B A single cell showing loose wall structure after a /+5 min

lysozyme-EDTA treatment (x 50,000)

c Spheroplasts after washing twice vrith 50mM Tris-HCl, 0.21"1 sucrose (pH 8.0) (x 5,000). FIG. 4

t a a- a aa r; ì a a a a a

a o a t a O a B a .i -¡ to 3 a: .'._ a tl .rl t. a

C 44

FIc.5: ELECTRON MICROGRAPHS 0F NTTROBACTER AGILTS MEMBRANE VBSICLES

Membrane vesicles prepared as described in Section 2.2.2 were negatively stained with 2% (w/v) phosphotungstic acid (pH 6.6) on carbon coated copper grids and examined in an electron microscope JBOL (Model JEM-100 cx) at an accelerating voltage of 60 kV (Section 2,2.16.I).

From top:

A. Nitrobacter membranes (x 100,000)

B. . A single membrane vesicle (x 150,000) C. Sectioned membrane vesicle (x 150,000) FIG. 5

it' I'

c 45

FrG.6: STOTCHTOMETRY 0F.NOt AND 02 UPT'^.KE n}ID UO' EXTRUSION By SPHEROPLASTS (a) AND MEMBRANE VESICLES (b). Bxperimental details as in Fig.2 and 3 except that the buffer also contained 0.2M sucrose and r^¡ashed cells were replaced by either spheroplasts (3.18 mg protein) or membrane vesicles (15 mg protein).

NOJ utilization (O) ¡ NO, nroducrion (O); 0Z uptake (tr). FlG. 6a l.o

* O.8 o o¡- Loo 0.6 4 3 0.4 z. /'o iõ 5-o.z

o 4 I t2 16 mtn

FlG. 6b

oc\¡ 4 oa- lF) 3 oz IN o 2 z. o a.Ê

o 4 I l2 t6 mtn 46

TABLB 1: EFFECTS OF VARIOUS METALLIC IONS ON O? DEPENDENT Noz UPTAKE AND N0ã PRODUCTIoN BY Ì"IEMBRANB VESr CLES. Experimental details as in Fig.2 except that the reaction mixture also contained the indicated metal salt at lmM final concentraEion.

% Inhibition Metal ion NO upÈake NO production 0, uptake 2 3

^2+le 0 3 Nd

F"3+ ( 2 6 Nd

cÐ2+ 0 0 0

Lfr_2+ 0 0 0

tt 12+ 3 5 6 2+ Mg o 0 0 2+ Ni 27 25 22 2+ Cu T7 19 T7 2+ Ca o 0 2

Nd - Noc deEermined because of interference h'ith determination. 47

some of these inhibitors on the utilization of both N0, and 0, and the pro- NO" by washed cells, spheroplasts and membrane vesicles of duction of 5 Nitrobacter agiTis were investigated. The effects of some metal inhibitors on N0, oxidation in washed ce1ls, spheroplasts and membrane vesicles are shown in Tables 2,3 anð 4 respectively. Thiourea, 8-hydroxy-quinoline, toluene dithiol and 2TMP all inhibited NOJ and 0, uptake and NOJ production by washed ce1ls (Table 2). DIECA which inhibits NHI oxidaÈion completely at. very low concentrations (i3UM) in Nitrosononas europaea (Bhandari and Nicholas, L979a) restricted N02 and 0, uptake by only 33 and 302 respectively at a much higher concenÈration (0.2M) and this inhibition r,ras not reversed by rhe addition of Cu2*. Sodium azide at 40¡rl,l inhibited NOJ poduction and 0, uptake completely in washed ce11s. The overall pattern of inhibition of NOI oxidation in washed cel1s (Table 2), spheroplasts (Tab1e 3) and membrane vesicles (Table 4) was similar but the extent of inhibitory effects varied. Thus azide kras more effective in washed cell than in membrane vesicles. In washed cells inhibitors can affect many metabolic reactions and this would account for the different responses between washed cel1s, spheroplasts and membrane vesicles.

The effects of inhibitors of electron transport, oxidative phosphorylation (uncouplers) and ATPase on NOJ and 0, uptake and N0! production by washed cells, spheroplasts and membrane vesicles are shown in Tables 5r6 and 7 respectively. The overall paLtern of inhibition was very similar for the NO, oxidising systemsbut again the extent varied. The inhibitors of electron transport viz, rolenone, amytal and HOQNO, all inhibited NOJ and 0, uptake production washed and spheroplasts (Tables 516) but had and NO]J' in ceIls little or no effect in membrane vesicles (Table 7). Nitrite oxidation has been shown Eo be sensitive to uncouplers (Cobley, L976a,b). The resttlts of this study also indicate that all the uncouplers used, strongly inhibited N0, and O, :uLÍ-J-i_zati-on and NOl nroduction either by washed ce1ls, spheroplasts or membrane vesicles. It is also evident from Tables 5,6 and 7 that CCCP at low concentrations restricted NO; and O, uptake and NOJ production, whereas 2.4.DNP, DBP and PCP did so at higher levels. The inhibitors of ATPase, namely DCCD and NBD chloride, also affected the uptake and extrusion processes in all preparations. OF NOã BY I,IASHED CELLS. TABLE 2: EFFESTS OF METAL INHIBIToRS ON THE UPTAKE OF NO; AND 02 AND PRODUCTION

The reaction mixture used is as in Fig.2 except that it

dissolved in 95Zo (u/") ethanol. Appropriate controls each inhibitor dissolved in ethanol. Inhibit Toluene KSCN Thiourea 2TMP 8-hydroxy DIECA Azide quinoline Dirhiol (mM) (ul"f (ull) (mM) mlul mll ) 200 0 0 50. 0.1 1 20 200

40 Ng, uptake 027 42 50 2t 50 47 L2 33 Nd Nd

100 47 N0^ production 027 50 59 23 49 Nd 17 Nd 100 J^

100 3l+ 0, uptake 010 40 46 t7 38 52 10 30 84

Nd - Not determined because of the interference wit,h determination.

s 00 49

TABLE EFFECTS OF METAL INHIBITORS ON THE UPTAKE OF NO AND O AND 3: 2 2 PRoDUCTTON 0F N05 BY SPHERoPLATS.

Reaction mixture in a total volume of 5m1 contained 50mM Tris-HCl, 0.2M sucrose (pH 7.5) and 2 to 5mg spheroplast protein. Experi- mental details as in Fig.2 and Table 2.

% Inhibition

2TT"IP 8-Hydroxy DIECA Toluene c0 quinoline dirhiol (15 lb for (rl{) (mM) (w) (uM) 15 min)

0.1 1.0 0.1 1.0 20 200 50 100

N0, uptake 49 100 45 s0 15 35 52 60 37

NO^ oroduction 70 42 J' 50 100 Nd Nd 17 Nd 56

0ruptake 56 r00 50 56 ls 35 53 68 39

Nd - Not determined because of inLerference with electrode. 50

TABLE 4: EFFECTS OF }'IETAL INHIBITORS ON THE UPTAKE OF NO2 AND 02 AND EXTRUSION OF NOã BY MEMBRANE VESICLES.

The reaction mixture in a final volume of 5m1 contained 50mM Tris -HCl,0.2M sucrose (pH 7.5) and 2 to 3mg vesicle protein. Experimental details as in Fig.2 and Table 2. % Inhibition

2T},fP 8-hydroxy Sodium Toluene quinoline azide dithiol ('M) (mM) (mM) (mM)

0.5 1.0 0.5 0.1 0.05

N0, uptake 52 65 75 57 33

NO production 50 62 Nd 55 40 3

0 uptake 55 6s 75 60 40 2

Nd - Not determined because of interference with electrode. (d-f) AND ATPase (g'h) 0N TABLE 5: EFFECTS OF INHIBITORS 0F ELECTRON TRANSP0RT (a-c), 0XIDATIVE PHOSPHORYLATION UPTAKE OF Not AND 02 AND THE PRoDUCTIoN 0F N0ã BY I'IASHED CELLS.

A1I inhibitors were dissolved in 957" (u/u) ethanol. Experimental details as in Table 2 and Fig'2' %I tion o h a b c d ò Rotenone Amytal H0QN0 cccP NBD Chloride DCCD 'l (ue) (uM) (ul"f ) (uMl (uM) (ul"f 50 20 60 615 28 54 25 50 100 10

31 69 N0, uptake 754153s2560235827633442 39 39

34 65 N0^ 2 60 12 39 28 65 22 56 30 66 36 45 33 36 J^ oroducrion

32 72 0, uptake 22 30 12 26 22 30 25 70 20 36 23 48 46 68

(,¡ì H TABLE 6: EFFECTS OF INHIBITORS OF ELECTRON TRANSPORT (A.C) OXIDATIVE PHOSPHORYLATION (d-f) AND ATPASC (g'h) ON THE UPTAKB 0F N02 AND 02 AND THE PRoDUCTION 0F N0ã BY SPHERoPLASTS.

All inhibitors except KCN were dissolved i¡ 95ir' (v/v) ethanol. KCN was dissolved in distilled water. Experimental details as in Fig.2 and Table 2.

% Inhibi ri on

o a b c d è f ô h Amytal H0QN0 KCN PCP CCCP 2,4-DNP NBD Chloride DCCD (uM) (uM) (uM) (uM) (uM) (ul"f ) (us) 10 20 20 50 150 200 20 40 510 50 100 50 100 s0 100

N0] uptake 10 38 16 51 2 60 100 25 75 62 71 26 42 43 s0 58 74

N0, production 1039L7s62NdNd2676627030464652 60 76

78 0 upLake 0 22 11 42 12 63 100 20 72 68 73 30 45 53 72 69 2

Nd - Not determined.

(¡ l\) (d-g) AND ATPASC (h'i) ON UPTAKE TABLE 7: EFFEcTS OF INHIBITORS OF ELECTRON TRANSPORT (a-c) OXIDATIVE PHOSPHORYLATION oF NO2- AND 02 AND THE PRODUCTION 0F N0ã BY I"IEMBRANE VESICLES.

The reaction mixture in a total volume of 5ml- contained 50mM Tris-Hc1, O.2M sucrose (pH 7.5) and 2-3ng vesicle protein. Experimental details as in Fig.2 and Table 2. " Inhibition

o l_ b c d e f. ö h Arnytal H0QN0 cccP 2,4-DBP 2,4-DNP PCP NBD Chloride DCCD (uM) (rnM) (uM) (uM) (uM) (ul'{) (ue) (ul'{) 20 0.5 1.0 50 1.0 10 20 50 100 50 100 50 10c 100 10

80 4I 46 NOJ uptake 24 36 10 5 62 76 85 100 27 43 51 85

40 45 N0, production 25 34 15 10 60 76 82 100 30 40 50 85 80

79 42 47 0 upuake 20 30 10 7 65 75 80 100 31 42 52 87 2

(¡ L^) 54

Sulfhydryl group inhibitors, NEM and pCMB which did not restrict N02 and 0, uptake and NO, production in washed ce1ls, inhibited these processes in spheroplasts and to a greater extent in membrane vesicles (Table 8) ' This would indicate that the bacterial ouLer membrane poses a barrier to the entry of NEM and pcMB into the cells. The inhibition indicates the involvement of -SH groups for 0, dePendent N0, utilization'

3.r.7 ATPase activitv in nenbrane vesicTes

1t L r a A Mg'+ dependent ATPase activity was located in the membrane vesicles- of-Ê Nitrobacter agi-7is. The time course of Pi production from ATP (ATP hydroly- vesicles isrpresenteci in The rate of ATP hydrolysis sis) by membrane IÌ*.t. lras approximately 4nmol min-i (mg protein)-'. Since the ATP hydrolysing Fl subunit of ATPase is located on the cyt,osol side of the cell membrane, and ATp is impermeable to membranes, the results indicate that the vesicles are ttinside outtt.

3.1.8 Effects of inhibitors on ATPase activitv in menbrane vesicTes

Unlike the ATpase of Nitrosomonas europaea (Bhandari and Nicholas, 1980) the uncouplers (CCCP, 2.4.DNP and oligomyci-n) dld not affect the ATPase activity in lVitrobac,ter agiTis (Table 9). However Nitrobacter ATPase was inhibited by known ATPase inhibitors. Thus at 10OUM, DCCD, the classical ATpase inhíbiror, resrricred rhe ATPase activity by 65% (Table 9). Sodium vanadare (10uM), diethylstilbestrol (2OUM) and NBD chloride (100u1'1) also inhibiued ATPase in rnembrane vesicles by 50, 45 and 9O7" respectively.

3.f.9 Effects of phosohoTioase A on nitrite oxidase and ATPase activities in nembrane ves cfes

Phosphalipase A, results in delipidation of membranes by breaking phospholipids into lysophosphatides and fatÈy acids:

membranes (phospholipiOs)PL-A rl-4 (lysophosphat.ides + fatty acids) membranes lysophosphatides and fat.ty acids can be removed by washing the membranes with serum albumin solution: 55

TABLE 8: BFFECTS OF NEM AND PCMB ON THE UPTAKE 0F N0? AND 0? AND THE PRODUCTION OF NOã BY SPHBROPLASTS AND MEMBRÃNE VESICLES.

Both NEl"l and pCMB were dissçlved in 957, (v/v) ethanol. Experirnental details as in Fíg.Z and Table 2.

% Inhibition

NEM (mla) pCMB (ml"f) 0.5 1.0 0.5 1.0

NO, upt.ake 0 16 26 39

Spheroplasts NOl nroduction 11 13 24 31 0, uptake 7 I4 20 36

NOl uptake 6s 81 32 72

Membrane N0, Production 58 80 30 7T vesicles 0, uptake 69 72 24 70 s6

FIG.7: ATPase ACTIVITY IN l"lEl'{BRANE VESICLES.

Membrane vesicles were prepared as described in Section 2.2.2. The reacÈion mixture in a total volume of I m1 contained 1.7 mg vesicle protein, 30 umol Tris-HCl (pH 7.3), 1.8 umol ATP (pH 7.3) and 3 Umol MgSOo. Incubation hlas at 30oC in a waterbath shaker. At the'indicated times the reaction was terminated by the addition of 0.5 ml 10% (w/v) TCA and then centrifuging at 10,000 x g for 15 min. Pi released from ATP was then determined in the supernatant fraction as described in Section 2.2.L7 ./+.

EFFECTS 0F PHOSPHOLIPASE-A? TREATMENT 0N ATPase AND N0" OXIDASE FIG.8 ¿ ACTIVITY IN MEMBRANE VBSICf,ES.

The reaction mixture in a total volume of I m1 contained 5 mg of vesicle protein, 25 pmol glycyl-glycine buffer (pH 8.9), 4 pmol CaCl), O.1 mg bovine serum albumin and 20 units of phospholipasë-Ar. Reaction mixture wiLhout phospholipase-A, was preincubateá for 5 min at room temperature (25'C) then pÉos- pholipase-4,, was added and incubation continued at 25"C in a waterbath sÉaker for the time period indicated. The reaction was terminaLed by adding 8 ml cold (4'C) I% (w/v) bovine serum albumin in glycyl-glycine buffer followed by cenÈrifugation at 144,000 g for 30 min. The pellet resuspended in cold bovine serum albumin solution and washed 4 times with the same solution. The washed pellet was resuspended in 0.25M sucrose and activities of ATPase and N0, oxidase were determined as described in Sections2.2.6.1 ãnd 2.2.L7.1 respectively. For the determination of lipid phosphate, aliquots of the reaction mixture were digested in 7O7" (v/v) perchloric acid and Pi deternined as described in Section 2.2.I7 .4.

NOI oxidase (o); ATPase (o); residual lipid phosphate (fl). ¿ o.4 FIG. 7

o.3

'çto É, oL if o.2 ct Er. o.l

o t5 30 45 60 incubation time (min)

FIG. 8

Lo {Jc, o 60 o I 40

o 20 40 60 incubation time (mirù 57

TABLE 9: EFFECTS OF SOII'IE INHIBITORS ON ATPASC ACTIVITY IN MEMBRANB VESICLES.

ATPase activity was determined as described in Section 2.2.6,I Reaction mixture in a total volume of funl contained 1mg vesicle protein in 50m14 Tris-HCl buffer (pH 7.3)' 1.Bpmol ATP and 3ml4 l{gt+. Incubation hlas at 30oC. Inorganic phosphate was determined as described in Section 2.2.17.4. In Ehe absence of inhibitors ATlase produced approx. 100 nmol Pi. (30 min)-l (rg protein)-I. Except for sodium vanadate which was dissolved in HrO, all ti¡e inhibitors were dissolved in 957" (v/v) ethanol.

Inhibitor Concentration (uM) % Inhibition

cccP 10 0

50 10

2,4.DNP 50 0 0ligomycin 50 0

DCCD 100 65

Sodium vanadate 10 50 Diethylstilbestrol 20 4s

NBD chloride 100 92 5B

serum albumin (lysophosphatides + fatty acids) membranes lipid deficient membranes + (lysophosphatides + fatty acids) serum albumin

This experiment was designed to determine the role of phospholipids and rnembrane conformaÈion in maintaining the activity of N0, oxidase and ATPase enzymes. The effects of phospholipase A, treatment on the activities of Èwo enzymes is shown in Fig.8. Phospholipase A, treatment of membrane vesicles resulted in loss of phospholipids from the membranes as shown by the release of Pi, and after 60 min incubation with the enzyme, membranes reEained only about IO% of. residual lipid phosphate (Fig.8). During this incubation time, membranes lost about 3Oi¿ of their NO, oxidising activity and 551^ of. their ATPase activity indicating a possible involvement of membrane sLructure in rnaintaining the activiLy of the two enzymes.

3.2 ASSIMILATION OF INORGANIC NITROGEN COMPOUNDS BY IVTTROBACTER AGTLIS

3.2.L Growth sËudies To study the effects of NHf on Èhe growth of Ni trobacter agiTis cultures hrere grown in a nitrite medium supplemented with various concentrations of NHOC1. The optimum concentration for growth was found to be 2mM (Fig.9). Exponent- ially growing culÈures of the bacterium (in 100m1 medium supplement.ed wih 2mM NH4CI) utilized 75 t 5mg NaNOrr(24h)-l as compared with cultures without NH4C1 which utilized 50 + 5mg (21+h) The lag period of growth, with or witout NH4C1, lras about 36h. After 6 days, cells from NHOC1 (2mM) supplemented cultures contained 0.55 t O.lmg protein ml-l culture as comparted with those wirhout NH4C1 (0.29 t 0.1mg) (Fig.10).

3.2.2 Inh iti NO oxidation b in washed ce77s Alfhough the growth experiment indicated that Nitrobacter can utilize small amounts of UUI (=2mM), high concentration resulted in an inhibition of grolrrh. From O, electrode traces of NOJ oxidation (Fig.11) it is clear thaL NHÏ inhibits NOj oxidation by washed cells of Nitrobacteî agiTis. The extent of inhibition increased wit.h the increasing levels of UHf when NOl concentration was kept constant (Fig.11a-c,e,f). Thus NHf at con cenErations equal ro rhose of NO, inhj.bited NO, oxidation by 2O7" (Fig.11b). The NH] inhibition could be reversed by increasing the N0, concentrations (Fig.lld, g) . s9

FIG .9: cRordrH 0F NTTROBACTER AGTLTS rN NH4C1 CONTATNTNG MEDTIJì,I.

Cells were grown in 250 m1 Erlenmeyer flasks containing 100 ml of culture medium (Section 2.2.I) and NH4CI at indicated con- centraLion. The sterile medium'was inoculated l,/ith 10 ml of an e:tponentially grohtn culture. Growth was monitored by following the rate of NO, utilization (Section 2.2.L7,1) which was found to be proportional to the increase in total cell nitrogen.

No NH4C1 (o); 0.1mM NHOCI (o); 0.5mM NHOC1 (û); 2ml'f NHOC1 (I).

FIG.10: CELL YIELDS FROM CULTURES OF NTTROBACTER AGILIS GROWN I,JITH VARIoUS CONCENTRATIoNS0F NH4C1.

Aft.er a 6 day incubation, cells grohrn with various concentrations of NHOC1, as described in Fig.9, were collected by centrifugation at 15,000 g for 15 min. Cell yields are expressed as rng protein _1 ml ' culture, determined by micro-biurer method (Section 2.2.I7.3). FIG. 9 200

60 E .= E (¡) E t20 Ect .;g

..H ! 80 .Ë c, çtt L 40

o 234 56 incubation time (d)

o.6 FlG. I O

o, E (,= =o E (o o.4 o T e .= c, o ct o.? It Le,

o 234 5 ruHå (mU)in the medh¡m 60

FIG.11: rNHrBrTrON 0F N02 oXIDATION rN r^rAsHED CELLS By NH4C1.

100U1 of washed'cells (10 mg wcr wt.) was added to a double walled glass vessel fitted with a Clark-type oxygen elecLrode, containing 0.25 mmol Tris-HC1 buffer (pH 7.8) in a final volume of 5 ml. The reaction mixture was maintained at 25"C by circulating hrater Lhrough the outer jacket of the vessel. Either KNO, or NHOC1 was added through a port in the lid of the vessel via a Hamilton microsyringe. The reaction mixt.ure r^¡as continuously stirred with a magnetic f1ea. The r.esponse of the electrode was recorded on a potentiometric recorder. The rates alongside the traces are in pmol 0, consumed min-l.

Additions (*): C, cel1s (100u1); N,KNO (umol); A, NH4C1 (¡rmol). , FrG. I I

7 æ

7 ro,N r0 I 20,4

(a) (b) (c) (d)

I Mht ro,A

roo,A t4

5,N

(e) (f) (s) 61

If the daLa from 0, traces are plotted as % j.nhlbition of N0, oxidation vs molar rario of UHI : nO, (Fig.12), it is clear that when NHf, concentration was increased keeping NO, concentration constant, the extent of inhibition of NOJ oxidation increased rapidly until UHI : NO, ratio was about 4 and increased + slowly thereafter. To ascertain whether NHi at iEs saturating concentration resulted in a complete inhibition of N0, oxidation activity, the data were plotted as double reciprocal plots of the fractional inhibition against the concentration of inhibitor (l,t/ecller et a7. 1976) (Fig.13). In this plot a complete inhibition at the saturating concentration of the modifier is i-ndica- t'ed when the curve intersects the Y axis aE a value of <1. Since the inter- cept for NHf was >1 (Fig.13), it only partially inhibited the oxidation of No;.

15N 3.2.3 Incorporation of 7ub"77ed conpounds into cefL nitrogen l5NHroH 15NHoc1 The incorporarion of trlst'to' Nul5NOr, und inro washed cells o1. Nitrobacter agilis was studied as described in Section 2.2.4 All these compounds were readily incorporated Ínto washed cel1s (Tab1e 10). The incorporation of tt*t,* was approximately 80 fold greater than that of 15UOj. tt*tl 15t't0r- The extenÈ of incorporation rn¡as in the order ,lsnurou r tlslror-. 15N.o*pounds Time course for incorporation of (Fig.14) indicated that the further incorporation of tt*tl und l5NH,OH ceased after about 4h of incubation, indicating that either ATP or NAD(P)H or both become rate-limiting. hlhen un- l5N labelled NO] was included as an energy source, in addition to substrates, 15u0, 1SNH,OH 15un[ the extent of incorporation or , ot was significantly increased (Table 10).

15N lub"1led compounds were incorporated far less rapidty into extracts of lYjErobacter than into washed ce1ls (Table 11), perhaps because disruption of the cells may result in loss of some components of the protein synthe- sizing system. The inclusion of NADH or ATP increased the incorporation of ttto; into protein but not that or lsrunf.

3.2.4 Enz of assiniTati

Enzymes of lifff assimilati:.on viz glutamine synthetase, glutamate synthase and glutamate dehydrogenase were detected in cell extracts of Nitrobacter agi7is, All the three enzynes were located in the cytosal fraction 62

FIG.12: COMPETITM INHIBITION 0F Not OXIDATIoN BY NH4C1.

The data were taken from experimenLs similar to those described in Fig.11.

FIG.13: DOUBLB RECIPROCAL PLOT 0F INHIBITION 0F NO OXIDATION AT 2 VARIoUS CoNCENTRATIoNS 0F NH4C1.

Rates of O, uptake byuashed cells were determined as described in Fig.ll. 1/I is the reciprocal of NHOC1 concentration and L/í, reciprocal of fractional inhibition of NO, oxidation by NH4C1. FlG. | 2

60 t---a c) lÉ (Ît 50 ô- = c\¡ 40 o (Fo 30 c, .9 e-Ë 20 .= I ro

o 2 4 6 I lO 20 molar rat¡o NH å/NOã )

FlG. I 3 4

1/i 2

o 2 3 1/l (mM NHäc0- ' 63

15I.I-T,RBNI,LEDA TABLE 10: INCORPORATION OF CO},IPOUNDS INTO IVASI]ED CBLLS.

!'Jashed cells (25mg protein), suspended in 6m1 0.lM phosphate buffer (pH 7.5).supplemented with 1.5mM KHCO" and lmg equivalent of rrN substrate were incubated ut 30"C for 2h in a 50m1 Erlenmeyer flask. l{here indicated, the reaction mixture also contained 10mM NaN02. The reaction was stopped by adding 30mI cold distilled water and samples for mass spectrometry were prepared as described in Section 2.2.4, Results are the mean values from three experiments from the same batch of cells, t S.E.M.

15N Substrate ir,.o.porated Iue (re pòtetn)-11

15 Na NO o.20 + o.o2 2 1 5¡¡o K 0.10 + o.o2 3 5tto K1 , + NaNO, 0.60 + o.o2 tt*t, OH 4.00 + 0.50 tt*t, 0H + NaNO 5.20 + 0. s0 2 l5tttt C1 8.30 t 0.80 4 15t¡tt Cl + NaNO 14.50 + 0.80 4 2

15N alnitial enrichment of the .ornpounds was as follows: lsllHocr (30 arom % excess), Nul5NO, und K15No3 (born l5ttHrOH 32.5 atom % excess) and (97 atom % excess). 64

15 tttoã, 15t'tttroH FIG. 14: T]ME COURSE FOR INCORPORATION OF NO2t AND tt*rl RESPECTIVELY INTO !'/ASHED CELLS.

The reaction mixture $/as as described in Table 10. At the indicaLed times the reaction was stopped by adding 30 m1 cold distilled water Eo the reaction mixture and sampl"" fot 15N analysis h¡ere prepared as described in Section 2.2.4. lsr'ruocr qo¡; l5nnron 1.¡, 15llo, {n); 15r,ro, {r). FlG. 14

o h 'õ +' Lo êL I g' E '9 +tQ' 6 G' o! CLL o 4 .= E çr)a. 2 O

o 24 6 incubation time (h) 6s

15N TABLE 11: INCORPORATTON OT' -LABELLED COMPOUNDS INTO PROTE]N OF CELL EXTRACTS (S5).

