The physiological function of gluconic acid production in acinetobacter species and other gram-negative bacteria: Implications for energy conservation

GLUCONIC ACID PRODUCTION IN

ACINETOBACTER SPECIES AND

OTHER GRAM-NEGATIVE BACTERIA

IMPLICATIONS FOR ENERGY CONSERVATION

TR diss

N

GLUCONIC ACID PRODUCTION IN

ACINETOBACTER SPECIES AND

GLUCONIC ACID PRODUCTION IN

ACINETOBACTER SPECIES AND

OTHER GRAM-NEGATIVE BACTERIA

IMPLICATIONS FOR ENERGY CONSERVATION

PROEFSCHRIFT

ter verkrijging van de graad van doctor aan de

Technische Universiteit Delft, op gezag van de

Rector Magnificus, prof. dr. J.M. Dirken, in het

openbaar te verdedigen ten overstaan van een

commissie aangewezen door het College van

Dekanen op dinsdag 2 juni 1987 te 16.00 uur

door

Bartholomeus Jozef van Schie

geboren te Wassenaar

TR diss

1547

Prof. dr. J.G. Kuenen

Dr. J.P. van Dijken heeft als begeleider in hoge mate bijgedragen aan het totstandkomen van het proefschrift. Het College van Dekanen heeft hem als zodanig aangewezen.

Overige leden: Dr. ir. J.A.M de Bont Prof. dr. ir. J.A. Duine Prof. dr. W.N. Konings Dr. J.D. Linton

Prof. dr. P. v.d. Putte

This study was carried out at the Department of Microbiology and Enzymology of the University of Technology Delft, The Netherlands, and subsidized by the Ministry of Economic Affairs.

(senryü, anoniem)

0cvv\ ^*5

A

Terwijl ik dit opschrijf blèrt uit de speakerboxen de heer Joe Cocker "With a little help from my friends" ja zo voel ik het ook. De vier jaar werk in Delft zijn voor wat de wetenschappelijke resultaten betreft beschreven in dit boekje en daarmee toegankelijk voor ieder die daarin geïnteresseerd is. Behalve dat ik veel van de bacterie heb leren begrijpen, heb ik ook een hoop mensen leren kennen. Iedereen van het laboratorium wil ik heel hartelijk bedanken voor hun bijdrage aan dit werk. Speciaal bedankt: Max, Nico, Joop en Jos voor de dagelijkse verzorging; Peter Arntz die met mij de kamer deelde en altijd geïnteresseerd en hulpvaardig was; Bas vanwege het computerwerk; Frieda voor het vele typwerk; de andere leden van het team-PQQ, Paul, Mario en Hans Dulne, jullie waren het biochemisch klankbord; de mensen van de werkplaats voor hun metaal, glas, hout en electronische diensten en de gezellige uurtjes in het keldertje, Klaas en Wil voor de verleende gastvrijheid in Groningen, Gijs door jou aandacht, belangstelling, heeft het onderzoek sterk gederepresseert kunnen verlopen. Een groot deel van dit werk is een co-produktie met Jackie, Robbert, Wiebe, Wilma, Oskar, Rene. Hans, van jou heb ik veel geleerd, door jouw inzet, energie, enthousiasme, geduld, humor en niet te vergeten vakkennis en vieuw, is het gelukt deze meneer enigszins te structureren zodat de vele proefjes en ideetjes ook nog zwart op wit kwamen te staan. Peter, behalve dat jij mij hebt kunnen overtuigen dat zand drijft, hebben wij regelmatig leuke gesprekken gehad met een drankje en een hapje.

I

Gezien de drastische effecten van de roersnelheid in een fermentor op het celvolume, de dikte van de celwand en de intracellulaire metaboliet concentratie van een uicro-organisme, is te verwachten dat dit grote gevolgen zal hebben voor het opschalen van bioprocessen.

Wase D.A.J., Ratwatte H.A.M., Appl. Microb. 22 (1985) 325-328 Wase D.A.J., Patel Y.H., J. Gen. Microb. 131 (1985) 725-736

II

Het vermogen van glucose dehydrogenase (EC 1.1.99.17) in Aj_ calcoaceticus LMD 79.41 om disaccharides te oxyderen, kan beschouwd worden als een in vitro artefact.

Dokter P., Pronk J.T., Schie B.J. van, Dijken J.P. van, Duine J.A., FEMS Microb. Letters (1987) in press

III

De aanwezigheid van denitrificerende enzymen in goed geaereerde tuinaarde, zoals aangetoond door Tiedje et al., kan verklaard worden door acceptatie van het bestaan van aërobe denitrificatie.

Tiedje J.M., Sexstone A.J. Myrold D.D., Robinson J.A., Antonie van Leeuwenhoek 48 (1982) 569-583

IV

Jong geleerd oud gedaan kan ook voor bacteriën gelden. Adler J., Biol. Chem. Hoppe-Seyler 368 (1987) 163-173

V

Het naken van een onderscheid tussen B-fructosidases: invertase (BC 3.2.1.26) en inulinase (BC 3.2.1.7) is onjuist.

Barman Th. E., Enzyme handbook (1974), Springer Verlag, Berlin-Heidelberg

VI

Het feit dat bakkersgist (Saccharomyces cerevisae) en voedergist (Candida utilia) dezelfde opbrengst hebben (0.5 g cellen, g substraat ) betekent nog niet dat beide organisnen met gelijke efficiëntie groeien. .

zodanig hoge concentratie aanwezig is dat het bijdraagt aan de kleur van dit produkt is onjuist.

Schlegel H.G., General Microbiology, p 329, Cambridge University Press

VIII

De berekeningen gemaakt door Muller en Babel aan de effiëntie van energie-generering uit glucose in A. calcoaceticus zijn onbetrouwbaar omdat niet is gecorrigeerd voor de gedeeltelijke verbranding van glucose tot CO».

Muller R.W., Babel W., Arch. Microbiol. 144 (1986) 62-66

IX

Met suikers houd je Acinetobacter calcoaceticus niet zoet. dit proefschrift

X

Het streven van de overheid om de onderzoeksinstellingen te revitaliseren door privatisering/verzelfstandiging zou in de eerste plaats op deze overheid zelf moeten worden uitgetest.

XI

De seizoensabonnementen voor theater en concertvoorstellingen maken een "spontaan avondje uit" tot een vaak maanden van te voren aangegane verplichting.

XII

Ook voor een proefschrift gaat op: 1'appétit vient en mangeant.

Chapter Page

I Introduction 3

II Oxygen and growth rate dependent regulation of glucose dehy- 35 drogenase activity in Acinetobacter calcoaceticus LMD 79.41.

III Non-coordinated synthesis of glucose dehydrogehase and its 53 prosthetic group PQQ in Acinetobacter and Pseudomonas species.

IV PQQ—dependent production of gluconic acid by Acinetobacter. 51 Agrobacterium and Rhizobium species.

V Energy transduction by electron transfer via a pyrroloquino- i\ line quinone dependent glucose dehydrogenase in Escerichia

coli. Pseudomonas aeruginosa and Acinetobacter ca1coaceticus (var lwoffi).

VI Glucose dehydrogenase-mediated solute transport and ATP 79 synthesis in Acinetobacter calcoaceticus.

VII An in vivo analysis of the energetics of aldose oxidation by 99 Acinetobacter calcoaceticus■

VIII Biochemical limits to microbial growth yields, an analysis ,,„ of mixed substrate utilization.

IX Silent genes of glucose metabolism in Acinetobacter 145 calcoaceticus LMD 79.41.

Summary \55 Samenvatting

I INTRODUCTION

A microbe can be considered a chemical engine operating at nearly neutral pH, relatively low temperature and pressure, and surrounded by water. The energy needed to maintain cellular functions can be obtained by chemical reactions or directly from light. Bacteria can be classified according to the nature of the primary energy source employed for growth, namely as chemotrophs or phototrophs. Among the group of chemotrophic organisms one may find lithotrophs, i.e. able to use energy derived from the oxidation of inorganic compounds, for example: hydrogen, sulfur, iron or ammonia, or organotrophs which use organic compounds as an energy source for growth. Irrespective the mode of energy generation either carbondioxide and/or an organic carbon source may be the major carbon source for the synthesis of cellular material.

Biological systems employ common intermediates to store energy derived from fuelling reactions. Energy is converted at the substrate level by scalar reactions into energy rich intermediates such as ATP or acetylphosphate, a process known as substrate level phosphorylation. Energy can also be stored in redox intermediates such as reduced pyridine nucleotides, flavins or quinones. Many bacteria possess an electron transport chain in which electrons from the redox intermediates are transferred to a final electron acceptor via dehydrogenases, quinones and cytochromes. In chemotrophs this acceptor can be oxygen or other oxidized compounds (such as: nitrate, carbon dioxide, sulfate, ferric ions, fumarate). In phototrophs the electron transport system is part of primary energy conservation during photosynthesis. The process of electron transport can either be coupled to vectorial proton translocation across the bacterial membrane, or accomplish charge separation through production and consumption of protons outside and inside the membrane, respectively. In both cases, a transmembrane proton gradient is generated. This gradient of protons, together with other gradients of ions across the membrane forms an electrochemical gradient often referred to as the proton motive force (pmf). The pmf can act as a driving force for several energy-requiring processes in the cell. The efficiency of vectorial proton extrusion and/or charge separation is determined by several factors, among others the concentration

of cytochromes in the membrane, the number of protons translocated per electron transferred and the impermeability of the membrane towards protons.

