Effect of anaerobic environment on the glutathione transferase isoenzymatic pattern in Proteus mirabilis

FEMS Microbiology Letters 147 (1997) 157^162

E¡ect of anaerobic environment on the glutathione transferase isoenzymatic pattern in Proteus mirabilis Nerino Allocati a *, Antonio Aceto b , Luigina Cellini a , Michele Masulli a , Beatrice Dragani b , Ra¡aele Petruzzelli b , Carmine Di Ilio b ;

a

é di Medicina e Chirurgia, Universita é `G. D'Annunzio', Via dei Vestini 31, I-66013 Chieti, Italy Istituto di Medicina Sperimentale, Facolta

b

é di Medicina e Chirurgia, Universita é `G. D'Annunzio', Via dei Vestini 31, I-66013 Chieti, Italy Istituto di Scienze Biochimiche, Facolta

Received 26 September 1996; revised 4 December 1996; accepted 10 December 1996

Abstract

When Proteus mirabilis was cultured anaerobically in the presence of nitrate as terminal electron acceptor, a dramatic reduction of glutathione transferase production occurred. The analysis of the glutathione affinity purified materials in terms of substrate specificity, SDS-PAGE pattern, IEF pattern and immunoblotting revealed that a significantly different glutathione transferase pattern also occurred: two new glutathione transferase forms with an isoelectric point at pH 4.8 and 5.0 appeared. Their N-terminal amino acid sequence analysis as well as the ability to bind to a glutathione affinity column indicate that major differences between anaerobic and aerobic glutathione transferase forms are mainly located in the C-terminal region of the primary structure. In contrast, no significant changes occurred in the production of glutathione transferase isoenzymes when P. mirabilis was grown anaerobically in the absence of a terminal electron acceptor. These results support the idea that bacterial glutathione transferase expression is not strictly related to the absence of oxygen stress. Keywords :

Aerobic respiration; Anaerobic respiration; Anaerobic; Glutathione transferase;

1. Introduction

Glutathione transferases (GSTs, EC 2.5.1.18) are a family of multifunctional dimeric proteins that catalyze the conjugation of reduced glutathione to a large variety of electrophilic compounds [1^4]. Glutathione transferases are also involved in intracellular binding and transport of hydrophobic compounds, such as heme, bilirubin, hormones and drugs, acting as intracellular proteins for the transport of various ligands [1^3]. * Corresponding author. Tel.: +39 (871) 355 281; fax: +39 (871) 355 282; e-mail: [email protected]

Proteus mirabilis

Glutathione transferases seem to be widely distributed in prokaryotes [5^13], but little is known about their biological functions, structures, and regulation. We have previously isolated and characterized glutathione transferases from Proteus mirabilis [5]. Using glutathione a¤nity chromatography and isoelectric focusing techniques we have found that P. mirabilis synthesizes multiple forms of glutathione transferase in the pH 5.8^6.7 range. For one of them (Pm-GST-6.0) the primary structure has been resolved [14]. Immunolabelling techniques have demonstrated that this enzyme is preponderantly localized in the periplasmic compartment [15]. Besides its conjugation capacity, a signi¢cant num-

0378-1097 / 97 / $17.00 Copyright ß 1997 Published by Elsevier Science B.V. All rights reserved PII S 0 3 7 8 - 1 0 9 7 ( 9 6 ) 0 0 5 2 1 - 6

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158

ber of glutathione transferases, including bacterial

cally, anaerobically or anaerobically with 40 mM

glutathione transferases, are able to catalyze the re-

KNO3 , using a fermenter (SET 20B ; Setric Genie

duction of organic hydroperoxides (selenium-inde-

Industriel, Toulouse, France). Each fresh medium

pendent glutathione peroxidase activity). For this

was inoculated with an appropriate volume (v/v :

capacity,

1/50)

it

has

been

proposed

that

glutathione

transferases, like many other glutathione-dependent enzymes,

are

involved

in

the

protection

of

cells

of

overnight

Additionally, it has been proposed that glutathione and glutathione-dependent enzymes, including

incubated

under

the

Aerobic conditions were obtained by air £ow. Anaerobiosis

against the products of oxidative metabolism.

culture

same atmospheric conditions.

was

maintained

with

nitrogen.

To

avoid exposure to oxygen, anaerobic cultures were chilled on ice before being harvested.

glutathione transferases, evolved in aerobic organisms in response to the requirements of inactivation of

toxic

products

of

oxygen

metabolism

[16,17].

