CURRENT MICROBIOLOGY VOI. 31 (1995), pp. 84-91 Current Microbiology An International Journal �9 Springer-Verlag New York Inc. 1995

Protein Profiles of Streptomyces aureofaciens Producing Tetracyclines: Reappraisal of the Effect of Benzyl Thiocyanate

Jana Novotmi, Li Xin-Ming,* Jitka Novotn~, Ji~f Vohradsk~, Jaroslav Weiser

Institute of Microbiology, Academy of Sciences of the Czech Republic, Videfiskfi 1083, 142 20 Prague 4, Czech Republic

Abstract. Cell protein profiles of submerged cultures of Streptomyces aureofaciens cultivated in the absence or presence of 12 p~M benzyl thiocyanate (BT) were analyzed by one-dimensional SDS polyacrylamide gel electrophoresis. Substantial increase in the intensity of the 13, 35, 37, 60, and 100 kDa protein bands was observed in cultures treated with BT. Similar increase in the 35, 37, and 60 kDa bands was found in a mutant blocked in the last chlortetracycline biosynthesis step. Effect of BT on the solid medium-grown cultures was also observed, with a more intensive substrate mycelium pigmentation and alteration in the spore size and shape as the most characteristic features. Earlier studies of BT effect involving those on the stimulation of chlortetracycline biosynthesis are summarized and a possible signal-transducing mechanism is discussed from the point of view of adaptation of S. aureofaciens to the uncoupling of oxidative phosphorylation.

Benzyl thiocyanate, C6HsCH2SCN, was widely used as stimulator of chlortetracycline (CTC) biosynthesis by Streptomyces aureofaciens in laboratory and indus- trial fermentations from the late 1950s [24]. When added at a concentration of 0.5-3 p,g m1-1 (3.4- 20.1 ~M) to young cultures (0-9 h after inoculation), it increased production of the antibiotic by more than 40% even in high-producing strains [24]. BT and about 40 analogs were examined for their influ- ence on tetracycline biosynthesis by S. aureofaciens var mediolanum and the observed structure-activity relationships determined the arrangement resulting in the maximal response with respect to benzene nucleus and the polar thiocyano moiety at the ends and the aliphatic chain interconnecting them [25]. Only 2-phenylethyl thiocyanate, C6HsCH2CHzSCN, produced a significantly higher stimulatory effect than BT.

Degradation of glucosinolates produced by plants (mainly from the order Brassicales, including impor- tant crop plants and vegetables) leads to the forma- tion of isothiocyanates, organic thiocyanates, and inorganic thiocyanate ions. The presence of BT was

* Present address: Department of Microbiology, Biozentrum, Uni- versity of Basel, 4056 Basel, Switzerland

Correspondence to: J. Novotn~

demonstrated after transformation of benzylglucosino- late in crushed Lepidium ruderale and L. sativum plants [20]. 2-Phenylethylglucosinolate is also one of natural glucosinolates and can therefore be consid- ered a potential source of 2-phenylethyl thiocyanate. All plants containing glucosinolates possess enzymes capable of hydrolyzing these compounds, presumably after a mechanical injury to the plant. Likewise, insects, fungi, and bacteria including Streptomyces were reported to be capable of hydrolyzing them [1, 20]. BT and its analogs are, therefore, common in natural environments, and S. aureofaciens, originally a soil microorganism [5], may encounter these volatile compounds in its ecological niche.

Attempts to explain the nature of the effect of BT on the producing organism started simultaneously with its practical use. However, the molecular events that take place in a streptomycete, intensifying its secondary metabolism, have not been fully under- stood to date. A series of papers by Hogt'~ilek [13-15], examining the relationships between carbohydrate metabolism and the biosynthesis of CTC, showed that the presence of BT in media during the early cultiva- tion phase led to an increase in the rate of phosphate utilization and respiration, whereas during the subse- quent cultivation the rates of respiration and carbohy- drate utilization were decreased in its presence. It