Samples (6m1) of celle xtracts (4rng protein m1-1) in O.lM phosphate buffer pH 7.8 * supplemenred wirh KHC03 (1.5mM) and 1mg equivalent o fl JN substrate, \.{ere incubated for 2h at 30"C in 50ml Erlenmeyer flasks. ATP or NADII was added as indicated and ATP was regenerated by adding creatine phosphate (100rng) and creatine kinase (50 Ug). The reaction h¡as stopped by adding 5m1 10% (w/v) trichloro acetic acid and after standing ol-ernight the contents \^/ere centrif uged and the protein pellet was washed ggce with Io% (w/u) t.i.hloro àceti. uãid. Samples for 15N enrichment analysis were prepared and analysed as described in Section 2.2.4. Results are the mean values from three determinations with S, from the same batch of cells, t S.E.M.

15N Substrate in.o.porated, lug (rg prãt"in¡-11 tt*ot 0.10 + o.o2 tt*o; + 0.5mM NADH o.L2 + o.o2 15ru0: + 1.0mM NADH o.24 + o.o2 tt*oí + 1.0mM ATP 0. 16 t 0.02 tt*oi + 1.0mM ATP + 1.0mM NADH o.27 + 0.03 ttrtl 0.49 + o,o2 ttrnl + 0.5mM NADH o.46 + o.o2 lsl¡ul + 1.0mM NADH o.47 + 0.03 tt*rÏ + 1.0mM ATP o.49 + 0.03 ttrrÏ + 1.0mM NADH + 1.0mM ATP 0.50 + o.o2 66

of the bacterium (Table 12). Glutamine synthetase (GS) required Mn2* fot its transferase activity and Mg2+ for biosynthetic activity. Glutamate synthase (GOGAT) was NADH dependent, while glutamate dehydrogenase (GDH) required either NADH or NADPH for its amination reaction, but the latter was more effective. Since the specific actj-vity of these enzymes may vary from one batch of cells to another, for experiments described in Table 12 two bottles of medium with and without 2mM NHOCI were inoculated wiÈh a single culture (divided into two equal volumes) and incubated for the same length of time under similar incubation conditions. The experiment was repeated hrith different batches of cel1s; although the absolute enzyme activities varied, the overall petEern h¡as as shrwn in Table 12. Glutamate synthase (G0GAT) rvas not detected in ce11s grown in a medium supplemented with 2mM NH4C1. However the GS activity rvas unaffectea Uy UH| and there was a substantial increase in GDH act.ivÍty compared with cells gro\{n with NOJ alone (Table 12).

3.2-5 Effects of L-methionine-DL-suTphoxinine (MSX) and azaserine on the activities of GS, GOGAT and GDH

MSX and azaserine are inhibitors of GS and GOGAT respectively (Stewart et a7., 1981). The effects of the two inhibitors on GS, GOGAT and GDH are shown in Fig.15. Neither llSX nor azaserine inhibited GDH but GS activíy was depressed by abouL 95Z^ in cells incubated with 250UM-MSX for 3h. Higher concentrations of MSX (up to lmM) inhibited GS completely. The preincubation of cells with 200pM azaserine for 2h or lmM azaserine for 30 min completly inactivated GOGAT. This indicates that both MSX and azaserine were taken up by the cells. In control experiment.s with untreated cells (Fig.15), the activities of GS, GOGAT and GDH remained constant over the period of incubation indicating thaL there l/as no apparent enzyme synthesis during preincubation. This would also suggest that MSX and azaserine inhibited the activit.ies of pre-existing GS and GOGAT respectively rather than repressing Èheir synthesis.

3.2.6 Effects of MSX and az 1ne t of ce77s on the inc ration of and IN o ce77 nit

Cel1s pretreated with MSX (250UM) or azaserine (200uM) or both were.used for 15N incorporation studies described in Table 10. The results in Table 13 indicate that there r¡ras no effect of preincubation of cells wíth l5nHf either MSX or azaserine or both together on the incorporation of tna 67

TABLE 12: SPECIF]C ACTIVITIES OF GLUTAMINE SYNTHETASE (GS), GLUTAMATE SYNTHASE (GOGAT) AND GLUTAMATE DEHYDROGENASB (GDH) IN CELL EXTRACTS (Sr+a). Cell extracts (5144) tere prepared by centrifuging crude S. fraction at I44,0009 for th at 4"C. Enzyme activities for" GS, GOGAT and GDH l{ere assayed in supernatant (5144) as described in Section 2.2.6. Results are the mean values from 5 deterrninations with S144 frorn the same baLch of cells t SEI'{.

Specific activitv Growth condition csa cocATb cDHc

NADPH NADH

Basal medium without NH4C1 12116 .0 810.5 17 .5t1 .5 6.0f1 .0

Basal medium with 2mM NH4C1 11015.0 0r0.5 38.5r1 .5 12 .511 .0

1 I anmol y glutamyl hydroxamate produced min (mg protein)- t bnmol NADH oxidised min (qtg protein)-r 1 cnmol NAD(P)H oxidised *ir,-r (mg protein) 6B

FIG.15: EFFECTS OF MSX AND AZASERINE ON GLUTAMINE SYNTHETÂSE, GLUTAMATE SYNTHASE AND GLUTAI{ATE DEHYDROGBNASE.

hlashed ce11s (1g wet weight in 10ml sodium phosphat.e buffer, pH 7.8) were incubated with 25OuM-l"fsx and 2o0uM azaserine at 30"c in a reciprocating water bath. At times indicated, ce1ls were harvested by centrifugation (15,0009 for 15 min), washed $rith buffer and disrupted by sonication as described in Section 2.2.3. The cell extract was centrifuged at 144,0009 and the supernatant was used for enzyme assays as described in Section 2.2.6 . Glutamine synthetase in treated (r) and untreated (tr) cells; glutamate synthase in treated (O) and untreated (Q) cells; glutamaÈe dehydrogenase in treated (e) and unÈreared (o) cells. nmol f GH formed min-r(mg protein)-r (¡ o o o o o

f\) (D=' =\'/ (^l

(¡ OI -Ì1 õ o (tl o a oì nmol NADG)H oxidised min - r(mg protein)-l 69

15 tt*rT TABLE 13: INCORPORATION OF NO, and rNTO cELLS IRETREATED r^/rrH I',ISX AND AZASERINE.

A suspension of washed cells (1g wet wt. in 10m1, 0.1M phos- phaÈe buffer, pH 7.8) was incubated at.30"C rnrith either 200¡r M azaserine or 250pM-MSX or both. Treated cells-yere harveste d by centrifugation at 10,0009 for 15 min. for l5ttttf, incorporation experimenÇg, reaction mixture \¡/as as described in Table 10. For ttl¡Ot incorporation studi-es, cells (25mg protein) hrere suspended in 75m1 culture meÇium (Section 2,2.I) in 12f m1r Erlenmeyer flasks and unlabelled t'NOt was replaced with Nar ,N02 (32.5 atom % excess). The incubaÈion was continued for 6h at 30oC in a water bath shaker. The cellg-collected by centrifugation were prepared and analysed fot 15N enrichrnent as described in Section 2.2.4. Results are the mean values of three determinati-ons with the same batch of cells I SEM.

l5N irr.orporate d Additions to cells IUe (*g protein)- 1 tt*o; 3.40 r 0.20 tt*o; Azaserine (200uM) + 3.00 r 0.20 lsllo, MSX (25ouu) + 2.80 r 0.20 15WO; Azaserine (20OuM) + MSX (250uM) + 3.10 r 0.30

It*nl 8.90 r 0.50 I Azaserine (200uM) + t*rl 8.80 r 0.40 15xn[ MSX (25ouu) + 9.00 r 0.20 l5uuf Azaserine (2OOuM) + MSX (25OuM) * 8.50 r 0.30 70

lsnoz respectively. This would mean that GS-GOGAT pathway is not the sole NHI AS similating system in the bacterium.

3.3 PURIFICATION, PROPERTIES AND REGULATION OF GLUTAMINE SYNTHETASE (GS) FROM NI?ROBACTER AGILIS AND NTTROSOMONAS EUROPAEA

3.3. 1 Purification of GS

The purification procedures for GS from Nitrobacter agiTís and Ni¿rosononas europaea are summarized in Tables 14 znd I5 respectively. The purified ênzymes fr,om lilitr.obacter agiTis and ilitrosononas europaea had specific activities of. 22O and 4.26umo1 yGH produced min-l (mg protein)-1 respectively. The details of purification procedures are described in Section 2.2.5.1. Nitrobacter enzyme was purified by Blue-Sepharose CL-68 affinity chromatography since this column binds enzymes which require nucleotides as cofactors. InteresLingly, Nitrosomonas enzyme did not bind to B1ue-Sepharose CL-68 so thaL more conven- tional Eechniques v{ere used for its purification. The elution profiles for Nitrobacter enzyme from Blue-sepharose CL-68 column and Sepharose-4B column are shown in Fig.16 and 17. Nitrosononas europaea grown with NHf had very little GS. Heat treatment of crude extracts at 65"C for 10 min resulted in the precipitaÈion of about 2O7" of the total proEein without any loss of GS activity. The pH precipitation step was essentially as described by Bhandari and Nicholas (1981) which resulted in 70 fold purified enzyme. Polyethylene glycol precipitaEion step was carried ouE as described by Stericher and Tyler (1980) and resulted in 710 fold purification. Purified GS from both Nitrobacter agiTis and iVi trosononas europaea appeared as one single protein band in polyacrylamide gels under non-denaturing electrophoretic conditions (Fig. 18 and 19 respectively).

3.3.2 Properties of GS Since the kinetic properEies of the enzyme from Nitrosononas europaea have been extensively studied by Bhandari and Nicholas (1981) this section will largely deal wit.h Ehe properties of the Nitrobacter enzyme.

3.3.2.I Molecular weight Under denaturing conditions in SDS-polyacrylamide electrophoresis the purified enzyme from Nitrobacter agilis moved as a single protein band 7T

TABLE 14: PURIFICATION OF GS FROI'4 NITROBACTER AGILIS.

All purification steps, except for heat treatment, v/ere performed at 4oC as described in Section 2.2.5J. Enzyme activity was determined by following the production of .¡ GH from L-glutamine and NH20H at pH 7.2 as described in Section 2. 2 .6.2. One unit is defined as Umol y GH produced min-I Specif activity is defined as nunber of units (mg proteins)- ]c

ø Purification Total Total Specific Fold /o sLep Protein units activity purifi- Recovery ('e) cation

Crude extract (S 0.s1 1.0 100 30 ) 148.56 7s.81 Heat treatment 100.81 69.92 0.69 1.4 94 Pooled Blue Sepharose CL-68 2.ro 45.33 2r.59 42.3 60 fractions

Pooled Sepharose-48 o.20 44.O 220.0 431 .0 58 fractions 72

TABLE 15: PURIF]CATION OF GS FROI"I NITROSOMONAS EI]ROPAEA. All purification steps were performed as described in Section 2,2.5.I. Enzyme activity was determined by following the production of '¡ GH from L-glutamine and NH20H al pH 7.2 (Section 2.2.ç.2). One enzyme unit is defined as pmol yGH produced gin-r and specific activity as nurnberof units (mg protein)-r.

Purífication Total Total Specific Fold /o step Protein units activity purifi- Recovery ('e) tion

Crude extract (SSO) s00.0 83.6 0.006 1.0 100

Heat treatment 399.0 9r.2 0.008 1.3 109 pH precipitation 12.50 156.4 o.420 70.0 187

PEG precipitation 0.20 25.6 4.26 710.0 31 73

FIG.16: ELUTION PROFILE FOR GS OF JVTTROBACTER AGILIS FROM BLUE SEPHAROSE CL-68 COLI]MN.

Heat treated cel1 extract. (section 2.2.5.1) was passed through a Blue sepharose cL-68 column (1.5 x 9 cm) pre-equiribrared vrith 10mM Tris-HCl, lml"f MnCl, buffer (pH 7.2)., The column was then washed hrith buffer (flow rate 50m1.h-t) until the absorbance (Ars¡) was close Eo zero. The enzyme (GS) was el uted from tñë'column with 2mM ADP in the sarne buffer. Enzyme activity was determined by following the production of Y GH from glutamine and NH2OH as described in Section 2.2.6,2. AZ'O (o); enzyme activiry (o).

FIG. 17 : ELUTION PROFILB FOR GS OF NITROBACTER AGILTS FRO}{ SEPHAROSE_ 48 COLUI'IN.

Pooled Blue-sepharose cL-68 fractions (section 2.2.5.1) were dialysed against 10mM Tris-HCl, ImM MnCl, buffer (pH 7 .2), concentrated on an Amicon PM-1 r membrane'filter and loaded onto a sepharose-4B column (2 x Tocn) pre-equilibrated with 10mM Tris-Hcl, lmM Mncl, buffer (pH 7.2)rThe enzyme was eluted with the same buffer (flows rate 12m1.h-'). Transferase activity was determined as described in Section 2.2.6.2 lrr' (o) i enzyme activiry (o). Arao Azeo o o o o o b o o o b ooo- o N cd s bb,bfÞ o) o o

o I' ++ a{ äo D õ' c) = ôa+ c= = e3 =lÞ r¡ô 5o o) -vo o I

Þ ..'-a-- +-l t- ! ta t (D (, o t o

'rl o f\) (.rl I õ T 6) I GH formed min- rml- | fraction : lmolË f o) s o o N (¡ à ynol lL GH formed min-¡ml-rfraction 7l+

FIG.18: POLYACRYLAMIDE GEL ELECTROPHORESIS OF PURIFIED GS FROM NITROBACTER AGILIS.

pAGE was carried out in 77" (w/v) gels as described in Section 2.2.10. Gel A was sLained for enzyme activity and gel B for prorein by coomassie blue method (section 2.2.10).

GS, glutamine syntheLase; BPB, bromophenol blue'

PURIFIED GS FROM FIG. 19: POLYACRYLAMIDE GEL ELECTRPHORESIS OF N ITROSOMON AS EUROP AEA.

PAGE was carried out in 57" (w/v) gels as described in Section 2.2.LO. ProEein band was stained using silver staining method (Section 2'2'IO)' GS, glutamine synthetase; BPB, bromophenol blue' FlG. å8

k <__GS

BPB

FIG. I 9 r-E-i H:È- l{ [-Í ¡i; - a. i' lr.fI ,i i' '- ;l ie .j þlf<- os

ê- gPe 75

(Fig.20). The molecular weight of enzyme subunit'was estimated to be 58,000 using standard SDS-t.reated protein markers (Fig.21). Thp molecular weight of the native enzyme determined by gel filtration using a Sepharose 6-8 column (1.6 x 100cm) r'¡as esLimated to be 700,000 (Eig.22). The results indicate t.hat th-e enzyme molecule (700,000) is composecl of l2 homologous subuniEs of approximately 58,000 each.

3.3.2.2 Substrate requirements for enzyme activity

. Both r¡ glutamyl transferase and biosyntheLic activities were recorded for the Nitrobacter GS. The subsLrate requirements for transferase activity are shown in Table 16. The results indicate that the transferase activity required a divalent cation (Mn2+). LiÈEle or no activity was recorded when either Mn2*, glutamine, NH2OH of arsenate vras omitted from the reaction mixture. When ADP was omitted, 207" of the activity of the complete reaction mixture was recorded.

The results in Table 17 indicat.e that the biosynÈhetic activity of the enzyme also required a divalent cation (Vg2+), glutamate, NHOCI and ATP. No activity !,/as recorded when eiEher of these compounds was omitted from the reacEion mixÈure.

3.3.2.3 Effects of metal ions

The effects of various meLal ions on transferase and biosynthetic activity of purified glutamine synthetase from Ni trobacter agiTis are shown in Tables 18 and 19. Optimum transferase activity vras obtained by using either Mn2t o, Cu2* but"een 0.1 and I0mM final concentration. Higher concentrations of either of the meÈal ions resulted in a decrease in transferase acEivity (Table 18). The order of effectivness of the divalent cations ,u" Mn2! cu2t > ¡4g2* > l¡i2+. optimum .it 'co2t biosynthetic activity was recorded with Mg-- and the order of effective- ness was lrg2* , Mn2+ > zn2* > co2+ > cu2+ > Ni2+ > cu2* , Fu2+ (Tuble 19).

3.3.2.4 K for substrates of transferase and biosynthetic reactions m

The effects of various concentrations of substrates of l'ln2* dependent transferase activit.y and Mg2+ d"p"ndent biosynthetic activity 76

FIG.2O: SDS_PAGE OF PURIFIED GS FROM NITROBACTER AGILIS

SDS-PAGE of enzyme was carried out in 102 (w/v) polyacrylamide gels as described in Section 2.2.IO. Left. hand lane contai_n- ed 15pg purified GS (SDS treated) and the right hand lane approximately 150pg of a mixture of SDS-treated prot.ein markers. From Lop to bottom - phosphorylase b, albumin, ovalbumin, carbonic anhydrase, trypsin inhibitor and bromophenol blue.

FIG.21: DETERMINATION OF SUBUNIT MOLECULAR I^IEIGHT OF PURIFIED GS FROM NTTROBACTER AGILIS.

Subunit molecular weight of GS was determined in SDS-PAGE as shown in Fig.20 and described in Section 2.2.L0. K^_- was calculated from electrophoretic mobilities of stun¿ål¿ proteins.

A. phosphorylase b (94,000) B. albumin (67,000) c. ovalbumin (43,000) D. carbonic anhydrase (30,000) E. trypsin inhÍbitor (20,100) GS. glutamine synthetase. 1-

Ç Ë t ,;ei.i

B s{J .9(¡, Ì u o ()= ot 0 4.4 Ë (tt o

4.2 E

4.O o o.4 0.6 0.8 Kav 77

FTG.22: DETERMINATION OF NATTVE MOLECULAR WEIGITT OF PURIFIED GS FROM A/TTROBACTER AGILIS.

The molecular weight of the native enzyme was determined by ge1-filtration in a Sepharose-68 column (1.6 x 10Ocm) equilibriated with 5Omlvf Tris-HCl buffer (pH 7.5) as described in Section 2.2.8. The column r^ras calibrated wíth the following standard proteins:

A. aldolase (158,000) B. 'catalase (232,000) C. ferritin (440,000) .D. thyroglobulin (669,000) GS. glutamine synLhetase. FIG.2?

6.O

D 5.8 P .C .9 c Q' 5.6 =! (tt =go o 5.4 B e oq, 5.2 A

5.O 2 3 VelVo 7B

TABLE 16: SUBSTRATE REQUIREMENTS FOR THE Y GLUTAMYL TRANSFERASE REACTION OF PURIFIED GS FROM NTTROBACTER AGILIS.

The complete assay mixture in a final volume of lml contained purified enzyme (5ug protein), L-glutamine (30mM), NH20H.HCl (neutralized rvith 2M NâOH) (30mla), MnCl2.t¡H2O (0.5mM), sodium arsenate (20mM), ADP (0.4m1"1) and imidazole-HCl buffer (40mM)'sodium pH 7.2. Ìn other test-tubes Mn2i, L-glutamine, NH?OH, arsenate and ADP were omittecl in turn from the reaõtion mixture. Enzyme activity was measured after a 15 min incubation at 37oC as described in Section 2.2.6.2.

Reaction mixture Activity %

Complete 100 )t Ornit Mn -' 0

Ornit L-glutamine 0

Omit hydroxylamine 0

Omit sodium arsenaÈe 5

Omit ADP 20 79

TABLE 17: SUBSTRATE REQUIREMENTS FOR THE BIOSYNTHETIC ACTIVITY OF PURIFIED GS FROM NITROBACTER AGILIS.

The complete assay mixÈure in a final volume of 0.2m1 contained purified enzyme (5Ug p6gtein), L-glutamate (100mM), NH¿,C1 (50mM), ATP (10mM), Mgt* (5mt't) aqd imidazole-HCt buffei (5OmM, pH 7.0). In other test-tubes Mg'+, L-glutamate, NH4C1 and ATP were omitted in turn from the reaction mixture. The Pi produced after a 15 min incubation period at 37oC was determined as described in Section 2.2.I7.4.

Reaction mixture % Activity

Complete 100

omir Mg2+ 5

Ornit glutamate 0

Gnir NH4C1 0

Omit ATP 0 80

TABLE 18: EFFECTS OF VARIOUS DIVALENT CATIONS ON THE Y GLUTAI"ÍYL TRANS- FERASE ACT]VITY OF PURIFIED GS FROM JVITROBACTER AGILIS.

An aliquot of purified enzyme hras desalted by passing through a Sephadex G-10 column (2.5 x 20cm) which had been previously equilibrated with 50mll Tris-HCl buffer (pH 7.2). Desalred enzyme (5-1OUg) was used in thç reacEion mixture described in Section 2.2.6-.2 except that Mn2* was replaced by either of thç cations listed below. Results are expressed as % of the Mnz+ dependent t,ransferase activity.

% actíviLv at cati on concentration (mluf) Metal ion 10 20 50

2+ Mn 100 77 40 2+ Mg 23 40 61 2+ Ca 0 0 0 2+ Ni 30 2 0 2+ Zn 0 0 Nd 2+ Cu 100 84 Nd 2+ Fe 0 2 Nd 2+ Co 5 45 Nd

Nd - NoÈ determined. B1

TABLE 19: EFFECTS OF VARIOUS DIVALENT CATIONS ON THE BIOSYNTHETIC ACTIVITY OF PURIFIED GS FROM NT?"ROBACTER AGILIS.

Purified enzyme was desalted as described in Table 18. Bio- synthetic activity was,rdeteimined as described in Section 2-.2.6.2 except that Mgz+ rvas replaced by either of the cations listed below. Results uru as % of Vg2+ dependent biosynthetic activitY. "*pràssed

7" acLi-víl' of cation con ntration mM Metal ion 1

Mrr2* 73 73 79 rqg2* 95 100 75 c^2+ Nd 42 48

^2+Ì¡e Nd 30 30

co2+ Nd 58 74

Ni2+ Nd 52 55 2+ Zn Nd 72 55 2+ Cu Nd 30 74

Nd - NoE determined. 82

ü¡ere considered. The effects of gl.utamine concentrations over a range of 0-5OmM are illustrated in Fig.23a. The K,n value of 14.6 t 1.5 was calculated from the double reciprocal plot (Fig.23b) of glutamine and Y glutamyl-hydroxamate produced.

The effect.s of various concentrations of NHrOH (0-4OmM) on transferase activity are shown in Fig.24a. The enzyme activity increased up ro 20mM NH,OH. The K, value for NH,OH was 2.6 t 0.8 calculated from double reciprocal plot (Fig.24b), as described in Section 2.2,7,

The effects of various concentrations of L-glutamate (0-50mM) on Mg2+ áependent biosynthetic activity are shown in Fig,25a. The enzyme activity increased up to 40ml"l glut.amate. The double recipocal plot (Fig.25b) of glutamaLe concenÈration against Pi produced gave a Ko' value of 6.3 t 1.6mM. The effects of NH4C1 over a range of 0-5ml'f are shown in Fig.26a. The biosynthetic activity increased up to about 2mM NHOCI. The Ko' value for NHOCI was 0.2 ! O.lmM as calculated from double reciprocal plot (Fig.26b) .

A summary of general properties of purified GS from Nitrobacter agiTis is given in Table 20.

3.3.2.5 Heat stability and effects of denaturing agents

The purified GS from lVi trobacter agiTis remained active after incubating at 5OoC for 15 min. At 60oC, the enzyme was inactivated by 607" within 10 min and at 70oC it was completely inactivated. The addition of glut¿rnine (30ila) and Mn2* (3m1"1) Prot.ected the enzyme by about 2O7" aL 60oC, while NH2OH (3OmM) accelerated its deactivation. The effects of NH20H were further investigated on both biosynthetic and transferase activities of the enzyme. The enzyme was preincubated for various periods with 10mM NHrOH. The data indicate that both transferase and biosynthetic activities decreased; Ehus afLer 20 min preincubation with NH2OH, the enzyrne lost about 75% and 85% of its biosynthetic and transferase activities respecti-vely (Fíg.27). The incubation of purifíed enzyme with 4M urea caused about B5Z loss of transferase activity within 20 min. 83

FIG.23: THE EFFECTS OF VARIOUS CONCENTRATIONS OF L_GLUTAMINE ON y-GLUTAMYL TRANSFERASE ACTIVITY OF PURIFIED GS FROM NITROBACTER AGILIS.

Aliquots of purified enzyme were added to the reaction mixture (final volume 1-ml) containing L-glutamine (0-50mù1), NH2OH-HC1 (neutralized with 2N NaOH) (30rnM), l"fr,2* (3mM), sodium arsenate (20mM), ADP (0.4mM) and imidazole-HCl buffer, pH 7.2 (40mM). Control tubes without glutamine were included. YGH produced was determined as described irr Section 2.2.6.2.

FIG.24: THE EFFECTS 0F VARIOUS CONCENTRATIONS 0F NH?OH ON y GLUTAMYL TRANSFERASB ACTIVITY OF PURIFIED GS FROM NITROBACTER AGILIS.

Experimental details as in Fig.2$ except t.hat NH'OH was varied from 0-40mM at a fixed concentraÈion of glutamine (30mM). FlG.23 î 6) 3o-o o >o.3t¡J oÉ. LL (b) (,TO.2 to !- Eo.r t

o o S (mM GLUTAMINE ) 5

-o.2 -o.l o o.o5 0. I o.2 t/s

FlG.24

(a) .o Iz o= l¡l (b) =É. o o.5 LL (,t t\ õ E _L o ro zo 30 40 S (mM NH2OH )

-2 -l oo.5 I 2 t/s

I B4

FIG.25: THE EFFECTS 0F VARIOUS CONCENTRATIONS 0F L-GLUTAMATE 0N THE BIOSYNTHBTIC ACTIVITY OF PURIFIED GS FROI',Í NITROBACTER AGILTS.

Aliquots of enzyme \{ere added to the reacLion mixture (0.2m1) final volume) qçntaining L-glutamate (0-50 mM), NH/,CI (50mM) ' ATP (iOmM), Mg'- (5mM) and imidazole-HCl buffer pH-7.0 (50mM). L glutamate vías omiÈted fron the control tubes. Afcer a 30 min incubation iieriod at 37oC the Pi formed was determined a- described in Section 2,2.I7.4.

FIG.26: THE EFFECTS 0F VARTOUS CONCENTRATToNS 0F NH¿Cl 0N THE Bï0- SYNTHETIC ACTIVITY OF PURIFIED GS FROM N.TTROBACTER AGILIS.

