ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 200, No. 1, March, pp. 45-54, 1980 Biosynthesis of Polymyxin by Bacillus Polymyxa Il. On the Nature and Interaction of the Multienzyme Complex with the End Product Polymyxinl R. BALAKRISHNAN,2 SATWANT KAUR,3 A. K. GOEL, S. PADMAVATHI, AND KUNTHALA JAYARAMAN4 Department of Molecular Biology, School of Biological Sciences, Madurai Kamaraj University, Madurai 625 021, India Received March 5, 1979; revised June 29, 1979 The interaction of polymyxin with the producer organism Bacillus polymyxa has been shown to be at the level of membranes, resulting in an enhancement of the activities of its own biosynthetic enzymes. This enhancement has been shown to be due to the solubiliza- tion of the membrane-associated multienzyme complex by polymyxin in a specific manner. The relevance of this physiological feature was also indicated by the appearance of the soluble multienzyme complex activity only in cells, which synthesize maximal amounts of polymyxin. Purification of the polymyxin released multienzyme complex from the membranes and the soluble form of the complex from the stationary phase cells has revealed several similarities between them. Both contain two major fractions of the molecular weights of 300,000 and 170,000, harboring all the polymyxin component amino acid-activating enzymes. Their multisubunit nature was established by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Using mutants blocked in sporulation and/or antibiotic synthesis, it was shown that the interaction of polymyxin with the producer organism was inoperative when antibiotic production was curtailed. This interaction has been suggested as one of the early sporulation-specific phenemenon. The physiological role of antibiotics on their producer organisms has remained un- clear hitherto. While the nature of the multi- enzyme systems involved in their biosynthe- sis is now well understood (1,2), the mecha- nism(s) of the extrusion of the antibiotics into the medium is not, nor the manner in which their production can be enhanced ’ We wish to place on record our most sincere thanks to Dr. J. Szulmajster, Laboratoire d’Enzymologie, C.N.R.S., Gif-sur-Yvette, France, for a very critical reading of the manuscripts. The expert typing of these manuscripts by Mrs. Jocelyne Mauger is gratefully acknowledged. Supported by the grant-in-aids of the Department of Science and Technology (SERC) and Council of Scientific and Industrial Research, India. * Present address: Department of Microbiology, TuRs School of Medicine, Boston, Mass. 3 Present address: Roche Institute of Molecular Biology, Nutley, N. J. 4 To whom all correspondence should be addressed. severalfold by a combination of genetic and physiological manipulations. In Bacillus polymyxa, the initial phase of sporulation is marked by the appearance of high amounts of extracellular polymyxin (3, 4), although we have observed that polymyxin synthe- sizing enzymes are present and functional even during the vegetative phase of the organism (5, 6). In the studies reported in the companion paper (6), we have demonstrated the associ- ation of the polymyxin synthesizing machin- ery with its end product, polymyxin. We have also shown that the activity of the enzymes involved in the polymyxin synthesis was at the start, associated with membranes and this activity was subsequently dis- placed to the soluble fraction of the cells during the rapid phase of polymyxin synthe- sis. The present work describes the inter- action of polymyxin with its biosynthetic 45 0003-986l/80/03904~10$02.00/0 Copyright 8 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
Transcript
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 200, No. 1, March, pp. 45-54, 1980
Biosynthesis of Polymyxin by Bacillus Polymyxa
Il. On the Nature and Interaction of the Multienzyme Complex
with the End Product Polymyxinl
R. BALAKRISHNAN,2 SATWANT KAUR,3 A. K. GOEL, S. PADMAVATHI, AND KUNTHALA JAYARAMAN4
Department of Molecular Biology, School of Biological Sciences, Madurai Kamaraj University, Madurai 625 021, India
Received March 5, 1979; revised June 29, 1979
The interaction of polymyxin with the producer organism Bacillus polymyxa has been shown to be at the level of membranes, resulting in an enhancement of the activities of its own biosynthetic enzymes. This enhancement has been shown to be due to the solubiliza- tion of the membrane-associated multienzyme complex by polymyxin in a specific manner. The relevance of this physiological feature was also indicated by the appearance of the soluble multienzyme complex activity only in cells, which synthesize maximal amounts of polymyxin. Purification of the polymyxin released multienzyme complex from the membranes and the soluble form of the complex from the stationary phase cells has revealed several similarities between them. Both contain two major fractions of the molecular weights of 300,000 and 170,000, harboring all the polymyxin component amino acid-activating enzymes. Their multisubunit nature was established by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Using mutants blocked in sporulation and/or antibiotic synthesis, it was shown that the interaction of polymyxin with the producer organism was inoperative when antibiotic production was curtailed. This interaction has been suggested as one of the early sporulation-specific phenemenon.
