MICROCYSTS OF MYXOCOCCUS XANTHUS

Transcription

1 JOURNAL OF BACTERIOLOGY Vol. 87, No. 2, p February, 1964 Copyright 1964 by the American Society for Microbiology Printed in U.S.A. ELECTRON TRANSPORT SYSTEM IN VEGETATIVE CELLS AND MICROCYSTS OF MYXOCOCCUS XANTHUS MARTIN DWORKIN AND DONALD J. NIEDERPRUEM Department of Microbiology, University of Minnesota, Minneapolis, Minnesota, and Department of Microbiology, Indiana University Medical Center, Indianapolis, Indiana Received for publication 29 August 1963 ABSTRACT DWORKIN, MARTIN (University of Minnesota, Minneapolis), AND DONALD J. NIEDERPRUEM. Electron transport system in vegetative cells and microcysts of Myxococcus xanthus. J. Bacteriol. 87: Respiration by intact cells of the fruiting myxobacterium Myxococcus xanthus is cyanide-sensitive and can be demonstrated in the vegetative cells but not in the microcysts. Cellfree particles from both vegetative cells and microcysts have cyanide-sensitive reduced nicotinamide adenine dinucleotide (NADH) oxidase, diaphorase, NADH cytochrome c reductase, and cytochrome oxidase activities. While the vegetative cell specific activities for NADH oxidase and diaphorase are slightly higher than those for the microcysts, the microcysts have ten times the cytochrome c reductase and cytochrome oxidase activities of the vegetative cells. Furthermore, the respiration of the microcyst particles is considerably less cyanide-sensitive than is that of the vegetative-cell particles. Difference spectra of the cellfree particles of vegetative cells and microcysts are qualitatively identical, showing the presence of b- and c-type cytochrome and flavoprotein. The a-type pigments are clearly present in the extracts of the vegetative cells and are suggested by the spectrum of the microcyst particles. The cytochrome oxidase activity of both extracts is consistent with the presence of a-type pigments in both. The spectra of the carbon monoxide-binding pigments were determined and, by this parameter, qualitative differences appear between the vegetative cells and the microcysts. A study of morphogenesis leads inevitably to the problem of the regulation of metabolic events. When the morphogenetic process can be described in biochemical terms, hypotheses of regulation based on mechanisms of control of enzyme activity or biosynthesis become a possibility. During the life cycle of the fruiting myxobacterium, Myxococcus xanthus, vegetative rods convert to round refractile microcysts. As part of a program to describe this cellular morphogenesis in biochemical terms, we have examined and compared some components of the electron-transport system in vegetative cells and microcysts of M. xanthus. MATERIALS AND METHODS Organism. M. xanthus FB was used for all the investigations to be described. This is a dispersedgrowing strain, and was described in previous publications (Dworkin, 1962; Voelz and Dworkin, 1962). Cultivation of vegetative cells. The culture was maintained as microcysts on CA medium and cultivated on CT (Casitone) medium as described in an earlier report (Dworkin, 1962). For the preparation of large quantities of vegetative cells, the organism was cultivated for 30 hr in Fernbach flasks containing 500 ml of CT medium. Incubation was at 30 C with shaking. The final Klett reading (no. 54 filter) was usually about 1,000 (approximately 2 X 109 cells per ml), at which point the cells were in the early stationary phase of the growth curve. Cultivation of microcysts. Under the proper nutritional conditions, vegetative cells will convert to microcysts. Optimally, these conditions involve starvation of the cells prior to placing them on a medium lacking or low in certain essential nutrients (Dworkin, 1963). Vegetative cells were grown in liquid CT