1.1. Incomplete oxidations

When a (chemo-organo) heterotrophic microorganism is cultivated in a mineral medium, the organic compound present serves both as a carbon and energy source for growth. This contrasts with chemolitho- or photoautotrophic growth in which energy conservation can be looked upon as a separate process. During heterotrophic growth various intermediates of central metabolic pathways such as glycolysis, pentose phophate route, Entner-Doudoroff route and TCA cycle, serve as precursors for biosynthesis but are also precursors for dissimilatory reactions. Micro-organisms exhibit an enormous diversity in primary metabolic pathways which convert the growth supporting substrate into intermediates of the central metabolic pathways. Also a diversity in the final step of electron transfer can be observed

(Fig. 1 ) .

During aerobic growth of heterotrophs, part of the substrate is normally oxidized to carbondioxide and the remainder is used to synthesize cellular material. A variety of microorganisms however, may oxidize their substrate incompletely under certain environmental conditions. In such cases the substrate is converted to a product which accumulates into the medium (Krumphanzl et al. 1982, Blanch et al. 1985). Mostly (but not always) this product is utilized in a later phase of growth. Well known examples of incomplete oxidations are the production of gluconic acid from glucose and of acetic acid from ethanol by Gluconobacter and Acetobacter spp. These latter oxidations involve membrane-bound dehydrogenases which are coupled to the electron transport chain. It seems therefore reasonable to assume that these processes lead to energy conservation. As the membrane-bound dehydrogenases often are not an integrated part, or in other cases, a short cut of central metabolic pathway's, the incomplete oxidation process may generate energy in an exclusively dissimilatory bypass of these central routes, and thus be separated from routes of carbon assimilation. Therefore, a strong analogy can be drawn between the incomplete oxidations mentioned above and chemolithotrophic oxidation in which also energy-generating reactions occur separated from the assimilatory routes (Fig. 1 ) . Analogous

terminal

cytochrome

oxidases

ELECTRON ACCEPTORS

g 1. Major pathways of energy conservation in chemotrophic bacteria.

is also the localization of the enzymes involved in these processes since in both cases they often are located at the periplasmic side of the membrane

(Hooper and DiSpirito, 1985; Wei Ping Lu et al. 1986).

Not only strict aerobes but also facultatively fermentative organisms may carry out incomplete oxidations. Certain yeasts, for example

Brettanomyces spp can convert glucose to a mixture of ethanol and acetic acid (Wijsman et al. 1985). Also, the aerobic conversion of glucose by Bacillus megaterium into acetate, pyruvate, acetoin and 2,3-butanediol has been called an incomplete oxidation (Gottschalk, 1979). Similarly the production of citric acid from glucose by Aspergillus niger and certain yeasts is commonly termed incomplete oxidation (Hütter, 1986). From a phenomenological point of view this is correct. However, the latter processes differ mechanistically from other incomplete oxidations such as the production of gluconic acid from glucose and acetic acid from ethanol. In; t)|e case of Brettanomyces spp and B^ megaterium, metabolism is mainly

fermentative, combined with a restricted oxidation of the intermediates of the fermentative route due to a bottle-neck in the central catabolic route (e.g. TCA cycle). In such cases, the incomplete oxidation of glucose involves a large number of reactions including decarboxylation and carboxylation reactions. This also holds for the formation of citric acid from glucose by the strict aërobe Aspergillus niger. In contrast, the formation of acetic acid from ethanol and the oxidation of glucose to gluconic acid, involves only one or two reactions (without no loss in carbon) which are catalyzed by membrane-bound periplasmic oxidoreductases (Loffhagen and Babel, 1984; Dokter et al., 1985). In further discussions on incomplete oxidations I will restrict myself to this latter type of (carbon conserving) periplasmic incomplete oxidations (Table IA, B ) . These processes are known to occur in Pseudomonadaceae, Enterobacteriaceae, Neisseriaceae and Acetobacteriaceae. These bacteria all are Gram-negative organisms and in all cases membrane-bound enzymes are involved in these reactions. These conversions have been known for more than a hundred years and have been applied in industrial processes (Milsom and Meers, 1985; Ghose and Bhadra, 1985). Two of these applications will be considered in some more detail, namely the oxidation of ethanol to acetic acid and the oxidation of glucose to gluconic acid.(Fig 2 en 3)

Table 1A. Some examples of periplasmic incomplete oxidations. (Gottschalk, 1986) SUBSTRATE PRODUCT propanol isopropanol glycerol glucose (aldose) gluconate ethanol acetaldehyde malate methylamine ribitol propionate acetone dihydroxyacetone

gluconate (aldonic acid) 2- or 5- hetogluconate acetaldehyde

acetic acid oxaloacetate

formaldehyde and ammonia ribulose

Table IB. Thermodynamic constants of some reactants of incomplete oxidations.

COMPOUND glucose gluconic acid ethanol acetaldehyde acetic acid FORMULA C6H12°6

W

w

^

►cytoplasm.membr. ►cytoplasm

ETHANOL ...ig!:DH,._>..ACETALDEHYDE . . . j ™ ™ . , . ACETATE

NADTPTNAD(P)H NAdPNADPH

TCA-cycle

bacteria. QPl = ethanol dehydrogenase QP2 = acetaldehyde dehydrogenase EtOH = ethanol dehydrogenase AHYDDH = acetaldehyde dehydrogenase

GLUCOSE VZZZÈfr GLUCONIC ACID ► periplasm

i ^ ^ g ^ ^ ^ ^ ^ « i ^ ^ ^ ^ ^ i ^ c y t o p ( a s m i c membrane

\7 _ ^

GLUC0SE6-P E T ) 6P-GLUC0NATE ►cytoplasm

r / \

EMBDEN OXIDATIVE ENTNER

MEYERHOF PENTOSE D0UD0R0FF

PATHWAY PHOSPHATE PATHWAY

CYCLE

Fig 3. Alternate metabolic pathways of glucose catabolism in Gram-negative bacteria

The oxidation of ethanol by Acetobacter spp. is a two-step process. The first reaction is the partial oxidation of ethanol to acetaldehyde. An aldehyde dehydrogenase completes the oxidation of acetaldehyde to acetic acid. Although it is generally assumed that membrane-bound ethanol and aldehyde dehydrogenase are responsible for acetic acid formation, definite proof has as yet to be given. The product acetic acid is accumulated at very high final concentrations (up to 2.5 mol/1) into the medium and at a very high rate (up to 4.5 g acetic acid/h/g cells). The conversion of ethanol into acetic acid is almost quantitative: an 98 procent conversion can be obtained in the industrial process.

The incomplete oxidation of glucose to gluconic acid is also a two-step process. The first step consists of the removal of two hydrogen atoms from D-glucose to yield D-glucono-8-lactone Fig. 5 and the second step is hydrolysis of the lactone to gluconic acid. In most cases studied this hydrolysis step is non-enzymic, however, a glucono-lactonase has been reported for E. coli (Hucoh et al., 1972), Azotobacter vinelandii (Brodie and Lipmann, 1955), Pseudoiaonas fluorescens (Jennyn, 1960) and P. fragi (Leroux and Tarr, 1963). In many cases gluconic acid can be oxidized further to 2-keto-gluconate or 5-keto-gluconate (Stubbs et al.f 1940). Again, these

reactions are catalyzed by membrane-bound dehydrogenases which probably are located at the periplasmic side of the cytoplasmic membrane. In Gluconobacter suboxydans. an organism which is being used in the industrial production of gluconic acid, glucose can also be oxidized (after transport into the cell) by an NADP-dependent glucose dehydrogenase (Ameyama et al., 1981). In this case gluconic acid is produced intracellulary. It is unknown until now which system is responsible for the extensive production of gluconic acid by G^ suboxydans■ This organism can produce gluconic acid in concentrations exceeding 1 mol/1. More established is the nature of the gluconic acid production process in Pseudomonas spp., Klebsiella aerogenes and A_^ calcoaceticus spp. In all these bacteria the conversion of glucose to gluconic acid is catalyzed exclusively by a membrane-bound glucose dehydrogenase, an enzyme belonging to the group of quinoproteins.

1.2. The cofactor PQQ

As early as 1955, a lack of coenzyme requirement and a stimulation of

Mg was shown for the oxidation of glucose to gluconic acid by the glucose dehydrogenase (GDH) present in cell-free extracts of Aerobacter aerogenes

Table 2. Some characteristics of purified pyrrolo-quinoline quinone midpoint potential at pH 7.0 absorbance coefficient molecular weight formula temperature s t a b i l i t y of PQQ pH s t a b i l i t y of POO s t a b i l i t y of POOH, structure POO structure POOH. + 90 mV 25.8 103 vT^.cm at 257 nm 330 C14 °8 N2 H4 stable at 120°C s t a b l e moderately low pH ( 2) not stable in an aerobic environment

Wovelength (nm)

F i g u r e 4

Absorbance spectrum of 'PQQ' (PQQ, PQQ-HeO) i n 0.1 M p o t a s s i u m

p h o s p h a t e , pH 7 . 0 , a t 17°C ( ) and a t 75=C ( ) .