The physiological role of antibiotics on their producer organisms has remained un- clear hitherto. While the nature of the multi- enzyme systems involved in their biosynthe- sis is now well understood (1,2), the mecha- nism(s) of the extrusion of the antibiotics into the medium is not, nor the manner in which their production can be enhanced
’ We wish to place on record our most sincere thanks to Dr. J. Szulmajster, Laboratoire d’Enzymologie, C.N.R.S., Gif-sur-Yvette, France, for a very critical reading of the manuscripts. The expert typing of these manuscripts by Mrs. Jocelyne Mauger is gratefully acknowledged. Supported by the grant-in-aids of the Department of Science and Technology (SERC) and Council of Scientific and Industrial Research, India.
* Present address: Department of Microbiology, TuRs School of Medicine, Boston, Mass.
3 Present address: Roche Institute of Molecular Biology, Nutley, N. J.
4 To whom all correspondence should be addressed.
severalfold by a combination of genetic and physiological manipulations. In Bacillus polymyxa, the initial phase of sporulation is marked by the appearance of high amounts of extracellular polymyxin (3, 4), although we have observed that polymyxin synthe- sizing enzymes are present and functional even during the vegetative phase of the organism (5, 6).
In the studies reported in the companion paper (6), we have demonstrated the associ- ation of the polymyxin synthesizing machin- ery with its end product, polymyxin. We have also shown that the activity of the enzymes involved in the polymyxin synthesis was at the start, associated with membranes and this activity was subsequently dis- placed to the soluble fraction of the cells during the rapid phase of polymyxin synthe- sis. The present work describes the inter- action of polymyxin with its biosynthetic
45 0003-986l/80/03904~10$02.00/0 Copyright 8 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
46 BALAKRISHNAN ET AL.
machinery under in vivo and in vitro condi- tions and the importance of this interaction in the processes of antibiotic synthesis and of sporulation in B. polymyxa.
MATERIALS AND METHODS
The wild-type strain of B. polymyxa 2459 and its Rif’, Spo- Ab- derivatives described previously (4) were used in these studies. Culture media used were described earlier (7).
The buffers used in these experiments were : TKM buffer: 0.1 M Tris-HCl buffer, pH 8.0, 0.1 M KCl, 10 mM Mg2+ TK buffer: 0.1 M Tris-HCl buffer, pH 8.0,O. 1 M KCl. The amino acid-dependent ATP-[32P]P, exchange activities were measured as outlined in the companion paper (6).
Preparation of membrane and soluble fraction. Spheroplasts obtained &er lysozyme treatment as outlined in our previous paper (6) were burst by passing through a French pressure cell, and the suspension was centrifuged at 39,000g for 30 min. The supernatant is referred to as the soluble fraction. The pellet was processed further for isolating pure membranes as described in the companion paper (6).
Release of membrane-bound amino acid-activating
enzymes involved in polymyxin synthesis. Purified membrane fractions were treated with polymyxin (28 nmol or 400 units/mg membrane protein) or TK buffer for 1 h at 0°C and the suspension was centrifuged at 39,OoOg for 30 min. The proteins in the supernatant were precipitated by the addition of ammonium sulfate to 60% saturation. The precipitate was resuspended in TKM buffer and dialyzed overnight against the same buffer and the dialysate contained the membrane- released proteins.