Experimental details as in Fig.25, except that NH^CI was varied from 0-5mlf at fixed glutamate concentratiori (100mM). o (a) FlG. 25 = ô td oÉ. LL o E 2 (b) o E L a

o ro zo 30 40 50 S (mM GLUTAMATE )

-o.2 -o. I o o.o5 0 I o.2 j /s

FlG. 26 (a) o. I = â ul o (b) oÉ u- fL to g? o

o ?34 5 S (mM NH4CI) 4

-5 2 3 4 5 -2 t/s 85

TABLE 20: PROPERTIES OF PURIFIED GS FROM NTTROBACTER AGTLIS.

Molecular weight of native enzyme 700,000

Subunit molecular weight in SDS gels 58,000

Number of subunits t2

K for s.lutamine L4.6 ! 1.5 mM m Km¿ for NH^OH 2.6 ! 0.8 mM K, for glutamaLe 6.3 t 1.6 mM

Kn, for NHI O.2 ! 0.1 mM 86

FTG.27: THE COURSE FOR INACTIVATION BY NH,OH 0FYGLUTAMYL TRANSFERASE AND BIOSYNTHETIC ACTIVITIES OF PURIFIED GS FROM N-TTROBACTER AGILIS.

Purified enzyme was preincubated with 10mM NHrOH.HCl (neutralized with 2M NaOH) for various times (0-20 min). Biosynthetic and transferase activities were determined as described in Section 2.2.6.2 except that the reacLion Í:lxture for latter activity conËained 10mM NH?OH. The results are expressed as percent of activitie5 without preincubation wiÈh hydroxylamine. Transferase activity (O), Biosynthetic activity (O).

FIG.28: NUÏ TNHTSITION OF 1 GLUTAMYL TRANSFERASE ÆTIVITY OF PURIFIED CS4 PROI',I NITROBACTER AGILIS.

Transferase activiEy of the purified enzyme was determined as described in Sectíon 2.2.6.2 except that NHOCI (0-50mlf) was also included in the reaction mixture. too FtG.27

80 .{i .l P() G' 60 ct L P oc, 40 o þ c, I 20

o 5 to 15 20 incubation time (min)

FlG. 2g 50 (a)

40 (b) z. o 30 ts e T z_ 20 a b a o t/i 4 o to 20 30 40 50 NH4CI (mM )

-o. I o o.o5 0. I o.2 I /hM NHICl) B7

3.3.2.6 Inhibition of transferase activity by NHOC1

The inhibiEion of transferase activit.y bv NH*CI (0-50mM) is shown in Fig.28 ; maximurn inhibition (40%) was at 40mM NHOCI. The type of inhibition was further investigated. The inhibitory effects of NHOCI at various concentrations of gluEamine and NH2OH are shown in Fig.29 and 30 respectively. The kinetic daÈa for double reciprocal plots indicate that. NHOCI was a competitive inhibitor of glutamine (Fig.29) and a mixed type inhibitor of NHrOH (Fig.30). Ammonium chloride reduced the affinity of glutamin= and NH,OH for the enzyme resultÍng in increase in K- values (Table 21). m

3.3.3 RepuTation of GS

3.3.3.1 Feed back inhibition

Glutamine synthetase from Nitrosononas europaea is inhibited by a variety of feed back inhibitors including various amino acids and nucleotides (Bhandari and Nicholas, 1981). The purified enzyme from Nitrobacter agiTis was also inhibited by amino acids and nucleotides. Thus at lOmM final concentration, alamine' serine, glycine and try- tophan inhibited Eransferase acEiviÈy by 65r45,40 and 332 and biosynthetic activiEy by 60,30,35 and 5% respectively (Table 22). The enzyme activity was litcle affected by the other amino acids sÈudied (Table 22). The effects of various concentrations of alanine, glycine and serine, on transferase activity are illustrated in Fig.31a. The extent of inhibition by these amino acids increased with an increase ín concentration. The results in Fig.3lb are expressed as double reciprocal plot,s of the fractional inhibition (calculated as described by l^ledler et a7¡, I976) against the concent,rations of the inhibitor. The daÈa indicate that glycine, serine as well as alanine only partially inhibited Ehe enzyme activity, because the curves intersect. the Y axis at a value of >1. A complet.e inhibition at saturaEing concentraEions of inhÍbitor is indicat.ed when the ploc inÈersects the Y axis at a value of <1. BB

FIG.29z COI"IPETITIVE INTIIBITION BY NH¿Cl OF Y-GLUTAMYL TRANSFERASE ACTIVITY OF PURIFIED GS FROM '/VTTROBACTER AGILIS.

Experimental details as in Fig.23 except that NH4CI (0-30mM) was also included in the assay mixture No NHOC1 (c); 10mM NHOC1 (o); 20mM NHOCI (I); 30ml'1 NHOCr (!).

FIG.30: MIXED TYPE INHIBITION BY NH/ Cl OF y-GLUTA},IYL TRANSFERASE ACTIVITY OF PURIFIED GS FROM ilTÍROBACTER AGILIS.

Experimental details as in Fig.24 except that NH4C1 (0-30mM) was also included in the assay mixture.

No. NH*CI (o) ; 10m1"1 NH*CI (o) ; 20mM NHOC1 (l) ; 30mM NHOC1 (!). FlG. 29 h .t E 6 E' (¡t c) tt= Lo CL 4 (9T à{- o e =- 2 \

-o. I o o. I o.2 l/S (mM glutamine)-l

t5 FlG. 30

âI I L '=L t. orlo o = -,c, ! CL (9- { E 5 E =- \

-o.5 o o.5 l.o I I /S (mM NH zOHf 89

TABLE 21: EFFECT OF NHÏ ON K- OF THE SUBSTRATES OF Y GLUTA}4YL TRANS- FERASE ACTIVÎTY OPMPURTT-TED GS FROM NTTROBACTER AGILIS

Transferase activity of the enzyme was measured as described in Section 2.2.6.2 except ,that the amounts of glutamine and NH20H were varied between 0 to 50mM and NH4Cf was included in the assay mixture as indicated. Kn values were calculated frorn double reciprocal plots (Sectlon 2.2.7).

J- NH; K, (mM) concentration (mM) Glutamine NH20H

0 14.6 2.6

10 r8.2 4.O

20 23.8 5.1

30 27.8 5.6 90

TABLE 22: EFFECTS OF VARIOUS AMINO ACIDS ON Y GLUTAMYL TRANSFERASE AND BIOSYNTHETIC ACTIVITIES OF PURIFIED GS FROM iVTTROBACTER AGILIS.

Transferase and biosynthetic activities of GS were determined as described in Section 2.2.6.2 except that reaction mixture also contained 10mM of the appropriate amino acid as indicated,

% Inhibition Amino acids Transferase activity Biosynthetic activit.y

L-GlutamaLe 0 L-Glycine 40 36 L-Aspartate t4 0 L-Iiistidine 4 6 L-AsparagÍne 4 0 L-Arginine l4 6 L-Leucine 0 0 L-Tyrosine 0 0 L-Serine 4s 30 L-Valine 0 72 L-Proline 0 o L-Lysine 10 L2 L-Tryptophan 33 0 L-Phenylalanine 0 0 L-Isoleucine 0 0 L-Alanine 65 60 91

FIG.31: INHIBITION OF Y-GLUTAMYL TRANSFERASE ACTIVITY OF PURIFIED GS FROM NTTROBACI'ER AGILIS BY VARIOUS CONCENTRATIONS OF ALANTNE (¡), GLYCTNB (o) AND SERTNE (o).

Transferase activity of the purified enzyme hras deLermined as described in Section 2.2.6.2 except that the reaction mixture also contained either alanine, glycine or serine (0-10mM). In Fig.31b, results of Fig.31a are plorred as double reciprocal plots of inhibitor (amino acid) concentration (I) against fractional inhibition (i). FlG. 3l

(a) 50

40 D

c o 30 ¿r- ! L F 20

o 2 468 to amino acid (mM)

7 (b)

1/, 4

o o.l o.2 0.3 o.4 0.5 1/t 92

The conrbined effects of various amino acids on Lhe Lransferase activity are shown in Table 23. The inhibition due to various combina- Eions of amino acids was lower than the sum of the inhibitions resulting from an individual amino acid. Thus the inhibitory effects due to alanine and glycine v/ere 40 and 277" respectively, but their combined inhibitory effect was 52% instead of. 67% (40 + 27%). The effects due to alanine, glycine and serine alone were 40 ,27 and 36% respectively but their observed combined inhibitory effect was only 692 instead of 103, the sum of the effects of the individual amino acids a1one, ie. 40 + 27 + 367". The values for the cumulative inhibitions were then calculated as dgscribed in Section 2.2.9. For all the pairs of amino acids studied, the observed inhibitions were j-n close agreernent with the values calculated for their cumulative inhibitory effects. Thus the combined inhibitory effect of. 52% for the pair of alanine and glycine was close Lo 56% calculated for cumulative effect, whereas the additive effect was 677".

The effects of various mono-, di- and triphosphate nucleotides on transferase activity was examined. The effects of various nucleotides presented in Table 24 i,ndícate that the enzyme was inhibited to a greater extent by di- and tri-phosphates nucleotides (except GDP and GTP) than by their mono-phosphate nucleotides (ecept AMP). Thus IDP, CDP and UDP inhibited the enzyme by 45,50 and 53% respectively and the inhibitory effects of ITP, ffP, TTP and ATP were 63,55,57 and 56% respectively. AMP restricted the enzyme activity by 657" but other mono-phosphate nucleotides viz. IMP, CMP, TW and UllP had liCtle or no effect.

3.3.3.2 Adenylylation and deadenylylation of GS.

In enteric bacteria (Stadtman et a7 ., 1970), photosynthetic bacteria (Johansson and GesL, 1977) and rhizobia (Darrow and Knotts, Ig77) Ehe extent of transferase actj-vity in the presence of 60mM MgCf, has been used as an indication of the degree of adenylylation of GS. The fully adenylylated enzyme is inactive in the presence of Mg2+ whereas the deaclenylylatecl one is not affected. Since the adenylylation state of the enzyme can change during harvesting of the bacteria (Bender et aj . t 1977) the addition of cet.yl trimelhyl ammoniun bromide (CTAB) to cultures stabilizes the adenylylation state of the enzyme' 93

TABLE 23: COMBINED EFFBCTS OF AMINO ACIDS ON Y GLUTAMYL TRANSFERASE ACTIVITY OF PURIFIED GS FROM N.TTROBACTER AGILIS. Glutamine synthetase activity htas determined as described in Table 22 and Section 2.2,6.2 except that each amino acid was at 5mM. Values for cumulative inhibiti-on lrere calculated as described in Section 2.2.9.

% Inhibition Inhibitor 0bserved Cumulative Additive

Ala 40

Glv 27

Ser 36

Ala + Gly 52 56 67

Ala + Ser 62 62 76

Ser + Gly 52 53 63 Ala+Gly+Ser 69 72 103 94

TABLE 24: EFFECTS OF NUCLEOTIDES ON Y GIUTAMYL TRANSFERASE ACTIVITY OF PURIFIBD GS FROM NITROBACTER AGILTS. Transferase activity ktas determined as described in Section 2.2.6.2 except thal Lhe ¡sss¡ionmixture also contained 20mù1 of the appropriate ¡ucleotide as indicated.

Nucleotides Z Inhibition

IDP 45

ITP 63

CDP 50

CTP 55

TTP 57

UDP 53

AMP 65

ATP s6 9s

The results shown in Table 25 indicate that the enzyme from normally grown Nitrobacter cells was severely inhibited by ¡lg2-*. The CTAB treatmenL of Ni trobacter cel1s prior to harvest had little effect on the extent of Mg2+ inhibition of transferase activity. Similar results were observed when cells harvested with or without CTAB were assayed for 1¡ vivo Eransferase activiEy. Based on a 12 subunit enzyme and applying the Shapiro.& Stadtman (t970U) formula (En = 12- tZ,*rtg27-Ug2*), an adenylylation state of 9 can be calculated for the Nitrobacter enzyme, and it varied beEween B and 11 in twenty separately grohrn batches of cells.

In Nitrosomonas europaea, for cultures harvested without added CTAB, an adenylylation state of 9 was recorded (Table 25), however when the cells were har-¡ested with CTAB (2.5Ug rf-l) the value decreased to 4. Í'lhen cel1s Brown at a 1ow ammonia concentration (20mM) were harvested after all the ruHf in the medium had been util lzed, Èhose' harvested with and without CTAB gave adenylylation states of I and -5 respectively. Although the Mg2+ effect on transferase acLivity (Table 25) nay be related to the adenylylat.ion state of the enzyme in Nitrosononas, the Shapiro & StadEman formula does not apPear to apply to crude extracts. If CTAB serves the same function as reported 6r other bacteria (Bender et a1., L9771 Johansson & Gest, L9771 Michalski et a7., 1983), its addition to cultures prior to harvest should prevent the adenylylation of the Nitrosononas enzyme. The Mgz+ effect on GS in crude preparations of Nitrosononas europaea varied from batch to batch and was dependent on the incubation period of the extracts. Freshly prepared extracE gave results shown in Table 25 but after a few hours Íncubation, either at room temperature or at 4oC, the Lrans- ferase activity of the enzyme in extracEs from cel1s harvested without CTAB was stimulated Uy t''tg2+.

Nitrosononas europaea is usually grown with high concentraEions of ammonia (about. 120mM) (see Bhandari & Nicholas, L979a) so t.hat at the end of exponential phase, the residual HHf is about 4Om!1. Thus in these cells, glutamine synthetase would be highly adenylylated. The results of an experiment in which cells were grown with 2OmM NHf, harvested in the presence of CTAB and the effect of Mg2+ on the transferase act.ivity of crude preparations deEermined are shown in Fig.32. 96

TABLE 25: EFFECTS O}- CTAB TREATMENT ON Y-GLUTAMYL TRANSFERASE ACTIVITY IN CELL EXTRACTS (Szs) FROM N/TROBACTER AGTLTS AND ivr7Ro- SOMONAS EUROPAEA.

Exponentially growing cultures (1 L) of Nitrobacter agiTis and Ni trosomonas europaea h/ere harvested in polycarbonate bottles (250m1 volume) in Sorvall GSA rotor ãt l0,OOOg for 20 min at {"C. As indicared, CTAB was added to cultuies (2.5Ug r1-1.) before harvesiing. Cells washed once with cold 10mM Tris-HCl, lml'f MnC12 buffer (pH 7.2) and finally suspended in a smal1 volume (approx. 5rn1) of the buffer. Cell suspen- sj-ons were sonicated Lo1 20 min and then centrifuged at 25,0009 for 15 min. The supernarant (SZS) was used ro determine the transferase activity with-ánd without 60mM MgC12, as described in Section 2.2.6.2. Jransferase activity iã ¿ãfi-ned as umol yGH produced (30 min)-l (rg prorein)-l.

CTAB Transferase activitv +14g2+ Bacterium /-Mg2+ treatment 2+ 2+ -Mg +60mM Mg Ratio

Nitrobacter agiTis

growing on 40mM N0, 10.50 2.ro o.20

+ 9.30 1.s0 0. 16

Nitrosononas europaea

growing on 3OmM NHf, 0.16 0.04 0.25

+ 0.16 0.11 o.69

NHf depleted cultures 0" 13 0.04 0.31

+ 0.08 0. 11 1 .38 97

FIG.32 : EFFECTS OF NHÏ CONCENTRATION IN CULTURE MEDIUI"I ON "¡_GLUTAMYL TRANSFERASB AôTIVITY OF GS IN CRUDE EXTRACTS OF NT?ROSOMONAS EUROPAEA AT VARIOUS STAGES OF GROWTH.

Cultures h¡ere grokrn in 10 4 batches (2 4 inoculum from a culture completely depleted of NHI) at 2B"C with vigorous aeration. Aliquots. (0.5-1 ¿) dispensed aseptically at the times indicated were harvested in polycarbonate tubes (250m1 volume) in Sorvall-GsA rotor at 10,0009 lor 20 min at 4"C in Ehe presence of CTAB (2.S8 mt-l). Cel1 exÈracts were prepared as described in Section 2.2.3. Transferase activit.y was determined with or without 60mM MgCl, at pH 7.4. Growth of bacterium was monitored by determining cell protein m1-1 culture and was found to be directly proportional to the raEe of lrlnl disappearance from the medium. Aliquots (1-2m1) were 4 withdrawn aseptically from growing cultures, centrifuged at 15r000g for 10 min to remove cells and supernatant used to deËermine NHf as described in Section 2.2.17.2. NHf utilization (O); gluÈamine synthetase specific acriviry (o) ; +tlgz+ /-Vg2+ rario (n) . FlG. 32 t2

I c. 20 2.O o '¡D o ê-L e trt, 1.6 E E r6 (t I + L ï €r¡, C\¡ .EL E ! E 2 1.2 6 Q' E =o + o ctt U C\I .= I o,8 4 I (,o (9 oc, lr\ +\tI E 4 o.4 2 E z L

o o o

o 20 40 60 -o incubation time (h) 9B

The medium (8¿) was ínoculated with 2l of a culture which had been + depleted of NHI (the ratio of transferase activity wiEh and v¿ithout 1.6). The oxidation of ammonia started immediately and after Mg2+ "u" 55h all rhe NHf was utilized. The specific activity of GS decreased from about 0.011 umol min-' (mg proEein) ' aL 15h to 0.004 umol *in-l _1 (mg protein) - at 40h and then increased thereafter. The +l'fg2+ /-vg2+ ratio increased from 0.1 at 15h to about 2.O aL 4Bh when the cultures that Mg2+ re- contained only 1mM residual ammonia indicating "ff"ct flected the adenylylation state of the enzyme.

The effects of snake venom phosphodiesterase treatment on transferase activity of GS with and without 60mM l{gCl2t arê. shown in Fig.33. In these experiments transferase assays hlere carried ouE at pH 7.4 in 1t the presence of 0.3mì,f MnClr. The Mg"- inhibition of transferase activity of purified enzyme from lVi trobacter agiTis was completely reversed after a 30 min treatment with snake venom phosphodiesterase (Fig.33a), however phosphodiesterase had no effect on Mgz+inhibited transferase activity of Nitrosononas enzyme (Fig.33b). Moreover, longer incubation times (up t.o 4h) and higher phosphodiesterase concentrations (up to -t )+ 2O0Ug nl-t) did not affect the extent of Mg-' inhibition.

The adenylylated and deadenylyted forms of GS have different pH optima for transferase activity; thus t.he adenyl ylated form has a lower pH optimum (Bender et a7., 1977). By treating purified enzyme from Nitrobacter with phosphodiesterase for defined periods, it was possible Eo prepare the enzyme at various stages of adenylylation as shown in Fig.34. The native enzyme (without phosphodiesterase treatment) had a pH optimum of around 7.0 and the deadenylylated form (phosphodiesterase treated for 60 min) at 7.8. The isoactivity pH of two acEivities of t,he enzyme was about 7.4 and this explains why the transferase activity of enzyme was not affected by phosphodiesterase treatment when assayed in the absence of Mgz+ (Fie.33a).

The two forms of enzyme have been shown to be inhibited differentially by feed back inhibitors (Gi-nsburg , Lg6g; Bender et a7., 1977) ' Similar results were obtained with the Ni trobacter enzyme (Table 26). The deadenylylated form of enzyme appeared to be inhibited to a grater | extent by alanine, glycine and serine and deadenylated enzyme by 5 AMP (Table 26). 99

FIG.33: EFFECTS OF PHOSPHOD]ESTERASE TREATMENT ON PURIFIED GS FROM NITROBACTER AGILIS (a) AND NITROSOMONAS EUROZAEA (b). The pH of an aliquot of purified enzyme was adjusted to g.g with M Tris. This was then divided into two equar volumes, one treated with phosphodiesterase (50Ug *1-1) at 37oC and the other taken as control. At the tj_mes indicated, aliquots of untreated and phosphodiesterase-treat,ed enzyme were wit.hdrawn and assayed for t,ransferase activity with and without 60mM l4gCI, aÈ pH 7.4. Enzyme activity is expressed u" 4540. The enzyme preparaËion was diluted initially so that 50U1 of enzyme produced enough yGH in 15 min to give an absorbance of approx. 0.15 (without 1t added Mg"' ).

Untreated enzyme wirh (o) and wirhour (O) l4g2*, treated enzyme wirh (tr) and wirhour (¡) Mg2*. transferase activitY Gs+o) o o o oa a a a I o o f\) o (¡ o (¡ o o

or\)

(¡lo ¿ ct c.crÀ ot* o õ' .'+ ='o(D 3 = o

of\)

(.rt o -r1

aõ g (rl oÀ (T 100

FIG.34: DEfERMINATION 0F IS0ACTIVITY pH FOR PURIFIED GS FROM NITROBACTER AGILIS.

Phosphodiesterase treatment (2OUg ,n1-1) of purified glutamine synthetase was as described in Fig.33. The transferase activity of treat.ed and untreated preparations h¡as determined at various pH values as described in Section 2.2.6.2. Untreated glutamine synthetase (O); phosphodiesterase treated for 20 min (o); 40 min (n) and 60 ¡nin (l) respectively. transferase activity (As+o) o ao P a f\) (¡l s { oa

a{ t\)

{ as E - a{ o)

\¡ a @

@ a -rl o 6) a scñ 10r

TABLE 26: EFFECTS OF SOI,IE AMINO ACIDS AND s'-AMP ON ADENYLYLATED AND DEADENYLYLATED FORI'{S OF THE PURIFIES GS FROM NITROBACTER AGILIS.

Deadenylylated form of glutamine synthetase vras prepared by phosphodiesterase treatment as described in Fig.33 except that. the incubation was for Ih. The adenylylçtion state of the enzyme was checked by determining the Mgz+ effect on transferase activity at pH 7.4 as described in Table 25. All amino acids !ùere at a final concentration of lOmlul and AMP at 20mM. The activities of untreated (GS-AMP) and phosphodiesterqse treated (GS) enzyne r.\¡ere 215 and 190 Umol YGH proãuced min-l (mg protein)-l respectively.

Z Inhibition of transferase activitv Additions GS_AMP GS

L-Alanine 70 85

L-Glycine 35 60

L-Serine 40 55

5I -A},IP 65 36 102

3.4 GLUTAMATE DBHYDROGBNASES OF /VT?ROBáCTER AGILTS

3.4. 1 Evidence for two isoz vnes of GDH in Nitrobacter apiTis

The crude preparations of Nitrobacter agiTis contain GDH which utilizes either NADH or NADPH for its amination reaction (Wa11ace and Nicholas, 1968; Section 3.2.4). To check whether the two activities were associated with two distinct proteins, electrophoresis of aliquots of the crude extract (SrO) was carried out in 5% (w/v) polyacrylamide gels (Section 2.2.10). The gels were stained for GDH activity using either NAD4 or NADP+ or both (Fig.35). The acriviry bands srained in gels A (NAD)+ and B (NADP+) had different electrophoretic mobilities and those in gel C (NAD+ + NADP+) also had two distincL bands. It is known that 2t-5tADP Sepharose-4B binds enzymes that require NADP+. l,lhen crude extracLs of lVitrobacter agilis were loaded onto a 2t5t ADP Sepharose-4B column and washed v/ith buffer (Section 2.2.5.2), the effluent contained NAD+-GDH only (get D). When the affinity column was eluted with 2mM NADPH, the eluare did not contain any NAD+-GDH (ge1 E) but NADP+ activity was detected (gels F and G). There are also two minor bands in gels F and G which may be either aggregates or active dissociated subunits of NADP+-GDH (see Smith et a7., Lg75). Only the NADP+ specific enzyme was detected in crude extracts (SgO) of lVitrosomonas europaea analysed in polyacrylamide gels. The results indicaÈe Èhat Nitrobacter agiTis has two dist.inct isozymes of GDH, one dependent on NAD* and the oEher on NADP+ while in lVitrosononas eutopaea one single NADP+ dependent GDH is present.

3.4.2 Purification of NADP+ -GDH

The NADP+-GDH from Nitrobacter agiTis was partially purified by affinity chromatography (Tabfe 27). The purified enzyme from'Nitrosononas europaea reported to be dependent on NADP+ only (Hoo per et a7., Lg67) was also prepared (Table 28). The crude extracts (SgO) of Nitrobacter agiTis contained borh NAD+ and NADP+ dependent activities (Table 27) while those of. Nitro- sononas europaea had NADP+-GDH with only negligible NAD+-dependent activity (Table 28). In SrrO fracrions the specific activity of NADP+-GDH of Nitro- sononas europaea was about 13 fold greater than LhaE of the NADP+ enzyme from Nitrobacter agiTis. The NADP+ enzymes from lVitrobacter agiTis and Nitro- sononas europaea were purified 52- and l42-f.old by affinity chromatography on 2r5t ADP Sepharose-4B. Both the purified preparations l/ere free from the NAD+_GDH. 103

FIG.35: DETECTION OF NAD+ AND NADP+ DE,PENDENT GDH IN NITROBACTER AGILIS

SrO fraction was loaded onto a 2r5rADP Sepharose-4B column (0.8 x llcm) and washed with 50m1"1 Tris/HCl (pH 7.5), then eluted krith 2m1"1 NADPH in the buffer. Aliquots of S30r2r5rADP Sepharose-4B buffer washings and NADPH eluted proteins were dialysed overnight against the same buffer and then loaded onto 52 (w/v) polyacrylamide tubes. Electrophoresis and GDH specific staining was carried out as described in Section 2.2.IO. Gels A to D were loaded with approx. 250pg protein +); and gels E to G, approx. 5ug protein. Gel A, (S¡O + NRD gel B, (S¡O + NADP+); gel C, (SgO + NAD+ +,NADP+); gel D, (2t5'ADP Sepharose-4B buffer washings + NAD+ + NADP+); gel E, (2'5iADP Sepharose-4B NADPH eluate + NAD+); gel F, (2'5'ADP Sepharose-4B NADPH eluate + NADP+); gel G, (2'5'ADP Sepharose-4B eluate + NAD+ + NADP+). FlG.35 ABCDEFG

t¡>Ð - - 104

TABLE 27: PURIFICATION OF NADP+-GDH FROM NTZROBACTER AGILIS.

Enzyme purification and assay of enzyme activiLy as described in Section 2.2.5.2 and 2.2.6.4. Activity was determined by following the oxidation of either NADH or NADPH at 340nm, One enzyme unit is defined as ntnol NAD(P)H oxidised min-r and specific activit.y as units (mg protein)-t.

Total Protein Total Units Specific Fold Purifi- Purification AcLivity cation step + + + f (me) NADP NAD NADP NAD' NADP-r NAD+

sso L2T 3111 2133 26 18 1 I st ro s9. s 2609 1649 44281 7 1.5 Pooled 2r5rADP Sepharose-48 1 1353 3 1353 52 fractions 105

TABLE 28: PURIFICATION OF NADP+-GDH FROI{ NTTROSOMONAS EUROPAEA. All purification sLeps and assay of enzyme activiLy as described in SecLins 2.2.5.2 and 2.2.6.4. Activity rr¡as determined by following the oxidation of NAD(P)H at,340nm. One enzyme unit is defined as nmol NAD(P)H oxidised rnin-r and specific activit.y as units (mg protein)-t.

