Interspecies interaction based on transfer of a thioredoxin-like compound in anaerobic chitin-degrading mixed cultures

FEMS Microbiology Ecology 62 (1989) 349-358 Published by Elsevier

349

FEMSEC 00223

Interspecies interaction based on transfer of a thioredoxin-like compound in anaerobic chitin-degrading mixed cultures R o e l Pel a n d J a n C. G o t t s c h a l Department of Microblolog3". Unieersi O' o["Groningen, tlaren, The Netherlands

Received 10 September 1987 Revision received 8 January 1989 Accepted 26 January 1989

Key words: Anaerobic polymer degradation; Chitin: Redox stimulation; Thioredoxin

1. S U M M A R Y Fermentation of chitin by mixed cultures of the chitinolytic Clostridium sp. strain 9.1 and various non-chitinolytic bacteria proceeded up to eight times faster than in pure cultures. The addition of spent media of such mixed cultures also resulted in a marked stimulation of chitinolysis in pure cultures of strain 9.1. Pure cultures fermented chitin much faster if supplemented with either spent media or cell-free extracts of the nonchitinolytic bacteria. The compound responsible for this stimulation was thermostable (10 rain at 85 ° C) and could not be removed by passage over Sephadex G-25, indicating a molecular weight of more than 1500. The heat stable enzyme thioredoxin (from Saccharomyces ceret, isiae) was shown to stimulate the chitin fermentation in a similar manner. Alkylation of this enzyme reduced its stimulatory action significantly indicating its (di)thiol : disulfide interchanging activity. It is hypothesized that essential sulfhydryl groups in the chitinolytic system of strain 9.1 are

Correspondence to: R. Pel, Institute for Soil Fertility, Oosterweg 92, 9751 PK Haren. The Netherlands.

reduced by thioredoxin a n d / o r similar thiol:disulfide transhydrogenases present in the cell-free extracts and spent media, resulting in an acceleration of chitin hydrolysis and fermentation. This stimulation may thus be the result of a new type of interspecies interaction in anaerobic mixed cultures.

2. I N T R O D U C T I O N In the course of our study on the involvement of anaerobic bacteria in the mineralization of chitin in the sediment of an estuarine environment we isolated various mesophilic, obligately anaerobic chitinolytic bacteria [1,2]. Although these chitinolytic species were able to grow in pure culture with chitin as the sole carbon and energy source the rate of chitin hydrolysis and fermentation was extremely low. However, further study of these chitinolytic species revealed that the anaerobic conversion of chitin proceeded up to eight times faster in cocultures with various saccharolytic facultatively anaerobic bacteria [3]. The fact that this stimulatory interaction was observed in cocultures with several unrelated species indicated its non-species specificity and its possible importance

0168-6496/'89,/$03.50 .~2,1989 Federation of European Microbiological Societies

350 for our understanding of anaerobic polysaccharide degradation in nature. This prompted us to investigate the nature of this interspecies interaction under controlled conditions in the laboratory. In a previous paper [4] the presence of essential sulfhydryl groups in the chitinolytic system of one of the isolated chitinolytic anaerobes, Clostridium sp. strain 9.1, was shown and we speculated on the importance of these groups for growth in the partially oxygenated upper layer of the sediment. Chitin fermentation by this anaerobe was significantly enhanced not only by the presence of several non-chitinolytic species but also by a supplementation of the N a , S - r e d u c e d medium with strong reductants. Addition of dithionite (0.2 mM) or T i ( I I I ) - N T A (0.3 mM) at the time of inoculation reduced the lag phase from 4 - 1 4 to 1-2 days and increased the rate of chitin fermentation by

50-300% [21. For a number of extracellular saccharases produced by different species of cellulose degrading anaerobes [5-8], the hydrolytic activity was shown to be strongly influenced by the reduction state of the sulfhydryl groups present in these enzymes. Sulfhydryl groups were also involved in the polysaccharide hydrolysis by xylanase of Ruminococcus flavefaciens [9] and the/~-amylase of CIostridium thermosulfurogenes [10], suggesting a general occurrence of thiol groups as functional components in extracellular saccharases of strict anaerobes. In this paper we will present data which strongly suggest that the thiol groups of the chitinolytic system of Clostridium sp. strain 9.1 play a role in a synergistic type of interspecies interaction resulting in enhanced chitin degradation in mixed cultures.