2.2. Preparation of cell extracts and glutathione a¤nity puri¢cation

Thus, glutathione transferases should be limited essentially to aerobic organisms and would not be ex-

Aerobic and anaerobic cultures were monitored by

pected to occur in anaerobic life. In this context we

600 nm with a Kontron spectrophotometer (Kontron

have previously found that the cytosol of the an-

Instruments, Milan, Italy). Cells were grown until

Bacteroides fragilis

is devoid of

OD = 0.6 and harvested by centrifugation. The pel-

glutathione transferase activity even though it was

lets were washed twice with 10 mM potassium phos-

able to synthesize a glutathione a¤nity binding pro-

phate bu¡er pH 7.0 containing 1 mM EDTA, resus-

tein

of

pended in the same bu¡er supplemented with 1 mM

aerobic glutathione transferases [18]. Although its

dithiothreitol (DTT) (bu¡er A) and disrupted by

role is unknown, it has been hypothesized that it

cold sonication with a Labsonic sonicator (Braun

might represent an evolutionary ancestor of the aero-

Milano SpA, Italy) in ¢ve bursts of 3 min each, at

bic glutathione transferases.

300 W. The particulate material was removed by

aerobic bacterium

with

structural

characteristics

reminiscent

It is well known that in addition to aerobic respiration,

P. mirabilis

is able to grow in the absence of

Ug

centrifugation for 60 min at 105 000

at 4³C and

the resulting supernatants were applied to a gluta-

molecular oxygen by anaerobic respiration with oth-

thione-Sepharose

er molecules as terminal electron acceptors, including

pre-equilibrated with bu¡er A. The column was ex-

a¤nity

column

[19]

which

was

P. mirabilis

haustively washed with bu¡er A, supplemented with

appears to be a good model to investigate the role of

50 mM KCl. The enzyme was eluted with Tris-HCl

glutathione transferase in the protection against the

bu¡er pH 9.6 containing 10 mM glutathione (GSH).

products of oxidative metabolism.

The fractions containing glutathione transferase ac-

nitrate, as well as by fermentation. Thus,

In the present paper, we have investigated whether

tivity were pooled, concentrated by ultra¢ltration,

alteration in the synthesis of glutathione transferase

dialysed against bu¡er A and subjected to further

occurs in

P. mirabilis

cytosol when cultivated in the

analyses.

absence of oxygen.

2.3. Enzyme assay Glutathione transferase activity with 1-chloro-2,4-

2. Materials and methods

dinitrobenzene

2.1. Bacterial strains and growth conditions

and [2,3-dichloro-4-(2-methylenebu-

tyryl)-phenoxy] acetic acid (ethacrynic acid) was assayed at 30³C in a Kontron spectrophotometer ac-

Proteus mirabilis throughout

3

this

strain AF 2924 [5] was used

work.

The

strain

was

stored

at

80³C, and fresh subcultures were regrown in Tryp-

The Se-independent glutathione peroxidase activity of glutathione transferase was measured with

K,K-

P.

dimethylbenzyl hydroperoxide (cumene hydroperox-

was grown at 37³C in TSB either aerobi-

ide) as previously reported [21]. Protein concentra-

tone Soya Broth (TSB ; Unipath, Milan, Italy).

mirabilis

cording to the methods of Habig and Jakoby [20].

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N. Allocati et al. / FEMS Microbiology Letters 147 (1997) 157^162

tions were determined by the method of Bradford [22], using Q-globulin as standard. 2.4. SDS-PAGE

SDS-PAGE in discontinuous slab gels was done by the method of Laemmli [23] using the Phast system on 12.5% (w/v) polyacrylamide precast gels, according to the manufacturer's instructions (Pharmacia Biotech SpA, Cologno Monzese, Italy). Phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa) and K-lactalbumin (14.4 kDa) were used as standards for determination of subunit molecular mass. Proteins were stained with Coomassie blue R-250. 2.5. Analytical isoelectric focusing and Immunoblotting