Total Protein Total Units Specific Fold Purifi- Purification Activity cation step (me) NADP+ NAD+ NADP+ NAD+ NADP+ NAD+

sgo 1360 448000 131 330 0.1 1 I srro 975 402000 0 412 0 1.3

Pooled 2r5r-ADP Sepharose-/+B 4.95 231000 0 46670 0 r42 fractions 106

3.4.3 Anination and deanination reactions in S I f racti.on

The NADP+-GDH from Nitrosononas europ¿s¿ functions in either direction ie. amination of o-ketoglutarate to form glutamate and deamination of glutamate to o-ketoglutarate (Hooper et a7. ¡ 1967). It is clear from Table 27 that the amination activity of NADP+-GDII from Nitrobacter agiTis r+¡as about Èwice that of NAD+-GDH. S.ince Lhe gels rvere stained following deamination reaction, it is clear that the NADP+-GDH of Ni trobacter agiTis predominantly operates in the direction of glutamate production. That this is indeed the case is supported by the data in Table 29. In the SrrO fraction of /Vi trobacter agiTis the aminaEion and deamination activities of the NAD+ enzyme were approximately equal, however the deamination activity of the NADP+-GDH was only about 47" of the amination activiuy (TabIe 29).

3.4.4 Properties of NADP* -GDH from Nitrobacter apiTis

Since the NADP+-GDH from Nl-trosononas europ¿s¿ has been sLudied in detail (Hooper et a7., 1967 ) thís section will deal with some properties of partially purified (52 fold) NADP+-GDH from Nitrobacter agiTis. The pH optima for amination and deamination reactions were 8 and 9 respectively (Fig.36). The rate of NADPH oxidation (amination reaction) was maximal with 10mM s-ketoglutaraue (Fig.37a). A double reciprocal plot of the rate of NADPH oxidation against substrate concentration gave a K, value of 3.57 for c-ketoglutarate (Fig.37b). The NADPH oxidation rat,e for NADPH increased rapidly up to 20mM NH4C1 and slowly thereafter so that the system appeared to be biphasic. Double reciprocal plots of the data (Fig.38b) produced two distinct K- values yiz. 33mM for )20mM NHf and 6.3mM for (2Omla NHf,. A m similar Eype of substrate stimulation of enzyme acËivity was observed with the NADPH (Fig.39). Two K, values of 100Ut1 and 7¡M r,/ere recorded for NADPH concentrations >50W and (50UM respectively.

The amination reaction of NADP+-GDH from Ni trosononas europaea is known to be inhibited by carboxylic acids and nicofinamide adenine nucleot.ides (Hooper et a7. t 1967). The effects of some of these compounds on the amination reaction of NADP+-GDH of Ni trobacter agilis were studied (Table 30). Thus fumaric acid inhibited the amination reaction appreciably (45% at 20m14) while malic and pyruvic acids (20m!1) and s-ketoglutarate (100mM) were without effect. Cis-oxaloacetic acid (OAA) slightly stimulated the reacti-on (18% at 5mM), but this effect was not enhanced by increasing the concentrations 107

TABLE 29: AMINATION AND DEAI',ÍINATION REACTION RATES IN EXTRACTS (S 110) 0F NITROBACTER AGILIS.

Crude extract o1. Nitrobacter agilis prepared as described in Section 2.2,3 was furtlìer centrifuged at 110,0009 and super- natanL (SftO) used for enzyme assays. Amination and deamination reactions of glutamate dehydrogenase r+ere determined at 25"C according to the methods described in Secti-on 2.2.6.4, Specific activity is expressed as nmol NAD(P)H oxidised or NAD(P) reduced min-r (mg protein)-t.

Reaction Specific Activity

+ Amination: NADPH + NADP 48.6

NADH + NAD+ 25.O

Deamination: NADP+ + NADPH s2.0

NAD+ + NADH 22.5 108

FTc. 36: DETERMINATION OF pH OPTII'{A FOR AÌ'ÍINATION AND DEA}4INATI0N ACTIVITIES OF PARÍIALLY PURIFIED NADP+_GDH FROM NTTROBACTER AGILIS. Amination (O) and dearnination (O) reactions of glutamate dehydrogenase \¡/ere determined at 30"C as described in Secrion 2.2.6.4 excepL that 50ml"f Tris-HCl buffer was replaced by 200mM Tris-HCl aL the pH indicated. For amination and deamination reactions, each assay rnixture received approximately 10 and 5OUg partially purified enzyme protein respectively.

FIG.37: THE EFFECTS OF VARIOUS CONCENTRATIONS OF A-KETOGLUTARATE ON THE AMINATION REACTION OF PARTIALLY PURIFIED NADP+-GDH FROM NITROBACTER AGILIS.

Amination reaction of partially purified NADP+-GDH (Table 27) was determined as described in Section 2.2.6.4, except that g-keLoglutarate concentration was varied over the range of 0-30mll. . The data in Fig.37b are plotted as reciprocal of reaction velocity (1/V) against reciprocal of o-ketoglutarate concentration. NMOI NADPH OXIDISED MIN-I +oN)

F Ã 6) nmol NADPH oxidised or f 3 NADP+ reduced min-r ñ Êt oÀ@ o) a I (¡ o o ao a o Ol p J. I @ CN a Ctl

(o q a -r1 (¡ -r1 6) õ N a (,¡ (¡l { o) 109

FIG.38: THE EFF-ECTS 0F VARIOUS CONCENTRATIONS 0F NH,Cl 0N THE AMINATION REACTION OF PARTIALLY PURIFIED NADP+-GDH FRöM NITROBACTER AGILIS.

Amination reacLion of parrially purified NADP+-GDH (Tabire 27) was determined as described in Section 2.2.6.4 except that NHOCI concentration was varied over t.he range 0-200mM. The data in Fig.38b are plotted as reciprocal of reaction velocity (1/V) against reciprocal of NHOC1 concentration (1/S).

FIG.39: THE EFFECTS OF VARIOUS CONCENTRATIONS OF NADPH ON THE AMINATION REACTION OF THE PARTIALLY PURIFIED NADP+_GDH FROM NJTR1BACTER AGILIS.

Amination reaction of parrially purified NADP+-GDH (TabIe 27)

was determined as described in Sect,ion 2.2.6.4 except that NADPH concentration was varied over the range of 0-200UM. The data in Fig.39b are plotted as reciprocal of reaction velocity (1/V) against recj.procal of NADPH concentration (1/S). I FtG. 38 z._ o l¡l I U) e (a) x 6 o (b) I o.6 IL o 4 z o o E E 2 o.4

3 S (mM NH4CI ) ì o.z

-o.2 o o.l o.2 0.3 0.4 0.5 t/s

o FlG. 3g Tz ô aL¡l (a) õ6 (b) oX E4 ô o.5 z. oz cE o.4 o 50 oo 200 o.3 S (¡M NADPH )

-o.2 -o.l o o.o5 0.1 o.2 t/s 110

TABLE 30: BFFECTS OF ORGANIC ACIDS ON THB AI'IINAT]ON ACTIVITY OF PARTIALLY PURIFIED NADP+-GDH FROM NTTROBACTER AGILIS.

Amination reaction of the NADP+ enzyme was determined at pH 8.0 as described in Section 2.2.6.4, except that the assay rnixture also contained an organic acid as indicated. The enzyme activity of the control was 1.32pmol NADPH oxidised min-r (mg protern,

Final concentration % Inhibition (-) or Additionsa (ml'f ) stirnulation (+)

Fumaric acid 10 -28 20 -4s

Cis oxaloacetic acid 5 +18 10 +13 20 +13

O-ketoglutaric acid 100 0

Dl-malic acid 20 -13

Pyruvic acid 10 -5 20 -10 uA11 th" acids were either neutralized to pH 7.5 before use or used as Na* sa1t. 111

of 0AA to 20mM. None of the nucl.eotides (NAD+, NADP+, NADPH, and ATP) affected enzyme activity substantially (Tab1e 31).

3.5 ENERGY CONSERVATION IN NITROSOMONAS EUROPAEA AND NITROBACTER AGILIS

3.5.1 Proton transTocation and oxvøen oulse exoerinents

Proton translocation during NHf and NHrOH oxidation by Nitrosononas europaea has been studied by a fluorescence quenching technique (Bhandarí and Nictrolas, I979a,b) and oxygen pulse technique (Drozd, 1976). Cobley (I976a,b) has shown by the oxygen pulse technique that the membrane parLicles of /VitroÐacter winogradskyi translocated protonsduring N0, oxidation.

Using fluorescence guenching technique (Bhandari and Nicholas, 1979a) in the present study, Nitrosononas europaea translocaLed protons during either Nfff or NH2OH oxidations (Fig.4Oa-c) and the results were similar to those reported earlier (Bhandari and Nicholas, L979a). The addition of NOl to a cel1 suspension of Nitrobacter agiTis resulted in a very slight change in quinacrine fluorescence (Fig,40d,e). Varying the concentration of either substrare (0.2 ro 2OrnM), cells (40 to t00mg) or quinacrine (0.05 to 0.2 ¡rmol) had no effect on the magnitude of response. Similar results were obtained with the spheroplasts of lvj trobacter agiTis. Thus attempts to study proton translocation in washed cells and spheroplasts of. Nitrobacter agi'Iis using fluorescence technique were largely unsuccessful under the experimental condit.ions described above.

For the quantitative estimation of proton translocation, experiments were done using the oxygen-pulse technique. The methodology and the details of apparatus used are described in Section 2.2.I2:2. This subsection will deal with the results of oxygen pulse experiments with NArosononas europaea and ¡Vitrobacter agiTis.

3.5. 1 . 1 Kinetic parameters of respiration in lVi trosornonas europaea and Ni trobacter agiTis

As the respiration activities and lcineEic parameters of a particular biological system play an imporLang role in overall energy meEabolism Lhese were considered for /Vj trosononas europaea and Nitrobacter agiTis before carrying out the oxygen pulse experiment. The kinetic data are summarized in Tables 32 and 33. IL2

TABLE 31: EFFECTS OF NUCLEOTIDES ON THE AMINATION ACTIVITY OF PARTIALLY PURIFIED NADP+-GDH FROM NT?ROBACTER AGILIS.

Amination reaction of the NADP+ enzyme was deterrnined at pH g.0 as described in section 2.2.6.4, except that the assay mixture also contained a nucleotide as indicated. The_ enzyme activitr of the control was 1.32pmo1 NADpH oxidised ,ntntl=iíË'ïr:;:i;;:Ï.

Final concentration Z Inhibit.ion (-) or Additio..s (mM) sLimulation (+)

NADPH 0;50 +5

NADP+ 0. 17 -5 0.35 -10 .l- NAD' 0.25 0 0.50 -10

ATP 0.50 0 113

FIG.40: PROTON EXTRUSION IN I^TASHED CBLLS 0F Nr?ROSOM)NAS EUROPAEA AND /VT?ROBACTER AGILIS DETERMINED BY FLUORESCENCE METHOD. Cell suspensions (20mg wet h't.) were preincubated for 5 min with 0.1¡rnol quinacrine and 25Umo1 Tris-HCl buffer (pH 7.5) in a final volume of 2.6m1 in a series of lcm cuvettes. Eíther NHOC1 (0.5mM) or NHrOH (0.5mM) were the substraLes for Nitrosomonas europaea and NaNO, (lmM) for fii Ërobacter agilis. The increase in fluorescence emission indicates an outr,r¡ardly directed movement of protons measured in a fluorimet.er aL 42]nm excitation and 485nm emission wavelength as described in Section 2.2.12.L. FlG. 40

NITROSOMONAS

NH4Ct NHz OH ct

UJ +2 pM CCCP

+5 pM CCCP

(d) (e)

. 2MIN TT4

TABLE 32: KINETIC PARAMBTERS OF RESPIRATION IN I^IASHED CELLS OF NITROSOMONAS EUROPAEA.

The respiration rates were determined at pH 7.5 by using a Clark-type oxygen electrode as described in Section 2.2.II. Oxygen upLake varues for a specific substrate ï/ere corrected for the endogenous rate. The K* for 02 was determined from electrode traces at low 02 concçntrations. Respiration rates are expressed as ng atom õ mln-l (mg protein)-l.

Km Substratea Respiration rate Substrate (rnM) 02 (uM)

Endogenous 20-60 10-20

NH4Cl 800-1s10 1.0 5-10 NH20H.HCl 400-690 0.3 r5-20

N2H4. H2S04 130-2s0 1.0 r5-20 a^, -'I'he given respiration rates are for 2mM concentration of each substrate. 115

TABLB 33: KINETIC PARAI'IETERS OF RESPIRATION IN hIASHBD CELLS ' SPHEROPLASTS AND I..IEI',IBRANE VESICLES OF NITROBACTER AGILIS. Bxperimental details as in Table 32. Spheroplasts and vesicles hrere prepared as descríbed in Section 2.2.2. Respiration rates (at pH 7.8) correçted for endogenous respiration are expressed à" n! atom 0 min-l (mg protein)-r.

Preparation Substratea Respiration rate N0¡ (mM) 0, (uM)

lrlashed cel1s Endogenous 20-40

NaN0, 400-800 0.8 15-20

Spheroplasts Endogenous 20-30

NaN0, 3s0-700 0.8 15-20

Vesicles Endogenous 0

NaNO, 30-40 0.8 Is-20

aThe given respiration rates are for 4mll NaNOr. 116

There were considerable changes in specific activities among several batches of bacteria used (Tables 32 and 33). The difference arose partly as the result of the length of storage of cells. It was observed for example that Nitrosononas eLtropaea progressÍvely lost its NHI oxidising activity, but gained in NH,OH oxj-dising activity over three days of storage (the maximum storage time) at 0-4oC. The K, values for specific substrates and for 0, were in reasonable agreement with the reported values (Drozd , 1916; Bhandari and Nicholas , I979a).

3.5.I.2 PermeanL ion requirement

For a number of bacteria, eg. denitrifiers (Kristjansson et a7. , 1978) valinomycin/K+ serves as a permeant ion in oxygen pulse experiments to collapse membrane potential (Aü) and al1ow proton ejection, but in Èhe case of Nitrosononas europaea LL was totally ineffecLi.ve in the range of 50 to 150ug valinomycin m1-1 even after incubation peri-ods of up to 24h at 0-4oC. It appears that valinomycin (molecular weight 1111.4) does not penetrate the outer membrane of this Gram negative bacterium. Valinomycin produced a small oxygen-p'ulse response after incubation periods of about lh at OoC in the case of Ni trosomonas europaea cel1s, pretreated with 1mM EDTA, but these results were not reproducable. Several salts of permeant ions were studied for their ability to promote oxygen pulse responses. These included KSCN, which has been used widely in oxidant pulse experiments (Scholes and Mit.chell, 1970b; Drozd, 1976), NaC1OO, sodium trichloroacetate (NaClrCrO2), t

Inhibition of S ration Bacterium Substrate KSCN -trichloroacetate 0.15M 0. 15I,f 0.30M 0.1M 0.2I,1

Nitrosononas europaea Endogenous 60 80 100 2rnM NHOC1 100 34 59

2mM NH'OH-HCI 75 23 39 2nl4 N,HO-H,SOA +2404

Nitrobacter agiTi-s Endogenous 75 100 4mM NaNO, 4L 36 35 50 astimulation of respiration.

ts 118

of N^H5+ oxidaLion. NaClO4 was an effective inhibitor of endogenous respiration in both bacteria but otherwise vùas a weak inhibitor. NaClrCrO, was used only in oxygen pulse studies with ili trobacter agiTis and so inhibition studies were carried ouL only with this organism. Effects of tipophilj-c cat.ion TPMPT on NHf and NHrOIì oxidation by Nitrosomonas europaea and N0, oxidation by Nitrobacter agiTis are shown in Table 35. TPMP+ v/as a potent inhibitor of oxidation of endogenous subsl-rates and of added llHf in Nitrosononas europaea aE low concentrations (5mM), but was much less effective for NHrOH oxidaLion. However TPMP+ even at very high concentrations ( 100 to 30OmM) only partially + inhibited NO! oxidation (15 to 207") by Nitrobacter agiTis. Thus TPMP was suitable for'0, pulse experiments wíth Nitrosononas europas¿ when NH20H was the oxidizable substrate and with Ni trobacter agiTis utilizing nitrite.

3.5.1.3 StoichiomeLric proton production

Stoichiometric protons were determined as described in Section 2.2.12.4 in reductant pulse experiments in the absence of a permeant ion and in oxygen pulse experiments in which 3 to 5¡rM CCCP was used to un- couple the system by rapid equilibration of protons between internal and external cell compartments (Fi-g.4td). From the following reactions the theoretical stoichiometric proton values for NHI, NH2OH and NrHr+ oxidation by Nitrosononas europaea and N0, oxidation by Nitrobacter

agiTis are 21 1 r 1 and 0 respectively for each mole of substrate oxidised:

t (i) Hnf + 30 --+ lroJ + u,O + 2H' 1 (ii) NH2OH + 20 --+. llOJ + HrO + H' -L (iii) NzHs++20_>lrf + 2HrO + H' 2 (iv) Nor+0----+N03

The results shown in Table 36 for the oxidation of wHf, UHrOH and trt{ by IVi trosononas europaea at pH 7 .4 and of N0, by Ni trobacter agiTis ¿¡ pH 7.8 are in reasonable agreement with Ehe values expected from equations (i) to (iv) and the following pK values (Jencks and Rogenstein, 1968):

*tl, 9.25; NH3oH+ , 5,96; NzHs*, 8. 1 ; HNO, , 3.2g . 119

TABLE 35: EFFECTS OF LIPOPHILIC CATION TPMP+ ON AEROBIC OXIDATION OF SUBSTRATES BY WASHED CELLS OF /VITROSOMONAS EUROP.AEA AND NITROBACTER AGILIS.

Respiration rates were determined by oxygen electrode as described in Table 32. The results for Nitrosononas europaea were recorded at pH 7.5 and for Nitrobacter agiTis at pH 7.8. The results for spheropl,asts and vesicles prepared from Nitrobacter agiTis were similar to those of whole ceIls.

f Bacterium TPI'{P' concentration Substratea Z Inhibition of (mM) respiration

Nitrosononas europaea 10 Endogenous 100 5 NH4C1 80 10 NH4C1 100

5 NH20H.HCl 5 15 NH20H.HCl 50

Nitrobacter agiTis 50 KNO2 5 100 KN02 T2 200 KNo2 25 300 KN02 50 aConcentration of substrates, NH4C1 and NHTOII were 5mM and for KNO, 2mM TABLE 36: PRODUCTION OF STOICHIOMETRIC PROTONS BY NTTROSOMONAS EI]ROPAEA AND NTTROBACTER AGILÏg.

The +H+/0 ratios in presence of CCCP were deternined as described in Section 2.2.I2.4. The substrate concentrations were over the range 2 t.o 4mM; CCCP, pulse; pE for NitrosoÍnonas eutopaea aL 7 -4 and for 0.15M SCN- except for NHi as substrate, when 0.2M C determined in reductant fiulse experiments (Section 6 to 50 nmol and the system \^ras vigorously stirred rnder pure 0r. The vaiues in parenthesis represent expected values for Nitrosononas europaea at pH 7.4 and for Nitiobacter agiTis at pH 7.8.

*tt+/O raÈio in +H+/substrate ratio in Bacterium Subst.rate presence of CCCP reductanL pulse experiments

Nitrosomonas europaea Endogenous 0 0. 15 NH4C1 0.68 + 0.04 (0.66) 7.9 t 0.1 (1.99) NH20H.HCl 0.48 + 0.05 (0. s2) 1.02 t 0.06 (1.04) N2H4-H2S04 0.46 t 0.05 (0.42) 0.89 r 0.07 (0.83)

Nitrobacter agiTis Endogenous 0 NaN0, 0.0 t 0.06 (0.0) undetermined

F N) O I2T

3.5.1.4 Oxygen pulse experiments tvith Nitrosononas europaea

Freshly harvested cells of ilitrosomonas europaea contained endogenous substrate which promoted proLon translocation. The oxygen responses were typically as reported earlier (Drozd, 1976) and shown in Fig.41. Endogenous substrare exhibited *H+/0 raLios of. 4.6 to 5.6 typically for the first few pulses oL O, (Fig.4lb) at pH 7.0 to 7..4 but decreased to 3.5 to 4.5 during subsequent pulses. It was frequently possible horvever to exhaust endogenous substrate for periods of 20 to 30 min by means of a series of large (100 to 200ng atom) pulses of. Or. In one batch of cells out of 13 the endogenous substrate becarne permanently exhausted following oxygen pulses. The *H+/0 raLios for endogenous substraEe oxidation were independent of Lhe amount of 0, per pulse from 5 to 40ng atom 0. The use of perchlorate (Table 34) and TPMP+ (Table 35), which totally inhibited oxidation of endogenous substrates, and use of cel1s lacking endogenous substrates permitted a clear distinction beEween proton-translocaÈion due to three amine-like subsÈrates and Lhat due to oxidation of endogenous substrates. It was found thaÈ the tfl+/O ratios obtained with the amine-lilce substrates at concentrations >2mM referred largely or entirely to the oxidation of these substrates and not to endogenous substrates. This result was consistent with the data in Table 32 which show that the rates of oxidation of endogenous substaLes were sma11 compared to those for specific substrates and wiÈh the results in Table 36 which indicaLe Ehat the yield of stoichiometric protons correlated well with the oxidative reaction of the specific substraÈe.

The +H+/0 ratio was essentially independent of the size of the oxygen pulse up to about 3Ong atom 0 in the case of NHI oxidation, but dependent on pulse size in the case of NHrOH (Fig.42) and NrH! oxidations. In part this effect may be a consequence of a moderately high Km (02) for the oxidation of NH2OH and NrHr+ (Table 32). For small pulses of oxygen, a larger fraction of the total reaction lies in the kinetically slow phase encountered at 0, concentration (K* (Où. The relevant +H+/0 ratio in the case of NH'OH and trtr* was taken to be the asymptotic value obtained with larger oxygen pulses.

The asymptotic ->H+ /O ratios in turn depended on the concentrations of llU[, nHrOH and *Znr* (Fig.a3). At. high concentrations of these substrates (20-40mM) the -)H+/0 ratios approached the stoíchiometric value (Table 36) lor the reaction, eg.0.7 for NH[. The effect was not due to substrate inhibition r22

FIG.41: OXYGEN PULSE RESPONSBS IN \IIASHBD CELLS 0F NTTROSM)NAS EUR)PAEA.

Freshly harvested cells were washed twice in 150m!1 KCl and finally suspended in the same solution (approximately 75mg wet _1 weight ml '). Oxygen pulse experiments were carried out as described in Secton 2.2.I2,2. Reaction mixture in a final volume of 1.5m1 contained approximately 105mg wet weight cells in 150mM KCl, 100ug carbonic anhydrase and 150n1"1 KSCN (where indicated). The initial pH of the cell suspension was adjusted to 7.4 with 50mM HCl or 50mM NaOH. Fig.4la illustrates the 0, pulse response in cells containing endogenous substrates, without any permeant ion in the reaction mixture; 41b shows the prot.on translocation associated v¡j-th endogenous respiration v/ith 150mM KSCN as permeant ion and 4lcrd show proton translocation associated with NHrOH oxidation in ce1ls depleted of endogenous substraÈes and with 150mM KSCN as permeant ion. Fig.41d illustrates Èhe fast equilibriation of pi:otons after addition of 4UM CCCP to the reacti-on mixture. -LnË I H

zc)

t. .Ots -..i.'_rj #€,rI -- ¡ r- f -r-

a I

IK l t

,kt

I I I I iilii l I

I L I I t I lliii iiii 'ltl \ :ril,I lr, ,tr 'I 'tÌ ll ii

iii

I r23

FrG.42| DEPENDENCE OF +H+/O RATIO ON THE SIZE OF THE OXYGEN PULSE FOR OXTDATION 0F NH20H BY IVI?ROSO\IONAS EUROPAEA.

The oxygen pulse experiments rvere carried out at 25"C and pH 7.4 as described in Sectíon 2.2.12,2. KSCN (I50mM) was used as a permeant ion. The NH20H concentrat.ions were (mM): 1.2(o); 3.l(tr) and 10.2 (^).

FIG.43: DEPENDENCE OF +H+/O RATIO ON THE SUBSTRATE CONCENTRATION FOR OXIDATION OF AMINE_LIKE SUBSTRATES BY NITROSOI4ONAS EUROPAEA.

The oxygen pulse experiments u¡ere carried out at 25oC and pH 7.4 as described in Section 2.2.12.2. The system con- t'ained O.2M perchloràte as permeant ion with NHf (O) and 0.15M KSCN as that with NH2OH (!) and N2H5+(^). Each poinr on the curves represents the asymptotic limit or plateau inferred from an experiment of the type illustrated in Fíg.42. FlG.42

.9 P G' L o 3 +\ -I E'(¡) L o) 2 CD €

o 20 40 60 ng atom O per pulse

FlG. 43

3 .o +' (tt C- o 2 +\ -I

o o ro 20 30 40 substrate conc.(mM) 124 of respiration. The respiratory proton pump would appear to be inhibited at high concentrations of these amine-like substrates. In this regard methyl- ammonium (CH3NI{3+) which is not oxidised by Nitrosononas euroPaea mimicked the effect of the amine-like substraEe in diminishing H+/0 ratios. For example, + 20mM CIlaNHr'lowt:red the +H+/O ratios from 3-4 for 2mM concentrations of these substrates to 0.9-1.5. Similar effects were also noted for the endogenous substraLe oxidaLion. Thus, therfl+/O ratios are taken to be the values extrapolated to zero substrate concent 4.4 f.or NH2OH and NrHr+. The data fo to those obtained with perchlorate as tl permeant cation (Table 37).

For the oxidation of nfff and NFITOH respectively, the possibility existed (Ritchie and Nicholas, L972) t.hat the product NOr- might itself serve as an oxidant as in the following dismutation reaction:

NH2OH + NO2- ---+ N2O + HrO + 0H Such a reaction could, if rapid, provide a different overall react.ion and possibly a different H*/O ratio:

NHzOH + O------+ å NZo + r å H20

Pulse experimenEs carried out with NO2- instead of O, wi-th NHf, and NHrOH as substrates indicaÈed that no proEon translocation occurred. Moreover the rate of anaerobic reduction of NOJ by NH,OH in Ni trosononas euroPaea was very low. Thus these reactions were not considered Eo be kinetically relevant.

