Effect of growth substrates on formation of alcohol dehydrogenase in Acetobacter methanolicus and Acetobacter aceti

JOURNAL OF FERMENTATION AND BIOENGINEERING

Vol. 83, No. 1, 21-25. 1997

Effect of Growth Substrates on Formation of Alcohol Dehydrogenase in Acetobacter methanolicus and Acetobacter aceti JITKA FRBBORTOVA,

* KAZUNOBU

MATSUSHITA,

AND

OSAO ADACHI

Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, 1677-l Yoshida, Yamaguchi 753, Japan Received 26 June 1996/Accepted 11 November 1996

Two diierent strains of acetic acid bacteria, Acetobacter methanoiicus and Acetobacter aceti, were grown on various carbon sources and their quinoprotein alcohol dehydrogenase (ADH) activities were measured. A. aceti was able to grow only on glycerol or glucose as a sole carbon source. The addition of another carbon source to a glycerol-containing medium promoted growth and increased the level of ADH activity. The results indicate that while ADH was constitutively produced under the growth conditions, an inactive form of ADH was formed besides the active form in cells grown in the medium containing glucose, acetic acid, or succinic acid. A. methanolicus was able to grow on a variety of carbon sources. ADH was formed in cells grown on glycerol or ethanol but not on methanol. ADH was also produced in cells grown on glucose or succinic acid, but the major part seemed to be the inactive form. The inactive form of ADH was thus shown to be produced under various growth conditions in both strains. [Key words: Acetobacter, alcohol dehydrogenase,

quinoprotein,

carbon source]

subunits I and II have been cloned and sequenced (9, 10). Like the ADH of A. methanolicus, the enzyme constitutes the ethanol oxidase respiratory chain, together with ubiquinone-9 and ubiquinol oxidase (8, 11, 12). The ADH activity in Acetobacter pasteurianus has been reported to increase more than lo-fold when the bacterium is grown in the presence of ethanol in a medium consisting of yeast extract, Polypepton, and glucose (13). Here, we report on the effect of a variety of growth substrates on ADH activities in two different acetic acid bacteria, A. aceti IF0 3284 and A. methanolicus JCM 6891. The results indicate that the ADHs of both A. aceti and A. methanolicus are constitutive, except when A. methanolicus is grown on methanol. The results also suggest that the ADH activities of both strains can be regulated by controlling the enzyme in its active and inactive forms.

Acetic acid bacteria are able to oxidize various alcohols and sugars using primary dehydrogenases localized on the periplasmic side of the cytoplasmic membrane and functioning by linking to the respiratory chain. Acetobacter methanolicus is a facultative methylotroph, able to grow at pH4.0 on methanol, glycerol, or glucose as a carbon and energy source by way of the ribulose monophosphate pathway (1). When grown on glycerol, the cells contain large amounts of quinoprotein alcohol dehydrogenase (ADH), while methanol-grown cells exhibit high methanol but low alcohol dehydrogenase activities (2). Both ADH and methanol dehydrogenase (MDH) have been purified from this organism and characterized (2, 3). ADH is a membrane-bound enzyme consisting of three subunits of approximately 80, 54, and 8 kDa (FrtbortovB, J. et al., unpublished results). Subunits I and II contain one and three heme c moieties, respectively, and subunit I also contains quinone cofactor, PQQ. MDH, on the other hand, is a soluble protein. Two different forms of MDH have been purified from A. methanolicus (3). Type I exhibits an a& conformation consisting of 62 and 8.5 kDa peptides, while type II has an a& conformation with a 7 subunit having a molecular mass of 32 kDa. The cofactor of both enzymes is PQQ. It has been shown that two different respiratory chains for ethanol and methanol oxidation are induced dependent on the carbon source (2). The ethanol oxidase system consists of ADH, ubiquinone-lo, and cytochrome bo ubiquinol oxidase, while the methanol oxidase system is composed of MDH, cytochrome cL, and cytochrome c oxidase. Acetobacter aceti is a widely used vinegar-producing bacterium. It can grow on ethanol, glucose, or glycerol as a carbon and energy source (4, 5). Quinoprotein ADH of A. aceti is involved in the oxidation of ethanol to acetic acid, together with aldehyde dehydrogenase (6, 7). ADH from A. aceti has been purified as a 3-subunit complex and characterized (5, 8). The genes encoding