culture, harvested, and resuspended to a Klett reading of about 100 in a buffer consisting of 0.1% MgSO4 and 0.01 M potassium phosphate, ph 7.6 (Mg-P). After about 18 hr of incubation at 30 C with shaking, the cells were harvested and resuspended in Mg-P to a concentration of about 5 X 109 cells per ml; 1 ml was pipetted over the surface of a medium containing 0.05% Tryptone, Mg-P, and 1 % Oxoid agar No. 3. Aluminum pie plates (24 cm in diameter) covered with aluminum foil were used in the place of petri dishes. After about 10 days of incubation at 30 C, the plates were covered with fruiting bodies, and the majority of vegetative cells had 316

2 VOL. 87, 1964 ELECTRON TRANSPORT IN M. XANTHUS converted to microcysts. The microcysts were washed off the plates with distilled water, and the suspension of vegetative cells and microcysts was centrifuged. The microcysts were separated from the vegetative cells by exposing the suspension to sonic vibration for 5 min with a 20-kc MSE sonic oscillator. This completely destroyed the vegetative cells but had no visible effect on the microcysts. The debris was removed by repeated recentrifugation of the pellet at 3,000 X g for 15 min. After about five such centrifugations, the pellet consisted almost solely of microcysts. The yield of microcysts was about 150 mg (wet weight) per plate. Viable counts performed on the microcyst suspension indicated about 85% plating efficiency. Preparation of cell-free extracts of vegetative cells. Cells from 1.5 liters of medium were harvested by centrifugation. This usually yielded about 10 g (wet weight) of cells. The cells were washed in a mixture of Mg-P, resuspended in 40 ml of 0.02 M phosphate buffer (ph 7.0), and then disrupted by one of three methods. (i) The cell suspension was passed through a French pressure cell at a pressure of 10,000 psi. (ii) The cells were disrupted in a Mini-Mill (Gifford-Wood Co., Hudson, N.Y.) as follows: 1 g of cells suspended in 10 ml of phosphate buffer was mixed with 10 ml of glass beads (acid-washed) 85 A in diameter; the mixture was ground in the Mini-Mill for 5 min, and the glass beads were then removed by filtration through Whatman no. 1 filter paper. (iii) Mill as described above and was then exposed to sonic vibration for 1 min in a 20-kc MSE sonic oscillator. All the above manipulations were performed at 3 to 7 C. In all cases, the suspension of disrupted cells was centrifuged at 6,000 X g for 10 min to remove intact cells and large debris. The supernatant fluid was then centrifuged at 144,000 x gma, for 90 min at 4 C. The resultant pellet was resuspended in 6 ml of phosphate buffer (0.02 M, ph 7.0) and represented the particulate fraction. The final supernatant fraction was also retained. Preparation of cell-free extracts of microcysts. The microcysts were resistant to breakage with a French pressure cell or by exposure to sonic vibration for 30 min. They were, however, effectively fragmented by 15-min treatment with the Mini- Mill, followed by exposure to sonic vibration for 1 min. This combination of breakage of the microcysts with the Mini-Mill followed by a brief The suspension was treated with the Mini- 317 period of sonic vibration proved to be quite effective. Comparisons of the activity of extracts of vegetative cells prepared by French pressure cell, Mini-Mill, and Mini-Mill plus sonic vibration were made and are presented in Results. Enzyme assays. All spectrophotometric measurements of enzymatic activity were made by use of a Zeiss PMQII spectrophotometer with semimicro cuvettes. The total volume of all reaction mixtures was 0.4 ml. The oxidation of reduced nicotinamide adenine dinucleotide (NADH) was measured spectrophotometrically by the decrease in the extinction at 340 m,u. The cuvette