3.5.1.5 Oxygen pulse experiments with Nitrobacter agiTis

Attempts to evoke respiration dependent proton translocation in Nìtrobacter agiTis cells were unsuccessful. The permeant ions employed in the study were valinomycin/K* (SO to lOopg d-1), thiocyanate (0.15 to 0.38M), perchlorate (0.2Y1), trichloroacetate (0.I to 0.25M) and Eripenyl methyl pho.sphonium cation (0.005 to 0.3M). Oxygen pulses were varied from 10 to 120ng atom O per pulse, NO; concenLraLion from 0 to 8mM, pH from 7.0 to 8.0 and the amount of cells from 100 Uo 150mg r,¡et wt. Experiments were also done in the absence of a permeant íon. A rapid transienÈ acidification occurring in the I to 5s range was nof observed and in fact the most consistent rapid response was a slight 725

TABLB 37: APPARENT *IT+/O RATIOS IN I^/ASHED CELI,S OF NT?ROSOI'IONAS EUROPAEA AND N.T?ROBACTER AGILIS DURING SUBSTRATE OXIDATION USING TPMP+ AS PBRI'ÍEANT ]ON. *U+/0 raLios were determined as described in Section 2.2.I2.2. The data are from approx. 10 experimenl-s. The resting pH in the oxygen pulse experiments was 7.4 lor both bacteria and the arnount per pulse was 40 ng atom.

TPMP+ concentration Substratea Apparent Bacterium (mÌ'{) +H+/O ratios

Nitrosononas europaea 5 endogenous b 5 NH4Cl b 5 NH20H 3. s-4.0 l5 NH20H 3. 5-4.0

50 KNO <0. 1 Nitrobacter agiTis 2 100-300 KNO <0.05 2

UNUOC1 and NHrOH concentrations were 2-5mM and that of KNO Z, 2mlf. ÞDifficult to estimate due to very slow reduction of Or. Apparent ratios were about 1.0. I26

alkalinization with +H+/0 ratio of about -0.15. This was usually followed by a slow but variable acidification that peaked within 20 to 35s, and had an apparent +H+/O ratio of 0.1 to 0.2. Neither the extent of acidification nor the time required for its completion was proportional to the amount of 0, injected. The response \^/as not clearly dependent on the prior acldition,of NOr. The responses were largely abolished by 41rll CCCP and so probably reprssented differences in proton concentration between inner and outer buffer compartments of cell.

A study was made of spheroplasts of Ni trobacter agiTis (prepared as described in Section 2.2.2) in a system containing valinc:ilycin _'l (50ug m1-'), 0.15M rhiocyanate or 0.2M perchlorate, 10mg of spheroplast pr.otein and 0 to 8mM NOj at pH 7.0 and 7.5. The only rapid response observed was a slight alkalinization with ->H+/0 ratio of about -0.06 (Fig.44). Lysis of spheroplasts was slight as deLermined by DNA release.

The study also included membrane vesicles of Nitrobacter agil.is in a system conraíning valinomycin (50ug r1-1)/r* or 0.15M KSCN,".17mg of vesicle protein and 0 to 4mM N0, at pH 7.5. In the absence of NOJ a transient alkalinization was observed with peak at 4 to 5s and an +H+/O rario of abour -0.2 (Fig.45a). I^¡ith 2 and 4mM NO, Èhe initial alkalinization was smaller 1+H+/0 = 0.l.-,1 and followed by a slow acidification in 30 to 50s range (Fig.45b,c). Both the amount of vesicle protein and the internal buffering capacity were similar to those found in systems conEaining intact cells. Thus vesicles were able, in principle, to translocate at least 100 nmol of proton before the þH might prohibit furEher prolon translocation. A nul1 result could have accrued if the vesicles had been half inverted (rinside oucr) and half right side ouL, but this was not the case. Vesicles bounded apparently by a single membrane hrere sÈudied to test t-he possibility Ehat the null result with cel1s and spheroplasts occurred because the proton pump in Nitrobacter agiTis translocated protons from one intracellular compart- ment to another as is the case in eukaryotic cells. t27

FIG.44: OXYGEN PULSE RESPONSES IN SPHEROPLASTS OE NITROBACTER AGILIS. SpheroplasLs r¡rere prepared as described in Section 2.2.2 except that lysozyme/EDTA treated cells were washed and suspended in 150mM KCl. Oxygen pulse experiments were carried ouL as described in Section 2.2.I2.2 with 150mM KSCN as a permeant ion. The reaction mixture in a final volume of 1.5m1 contained spheroplasts (70mg wet weight m1-1) in 150m1'{ KCl, carbonic anhydrase (80ug t1-1) and l5OmM KSCN. The resting pH of the suspension v/as 7 .55. The sysLem rrtas calibrat.ed with 5ml'1 HCl. FlG. 44 c

lL I i I t r28

FIG.45: OXYGEN PULSE RESPONSES IN MEMBRANE VESICLES OF NITROBACTER AGILIS. Membrane vesicles, prepared as described in Section 2.2.2, washed twice in 150mM KCl and finally suspended in the same solut.ion (fl.4mg protein r1-1). The reaction mixture in a final volume of 1.5m1 contained vesicles (=17 mg protein), carbonic anhydrase (100Ug) and either valinomycin (75U8) or KSCN (l50mM). The initial pH of the suspension at 8.2 was tiEraEed Eo 7,5 with 50mM HCl. The system was calibrated u/ith 5mM HCl. I

i I 'uTu r I

(r)

Zg T 40 i l I I I I l. I I

I 'tl I ; I il rl tl I rli: "o/ J)] 1rt a tl J I

I I I . I I :- ;; -.L .

\| I

I :l ON (q) S S F

ql ,toÌ

T T 11g ,. zo/ 11g q J

l 1 r29

3.5.1.6 Duration of respiration after an oxygen pulse

The slor,rest rates of respiration were those suPported by endogenous sulrsÈrates in cells and Uy NOJ in vesicles from Ni-trobacter agiTis (Table 33). These rates were taken as l5ng atom O *in-l (*g _1 protein)-' to account for partial inhibition of respiration by permeant ions. Because the amount of proteins v¡as about 15mg, the overall rate The durâtion of 2Ong atom pulse of 0, rvould was about 4ng atom O "-1. have therefore been 5s. In all other systems rePorted in Tables 32 and 33 the respiration time would have been so short as to lie always within the mixing time.

3.5.2 Proton eTectrochenica7-pradients in v,ashed ce77s of Nitrosononas euroDaea and Nitrobacter apiTis

3.5.2.L Uptake of radioactive probes

All probesr listed in Table 38, used to determine mernbrane potential (Aü) and transmembrane pH gradient (^pH) were readily taken up by the cells of Ni trosomonas europaea and Nitrobacter agiTis and an equilibrium state was reached wiUhin 5 min. The EDTA treatment of the cells was necessary in order to make them permeable to radioactive compounds, indeed without this treatment the results vrere variable and erratic. High concentrations of EDTA (5mM for Nitrosononas and lOrnÙl for Nitrobacter) relative to Ehose used for E. coli (Padaç¡ et a7 ,, 1976) were employed because of the complex cell membrane structures of these bacteria (Murray and hlatson, 1965). EDTA treated cel1s \n/ere metabolically active since oxygen uptake values were similar to those of untreated cel1s.

3.5.2.2 Measurement of apH as a furiction of external pH (pHe)

The uprake of [1aC] benzoic acid, tlaC] acetylsalicylic acid and tlaC] meEhylamine respectively by Nitrosononas europaea and Nitrobacter agiTis has been plot.ted against pHe in Fig.46a and b. The uptake of all three conpounds was pHe dependenÈ. ¡/i trosononas europaea accumulated the Lwo weak acids only when the pHe was below 7.0 and the weak base methylamine when the pHe was above 7.0 (Fig.46a), indicating thaL the internal pH (pHi) of bacterium was maintained around neut-rality. 1¡s ApH was almost zero aL pHi of 7.0, but when pHe was )7.0 the pHi becanle acidic in relatj.on to pHe (inside acidic). For /Vjtrobacter agiTis (Fig.46b), TABLE 38: PROBES FOR DETERMINING AÜ AND ApH IN \^IASHED CELLS OF NITROSOMONAS EI]ROPAEA T\ND NTTROBACTER AGILIS.

\^lashed cell suspensions in 5Omlf Na-phosphate buffer (pH 6.0) were used to deLermine ApH and Arþ as described EDTA the cells .uriied ouÈ at 37"C for 10 min (Section 2-2-73-I)- ApH SecÈion 2.2.13. treatment of "us and A$ are the mean values of 10 det.erminations with the same batch oi cells.

(mV) AoH + SEM Arf I SEM Untreated EDTA-Ireated Bacteria Probe Untreated EDTA-treated ce1ls cells cells cells

lacl Nitrosononas europaea t Benzoic Acid 0.22 ! O.r2 0.33 10.03 [1ac] Acetyl salicylic 0.20 ! 0 .12 0.28 r 0.03 acid lac] [ Methylamine-HC1 0.0 t 0.01 0.0 r 0.01 I 1t( + q t3nl TPP' 100 I 15 lacl Nitrobacter agiTis t Benzoic Acid 0.80 r 0.10 1.36 r 0.05 tlacl Acetyl salicylic 0.72 ! O.l2 t.22 ! 0.06 acid lac] [ Methylamine-HC1 0.00 t 0.00 0.0 r 0.0 I t3Hl TPP' 82!B 105r5

UJ O 131

FIG.46 : uprAKE.oF [14c] snNzorc AcrD, [14c] AcETyL sAl,rcylrc ACrD AND [rac1 uetHyLAMrNE AS A FUNcrroN 0F pHe BY EDTA TREATED CELLS OF (a) NITR2S?MONAS EUROPAEA AND (b) /VTTROBACTER AGILIS.

EDTA treated ce1ls of Nj trosomonas europaea and Nitrobacter agilis hrere prepared as described in Section 2.2.13.1. Treated cells were suspended in 50m14 Na-phosphate buffer at pH values indicated. Uptake sLudies v/ere carried out as described in Section 2.2.13.3 [lac] benzoic acid (o); tlacl acetyl salicylic acid (o); and tlacl methylamine (o). FlG. 46

6 ( a)

4 +¡ o= (J .s- to L 2 -= s()

o o

6 7 I 6 7 I

pHe PHE r32

the pHe at which neither of the two weak acíds nor rveak base was taken up by the cells, was about 7.5. Benzoic and acetyl salicylic acids r{ere not metabolized by either bacterium. Nitrosononas europaea however slowly utilized the weak base methylamine when the external pH was greater than 7.5. Because methylamine \^Ias utilized slowly by Nitrosononas europaea, Èhe uptake studies which were completed within 5 min were unaffected by this metabolism.

Nitrosononas europaea had a limited capacity to maintain a constant pHi and thus it increased from 6.3 to 7.8 when Lhe pHe was varied over the range 6.0 to 8.5 (Fig.47a). 0n the other hand in Nitrobacter agiTis (Fig.47b) pHi increased from 7.3 to 7.8 when the pHe was increased from 6.0 to 8.5. Thus at pHe 6.0 Nitrosononas europaea and Nitrobacter agiTis had a ApH of 0.3 and 1.3 pH units respectively (Table 38). As these bacteria respire optimally between pH 7.5 to 8.0, it appears that they do noL have u ApH (inside alkaline) but instead the pHi- are either similar to pHe or more acidic (inside acid)' Over a range of pHe (6.0 to 8.5), the pHi in Nitrobacter agiTis increased only by about 0.5 uníts, whereas in Nitrosononas europaea it increased by 1.5 units.

3.5.2.3 Measurement of AÜ as a function of pHe

The variations in Aü as determined by [3U]tpp+ uptake over a pHe range 6 to 8.5 are shown in Fig.47. TPP+ tends to bind to cellular components so that daLa were corrected for this non-specific binding (Section 2.2.13.3). In both bacteria Arþ incr.eased r"iEh increasing pHe. Thus in Nitrosomonas europaea AÙ increased from 125mV at pHe 6.0 to 178 at pHe 8.5. In Nitrobacter agiTis the effect. of pHe on Atþ was less pronounced than in Nitrosononas europaea, thus iE increased from 105mV at pHe 6.0 to 135mV at pHe 8.5, an increase of approximately 10mV for each pH unit. The increase in Atþ ;-n Nitrosononas europaea l,/as non- linear and approached a plateau at pH 8.0, while in /Vjtrobacter agilis the increase hlas almost linear.

3.5.2.4 Total proton-motive force (Ap)

Since Ap i" a function of Ar! and ApH, it is clear fton Fíg'47a that Ap remained almost constant (135-145mV) in Nitrosononas europaea 133

FTG.47: BFFECTS OF PHC ON PHi, APH, AÜ AND AP IN EDTA-TREATED CELLS 0F (a) NITR)S)MONAS EUR)PAEA AND (b) NITROBACTER AGILIS.

EDTA treated ce1ls were suspended in 50mM Na-phosphat.e buffer at pH values indicated. Uptake studies were carried out as described in Section 2.2.13.3. Intracellular pH (^) and ApH (e) represented in terms of mV (59 x ApH) were determined from the uptake of tlaC] benzoic acid and tlaC] methylamine. Aü values (o) were calculated from rhe uprake of [3U] fpp+. Ap (O) was calculated from ApH and Ar! as described in Section 2,2.I3.4. FlG.47

-r50

-roo

MV -50 I

7 0 pHi

6 +5 6 7 I 6 7 8 PHE PHE 134

over a range of external pH. This was largely the result of an increase in Aù and a decrease in ¡pH when t.he pHe was increased from 6.0 to 8.5, Ehus a decrease in ApH was comPensated by an increase in Aü. In /Vitrobacter agiTìs (Fig.47b), hotvever, the conÈribution of ApH decreased rapidly when the pHe was increased (-73mV aE pH 6.0 to + 40mV ar pH 8.5), while arþ increased by 30mv only from pHe 6 to 8.5, thus decreasing the total proton-motivè force from 177mV at pHe 6.0 to 95mV at pHe 8.5.

3.5,2.5 Proton - moLive force in cells of /Vitrosononas europaea harvesLed at various stages of growth

Nitrosomonas europ¿s¿ grok¡S slowly (nean generation time 10-i2h). About 24h after inoculation the exponential stage of growth sEarEed and it lasred for another 4 days (Fig.48a,b). Because the cell yields were low, it was not possible to conduct uptake studies with the probes to determine ApH and ArÞ in growing cultures as described by Kashket for a number of bacteria (Kashket et a7., 1980; Kashket, l98la,b). To assess ',.¡hether there were any changes in ApH and Atþ at various stages of growth, cultures (11) were harvested at various times as shown in Fig.48a and b. Thus ApH and Arþ were determined at two pHe values (6 and 8) after suspend- ing the cells in fresh culture medium. The intracellular water volume was reasonably constant during growth (1.6 t 0.2¡rt (mg dry rt.)-l). Cells harvested at different stages of growth maintained a fairly constant pHi (Fie.48a,b). Thus aE pHe 6.0, Aü h,as approximately I22nY and ApH 0.3 uniEs (inside alkaline) and at pHe 8.0, AÙ was approximately I65mV and ApH 0.5 units (inside acid). A similar experiment. with Nitrobacter agilìs was not possible because of exceptionally low cell yields (40mg wet v/t . ¿-I culture after 5 days of growth).

3.5.2.6 Effects of some inhíbitors on the components of ÀP

To deEermine the relevance of respiratory potential to Ap main- tenancerthe effects of respiraÈory inhibitors on ApH and Atf in NitrosomonaseuroPaea and' Nitrobacter agiTis were invesEigaLed' Sodium dieLhyldithiocarbamal-e (DIECA), a Porent inhibitor of UHf oxidation by Nítrosononas eurr.paea (Bhandari and Nicholas, L979a,b) completely in- (Tab1e 39)' híbit.ed respiration aL 2OUM but did not have any effect on Ap In Ni trobacter agiTis (Table 40), sodiurn azíde at 50uM cornpletely 135

FIG .48: INTRACELLULAR pH AND IN CELLS 0F /VTTROSOMONAS EUROPAEA VARIOUS STAGES 0F GROWTH^{r DETBRMINBD AT pHe VALUES 0F (a) 6.0^T AND (b) 8.0.

Cultures (IB ¿ ) were grohrn ín 20 A Pyrex glass bottles at a constant temperature (2BoC) and pH (8.0). Growth of the bacterium (O) was monitored throughout the incubation period by the rate of NH] oxidation and by determining the protein contents of the cells. At the times indicated, L-to 2¿ cultures withdrawn aseptically were harvested by centrifugation (10,0009 for 30 min) in 250m1 polycarbonate bottles. The ce1ls were Lhen washed and resuspended in growth nedium at either pH 6.0 (a) or pH 8.0 (b). Uptake studj-es h¡ere carried out as described in Section 2.2.73.3. Aü (O) was calculated from the uptake of ¡3U1 ffe+. Intracellular pH (D) was deterrnined by the uptake of t14C] benzoic acid and [laC] methylamine at pHe 6.0 and B.O respectively. FlG. 48

(a) o b) o o I -r50 PH¡

7 O.) G- 3 MV 6 I (¡) c- -too = o.3 +t o= E o a 2 9' E

-50 0. 1 (¡) +t (-o 0 è o 40 80 t20 40 80 t20 incubation ( h) 136

TABLE 39: EFFECTS 0F S0l'{B INHIBITORS 0N RESPIRATION AND PR0T0N-Ì'{0TIVE FORCE IN NITROSOMONAS EUROPAEA.

hlashed cell suspensions in 50mM Tris-HCl at pH 6.9 were employed for Arþ and ApH {eterminations,. Atþ and ApH rvere calculated from lrtã uptár." oi [3u1 rtr+ ana tlac] benzoic-acid repectively as described in Section 2,2.I3, Respiration rates were determined at pH 7.8 by oxygen electrode technique (Section 2.2.1I). Conlrol ratã qf õ2 uptake rvas approxitatòly B5O ng aLom 0 min-l (mg protein)-1.

,a b c Concentration % Inhibition of ApH Ap Inhibitor (ull¡ 1t'ttl.+) respiration ^qr(mv) ('v)

None L47 0.18 158

DIECA 20 100 148 0.00 148

CCCP 10 80 110 0. 10 116 100 100 80 0.00 80

DCCD 200 50 IT7 0. 16 I28

DESB 50 25 r87 0.50 2L6 alnside negative blnside alkaline cfnside negative 137

TABLE 40: BFFECTS 0F SOME INHIBITORS 0N RESPIRATION AND PROTON-MOTIVB FORCE TN ATT?ROBACTER AGILIS.

lnlashed cell suspensions in 50mM Tris-HCl at pH 7.0 were employed for Aù and ApH determinatioris., AÜ and ApH were calculated from the uptake of [JU] tlt+ anA Ir4C] benzoic acid respectively as described in Section 2.2.I3, Respiration rates were determined at pH 7.8 by oxygen electrode technique (Section 2.2.1I). Control rate çf'ö, uptat e was .ppto*i*utely 650 ng atom 0 min-1 (mg protein)-1.

b Concentration % Inhibition of AUa apH Inhibitor (uu) respiration (tnv) ^p'(mv)

None 115 0.34 138

Sodium azLde 50 100 TT4 0.05 tL7

CCCP 10 70 82 0.20 94 50 100 75 o.r2 82

DCCD 100 55 116 0.38 138 250 100 118 0.08 113

DESB 20 45 L24 0.31 t42 50 85 133 0.09 139 alnside negative blnside alkaline clnside negaEive 138

irihibited NOl oxidation (also see 0'Kelly et a7,, 1970 and Sect.ion 3.1.6) and although it had no effect on Arþ it dissipated ApH completely, thus lowering ¡p by about 20mV. Because respiration in nitrifying bacteria has been shown to be inhibited by uncouplers (A1eem, L977; Bhandari and Nicholas, I979a,bi Aleem and Sewell, 19Bf; Section 3.1.6) and this effecL rvas related to the collapse of AtÞ in Nitrobacter (Cobley, L976a,b), the effecls of the classical uncouþler CCCP on Ap in both Nj trosonlonas europaea (Table 39) and Nitrobacter agiTis (Tab1e 40) were investigated. The respiration of both nitrifiers was completely inhibited by 50¡-uY CCCP but Ap was only partially collapsed (Tables 39'40). As shown in Section 3.1.6 several ATPase inhibitors restrict respiration in Nitrobacter agilis to an extent similar to ATPase itself. To investi- gate whether this effect rvas related to a collapse of Aprtwo ATPase inhibitors, DCCD and DESB' were usecl in studies on atþ and apH' DCCD at high concentrations ()200W) affected Ap in both nitrifiers by lowering Arþ in Nitrosononas europaea (Table 39) and ApH in Ni trobacter agilis (Table 4O). DESB had litt.le or no effect on Ap in Ni trobacter agilis (Table 40) but at SOUl,l it elevated Ap by about 60mV (negative inside) in Nitrosononas europaea.

3.5.2.7 Effects of ruuf and NH,OH on Arþ and ApH

Permeant amines and amine-like compounds have a tendency to redistribute across the membrane tohlards the acidic side in response Lo a pH gradient (Krogman et a7., L959¡ Good, 1960). It has been shown that high concentrations of substrate (NHl, NH2OH or NrHr+) in Nj trosononas europaea tended to diminish proton pumping (Section 3.5.f.4). As shown in Fig.49, both NHf and NH,OH at high concentrations diminished the smal1 ApH across the ce11 membranes of Nitrosomonas europaea completely. Moreover, *nl also decreased Aü (17OmV to l4OmV at 100mM Ufff) Uut relatively small concentrations of NHrOH (=20mt"t) rapidly dissipated Aü by abouE 60mV. Increasing the NH20H beyond 20mM did not dissipate Atf any further. ivitrobacter agiTis does not oxidise NHI or NHTOH, but it can assimilate small amount of NHOC1 (Sect.ion 3.2.I). High concentraions of Nu[ ()10mM) clissipaLed ApH completely and A$ partially (20mV at toomM NHI). 139

FIG.49: EFFECTS OF NHÍ AND NH?OH ON AÛ AND APH IN I^/ASHED CELLS OF N TTROSOMON AS ÊUROPAEA:

EDTA treated cells r{ere suspended in 5omM Tris-HCl, pH 6.9. arþ was determined from the uptake of [3H] rpp+ after treatment. of the suspensions wit.h eirher NHOC1 (O) or NH2OH (e) at indicated concentrations. apH was calculated from the distribution of tlac] benzoic acid in the presence of either NH4c1 (v) or NH20H (^). For derairs of uprake srudies see Section 2.2.I3.3. FlG.49

-r50

-roo

o. I -50 apH

o o o 50 roo

rrt\,,l( NH{or N jwH) 140

3.5.3 ATP biosvnthesis dri ven by artificiaTTy induccdproton notive force

3.5.3.1 ATP biosynthesis in .Mitrosononas europaea

By use of K* ionophore valinomycin, ATP biosynthesis has been demonstrated in intact bacterial cel1s, under the condiÈíons of artificially created proton-mol-ive force (Maloney et a7., I974i hlilson et a7., Lg76). The addition of valinomycin to a cell suspension in Kt free medium results in K* efflux creating a Arl.r (inside negative) which in turn can drive ATP biosynthesis. A similar approach has been used in the present study to demonstrate ATP synthesis in Nitrosononas europaea. Changes in intracellular ATP levels were monitored using the firefly luciferin-luciferase method of ATP determination (Stanley and lrlilliams , 1969).

The addition of NH4C1 to a cell suspension at pH 7.5 in 0.11'{ sodium phosphate buffer resulted in a rapid increase in intracellular ATP levels (Fig.50). trlithin 5 min of adding the substraLe there was a 2.5 f.oLd increase in ATP levels and this value decreased slowly, levelling out after 10 min (Fig.50). At pH 6.0 however, the addition of NH4C1 did not produce ATP since at. this pH substate oxidation is completely inhibited. The addition of valinomycin (100ug) did not induce any A'IP bi osynt.hesis in washed cells indicat.ing that this compound is probably impermeable to the cells (also see Section 3.5.1.4). Thus experiments were conducLed with spheroplasts prepared from washed ce1ls according to the method of Bhandari and Nicholas (1979b). The addition of NHOC1 to a suspension of spheroplasts produced a synthesi-s of ATP at about half the rate of washed cells (Fig.51). The addition of valinomycin to spheroplasts in a potassium free medium resulted in a rapid increase in intracellular ATP levels followed by a slow decline (Fig.51). ATP synthesis reached a maximum within 1 min of adding valinomycin. Similar results were obtained when cells were pretreated with either DIECA (5qrM), 2-chloro-6-trichloromethyl pyridine (N-serve) (100uM) or CO (20 min bubbling) to inhibit NHf oxidation, indicating that valinomycin incluced ATP biosynthesis was not associated with endogenous Ufff respiration or a possible lfU[ contamination. ATP synthesis induced by valinomycin was inhibited completely by DCCD (Fig.52) an inhibitor of cuz*, Mg2+ lTpuses indicating that a functional ATPase is required for I4T

FIG.50: ATP SYNTHESIS IN I^/ASHED CELLS OF /VI?ROSOLIONAS EUROzAEA.

['/ashed cells (500mg wet weight) were suspended in 200n1 of NII¿C1 free growth medium (Secrion 2.2.r) and aerared for 2h. cel1s were harvested by centrifugation at 15,ooog for 10 min, washed once with 0.lM soclium phosphare (pH 7.5) and then s'rspen$ed in 100m1 of the same buffer at appropriate pH. For NHj induced ATP synrhesis rhe buffer at pH 7.5 and in all ottier experiments it was.,at pH 6.0. "a"Five nl of each suspensions (5mg wet weight m1-') was used for each experiment. At. zero time, 0.4m1 aliquot k/as renoved to determine the basal ATP level, then either 2mM NHrCI (e) or lOOyg valinomycin (o) was added. At the times*indicared 0.4mi aliquots were removed for ATP assay by firely method as described in section 2.2.r7.5. All experiments v/ere carried out at 3ooc in a waterbath shaker.

FIG.51: VALINOMYCIN TNDUCED ATP SYNTHESIS IN SPHEROPLASTS OF NITROSOI4ONAS EUROPAEA.

spheroplasts (400mg hret weight) prepared from starved cel1s (Fig.50) by the merhod Of Bhandari and Nicholas (Lglgb) were sus_ pended in 100m1 of 0.1M sodium Bhosphare conraining O,2M sucrose and 1mM MgClr. For ttH] inãuced ATp biosyñrhesis experiments the pH of the suspeñding medium was 7.5 while for valinomycin experiments it was 6.0. Five ml of spheroplast suspension was used for each experirnent. At zero time, either 2mM NHr,Cl (O) or 100pg valinomycin (O) was added. Further detaiÏs are given in Fig.50. pmol ATP produced (mg wet wt.)-l ATP produced (mg wet wt.)-l ¡rmol o o o a oa a o a I o a aN (,¡ s N À o) @ o o

('l Ul .+ -t. J 3 (D (D ¿ =.J =' o õ

l l 6) 6) a (¡a (¡ ; t o r42

FIG.52z EFFECT OF DCCD ON VALINOMYCIN INDUCED ATP SYNTHESIS IN SPHBROPLASTS OF NITROSOMONAS EUROPAEA.