MATERIALS

AND METHODS

Bacterial strains, growth conditions, and preparation A. methanoiicus of soluble and membrane fractions JCM 6891 was grown aerobically at 30°C in a basal medium consisting of 3 g (NH&S04, 1 g KH2P04, 0.16 g 0.7 g MgS04.7H20, 0.5 g NaCl, 0.4g K2HP04, Ca(NO&, and 5 g yeast extract per liter, supplemented with 1% methanol or 0.5% of another carbon source (14). When succinic or pyruvic acid was used as a carbon source, the pH of the medium was adjusted to 4 using 1 M NaOH. In other cases, the pH was adjusted to 4 using concentrated HCl. A. aceti IF0 3284 was grown aerobically at 30°C in a basal medium containing 3g yeast extract and 2 g Polypepton per liter of 50mM potassium phosphate buffer (pH 6.5), which was supplemented with 1% glycerol or 0.1% glycerol with 0.5% of another carbon source. The pH was adjusted to 5 with 1 M NaOH in the case of growth on succinic acid or pyruvic acid as another carbon source. Both strains were cultured first for 24 h in a 20-ml tube with 5 ml of the basal medium containing an appropriate carbon

* Corresponding author. 21

22

FREBORTOVA ET AL.

source. Two milliliters of the broth were then inoculated into a 500-ml flask containing 1OOml of the same medium. Cells were grown to the late logarithmic phase and then harvested. The cells were disrupted and the membrane and soluble fractions were prepared as described previously (2, 8). Enzyme activities were measured at Enzyme assays 25°C. The ferricyanide reductase activity of ADH was measured calorimetrically with Dupanol reagent (6). MDH and GDH activities were measured spectrophotometrically with phenazine methosulfate (PMS) and 2,6-dichlorophenol indophenol (DCIP) as electron acceptors by monitoring the decrease in the absorbance at 600nm. The extinction coefficients of DCIP at pHs6.0 and 9.5 are 11,130M~r cm-’ and 15,9OQM-’ cm-l, respectively. The reaction mixture (total 1 ml) for MDH contained 45.5 mM glycine-NaOH buffer (pH 9.5), enzyme solution, 0.11 mM DCIP, 0.2 mM PMS, 51 mM ethylamine hydrochloride, and 10 mM methanol. GDH was assayed in a reaction mixture (total 1 ml) consisting of 45 mM potassium phosphate buffer (pH 6.0), enzyme solution, 0.11 mM DCIP, 0.2 mM PMS, 8 mM NaNs, and 10mM glucose. When the membrane fraction was used as an enzyme source, 1 mM KCN was included in both reaction mixtures. One unit of enzyme activity was defined as 1 pmol of substrate oxidized by the enzyme per minute. SDS-PAGE and immunoblot analysis SDS-PAGE (15) was performed on a slab gel (12.5%) in Tris-glycine running buffer. Before application, samples were treated at 60°C with 3% SDS and 50mM dithiothreitol for 30min. The standard marker proteins were the prestained molecular weight markers (low molecular weight Immunoblotting was performed as range, Bio-Rad). described (16) using antibody raised against ADH of A. aceti (17). Heme staining was performed as heme-catalyzed peroxidase activity (18). Heme content was determined Other procedures from the dithionite-reduced minus ferricyanide-oxidized difference spectra of its pyridine hemochrome using a Hitachi 557 dual-wavelength spectrophotometer. The sample was prepared by mixing with pyridine to 20% and NaOH to 0.2 M final concentrations. The heme content was calculated using an extinction coefficient of 24,300 M-r cm-r (549-535 nm). Protein content was determined by a modified version of Lowry’s method (19) with bovine serum albumin as a standard protein.

J. FERMENT. BIOENG.,

0

10

20

30

40

50

0

10 20 30 40

50 60 70

Time (h) FIG. 1. Growth of A. uceti in media containing various growth substrates. (A) Growth in the basal medium containing 1% glycerol (0) or 0.5% glucose (0). (B) Growth in the basal medium containing 0.1% glycerol (m), or containing 0.1% glycerol together with 0.5% glucose ( q), 0.5% ethanol (A), 0.5% acetic acid (v), 0.5% lactic acid (A), or 0.5% pyruvic acid (0). The growth in the presence of 0.1% glycerol with 0.5% succinic acid was the same as that in the presence of only 0.1% glycerol.