contained 0.1,umole of NADH, 0.6,mole of phosphate buffer (ph 7.0), and either mg of vegetative-cell particle protein or 0.09 mg of microcyst particle protein. The NADH cytochrome c reductase was measured by the increase in optical density (OD) at 550 m,u of mammalian cytochrome c in the presence of cyanide. The cuvette contained 0.1,umole of NADH, 75,ug of mammalian cytochrome c (bubbled with air for 5 min prior to use), 1.6 X 10-3 M KCN and either 0.67 mg of vegetative-cell particle protein or 0.09 mg of microcyst particle protein. Cytochrome oxidase was determined by following the decrease in OD at 550 mu of chemically reduced mammalian cytochrome c. The reaction mixture contained 75 MAg of cytochrome c (reduced by adding a few crystals of Na2S204, then bubbled with air for 10 min to remove the excess Na2S204) and either 0.75 mg of vegetative-cell particle protein or 0.09 mg of microcyst particle protein. Diaphorase activity was determined spectrophotometrically by changes in the extinction at 600 m,u, with 2,6-dichlorophenolindophenol (DIP) as the hydrogen acceptor and NADH or succinate as hydrogen donor. The cuvette contained 0.1,umole of NADH or 2,umoles of succinate, 0.02,umole of DIP, 1.6 X 10-3 M KCN, 0.4,umole of phosphate buffer (ph 7.0), and either mg of vegetative-cell particle protein or 0.09 mg of microcyst particle protein. Determinations of oxidase activity of cell-free particles and respiration by intact cells were based on conventional manometric measurements of oxygen uptake at 30 C. Warburg vessels contained 3.15 ml of final reaction mixture buffered with 0.02 M phosphate at ph 7.0. When cyanide was used in manometric experiments, it was prepared as described by Robbie (1948).

3 318 DWORKIN AND NIEDERPRUEM J. BACTERIOL. In all cases where rates of enzyme activity were determined, the concentration of the extract was varied until a rate-limiting concentration was reached. Absorption spectra. Difference spectra were obtained with a Cary recording spectrophotometer equipped with an expanded scale (O to 0.1 OD). The particles which were reduced with Na2S204 were solubilized by the addition of sodium cholate to a final concentration of 1 %. When the particles were enzymatically reduced by the addition of an excess of NADH, they were not solubilized with sodium cholate. For the determination of carbon monoxide-binding pigments, a cuvette containing the particles reduced with Na2S204 was bubbled with carbon monoxide for 30 sec. Protein was determined by the method of Lowry et al. (1951). All determinations of spectra and enzyme activity were performed on the same day that the extract was prepared. z I _ RESULTS Respiration of intact vegetative cells and microcysts. An examination of the respiratory characteristics of the intact vegetative cells of M. xanthus was a necessary preliminary to studies dealing with the enzymatic activities of cell-free extracts. With Casitone as the substrate, the cells had a Qo (uliters of 02 per mg per hr) of 12.0, and this activity was markedly sensitive to cyanide (Fig. 1). The oxygen consumption was inhibited 57% with 10-3 M cyanide, suggesting the involvement of cytochrome components in the normal respiration. It was also apparent that cyanide (10- M) had no detectable effect on the endogenous respiration. Over a 7-hr period, there was no measurable endogenous respiration by the microcysts (Klett reading of the suspension in the flask was 450) nor was there any stimulation of the respiration when 2% Casitone was added. CT KCN 104M CT+KCN 4.6x10-4m TIME IN MINUTES FIG. 1. Oxygen uptake by vegetative cells of Myxococcus xanthus. Each Warburg flask contained 16.0 mg (dry weight) of cells. Casitone to a final concentration of 2% was tipped in from the side arm at 10 min.