Treatment of membrane fraction with polyamines and o!&rgents. The purified membrane fraction was resuspended in TKM buffer (1 mg/ml), and was treated separately with Brij 35 (O.l%), SDS (O.l%), Triton X-100 (O.l%), Putrescine (100 nmol/ml), Spermidine (100 nmol/ml), and DAB (100 nmol/ml) and left at 0°C for 1 h. The suspensions were centrifuged at 39,000g for 30 min and the supernatant proteins were precipitated by ammonium sulfate at 60% saturation. Each precipitate was dissolved in TKM buffer, dialyzed overnight against the same buffer, and assayed for the DAB activation.
Sepharose /B column chromatography. Sepharose 4B beads were suspended in TKM buffer, containing 10% (v/v) glycerol. This suspension was poured into a glass column (1 x 40 cm) and the gel was settled by
s Abbreviations used: TKM buffer, 0.1 M Tris-HCl buffer, pH 8.0, 0.1 M KCl, 10 mM MgS+; TK buffer, 0.1 M Tris-HCl buffer, pH 8.0, 0.1 M KCI; SDS, sodium dodecyl sulfate; DAB, LB&diaminobutyric acid.
washing it overnight with the same buffer. The con- centrated membrane-released fractions or the soluble fractions (5 mg protein/ml) were loaded on the column and the proteins were eluted by TKM buffer, contain- ing 10% (v/v) glycerol at a flow rate of 15 ml/h. Using proteins of known molecular weight, the V, of the column was determined.
Polyacrylamide gel electrophoresis of the proteins releasedfrom membranes by polymyxin. Native acryl- amide gels (5%) were prepared in tubes (0.6 x 11 cm) with 0.1 M Tris-glycine buffer, pH 8.3, and the protein samples (50 Kg/tube) were layered on a stacking gel (3%), made with 0.1 M Tris-glycine buffer, pH 6.5. The electrophoretic run was carried out at 2 mA/tube for 4-5 h with bromophenol blue as the marker dye. Electrophoresis under dissociating conditions was carried out in slab gels (1 mm thick) containing 0.1 M
Tris-glycine buffer, pH 8.0, 2% SDS and 10% poly- acrylamide. The samples were dissolved in 0.1 M Tris- glycine buffer, pH 8.0, containing 0.8 M fl-mercapto- ethanol, 2% SDS, 20% glycerol, and 0.002% bromophenol blue. They were heated for 5 min at 100°C prior to their loading on the slab gels. A current supply of IO- 15 mA was maintained until the completion of the run (5-6 h).
The protein bands were stained with a 0.1% solution of amido black in 7% acetic acid and 50% ethanol solu- tion for 2 h and destained in the same solvent. Standard proteins were used to calibrate the gels.
RESULTS
Effect of Addition of Polymyxin on the DAB-Activating Enzyme Activity
Whole cells. We have observed that the levels of the membrane-bound and soluble DAB-activating enzyme activities increased during the later log phase of growth of B. polymyxa, when high amounts of polymyxin was accumulated in the medium (5, ‘7). It was interesting to know whether this situa- tion could be simulated in cells at their early log phase of growth by the addition of poly- myxin to these cells at physiological con- centrations. When these cells were exposed to polymyxin, we observed a sharp increase in the specific activity of the membrane associated DAB-activating enzyme, which is characteristic of polymyxin-synthesizing complex (6). The data presented in Fig. 1 shows this to be a concentration-dependent phenomenon. The low levels of the DAB activation in the soluble fraction of the cells was not influenced by the addition of poly- myxin.
POLYMYXIN BIOSYNTHESIS: PHYSIOLOGICAL ROLE 47
I -.A--------* -.‘Lu.
FIG. 1. Effect of polymyxin addition on the subcel- lular DAB activation in the vegetative cells of B. polymyxa. Cultures of B. polymyxa (200 ml) in their early log phase of growth were exposed to different concentrations of polymyxin indicated, for 2 h at 30°C with aeration. Membrane and soluble fractions were prepared from these cells as outlined under Materials and Methods, and their DAB-activating enzyme activity was measured as nmol of ATP exchanged with [32P]Pi dependent on added DAB/mg protein. 0, Soluble fraction; 0, membrane fraction.
Isolated membrane fractions. When puti- fied membrane fractions from cells grown up to the midlog phase were treated with different concentrations of polymyxin a dou- bling of the DAB-activating enzyme activity was observed (Fig. 2). This result substanti- ated our observations with the whole cells and indicated a direct involvement of poly- myxin with at least one of its biosynthetic enzymes, with a strong stimulation of its activity.