Spheroplasts hrere suspended (4mg wet wt. mf-l) in a reaction mixture described in Fig.51, supplemented with DCCD (50-100UM). Valinomycin (100¡tg) was added aL zero time. Experimental deLails as in Fig.50 and 51.

v:j-rhour DCCD (6); wirh DCCD (O).

FIG.53: EFFECT OF KT ON VALINOMYCIN INDUCED ATP SYNTHESIS IN SPHEROPLASTS OF NITROSOMONAS EUROPAEA.

The reaction mixture described in Fig.51 was supplemented with 2,5,7 or 10mI4 KCl. Reaction v/as started by the addition of 100yg valinomycin at zero time. Experimental details as in Fig.50 and 51.

no KCI (o); 2mM KCl (o); 5mM KCl (a); 7mM KCl (a); 10mM KCl (v). pmol produced (mg l ATP wet wt.f ¡r mol ATP produced(mg wet wt.)-l

ao ao ao o o (¡l o a I N s tu C^l + o o

ru Jfu (D=' (D ol clr ==. =. s s .T Tl (Jì 6) (¡ c) a a (¡ C'l (¡t N 143

ATP biosynthesis in Nitrosononas europaea,

The synLhesis of ATP is dependent on t,he magnitude of the membrane potential (AU). In the absence of K+, the addiLion of a Kt ionophore (valinomycin) results in rapid exLrusiorr of K* creating a proton-motive force towards the inside of cell. An inhibition of ATP synthesis mediated by valinomycin would be expected should the ¡q collapse on adding external K*. The results in Fig.53 clearly show that this is so, since K* reduced valinomycin induced ATP biosynthesis and cornpletely inhibited iL at 10m1"1 concentration.

As these experiments were conducted at pH 6.0 the cells would also have a ÀpH (inside alkaline) to add to tocal Ap.

Nigericiq another K+ ionophore which results in K+/H+ exchange across the membranes, would create a ApH (Ínside acid) if added Lo cell suspension in K* free medium, Thus theoreLically the addition of nigericin to cells should result in a decrease in intracellular ATP because inside acid ApH would lower the Arþ (inside negative) so that ATPase would work in the direction of ATP hydrolysis instead of ATP synthesis. The results were, however, contrary to this anticipaLed result since nigericin appeared to acE like valinomycin. In spheroplasts, nigericin induced ATP synthesis in K* free medium which reached a maximum at 3Os after its addition, followed by a rapid decrease. hlhether this un- expected result was due to membrane compartmentation and some localized pH gradient (inside alkaline) or due to some non-specific ion upLake is not clear. However in a fluorescence quenching experiment (Fig.54) nigericin appeared to carry out classical K+/H+ exchange. The addition of nfff to a spheroplast suspension resulted in fluorescence increase (Fig.54a) typical of results of Section 3.5.1, and as described by Bhandari and Nicholas (1979b). Addition of nigericin produced a slow decrease in fluorescence indicating an inwardly directed proton movement (Fig.54b). tJhen nigericin was added first, followed by NHOCI the proton extrusion was inhibited but this effect was reversed by adding KCI (Fig.54b). When KCI was added first, subsequent addition of nigericin resulted in proton extrusion (Fig.54c). The addition of KCI to nigericin-treated spheroplasts produced an extrusion of protons (Fig.54d). The possible j-on exchange reac¡ions observed in the experi- r44

FIG.54: NIGERICIN },ÍEDIATED K+/H+ EXCHANGE IN SPHEROPLASTS OF NITROSOMONAS EUROPAEA.

Spheroplast.s were prepared according to the method of Bhandari and Nicholas (1979b). To 2.5m1 0.lM Tris-HCl buffer (pH 7.5) supplernented with 0.214 sucrose and lml"l Mgcl, in a 1cm cuvette were added 0.lumo1 quinacrine hydrochloride and 0.lm1 spheroplasts (50mg wet weight). The following additions (*) were made: NH4CI (70umo1), nigericin (NIG) (5oue) or KCl (200¡rmol). Fluorescence emission was determined (arbitrary units) in a Fluorispec model SF-l fluorimet.er (Baird AÈomic, Mas.sachussetts, U.S.A.) as described in Section 2.2.!2.I.

FIG. 55: SCHEMATIC REPRESENTATION OF NIGERICIN MEDIATED K+/H+ EXCHANGE ACROSS THE SPHEROPLAST MEMBRANE. FlG. 54

(a)

+ NH 4 I (b) KCt

NIG N IJ I g) T* ÉttJ NTå zO (c) trJ ()z KC NG IJ O a UJ oÉ. (d) l J l! NIG KCI

FlG. 55

out ln

UJ z K+ É. H+ co NIG H+ H+ UJ K+

(a) (b) (c) 145

mellt are shown in Fig.55. In K* free medium, the addition of nigericin would result in loss of intracellular K* and its replacement by H+ (Fig.55b) resulting in decrease in quinacrine fluorescence. However in K* containing medium, nigericin addition would result in K* uptake associated with H* extrusion (Fig.55c) and hence increase in quinacrine f luorescence

3.5.3.2 ATP biosynthesis in /Vitrobacter agilis

Attempts to show valinomycin induced ATP s)'nthesis were largely unsuccessful. Cells usually maintained high levels of intracellular ATP. Aerating cells for up to lOh in a NOJ free medium had little effect on the intracellular ATP pool. The addition of N0, to a starved cell suspension resulted in an increase i-n intracellular ATP similar to that shown for lVitrosononas euîopaea (Fig.50) but about half'the magnitude. The endogenous ATP varied between 0.5 to 2.Omlul in ten separate batches of cells used in the study. Addition of valinomycin (10-50¡rg *l-1) to a cell or spheroplast suspension in a K* free medium resulted in a slow and variable response. This response plus the N0; induced intracellular ATP changes were completely abolished by preincubating cells with 50- lOOUl'f sodium az]rde, an inhibitor of N0, oxidation (Section 3.1.6) indicating that valinomycin response was probably associated with some phenomenon other than the generation of an artificial AÙ. Preincubation of ce11s with eithr DCCD (100-200U1"1) or DESB (20-100U1'f) drastically reduced the intracellular ATP levels (0.05 - 0.O8mlf) and these treated cells did not synthesize ATP when either N0, or valinomycin were added to a K* free medium.

+ + 3.5.4 Na and K trans rt

The apparenL absence of proton translocation in Nitrobacter agiTis posed a problem : how is energy conserved in this bacterium? One of the possible alternative mechanisms involves a respiration dependent primaty N.+ pump as has been reported in Vibrio aTgirtoTyticus (Tokuda and Unemoto, 1981). HaTobacteriun haTobiurn also exlrudes Na+ by either a Na+/H+ antiporter (Lanyi and MacDonald, Lg76) or a Na* pump, halorhodopsin (Lindley and MacDonald, L979)' In this Section the existence of such systems in Nitrosononas europ¿¿y and Nitrobacter agilis is explored. Since Na* and K+ transport systems play an r46

important role in overall bioenergetics of the cell' their effects on Âp have been investigated.

3.5.4,I Preparation of K+ depleted cells

Bacterial ce11s usually contain high concentrations of K* and comparatively low amounts of Naf. To cfraracterize the K* and Nt* tran=- port systems ít is necessary to deplet.e cellular K* and to manipulate the internal cation coucentrations of cells without damaging the transport systems. A simple and novel method of K* depletion of bacterial ce1ls has been described by Nakamura et a7. (1982). The n,ethod involves treating cells $/ith DEA at alkaline pH (see Section 2,2.L4,I), The intracellular concenLrations of K* and Na* in both Nitrosononas europaea 6nd Nitrobacter agiTis determined by atomic absorption spectroscopy varied greatly from one batch of cel1s to another (Table 41). The variation was due in part to the number of cell washings and duration of storage prior to deLermining the K* and Na* contents of cells. When the cel-ls of either Nitrosononas europaea or {itrobacter agiTis.klere suspended in 50mM Tris-HCl (pH 7.5 - 9.0) containing 150mM NaCl, a slow extrusion of intracellulor K* occurred (Fig.56). In the presence of SOmlvl DEA-HCf (pH 9.2), both Nitrosononas europaea and Nitrobacter agiTis rapidly lost intracellular K*. Thus after 10 min of DEA treatment there r¡ras a loss of about 80 and 952 intracellular K* from Nitrosononas europaea and Nitrobacter agilis respectively (Fig.56). There v/as no net entry of Na* even when Ehe reaction mixLure conEained 150mM NaCl in addition Èo 50mM DEA. The intracellular concentration of Na* in both bacteria remained constant during DEA treatment. The DEA trated ce11s contained (5mM K+.

3.5.4.2 Respiration in K+ depleted ce1ls

The respiration rates of untreaEed and DEA treated ce1ls of both Nitrosononas europaea and Nitrobacter agiTis with and without KC1 are shown in Table 42. The K+ depleted (DEA treated) cel1s of Ni¿r;osononas europaea retained about 837" of the NHf oxidising capacity and, 747. of the NHrOH oxidising activity of the untreated ce1ls. The NOj oxídising activity of Nitrobacter agilis was little affected by K+ depletion. The addition of K+ hud no effect on NIlrOH oxidation by NitrosonplaP r47

TABLE 41: INTRACEI,T,ULAR CONCENTRATIONS OF NA+ ANd K* IN I'/ASHED CBLLS OF NTTROSOTTONAS EUROPAEA AND AIT?ROBACTER AGILIS.

Freshly harvested cells were washed once in 50rnM Tris-HC1 buffer (pH 7.5) and then suspended in the same buffer (25 mg wet wE. ml-r). Aliquots (100-200u1) filtered through Millipore filters (O.22ù were washed once with 2m1, 0.2M choline chloride and Na* and K* r+ere determined in TCA extracts of ce1ls by atomic absorption specLroscopy, as described in Section 2.2.I4.2.

fntracellular conc.entration (mll) Batch Nitrosononas europaea Nitrobacter aqilts Nar ¡+ Na+ 1+

1 80 85 25 160

2 s9 r46 37 290

3 73 170 30 270

4 62 99 45 310

5 65 135 Nd Nd

Nd - Not determined. 148

FIG.56: TIME COURSE FOR K+ EXTRUSION FROM THE CELLS OF (a) NrrRosoMoNAS EUROPAEA AND (b) NITROBACTER AGILIS DURING DIETHANOLAI"I]NE (DEA) TREATMENT.

I'reshly harvested cells (approx. 500mg wet wt.) washed twice with lOOml"f K+ phosphate buffer (pH 7.5) were suspended in 4ml of'10mM HEPES-NaOU (pH 8.0). To start the reaction, Lhis cell suspension was added to 2lml of 50mM DBA-HCI, 150mM NaCl (pH 22NuCt. 9.2) containng 4 UCi *t-1 Aliquots withdrawn ar times indicated were dispensed into lml of 0.4M choline chloride and filtered immediately through llillipore filters (0.45U). The cells on the fi-lter were washed twice with 2m1 of 0.4M choline chloride. K+ was determined in TCA extracts as described in Section 2.2.14.2. Tn control experiments (O) the cel1 suspension in HEPES buffer was diluLed wiLh 50mM Tris -HCl buffer (pH 7.5-9.0) instead of DEA-HCI (O). The uptake of 22*u+ (¡) was determined after drying the filters for th at 100'C and then immersing them in 10m1 of toluene based scinti- llaËion counting fluid (O.37" w/v PPO and 0.O37" w/v P0P0P in toluene). Radioactivity (22Uu*) was measured in a Packard Tricarb 460 CD scintillation spectrometer. FlG. 56

( (b) too a)

80 .Ë (l,x + )¿ 60 o oL (¡) l< ((t {-t CL 40 += ct .F 20 ie

o o to 20 30 40 0 to 20 30 40 incubation time (min) r49

TABLE 42: BFFECTS OF K+ DEPLETION AND K+ ADDITION ON RESPIRATION IN NITROSOMONAS EUROPAE,A AND NITROBACTER AGILIS.

K+ depleted cel1s were prepared by DEA treatment as described in Section 2.2.14.1. Respiration rates of bacteria determined by Clarke-type oxygen electrode (Section 2.2.II) are expressed as ng atom t0t nlin-r (mg protein)-t.

Respiration Bacterium DEA K* additionb Treatment Substrateu raLe

Nitrosononas europaea NH4Cl 240

NH4C1 + 160

NH20H 760

NH20H + 7 /+O

+ NH4Cl 200

+ NH4C1 + 140

+ NH20H 560

+ NH20ll + 540

Nitrobacter agiTis NaN0, r20 NaN0, + r20 + NaN0, 110 + NaN0, + 110 tTh" .on.entration of each substrate was 2.5mM bK* KCI (25ml"f). "u" added as 150

europaea and N0; oxidation by /Vitrobacter agilis in either untreated or DEA t.reated cells, but it inhibited NHf oxj claLion j.n both treated and untreated cells of Ni trosononas europaea. Thus 25mM KCl inhibited NIII oxidation to a similar extent in untreated and DEA treated cells of

N itrosono'nas euro paea,

. ,.+ 3.5.4.3 ProLon-mot ive force in K' depleted ce1ls and the effecL of added K

The changes in the components of Ap, viz. ApH and Arþ, in K* depleted ce1ls of Nitrosomonas europaea and Nitrobacter agiTis as a function of external pH (pH.) are illustrated in Fig.57. The overall pattern of variation in ApH and Atþ in response to pHe in untreated ce11s of both bacteria was similar to that shor+n in Section 3.5.2. In K+ depleted cells of Nitrosomonas europaea A'J.r varied from 126mV at p$e 6.2 Èo 155mV at pH 8.2 while ínNitrobacter agiTis it varied from 100mV to 135nrV over the pHe range 6.2 Eo 8.2. The addition of 20mM KCl resulted in depolarization of Ârþ by about 5mV in l,litrosononas europaea and 10mV in ¡{i tròbacter agilis This depolarization of AÜ was independent of pHe in both bacteria (Fig.57). During the pHe changes frorn 6.2 Eo 8.2, ApH in K+ depleted cells of Nitrosononas europaea and Ni'trobacter agiTis varied from -34 Eo -5mV and from -44 to +12mV respectively. The addition of K+ (2OmM KCI) to K+ depleted cells resulted in a concomitanE increase in ApH (alkaline inside) by about 5mV in N.ítrosononas europaea and 10mV ín Nitrobacter agì1is Again these changes were independent of pHe. Thus the partial depolarization of Aü by K+ in K+ depleted cells of bot.h bacteria was compensated by an equivaleht increase in ApH so that the total proton-motive force remained almost constant in Kt depleted cells and in those supplemented with f+ lFig .57). These results indicate that t.he inward movement of K+ in boEh bacteria is electrogenic which leads to depolarization of the membrane (decrease in Âü, inside negative), which in turn allows the cel1s to pump more protons into the medium (increase in ApH, inside alkaline).

3.5:4.4 22Na+ loading of Kt depleted cells

The results in Fig.56indicate tha¡ there was no intake of Na* associated with K* exiC by the cel1s of either Nittosononas europaea or 151

FIG.57 : F.FFBCTS OF EXTERNAL pH (pHe) ON ApH, ATI RND Ap IN T+ onpLpTeI CELLS OF (A) NITROSOMONAS EUROPAEA AND (b) NITROBACTER AGILIS AND THB EFFECTS OF ADDED K+.

K+ depleted cells h'ere suspended in 5omM sodium phosphate at the pH values indicated. uptake studies were carried out as described in secrion 2,2.13.3. apH, represented in terms of mv ie. 59 x apH(o¡ )was derermined wirh tlac] benzoic acid; arü values(aa)were carculared from the uprake of [3tt1tle*; Ap(nl )was determined from rhe ApH and ArJ.r values (Ap = Atþ-59ApH). Open symbols, without KCl; closed symbols, + 20mM KCl. FlG.57 (a) (b) -t50

- too

MV -50

I I I 6 7 I 6 7 I pHe pHe L52

Nitrobacter agilis during DEA treatment. If DEA is removed fron the reaction mixture by rvashing with a Nat containing buffer (eg. Tris, Hepes or phosphate) the cells readily take up Na+. The tirne course of 22Nu* uptake of by DEA Ereated cells of Ni trosononas europaea and Nìtrobacter agilis respectively are shown in Fig.58. Thus when the 22*u+ amine loaded cells of either bacterium were exposed to there was 22Nu+. 22Nu+ an immediate uptak" of The exLent of accumulation of was dependent. on the concentration of DEA in the exEernal medium. Thus h,ith 50mM DEA, no net entry of.22Nu was observed in either bacteriurn. Ih t.he absence of DEA however, both Nitrosononas europaea and Nitrobacter agilis accumulated Na*. Thus after about 20 min incubation with 22Na* (*t'¡r*) cells reached an equilibrium state and contained 150- 200mM intracellular Na*.

3.5.4.5 22Nu* extrusion fro* 22Nu* loacled cells 22Nu* loaded cells were prepared as described above (Fig.58) and 22Nu+ an ac¡ive extrusion of from the ce1ls was determined by a filtration method at ZSoC (fig.59). Tn Nitrosononas europaea only aboút 20% 22*^+ 22N.+ r,¡as extruded from cells when K*rvas added to loaded cel.ls 22Nu* (Fig.59a). In r.he presence of CCCP, the addition of K+ to loaded 22N"+. cells ef NiËros omonas euroPaea did noÈ result in an extrusion of 22Nu+ In Ni trobacter agiTis 22fu+ loaded cells, the extrusion of. (Fig.59b) required K+ as a counter ion permitting overall neutrality. Thus the addition of K* resulted in about 80% loss of Naf from the cells 22Na+ in 15 min. The a,ldition of CCCP completely inhibited extrusion from Èhe cells of /Vi trobacter agiTis indicaLing that the driving force for Na* exl-rusÍon is Âp. To check whether there was any respiration driven Na+ pump in these bacteria, the effects of NHf onNitrosononas 22fu+ europaea and NO, in IVi trobacter agiTis on the were studied. 22*u+ "ystem The addition of NHOC1 to loaded ce11s of eirher Nitrosononas 22Nu* europaea or Ni trobacter agiTis, resulted in a rapid extrusion of (Fig.59a,b). However this loss was not respiration dependent because t.he addition of diéthyldithiocarbanate, an inhibiÈor of NHf oxidation by Nitrosononas europaea (Table 39), prior to NHOC1 did not prevent the 22Nu* ext.rusion of in Ni trosomonas europaea . This was also confj-rmed by replacing NHf v/ith CH3NH, (which is not oxidised by the cells), which 153

')) r FIG.58: UPTAKE 0F --Na' BY DIETHANOLAI'{INE (DEA) LOADED CELLS 0F (a) NTTROSOMONAS EUROqAEA AND (b) NITROBACTER AGTLIS.

K+ depleted cel1s, prepared by DEA treatment as described in Section 2.2.f4.1, hrere washed once in 50mM Tris-HCI (pH 7.5) and then suspended in the sarne buffer (45Omg wer wt. ml-1))a '). For --Na' uptake studies 100p1 of this cell suspension v/as diluted to lml with 100mM, Na-HEPES buffer 22NuCt (pH 7.5) conraining (4 pCi ,nt-l). Aliquors (IOOu1) were t.hen withdrawn at various times and diluted with lml of cold 0.2M choline-chloride, 10mM Tris -HCl (pH 7.25) and filtered immediately through Millipore filrers (0.45U) and washed with 2nL of choline chloricle buffer. The filters, dried at 100oC for th were immersed in 1Oml scintillation fluid and radioassayed as described in Fig.56. Radioactivity was corrected for filter bound 22Nu*. FlG. 58

(a) ( b) roo

60 @ J C' +tè 40 += ct z, OJ N E ?o

o o ro 20 õo 40 0 lo 20 30 40 incubation time (min) rs4

22Nu* 22uu+ Frc.59, ExrRUSroN tr'Rot'r Lo¡,reo CELLS oF (a) NrrRosoMoNAS EUROPAEA AND (b) NITROBACTER AGILIS. 22*u+ loaded cells were prepared as described in Fig.58. 1tr The arnounts of "Na- retained by the ce1ls were determined by the filtration method described in Fig.56 and Fig.58. The following compounds were added at zero time:

20mM KCI (r); 20ml"l KC1 + 20uM CCCP (tr); 10mM NHOC1 (o); 5mM llaNO, (o); 10mM NHOC1 + 20uM CCCP (v); 10mM NHOC1 + 200uM DIECA (r); IOmM methylamine-HC1 (O). In the absence of any additions there $/as no net loss of 22Nu* during the period of incubation. FlG. 59 t20 (a) ! I (¡t () qt -Ê 80 I

-c¡ '9 c, 'ãL c,

I 'ctz 40 N E

o 5 ro 15 20 0 5 ¡o 15 20

incubation time (min) 15s

22fu+ also resulted in extrusion from the cells. This, together with 22fu+ NH4C1 dependent loss from Nitrobacter agilis cells indicates that + NHi acts as a permeant amine lilce diethanolamine and methylamine and is transported into the cells of both Ni-trosononas europaea and fii trobacter agiTis in its unprotonated form (NH3) by passive diffusion. There was no evidence of a respiratory driven Na* pump in Nitrobacter agiTis 22Nu* because the addition of NO, to loaded cells did not result in the 22Ñu+ exLrusion of (Fig.59;). Ir should be nored here rhar Na* loaded cells of boEh bacteria actively respire in the presence of an appropriate oxidisable nitrogen substrate, so the apparent lack of a respiraLion dependent Na+pump could not have been ;aused by respiration Ioss.

15N 18 3.5.5 StabTe isotope experinents with and 0 7abe77ed compounds to

studye¿ NOí pZtiellpyby_cel_1_s _oJ Njtlpþqcter asiLis

The inability of Nitrobacter agiTis to translocate protons during respiration led to postulation of alternate energy.conserving mechanisms in this bacterium. Dr. T.C. Hollocher of Brandeis University (U.S.A.) suggested (private communication) that the bacteriurn might synthesize ATP by a substrate type phosphorylation involving a mixed anhydride between either NOa and ,OO'- or NO, and ADP. Should this concept be correct., the t0t in NO^ oroduced by N0, oxidation in Nitrobacter agiTis would come from ,OO'- as fotlo"i:

Pi as nucleophile

t+ 9- og -----+ 0^P-0-N /lo ATP + 0-N0 ,\ \o- 2 ADP-O- ADP oxygen as nucleophile

a rù +,zo x- ADP-O + N0^ _> ADP-O- ADP-O-Po N03 z *\a- 3 + \ 03P0

Cellu1ar ATP 6_ ADP_O- Pos hydrolysis ADP_O +Pi ! ßx+ ADP_Ô_ + NOt ADP_O-N /o ---+ nneöro, * ðno; t \ --> 03P0 1s6

Thus this mechanj-sm \{as suggest-ed to involve NOj as an intermediate during NOj oxidation. It has been shorvn that the source of oxygen during N0, oxidation by ['litrobacter is HrO (Aleem et al., 1965), based on the incorporation of 0.044 180'Ín.o to 0.078 aLom % NO] from 82 aLont % nrtuo. The following exper:i- tOt ments were designed to check the source of in NOJ produced as a result of el_1s were j_ncubated with nitrite (NO; ompounds , ur18o (90-97 atom %) ,'uoi 0 atom Z). Two distj.ncr mel,horls (GC/ alysis of the product of N0, oxidation (nitrate). For GC/MS studies, nitrate was converted to NrO and phosphate to trimethyJ phosphate which were then analysed as described in detail in 151¡-l,ll"1R Section 2.2.15.I. For analysis, the reaction rnixtures were analysed as such, after separation of ce1ls by centrifugation (Section 2.2.L5,2).

3.5.5.1 GCIMS studies NO oxidation of 2

18 3.5.5.1.1. Changes in 0 contents of phosphate duri-ng N0, oxidation

The mass spectra of a comrnercially available sample of t.rimethyl phosphate and one prepared by the methylation of Hrel8oo are shown in Fig.60A and B respectively. Fig.60C illustrates the spectrum of trimethyl phosphate prepared ftot el8Of- which had previously been incubated wirh N0, and the cells of Nitrobacter agiTis (Section 2.2.r5.Ð, By esrimar- ing the abundance of trimethyl phosphate molecule (M+) peak at 140 amu in Fig.60A and five other fragrnentation ions at 110,109,95,80 and 79 amu and comparing them with corresponding amu values in Fig.608 and 6OC, it was 180 established Ehat there was no apparent loss of .ont"nt of phosphate during N0, oxidation by Nitrobacter.

15N180 3.5.5.r.2 contents of nitrate produced by the oxidation of nitrite

A summary of the results of isotope experiments in which the product, 15N 180 nitrate, was analysed for und following its conversion to NrO gas (section 2.2.15.1) is given in Table 43. I{hen unlabelled NO, was I80 oxidised by ce1ls of l/j trobacter agil is the only source of which was significantly incorporared v/irh NO; r." Hrl8o (Fie. 6l). rn rhis experiment, the normarized integral at 46 amu(Nr1B0) of 7.48 was in reason?ble agreement with the value of 7.BB expected if the Hr1B0 ,hr.h was used in 157

FIG.6O: MASS SPECTRA OF TRIMETHYL PHOSPHATE.

Commercíal sample (A) was purchased from Sigma Chemical Co., ttO, St. Louis, U.S.A. trimethyl phosphate (B) was prepared by the methylation of HrelBOO wiLh diazomethane as described in Section 2.2.15.1. Spectrurn (C) r+as obtained 180 for trimethyl phosphate prepared from the pho=phutu recovered frorn a NO, oxidising system (Section 2.2.L5,1) at the end of incubation period. For further details see Section 2.2.I5.I.

a FlG. 60

(A)

''i " "i ' :""'-i - "1 " : 'i ""-'¡"- 'i " 1"

I r... r .r I r 1.. ! :i ¡,ii l i:i

¡ I Íti n it { I I t: ll I I : ""'!"' 'i'" --l-'-"!'-"'¡ '"-l ¡ I f"' ,_. . lf ! I li.

(c)

riifi irl ¡, i ä l il U ii ..i!,.-i¡-"'l i¡li ,'i.""' " ' r '! "''i" - i'-' 1-'1 I a t::¡.¡ l::. ti I i ;1 ¡::l .t TABLE 43: STABLE ISOTOPE EXPERIMENTS LIITH \^IASHED CELLS OF A/TTROBACTER AGILIS. The incubation mixtures \.{rere prepared as described in Section 2.2.15.1. Each mixture contained 100mg (wet wt. ) of cel1s. Following the complete oxidation of N02, the NOj was recovered and converted to Ñ20 as dáscribed in Secr.ion 2.1.15.1. The derails of GC/MS'technieuõ are described in Section 2.2.I5.I. Dãta were collected, background corrected and processed under the Selected Ion Monitor or Peakfinder programme which assigns an iãtegral of 100 to the largest (base) peak of the set. For comparison values i,ere calculated (in parenthes:-s) assuming that,only one of.,5he three 0 atoms arose directly from water during oxidation'of ÑO¡ and that of stocks of r)NO2 and H2lõO used were as specified in Section 2.L.2. Null values at the fivã amu values indicated were typically 0 to 0.2 after background correction.