3. M A T E R I A L S A N D M E T H O D S 3.1. Organisms and medium Clostridium sp. strain 9.1 was grown in a medium as described previously [2] except for the applied concentrations of yeast extract (0.008,.% w / v ) and Na~S (0.3-0.4 raM). For all experiments strain 9.1 was pre-grown in a medium

amended with 1 mg chitin per ml and from such precultures media were inoculated (0.8%, v / v ) resuiting in approximately 2 × 10 6 cells per ml. Five different species of non-chitinolytic sugarfermenting bacteria were used in coculture experiments with strain 9.1, i.e. Klebsiella aerogenes strain A T C C 15380, Clostridium acetobutvlicum strain A T C C 824 and a wild type Escherichia coli strain obtained from the culture collection of our department (identification according to API 20E testsystem for Enterobacteriaceae Montalieu Vercieu, France). Two facultative anaerobes, strains HA 8.1 and G H 8.2, were isolated from the original chitinolytic community containing strain 9.1 as one of the predominant primary chitinolvtic bacteria [2]. Strains H A 8.1 and G H 8.2 are capable of fermenting N-acetylglucosamine (NAG) and NAG-oligomers (a more detailed description of these strains has appeared elsewhere [3]. The saccharolytic bacteria were grown in the same medium used for the cultivation of strain 9.1 except that chitin was replaced by 5 mM N A G . From these cultures, after reaching early stationary phases, pure cultures of strain 9.1 were inoculated (0.8.~, v / v ) resulting in a starting cell number of approximately 4 X 10 ° cells per ml. Since both fermentation and chitinolysis bv strain 9.1 proceed in a nearly linear fashion the rates of these processes can be expressed as mmol -d ~.1 ~or m g . d ~-ml ~, respectively. These rates have been obtained from the mid-growth phases of the various cultures. All supplementations were made aseptically from stock solutions or preparations that had been filter-sterilized.

3.2. Chemical analyses Fermentation products and chitin hydrolysis products were determined as described previously [2,4]. Protein was determined using the method of Lowry [11]. Residual chitin in culture samples was determined by total organic carbon (TOC) analyses [2] after solubilization of the cells. Samples were centrifuged at 9000 × g for 5 min and the pellet was resuspended in 1 N N a O H at 100°C. After 30 rain the chitin was collected by centrifuging at 9000 × g for 5 rain, washed twice with distilled

351 water and resuspended in de-gassed distilled water to its original volume.

3. 3. Preparation of cell-free extracts and membrane fractions Cell-free extracts of N A G - g r o w n secondary bacteria were prepared as described by Adler et al. [12], however, rupture of the cells was achieved by sonication under an atmosphere of N,. The extracts were filter-sterilized (0.2 ffm), kept under an atmosphere of N~ and frozen ( - 2 0 ° C ) until needed. No loss of activity was observed after repeated thawing and freezing. For preparation of the membrane fraction crude cell-free extracts were centrifuged for 2 h at 225 000 x g at 4 ° C. The supernatant was saved, and will be referred to as 'cytosolic fraction'. The pellet obtained after this high-speed centrifugation step was thoroughly washed and resuspended in the same buffer (0 ° C) as used for the preparation of crude extracts. In order to facilitate filter sterilization the membrane fraction was sonicated two times for 15 s under an atmosphere of N 2. The cytosolic and membrane fraction were filter sterilized (0.2 fire) and kept frozen ( - 2 0 ° C ) under Nz until needed. 3.4. Removal of low molecular compounds from cell-free extracts and spent media Removal of low molecular weight compounds from cell-free extracts was accomplished by passage over Sephadex G-25 (fine grade). Because of the unknown nature of the active elements in the extracts, all manipulations were performed under anoxic conditions to minimize inactivation of oxygen-labile compounds. A centrifuge desalting technique was applied using small glass columns. The columns with an internal volume of approx. 7 ml (13 m m wide and 55 m m long), were equipped with a needle as outlet and a butyl rubber septum at the top to permit easy aseptical manipulations. The columns were packed with 5 ml Sephadex G-25 swollen in 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer, p H 7.5, and sterilized by autoclaving. The columns were then thoroughly rinsed with deoxygenated sterile HEPES buffer. Subsequently the void volume was spun out at 1000 x g for 5 rain. All