while ethanol and succinic acid showed no such promotional effect. With acetic acid, growth occurred after a prolonged lag phase (Fig. 1B). Glucose and ethanol increased the total growth, lactic, pyruvic, and acetic acids did so to some extent, but succinic acid did not do so at all. A. methanolicus grew best on methanol or glucose as carbon sources. Growth on glycerol and succinic acid occurred only after a prolonged lag phase. Poor growth was observed on ethanol, and no appreciable growth on pyruvic acid or without any carbon source (Fig. 2). ADH activity in A. aceti Approximately the same ADH activity was detected in the membranes of A. aceti when grown on glycerol and glucose (Table 1). However, the heme c content was two-fold higher in the mem-

RESULTS AND DISCUSSION Effect of carbon source on growth of A. aceti and A. In the case of A. aceti, growth was observed only on glycerol and glucose among the carbon sources tested, though the growth on glucose was low (Fig. 1A). Although all Acetobacter strains are reported to grow on a solid ethanol medium (20), our strain did not grow in an ethanol-containing liquid medium (data not shown). The reason for this may be the difference in the medium composition; the medium used in this study contained a relatively low concentration of yeast extract (0.3%) and no calcium carbonate. A. aceti also did not grow on acetic, lactic, succinic, or pyruvic acid as a sole carbon source (data not shown). When the effects of the above carbon source compounds were examined in the presence of 0.1% glycerol, glucose, lactic acid, and pyruvic acid were found to promote the growth rate,

methanolicus

0

10

20

30 40 Time (h)

50

60

70

FIG. 2. Growth of A. methanolicus in media containing various growth substrates. A. methanolicus was aerobically grown at 30°C in the basal medium (0 ) and in the basal medium containing 0.5% glycerol (0), 1% methanol ( n ), 0.5% glucose ( q ), 0.5% ethanol (A), 0.5% succinic acid (A), or 0.5% nvruvic acid (0). When the bacterium was grown in the presence of bbih 0.5% glycerol and 1% methanol, the growth was the same as that in the presence of methanol.

FORMATION OF ALCOHOL DEHYDROGENASE

VOL. 83, 1997 TABLE 1.

ADH activities and heme c contents in membrane and soluble fractions of A. a& grown on various carbon sources

1% 0.1% 0.1% 0.1% 0.1% 0.1% 0.1% 0.1%

+ glucose + ethanol + acetic acid +lactic acid +pyruvic acid + succinic acid

5.92 6.39 6.60 5.60 4.80 5.60 6.85 5.76 5.90

Soluble fraction

Membrane fraction

Final pH

Carbon source

Glucose Glycerol Glycerol Glycerol Glycerol Glycerol Glycerol Glycerol Glycerol

23

ADH activity

heme c

ADH activity

U/mg

U/nmol of heme c

nmol/mg

U/mg

3.15 3.04 3.44 4.19 6.19 4.48 5.06 4.65 4.48

7.95 15.75 14.60 9.21 17.19 11.20 15.62 15.09 11.80

0.396 0.193 0.235 0.452 0.360 0.400 0.324 0.308 0.379

0.33 0.71 0.79 0.67 0.64 0.43 0.45 0.49 0.45

of glucose-grown cells than in those of glycerolgrown cells, and thus the ADH activity expressed as units per nmol of heme c in the membranes of cells grown on glucose was about half that in the membranes of glycerol-grown cells (Table 1). SDS-PAGE with heme staining showed the heme c in the membranes of A. aceti to be derived mainly from subunits I and II of ADH irrespective of the carbon source used for growth (Fig. 3), suggesting that an inactive form of ADH may be formed besides the active one. Such an inactive form of ADH observed in Gluconobacter suboxydans grown at low pH and under high-aeration conditions has been purified and characterized (21). As detailed above, A. aceti was not able to grow in the minimum medium or in the medium containing ethanol, or acetic, lactic, pyruvic, or succinic acid as the sole carbon source, whereas growth occurred in the presence of 0.1% glycerol. The effects of the above-mentioned carbon sources on ADH activity were thus examined in cells grown in the presence of each of these carbon sources together with glycerol. The ADH activity increased 1.8-fold in the membranes of cells grown on both glycerol and ethanol compared to that in the membranes of cells grown on glycerol. Glucose, and acetic, branes

FIG. 3. SDS-PAGE with heme staining of the membrane fraction of A. aceti grown in the presence of various carbon sources. The membrane fraction (50 pg of protein) was electrophoresed in 12.5% SDS-polyacrylamide gel and the gel was stained for heme c. Lane 1, 1% glycerol; lane 2, 0.1% glycerol; lane 3, 0.1% glycerol and 0.5% glucose; lane 4,0.1% glycerol and 0.5% ethanol; lane 5,0.1% glycerol and 0.5% acetic acid; lane 6, 0.1% glycerol and 0.5% lactic acid; lane 7, 0.1% glycerol and 0.5% succinic acid, lane 8, 0.1% glycerol and 0.5% pyruvic acid. I and II indicate the heme c bands derived from ADH subunits I and II, respectively.