J. Novotnfi et al.: Altered Protein Profiles of Streptomyces aureofaciens 85

was also found tha t BT, when a d d e d to washed mycel ia at levels up to 6 x 10 -4 M, inh ib i t ed only

ox ida t ion of the sugar p r e s e n t in the m e d i u m dur ing its p r e c e d i n g growth. T h e ox ida t ion o f o t h e r sugars or e n d o g e n o u s r e sp i r a t ion were not a f fec ted by BT. A t h igher concen t r a t i ons of BT, e n d o g e n o u s r e sp i r a t i on was also lower, even t hough the ox ida t ion of a m i n o acids and o rgan ic acids was no t inh ib i ted at all. In the case of glucose ox ida t ion in the p re sence of BT, the C 1 / C 6 quo t i en t was found to increase . This observa- t ion, t oge the r wi th the reverse effect of inorganic

p h o s p h a t e on the process , sugges ted tha t the deg ree of pa r t i c ipa t i on of pen to se p h o s p h a t e pa thway in the ca rbon flow may be impl ica ted . In addi t ion , a de- c rease in the activity of the enzymes o f the t r icarbox- ylic acid cycle was obse rved in mycel ia growing in the p r e s e n c e o f BT [16]. In the 1960s, o t h e r effects of BT on S. aureofaciens were observed . I t r eve r t ed the inh ib i tory effects of i n t e r r u p t e d ae ra t i on [13] and b io t in [26] on C T C accumula t ion .

M o r e r ecen t inves t igat ions have shown tha t BT

inc reased the activi ty o f anhydro te t r acyc l ine oxygen- ase (ATCox) , an enzyme cata lyzing the p e n u l t i m a t e r eac t ion in the te t racyc l ine b iosyn the t i c pa thway of S. aureofaciens [4]. T h e f inding tha t the A T C o x level was inverse ly r e l a t ed to the ra te of sugar u t i l i za t ion even

in the absence o f B T a p p e a r e d to fu r the r conf i rm the hypothes is suggest ing BT to be effective via carbohy- d r a t e u t i l i za t ion [6]. However , the same s tudy also showed some cases, in which A T C o x activi ty signifi- cant ly i nc reased as well as the a m o u n t of C T C

p r o d u c e d , when the ra te of sugar c o n s u m p t i o n in the p r e s e n c e o f BT was no t changed .

BT was also found to affect the loca l iza t ion of the b iosyn the t i c p rocess in S. aureofaciens. Its p r e s e n c e in the m e d i u m resu l t ed no t only in a to ta l inc rease of the A T C o x activity, bu t also in a change of the d is t r ibu- t ion of this enzyme in individual subce l lu la r f rac t ions [7]. S imi lar ly to some o t h e r m e m b r a n e - b o u n d en- zymes, A T C o x was r ed i s t r i bu t ed f rom m e m b r a n e s t ruc tures to the n o n - m e m b r a n e c o m p a r t m e n t s [7,

27]. In addi t ion , a p r o d u c t b ind ing in the in t r ace l lu l a r c o m p a r t m e n t s was signif icantly r e d u c e d in BT-grown cells as well as in the h igh -p roduc ing s t ra in , whi le C T C depos i t s in the nuc leo id reg ion of the wi ld- type, l ow-produc ing s t ra in cells were obse rved [17, 18].

In an a t t e m p t to r econs ide r var ious aspects of the BT effect on S. aureofaciens, we used an a p p r o a c h to ob ta in a m o r e gene ra l view of a l t e ra t ions which BT caused . Here , we c o m p a r e o n e - d i m e n s i o n a l cell p ro- te in prof i les f rom mycel ia grown with and wi thou t BT in s u b m e r g e d cul tures l W e also desc r ibe its inf luence on the d e v e l o p m e n t a l cycle o f cu l tu res grown on the

solid a g a r med ium. Overviewing the resul ts of these

and prev ious studies, we suggest a novel hypothes is

expla in ing the effect o f BT on S. aureofaciens as a pa r t i a l d i so rgan iza t ion of m e m b r a n e funct ions induc- ing a stress r e sponse o f the mic roorgan i sm.

Mater ia l s and M e t h o d s

Bacterial strains, media, and culture conditions. The strains of Streptomyces aureofaciens ATCC 10762 (A-377) [5], maintained in the collection of the Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, and S. aureofaciens ATCC 12748 (S-1308) [22], obtained from I.S. Hunter, Robertson Labora- tory for Biotechnology, University of Glasgow, Glasgow, UK, were used throughout the study.