these manipulations were carried out aseptically with the needle of the column positioned through the septum of an empty N,-flushed sterile sert/m bottle or vial to protect it from contamination. The columns were loaded with 0.5 ml cell-free extract and the vials were replaced by empty ones for collection of the samples. After an equilibration for 10 rain at 60 x g the desalted sample was spun out at 1000 x g. Spent media of cocultures were freed of low molecular weight compounds in a similar way as the cell-free extracts. Culture fluid of cocultures that had just reached the stationary phase were passed over 0.2 # m membrane filters prior to their application to Sephadex columns. Columns packed with 30 ml Sephadex-G25 were loaded with 5 ml spent medium. The desalted spent medium was filter-sterilized (0.2 /~m) and heated to 8 5 ° C for 10 rain under an atmosphere of N~. The heat treatment was included since a well known class of disulfide reductases has been shown to be resistant to temperatures from 85 to 1 0 0 ° C [13,14]. Spent medium treated in this way was added immediately to the culture medium in a ratio of 1:1. Corrections were made to ensure that the final concentration of the other medium constituents remained unchanged. The efficiency of removal of low molecular weight compounds from the extracts and the spent media was estimated using strong chromophoric standards. Cresyl violet acetate (MW 321), Janus Green B (MW 511) and Vitamin B12 (MW 1355) were dissolved in crude cell-free extracts to a final concentration of approx. 10 m M and the absorbance recorded at the respective X m~,-values before and after the desalting treatment. The efficiencies of removal were > 99.9%, 99.5% and > 99.9% respectively.

3.5. Thioredoxin and thioredoxin reductase preparations Partially purified preparations of thioredoxin (0.12 mg p r o t e i n / m l ; 1.8 U / m g protein) and thioredoxin reductase (0.15 mg protein; 2.8 U / r a g protein) from Saccharomvces cereuisiae were a gift of Dr. P. Large (Univ. Hull, U.K.). The procedure employed for the purification of thioredoxin was essentially the one described by Gonzalez Porqu6

352 et al. [15] up to the chromatography step on Sephadex G-50. The presence of GSH-disulfide transhydrogenase activity in the thioredoxin preparation could not be demonstrated using an assay which employs 2-hydroxy-ethyldisulfide as an artificial substrate ([16]: in our crude E. coli extract and the extracts passed over Sephadex G-25 the specific activities were +_0.3 and _+0.22 U / m g protein, respectively). Alkylation of the thioredoxin preparation in order to block its transhydrogenase activity was performed with iodoacetate (IoAc) [17]. Alkylation was achieved by aseptic incubation of thioredoxin with approx. 4 mM dithiothreitol at 3 7 ° C for 1 / 2 h under an atmosphere of N~, and subsequent addition of IoAc in 50 mM Tris-C1. pH 7.5 to a final concentration of 15 raM. This mixture was incubated for 1 h at room temperature before addition to cultures of strain 9.1. The unreacted IoAc in this mixture had no noticeable effect on growth of the bacterium, probably as a result of the low final concentration in the cultures (approx. 0.1 mM) and the presence of sulfide in the medium (0.3-0.4 mM). The addition of a mixture without thioredoxin to cultures of strain 9.1 was used as a control.