lactic, pyruvic and succinic acids also increased the ADH activity 1.2- to 1.6-fold (Table 1). Judging from the ADH activities (units per nmol of heme c, Table 1) and the heme staining in SDS-PAGE (Fig. 3), an inactive form of ADH may also be produced in cells grown on glycerol together with glucose, acetic acid, or succinic acid. In contrast to A. ADH activity in A. methanolicus aceti, A. methanolicus can grow on a variety of carbon sources, and thus the ADH activities in cells grown on different carbon sources can be compared. A more than 50-fold increase in ADH activity was observed in cells grown on glycerol, a 30-fold increase in ethanol-grown cells, and a 6-fold increase in methanol or succinic acidgrown cells, compared with that in glucose-grown cells (Table 2). Although the presence of inactive ADH can be estimated from the ADH activity expressed as units per nmol of heme c, unlike in the case of A. aceti, this method is not accurate for A. methanolicus since heme c present in the cells is derived not only from ADH but also from soluble cytochrome cL and other membranebound heme c coming from cytochrome co (2, 22, 23; see Fig. 4). Thus, to elucidate whether low ADH activity was due to the formation of inactive ADH or to a lack of ADH protein, the soluble and membrane fractions were further analyzed by immunoblot analysis with antibody raised against ADH of A. aceti. As can be seen in Fig. 5, subunits I and II, but not subunit III, of ADH cross-reacted with the antibody, which has been confirmed also with purified ADH (FrkbortovB, J. et al., unpublished results). Two additional bands were observed between the bands for subunits I and II in some samples (Fig. 5). The upper of the two bands (62 kDa) was identified as the a subunit of MDH using purified MDH of A. methanolicus, which may have a high degree of similarity in its amino acid sequence to subunit I of ADH (24). Immunoblot analysis showed that the low ADH activity in methanol-grown cells was due to the low amount of ADH protein, while inactive ADH seemed to be produced in cells grown on glucose or succinic acid. Formation of inactive ADH in glucose-grown cells was accompanied by the release of subunit I into the soluble fraction (Fig. 5). This phenomenon has been observed also in the cases of G. suboxydans (21) and A.

pasteurianus ( 13). Since the amount of ADH protein in methanol-grown cells was considerably lower than that in glycerol- or ethanol-grown cells, and lower even than that in glucoseor succinic acid-grown cells, we investigated the possibility that methanol can repress the production of ADH protein. For this purpose, the bacterium was grown in

24

J. FERMENT. BIOENG.,

FREBORTOVAETAL.

TABLE Carbon

2.

ADH

source

activities

and heme c contents

Final pH

3.6 3.7 3.5 3.7 3.5 5.8

and soluble

Membrane

fractions

3.65 0.42 1.50 0.07 2.15 0.40

of A. methanolicus grown

heme c

U/nmol

of heme c 5.57 2.47 6.02 0.33 6.64 1.18

the presence of both glycerol and methanol. The growth rate and the total growth in the presence of both carbon sources were the same as in the presence of only methanol (data not shown). The ADH level was between the levels induced during the growth on the respective substrates (Table 2). This result suggests that methanol represses ADH production while glucose and succinic acid accumulate inactive ADH. It is noteworthy that although ADH is a membranebound protein and subunit II has been shown to be always localized in the cytoplasmic membrane (25), ADH activity and ADH subunits could be detected in both the membrane and soluble fractions, especially in the case of A. methano~icus (Tables 1 and 2; Figs. 4 and 5). This is attributed to inefficient separation of the fractions during ultracentrifugation. However, the fractionation was not improved even when double ultracentrifugation of the cell free extract was used and the cells were harvested in the early or mid logarithmic phase (data not shown). The results obtained in this study Conclusions suggest that ADH of A. aceti, as well as that of A. In A. methanolicus, is produced constitutively. methanolicus, however, methanol seems to repress ADH production. In the case of A. aceti, a high level of ADH activity is present even in glucose-grown cells, although