Solubilization of the Membrane-Bound DAB-Activating Enzyme by Polymyxin
The stimulation of membrane-bound DAB- activating enzyme activity by polymyxin could be due to either a change in the con- formation of the membrane by polymyxin and/or an increased accessibility of substrate for the enzyme. When polymyxin-treated membranes were resedimented, the DAB- activating enzyme activity appeared in the supernatant (Fig. 3). The increase in the observed specific activities of the membrane- released fractions indicated that the enhance- ment in the enzyme levels of polymyxin was mostly due to the release of the DAB-ac- tivating enzyme from the membranes. Figure
FIG. 2. Effect of polymyxin on membrane-associated DAB activation. Purified membrane fractions (1 mg/ml) prepared from the cells of& polymyxa at their midlog phase of growth (120 Klett units) were treated with different concentrations of polymyxin at 0°C for 1 min. The DAB activation of the treated membranes was assayed in the presence of polymyxin.
3 shows that this release was also a concen- tration-dependent phenomenon. At higher concentrations of polymyxin, where its surfactant property was manifested, the enzyme activity was lost, although more proteins were released.
Specijkity of Polymyxin Effect
In order to see if the release of membrane- bound DAB-activating enzyme activity was
FIG. 3. Release and enhancement of the membrane- bound DAB-activating enzyme by polymyxin. Isolated membrane fractions from cells ofB. polymyxa at their midlog phase of growth were treated with different concentrations of polymyxin and after 1 h at 0°C the membranes were resedimented. The DAB activation and protein concentrations in the supernatants were determined. 0, DAB activation/mg protein of the supernatant; 0, pg protein released/mg membrane protein.
48 BALAKRISHNAN ET AL.
TABLE I
EFFECT OF POLYMYXIN, POLYAMINES, AND DETERGENTS ON THE RELEASE OF MEMBRANE-BOUND DAB-ACTIVATING ENZYME”
DAB activation in
Addition
Membrane Supematant
Total Specific Total Specific activity activity activity activity
a Membrane fractions, prepared from the midlog phase cells of B. poolymyxa 2459, were treated with the various agents at concentrations indicated. After an hour at 0°C the membranes were spun down, and the DAB activation was measured both in total membranes and supematants obtained. Enzyme activities are expressed as nmol of ATP exchanged with [“PIP, dependent on added DAB/mg protein.
b Not detectable.
a specific property of polymyxin, the purified membranes were treated with DAB, putres- tine, and spermidine. These compounds were chosen for their basic properties and could be expected to simulate polymyxin. As seen from the results outlined in Table I, only polymyxin was effective in the release of the membrane-bound activity and also stimulated both the membrane-bound and the membrane-released enzyme activities. This stimulation was never observed with any of the other compounds tested. Actually, putrescine and spermidine rather inhibited the DAB activation, and only a small amount of the membrane-bound enzyme was re- leased. Variation of the concentrations of these polyamines had no effect (data not shown). The release of DAB activation from the membranes could also be accomplished, albeit to a smaller extent, with buffers devoid of Mg2+ (Table I).
To test the possibility that the surfactant nature of polymyxin could have mediated the release of the membrane proteins, other surfactants like Brij 35 and Triton X-100 were used. Although a considerable amount of protein was released from the membranes
by these agents, the DAB-activating enzyme activity was totally lost in their presence (Table I).
Further evidence for the specificity of polymyxin effect was obtained from the dialysis experiments. When polymyxin- treated membranes or the soluble fractions from the stationary phase cells were dialyzed free of the added polymyxin, the DAB- activating enzyme activity was considerably reduced. Readdition of polymyxin, or dialy- sis in presence of polymyxin in the buffer restored the activity (Table II).
Release of Multienzyme Complex from Membranes by Polymyxin
When membranes were treated with polymyxin, we observed the release of all the polymyxin component amino acid-activat- ing enzyme activities in addition to DAB- activating enzyme activity (Table III). The presence of these enzyme activities in the membrane-released fractions was not due to the contamination from the soluble fraction, as revealed by the absence of activation of the noncomponent amino acids such as L-
POLYMYXIN BIOSYNTHESIS: PHYSIOLOGICAL ROLE 49
Val and L-Ala. Like for DAB, the activation of other component amino acids was also en- hanced by the addition of polymyxin.