3.6 Chemicals Dithiothreitol was purchased from BDH Chemicals. N A D H , N A D P H , reduced glutathione (GSH) and oxidized glutathione (GSSG) were from Boehringer, Mannheim, F.R.G. Iodoacetate was from Merck, Darmstadt, F.R.G. 2-hydroxyethyl disulfide was from Janssen Chimica, Beerse, Belgium. Cresyl violet acetate, Janus Green B, Vitamin Bl~. N A G and chitobiose (NAG-dimer) were from Sigma, Poole, U.K. Chitotetraose and chitobiose used as substrates in growth experiments were prepared as described previously [2].

4. RESULTS

4.1. The effect of dithionite on the growth of Clostridium sp. strain 9.1 Although the addition of dithionite enhanced the rate of both fermentation and hydrolysis of the polymer, chitinolysis was affected most, espe-

cially at low concentrations of autoclaved yeast extract in the medium (0.005-0.016%, w/v). This was indicated by the transient appearance of chitobiose in the culture fluid up to 500-700 ~M. At yeast extract concentrations higher than 0.07% ( w / v ) , accumulation of chitobiose did not occur. Stimulation of chitin fermentation could not be achieved by increasing the concentration of sulfide in the medium to 2-3 raM. Replacement of resazurine by Janus Green B as a redox indicator (E~ = 260 mV [18]) revealed that dithionite poised the redox potential (E h) of the cultures at a value of less than -300 mV [18]. After approximately 6 hours the culture E h increased to a more positive value than 300 inV. But as soon as chitin hydrolysis started (judged from the drop in culture turbidity [4]) the Eh-indicator returned to its fully reduced state. In the absence of dithionite the same reduction of the E~, was observed at the initiation of chitinolysis, indicating that attainment of this very low Eh-value during the active growth phase did not depend on supplementation with dithionite.

4.2. Chitin [ermentation in the presence of secondary bacteria, spent media and cell-free extracts In cocultures of Clostridium sp. strain 9.1 and non-chitinolytic bacteria, chitin fermentation proceeded at a 4- to 8-fold higher rate than in cultures of strain 9.1 alone. Interestingly, arbitraril'¢ chosen secondary bacteria (E. coli, K. aerogenes and C. acetobutvlicum) stimulated equally well or even better than saccharolytic species isolated from the original chitinolytic community (strains HA 8.1 and G H 8.2). The results of a coculture with C. acetobutvlicum as the secondary species are given in Fig. 1, showing an 8-fold increase in the rate of chitinolysis. C. acetobutvlicum is not capable of utilizing NAG-oligomers and therefore growth of the organism in coculture depends on the NAGmonomer liberated by strain 9.1. The appearance of some butx'rate indicated slight growth of the secondary ~pecies (fermentation of N A G by C. acetobutvlicum in pure culture yielded only 0.45 mmol of butyrate per rnmol of N A G utilized). Stimulation of chitin degradation was al~o observed when spent medium from NAG-grown cultures of the non-chitinolvtic bacteria was added to

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pure cultures of strain 9.1 (Table 1). Crude cell-free extracts of the non-chitinolytic bacteria also enhanced chitinolysis. The cytosolic and membrane fractions prepared from these extracts by ultracentrifugation differed significantly in this respect (see Table 1). Heating of the spent media and the cell-free extracts for 10 rain at 8 5 ° C did not influence their stimulatory activity. Addition of a small amount of GSH (50 /~M) together with the crude extracts shortened the lag phases of these cultures by 50-70%, though GSH alone had very little effect. Enhancement of chitinolysis by E. coli crude extract was detectable at protein concentrations in the culture fluid as low as 0.6/.tg/ml, and 4 times repeated passage of this extract over Sephadex-G25 to remove low molecular weight constituents did not eliminate its activity (Table 1). The involvement of (a) thermostable and high molecular compound(s) was implicated further by the enhancement of chitin degradation observed