ADH activitv

nmol/mg 0.655 0.170 0.249 0.214 0.324 0.339

carbon

sources

fraction heme c

U/mg

nmol/mg

1.73 0.22 0.70 0.02 0.64 0.26

0.465 0.258 0.212 0.209 0.243 0.446

part of this ADH appears to be produced as an inactive form. In contrast, A. methanolicus is unable to produce any active ADH in a glucose medium, like another acetic acid bacterium, A. pasteurianus (13). In both A. methanolicus and A. pasteurianus, the ADH activity is greatly enhanced in the presence of ethanol, and, as shown in this study, glycerol has an even greater effect on ADH activity in A. methanolicus. However, the same amount of ADH protein can be produced both in the absence and presence of ethanol in the case of A. pasteurianus. It has been suggested that the low ADH activity in cells grown without ethanol is due to the incorrect localization of subunit I, which is partly detected in the soluble fraction (13). The release of subunit I into the periplasm is also observed in glucose-grown cells of A. methanolicus (Fig. 5). Recently, an “inactive” form of ADH having an activity 10 times lower than the active ADH has been isolated from G. suboxyduns grown under acidic or high-aeration conditions (21). Under these conditions, subunit I could be detected in the soluble fraction as well as in the membrane fraction. However, the subunit compositions, molecular masses, and heme c and PQQ contents were the same in both the inactive and active forms. It is therefore suggested that the release of subunit I into the soluble fraction is not responsible for low ADH activity, but that it occurs in the presence of inactive ADH. This is consistent with the findings that inactive ADH has a relatively loose conformation due to some defect in the interaction among subunits (21), and that the subunit I/III complex is bound to the membrane via subunit II embedded in the membrane (25). Since subunit III is essential for ADH activity, and is speculated to maintain the right conforkDa

FIG. 4. SDS-PAGE with heme staining of the soluble and membrane fractions of A. methnnolicus grown in the presence of various carbon sources. The soluble (40 pg of protein) and membrane (60 /Ig of protein) fractions were electrophoresed in 12.5% SDS-polyacrylamide gel and the gel was stained for heme c. Lane 1, Soluble fraction of cells grown on glycerol; lane 2, soluble fraction of cells grown on methanol; lane 3, soluble fraction of cells grown on glycerol and methanol; lane 4, soluble fraction of cells grown on glucose; lane 5, soluble fraction of cells grown on ethanol; lane 6, soluble fraction of cells grown on succinic acid; lane 7, membrane fraction of cells grown on glycerol; lane 8, membrane fraction of cells grown on methanol; lane 9, membrane fraction of cells grown on glycerol and methanol; lane 10, membrane fraction of cells grown on glucose; lane 11, membrane fraction of cells grown on ethanol; lane 12, membrane fraction of cells grown on succinic acid. I and II indicate the heme c bands derived from ADH subunits I and II, respectively, and cr and co the bands derived from cytochromes cr and co, respectively.

on various Soluble

fraction

ADH activitv U/mg

Glycerol Methanol Glycerol+methanol Glucose Ethanol Succinic acid

in membrane

1

2

3 4

5

6

7

8

9

10 II 12

FIG. 5. Immunoblot analysis of the soluble and membrane fractions of A. methanolicus grown in the presence of various growth substrates. The soluble and membrane fractions (20 ,ug of protein) were electrophoresed in 12.5% SDS-polyacrylamide gel and the proteins were electroblotted to a polyvinylidene difluoride membrane. The membrane was stained with antibody raised against ADH of A. uceti. Samples applied to the lanes l-12 correspond to the descriptions in Fig. 4. I and II indicate the bands of ADH subunits I and II, respectively, and CYindicates the (Ysubunit of MDH.

FORMATION OF ALCOHOL DEHYDROGENASE

VOL. 83, 1997

mation of the ADH complex (29, incorrect interaction of subunit III with the other two subunits may be the cause of the ADH inactivation. In G. suboxydans, the inactive form of ADH could be converted to the active form by changing the growth conditions (21). The data from this previous study together with the present results suggest that in acetic acid bacteria in general, conversion from the inactive to the active form or vice versa occurs as a result of a change in the growth conditions. Therefore, the ADH activity of acetic acid bacteria is able to be regulated not only by de nova synthesis of the enzyme, but also by controlling the level of the inactive form under various growth conditions. Inactive ADH of G. suboxydans proved useful in studying the intramolecular electron transport of the ADH complex (17), and thus isolating such an inactive ADH from other organisms would be of interest. In A. aceti, inactive ADH has been found to be produced under conditions similar to those of G. suboxydans (Matsushita, K. et al., unpublished results) and in cells grown on glucose (this study). In the case of A. methanokus, glucose-grown cells may be the source of inactive ADH. ACKNOWLEDGMENT

This work was supported Society (No. 7-131) to J.F.

by a grant from The Japan Science

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