We can then assume that it was the as- sociation of the multienzyme complex with polymyxin, that was responsible for the observed stimulation of the activity of the complex.
Nature of the Multienzyme Complex involved in Polymyxin Synthesis
In order to understand the nature of the two forms of the multienzyme complex en- countered in the cells, the complex released from the membranes and the soluble form were fractionated on Sepharose 4B columns. The fractions were monitored for DAB activation and were counter-checked for other polymyxin component amino acid ac- tivation and bioassayable polymyxin.
As seen in Fig. 4A the multienzyme complex released from membranes by poly- myxin was made of two species: Frac- tion I contained DAB-, L-Leu-, L-Phe-, and L-Thr-activating enzyme activities and was eluted in the molecular weight range of 300,000. Fraction II had low DAB activation but comparable L-Thr, L-Phe activation and high L-Leu activation and was eluted in the molecular weight range of 180,000.
As seen from the figure, only fraction I had bioassayable polymyxin. It may be added here that at this stage only the membrane- associated multienzyme complex was present in the cells.
Since high levels of the DAB-activating enzyme was present in the soluble extracts of the cells at later stages of growth, the
TABLE II
EFFECT OF POLYMYXIN ON DAB-ACTIVATING ENZYME OF MEMBRANE AND SOLUBLE
FRACTIONS OF B. polymyxa”
DAB activation of
Treatment Membrane Soluble
fraction fraction
Nil Dialysis Dialysis followed by
1256 100 625 50
readdition of polymyxin (400 unitslmg protein) 1250 150
a Membrane and soluble fractions from late logarith- mic cells of B. polymyxa were used. Isolated mem- brane fractions and the soluble proteins were dialyzed against TKM buffer. DAB activation was assayed in the dialysates before and after readdition of poly- myxin (400 units/mg protein). Enzyme activities are expressed as indicated in Table I.
soluble fractions were also fractionated on a similar Sepharose 4B column (Fig. 4B). It was observed that the fractions harboring DAB activation also contained other compo- nent amino acid-activating enzymes. More- over, the fractionation profiles of the soluble multienzyme complex was largely identical to the complex released from membranes by polymyxin. Once again, the fraction I of the complex had polymyxin associated with it (Fig. 4B).
It was mentioned earlier (Table I) that a small amount of the multienzyme complex could be released from the membranes by buffers devoid of Mg2+. On the other hand, it has been known that Mg2+ is essential for
TABLE III
RELEASE OF THE AMINO ACIDACTIVATING ENZYMES FROM THE MEMBRANES"
Activation of Treatment of membranes DAB L-Leu LThr L-Phe L-Ala L-Val
d Membrane fractions were prepared from midlog phase cells. Amino acid activations of the soluble fiat- tions obtained after treatment of the membranes as indicated, are expressed as in Table I.
BALAKRISHNAN ET AL.
FRACTION NUYl2R
FIG. 4. Sepharose 4B column fractionation profiles of the polymyxin biosynthetic complex from. (A) Poly- myxin released proteins from membranes; (B) soluble extracts of cells at stationary phase; (C) TK buffer released proteins &om the membranes. The various extracts concentrated to 10 mg protein/ml, were loaded on a Sepharose 4B column (40 x 1 cm) and were separated by developing the columns with TKM (A
membrane-associated DAB activation (8). These results suggested that the association of the multienzyme proteins with the mem- branes was maintained by ionic forces and required the presence of Mg2+ for function. We therefore fractionated the proteins re- leased from membranes by TK buffer on a Sepharose 4B column and the fractions were monitored for DAB and other component amino acid activation in presence of added Mgz+. As seen from Fig. 4C several species of the multienzyme complex were eluted from this column, although the specific activities of these materials were much lower when compared to the materials released by poly- myxin (Fig. 4A). It must also be mentioned that the addition of Mg2+ or polymyxin to the materials released by TK buffer did not restore the pattern observed with the poly- myxin-released multienzyme complex nor was any polymyxin detected in any of the column fractions.