Normalized inteqrals for N 2 Incubation mixÈ.ure 44 45 l8o 15N 15N l5N lB0 N 0 n15l,lo N + o Nr5N18o 2 2 2

(0.73) (0.20) (o) (0) NO 't*Hzo+rof + alr 100 0.20 0.rs o.o 0.0 _ tq 2- NOr+Hr^"O+P0 + air 100 0.20 (o.72) 7.48 (7.88) 0.0 (0.0s) o.z (0) 4 ')_ ¡i0 f Hzo + ptSo + air 100 0.80 (0.73) 0.21 (0.20) o.o (o) 0.0 (0) 2 4 NOr+Hro+rOf+180, 100 0.42 (0.73) 0.18 (0.20) o.o (o) 0.0 (0) lq _ t- '-NOz +Hr1 +P0; +air 0.82 (I.oo) I00 0.20 (0.37) o.o (0.20) 0.0 (0) 15Ho- 180 *H2 * ro! + air 0.79 ( I .02) 100 3.504(0.44) 7.80(7.95) 0.5 (0.03 ) tt*ot2 1a ,_ 0.82 ( 1 .oo) 100 r.2oa(0.37) 0.41 (0.20) 0.0 (0) + H^0+P^"0;¿4 +air 't<-No, 2- 18 ^ + HrO + P0 T 0 0.83 ( 1 .oo) 100 i . loa(0.37) 0.31(0.20) 0.0 (0) 4 2

aDue largely to spillover from the base peak at 45 amu as the result of drifts in tuning. This was confirrned by use of the mode of data collection (Peak Finder Programme). =r""p (¡ oo 159

FIG.61 : MASS SPECTRA 0F N20 cAS

(A) a comrnercial sample (B) and (C) prepared by rhe pyrolysis of ammonium nitrate obtained from a lrIO, oxidising syslem with HrO und Hr180 respectively (Section 2.2.15.I), Data were obtained under the peak Finder programme. Spectra do,not include ions below 35 amu (lower cut off level was 35 amu) and thus fragments of NrO ie. NO+(i-on 30) and f,fj (ion 2g) are absenr in rhese specLra. Details of the experiments and GC/MS technique are described in Section 2.2.I5.I. FlG. 6 I

{-l *- - -'" -' --' "'' "" '-'l '* '''l -''l -- ^'r --' -¡' '" -1 -- -'l ^- I -' -'r '-r -' -r -- -'l tr. ¡ i t.1 ..-l l:.1 .!. ¡:-¡ ti ïi rll .i r;i i;::r ï;rjr 1 l¡ t:] G) G) (c) 160

1B the experiment had contained 95 atom % 0, assuming that only one oxygen atom of N0, had been derived from water and that the N0, stock contained

10% (w/rv) N0. ini-tially. Similarly in experiments, when 99 a:om 7" tt*o; 180 u=Jo instead of.unlabelled NOrr only fror rrtuo rvas signi- "u" tt*Oã. ficantly incorporated in The observed value (7.80) and calculated value (7.95) are inclose agreement (Table 43).

3.5.5.2 l5l¡-¡¡ltR srudies of NOj oxidarion

One of the advantages of using l5N-ttttqn is that rvith the aid of stable 1BO¡ isotopes 1151i una at" reactants and the products of a biochemicalreaction can be analysed directly. This overcomes any dilution or exchange reactj-ons associated rvith the processing of the samples. The results in Fig.62 are for an experiment similar to the one recorded in Table 43, except that 15t',t-tlt"tR t.he samples vere analysecl directly Uy (Section 2.2.15.2). The signals of variou" 15N und 180 ,,iatrte standards (Fig .62A) are essentially as reported by Andersson et a7. (1982) but only three peaks were observed, tt*tuo;, l5N160rl8o- 15N160180, corresponding to rnd as confirmed by tt*t6O; spiking resonance. The peaks were well resolvecl and separated by I.7L Hz (0.0563 ppm). A visible signal was observed after a few scans tt*0, when the concentration of v/as more than 40mM. Smaller concentrations required longer accumulation time.

15N0, lrJhen Èhe cel1s were incubated wirh with trt6o in 100mM pL6o?- buffer, only one resonance was observed which corresponded to tt*tuoã. tt*ot rn Fig.62B, the NMR spectrum of the product of oxidation 1BO, in the presence of 100% ," shoryn. Again only one peak was observed tt*t6O; with an isotopic configuration of indj-cating that^none of the 'O' l5tlO* in nitrate producea Uy oxidation was derived frortlSOr. I^lhen the 15N0, cells were incubated with and ttrl8O two major peaks and a minor one t5*t6ort8o- were observed, separated by 1.lL Hz and representing tt*tuo;, 15Nl6ottoã und respectively (Fig .62c). The ratio of areas of three peaks 15N160r180- 15NI60180, was 31.6 : 6: l. Thu" und isotope combina.tions t5,.t160;. were abou t L9% and 3 .27" respectlvely, of The spectrum of the 15UO; 1dO product of oxidation in the pr3sence o¡ phosphate (a11 4 'O' lBO) atoms labelled is shown in Fig .62D. Only one peak was observed "itf, i5N160- 15t'tO, which corresponded to jndicating that none of the '0' in are derived from pLgOl- durilg nítrite oxiclation by Ni trobacter ag+Li1 Iiì another experiment in which ce1ls were incubated for l8h with pLBOI- 161

180/160 FIG.62z NMR SPECTRA OF DERIVATIVES OF NITRATE.

15N-180 (A) lO0mM standard nitrate deríva.tives produced by chemical exchange (Secuion 2.I.2). 15N160- (B) 4omM, produced l5llluoã- 15N16 (c) zo*t't * duced by cells in pres t5¡r16og 2- (D) 5omM, produced 4 Experirnental details are described in Section 2.2.I5.2. FlG. 62

(A)

(B)

(c)

(D)

4 o-4 I Hz r62

15UO' ..ra the NMR spectrum vras similar to that observed in Fig.62D. Thus there appeared to be no measurable biological or chemical exchange l8o 15uoJ 15nor. of between either p18o1- and Hro, prSoî- una o. el8of-una

The observaLions of GC/MS and Nl4R studies taken l-ogether indicate that one and only one oxygen in NO, produced as a result of NOrroxidation by Nitrobacter agiTis originates from HrO and not from O, or POi , ruling out the possibilit.y of substrate level oxidative phosphorylaLion involving a P-O-N type intermediate. ,

r63

4, DISCUSS]ON

4.r NITRITE OXIDATION BY I^IASHED CELLS, SPHEROPLASTS AND I'{EMBRANE VESICLES OF N-TTROBACTER AGILIS

The oxidation of N0, by Nitrobacter has been studied in intact cells (Lees and Slrnpson, 1957), cell free extracts (A1eem and Nason, 1959) and membrane particles (0tKelly et a7,, L97O; Cobley, L976a,l:; Aleern and Sewell, 1981). In most of these sLudies, the utilization of N0, has been followed by a colorimetric method for N0, or by 0, uptake. In the present work electrode methods have. been developed lo monitor 0, uptake and NO, lroduction simultaneously and continuously during NOI oxidation by ce1ls, spheroplasts anC membrane vesicles respectively. Utilization of N0, has been followed by a colorimetric method (Nicholas and Nason, 1957). This approach to study NOI oxidation is parti-cularly advantageous because all the three paraneters of N0, oxidation, l.e. N0, and O, utilization and N0, production can be measured conveniently in the same reaction mixture.

The following stoichlometry of N0, oxidation by washed ce11s, 1N02 : 0.502 :0.75 NOJ indicates that some of the N0ã (or NOl) is retained by the cells and probably reduced to NHI vía assimilatory nitrate and nitrite reductases (l'lallace and Nicholas, f968). The different stoichiometry for N0, in spheroplasts and vesicles (INOJ : O.5OZ : lNOr) as compared wj.th that for washed ce1ls (1N0;:0.50r:0.75 NOJ) maV be associated with impairrnent of the nitrate and nitrite reducing systems i-n spheroplasts and vesicles.

0rKe11y et a7, (1970) found that the oxidation of N0, by rnembrane particles of Ni trobacter agilis was sensitive to a variety of metal inhibitors, however Aleem (1977) reported that the metal binding agents,B-hydroxy quinoline (8HQ), O-phenanthroline, o-otdipyridyland salicylaldoxime had little or no effect on NO] The recorded although z oxidation. results in this thesis indicate that N0, oxidation was sensitive to inhibitors such as 2-trichloromethyl-pyridine (2TMP) and BHQ, their effects could not be reversed by the addition of any metal salts and thus may be non-specific. The jnhibitors of cytochrome oxidase viz. azide and C0 also inhibited N0, oxidation in washed ce1ls, spheroplasts and membrane vesicles, and these results are consistent with earlier findi-ngs (0rKe11y et a7.¡ I97O; Aleem, 1977; Aleem and Sewell, 19Bf). In washed ce11s and in spheroplasts NO; oxi-clation was restricted by inhibitors of electron transport but these effects were not observed in membrane vesicles 164 indicating that in cells and spheroplasL these inhibitors can affecL a variety of metabolic functions. The fact that electron transporL inhibitors do not affect N0, oxidaLion in membrane vesicles substantiates the idea that N0, does not enter the electron transport cha:'.n at the level of either NADH, ubiquinone or cytochrome b (Aleem, 1977; AJ-eem and Seruell, 1981). The observations in the present rvork that N0, oxidation by washed cel-1s, spheroplasts and nembrane vesicles was inhibited by uncouplers are similar to l,hose of Cobley (1976a), Aleem (1977 ) and Aleem and Sewell (1981).

).t A Mg"' dependent ATPase has been detected in nembrane vesicles of Nitrobacter agiTis (Section 3.1.7). ATPase activity requiring either Mg2+ ot CuZ* has been found i-n a large number of bacteria (l"lachtiger and Fox, 1973; Abrams and Smith, L914). The enzyme, associated with menibranes, plays an important role in ion tr:ansport and ATP synthesis. Unlike the ATPase of Nitrosononas europaea (Bhandari and Nicholas, 1980), that of Nitrobacter agiTis was unaffected by uncouplers (egs. CCCP, DNP) but was strongly inhibited by classical ATPase inhibitors (eg. DCCD). One important observation in the present study is that the ATPase inhibitors (DCCD, vanadate, diethylstilbestrol and NBD chloride) also restrict N0, oxidation to approximately the same extent as for ATPase itself. The ATPase however was not affected by the inhibitors of N0" oxidation (eg. uncouplers). This indicates a possible functional ¿ relationship betvreen N0, oxidase and ATPase in the bacterium. The delipidation of membranes by phospholipase A, treatment results in a loss of ATPase activity suggesting a possible role of membrane conformation and phospholipids in maintaining the activity of this enzyme.

4.2 ASSIMILATION OF INORGANIC NITROGEN COMPOUNDS IN NT?ROBACTER AGILIS AND ]VTTROSOI"IONAS EUROPAEA

4.2.1 Pathways of nitrog en assiniTation in Nitrobacter aailis

Although numerous studies have been done on the mechanism of N0, oxidation by Nitrobacter agilis, the further assimilation of NOJ (or NOr) into cell nitrogen is not well understood. An attempt has been made to delineate the pathways of assirnilation of inorganic nitrogen compounds in this bacterium.

The experiments reported in Section 3.2 clearl-y show that washed cells of Nitrobacter agiTis incorporatuA l5llOJ, tt*Oã, l5NHrOH and tt*nl respectively tt*t} into cell nitrogen; of these was most readiiy utilized. These results are in agreement with those of In/allace and Nicholas (f968). This also 165

confirms the finding that NH) enhances the grotvth of this bacterium in pure culLures (Section 3.2.1). Thus in i,ts natural habitat,'4 NI{} wouJ.d be an important source of nitrogen for Nitrobacter agi.7is.

Cobley (I976a) reported that N0; oxidation by electron transport particles of Ni trobacter winogradskyi was stimulated by NHOC1 (maxirnum stinu- lation 357" at 2mM). In the present study with washed ce1ls by /Vitrobacter agiTis, no such stimulation was observed. Although 2m}'l NH4CI stimulated the growth of the l¡acterium, higher concentrations inhibited N0, oxiclation. This effect !/as reversed by increasing the concentration of N0,, indicating that it vras competitive (Section 3.2.2). This phenomenon could have ecological significance in regulating the overall nitrification of ammonia to nitrite" High concentrations of NHf would inhibit NO, oxidation until such time as enough N0, r,,as produced (eg. from the oxj-dation of nHf by Ni trosomonas) to overcome the inhibition. Since NHf, even at relatively high concent.rations does not inhibit N0, oxidation completely (Fig. 12) , its accurnulation would only retard and not completely inhibit the rate of NOJ conversion to NOJ.

In most bacteria assimilating ammonia directly as the source of nitrogen, glutamate dehydrogenase (GDH) is usually a key enzyme, producing glutamate. Under these conditions glutamine synthetase (GS) has a relatively low activity (trdoo1fo1k et a7., 1966; Bender et a7., L977; ELy et a7., f97B). In Nitrosononas europaea, GDH funcÈions primarily in the direction of glutamate production (Hooper et a7. , 1967). This was confirmed by Bhandari and Nicholas (1981) who also found that GS activiEy in Nitrosononas europaea \^¡as relatively low compared with that of GDH whereas glutamate synthase (GOGAT) was not detected. In the present study with Nitrobacter agiTis although GS had appreciabl.e activity (transferase), relatively 10ï activity was recorded for GOGAT. Glutamate is the major amino acid in the cytoplasm of Nitrobacter agiTis¡.viz. about 25% (w/w) of the total amino acids (\rlallace et a7.t 1970). It is likely that GDH is the main pathway for the synthesis of glutamate in this bacterium. The evidence in support of this conclusion comes from experiments reported in Section 3.2.5. The inhibil-ion of both GS and GOGAT by L-methionine Dl-sulfoximine (MSX) and azaserine respectively did not have any effect on the incorporation of tttO; or ttttT into ce11 nitrogen indicating that either this pathway is not mandatory or following iLs inhibition, GDH takes over the adequate production of glutamate for cell growth.

Nitrobacter has active nitrate, nitrite and hydroxyl-amine reductases (ln/al1ace and Nicholas, 1968) and these enzymes are probably required for t-he 166

assimilation of Noz and NO, via NHf rvhen lhere is no exogenous NI{[ available to Lhe bacterium. Based on the availabl-e information, the following schene is proposed for the assinilation of inorganic nitrogen conpounds by Nitrobacter agilis.

NHpH pr otcins l) nitrite oxidase NADH 2) nitrate reductase aa 3) nitrite reductase Noã NADH NAD NADH NAO NADPH NAOP (J I 4) hydroxylanine reductase o2 Glu 3 5 5) gTutanate dehydrogenase ATP NAOI{ 6) glutantine synthetase aza m9x 7 ) gTutanate synthase :" cMBRANE CYTOPLASM ln 'dher compounds

4.2.2 Purification, oroDerties and re uTation of pl-tttani,ne svnthetase and gTutanate dehyd¡ogep4sq fron Nitrobacter a7ilis and Nitrosononas europaea

Glutamine synthetase (L-glutamate : ammonia ligase ADP forming, EC6. 3.I.2) j-s a key regulatory enzyme of inorganic nitrogen metabolism in many organisms (Tyler, 1978). The enzyme has been characterized and its properties determined in a variety of bacteria (Nagatati et a7., l'97I; Brown et a7., 1974; Kleiner and Kleinschmidt , 1916; Bender et a7. , 1977; Stacey et a7. , 1977; Johansson and Gest, L977; Darrow and Knotts, 1977; Siedal and Shelton, L979; Alef et a1., 1981; Alef and Zumft, 1981; Bhandari and Nicholas, 1981; Khanna and Nicholas, l983a; Murrell and Dalton, 1983). There is no information available about this enzyme and iËs possible role in the nitrogen metabolism of the nitrifying bacterium Nitrobacter agilis.

The properties of GS purified from Ni trobacter agiTis described in this thesis are simil,ar to those for a variety of bacteria in terms of requir:ernenE for divalent cations, molecular weight, number of subunits and inhibition by (Hubbard Deuel and NHL4' amino acid and nucleotides and Stadtman, 1967; Stadtman, L97O; Hachimori et a7., 1974; Bhandari and Nicholas, 19Bl). The K_ values for the substrates of the transferase and biosynthetic reactions of m the purified /Vi trobacter enzyme are comparable to those reported for other bacteria (eg. Shapiro and Stadtman,1970a). The molecular weíght of GS from Nitrobacter agiTis was found to be 700,000 which is similar to that reported for this enzlyme from Rhodopseudononas paTustris (=700,000) (Alef et a7.,198f) but higher than that of the Escherichia coTi enzyme (592,000) (Shapiro and Stacltman, l970a). The subunib molecular weight of 58,000 for the enzyme fron r61

Nitrobacter indicates that tl-re native enzyme is contposed of 12 homologous subunits as in other bacteria eg. E. coTi (Shapiro and Stadtman, l970a).

The inhibition by NHOC1 of the transferase activity of GS from Nitrobacter agiTis (Section 3.3.2.6) supports a postulated model that glutamine reacts with the enzyme in such a way that its -NH, Eroup occupies the NHf binding site, while the |toxygen-bindingt', site Lo which glutamate is normall.y bound is required for the attachment of the corresporlding oxygen group of glutamine (Gass and luleister, 1970). The results also Índicate that the inactivation of the llUf Uinaing si.te by glutamine would preclude the binding of NH20H at this locus.

The GS from E. coTi is regulated by a complex set of mechanisms (Magasanik et a7., 1974) involving feed back inhibition, repression/derepression and adenylylation/deadenylylation (\^/oolfolk et a7. , 1966; Ginsburg and Stadtman , 1973; tr^iohlhueter et al., L973). SubseÇuent reports indicate that a similar type of regulation exists in a variety of other bacteria (Bender er a7., L977; Johansson and Gest, L977; Darrow and Knotts, 1977; Alef and Zumft, 1981; Khanna and Nicholas, 19B3a,b; Michalski et a7 . , 1983) . Bhandari and Nicholas (1981) reported that the enzyme from Nitrosononas europaea is inhibited by several feed back inhibitors. The inhibition of GS from Nitrobacter agiTis by amino acids and nucleotides reported in this-thesis indicates that this enzyrne is also regulated by similar feed back mechanisms. Mixt,ures of various amino acids showed cumulative inhibition of enzyme activity suggesting that each rnodifier is com¡letely independent in its action and thus it is possible thaL separate binding sites on the enzyme are present for each of the feed back inhibitors as proposed for the E.' coTi enzyme (Stadtman et a7., 1968)

In general, GS from Gram-negative bacteria are regulated by adenylylation /deadenylylaÈion (Ginsburg and Stadtman, 1973). The adenylylated and deadenylylated fornsof GS differ in their regulatory properLies (Bender et af., 1977). It is well known that the adenylylation state of the enzyme depends on the nitrogen source in the growth medium. Thus bacterial cel1s growing with NHf, contain GS largely repressed and in an adenylylated form (Wohlhueter et â7., Ig73). As expected Nitrosononas europaea grov/n with NHf contain.s GDH as the nain enzyme for the assimilation of rufff (Hooper et a7, , 1967) and has relatively little GS activity (Bhandari and Nicholas, 19Bl and the present study). 0n the other hand Nitrobacter agiTis has appreciable activirties of GS and GDH but relatively low GOGAT (Section 3.2). 168

It is of interest that the Ni trobacter enzyme rvas highly adenylylated even rvhen the cells r.{ere grown with NO' without any added NIlf in the culture medium. Cetyl trimeLhyl ammonium bromide (CTAB) treatment of the cul-tures prior to harvest hacl no substantial effect on the state of adenylylation of GS. Relatively low concentrations of CTAB (2.5Ug t1-1) ruere used compared to those employed for oEher bacteria (Bender et a7., \977; Davies and Ormerod, 1982., Michalski et a7. , f9B3) because higher concentrations resulted in cell 1ysis. This lysis may be associated with the 1ow cell density of exponentially gro\4/n cultures of njtrifiers. The native adenylylated form of GS from Nitrobacter agilis could be deadenylylated by treaLment with snalce venom phosphodiesterase. This confirms that the Ni trobacter enzyme is i.ndeed regulated by an adenylylation/deadenylylation mecha.rism. Another line of evidence to support this conclusion is that differentially adenylylated forms of Nitrobacter GS, prepared by controlled phosphodiesterase treatments of GS differ in their pH optima. The isoactivity pll of 7,4 for GS fron Nitrobacter agiTis lies between 7.15 for B. coTi (Stadtman et a7., f970) and 7.55 for KTebsiaTTa aerogenes (Bender et a7., L977). This isoactivity pH of Nitrobacter GS was independent of the purification stage of the enzyme as \^/as also found in K. aerogenes (Bender et a7,ç l9l7).

Nitrosononas europaea growing with high concentrations (=120mì4) of llH[ would be expected to have a highly adenylylated forrn of GS. The results presented in this thesis indicaÈe that the enzyme from Nitrosononas europaea has some unusual features, viz. stimulation of transferase activity by Mg2i- in )t crude extracts, and the inability of phosphodiesterase to reverse the Mg"' effect. However there are three lines of evidence indicati-ng that the enzyme is adenylylated: (a) the biosynthetic activity tu= Mn2t dependent, (b) the 1t Mg-- effect on the transferase activity was dependent on the amount of NHI present in the medium (Fig. 32), and (c) the enzyme was progressively inhibited by increasing concentrations of urea (Bhandari, 19BI). The biosynthetic activity of a fully adenylylated GS from E. coTi has been shown to be l"fn2* dependent (Shapiro and Stadtman, 1970b). Moreover in E. coTi (Kingdon et a7. 1967) and in Rhodopseudononas capsuTata (Johansson and Gest, I977) adenylylation of the enzyme resulted in a change in metal specificity from Mg2+ to Mnz+ for the biosynthetic activity. The Mg2+ effect on transferase activity in Nitrosomonas europaea may be related to the adenylylationstate of the enzyrne since it was directly affected by the amount of NH; present in the growth medium' It should be noted that Mg2+ stirnulation of transferase activity was independent <¡f l-he concentration of M;2+ in the assay mi-xture because an increase in Mn2* r69

(up ro 2OrnM) did not modify the l'1g2+ (20-1OOnM) effect on GS transferase activity. There \^ras no ef f ect of Mg2+ on transf erase activity in purif iecl enzyme preparations. It is conceivable however that during the purification procedure the adenylylation state of the enzyrne nay have changed.

The observation that snake venom phosphodiesterase did not affect the )t Mg-' effect on t,ransferase activity of GS from Nitrosononas europaea, is comparable to recent reports for ChTorobiun TimicoTa (Davies and Ornerod, 1-9BZ) and C. vibrioforme (Khanna and Nicholas, 19B3a,b). Davies and Ormerod (1982) reported that although the transferase activity of GS from C. Tinicola r'¡as inhibited ¡y ùlg2+, phosphodiesterase treatment had no effect and they concluded thaL the enzynìe was not regulated by an adenylylation/deadenylylation type mechanism. Khanna and Nicholas (f983b) however have shown that when toluene permeabilized cells of C. vibrioforme rvere incubated with tlaC] ATP followed by an ammonia shock, the tlaC] labe1 lüas associated with GS, but phosphodiesterase treatmenÈ of this enzyme clid noL remove the IIaC] adenine rnoiety. In a similar experiment with toluene treated cells of Ni trosononas europaea [laC] ATP did not label GS, presumably because of very lorv amounts of enzyme in this bacterium. However from the results for ChTorobium (Khanna and Nicholas, 19B3a,b) and for Nitrosononas europaea (present study) it would appear that the inability of phosphodiesterase Lo reverse the Mg2+ inhibition of transferase activity does not necessarily imply that the enzyme is not adenylylated. It is of interest that both ChTorobiu¡¡ ancl Nitrosononas enzymes did noL interact with Ctbacron Blue F3G-A dye (in Blue Sepharose CL-68 column) which normally binds to the nucleotide binding site of GS. It is possible therefore that the nucleotide bi-nding site of the enzymes from ChTorobium and Nitrosononas differ frorn those of GS from other bacteria (eg. Nitrobacter).

As discussed in Section 4.2.L the cell-free extracts of Nitrobacter agilis contained NAD+ and NADP+ dependent glutamate dehydrogenase (GDH) activities. It is clear from the results reported in this Lhesis that these two activities were associated wit.h two distinct protein fractions. This report is similar to those f.or ThiobaciTTus noveTTus (LerJohn et a7., 1968), Hydrogenononas HI6 (Krämer, 1970) and Micrococcus aerogenes (Kew and Woolfolk, I97O; Johnson and lr/estlake , lg72) which also have two GDH isozymes dependent on NAD* and NADP+ respectively. The NAD+-GDH of Nitrobacter agiTis appears to operate in either clirection ie. amination of o-ketoglutarate to glutamate and deamination of glutamate to a-ketoglutarate, whereas NADP+-GDH functions mainly in the direction of glutamate (ammonia assimilation). It is of interest that the r70

amination reaction of NADP+-GDH from Nitrobacter agiTis was stimulated by NHÏ and NADPH (substrate stimulation) so Lhat Lwo distinct K,o values were obtained for either subsLrate. Cells of lVi trobacter agiTis contained a high cöncen- rrarion of llH[ (approximately 3Om!1) and this explains why (a) GS is highly aclenylylarecl (Secrion 3.3.3) and (b) NADP+-GDH (Km NHl,6.3 to 33mM) operates in the direction of glutainate production even rvhen no exogenous NHf is available to the bacterium. Nitrosono1as europaea has only biosynthetic GDH activity (NADP+-GDH) which can function in either direction ie. arnination and deamination (Hooper et a7, , 1967). It is likely horvever that under physiological growth conditions, the enzyrne is essentially unidirectional since the dearninarion reaction is almost completely inhibited (80 to 9O%) by 10ml'4 NHOC1 (Hooper et a7., 1967). No such regulation of NADP+-GDH from Nitrobacter seems necessary because the dear¡ination activity of the enzyrne is only about 4% of that of the amination activity and Lhe enzyme tvould thus appear largely biosynthetic. Unlike the NADP+-GDH of Nitrosononas europaea (tlooper et a7.g 1967) the amination reaction of NADP+-GDI{ frorn Nitrobacter agiTi.s rvas unaffected by high concentrations (100mM) of q-ketoglutarate and by nucleotides. In fact NADPH stimulated the amination activity as shown in Fig. 39.