4 VOL. 87, 1964 ELECTRON TRANSPORT IN M. XANTHUS 319 Effectiveness of various techniques of disruption of cells. Vegetative cells were easily disrupted by any of a variety of techniques (e.g., sonic vibration, French pressure cell, and grinding in a Mini- Mill). The microcysts, however, resisted disruption by the French pressure cell and sonic vibration (30 min), but were effectively disrupted by 15 min of grinding in a Mini-Mill. Microscopy of the debris revealed that, while the microcysts lose their refractility and are obviously disrupted, the hulls remain essentially unfragmented. Sonic vibration of the hulls for 1 min effectively fragmented the cells. To determine whether this treatment resulted in any drastic loss of enzymatic activity, vegetative cells were disrupted by the French pressure cell, grinding with the Mini-Mill, and grinding with the Mini-Mill followed by 1 min of sonic vibration. The activity of the resultant particulate preparations is shown in Table 1. NADH oxidation, diaphorase (with NADH), and height of the 552- and 425- m,u peaks (reduced with Na2S204) were compared. In all cases, the preparation ground in the Mini- Mill and exposed to sonic vibration for 1 min had a comparable activity with the preparation disrupted by the French pressure cell. We felt confident, therefore, in using this method for preparing both vegetative-cell and microcyst extracts. Activity of cell-free extracts. All of the activities to be compared were determined on preparations obtained by the Mini-Mill-sonic vibration technique. The particulate fraction isolated from vegetative cells of M. xanthus oxidized NADH with the uptake of oxygen and had a Qo, of 29.0 (Table 2). (This rate of oxidation could more than account for the rate of cellular respiration observed.) In addition, the NADH oxidase activity, like the oxygen consumption by intact cells, was markedly sensitive to cyanide (Table 3). Despite the absence of any significant respiration by the intact microcysts, the particulate TABLE 1. fraction isolated from microcysts oxidized NADH with the uptake of oxygen and had a Qo. of 23.0 (Table 2). This activity was inhibited 34% by cyanide (Table 3). The NADH oxidase activity was also measured spectrophotometrically by the oxidation of NADH. Table 4 indicates the activities obtained. The specific activity of the microcyst particles was 36% that of the equivalent vegetative-cell fraction. The supernatant fractions of both the vegetative cells and microcysts also oxidized NADH, but the specific activity was only 11% of the particulate fraction in the case of the vegetative cells, and 39% in the case of the microcyst preparation (Table 4). Both the vegetative-cell and microcyst soluble fractions had approximately equal specific activity. The activity of the supernatant fluid was cyanide-resistant in both preparations. In the particulate fraction prepared from the vegetative cells, diaphorase activity was present with either NADH or succinate as substrate. TABLE 2. Comparison of respiratory activities of vegetative cells, microcysts, and their respective particulate fractions Prepn Qo, Intact cells* Vegetative cells Microcysts... 0 Cell-free particlest Vegetative cells Microcysts * Casitone (2%) as substrate. Each Warburg flask contained 15.0 mg (dry weight) of washed vegetative cells or 7.5 mg (dry weight) of washed microcysts. t NADH (5,imoles) as substrate. Warburg flasks contained either 4.5 mg of vegetative-cell particle protein or 3.5 mg of microcyst particle protein. Comparison of cell-free particles of vegetative cells disrupted by different methods Height of 552-mpA Height of 425 m NADHoxdation Diaphorase with peak (575 mp as "ek(5 p Method Metbod of disruption (OtDs4N per mg of NADH as substrate base) in difference serence bspe fdisrption (&Mw pe mg of (AOD6oo per mg of spectrum. OD increprotein inoitrnc per min) pcrm protein per min) ment (cm-' per g OD increment (cm-s of protein) per g of protein) French pressure cell Mini-Mill Mini-Mill and sonic vibration As ae