Subunit Nature of the Polymyxin Biosynthetic Complex
Electrophoretic analysis of fractions I and II of the materials released from mem- branes by polymyxin is shown in Fig. 5. In the native gels, the two fractions aggregated and appeared as a single band, while in SDS-gels, at least six major bands were de- tectable. These results confirmed the multi- subunit nature of the polymyxin synthetic complex.
Effect of Polymyxin on Its Biosynthetic Complex in Asporogenic Mutants of B. polymyxa
Asporogenic mutants of B. polymyxa, blocked at stage 0 of sporulation, do not produce extracellular polymyxin (4). But mutants that were blocked after this stage
and B)/TK (C) buffer containing 20% glycerol. The individual fractions (2 ml) were monitored for DAB activation to begin with and were counter checked for activation of other component amino acid activation and bioassayable polymyxin (only the presence and absence was scored). 0 - l , DAB; 0 - - - 0, Leu; 0 - 0, Thr; 0 - - - 0, Phe; q , polymyxin.
POLYMYXIN BIOSYNTHESIS: PHYSIOLOGICAL ROLE
FIG. 5. Native and SDS-acrylamide gel electrophoresis of polymyxin-released proteins from membranes of B. polymyxa. The fractions I and II from the Sepharose column (Fig. 4A) of the materials released from membranes by polymyxin were pooled (50- 100 Fg protein/slot) and analysed in: (a) 5% native acrylamide tube gel; (b) 10% SDS-acrylamide slab gel.
do produce normal levels of polymyxin (4). In one of the latter class of mutants, NRS 12, we observed a highly reduced (only 25% of the wild type) production of the antibiotic (Table IV). As shown in this table, although the levels of the membrane-bound polymyxin component amino acid activation of this mutant was normal, in contrast to the wild type, there was less delocalization of the complex from membranes by polymyxin. In another mutant, NRS 18, blocked at late stage of sporulation, the antibiotic produc- tion was normal and the solubilization of the membrane associated complex of poly- myxin was compared to the wild type (Table IV).
A comparison of the membrane and soluble DAB activation during growth and sporula- tion in the wild type and the mutants is outlined in Table V. It is evident that in the
low antibiotic producing mutant, NRS 12 (Table IV), the soluble DAB activation at the stationary phase was considerably less, although its membrane-bound activity during growth was normal. The mutant NRS 18 with its normal level of antibiotic production showed high levels of DAB activation in the soluble extracts of the stationary phase cells, similar to the wild type. These latter results indicate that the delocalization of the multi- enzyme complex from the membranes was a relevant physiological phenomenon re- quired for higher antibiotic production.
DISCUSSION
Synthesis of polymyxin, which reaches a maximum at the stationary stage of growth in B. polymyxa can be governed by:
(a) the availability of the precursor amino
BALAKRISHNAN ET AL.
TABLE IV
EFFECT OF POLYMYXIN ON THE MEMBRANES ISOLATED FROM WILD TYPE
(1 Purified membrane fractions of the strains were prepared from the cultures of midlog phase of growth as outlined under Materials and Methods. The membranes were treated with 400 units of polymyxin/mg protein and incubated at 0°C for 60 min and centrifuged at 16,000 rpm for 30 min. The membranes and supernatants were assayed for the amino acid activations. The values are expressed in nmol of ATP exchanged with [“P]Plmg protein.
acids, facilitated by the slow down of protein synthesis during the stationary phase and
(b) the induction of the biosynthetic machinery during the stationary phase or an enhancement of this machinery that was already present during the vegetative phase of growth.
Our studies have established that the polymyxin synthesizing enzyme complex was located in the membrane fractions of the cells and can be functional even at the
vegetative phase of growth in the presence of chloramphenicol. We have further shown that when cells at the preearly stage of antibiotic synthesis were treated with poly- myxin, there was a sharp increase in the membrane-associated DAB-activating en- zyme activity, possibly by simulating the in vivo conditions observed at the later stage of growth (Fig. 1). This increase of activation of DAB was also demonstrated with isolated membrane preparations (Fig.