Attempts to purify NAD+-GDH from Ni trobacter agiTis by affinity chromatography on Blue-Sepharose CL-68 r,{ere unsuccessful. Low activity of the NAD+ enzyme as well as minimal cell yields of Nitrobacter agiTis make it difficult to purify this enzyme in sufficient amounts to study its properties and regulation. The bacterial NAD*-GDH are usually catabolic in function ie. they utilize glutamate to produce o-ketoglutarate required as a substrate for transamination reactions and for the Lricarboxylic acid cycle (Smith et a7. t Ig75). Although NAD+-GDH from Nitrobacter agiTis can carry out both deamination and arnination functions, its main role may be the production of c-ketoglutarate .

The high intracellular NHf concentration observed under laboratory growth conditions support the view that GDH is the main pathway for NHI assimilation ln Ni trobacter agiTis. Accumulation of Nff] via the assimilatorV NO, reductase (Wa11ace and Nicholas, f96B) appears to be under minimal regulatory control. Under natural growth conditions in the soil, the availability of N source (N0, analor NHI) for Nj trobacter agilis can vary greatly and this may account for the fact tha r the bacrerium has rwo NHI assirnilation pathways ie. GDH and GS/ The schemes are suggested assuming that NOj reductase is GOGAT. following I responsible for the production of NHf in ili trobacter agilis when exogenous NH¿ 171

is unavailable to bacLerium. \^lhen N0, is limitingr'the nitrite oxidase would predominate, generating ATP and reducing equivalents, resulting in minimal reduction of NOJ to NHÏ. Under these conditions GS would be unadenylylated (active form) and r,¡oulcl readily assimilate the small anìounts of Nfif available (K*NH4 = 0.2mM).

N¡ p low GDH low N i'------. N Hl------::l--^ --¡ Glu- amino acids

GS GOGAT octive

G I n

NO; other compounds

OUT IN

IVhen an ample supply of NO, is available (eg. culture growth conditions), N0; reductase would produce suffícient amounts of lfUf that would result in adenylylation of GS. ffre NHf thus produced would then be predorninately assimilated by the GDH pathway.

N¡R high c Dtl h lgh NH; Glu- amino acids I ,. GS.A M P ,'cocAT lnactive

a G ln I I Noã tI other compounds

OUT IN

4,3 ENERGY CONSERVATION IN NITROSOMONAS EUROPAEA AND N.TTROBACTER AGILIS

4.3.1 Respiration driven proton transTocation

ft is well known thaE the operation of the respiratory chain results in the translocation of protons across the cel1 membrane thus developing an electrical potential (AU, inside negative) as well as a pH gradient (ApH, inside alkaline). This resulLs in a proton-motive force (Àp) whj-ch drives active transport and ATP synLhesis (Mitchell, 1966). 172

The subject of respiratory driven proton translocation in nitrifying bacteria is relatively unexplored . Drozd (1976) reported *H+/0 ratios of about 2 f.or the oxidation of NI{[ and NH20l{ by Nitz'osomonas europaea. The + results for NHj appear Lo be in error, becattse 0.15M KSCN, which rvas çtsed as the permeant ion, completely inhibiLs the oxidation of lfHf (Bhandari and Nicholas , I979a, and present study). Although the value reported for the oxidaLion of NHrOH is correct for the particular NH2OFI concentration used in the 0, pulse experinents, no account uas taken of phenomenon Lhat might diminish the -tH+/O ratio, namely the tendency of permeant amines to follorr protons across the membranes.

The results reported in this thesis i-ndicate that Nitrosononas europaea responded in a classical fashion in oxygen pulse experinents and showed efficieniies of proton translocation with amine-1ike substrates (+¡1+70â¿ 4) comparable to those for enteric bacteria (Garland et a7., L975) and for the reduction of nitrogen oxides by denitrifying bacteria (Kristjansson et a1. , 1978). The +H+/0 ratios for the oxidation of endogenous substrate, which is presumed to represent a set of organic compounds, were j-n the range of 4 to 6. High concentrations of NHI, NH20H and NrHr+ appeared to diminish the proton pumping activity of the cells. The most likely cause of this is the well known tendency of the permeant amines to redistribute across the membrane towards Lhe acidíc side in response Lo a pH gradient (Krogman et a7. , 1959; Good, 1960; Cobley, L976a,b).

1¡s *H+/0 ratios recorded f.or Nitrosononas europaea are quantitatively in accord with the finding that the first step in NH| oxidation (NH|+O -> NH20H + H+) is mediated by a mono-oxygenase (Dua et al., 1979; Hollocher et êt7. , l9B1; Andersson et a7 . , 1982) and could noL thermodynamically support proton translocation (AGo' = -0.7 kcal). The observed +H+/O ratio for NI{[ is 3.4. Substracting 0.66 stoichiometric protons per 0 atom gives a ratio of 2.74. Inasmuch as the first of the three 0 atoms is utilized in an 0 insertion reaction, the effective +H+/0 ratio becornes 3/2 x 2,74 = 4.1 for the two 0 aÈoms reduced yia electron transport system. The corresponding ratio for NH20H and NrHr+ oxidation by Nitrosononas europaea would be 4.4-0.5 = 3,9. Thus the *H+ /O ratio ascribable to pumped protons is about the same for these three substrates. These data also indicate that NHl, NH2OH and NrHr+ enter 173

the cel1s of Nitrosomonas europaea as neutral specj-es and N0, exits as an electroneutral species lvith one proton. This pattern i.s analogous 1-o that inferred from oxidant pulse studies of N0, reduction by denitrifying bacteria (Kristjansson et a-l., f978) and rvould prevent the internal acidification of Nitrosononas europaea.

If the reaction NIJTOH + 20 + NOt + H2O + H+ (AG" = -83 kcal or _1- -347 .4 kJ mol ) is the rel.evant energy yielding reaction in llU[ oxiclation by Nitrosonolâs europãeât the average free energy availal¡1e to each of the 8.2 protons translocated is about 10 lccat (approximately 42 l

In Ni trobacter the free energy available in the aerobic oxidation of a high potential reductant N0; (NOt + 0 + NOr; AG": -18 kcal or -75.4 kJ *o1-l) is used to drive the reduction of a low poten tial oxidant NAD* and to produce ATP (Aleem, 1968, L977).

I NOr+NAD'+H20*N03 + NADH + tl+ AGo' = 38 kcal

It has been shown with membrane vesicles that the aerobic oxidation of both N0, and NADH is linked to ATP synthesis (Aleem and Al.exander, 1958; Aleem and Nason, 1960; Butt and Lees, 1960; Cobley, I976b). The coupling of N0, respiration to phosphorylation and NAD+ reduction is generally assurned to be by way of the proton-motive force (Cobley, I976a,b; A1eem, 1977; Aleem and Sewell, 1981; Ferguson, l9B2). Thus the apparent inability of Nitrobacter agiTis to translocate protons in 0, pulse studies is remarkable and interesting.

The only report of resplration dependent proton translocation in Nitro- bacter is that of Cobley (1976b) who demonstrãted with electron transport particles of. Nitrobacter winogradskyi EhaE the aerobic oxidation of N0, resulted in a small but reversible alkalinizalion of the medium. Although the effect was abolished by agents known to collapse Ap , the amplitude of the effect showed saturation with respect to O, was not appreciably enhanced by valinomycin/K+, and shorved an extremely low H+/O ratio of -(-0.1. hlhether this effect resultecl dírectly from the operation of a respiratory proton pump in inverted membrane particles is uncertain. The data reported in this thesis'also show small respir:ation rlependent changes in external pH. These changes and the rt4

subsequent slower ones in external proton concentration rvere not nuch enhanced by permeant ions and in general exhibited properties and kinetics distinctly different from the classical picture. No convj-ncing evidence is thus available I for a respiratory proton pump in Nitrobacter agiTis to support Cobley s predicrion (Cobley, 1976b) rhat the + lI+/0 ratj-o should be 1. The data of Cobley (L916a,b) show that N0, oxidation in Njtrobacter winogradskyi membrane vesicles is inhibited by agents that collapse the membrane potential, but whether the effect is mediaLed entirely through the membrane potential is not clear. The extent of inhibition imposed by the uncoupler CCCP at higl'r concentrations (lBOUl,4) and the K* carrier valinomycin was not complete (Cob1ey, Ig76b). The uncoupling concentration of CCCP is usually between 5 to 20UM. In any case, the rates of NO, oxidation in the Nitrobacter agiTis preparations used in this thesis were adequate for oxygen pulse studies even in the presence of permeant ion concentrations lcnown to be effective in collapsing the membrane potential in Ni trosomonas europaea, There hras no evidence in these studies that the membranes of Nitrobacter agilis were abnormally permeant to protons (uncoupled). The relaxation of protons across the membranes follorving acid pulses was slow (Fig. t+4) and typical of that observed with Nltrosononas europaea (Fig. 41) and other bacteria (Kristjansson et al., 1978). ülhile it permeanL ion was in fact permeant is conceivable that none of the nominally ' this requires lVi trobacter agiTis to possess a unique membrane system, which is unlikely. l4oreover the lipophili-c cation TPMP+, which is a well known permeant ion and has been used to determine membrane potential in bacteria (Rottenberg, L979) and Èo collapse the membrane potential in oxidant pulse experiments (Boogerd et a7., 1981; Castignetti and Hollocher, 1983)' was effective with Nitrosononas europaea but not with Nitrobacter agiTis. Valinomycin should have penetrated the cytoplasmic membrane in spheroplasts and membrane vesicles of Nitrobacter agiTis even if it could not do so in intact cells. Based on the results for Nitrosononas europaea and a variety of other bacLeria there is no evidence that permeant ions inhibit the respiratory Proton PumP Per se.

4.3.2 Proton-notive f orce and ATP biosvnthesis

According to Mirchellrs chemiosrqotic hypothesis (Mj-tcheL]-, 1966), the electrochemical gradient of proton" (ür+) gives rise to a proton-motive force (Ap). This proton gradient consists of an electrical potential (Arþ) and a pH gradienU (ApH). The two comPonents of Âp have the following relationship: bacterj-a Ap = - 2.3RT/F ApH. Whereas ¡p has been determined in a variety of ^ü 175

(padan et a7., 1976; Guffanti et a7., L91B; Deutsch and Kula, 1978; Kashket et a7 . , 1980; Kashket, 198la , b; Jarrel I and Sprott , 1981; l"latin et al., f9B2) no information is available for the nitrifying bacteria. The rneasurement of Ap in Nitrosononas europaea ancl Nritrobacter agilis is reported in this thesis.

The results in Sectjon 3.5.2 indicate that at an external pH (pHe) of 7.0 for Nitrosononas europaea and 1"5 lor Nitrobacter agiTis there rvas no ApH (inside alkaline) because at these pH values neiLher weak acids nor: weak base were accumulated by these bacteria. The pH optima for NHI and N0, oxidation by Nitrosononas europaea and Nitrobacter agilis respectively were betr'¡een 7.5 and 8.0, indicating that optimally respiring cel1s of these bacteria do not have a ApH. Nitrosononas europaea appeared to have a limited capaci-ty to maintain a constant intracellular pH (pHi). This result contrasts with those reported for ylicrococcus lysodeikticus (Friedberg and Kaback, l9B0) and E. coTi under aerobic conditions (Padan et a7, , 1976), but is comparable to anaerobic bacteria, namely, MethanospiriTTun hungaetei (Jarrell and Sprott, 19Bl)' Clostridiun pasteurianum (R:-ebeling et a1., L975) and even E. col-i grown under anaerobic conditions (Kashket and trrlong, 1969). In Nitrobacter agilis pEi rema1ned relatively constant (7.3 to 7.8) over a range of pHe values (6 to 8.5) (Padan et a7,, \976) which is in agreement with the results for E. coTi ' Micrococcus Tysodeikticus (Friedberg and Kaback, 1980), ThiobaciTTus acidophiTus (Matin et a7., Ig82) and BaciTTus subtilis (Khan and Macnab, f9B0) (for a reviers see Padan et a7., 19Bl).

The weak base methylamine, used as a probe for the determination of ApH, is noL oxidised by either nitrifier. Since ammonia and its analogues are probably taken up by Nitrosononas europaea as neutral 'species (Drozd, 1976) it is unlikely that the cells would accumulate methylamine in response to a AÜ (inside negative) as reported for Azotobacter vineTandii (Laane et a7.,1980).

At a pHe of 6.0, neither Nitrosononas europaea îor Nitrobacter agiTis oxidised its respective substrate, but they still maintained a reasonable ApH and Aü, and thus Ap. In fact, in Njtrobacter agiTis, Ap was maximal at pHe 6.0 (or less than 6.0), and it decreased linearly with an increase in pHe' However at pH 7.0 when both Nitrosononas europaea and Nitrobacter agiTis re' tained about hatf of their respiratory activities, the sma1l A pll was dissipated by uncouplers and compounds which inhibj U respi.ration. It is known that Nitrosononas europaea and Nitrobacter agiTis have appreciable rates of endogenous respiration (Section 3.5.1; Drozd, 1976; Sewe11 and Aleem, 1979; 176

llyman and fiood, 1983) involving cornplex organic substrates. In Nitrosononas europaea endogenous respiration has been shown to be coupled to pl:oton translocation (Secti.on 3.5.1 and Drozd, 1976). Tt is likely that this endcigenous respiration enables the ce1ls to maintain a reasonable Ap in the absence of exogenous sullstïates, or when the exogenous respiration is inhibited. This phenomenon could have ecological significance for nitrifiers, because these Lhat preclude soil bacteria in their natural habitat ,may encounter conditions respiration for extended periods of time.

In Nj trosononas europaea the uncoupler CCCP severel)¡ inhibited respiration (80% ar 10UM CCCP) bur lowered Ap by abouL 40mV. At higher concentratins (100 pla CCCP) the respiration was completely inhibited but Ap was reduceC by only 7BmV. In Nitrobacter agí7is; CCCP (50UM) completely restricted respiration, but non-respiring cells still maintained a Ap of B2mV (inside negative). Un- couplers are known to restrict respiration in nitrifying bacteria (Cobley, I976b; Aleem, Ig77; Bhandari and Nicholas, I979a,b, 1980; Aleem and Sewe11, 19Bf) but it is only recently they have been shown to ínhibit respiration in other bacteria eg. ThiobaciTTus acidophiTus (lfatin et a7. , I9B2) and denitrification in Pseudononas denitrificans and Pseudononas aeruginosa (l'/alLer et a7., 1978). The effects of uncouplers on denitrification were noL linked to Èhe collapse of Ap buL rather to their detergent-like effects on the cell membranes (Irtalter et a7 . , 1978). It is possible that the mechanisrn of inhibition of respiration by CCCP and other uncouplers in lVitrosomonas europaea and Nitrobacter agiTis is similar to that in the denitrifying bacteria.

Because the inhibitors known to collapse Arþ also inhibited N0, oxidation by membrane particles of Nitrobacter winogradskyi, Cobley (I976a,b) predicted rhar NOt oxidarion requires A$. The way in which NOI oxidation is mediated by Arf is however not understood (discussed in Section 4.3.i). Cobley also re- porred rhar NHI srimulated N0; oxidation by collapsíng Apll. The results of rhe presenr study also indicate that ApH is collapsed Uy }{H[, but NHI also lowered Aü. Thus Cobley's prediction (Cobley, Ig76b) that NHf stimulation of NgJ oxidation resulted from a collapse of ApH only is not substantiated by the data presented in this thesis.

As discussed in Section 4.1, ATPase inhibitors strongly inhibi-ted N0, oxidation by Ni trobacter agiTis. Similarly NHI oxidation by Ni trosononas euÍopaea was also resËricted by ATPase inhibj-tors but to a lesser extent (Section 3.5.2). This inhibition did not appear to be associated rvittr a collapse of Ap, because DESB elevated Âp in Ni trosononas europaea rather than lowering it. 17l

The results of the present study indicate that the toLal Ap at pH 7.5 wou1rl be approximat.ely 150mV and 125rnV (inside negatìve) in Nitrosotnonas europaea and Nitrobacter agiTis respectively. In E. coli Ap is cornposed of both Arþ and ApH (Padan et a7., L976); however ÀpH appears to be absent in Met-hanobacteriun thernoautotrophicun and MethanospirifTum hungatei (Jarrell and Sprott, 1981). The overall behaviour of ApH aud Atþ in nitrifying bacterià is quite similar to that repor:ted for other bacteria êg E. coTi (Padan et a7,, r976).

In this thesis, it is shorvn that spheroplasts of Ni trosononas europaea synthesize ATP in response to an artificially created Atl.t. It was shown earlier (Reid et a7., L966) that rapid immersion of mitochondria in an acidic mediurn resulted in ATP synthesis. Subsequently, l"laloney eta7. (L974) and Wilson et a7. (1976) dernonstrated that both Streptococcus factis and E. coJí synthesize ATP in response to a valinornycin induced Arþ. The results of ATP.synthesis by Nitrosononas europaea are comparable to those for S. Tactis and E. coTi (lufaloney et a7. 1974; Wilson et a7., 1976). The ATP synthesis induced by valinomycin was inhibited by DCCD indicating the involvement of ATPase. The inhibition of valinomycin índuced ATP synthesis in spheroplasts of Nitrosononas europaea by increasing concentrations of KC1 (Fig. 53) indicated that it was dependent on the magnitude of AV as reported for E. co-Li and S. Tactis (Maloney et a7., 1974; I^lilson et a7., L976). Valinomycin induced ATP synthesis was always transient presumably because of (a) ATP hydrolysis following the increase in intracellular ATP levels and (b) collapse of Ar! by inward movement of cations. Thus the results reported in this thesis provide evidence that, as in other bacteria, Nitrosomonas europaea also has a respiratory driven Prot.on- motive force coupled to an energy conserving system as expected from Mitchell I s chemiosmotic hypothesis (l'{itchell , 1966). For reasons given in Section 3.5.3.2 similar experiments of valinornycin induced ATP synthesis with washed ce11s and spheroplasts of Nitrobacter agiTis were inconclusive.

+ + 4.3.3 Na and K transport

In recent years a substantial amount of information has accumulated on the role of Ap in living systems. In addition to pumps which extrude protons to generat.e a Ap, bacteria also possess several genetically distinct cation transport systems (Harold and Atendorf, L974; Rhoads et a1.t 1976; Epstein and Laimins, 19BO). Transport systems which derive their energy from previously formed electrochemical gradients utilize three basic mechanisns for energy coupling, as described by Mitchell (1973), namely, symports, uniports and anti- 178

ports . Of the sevel:al ion transport -systems, those f or Na* and K* play an importernt role in regulating intracellular pH (Krulwich et al., 1979; Beck and Rosen, L919; Brey et a7.,1980; Plack and Rosen' 1980; Tokuda et ai.,19Bl) and active transport of nutrients (Stock and Rosenan, L97I; Thomson and Mcleod, L97L; Lanyi et a7,, 1976; Tokuda and Kaback, L977; Eddy, 1978). As there is no infornation available on these transport systems in nitrifying bacteria, an attenpt has been made to characterize some of'these cation Lransport systems in Nitrosononas europaea and Nitrobacter agiTis.

The results of K+ depletion experiments with the cells of Nitrosononas europaea and Ni trobacter agiTis respectively are similar to those reported for Vibrio alginoTyticus and E. cc¡7i (Nakamura et a7.¡ L9B2). The atniue treatment method for K* depletion was effective for both nitrifiers. There was no net entry of Na* tluring the extrusion of Kf in either nitrifier indlcating that the uptake of unprotonated amine into the cells by passive diffusion and its subsequent protonation inside a11ows for the K* extrusion via t<+/H+ antiporter even in the absence of Na+ "nttY.

OUT IN

N 4

H+ + K

Since the internal pH is decreased by the extrusion of K* and the antiporter is relatively inactive at lower pH (Nakanura et a7., I9B2), a high concentration of amine is required for the bulk release of cel1u1ar K*. It appears that ttre cells of Ni trosononas europaea grov/n with high NHf have relatively low and variable intracellular K* (Table 4I) which could well be associated with K+/H+ antiporter system and NHf transport into the cell.

1¡g depol arizat:on of Arþ by K+ has been shown in streptococcus faecajis and E. coTi (Bakker and Mangerich, 1981). These authors reported that the addition of Kt to K* depleted cells of S. faecaTis resulted in depolarLza1ion of Atþ by about 60mV but this depolarization of Aü $/as compensated by an approximately equivalent increase in AplJ so that the total proton-motive force remained reasonably constant. Thus the electrogenic K* influx results in an interconversion between the components of Ap. Similar results \,{ere obta j-ned lor E. cof i but the extent of depolarization of AÜ by K+ was much lower than in S. faecafis 119

(Bakker a¡d Mangerich, 19Bf). It appears that the extent of depolarization of Arl.r by K+ depends on the ability of cells to take up K{-. Thus the results reported in this thesis indicate that a sinilar mechanistn exists in both Nitrosononas europaea and Nitrobacter agili.s. In the absence of other permeant ions the prol-on pumps of the cytoplastric menbranes tend to develop a large outwardly directed Aü and a srna11 ApH (Mjtchell, f966). Inwar:d movement of Kt r,,i11 decrease Arþ, which a11ows more protous to be purnped out with Lhe result that in steady sLate AÜ is partially converted into ÂpH. Such interconversions are well known in energy transducing membranes eg. lipid soluble catj-on TPMP+ causes an extensive conversion of AÜ Ínto ÂpH in illu- ninated cell suspensions of HaTobacteriunt haTobittn (Bakker et a7. t Lgl6). Thus increasing concentration of a p--rneant ion continues to decrease ÂÜ. Up to a certain point the depolarizaLion of Arþ can be compensated by an increase of ApI{ until the membrane becomes leaky to protons due to secondary effects such as swelling (Padan and Rottenberg , I973).

A respiration dependenL prirnary Nat extrusion system functioning at I alkaline pH has been demonstrated in the marine bacterium Vibrio alginoTyticus

i (Tokuda and Unemoto, f9B1) . HaTobacteriun haJ-obium also extrudes Na* eiLher by a Na+/H+ antiporter system (Lanyi and lulacDonald, 1916) or a Na* PumP, 22fu+ halorhodopsin (Lindley and MacDonald, LgTg). Using loaded cells it is tVj and Nitrobactet agíJis shown in the present work that both trosononas europaea ' 22Nu+ 22Nu* lack the respiration dependent Na* pump. Extrusj-on of f.ot loaded cells required K* as a counter-ion in both Nitrosononas euîopaea and Nittobacter agilis but the extent of 22N"* extrusion by K+ addition in Nj trosononas europaea was much less than in ffjtrobacter agiTis (Fig. 59). It appears that the K* uptake system of /Vi trosononas europaea is not as efficient as in Nitrobacter agiTis and thi-s observation is supported by the fact that Kf uptake results in about twice the depolari-zai'ion of Arþ in Nitrobacter agilis than in Nitrosononas europaea (Fig.57). Amines and NHOCI also resulted in 22Nu*, the extrusion of probably via a Nu+/H+ anLiporter systen.

fn summary the results reported in Section 3.5.4 indicate that Nitro- sononas europaea and ffi trobacter agiTis have several distinct cation transport syst-ems inclucling antiporters for r+/u+, K+/N.+ and Na+/H+. At least one of these antiporters (K+/Na+) requites an electrochemical gradient of protons for its operation. K* can also be transported by an electrogenic mechanism. The possible cation transport systems in Nj trosononas europaea and Nitrobacter agiTis are summatr'zed in the follorving scheme'. 180

ouT IN

6+ H+

a Na'

+ K

L Na' ¿ + K K. + H proton conduclor

NHs

resP irolion dePendent H+ ( Nitroboc ter ? |

ADP+ Pi H+ ATP + H ¡pp+pi

15 1B 4.3,4 /V 0 Íso studies of NO oxidation b Nitrobacter iTis

The results reported in this thesis clearly indicate that Nitrosomonas europaea follows the classical chemiosmotic pattern for energy conservation. Although in Nitrobacter agiTis the pattern of components of proton-motive force (Arþ an¿ ApH) and cation transport systems appear quite similar Eo those in other bacteria (See Paclan et aI.r 1981 and the references therein), the apparent absence of demonstrable proton translocation in fluorescence quenching and oxygen pulse experiments is unclear. During the course of this study, Dr. T.C. Hollocher (Biochemistry Department, Brandeis University, U.S.A.) suggested (personal communication) that in Ehe absence of a respiratory proton 181

pump the bacterium might synthesize ATP by substrate level phospllorylation involving a mixed anhydricle betleen either NOJ and fO2O or NO! and ADP, b)' analogy with the oxidation of sulfite by Thiobacil.Tus and reverse of clis- similatory reduction of sulfate by DesuTfovibrio' both of r+hich proceed by way of adenosine 5r-phosphosulfate (Roy and Truclinger, 1970)" The validity of 15N-NI'{R this hypot-hesis was checked by means of and GC/MS studies using stable 15N-NÌ'1R i"oropu" of 15N und 180 (Section 3.5.5). The results of both und t0t_in GC/Ì'{S stuclies clearly indicaLe that the third NO, lroduced fronr N0, originated from HrO and not frorn either O, or PO'j . These findings are in agreement wit.h those of Aleem et a-1. (1965). The GC/MS studies of NOt oxidaLion reported in Section 3.5.5.1 indicate that there hras no apparent ex- 18ò lBO change of between.HrO and plBO?- so that it is unlikely tl'rat would have been lost from PISOî- during the experinents. The results in Section 3.5.5 also rule out the possibi-lity of the formation of a P-O-N type interrnediate as suggested by Hollocher.

15N-NMR The results of th" studY (Section 3.5.5.2) indi-cate that when t

tOt to the theoretical value (100 z 32). This result proves that all the in nitrate was derived from water. The oxidation of N0, to N0! by Ni trobacter agilis requires only one oxygen atom which is supplied by water (Aleem et a7., 15N160180, 1965 and the present study). The appearance of a resonance in 180 tne l5N-NMR spectrum (Fig.62) is thus unusual. A chemical exchang" of ne pH is unlikely. A possible explana- uld be associated with the recycling of ilatory type nitrate reductase (II) (Sewell and Aleem, L979) in thick cell suspensions which tend to become anaerobic 1 Bo H 2 15n16oz (1 ,\t-- 15N160 180- 2

tt*tuot 150¡t6o11Bo + ttr18o (rl ) II

15N160180- I 15N16018 oz

1Bo H2 182

In conclusion, it is possible that Ni trobacter agil.is conforms to the classi-ca1 chemiosrnotic coupling nechanisn for ATP biosynthesis but the non- detection of a resp]-ratory proton pump in this bacteriurn is unusual and further work is awaited to resolve this interesting anomaly '

l.OJ

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