Sucrose-soybean meal extract medium [13] was prepared by boiling 30 g of soybean defatted meal in 750 ml of water for 10 rain. After filtration and a separate sterilization in 40-ml portions, 20-ml sterile portions of other components (sucrose 30 g, sodium chloride 5 g, calcium carbonate 4 g, ammonium sulfate 2 g, beet molasses 2 g, corn steep 5 g in 250 ml of water) were added. Flasks with 60 ml of the medium inoculated by spores to produce vegetative inocu- lure or by 5% vegetative inoculum to obtain tested cultures were shaken at 28~ Concentration of BT added immediately before the second inoculation was 12 ~M; the control cultures were supplied with the same volume (0.5 ml) of water.

The sporulation agar medium was as described [24]. Spore suspensions for the inoculation of Petri dishes were prepared by washing spores from slants with distilled water in the presence of 3-ram-diameter glass beads and by subsequent filtration through cotton. The spore counts were determined as the optical density of the suspension at 580 nm and calculated on the basis of a calibration obtained by phase contrast microscopy in the counting chamber (Thoma). BT was added to the agar medium inoculated by ca. 2 x l0 s spores as a water solution (0-20 ~,g/0.2 hal) diffusing from a 6-ram-diameter well cut in agar in a center of the 9-cm-diameter plate. Alternatively, solid BT-paper discs (1-cm diameter) were prepared by dropping BT dissolved in ethanol and evaporating the solute before placing discs onto the inner side of a lid of the plate in the reversed position. Instead of BT-paper discs, small crystals of solid BT were applied in some experiments. The plates were incubated at 28~

Preparation of cell-free extracts. Mycelia grown in submerged cultures and harvested by centrifugation were washed twice with distilled water and stored frozen. Disintegration was carried out by grinding frozen mycelium with the same amount of ballotina in a mortar. Broken mycelium was suspended in the extraction buffer (0.1 M Tris-HCI buffer, pH 7.4, 0.2 mM EDTA, 15% glycerol, 1 mM monotbioglycerol, and 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and centrifuged at 12,000g for 30 rain. All steps were carried out at 4~

Electrophoresis. Sodium dodecyl sulfate-polyacrytamide gel electro- phoresis (SDS-PAGE) was as described [19] with 8%, 10%, and 12% acrylamide in the separation gels. Electrophoresis was carried out at 17-20 mA per gel with a Midget Electrophoresis Unit (LKB). Proteins were stained with Coomassie Brilliant Blue R-250 (Sigma), and the following molecular weight standards (Sigma) were used: [3-galactosidase 116,000, phosphorylase b 97,400, and Dalton Mark VII-L (bovine albumin 66,000, egg albumin 45,000, glyceraldehyde-3-phosphate dehydrogenase 36,000, carbonic anhy- drase 29,000, trypsinogen 24,000, trypsin inhibitor 20,100, and a-lactalbumin 14,200). ATCox was isolated as described [28]. Gels, which were air-dried between two layers of cellophane, were

86 CURgENT MICROBIOLOGY Vol. 31 (1995)

scanned and analyzed as described [29]. The densitometric profiles were expressed in OD-proportional units.

Enzyme assay. ATCox activity was assayed by the modified method of B~hal et al. [3]: 30-200 t~1 of cell-free extracts were combined with 80 mM Tris-HCl buffer (pH 7.4) containing 0.24 m s NADP, 0.6 mM glucose-6-phosphate, 1.1 U glucose-6-phosphate dehydro- genase (Serva), and 40 I~M anhydrotetracycline (ATC) in a final volume of 0.5 ml. The mixtures were incubated at 37~ and changes in absorbance monitored in the range of 250-550 nm in Hewlett- Packard 8451A diode-array spectrophotometer by following the overlapping spectra of individual tetracycline derivatives. Initial slopes of reaction curves at 440 nm and the molar absorptivity of e_440 = 966 M -1 mm -1 were used for the calculation of ATCox activities. Protein content was determined by the Coomassie Blue binding assay [2] with bovine serum albumin as the standard.

E l e c t r o n m i c r o s c o p y . Morphological properties of the spore chains were observed in preparations obtained by a direct impression of the surface cultures. Transmission electron microscopy observa- tions were carried out by using a JEM 100B microscope at an accelerating voltage of 60 kV.