The d i f f e r e n c e s o r e due t o f o r m a t i o n of a h i g h e r amount of PQQ-He0 a t low t e m p e r a t u r e . ( r e p r o d u c e d from Duine e t e l . , 1986).

H

0

glucose gluconolactone - ^ ► gluconate+H*

2H

y 0

H

0

(Dalby and Blackwood 1955). In the sixties Hauge showed that the prosthetic group of glucose dehydrogenase from Bacterium anitratum exhibited unique properties (Hauge, 1961a; 1961b; 1964; Hauge and Miirer, 1964). This cofactor was also present in cell-free extracts of Bacterium anitratum in a free form and could be used to reconstitute the apo-GDH present in cells of Rhodopseudomonas spheroides grown anaerobically in the light (Niederpruem and Doudoroff, 1965).

Not only glucose dehydrogenase but also the methanol dehydrogenase of Gram-negative methylotrophic bacteria was found to contain a cofactor with similar unique properties (Anthony and Zatman, 1967; Anthony, 1986). Originally this cofactor was considered to be a pteridine (Urushibara et al., 1971) and later on a lumazine derivative (Sperl et al., 1973). However, ESR spectroscopy showed that the cofactor of methanol dehydrogenase was a nitrogen-containing quinone (Westerling et al., 1979). Its chemical structure was resolved independently by Duine et al. (1980) and Salisbury et al. (1979). Salisbury et al. proposed the trivial name "methoxatin" for the structure: 4,5-dihydroxy~4,5-dioxo-lH-pyrrolo(2,3-f)quinoline-2,7,9-tricarboxylic acid (Salisbury et al. 1979). Duine et al. (1980) proposed the semisystematic name pyrrolo-quinoline quinone (PQQ). Enzymes which carry this cofactor are now commonly termed quinoproteins. It was shown that glucose dehydrogenase of Acinetobacter calcoaceticus contained the same cofactor as methanol dehydrogenase (Duine et al., 1979).

The chemical structure of PQQ and some physical properties are given in Fig. 4 and table 2. After its discovery it became necessary to produce the cofactor in substantial amounts via chemical synthesis. By now several routes to synthesize PQQ are known (Corey and Tramontana 1981). Also a convenient determination method for PQQ using HPLC technique and a biological test system with apo-GDH have been developed by Duine and coworkers (1983) and later by Ameyama et al. (1985). A sensitive biological detection method using commercially available Escherichia coli cells is described in this thesis (Van Schie et al., 1987).

Identification of oxido-reductases containing PQQ as a cofactor is now relatively easy and the list of quinoproteins is expanding rapidly (table 3 ) . Quinoproteins are not only found in Gram-negative prokaryotes, but also in the Gram-positive Arthrobacter PI in which the methylamine oxidase was found to be a quinoprotein (Iersel van, et al. 1986). Also in eukaryotic cells quinoproteins are present. For instance mammalian serum amine oxidase and choline dehydrogenase are quinoproteins (Lobenstein-Verbeek et al.,

ENZYME ORGANISM/SOURCE REFERENCE Methanol dehydrogenase Glucose dehydrogenase Ethanol dehydrogenase Glycerol dehydrogenase Methylamine dehydrogenase Aldehyde dehydrogenase Methylamine oxidase Polyethylene glycol dehydrogenase Choline dehydrogenase Amine oxidase Nitroalkane oxidase Diamine oxidase Lysyl oxidase Polyvinylalcohol dehydroge-Hyphomicrobium sp. see table 4 + 5 Pseudomonas aeruginosa Gluconobacter industrius Pseudomonas AMI

acetic acid bacteria Arthrobacter PI

Synergistic culture of a Pseudomonas and a Flavo-bacterium species Mammalian liver Mammalian bovine serum

Fusarium oxysporum Pore kidney Human placenta Pseudomonas sp.

Duine and Frank, 1979

Groen et al., 1984 Ameyama et al., 1985 De Beer et al., 1980 Ameyama and Adachi, 1982 Iersel van et al., 1986 Kawai et al., 1985

Ameyama et al., 1985

Lobenstein-Verbeek et al.,1984; Moog et al., 1986

Kido and Soda, 1984

Meer, van der et al., 1986a Meer, van der et al., 1986b Shimao et al., 1986

1984; Ameyama et al., 1985). We have provided evidence that several bacteria do not synthesize PQQ under a wide range of growth conditions, suggesting that these bacteria are unable to synthesize PQQ (see Table 4) (Van Schie et al. 1984; 1985; 1987). We have proposed that PQQ can be regarded as a vitamin for organisms which synthesize a quinoprotein apo-enzyme but are unable to produce PQQ. Indeed addition of PQQ to the growth medium resulted in growth of a Pseudomonas sp. on polyviny] alcohol (Shimao et al., 1984; 1986), of P_;_ testosteroni on ethanol (Groen et al ., 1986) and of PTS-negative mutants of E. coli on glucose (Hommes et al., 1985) and galactose

(Hommes et al., 1986). Further support for the assumption that in these cases PQQ may be regarded as a vitamin is provided by the observation that PQQ can be encountered as a naturally occuring compound in the environment (Ameyama et al., 1985).

For example, Hyphomicrobium spp. excrete substantial amounts of PQQ (Ameyama et al., 1984) and PQQ can be found in vinegars obtained from acetic acid bacteria (Duine et al, 1985). So far the enzymic basis for the inability of certain bacteria to synthesize PQQ is not known. The elucidation of this problem is to be awaited in the near future since studies on the biosynthetic route for PQQ synthesis are well under way.

1.3. The quinoprotein glucose dehydrogenase

In 1864, Pasteur first reported on the incomplete oxidation of ethanol to acetic acid with Mycoderma aceti. The first report on microbial production of gluconic acid was published by Boutroux (1880) who isolated calcium-gluconate from cultures of Mycoderma aceti (which is likely to have been a species of Acetobacter). Pseudomonas species have also been reported to produce gluconic acid from glucose (Lockwood, 1941; Stokes and Campbell, 1951; Entner and Stanier, 1953). Direct oxidation of glucose to gluconic acid by an Aerobacter sp. was described in 1949 (De Ley, 1949; De Ley and Cornut, 1951).

The enzyme catalyzing glucose oxidation in Aerobacter aerogenes (now known as Klebsiella aerogenes) was characterized by Dalby and Blackwood (1955). It was found to be present in the particulate fraction of the cell-free extract and "appeared to be specific for sugars having the sane configuration as glucose at the second and fourth carbon atoms". The enzyme-system was not stimulated by any known coenzyme but required divalent cations for optimal activity (Dalby and Blackwood, 1955). In the early

Table 4. Organisms in which the oxidation of glucose to gluconic acid is partially or completely dependent on exogenous PQQ.

ORGANISMS REFERENCES Acinetobacter lwoffi Acinetobacter calcoaceticus LMD 82.3 Rhodopseudomonas sphaeroides Pseudomonas acidovorans Pseudomonas aeruginosa Escherichia coli Agrobacterium radiobacter Agrobacterium tumefaciens Rhizobium .japonicum Rhizobium leguminosarum Azotobacter vinelandii Klebsiella aerogenes Salmonella thyphimurium v. Schie et al., 1984 v. Schie et al., 1987 v. Schie et al v. Schie et al v. Schie et al Homines et al.,1985 Linton et 1985 v. Schie et v. Schie et v. Schie et v. Schie et Ameyaraa et al., Hommes et al., 1984 1984 1984; 1985 a l . , 1 9 8 4 ; v . S c h i e e t

al.,1987; Ameyama et al.

al, al. al. al, , 1987 , 1987 , 1987 , 1987 1985 1986

Table 5. Bacteria reported dehydrogenase.

to possess active membrane-bound glucose

ORGANISM REFERENCE Pseudomonas fluorescens Aerobacter aerogenes Azotobacter vinelandii Micrococcus sodonensis Pseudomonas pseudomallei Gluconobacter suboxydans Pseudomonas quercito-pyrogallica Acinetobacter calcoaceticus Pseudomonas aeruginosa Pseudomonas fragi Rhodopseudomonas sphearoides Pseudomonas cepacia Pseudomonas putida Acetobacter pasteurianum Klebsiella aerogenes Serratia marcescens 1)

Wood and Schwerdt, 1953 Dalby and Blackwood, 1955 Brodie and Lipmann, 1955 Perry and Evans, 1956 Dowling and Levine, 1956

King and Cheldelin, 1957; Stouthamer, 1960

Bentley and Schlecta, 1960 Hauge, 1960

Campbell et al. , 1962 Leroux and Tarr, 1963

Niederpruem and Doudoroff, 1965 Lessie et al., 1973

Vincente and Canovas, 1973 Kieslich, 1976

Neijssel et al., 1983 van Schie et al., 1984

sixties the GDH from Bacterium anitratum was purified and characterized by Hauge (Hauge, 1960a 1960b; 1964; Hauge and Hallberg, 1964; Hauge and Mu'rer, 1964). A comparative study on GDH in bacteria by Hauge (1961b) revealed a strong homology with the enzymes present in Acetobacter suboxydans. Pseudomonas fluorescens, Azotobacter vinelandii. Aerobacter aerogenes and Bacterium anitratum. Glucose dehydrogenase has been detected in a wide variety of bacteria (table 4 and 5) and has been purified to homogenity from

several sources (table 6 ) .