" Enhancement of chitinolysis was estimated by comparing the rates of overall chitin hydrolysis (drop in culture turbidity at 660 nm, see [4]) in the mid-growth phase of the cultures. Rate in unsupplemented culture was approx. 7 × 1 0 -2 mg chitin.d-n.m1-1 (see Fig. 1A). b 4% (V/V) filter-sterilized spent medium added from culture grown with 5 mM NAG. c Figure in parentheses represents the amount of protein added (~g per ml culture fluid). d Not determined. e Extract passed over Sephadex-G25 4 times.

with heated (10 min at 8 5 ° C ) Sephadex-treated spent medium of cocultures of strain 9.1 and sugar-fermenting species (Fig. 2). The observed stimulation in these experiments was not due to the presence of residual chitinase activity in the spent media since addition of spent medium to 10

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culture medium in the absence of strain 9.1 did not result in measurable hydrolysis of chitin. This is due to the fact that any chitinase activity present in the spent medium is lost by: (1) adsorption to the cellulose-acetate membrane filters used to sterilize the spent medium and (2) the heat-treatment at 85 o C. Stimulation of chitin fermentation could not be achieved by supplementation of the medium with crude cell-free extract prepared from strain 9.1 itself (final conc. 45 fig protein/ml). Nor did the addition of GSH (0.05-0.4 mM), GSSG (0.8 mM), cysteine (1-3 raM), 2 hydroxy-l,4 naphthoquinone (0.05 mM), 1,2 naphthoquinone (0.05 mM), casamino acids (0.02% w / v ) or tryptone (0.02% w / v ) have a measurable influence. 4. 3. The effect of thioredoxin on the fermentation of chitin and NA G-oligomers On the basis of the available information, i.e.

the presence of essential SH-groups in the chitinolytic system of Clostridium sp. strain 9.1 [4], the enhancement of chitinolysis by strong reductants (this paper and [2]) and the high molecular weight and thermostability of the stimulatory compound(s) in the cell-free extracts and spent media of cocultures (this paper), it was hypothesized that the secondary bacteria supplied strain 9.1 with (a) redox-active compound(s). Thioredoxin and glutaredoxin, small thermostable enzymes (MW approx. 12 000) exhibiting disulfide-reducing activities and known to be present in many organisms [16,19], were interestingly candidates. A thioredoxin preparation from Saccharomyces cerevisiae applied at an appreciably lower protein concentration than the cell-free extracts enhanced chitin fermentation 5- to 6-fold (compare Figs. 3A + B and 1A). Addition of this thioredoxin preparation did not affect the sulfide poised redox value ( - 140 mV) of culture media, as measured

355 Table 2 Fermentation end products a and growth yield b of Clostndium sp. strain 9.1 depending on growth conditions Addition

no addition thioredoxin (0.5) a C. acetobuo'licum e E. coil extract (2) d no addition thioredoxin (0.7) a no addition thioredoxin (0.7) a

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chitin c chitin c chitin ~ chitin ¢ NAG2 NAG, NAG4 NAG4

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Formate

Acetate

Ethanol

Acetate/ Ethanol ratio

125 200 200 190 153 181 128 184

19 19 16 16 18 14 22 29

175 197 188 200 195 186 199 209

81 69 77 78 97 84 82 68

2.16 2.86 2.44 2.56 2.00 2.21 2.43 3.07

Yield

13.8 24.4 24.7 28.1 11.2 24.2 12.3 23.7

Expressed as rnrnol of product per 100 mmol of NAG-equivalents fermented. CO2 could not be determined due to the use of HCO3-buffer. b Expressed as g protein per mol NAG-equivalent fermented. c 65% of the initial amount of chitin is actually fermented (see [2]). d Figure in parentheses represents the amount added expressed as /Lg protein per ml culture fluid. Mixed culture fermentation in the presence of C. acetobutylicum ATCC 824.