TABLE V
DISTRIBUTION OF DAB-ACTIVATING ENZYME ACTIVITY IN THE MEMBRANE AND SOLUBLE FRACTIONS
AT VEGATATIVE AND SPORULATING STAGES OF B. polymyxa 2459 AND ITS MUTANTS
a Represented as nmol ATP-[3*P]P exchange/mg protein. The cultures were grown in K medium and harvested at midlog and sporulating phase (tz). Membrane and soluble fractions were prepared from these cells as described under Materials and Methods and were assayed for DAB activation as outlined in Table I.
b Not detectable.
POLYMYXIN BIOSYNTHESIS: PHYSIOLOGICAL ROLE 53
TIME IN HOURS AFTER POLYMYXIN
ADDITION
FIG. 6. Effect of addition of polymyxin to the vegetative cells of B. polynzyza wild-type strain and one of its Rif’, SpoO, Ab- mutant, RSO 5. Polymyxin at indicated concentration was added to cells grown up to an initial Klett of 30 (arrow indicates time of addition) and their growth pattern was observed for 5 h at half-hourly intervals. 0, wild type; 0, RSO 5.
Z), and it was shown to be due to the release of DAB and other polymyxin component amino acid-activating enzymes from the membranes by polymyxin. Moreover, the interaction of polymyxin with its biosyn- thetic complex is specific as revealed by the inability of other polyamines or cationic detergents to mimic this effect (Table I).
The multienzyme complex solubilized by polymyxin from the membranes has a high molecular weight range of 180,000 to 300,000 as determined by Sepharose 4B chromatog- raphy (Fig. 4A). The membrane-bound and soluble forms of this complex are similar if not identical (Fig. 4B). The multienzyme complex appears to be the major component of the proteins released from the membrane by polymyxin, as there occurs considerable
enhancement of the specific activity of the component enzymes (Table I). The single band native gel profile of the complex after purification on Sepharose 4B column strength- ens this contention. On electrophoresis under dissociation conditions, the purified complex appears to be composed of at least six polypeptides (Fig. 5).
High levels of DAB activation in the soluble form in the wild type is accompanied by a concomitant increase of extracellular polymyxin. In the asporogenic mutant (NRSE) in which the production of poly- myxin was decreased, such an increase was not observed, indicating a correlation be- tween the appearance of soluble form of the multienzyme complex and active antibiotic synthesis.
54 BALAKRISHNAN ET AL.
Inhibition of vegetative growth of the producer organism by different antibiotics is well known (9, 11, 12). We also have re- ported (10) that the cells of B. polymyxa are sensitive to polymyxin during the early log phase of growth and became resistant to the antibiotic at the later stages of growth (Fig. 6). This can now be explained by the development of the ability of the organism for an effective interaction with polymyxin. The gradual rise in the membrane-bound biosynthetic complex during growth, prior to its appearance in the soluble fraction, indicated that this complex was specifically involved in the interaction with polymyxin. At this stage, in which the resistance to polymyxin was also maximal, there was considerable increase in intracellular poly- myxin, 50% of which was present in the protein-bound form (Table 1 of (6)).
In the case of Ab- mutants, deficient in the biosynthetic machinery addition of polymyxin resulted in an immediate lysis of the cells (Fig. 6). These mutants possibly do not possess the specific sites for interac- tion with polymyxin. Since it has been shown (5, 6) that these mutants are pleiotropically regulated for both sporulation and antibiotic expressions, it can then be assumed that membrane interaction with the antibiotic is of relevance to both processes of the producer organism.
This observation with B. polymyxa is in general agreement with other studies re- ported in Bacilli, which demonstrated that mutations in SpoO loci result in pleiotropic effects such as loss of antibiotic production and early sporulation function (13, 14). It has also been shown that revertants of these mutants selected for Spa+ traits are also resistant to membrane specific antibiotics (15, 16).
The results of our investigations described here assign a specific role for the peptide antibiotic polymyxin, in the producer organ- ism by its interaction with the membranes, resulting in the development of resistance toward its own antibiotic. A direct implica-
tion of these studies is that for the success of selection of mutants for high antibiotic production it must be combined with muta- tions that confer resistance to the same antibiotic.
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