It is noteworthy that the quinoprotein GDH is not the only enzyme capable of production of gluconic acid: several other glucose oxidizing enzymes are known (table 7 ) .

Table 6. Organisms from which quinoprotein glucose dehydrogenase ha s been purified.

ORGANISM REFERENCE

Bacterium anitratum Hauge, 1964

Gluconobacter suboxydans Adachi and Ameyama, 1981 Pseudomonas fluorescens Matsushita and Ameyama, 1981 Acinetobacter calcoaceticus Dokter et al., 1986

Escherichia coli Ameyama et al., 1986

The GDH in Acinetobacter spp. (and probably in other bacteria as well) is localized at the periplasmic side of the cytoplasmic membrane (Fig. 5 ) . This can be concluded from the fact that whole cells (and membrane vesicles) show high rates of glucose oxidation, whereas the rate of transport of glucose or gluconate into the cell is very low. Other evidence for the periplasmic localization is the finding that cultures growing in the presence of low concentrations of Triton-X-100 excrete GDH into the culture fluid (Dokter et al., 1985) and the observation that inside out vesicles oxidize glucose at a rate 30 times lower than the oxidation rate observed with right-side out vesicles (Kitagawa et al., 1986). More direct evidence for an extracellular localization such as presented for methanol dehydrogenase (Kasprzak and Steenkamp, 1984) and ethanol dehydrogenase (Loffhagen and Babel, 1984) has now been reported for the E.coli glucose dehydrogenase (Matsushita et al., 1986).

The GDH of Acinetobacters is coupled to the respiratory chain at the level of cytochrome-b as was already shown by Hauge (1960; Hauge and Hallberg, 1964). Hauge showed that isolation of GDH is accompanied with a

ENZYME EC NUMBER COFACTOR SOURCE REFERENCE

Glucose dehydrogenase 1.1.1.118 NAD

Glucose dehydrogenase 1.1.1.119 NADP

Glucose dehydrogenase 1.1.99.10 FAD

Glucose dehydrogenase 1.1.99.17 PQQ

Glucose oxidase 1.1.3.4 FAD

Bacillus megaterium Broberg et al.; 1968

Gluconobacter suboxydans Ameyama et al., 1981

Pediococcus pentosaceus Lee and Dobrogosz, 1965 Aspergillus oryzae Bak, 1967

Acinetobacter calcoaceticus Hauge, 1966

Aspergillus niger Pazur, 1966

cytochrome (Hauge, 1960b), this cytochrome has now been isolated and purified by Paul Dokter of our department. This cytochrome b-562 might be essential for GDH activity and possesses a molecular weight of 18,000 (Dokter et al.,.1987). Like GDH it is located at the periplasmic site of the membrane. The absorbance spectrum of the purified cytochrome b-562 of A. calcoaceticus (Dokter et al., 1987) was found to be similar to that of the recently purified cytochrome b-562 of a Rhodopseudomonas spheroides (Koh-Iba et al., 1986) an organism which also possesses GDH. Such a cytochrome b-562 has also been purified for E^ coli (Bullock and Meyer, 1978; Kita et al., 1984). This cytochrome might be the natural electron acceptor for PQQH„ and,

in analogy with NADH dehydrogenase, may be called PQQ-dehydrogenase. Electron-transfer from glucose is mediated via a quinone (Beardmore-Gray and Anthony, 1986) which transfers the electrons to the bulk cytochrome b. Matsushita et al. (1987) have provided evidence for the coupling of GDH to the E_^ coli respiratory chain. Electron transfer via GDH is mediated via ubiquinone 8 towards cytochrome o or d. They have shown that proteoliposomes reconstituted with GDH, Q8 and cytochrome oxidase function as effectively as native membrane vesicles with respect to turnover rate and generation of a proton gradient.

1.4. Physiological functions of incomplete oxidations

The terminology used in the past to describe incomplete oxidations reveals a lack of detailed knowledge on the physiological function of these processes. Terms as overproduction of metabolites, uncoupled growth, metabolic slip reactions, overflow metabolism, shunt metabolism, energy-spilling reactions and energy-dissipating processes can be found in literature. It is of interest to note that all these terms carry a more or less negative tang. This is even the more striking in descriptions of incomplete oxidations as: "The best explanation seems to be that the metabolism of the organism becomes deranged. It becomes, so to speak, pathological. This pathological behaviour is a direct result of the influence of abnormal environmental conditions." (Foster, J.W., 1949), and "these reactions are effective mechanisms for spilling the excess energy initially generated when a growth limitation is temporarily relieved" (Neijssel and Tempest, 1979) and "pathophysiology of production mechanisms" (Vanic, 1982). From several studies on the direct oxidation of glucose into gluconate and ketogluconate

it was concluded that these reactions did not provide energy in Pseudomonas aeruginosa (Campbell et al., 1956; Mackechnie and Dawes, 1969) and Gluconobacter oxydans (Uspenskaya and Loitsyanskaya, 1979). However, this view has changed in the past few years. From studies on A^ calcoaceticus IMD 79.41 the role of incomplete oxidations in energy metabolism became evident (this thesis). Also in the case of Klebsiella aerogenes it is now accepted that gluconic acid production in this organism is an adaptation to situations of energy stress (Hommes et al., 1985).

Besides a role in energy metabolism several other physiological functions of incomplete oxidations have been suggested. For instance the incomplete oxidation of glucose to gluconic acid has been assumed to confer the ability of Pseudomonas spp to sequester the sugar as gluconate, a compound suggested to be not readily used by various other organisms (Dawes, 1981). However, this explanation cannot hold in view of the fact that many micro-organisms are known which rapidly degrade gluconate. Another function could be that by acid production an organism creates a micro environment which is beneficial to itself.

Although, as mentioned above, production of gluconic acid from glucose may make a positive contribution to the energy budget of the organism this does not hold in all cases. Gluconic acid production by the fungus Aspergillus niger does certainly not yield energy since this reaction is catalyzed by glucose oxidase that via FAD directly reacts with oxygen according to the equation:

glucose + 0„ -» gluconic acid + H„0„

It is also noteworthy that in many organisms oxidation of glucose into gluconic acid can function in many organisms as a bypass leading to another metabolic route for glucose. In these cases glucose dehydrogenase functions as a low affinity system for glucose metabolion (Lessie and Phibbs, 1984). This has been clearly demonstrated with Pseudomonas spp. These organisms always produce gluconic acid when glucose is present in excess (Dawes, 1981).

This thesis is focused on only one feature of the incomplete oxidation of glucose, namely energy conservation.

The contribution of the GDH catalyzed reaction to the energy conserving processes is difficult to quantify in organisms such as Klebsiella spp., which possess multiple catabolic routes for glucose breakdown (Fig. 3 ) . The

bacterium Acinetobacter calcoaceticus is an exception in this respect: several strains possess GDH as the only glucose-metabolizing enzyme. This organism is therefore an ideal model system to investigate the physiological function of GDH (Fig. 6 ) .

GLUCOSE EgSSJ>GLUCONATE

6P-GLUC0NATE

E.D. PATHWAY

Fig 6. Glucose metabolism in Acinetobacter calcoaceticus LMD 79.41

1.5. Acinetobacter calcoaceticus: model organism for a study on incomplete oxidation of glucose

The bacterium called Acinetobacter calcoaceticus was originally isolated in our laboratory by M.W. Beijerinck (Beijerinck, 1911) and named Micrococcus calcoaceticus (calx = chalk; acetum = acetic acid: calco­ aceticus = calcium acetate, which was used by Beijerinck in the enrichment medium from which he isolated the organism). The genus Acinetobacter (akinetos = unable to move; bactrum = a rod) includes two species (Juni, 1984): Acinetobacter calcoaceticus and Acinetobacter Iwoffi. The aerobic acidification of a glucose-containing medium has been used as a taxonomie criterion: acid producers are called A_^ calcoaceticus formerly known as strains of Bacterium anitratum or Here11a vaginicola. Those Acinetobacter spp. unable to produce acid belong to A^ Iwoffi formerly named Moraxella Iwoffi or Mima polymorpha.

Acinetobacters are rods of 0.9-1.6 jim in diameter and 1.5-2.5 pm in length, which become spherical in the exponential phase of growth. They do

not form spores, are Gram-negative and have a strict respiratory metabolism with oxygen as the terminal electron acceptor. All strains are able to grow

at 20-30 C, with most strains having temperature optima of 33-37°C. They are oxidase-negative, lack cytochrome c, are catalase positive and the mol % G+C

of the DNA is 38-47.