with a p l a t i n u m redox electrode. The presence of N A D P H (50 I~M) plus thioredoxin reductase in a d d i t i o n to thioredoxin resulted in a r e d u c t i o n of the lag-phase b u t not in a further increase in the rate of chitin degradation. T h i o r e d o x i n reductase alone had n o s t i m u l a t o r y activity at all a n d in fact decreased the rate of chitinolysis by approximately 25%. Boiling of the thioredoxin preparation for 10 m i n did not affect its activity. Stimulation by the thioredoxin p r e p a r a t i o n was reduced by 50-70% after a n alkylation with IoAc, indicating that its activity was based o n t h i o l : d i s u l f i d e interchange. T h i o r e d o x i n was far less effective in e n h a n c i n g the f e r m e n t a t i o n of chitobiose t h a n of chitin in pure cultures of strain 9.1. The rates of substrate utilization a n d of p r o d u c t f o r m a t i o n were increased by only 50% in the presence of thioredoxin (Fig. 3C + D). W h e n chitotetraose was used as g r o w t h s u b s t r a t e the s t i m u l a t o r y effect of thioredoxin was more p r o n o u n c e d , resulting in a 2-fold increase in the rate of f e r m e n t a t i o n . E n h a n c e m e n t of N A G - o l i g o m e r f e r m e n t a t i o n was not observed after s u p p l e m e n t a t i o n of the cultures with dithionite, although the lag phases were reduced. T h i o d o x i n n o t only e n h a n c e d the rate of ferm e n t a t i o n b u t also c h a n g e d the cell yield a n d the

ratio of f e r m e n t a t i o n e n d p r o d u c t s of strain 9.1 grown o n chitin or N A G - o l i g o m e r s (Table 2). Moreover, the m o r p h o l o g y of the Clostridium was affected d u r i n g its growth o n N A G - o l i g o m e r s . In the absence of t h i o r e d o x i n the cells were filam e n t o u s a n d occurred in small clusters responsible for the irregular course of the optical density m e a s u r e m e n t in Fig. 3C. However, in its presence the cells b e c a m e short a n d h o m o g e n o u s l y susp e n d e d after a n initial f i l a m e n t o u s stage (OD660 < O.O2).

5. D I S C U S S I O N The f e r m e n t a t i o n of chitin by Clostridium strain 9.1 in pure culture proceeded m u c h slower than in mixed culture with either one of various nonchitinolytic bacteria such as E. coli, K. aerogenes, C. acetobutylicum or the facultative a n a e r o b i c strains H A 8.1 a n d G H 8.2. A l t h o u g h the seco n d a r y species c o n s u m e the N A G liberated by strain 9.1, the e n h a n c e m e n t of chitin d e g r a d a t i o n is n o t due to the removal of potentially i n h i b i t o r y sugars because a d d i t i o n of N A G or N A G oligomers (up to 10 × the N A G c o n c e n t r a t i o n f o u n d in pure culture) did not affect the rate of chitin f e r m e n t a t i o n in pure culture of strain 9.1