T e t r a c y c l i n e assay. Tetracyclines (CTC as a dominant antibiotic product representing approximately 90% in the mixture with tetracycline) were determined spectrophotometrically and cali- brated with CTC [21].

Changes in the accumulation of dehydrochlortetracycline (DHCTC), a final product of the blocked mutant of S. aureofaciens ATCC 12748, were estimated spectrophotometrically in the range of 300-500 nm (absorption maximum at 400 nm [23]) in diluted culture liquids after centrifugation of mycelia. Water was used as a reference sample.

Results

Changes in protein profiles of submerged cultures. In previous studies ATCox was expressed to reach higher levels in BT-grown cultures [4, 6, 7]. The analysis of whole-cell extracts from cultures grown with and without BT was performed with one- dimensional SDS-polyacrylamide gel electrophoresis to find out whether the increased expression was specific for ATCox or involved more individual pro- teins.

Figure 1 shows densitograms of 10% acrylamide gels separating approximately 30 discrete protein bands of a molecular size of 24-150 kDa from cell-free extracts of wild-type strain S. aureofaciens ATCC 10762. Three substantially stronger protein bands were visible in extracts from the cells grown with BT. Their apparent size was 60, 37, and 35 kDa. The increase occurred in the cells throughout the whole cultivation period, but the most visible re- sponse was observed in 24-, 33-, and 48-h mycelia (Fig. lb,c,d). Similar results were obtained with 12% gels, where proteins larger than 10 kDa could be seen. An additional intensive band (13 kDa) was observed that was expressed at higher levels similar to those of the above-mentioned proteins. A densitogram from

0.1

0.0

116 66 4-5 29 Mr(kDQ ) I I I I

~,. ~. <:1

I I I I I

0 . 0 , , , , - ' " i

OD

O. 1 ;; c

0.0 - " " I I I I I

O. 1 :" d

0.0 " ............ i I i I I

011 0.0

0.0 0.2 0.4 0.6 0.8 ] .0

Rf

Fig. 1. Densitograms of S. aureofaciens ATCC 10762 cell-free extracts separated on 10% acrylamide gels by SDS-PAGE. Dotted line, a control culture; solid line, a culture grown in the presence of 12 p~M BT; a, l l -h-old; b, 24-h-old; c, 34-h-old; d, 48-h-old; and e, 72-h-old cultures. As Mr standards were used, [3-galactosidase (116,000), bovine albumin (66,000), egg albumin (45,000), and carbonic anhydrase (29,000) and their positions are shown on the top with numbered bars.

24-h-old mycelia is shown for illustration (Fig. 2). The 60-kDa band represents ATCox (Mr subunit = 57 kDa [28]), whose activity was increased 2- to 5-fold in samples removed at different cultivation times (Table 1). However, the increased expression of other pro- tein(s) of this size is likely also to occur. As can be seen on 8% gels allowing better resolution in that range (Fig. 3), the most intensively responding band from this size range had slightly lower mobility than purified ATCox (Fig. 3a,b). In addition to the above- mentioned bands, another responsive protein of 100- kDa size was observed on the 8% gel (Fig. 3a), but only in the 24- and 48-h mycelia (Table 1). The

J. Novotn~ et al.: Altered Protein Profiles of Streptomyces aureofaciens 87

0.1

OD

0,0

0.0

66 45 56 29 24- 20 14- I I I F I I I M r (kDo)

i j i I I

0.2 0.4 0.6 0.8 1.0

Rf

Fig. 2. Densitograms ofS. aureofaciens ATCC 10762 cell-free extracts from 24-h-old cultures grown with (solid line) and without (dotted line) BT, Separated on a 12% acrylamide gel. Mr standards: bovine albu- min (66,000), egg albumin (45,000), glyceraldehyde- 3-phosphate dehydrogenase (36,000), carbonic an- hydrase (29,000), trypsinogen (24,000), trypsin inhibitor (20,100), and ct-lactalbumin (14,200).