The nutritional versatility of Acinetobacter spp. is enormous, utilizable carbon sources in mineral media include: sugars, fatty acids, aliphatic alcohols, dicarboxylic acids, various amino acids, unbranched hydrocarbons, many aromatic and alicylic compounds (Baumann et al., 1968). Some Acinetobacter strains are well known producers of an extracellular polysaccharide which may amount up to 30* of the total dry mass (Kaplan and Rosenberg, 1983). Their natural habitat is soil, water and sewage

(Acinetobacters are important inhabitants of waste water treatment systems) (Baumann et al., 1968). Although, isolated frequently from several areas of the human body, there is some uncertainty whether Acinetobacters are present as contaminants rather than as commensals (Rosenthal 1978). Although Acinetobacters are generally considered to be non-pathogenic, they are causative agents of nosocomial infections, particularly in debilitated

individuals (Rosenthal 1978; Hoffman 1982).

The taxonomical position of Acinetobacters has been subject to various investigations and from these studies it became clear that this group of bacteria is phenotypically and genetically heterogenous (Baumann et al.,

1968; Johnson et al., 1970). Recently, it was proposed to divide the genus Acinetobacters into 12 genospecies on the basis of a DNA-hybridization study

(Bouvet and Grimont, 1986).

The ability of Acinetobacters to form acid from D-glucose as well as from 2-deoxy-D-glucose, D-mannose, D-allose, L-arabinose, D-xylose,

D-ribose and D-galactose (Dokter et al., 1987) depends on the presence of the quinoprotein aldose dehydrogenase (EG 1.1.99.17) that catalyzes the oxidation of these aldoses to their corresponding aldono-lactone (which hydrolyzes spontaneously to aldonic acid). The majority of Acinetobacters is unable to oxidize these aldonic acids further and this offers therefore a unique model system to study the physiology of these imcomplete oxidations (Fig. 6 ) . This property combined with the relatively high growth rates in mineral media as well as their nutritional versatility and the accessibility towards genetic techniques (Juni, 1978) made us decide to chose A. calcoaceticus as a model organism.

OUTLINE OF THIS STUDY

In this thesis, some physiological characteristics of the incomplete oxidation of glucose to gluconic acid are presented. Attention is mainly focused on the central question: can this incomplete oxidation contribute to the energy-budget of Acinetobacter calcoaceticus and other Gram-negative bacteria?

The results presented in Chapter II show that GDH in A^ calcoaceticus LMD 79.41 is synthesized constitutively but that the synthesis of the enzyme is strongly derepressed during culture conditions which demand a high energy output of dissimilatory processes.

The enzyme is also present in Acinetobacter spp. unable to form acid from aldose sugars, these strains possessing an apo-GDH which can be reconstituted to active holoenzyme by addition of the cofactor PQQ to the growth medium (Chapter III). This phenomenon is not restricted to A_^ lwoffi: several micro-organisms contain apo-GDH as is demonstrated in Chapter IV.

The incomplete oxidation of glucose to gluconic acid leads in several bacteria to the generation of a proton motive force which can drive solute transport (Chapters V and VI).

Not only in vitro but also iri vivo studies reveal that aldose oxidation by GDH leads to the formation of usefull energy for the cell. Gluconic acid formation from glucose resulted in a rapid accumulation of ATP in whole cells of A_^ calcoaceticus (Chapter VI).

Moreover, oxidation of glucose and of other GDH substrates such as glucose by acetate-limited chemostat cultures of A_^ calcoaceticus LMD 79.41 resulted in an increase of the molar growth yield on acetate (Chapter VII).

Although originally only a function of GDH in energy metabolism was foreseen, it became clear in the course of continuous culture studies that A. calcoaceticus LMD 79.41 is intrinsically able to grow on glucose. Prolonged cultivation of the organism in carbon-limited chemostats on acetate/glucose mixtures resulted in the appearance of a new metabolic potential: the ability to grow on glucose as a sole carbon and energy source for growth (Chapter VIII).

This thesis is concluded with a theoretical contribution on the bioenergetics of microbial growth in the presence of an auxiliary energy source. The bioenergetics of the oxidation of glucose to gluconic acid by A. calcoaceticus LMD 79.41 is compared to the oxidation of formiate and thiosulfate by chemolithotrophic bacteria (Chapter IX).

The outline of this study is schematically drawn in figure.7.

APO-GDH

PQQ qlucose qluconic acid

, ^ § ^ 7 Chap. I I + IX

Chap. I l l + IV

HOLO-GDH

< ^

solute A - - J

p m

f j — x ATP synthesis

transport

N v c

.

VT

' r k „ „ UTT j . VTTT U i a p . VI Chap. V + VI

Chap. VII + VIII C h aP '

REFERENCES

Adachi 0. and Ameyama M. (1981) D-glucose dehydrogenase from Gluconobacter suboxvdans. Methods of Enzymology 89, 159-163.

Alefounder P.R. and Ferguson S.J. (1981) A periplasmic location for methanol dehydrogenase from Faracoccus denitrificans: implications for proton pumping by cytochrome aa3 Biochem. Biophys. Res. Commun. 98, 778-784.

Alsberg C.L. (1911) The formation of D-gluconic acid by Bacterium savastanoi. J. Biol. Chem. 9, 1-7.

Ameyama M. and Adachi 0. (1982) Aldehyde dehydrogenase from acetic acid bacteria, membrane-bound. Methods of Enzymology 89, 491-497.

Ameyama M., Hayashi M., Matsushita K., Shinagawa E. and Adachi 0. (1984) Microbial production of pyrrolo-quinoline quinone. Agric. Biol. Chem. 48, 561-565.

Ameyama M. , Nonobe M., Sinagawa E., Matsushita K. and Adachi 0. (1985A) Mode of binding of Pyrroloquinoline Quinone to apoglucose dehydrogenase. Agric. Biol. Chem. 49, 1227-1231.

Ameyama M., Shinagawa E., Matsushita K. and Adachi 0. (1985B) Growth stimulating activity for microorganisms in naturally occuring substances and partial characterization of the substance for the activity as PQQ. Agric. Biol. Chem. 49, 699-709.

Ameyama M., Shinagawa E., Matsushita K., and Adachi 0. (1985C) Solubilization, purification and properties of membrane-bound glycerol dehydrogenase from Gluconobacter industrius. Agric. Biol. Chem. 49, 1001-1010.

Ameyama M, Shinagawa E., Matsushita K., Takimoto K., Nakashima K. and Adachi 0. (1985D) Mammalian choline dehydrogenase a quinoprotein. Agric. Biol. Chem. 49, 3632-3626.

Ameyama M., Nonobe M., Shinagawa E., Matsushita K., Takimoto K. and Adachi 0. (1986). Purification and characterization of quinoprotein apo-D-glucose dehydrogenase from Escherichia coli. Agric. Biol. Chem. 50, 49-57.

Anthony C. (1986) Bacterial oxidation of methane and methanol. Adv. Microbiol. Phys. 27, 113-203.

Anthony C. and Zatman L.J. (1967) The microbial oxidation of methanol. Purification and properties of the alcohol dehydrogenase of Pseudomanaa sp. M 27. Biochem. J. 104, 953-959.

Bak T.G. (1967) Studies on glucose dehydrogenase of Aspergillus orzyae II. Purification and chemical properties. Biochem. Biophys. Acta 139, 277-293.

Baumann P. (1968) Isolation of Acinetobacter from soil and water. J. Bact. 96, 39-42.

Baumann P., Doudoroff M. and Stanier R.Y. (1968) A study of the Moraxella group II. Oxidative-negative species (genus Acinetobacter). J. Bact. 95, 1520-1541.

Beardmore-Gray N. and Anthony C. (1986) The oxidation of glucose by Acinetobacter calcoaceticus: the interaction of the quinoprotein glucose dehydrogenase with the electron transport chain. J. Gen. Microbiol. 132. 1257-1268.

Beer de R., Duine J.A., Frank Jzn.J. and Large P.J. (1980) The prosthetic group of methylamine dehydrogenase from Paeudomonas AMI. Biochem. Biophys. Acta 622, 370-374.

Bentley R., Schlecta L. (1960) Oxidation of mono- and disaccharides to aldonic acids by Paeudomonas species. J. Bac. 79, 346-355.

Beijerinck M. (1911) Pigments as products of oxidation by bacterial action. Proc. K. Ned. Akad. Wet. 13, 1066-1077.

Blanch H.W. (1985) Industrial chemicals, biochemicals and fuels. In: Comprehensive Biotechnology (Ed. M. Moo-Young). Pergamon Press Ltd., Oxford. 3, 659-1045.

Boutroux L. (1880) Sur une fermentation nouvelle du glucosee. C.H. Hebd. Seances Academica Sciences 91, 236-238.

Brodie A.F. and Lipmann F. (1955) The identification of a gluconolactonase. J. Biol. Chem. 212, 677-685.

Broberg P.L., Welsch M. and Smith L (1969) Glucose dehydrogenase of Bacillus megaterium KM, coupling of the cytoplasmic enzyme with membrane-bound cytochromes. Biochim. Biophys. Acta 172, 205-215.

Campbell J.J.R., Ramakrishna T., Linnes A.G., Eagles B.A. (1956) Evaluation of the energy gained by Pseudomonaa aeruginosa during the oxidation of glucose to 2-ketogluconate. Can. J. Microbiol. 2, 304-310.