5 320 DWORKIN AND NIEDERPRUEM J. BACTERIOL. Diaphorase activity with NADH as the substrate was also present in the particulate fraction from the microcysts. Succinate was not tested as an electron donor with the latter. The presence of cyanide was required to achieve maximal rates in both cases, presumably because of the oxidase activity in the extracts. The microcyst preparation was 58% as active as the vegetative-cell preparation (Table 4). The particulate fractions of both the vegetative cells and microcysts exhibited NADH cytochrome c reductase activity in the presence of 1.4 X 10-3 M KCN. The activity of the microcyst particles was ten times greater than that of the vegetative cells (Table 4). Similarly, both preparations showed cytochrome oxidase activity. Once again, TABLE 3. Effect of cyanide on some activities of vegetative cells, microcysts, and their respective particulate fractions Concn of 10-s me Metabolic activity CoCnN Xo Per Prcn cent Respiration by intact vegetative cells Oxygen uptake by vegetative cell particles with NADH Oxygen uptake by microcyst particles with NADH Oxidation of NADH by particles of vegetative cells Oxidation of NADH by particles of microcysts TABLE 4. Comparison of enzyme activities of vegetative-cell and microcyst preparations,od per mg of protein per min Enzyme system Vegetative Microcyst cell particles particles NADH oxidation Diaphorase NADH as substrate Succinate as substrate 0.32 Cytochrome c reductase Cytochrome oxidase Soluble fraction of vegetative cells Soluble fraction of microcysts NADH oxidation * Not performed. E 0.10 o 0.09 E c Microcyst Particles 0.01 o.ooc1 Ii Wavelength (mp) FIG. 2. Difference spectra (reduced minus oxidized) of cell-free particles of vegetative cells and microcysts of Myxococcus xanthus. Cuvettes (3 ml) contained either 12.0 mg of vegetative cell particles or 2.0 mg of microcyst particles in 0.1 m phosphate buffer (ph 7.0). The particles were solublized with sodium cholate at a final concentration of 1% and were reduced with Na2S204. the microcyst preparation was ten times as active as the vegetative cell preparation (Table 4). Difference spectra. The demonstration of a cyanide-sensitive cellular respiration, as well as NADH oxidase activity in cell-free extracts of vegetative cells of M. xanthus, suggested that a respiratory chain involving cytochrome components was operative. When Na2S204 was used as the reducing agent, the difference spectrum of the particulate fraction of the vegetative cells showed characteristic absorption peaks indicating a-, b-, and c-type cytochromes at 602, 562, and 552 mju, respectively (Fig. 2). A small peak was also apparent at 587 ma. The absorption at 524 m,u resembled the beta bands of b- and c-type cytochromes. The Soret region exhibited a shoulder

6 VOL. 87, 1964 ELECTRON TRANSPORT IN M. XANTHUS 321 at 442 m,i and a main peak at 425 m,a; the former resembled cytochrome a plus a3, and the latter may represent a mixture of b- plus c-type components. The decrease in absorbance at about 460 m,u is indicative of flavoprotein. When NADH was used to reduce the particulate fraction enzymatically, the spectrum was not as well defined as when the particles were reduced with Na2S204. Distinct peaks and shoulders were present, however, at 562, 552, 524, 442, and 425 m,u. This supports further the notion that respiration of vegetative cells of M. xanthus involves cytochrome components. The spectrum of the microcyst particles was qualitatively identical with that of the vegetative cells. Figure 2 illustrates the difference spectrum of the microcyst particles. Absorption peaks are present at 602, 587, 552, 524, 442, and 425 m,u. An attempt was made to delineate further the a-type components by determining the spectra of the CO-binding pigments. The difference spectrum of the vegetative-cell particles (reduced plus CO) - (reduced) is presented in Fig. 3. A marked decrease in absorption was noted at 425, 445, and 552 m,, while new peaks were apparent at 431 and 415 mu. In the microcyst preparation, distinct troughs appeared at 552 and 425 m,u with a new peak appearing at 405 ma (Fig. 3). DIscussIoN Vegetative cells and microcysts of M. xanthus have a functional, classic-type cytochrome system, similar in most respects to that found in many aerobic bacteria (Smith, 1954). Cytochromes of a-, b-, and c-types were tentatively identified, and the nature of the carbon monoxidebinding pigments adds to the evidence suggesting the existence of cytochrome oxidase. The presence of diaphorase activity and the trough at 460 m,i in the difference spectrum suggests the participation of flavine-linked enzymes in the respiratory chain. This must, however, await a more critical examination. It is quite tempting to try to equate the various peaks and troughs of the difference spectra with specific cytochrome components, as was done by Smith (1954). The