Table 1. Changes in expression of BT-responsive proteins, a ATCox activity, b and CTC c or DHCTC a production provoked by the addition of 12 p~M BT at the start of culture found during cultivation of the wild-type strain S. aureofaciens ATCC 10762 and of the mutant strain ATCC 12748

Culture ATCox CTC age (h) Strain 13 kDa 35 kDa 37 kDa 60 kDa 100 kDa (fold) (fold) DHCTC

11 10762 + + + + nd e nd 1 12748 + + + nd 1.1 nd

24 10762 + + + + + + + + + + 4.2 5.7 12748 + + + + + + nd 2.3 nd

34 10762 + + + + + + + nd 3.7 12748 + + + + + + nd 3.8 +

48 10762 + + + + + + + + 3.2 4.2 12748 + + + + + + nd 3.5 +

72 10762 + + + + nd 4.0 4 12748 + + + nd 1.4 nd

a Increase in band intensity. b Ratio of specific activities of enzyme in BT-treated and nontreated cultures. c Ratio of the antibiotic accumulated in BT-treated and nontreated cultures. a Increase in A40o of 10• diluted culture liquids. end, not determined.

i nc reased express ion o f four to five p r o t e i n bands af fec ted by BT was always in co r re l a t ion with the intensif ied C T C p roduc t ion and A T C o x activity (Tab le 1), the r e l a t ion of those two l a t t e r having b e e n desc r ibed ea r l i e r [4, 6].

A m u t a n t s t ra in S. aureofaciens A T C C 12748, wi th its or igin in a h igh -p roduc ing strain, b locked in the last t e t racyc l ine b iosyn the t i c s tep owing to its def ic iency in a 8-hydroxy-5-deazaf lavin coenzyme [22; Novo tmi et al. u n p u b l i s h e d resul ts] , was also used for c ompa r i son of p r o t e i n prof i les in cu l tu res grown wi th and wi thou t BT. T h e p r o t e i n b a n d s of the 60, 37, and 35 k D a sizes r e s p o n d e d to BT as in A T C C 10762; levels o f A T C o x were also i nc reased (Tab le 1). Changes in o t h e r size ranges were not significant; e.g., the in tens ive 13-kDa band was obse rvab le even in the absence of BT ( d a t a not shown). A b s o r p t i o n spec t ra of cu l tu re l iquids exhibi t ing inc reased va lues in the reg ion o f 400 nm, when BT was added , also suggest

h igher accumula t ion of the final p r o d u c t D H C T C ,

c ha ra c t e r i z e d by abso rp t ion m a x i m u m at this wave- length (Fig. 4).

M orpho log i c a l changes of sur face cultures;. BT, an

i m p o r t a n t effector o f t e t racyc l ine p r o d u c t i o n in sub-

m e r g e d cul tures , has not yet b e e n s tud ied as a

subs tance tha t could affect cu l tu res grown on surfaces of sol id media . As genes for an t ib io t ic p roduc t i on

may be subject to r egu la to ry m e c h a n i s m s involved in

aer ia l myce l ium f o r m a t i o n and in the d e v e l o p m e n t of spore chains in s t r ep tomyce te s [12], we asked w h e t h e r the a p p e a r a n c e of mycel ia l lawns or the s t ruc ture of spore chains cou ld be a l t e r ed by the p re sence of BT in the env i ronmen t of d i f fe rent ia t ing mycel ia .

Wi ld - type S. aureofaciens A T C C 10762 ( spore suspens ion) was i nocu la t ed on to the surface of sporu- la t ion agar in Pe t r i dishes. In one set of exper iments ,

BT was a d d e d as a so lu t ion in w a t e r (0-20 ~g/0 .2 ml)

88 CURRENT MICROBIOLOGY Vol. 31 (1995)

OD

0.1

0.0

0.03

0.00

116 97 66 45 29 I I I 1.~ I Mr (kDo)

I i i i I

i I * ~ L

0.0 0.2 0.4 0.6 0.8 1.0

Rf

Fig. 3. Densitograms of S. aureofaciens ATCC 10762 cell-free extracts from 48-h-old cultures grown with (solid line) and without (dotted line) BT (a) and of partially purified ATCox (b). Separated on an 8% acrylamide gel. Mr standards: [3-galactosidase (116,000), phosphorylase b (97,000), bovine albumin (66,000), egg albumin (45,000), and carbonic anhy- drase (29,000). The arrow indicates the position of the 60-kDa BT-responsive band.