Corey E.J. and Tramontano A. (1981) Total synthesis of quinonoid alcohol dehydrogenase coenzyme of methylotrophic bacteria. J. Am. Chem. Soc. 103, 5599-5600.

Dalby A. and Blackwood A.C. (1955) Oxidation of sugars by an enzyme preparation from Aerobacter aerogenes. Can. Microbiol. 1, 733-742.

Dawes E.A. (1981) Carbon metabolism, pp. 1-38. In: P.H. Calcott (ed.) Continuous culture of cells Vol.2, CRC Press Inc. Boca Raton, Florida.

De Ley J. (1949) Enzyme activity and nitrogen content of E. coli. Nature 164, 618-620.

De Ley J. and Cornut S. (1951) Direct oxidation of glucose by Aerobacter sp. Nature 168, 515-516.

Dokter P., Kleef van M.A.G., Frank Jzn.J. and Duine J.N. (1985) Production of quinoprotein glucose dehydrogenase in the culture medium of Acinetobacter calcoaceticus. Enzyme Microbiol. Techn. 7, 613-617.

Dokter P., Frank Jnz.J. and Duine J.A. (1986) Purification and characterization of quinoprotein glucose dehydrogenase in Acinetobacter calcoaceticus IMO 79.41. Biochem. J. 239, 163-167.

Dokter P., Pronk J.T., Schie van B.J., Dijken van J.P. and Duine J.A. (1987) In vivo and in vitro substrate specificity of the quinoprotein glucose dehydrogenase in Acinetobacter calcoaceticus IMD 79.41. FEMS Microbial Letters (submitted).

Dokter P., Frank Jzn, J. and Duine J.A. (1987) The purification and characterization of cytochrome 6562 of Acinetobacter calcoaceticus LMD 79.41. Biochem. J. (in preparation).

Dolin M.I. and Juni E. (1978) Utilization of oxalacetate by Acinetobacter callcoaceticus. Evidence for coupling between malic enzyme and malic dehydrogenase. J. Bac. 133, 786-793.

Duine J.A., Frank Jzn.J. and Zeeland J.K. (1979) Glucose dehydrogenase from Acinetobacter calcoaceticus: a quinoprotein. FEBS Letters 108, 443-446.

Duine J.A., Frank Jzn.J. and Verwiel P.E.J. (1980) Structure and activity of the prosthetic group of methanol dehydrogenase. Eur. J. Biochem. 108, 187-192.

Duine J.A., Frank Jzn. J. and Jongejan J.A. (1985) The coenzyme PQQ and quinoproteins, a novel class of oxidoreductase enzymes. Proceedings of the 16th FEBS Congres, partA, 79-88. VN13 Science Press.

Duine J.A., Frank Jzn.J., Jongejan J.A. (1986) PQQ and quinoprotein enzymes in microbial oxidations FEMS Microbiol Rev. 32, 165-178.

Duine J.A., Frank Jzn.J. and Jongejan J.A. (1983) Detection and determination of pyrroloquinoline quinone, the coenzyme of quinoproteins. Anal. Biochem. 133, 239-243.

Foster J.W. (1949) Chemical activities of Fungi, pp. 164-169 Academic Press, New York.

Ghose T.K. and Bhadra A. (1985) Acetic acid In: Comprehensive Biotechnology (Ed. M. Moo-Young) Pergamon Press Ltd., Oxford 3, 701-729.

Gottschalk G. (1986) Bacterial metabolism. Springer-Verlag New York Inc., 169-174.

Groen B.W., Kleef van M.A.G. and Duine J.A. (1986) Quinohaemoprotein alcohol dehydrogenase apoenzyme from Pseudomonas teatosteroni. Biochem. J. 234. 611-615.

Glew R.H., Moellering Jr. M.D. and Kunz L.J. (1977) Interactions with Acinetobacter calcoaceticus: Clinical and Laboratory Studies. Medicine 56, 79-97.

Hauge J.C. (1960a) Kinetics and specificity of glucose dehydrogenase from Bacterium anitratum. Biochem. Biophys. Acta 45, 263-269.

Hauge J.G. (1960b) Purification and properties of glucose dehydrogenase and cytochrome b from Bacterium anitratum. Biochem. Biophys. Acta 45, 250-262.

Hauge J.G. (1961a) Mode of action of glucose dehydrogenase from Bacterium anitratum. Arch. Biochem. Biophy. 94, 308-318.

Hauge J.G. (1961b) Glucose dehydrogenase in bacteria. A comparative study. J. Bact. 82, 609-614.

Hauge J.G. (1964) Glucose Dehydrogenase of Bacterium anitratum: an Enzyme with a Novel Prosthetic Group. J. Biol. Chem. 239, 3630-3639.

Hauge J.G. and Mürer E.H. (1964) Studies on the nature of the prosthetic group of glucose dehydrogenase of Bacterium anitratum. Biochem. Biophys. Acta 81, 244-250.

Hauge J.G. and Hallberg P.A. (1964) Solubilization and properties of the structurally bound glucose dehydrogenase of Bacterium anitratum.Biochem. Biophys. Acta 81, 251-256.

Hauge J.G., King T.E. and Cheldelin V.H. (1955) Alternative conversions of glycerol to dihydroxy acetone in Acetobacter susoxydans. J. Biol. Chem. 214, 1-9.

Hauge J.H. (1966) Glucose dehydrogenase - Particulate 1. Pseudomonas species and Bacterium anitratum. Methods of Enzymology 9, 92-98.

Hoffmann S., Mabeck C.E. and Vejlsgaard R. (1982) Bacteriuria caused by Acintobacter calcoacet icus biovars in a normal population of general practice. J. Clin. Microbiol. 16, 443-451.

Hooper A.B. and DiSpirito A.A. (1985) In bacteria which grow on simple reductants generation of a proton gradient involves extracytoplasmic oxidation of substrate. Microbiol. Rev. 49, 140-157.

Hommes R.W.J., Postma P.W., Neijsel O.M., Tempest D.W., Dokter P. and Duine J.A. (1984) Evidence of quinoprotein glucose dehydrogenase apoenzyme in several strains of Escherichia coli. FEMS Microbial Letters 24, 329-333.

Hucho F. et.al. (1972) Glucono - 6 - lactonase from Escherichia coli Biochem. Biophys. Acta 276, 176-179.

Hu'tter R. (1986) Overproduction of microbial metabolites. _in: Biotechnology vol 4, Ed. H. Pape and H.J. Rehm. VCH Verlagsgesellschaft, Weinheim.

Iba K., Takamiya K. and Arata H. (1985) Isolation and characterization of cytochrome b-562 from cytochrome b-c, complex of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26. FEBS Letters 183. 151-154.

Iersel van J., van der Meer R.A. and Duine J.A. (1986) Methylamine oxidase from Arthrobacter PI: A bacterial copper-quinoprotein amine oxidase. Eur. J. Biochem. 261, 415-419.

Jermyn M.A. (1960) Studies on the glucono - 8 - lactonase of Pseudomonas fluorescens Biochem. Biophys. Acta 37, 78-92.

Juni E. (1978) Genetics and physiology of Acinetobacter. Ann. Rev. Microbiol. 32, 349-371.

Juni E. (1984) In: Krieg N.H., Holt J.G. (eds) Bergey's Manual of Systematic Bacteriology, Will iams and Wilking, Baltimore London, 1, 303—307.

Kaplan N. and Hosenberg E. (1982) Exopolysaccharide Distribution of and Bioemulsifier Production by Acinetobacter calcoaceticus BD4 and BD413. Appl. Environ. Microbiol. 44, 1335-1341.

Kasprzak A.A. and Steenkamp D.J. (1983) Localization of the major dehydrogenases in two methylotrophs by radiochemical labeling J. Bact. 156. 348-353.

Kawai F., Yamanaka H., Ameyama M, Shinagawa E., Matsushita E. and Adachi 0. (1985) Identification of the prosthetic group and further characterization of a novel enzyme, polyethylene glycol dehydrogenase. Agric. Biol. Chem. 49. 1071-1076.

Kido T. and Sada K. (1984) Seikagaku 56, 952.

Kieslich K. (1976) Microbial transformations of non-steroid cyclic compounds. George Thieme Publishers, Stuttgart.

King T.E. and Cheldelin Y.H. (1957) Glucose oxidation and cytochromes in solubilized particulate fractions of Acetobacter suboxydans. J. Biol. Chem. 224. 579-590.

Kita K. and Anraku Y. (1981) Composition and sequence of b cytochromes in the respiratory chain of aerobically grown Escherichia coli K-12 in the early exponential phase. Biochem. Int. 2, 105-112.

Kita K., Konishi K. and Anraku Y. (1984) Terminal Oxidases of Escherichia coli aerobic respiratory chain. J. Biol. Chem. 259, 3368-3374.

Kitagawa K., Tateishi A., Nakano F., Matsumoto T., Morohoshi T., Tanino T.

and Usui T. (1986). Sources of energy and energy coupling reactions of the active transport systems in Acinetobacter calcoaceticus. Agric. Biol. Chem. 50, 2939-2940.