356 [3]. Evidence has been presented indicating that the secondary bacteria in the mixed cultures produce a thermostable and relatively high molecular weight ( > 1500) factor (Fig. 2). This factor was not only demonstrated in the spent medium of the mixed cultures but it was also present in cell-free extracts and in spent media of the secondary bacteria grown in pure culture on N A G (Table 1). Acceleration of chitinolysis is also observed with strong reductants such as dithionite and Ti(III)-NTA. The presence of essential SH-groups in the chitinolytic enzyme system of strain 9.1 [4] suggests that the stimulatory effect of these reductants is based on a redox interaction with these sulfhydryl groups. Thioredoxin, a small thermostable dithiol protein found in many bacteria and located at the cell periphery (either in the periplasmic space [20] or at the inner side of the cytoplasmic membrane [21]), is capable of reducing a broad spectrum of protein disulfides [19]. For this reason thioredoxin (from Saccharomw'es cereeisiae) was used to test its effect on chitin hydrolysis. Indeed the enzyme proved to be very effective in accelerating the fermentation of chitin. Considering the thermostable and high molecular nature of the stimulatory factor(s) it is very likely that the compound(s) involved i s / a r e identical or closely related to thioredoxin. It is unlikely, for at least two reasons, that the enhancement of chitin fermentation by thioredoxin results from a direct lowering of the initially sulfide poised culture-E h ( - 1 4 0 mV). Firstly, the thioredoxin is added mainly as a protein-disulfide (due to exposure to oxygen during its purification [22]). Secondly, the applied concentration of the enzyme (approx. 50 nM) is extremely low compared to sulfide. Thus, if in spite of this thioredoxin is stimulatory because of its reducing properties, the enzyme must be rendered active through reduction by strain 9.1 itself. Strain 9.1 is likely to be capable of doing this given its capacity to lower the redox value of its own medium to below - 300 mV and to reduce the membrane-impermeable oxidant ferricyanide [4]. Assuming that also extracellular disulfides can be reduced as has been reported for Streptococcus rnutans [23], it is hypothesized that thioredoxin and the presumed redox-active compounds in the spent media and

cell-free extracts, function in the transfer of reducing equivalents from strain 9.1 to the chitinolytic enzyme system located outside the cytoplasmic membrane. The involvement of such a thiol:disulfide transhydrogenating activity is supported further by the observed drop in stimulation by thioredoxin after treatment with the SH-blocking reagent IoAc (see above). The fact that stimulation of chitobiose or chitotetraose fermentation by thioredoxin is much less than with chitin indicates an interaction of this enzyme on the level of polysaccharide hydrolysis. This is also supported by the absence of stimulation by dithionite in the oligomer-grown cultures. Therefore, in a tentative interpretation of the current data, the chitinolytic system is thought to be synthesized by Clostridium sp. strain 9.1 in a rather inactive conformation due to the presence of intramolecular disulfide or mixed-protein disulfide bonds. The importance of thioredoxin for growth of strain 9.1 is further reflected in the increase in the yield of the organism (see Table 2). Although redirection of the fermentation towards more acetate and H 2 production may have contributed to an increase in yield, this cannot explain an approximately 2-fold increase in biomass. An explanation for these changes has to await further studies. In cell-free extracts of E. coli and K. aerogenes the stimulatory compound(s) appeared to be located exclusively in the cytoplasm. This is in agreement with the characterization of thioredoxin as a cytosolic enzyme in E. coli [21]. In contrast a significant activity was located in the membrane fraction of extracts of the secondary bacteria G H 8.1 and HA-8.2. This may indicate the presence of additional high molecular stimulating compounds. Indeed in chicken embryo, wheat endosperm and mammalian liver-preparations, membrane-bound thioltransferases with a fairly broad substrate specificity have been found [24]. A dependence on sugar-fermenting secondary bacteria has been reported earlier for a number of anaerobic cellulose-degrading species [25]. Although the observed enhancement of cellulose breakdown in these cases has been explained in terms of a removal of i n h i b i t o ~ compounds (such

357 as r e d u c i n g s u g a r s ) , it w o u l d b e i n t e r e s t i n g to e x a m i n e t h e s e i n t e r a c t i o n s in m o r e d e t a i l as m o s t o f t h e c e l l u l o l y t i c s p e c i e s a p p e a r to p o s s e s s e s s e n tial t h i o l g r o u p s in t h e i r h y d r o l y t i c e n z y m e s [ 5 - 8 ] .

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