2 . 0

1 . 5

0 0 C 0 n L 1 . 0 0

n

. 5

o.n, I l i ~o 0 C3 0 (3 0 LO 0 tO [] 03 CO ~ ~ LO

WovelenBth (rim)

Fig. 4. Comparison of absorption spectra of culture liquids from 46-h-old S. aureofaciens ATCC 12748 cultures grown with (1) and without (2) BT. Samples were diluted 10x.

into a well cut in the agar. In the other set, paper discs containing various amounts BT (0-2 mg) were placed on the inner side of a plate lid at a distance from the agar surface. In both cases BT was applied immedi- ately after inoculation; therefore, effects on spore germination and early growth phases could also be involved, while in submerged cultures BT was added simultaneously with the vegetative inoculum.

BT in the amount of 20 Izg per dish, which is similar to the concentration used for stimulation of CTC biosynthesis in submerged cultures, induced a yellow-orange color of substrate mycelia shown on

the reverse bottom of dishes after 2 days. Similar intensification of pigment formation accompanied CTC biosynthesis in liquid cultures grown with BT. In control cultures older than 3 days, rich grey-colored sporulation lawns were observed, while only faint grey to white color with pink tint developed in the presence of BT. Their aerial mycelium formation could be observed 1-2 days later. Amounts of 2 Ixg BT per dish or less were without any visible effect on substrate mycelia and sporulation lawns.

Volatile BT applied from paper discs through air exhibited a similar effect; 200 or 2000 ~g BT per disc caused growth in an intensively yellow-orange col- ored ring with a faint fluffy white surface surrounding a cream ring of only substrate mycelia and a clear zone without any growth in the center (inhibition zone of 3.5-cm diameter for the highest amount of BT). Thus, gradient in concentration of BT formed across the dish diameter has shown effects from complete inhibition of growth and impairment of aerial myce- lium and sporulation to intensification of secondary metabolism represented by pigment formation.

Spores formed after BT treatment were able to give colonies with a normal development of aerial mycelia and spores, when re-inoculated in the ab- sence of BT.

Electron micrographs of impression preparations of cultures sporulating in the presence of BT revealed lower numbers of spore chains that were shorter with spores of altered dimensions (Table 2).

In conclusion, no stimulation of development of aerial mycelium and spore chains was observed in the

J. Novotnfi et al.: Altered Protein Profiles of Streptomyces aureofaciens 89

Table 2. Effect of BT ~ on spore dimensions of S. aureofaciens ATCC 10762

Dimensions 0xm) Control BT

Length 1.12 _+ 0.16 (70) b 1.25 _+ 0.13 (36) Width 0.68 _+ 0.05 (12) 0.99 _+ 0.1l (32) Length/width ratio 1.65 1.26

a A crystalline BT ( < 1 mg per dish) was applied on the plate lid. b Values in parentheses give the number of spores checked.

presence of BT, the only effect being less abundant and immature (lacking grey pigment) spore chains containing spores of lower length-to-width ratio.

Discuss ion

One-dimensional SDS-PAGE was used to establish the existence and number of the most intensively BT-responsive proteins that allow quick comparison of strains and easy preliminary search for optimal response to the BT challenge. However, high sensitiv- ity determination of the number of induced proteins and their identification accompanied, for example, by N-terminal amino acid sequencing should be carried out with two-dimensional electrophoresis to decrease substantially the probability of occurrence of two (or more) proteins in one location of the gel. Such a detailed analysis of BT-responsive proteins, which is under way, could answer the question of how specific its effect is. The question consists of two parts: (i) do all or only several key enzymes of the CTC biosyn- thetic pathway respond to BT?; (ii) is the expression of proteins other than those directly involved in CTC biosynthesis sensitive to this signal, and, if so, mem- bers of which sets are tightly co-regulated and thus perhaps functionally related to the BT-enhanced antibiotic formation? Moreover, a subcellular fraction- ation of total proteins should be used for characteriza- tion of the location of the induced proteins in cell compartments.

Under our conditions, S. aureofaciens ATCC 12748 used by McCormick et al. [22] also responded to BT. It exhibited an increased formation of several proteins of similar sizes as the wild-type strain, a higher activity of ATCox, and a higher accumulation of its final product, DHCTC. The absence of the BT effect in some strains of S. aureofaciens mentioned by Goodman [11] could be caused by a difference in the strains (non-sensitive mutants) or by different cultiva- tion conditions.