Kluyver A.J., De Ley J. and Rijven A. (1951) The formation and consumption of lactobionic and maltobionic acids by Pseudomonas species. J. Microbiol. Serol. 17, 1-14.

Krumphanzl V., Sikyta B. and Vanek Z. (1982) Overproduction of microbial products. Academie Press, London.

Matsushita K., Nonobe M., Shinagawa E., Adachi 0. and Ameyama M. (1987) Reconstitution of purified pyrroloquinoline quinone-dependent D-glucose oxidase respiratory chain of Escherichia coli with cytochrome o oxidase. J. Bac. 169, 205-209.

Lee C.K. and Dobrogosz W.J. (1965) Oxidative metabolism in Pediococcus pentosaceus III. Glucose dehydrogenase system. J. Bac. 90, 653-660.

Leroux M. and Tarr H.L.A. (1963) Glucose and ribose oxidation by Pseudomonas fragi. Can. J. Biochem. Physiol. 41, 1023-1034.

Lessie T.G. and Phibbs P.V. (1984) Alternative pathways of carbohydrate utilization in Pseudomonas. Ann. Rev. Microbiol. 38, 358-387.

Linton J.D., Woodard S. and Gouldney D.G. (1987) The consequence of stimulating glucose dehydrogenase activity by the addition of PQQ on metabolite production by Agrobacterium radiobacter NCIB 11883. Appl. Microbiol. Biotechnol. 25, 357-361.

Lobenstein-Verbeek C.L., Jongejan J.A., Frank Jzn.J. and Duine J.A. (1984) Bovine serum araine oxidase: a mammaliaro enzyme having covalently bound PQQ as prosthetic group. FEBS Lett. 170, 305-309.

Lockwood L.B., Tabenkin B. and Word G.E. (1941) The production of gluconic acid and 2-ketogluconic acid from glucose by species of Pseudomonas and Phytomonas. J. Bact. 42, 51-61.

Loffhagen N. and Babel W. (1984) Localization of primary alcohol-oxidizing enzymes in the facultative methylotrophic Acetobacter sp. MB 70. Zeitschr. Allg. Microbiol. 24, 143-149.

Mackechnie I. and Dawes E.A. (1969) An evaluation of the pathways of metabolism of glucose, gluconate and 2-oxogluconate by Paeudomonas aeruginosa by measurement of molar growth yields. J. Gen. Microbiol. 55, 341-349.

Matsushita K. and Ameyama M. (1981) D-glucose dehydrogenase from Pseudomonas fluorescens. Methods of Enzymology 89, 149-154.

Matsushita K., Shinagawa E., Inoue T., Adachi 0. and Ameyama M. (1986) Immunological evidence for two types of PQQ-dependent D-glucose dehydrogenase in bacterial membranes and the location of the enzyme in Escherichia coli. FEMS Microbiol Letters 37, 141-144.

Meer van der R.A. and Duine J.A. (1986a) Covalently bound pyrroloquinoline quinonee is the organic prosthetic group in human placental lysyl oxidase. Biochem. J. 239, 789-791.

Meer van der R.A., Jongejan J.A., Frank Jzn.J. and Duine J.A. (1986b) Hydrazone formation of 2,4-dinitrophenyl-hydrazine with pyrroloquinoline quinone in porcine kidney diamine oxidase. FEBS Lett. 206, 111-114.

Milsom P.E. and Meers J.L. (1985) Gluconic and Itaconic acids in: M. Moo-Young, Comprehensive Biotechnology, Vol 3, 681-700 Pergamon Press Ltd., Oxford.

Moog R.S., Mc. Guirl M.A., Cote C.E. and Dooley D.M. (1986) Evidence for methoxatin (pyrroloquinoline quinone) as the cofactor in source plasma oxidase from resonancee Raman spectroscopy. Proc. Nat. Acad. Sci. USA 83, 8435-8439.

Niederpruem D.J. and Doudoroff M. (1965) Cofactor-dependent aldose dehydrogenase of Rhodopseudomonas spheroides■ J. Bact. 89, 697-705.

Neijssel O.M. and Tempest D.W. (1979) The physiology of metabolite overproduction, in: "Proc. 29 Symp. Soc. Gen. Microbiol. Cambridge", pp. 53-82. Cambridge University Press.

Pasteur L. (1862) Etudes sur les mycodermes role de ces plantes dans la fermentation acetique. Compt. Rend. Acad. Scienc. 54, 285-270.

Pazur J.H. (1966) Glucose oxidase from Aspergillus niger. Methods of Enzymology 9, 82-87.

Hohr M., Kubicek C.P. and Kominek J. (1985) Gluconic acid, in Biotechnology vol.3 (Ed.) Rehm H.J. and Reed G., Verlag Chemie, Weinheim.

Rosenthal S.L. 1978 Clinical role of Acinetobacters and Moraxella in: Glucose non-fermenting gram-negative bacteria in clinical microbiolgy. (Ed.) G.H. Gilardi, CRC Press, Florida.

Salisbury S.A., Forrest H.S., Cruse W.B.T. and Kennard 0. (1979) A novel coenzyme from bacterial primary dehydrogenases. Nature 280. 843-844.

Sar N. and Rosenberg E. (1983) Emulsifier production by Acinetobacter calcoaceticus strains. Current Microbiol. 9, 309-314.

Schie van B.J., Dijken van J.P. and Kuenen J.G. (1984) Non-coordinated synthesis of glucose dehydrogenase and its prosthetic group PQQ in Acinetobacter and Pseudomonas species. FEMS Microbiology Letters 24, 133-138.

Schie van B.J., Hellingwerf K.J., Dijken van J.P., Elferink M.G.L., Dijl van J.M., Kuenen J.G. and Konings W.N. (1985) Energy transduction by electron transfer via a pyrrolo-quinoline quinone dependent glucose dehydrogenase in Escherichia coli. Pseudomonas aeruginosa and Acinetobacter calcoaceticus (var. lwoffi). J. Bact. 136, 493-499.

Schie van B.J., Mooy de O.H., Linton J.D., Dijken van J.P. and Kuenen J.G. (1987) PQQ-dependent production of gluconic acid by Acinetobacter. Agrobacterium and Rhizobium species, J. Gen. Microbiol. 133 (in the Press).

Shimao M., Nishimura Y., Kato N. and Sakazawa C. (1985) Localization of polyvinyl alcohol oxidase produced by a bacterial symbiont, Pseudomonas sp. Strain VM15C. Appl. Env. Microbiol. 49, 8-10.

Shimao M., Ninomiya K., Kuno 0., Kato N. and Sakazawa S. (1986) Existence of a novel enzyme, PQQ-dependent polyvinyl-alcohol dehydrogenase, in a bacterial symbiont, Pseudomonas sp. Strain VM15C. Appl. Env. Microbiol. 51,

258-275.

Shimao M., Yamamoto H., Ninomiya K., Kato N., Adachi 0., Ameyama and Sakazawa S. (1984) POQ as an essential growth factor for a polyvinyl alcohol degrading symbiont, Pseudomonas sp. VM15C. Agric. Biol. Chem. 48, 2873-2876.

Spert G.T., Forrest H.S. and Gibson D.T. (1973) Chemical characterization of the cofactor isolated from the alcohol dehydrogenase of methane and methanol oxidizing bacteria. Bacteriol. Proc. 151.

Stouthamer A.H. (1960) Koolhydraatstofwisseling van de azijnzuurbacteriën. Thesis, University Utrecht.

Stubbs J.J., Lockwood L.B., Roe E.T., Tabenkin B. and Ward G.E. (1940) Bacterial production of ketogluconic acids from glucose. Ind. Eng. Chem. 32, 1626-1634.

Urushibara T., Forrest H.S., Hoare D.S. and Patel R.N. (1971) Pteridines produced by Methylococcus capsulatus. Biochem. J. 125, 141-146.

Uspenskaya SLN., Loitsyanskaya M.S. (1979) Effectiveness of the utilization of glucose by Gluconobacter oxydans. Microbiology 48, 306-310.

Vanek Z., Cudlin J., Blulmauerova M., Hostalek Z., Podojil M., Rehacek Z. and Krumphanzl V. (1981). Physiology and pathophysiology of the production of excessive metabolites. Inst. Microbiol., Czechoslovak Academy of Sc i ences, Prague.

Weimberg R. (1961) Pentose oxidation by Pseudomonas fragi. J. Biol. Chem. 236. 629-635.

Wei-Ping-Lu, Swoboda B.E.P. and Kelly D.P. (1985) Properties of the thiosulphate-oxidizing multi-enzyme system from Thiobacillus versutus. Biochem. Biophys. Acta 828, 116-122.

Westerling J., Frank Jzn. and Duine J.A. (1979) The prosthetic group of nethanol dehydrogenase from Hyphomicrobium X: electron spin resonance evidence for a quinone structure. Biochen. Biophys. Res. Commun. 87, 719-724.

Wood W.A. and Scbwerdt R.F. (1953) Carbohydrate oxidation by Pseudomonas fluorescens. I. The mechanism of glucose and gluconate oxidation. J. Biol. Chem. 201, 501-511.