Samples for isoelectric focusing (IEF) were incubated with 1 mM DTT for 30 min at room temperature. IEF in the broad range was performed according to the manufacturer's instructions on a 5% (w/v) polyacrylamide horizontal slab gel with 2.2% (w/v) pH 3.5^9.5 ampholine (Ampholine PAGplate; Pharmacia Biotech). Gels were prechilled at 15³C and run in a horizontal IEF apparatus (Multiphor II, Pharmacia Biotech). 60 Wg of each sample was loaded onto prefocused gels (15 W, 1500 V, 20 min) and focused to equilibrium (30 W, 1500 V). The IEF process was routinely monitored with methyl red containing pI markers (Pharmacia Biotech). Gels were ¢xed and stained in 0.04% Coomassie blue G-250 in an aque-

ous solution of 6% perchloric acid (w/v). The isoelectric point of the proteins was estimated according to the manufacturer's instructions. Proteins were electrophoretically transferred from polyacrylamide gels to nitrocellulose membranes following the manufacturer's instructions using a transfer unit (Bio-Rad Laboratories, Milan, Italy) for 16 h at 5³C with a constant voltage of 30 V in 0.7% acetic acid. All incubations were for 1 h at 25³C with intermediate rinses in 50 mM Tris base bu¡er, pH 7.5, 400 mM NaCl (bu¡er A) containing 0.05% Tween 20 (bu¡er B). Non-speci¢c binding was blocked by placing membranes in bu¡er A supplemented with 3% (w/v) bovine serum albumin. Membranes were incubated with primary antiserum (antiserum raised against Pm-GST-6.0 at optimum dilution, 1:250), in bu¡er A containing 3% bovine serum albumin. The membranes were washed with bu¡er B and then incubated for 1 h at room temperature, with gentle shaking, in the same bu¡er containing 1% (w/v) gelatine and a horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad) diluted 1:3000. After treatment, the membranes were washed three times in bu¡er B and twice in bu¡er A, then immersed in developing solution (100 ml bu¡er A containing 60 mg 4-chloro-1-naphthol and 60 Wl 30% H2 O2 ). The blot was then washed once with distilled water, air-dried and photographed. Antiserum raised against Pm-GST-6.0 was available in our laboratory and was the same as that used previously [5]. 2.6. Determination of N-terminal amino acid sequence

After IEF, the bands from the gels were trans-

Table 1 Puri¢cation of glutathione transferases from P. mirabilis grown under di¡erent conditions by glutathione a¤nity chromatography Total protein (mg) Speci¢c activity (units mg31 protein) Total activity (units) Growth conditiona Step Aerobic Cell extract 3400 ^ ^ A¤nity chromatography 1.74 0.68 1.18 Anaerobic

Cell extract A¤nity chromatography

3000 0.99

^ 0.9

^ 0.89

Cell extract A¤nity chromatography a Bacteria were grown until OD600 = 0.6.

3320 0.189

^ 0.07

^ 0.013

Anaerobic+NO3

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ferred onto PVDF membranes (Bio-Rad) in 1.5% (v/v) acetic acid and 20% (v/v) methanol. The PVDF membranes were stained with 0.1% Coomassie blue in 50% methanol. The protein bands were excised and subjected to automated Edman degradation analysis on a 476 gas-phase Sequencer (Applied Biosystem) according to the manufacturer's instructions. 3. Results and discussion

Table 1 shows the results of the puri¢cation by glutathione a¤nity chromatography of the glutathione transferase from Proteus mirabilis grown in three di¡erent environmental conditions, i.e. in the presence of atmospheric oxygen (Aerobic), in the absence of oxygen (Anaerobic) and in the absence of oxygen with 40 mM KNO3 as electron acceptor (Anaerobic+NO3 ). The glutathione transferase content recovered from glutathione a¤nity chromatography of the anaerobic+NO3 sample was lower than that obtained from bacteria grown in aerobic (10-fold) and anaerobic without electron acceptor (5-fold) conditions. Taking into account that the extract of an identical number of bacteria was loaded on the glutathione a¤nity matrix, the results presented in Table 1 clearly indicate that the content of glutathione transferase protein produced by the bacteria grown under anaerobic conditions is lower than that produced during aerobiosis. The results presented in Table 2 show that the speci¢c activity of glutathione transferase puri¢ed from the anaerobic+NO3 sample towards three model substrates is signi¢cantly di¡erent from those of the glutathione transferases in aerobic and anaerobic samples. We have previously found that multiple forms of glutathione transferase are present in the cytosol ex-

Fig. 1. SDS-PAGE of glutathione transferases, resolved by glutathione a¤nity chromatography, from P. mirabilis grown under di¡erent conditions. Lane 1, molecular mass markers; lane 2, aerobic; lane 3, anaerobic; lane 4, anaerobic+NO3 .