Existence of a hypothetical receptor for BT was suggested whose extracytoplasmic (membrane) loca-

tion was deduced from the concentration dependence of the inhibition effect and from reversion of its effect by washing mycelia [14]. Judging from the structure- activity relationships of BT analogs [25] and from the distribution of BT response in strains of S. aureofa- ciens [11], the hypothetical receptor would be of rather high specificity for BT, and its occurrence would be limited. A signal mediated by this receptor would obviously result in increased levels of several BT-responsive proteins. Formation of the antibiotic as well as of a vegetative pigment would be thus enhanced. However, an antibacterial effect was ob- served at high concentrations of BT, resulting in a severe inhibition of respiration and sporulation and even in a complete cessation of growth [14, this work].

We review previous studies of physiology of S. aureofaciens affected by BT to produce higher amounts of CTC (see Introduction). These studies imply, together with the changes in the content of cell proteins described in this study, that both an immedi- ate reaction and a later adaptation are involved. First, cytoplasmic membrane responsible for the contact of the bacterium with the environment and mediating uptake of nutrients and export of products may be the primary target site. Inhibition of sucrose respiration was observed 15 min after addition of BT [14]. Alterations in the sugar consumption rate and the antibiotic export were, however, also later conse- quences of BT presence [14, 17, 18]. Second, a significantly increased synthesis of a set of several unidentified proteins and ATCox [cf. 4,6] is the result of the BT effect, which can also be demonstrated in different translocations of membrane proteins [7, 27]. Third, ATP level was decreased in BT-grown cultures (Hogt'filek, personal communication).

On the basis of the BT effects discussed above and on a partial structural similarity between BT and CCCP (carbonyl cyanide m-chlorophenylhydrazone) molecules, it can be speculated that BT affects primarily energy transduction on the membrane. Thus, nutrient limitation, uncoupling of ATP synthe- sis, and inhibition of translocations of membrane- and extracellular proteins as consequences of a par- tial collapse of membrane functions could evoke an adaptation of the microorganism (mediated by the formation of proteins de novo) to a stress factor [10]. This adaptation could also involve a production of antibiotic compounds protecting sources of energy against competitors with which the environment is shared. In other words, BT might act, directly or indirectly, as an uncoupler of oxidative phosphoryla- tion.

At the time this publication was prepared, Fuska

90 CURRENT MICROBIOLOGY Vol. 31 (1995)

et al. [8] independently presented more direct evi- dence suggesting uncoupling of oxidative phosphory- lation by BT, benzyl isothiocyanate, and dibenzyl disulfide (an in vivo transformation product of BT [9]) as these agents at 1 IxM concentrations stimulated the oxygen respiration of S. aureofaciens. In addition, they have shown that BT (30 ~LM), benzyl isothiocya- hate (30 txM), dibenzyl disulfide (8 ~LM), and even CCCP (3 txM) increased CTC biosynthesis in an industrial strain by 43, 34, 30, and 14%, respectively. However, in an in vitro system (proteoliposomes where membrane potential and proton gradient across the liposomal membrane was generated by proton- pumping activity of cytochrome c oxidase), the proto- nophoric activity of BT (16 and 120 IxM) was not proved [Opekarov~i and Novotn~i unpublished].

In any case, further studies are needed to estab- lish whether BT and benzyl isothiocyanate act di- rectly or only after transformation into active deriva- tives (e.g., dibenzyl disulfide [9]), and whether or not the stimuli of the true uncouplers of oxidative phos- phorylation on one hand and BT and its derivatives on the other hand are responded to similarly. One- dimensional SDS-PAGE, providing protein profiles of cultures grown in the presence of compounds in question, will be used as an efficient and quick way for comparison of respective responses.

ACKNOWLEDGMENTS

This work was supported in part by a research grant from the Grant Agency of the Czech Republic to J.W. The authors thank Dr. V. Erban for technical help with impression preparations of sporulat- ing cultures and Drs. Z. Hogt'filek, M. Opekarovfi, and J. Neus for stimulating discussions and critical reading of the manuscript.

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