Wijsman M.R., van Dijken J.P., van Kleeff B.H.A. and Scheffers W.A. (1984) Inhibition of fermentation and growth in batch cultures of the yeast Brettanomyces intermedius upon a shift from aerobic to anaerobic conditions (Ousters effect). Antonie van Leeuwenhoek 50, 183-192.

Chapter II

Effects of growth rate and oxygen tension on glucose dehydrogenase activity

in Acinetobacter calcoaceticus 1MD 79.41

B.J. van Schie, J.P. van Dijken and J.G. Kuenen.

SUMARY

The regulation of the synthesis of the quinoprotein glucose dehydrogenase (EC 1.1.99.17) has been studied in Acinetobacter calcoaceticus LMD 79.41, an organism able to oxidize glucose to gluconic acid, but unable to grow on both compounds. Glucose dehydrogenase was synthesized constitutively in both batch and carbon-limited chemostat cultures on a variety of substrates. In acetate-limited chemostat cultures glucose dehydrogenase levels and the glucose-oxidizing capacity of whole cells were dependent on the growth rate. They strongly increased at low growth rates at which the maintenance requirement of the cells had a pronounced effect on biomass yield.

Cultures grown on a mixture of acetate and glucose in carbon and energy-limited chemostat cultures oxidized glucose quantitatively to gluconic acid. However, during oxygen-limited growth on this mixture glucose was not oxidized and only very low levels of glucose dehydrogenase were detected in cell-free extracts. After introduction of excess oxygen, however, cultures or washed cell suspensions almost instantaneously gained the capacity to oxidize glucose at a high rate, by an as yet unknown mechanism.

INTRODUCTION

The biochemistry of glucose dehydrogenase, (GDH; EC 1.1.99.17) from Acinetobacter calcoaceticus has been studied extensively by Hauge in the early sixties. The enzyme was found to contain an unknown cofactor (Hauge 1960), later identified as PQQ (Duine et al. 1986). A detailed characterization of this enzyme from A^ calcoaceticus showed that glucose dehydrogenase is a membrane-bound perlplasmic enzyme, containing two molecules of PQQ per enzyme molecule (Dokter et al. 1986).

Regulation of the synthesis of the quinoprotein glucose dehydrogenase has mainly been studied in Pseudomonas spp. The physiological role of the enzyme is difficult to deduce from such studies due to the presence of alternative routes of glucose catabolism (Lessie and Phibbs 1984). Glucose dehydrogenase is synthesized constitutively in Pseudomonas cepacia (Berka et al. 1984) but is inducible in Pseudomonas aeruginosa (Hylemon and Phibbs 1972). GDH activity in P^. aeruginosa grown on glucose, decreased at low dissolved oxygen tensions (Mitchell and Dawes 1982) and was absent during

anaerobic growth on glucose (Hunt and Phibbs 1983). This, however, is not due to the absence of GDH protein, but results from apparent inability of P. aeruginosa to synthesize PQQ anaerobically (Van Schie et al. 1984). Whether such a modulation of GDH activity via PQQ synthesis is a general phenomenon in Pseudomonas spp. is not known at present. GDH activity in P. fluorescens was shown to be affected by several growth conditions. Synthesis of the enzyme is favoured by growth at low temperatures (Lynch et al.1975a, b ) , low pH (Quay et al.1972) or high water activity (Prior and Kenyon 1980).

Although A_^ calcoaceticus LMD 79.41 is unable to grow on glucose or gluconate, glucose is oxidized quantitatively at a high rate to gluconic acid (Van Schie et al. 1984). Preliminary results indicated that GDH is synthesized constitutively in this organism (de Bont et al. 1984), but that GDH synthesis is affected by growth rate (Visser et al. 1985). In view of the important role of this enzyme in the utilization of glucose as an auxiliary energy source by A_;_ calcoaceticus (Van Schie et al. 1987c; Muller and Babel 1986) it was decided to investigate regulation of GDH activity in more detail.

MATERIAL AND METHODS

Media and growth conditions

Chemostat cultivation of Acinetobacter calcoaceticus LMD 79.41 was performed in Applicon laboratory fermenters with a working volume of 1 L at pH 7.0 and 30 C. Dissolved oxygen was measured with a galvanic oxygen electrode, and controlled at the desired value by the stirring rate. The mineral salts medium was prepared according to van Schie et al. (1984). It contained one of the following substrates as a carbon and energy source: sodium acetate, 30 mM; sodium p-hydroxybenzoate, 15 mM; alanine 15 mM; sodium adipate 10 mM; sodium succinate 15 mM; ethanol 30 mM. Oxygen-limited cultures were obtained by adjusting the stirring rate to such an extent that approximately 10* of the input concentration of the carbon source was detected in the culture fluid. Batch cultivation was performed in erlenmeyer flasks of 100 ml, containing 25 ml of mineral medium, on rotatory shakers at 150 rpm and 30*C.

Cells were washed with a 100 mM phosphate buffer, pH 7.0 containing 10 raM MgSO. and resuspended in 4 ml of the same buffer. Cells were disrupted by sonication at 4*C with an MSB 150 W sonicator for 3 min with intermittant periods of cooling. Whole cells and debris were removed by centrifugation for 20 min at 13.000x g. The clear supernatants were used as cell- free extracts.

Enzyme assays.

Glucose dehydrogenase assays were carried out at 30*C with freshly-prepared extracts using a model 100-60 Hitachi spectrophotometer. In all assays, the reaction was linearly proportional to the amount of extract present. The assay mixture used contained: potassium phosphate buffer, pH 7.0, 100 mM; MgSO., 10 mM; KCN, 1 mM; DCPIP (2,6- dichlorophenolindophenol), 60 (iM and PBS (phenazine ethosulphate) 0.3 mM. The reaction was started by addition of glucose to a final concentration of 20 mM. A molar extinction coefficient for DCPIP 18 mM was used to calculate enzyme actvity (Armstrong 1964).

Measurement of glucose-oxidizing capacity of whole cells.

Glucose oxidation by whole cells was assayed by following the rate of oxygen consumption at 30 C with a Clark type oxygen electrode. In the case of oxygen-limited cultures, cells were washed twice at 4*C with 100 mM potassium phosphate buffer pH 7.0 containing 10 mM MgSO. by centrifugation to remove residual substrate. The reaction was started by addition of 20 mM glucose and oxygen-consumption rates were calculated on the basis of an oxygen concentration in air saturated buffer of 225 jiM.

Analytical assays

Glucose was measured with the GOD/PAP method, gluconate with gluconate kinase/6-P-gluconate dehydrogenase, acetate with acetyl-coA-synthetase, citrate synthase and malate dehydrogenase (testcombinations Boehringer). Protein was measured by the Bradford method (Bio-Rad Laboratories) and by a modified Lowry method (Pierce Chemical Company) with bovine serum albumine as a standard, according to the instructions of the manufacturers. A Beekman Model 915B Tocamaster Total Organic Carbon Analyser was used to determine the carbon content of whole culture and culture supernatants, the carbon

content of bacteria being obtained from the difference. Bacterial dry weight was calculated assuming a cellular carbon content of 50%.

Chemicals

DCPIP and PES were obtained from Sigma Chemical Co., PQQ (2.7.9-tricarboxy-lH-pyrrolo [2.3.f] quinoline-4.5- dione) was a gift of Dr J.A. Duine from our department (PQQ is now commercially available from Fluka AG, Buchs, Switserland.)

RESULTS

Like the majority of Acinetobacter species A^ calcoaceticus LMD 79.41 is able to produce acid from several aldose sugars but is incapable of growth on these sugars or on the corresponding aldonic acids (Baumann et al., 1968; Juni, 1978). Cells grown in batch culture on acetate, alanine, citrate, glutamate, lactate, malate, or pyruvate and harvested in the stationary growth phase oxidized glucose with activities varying between 35-80 nmol

-1 -1

oxygen consumed, min .mg dry wt (Table 1 ) . The product of glucose oxidation was identified as gluconic acid. During carbon-limited chemostat cultivation on several substrates at a relatively low growth rate of

0.15 h , cells possessed a much higher glucose oxidation capacity. Activities varied between 390-775 nmol oxygen consumed.min .mg dry wt

(Table 1 ) . Also these cells quantitatively oxidized glucose to gluconic acid. It can thus be concluded that GDH is synthesized constitutively by A. calcoaceticus LMD 79.41. In order to further investigate the cause of the 10-fold difference in glucose-oxidizing capacity between batch and chemostat cultures, the effect of growth rate on GDH synthesis was investigated.

A. calcoaceticus LMD 79.41 grown acetate- limited in chemostat cultures exhibited a growth rate-dependent cell yield (Fig 1 ) . Below a dilution rate of 0.2 h a marked decrease in cell yield was observed. At a dilution rate of 0.025 h the cell yield amounted less than 25% of the value obtained at high dilution rates due to a high maintenance requirement. From a double reciprocal plot of cell yield versus growth rate (Fig 2 ) , a maintenance coefficient of 3.2 mmol acetate (g dry wt) h and a maximum molar growth yield of 26.1 g dry wt (mol acetate) was calculated. A similar pronounced