tract of P. mirabilis. These isoforms are composed of subunits having the same apparent electrophoretic mobility (22.5 kDa) and a similar immunological determinant. The majority of these forms focused in the pH 5.8^6.7 range when analysed by IEF [5]. To investigate whether, as suggested by the results presented in Table 2, changes in the glutathione transferase subunit composition have occurred in the bacterium as a consequence of their di¡erent growth conditions, the glutathione a¤nity fractions were subjected to SDS-PAGE and IEF analysis. When the glutathione a¤nity puri¢ed fractions of the three samples investigated were analyzed by SDS-PAGE the results presented in Fig. 1 were obtained. A single band with an identical electrophoretic mobility (22.5 kDa) was obtained. This result would indicate that no marked changes

Table 2 Comparison of speci¢c activities from glutathione transferases of P. mirabilis grown under di¡erent conditions towards various substrates Substrate Speci¢c activity (units mg31 protein) Aerobic Anaerobic Anaerobic+NO3 1-Chloro-2,4-dinitrobenzene 0.68 0.9 0.07 K,K-Dimethylbenzyl hydroperoxide (cumene hydroperoxide) 0.172 0.21 0.04 [2,3-Dichloro-4-(2-methylenebutyryl)-phenoxy] acetic acid (ethacrynic acid) 0.017 0.026 0.009

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161

Fig. 2. Analytical isoelectrofocusing (A) and immunoblots (B) of glutathione transferases, resolved by glutathione a¤nity chromatography, from

P. mirabilis

grown under di¡erent conditions. A :

Lane 1, pI marker proteins ;

lane 2, aerobic ;

lane 3, anaerobic ;

lane 4,

anaerobic+NO3 . B : lane 5, anaerobic+NO3 ; lane 6, anaerobic ; lane 7, aerobic.

in the glutathione transferase subunit expression has

Pro-Gly-Ser-Cys-Ser-Leu which matched the N-ter-

occurred as a consequence of di¡erent growth con-

minal of Pm-GST-6.0 [14].

ditions.

The results of the present investigation clearly sup-

However, to allow a more accurate estimation of

port the idea that bacterial glutathione transferase

glutathione transferase content, the glutathione-a¤n-

synthesis is not strictly related to the absence of oxy-

ity preparations were subjected to IEF, in the pH 3^

gen stress. In fact, when

10 range. All three samples showed multiple bands

aerobically without a terminal acceptor of electron,

(Fig. 2A). In all samples the majority of the bands

no dramatic qualitative and quantitative di¡erences

were found in the pH interval between 5.60^6.60. A

in glutathione transferase content occur. In contrast,

signi¢cantly di¡erent glutathione transferase pattern

when the bacterium is cultured anaerobically in the

was found in the anaerobic+NO3 sample when com-

presence of nitrate as terminal electron acceptor a

pared with the other two samples. Particularly in the

much lower amount of glutathione transferase is syn-

anaerobic+NO3 sample a micro heterogeneity of new

thesized. Moreover, in these conditions, new gluta-

forms was observed in the acidic region with two

thione transferase forms are also produced. The iden-

predominant bands having isoelectric points at pH

tity of N-terminal sequences and the ability to bind

4.8 and 5.0 respectively. Considering that only one

to glutathione a¤nity column suggest that major dif-

native 22.5 kDa band was obtained in SDS-PAGE,

ferences between new anaerobic glutathione transfer-

we exclude that the two acidic forms observed in IEF

ase forms and Pm-GST-6.0 are mainly located in

experiment (Fig. 2) could be the result of speci¢c

their C-terminal region. At this stage we can not

protein degradation under anaerobic conditions.

exclude that anaerobic growth conditions could not

P. mirabilis

is cultured an-

When these preparations were transferred to a ni-

only result in gene regulation but also in protein

trocellulose membrane and probed with Pm-GST-6.0

modi¢cation. It is likely that new glutathione trans-

antiserum a positive reaction was detected with the

ferase forms observed in anaerobic nitrate respiration

isoforms focused in the pH 5.60^6.60 range as well

are related to speci¢c detoxi¢cation functions.

as with the acid forms (Fig. 2B). To further characterize the new anaerobic forms, the two predominant acidic bands were also electro-

Acknowledgments

blotted from IEF and identi¢ed by Edman degradation analysis. Their N-terminal amino acid sequences gave the same sequence Met-Lys-Leu-Tyr-Tyr-Thr-

We thank Mrs. Gabriella Toro for excellent technical assistance.

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162

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