fact that Smith's data were obtained from living intact cells prompts caution, however, in relating such data to those obtained with Na2S204-treated fragments of disrupted cells. The most significant information to emerge E 0.10h C E,9 Vegetative Cell Particles Microcyst Pdrticles 'Wavelength (mu) FIG. 3. Difference spectra of CO-binding pigments from cell-free particles of vegetative cells and microcysts of Myxococcus xanthus. Cuvettes (8 ml) contained either 12.0 mg of vegetative cell particles or 2.0 mg of microcyst particles in 0.1 m phosphate buffer (ph 7.0). The particles were solublized with sodium cholate at a final concentration of 1% and were reduced with Na2S204. The cuvettes were bubbled with carbon monoxide for 30 sec. from this investigation is that the microcyst has a functional electron-transport system which is fundamentally similar to that of the vegetative cells. Differences do exist, however, and may be summarized as follows. (i) Although intact vegetative cells respire with a Qo2 of 12.0, no oxygen uptake could be detected with the intact microcysts. It is possible that some oxygen uptake might have been detected with a considerably more dense suspension of microcysts. (ii) The particulate fraction of the vegetative cells has slightly higher NADH oxidase and diaphorase activities than does the particulate fraction from the microcysts. Since, however, these are relatively crude preparations, it is unwise to ascribe too much significance to these differences. (iii) Although NADH oxidation by the vegetative-cell particles (measured spectrophotometrically) is inhibited 78% by 1.4 X 10-3 M cyanide, that of the microcyst particles is inhibited only 29%. Since both vegetative cell and microcyst extracts possess a soluble, cyanide-resistant NADH dehydrogenase, the presence of contaminating amounts of this enzyme may be responsible for some of the observed cyanide-resistant activity of the particles.

7 322 DWORKIN AND NIEDERPRUEM J. BACTERIOL (iv) The microcyst particles have ten times the NADH cytochrome c reductase and cytochrome oxidase activities of the vegetative-cell particles. (v) CO-binding peaks appear at 405 m, in the microcyst preparation and at 415 and 431 m,u in the vegetative-cell particle preparation. It is difficult at this point to assess the significance of these differences. Although precautions have been taken to eliminate preparational artifacts, they may still play a role. A recent morphological examination of microcyst formation, by means of electron micrographs of thin sections of cells of M. xanthus (Voelz and Dworkin, 1962), suggested that microcyst formation, in contrast to endospore formation, does not involve extensive breakdown of existing vegetative-cell structures and resynthesis of new microcyst material. It was of considerable interest, therefore, to discover that the respiratory apparatus of the microcyst does not differ in any significant qualitative sense from that of the vegetative cell. We are being led to the conclusion that the myxobacterial microcyst is essentially a round vegetative cell surrounded by a dense capsule. The capsule may be responsible for the enhanced resistance to desiccation, in addition to serving as a barrier to the penetration of nutrients. That such a penetration barrier exists is suggested by the lack of microcyst respiration despite the presence of an active NADH oxidase system. If the vegetative cells and microcysts differ principally by virtue of their shape and the presence or absence of a capsule, the cellular morphogenesis may primarily involve the nature of the cell wall and, therefore, becomes considerably simplified. ACKNOWLEDGMENT This work was supported, in part, by research grant NSFGB9 from the Developmental Biology Program of the National Science Foundation. The senior author is a U.S. Public Health Service research career awardee (GM-(1-K3) ) of the National Institute of General Medical Sciences. LITERATURE CITED DWORKIN, M Nutritional requirements for vegetative growth of Myxococcus xanthus. J. Bacteriol. 84: DWORKIN, M Nutritional regulation of morphogenesis in Myxococcus xanthus. J. Bacteriol. 86: LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: ROBBIE, W. A Use of cyanide in tissue respiration studies. Methods Med. Res. 1: SMITH, L Bacterial cytochromes. Difference spectra. Arch. Biochem. Biophys. 50: VOELZ, H., AND M. DWORKIN Fine structure of Myxococcus xanthus during morphogenesis J. Bacteriol. 84: