JOURNAL OF BACTERIOLOGY, Jan. 1971, p. 185 189 Vol. 105, No.Copyright 1971 American Society lor Microbiology Printed in U.S.A.

Physiological Studies of Encystment inAzotobacter vinelandiilH. L. SADOFF, E. BERKE,2 AND B. LOPERFIDO

Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan

Received for publication 3 August 1970

Azotobacter vinelandii, in late exponential growth phase, encysts when the glu-cose in the medium is replaced with j.-hydroxybutyrate. A final cell division thenoccurs without apparent deoxyribonucleic acid (DNA) synthesis, resulting in a re-duction from two to one nucleoids per cell and a final DNA content of 3.2 x 10-14g per cell. This is also the DNA content per cyst. A f3-hydroxybutyrate dehydroge-nase is derepressed by the addition of the inducer and is identical to the enzyme inacetate-grown cells in its pH optimum, Michaelis constant for substrate, tempera-ture-activity response, and mobility during electrophoresis in acrylamide gel.

Cysts of Azotobacter vinelandii are restingcells which are analogous to endospores of bacilliin that they are considerably more resistant thanvegetative cells to deleterious physical and chem-ical agents (17). Cultures of this organism maybe induced to encystment by growth on agarplates of Burk's nitrogen-free medium supple-mented with 0.2% n-butyl alcohol as the carbonsource ( 18, 23). Alternatively, cultures may begrown to late exponential phase in liquid mediumwith glucose as the carbon source; the cells arethen induced to encystment by the replacement ofthe glucose with either p-hydroxybutyrate (BHB)or crotonate (9). This two-step process can becarried out in relatively large culture volumesand has been applicable to the study of the timecourse of this unique cellular differentiationprocess at the physiological (9) or fine structurallevel (6). We have been concerned with the bio-chemical events which occur upon the inductionof encystment and have examined the immediateeffects of the addition of BHB on cell growth,cell division, and enzyme induction.

MATERIALS AND METHODS

Strain and cultivation. A. vinelandii ATCC 12837,which was used throughout these experiments, wascultivated in Burk's nitrogen-free medium (22) at 30 C.In the two-step method for cyst production (9), cultureswere grown to late exponential phase (2 x 108 cells/ml)with 1% glucose as the carbon source. The cells werecentrifuged from the medium aseptically, suspended insterile Burk's medium with 0.2% BHB as the carbon

Journal article 5177 Irom the Agricultural Experiment Station,Michigan State University.

2 Public Health Service predoctoral trainee, grant GM-0191 1.

source, and incubated with aeration at 30 C. Aqueoussolutions of both glucose and BHB (sodium salt) weresterilized separately by autoclaving.

Preparation of cell extracts. Vegetative and encyst-ing cells of A. vinelandii were suspended in 0.05 M tris(hydroxymethyl)aminomethane (Tris) butter (pH 7.5)containing 10 -3 M ethylenediaminetetraacetic acid(EDTA), and were then disrupted in a sonic oscil-lator (Measuring & Scientific Equipment, Ltd.). Thechelating agent (EDTA) was necessary to achieve rapidand reproducible cell rupture.

Analytical procedures. The nitrogen content of vege-tative and encysting cells was determined by the micro-Kjeldahl analysis (19). Deoxyribonucleic acid (DNA)was determined by the method ot Burton (2). Cellswere extracted with 10% trichloroacetic acid at 95 Cfor 15 min, and the deoxynucleotides were assayed byusing highly polymerized calf thymus DNA as a stand-ard. Proteins were estimated spectrophotometrically(20). CQmpounds containing amino sugars were assayedby the Rondle and Morgan (15) procedure with gluco-samine as the standard. The D-13-hydroxybutyric aciddehydrogenase of encysting cells was assayed by theprocedure of Jurtshuk et al. (7). The rate of oxidationof BHB was equal to the rate of reduction of nicotina-mide adenine dinucleotide (NAD), measured at 340nm. One unit of enzyme oxidized I Amole ot substrateper min at 37 C, and specific activity was expressed inunits ot' protein per mg.

Photomicroscopy. Time-lapse phase-contrast photo-microscopy of encysting A. vinelandii was carried outwith a Zeiss Photomicroscope by using slide culturesof the organism on Burk's nitrogen-free agar mediumcontaining 0.2% BHB. Nuclear staining ot'A. vinelandiiat various stages in its life cycle was performed by theprocedures of Piekarski (14). Direct cell counts, whichwere made with a Petroff-Hauser counting chamber,corresponded well with viable counts obtained by stand-ard plating techniques.

Gel electrophoresis. The relative mobilities of BHB

185

on June 21, 2017 by guesthttp://jb.asm

.org/D

ownloaded from

SADOFF, BERKE, AND LOPERFIDO

dehydrogenases in acrylamide gel electrophoresis atpH 8.3 and 4 C were tested by the method of Davis(4). Vertical gels of 5% Cyanogum 41 (10 by 20 by 0.3cm) were prepared in a vertical gel electrophoresis cell(E-C Apparatus Corp., Philadelphia, Pa.). Tris-glycinebutter (pH 8.3) was employed, and a constant currentot 6 ma was maintained. Bromophenol blue, the anionicmarker, migrated to within I cm of the anionic end otthe gel in 1.75 hr. The BHB dehydrogenases werestained specitically by coupling nitroblue tetrazoliumreduction to the oxidation of the substrate ( 16).

RESULTSGrowth and encystment. The two-step encyst-

ment procedure (9) was scaled-up to a 3-litervolume in the following manner. Cultures of A.vinelandii were grown in 100-ml volumes ofBurk's medium (1% glucose) per 500-ml Erlen-meyer flask. When a cell concentration of 2 x 108per ml was achieved (approximately 18 hr at 30C with shaking), 200 ml of culture was intro-duced into 2,800 ml of the same medium at 30 Cin a 3-liter New Brunswick fermentor. The cul-ture was stirred at 400 rev/min and aerated at3 liters/min. Samples of this culture were takenat intervals for analyses of turbidity, cell count,total nitrogen, DNA, and BHB dehydrogenase.When the cell concentration reached 2 x 108 perml, the cells were harvested by centrifugationand suspended in fresh Burk's nitrogen-free me-dium containing 0.2% BHB. Stirring, aeration,and sampling were continued through the initialstages of encystment.

Figure I presents growth data for A. vinelandiiunder the conditions described. After the induc-tion of encystment by BHB, the culture appearedto undergo one cell doubling. This was apparentfrom both the turbidity and total nitrogen dataand was substantiated by time-lapse phase-con-trast micrography (Fig. 2). A cluster of six vege-tative cells on a slide culture was observed overa period of 8 hr. These cells had been grown inliquid Burk's nitrogen-free medium containingglucose. They were washed once in buffer andspread over the BHB-containing medium. Theinitiation of cell division was readily apparent at2.5 and 3.5 hr. At 8 hr, five of the cells had di-vided, and their progeny appeared to be in theearly stages of encystment. At that time, thesixth cell was in its final division.Concomitant with the last cell division, an ar-

rest of nitrogen fixation occurred. The total nitro-gen of the culture remained constant with ap-proximately 95% in the cellular material. DNAsynthesis stopped at the time of the last cell divi-sion. The DNA content per cell dropped from15 x 10-14 g per cell in exponentially growingcultures to 3.4 x 10-14 g per encysting cell (Fig.

1.00.8O.6

0W0Q40

TIME- HOURSFIG. 1. Growth curve of A. vinelandii in Burk's ni-

trogen-free medium containing 1% glucose, in whichturbidity (optical density at 600 nm) or micrograms ofnitrogen per milliliter are plotted as functions of time.Cells in late exponential growth were induced to enc.vst-ment with 0.2% BHB with subsequent inhibition of ni-trogen fixation and cell growth.

3). The latter value is precisely the DNA con-tent per cyst of A. vinelandii. Nuclear stainingshowed that cells in early exponential growthhad as many as four nucleoids, whereas late ex-ponential-phase cells had two. Cysts containedonly one nucleoid.An alternative procedure for initial extraction

(3) and then DNA analysis was performed tovalidate the observations of DNA content percell. A 100-ml culture of A. vinelandii was grownin Burk's medium (1% glucose) to a concentra-tion of 1.8 x 108 cells/ml. The culture waschilled to 4 C; the cells were harvested, washedin 0.1 M Tris-0.1 M NaCl buffer (pH 9.0), andresuspended in 18 ml of the same butfer. Twomilliliters of cold 10% sodium dodecyl sultfatewas added, and the suspension was stirred for20 min in an ice bath. An equal volume (20 ml)of water-saturated phenol was added, and themixture was slowly stirred for an additional 20min. The aqueous and phenol layers were sepa-rated by centrifugation, and the DNA in theaqueous layer was precipitated by the addition ofthree volumes of ethanol. The DNA precipitateswere collected and suspended in 0.1 M NaCI. Thetotal DNA recovered from 1.8 x 1010 cells was840 ug, amounting to 4.7 x 10-14 g/cell. Theamino sugar content of the DNA preparationwas used as a measure of its contamination withcell envelope constituents (21). The DNA prepa-ration contained an equivalent of 25 ,g of glu-

186 J. B ACTl RIOI..

on June 21, 2017 by guesthttp://jb.asm

.org/D

ownloaded from

ENCYSTMENT IN A. VINELANDII

FIG. 2. Time-lapse, bright phase-contrast photomicrographs of encysting A. vinelandii. Cells were grown to lateexponential growth, and slide cultures were prepared on Burk's agar containing 0.2% BHB. The marker indicates Snm.

cosamine whereas the supernatant solution con-tained 1860 jig of glucosamine.The addition of BHB to late exponentially

growing cultures of A. vinelandii resulted in theinduction of a soluble fl-hydroxybutyric aciddehydrogenase which was NAD dependent. Thisenzyme was not found in young cells; growing onglucose however, within 1 hr of the addition ofBHB, a specific activity of 0.03 units/mg of pro-tein was noted in cell extracts (Table 1). Be-cause of the relationship of BHB metabolism toencystment, we considered the possibility thatthe dehydrogenase was a unique "encystmentenzyme" analogous to several which occur incells with the onset of sporulation in Bacillusspecies (1, 5). A soluble BHB dehydrogenasehas been studied in cells of A. vinelandii grownon acetate as the sole source of carbon and en-ergy (7). We grew 3-liter cultures of the organismin Burk's nitrogen-free medium containing 1%sodium acetate, harvested the cells, and preparedextracts. The specific activity of the BHB dehy-drogenase was 0.025 units/mg. This enzyme wascompared to that occurring during encystment byobserving enzyme activities over the temperaturerange 26 to 48 C, pH responses, Michaelis con-stants for substrate, and relative electrophoreticmobilities in acrylamide gels. The results of the

20

w

za

0

16 .

121-

4

0

9.0

-J

8.6 "..

-J

8.2 w

0

7.8 J

74

-10 0 10 20 30TIME-HOURS

FIG. 3. Cell concentration and DNA content per cellof encysting A. vinelandii as a function of time.

comparisons are presented in Fig. 4 and Table 2and indicate that the two enzymes are very simi-lar.

DISCUSSIONThe replacement of glucose with BHB in the

two-step induction of encystment is similar in

I

BHB

187VOL. 105, 1971

8

on June 21, 2017 by guesthttp://jb.asm

.org/D

ownloaded from

SADOFF, BERKE, AND LOPERFIDO

TABLE 1. Specific activity of 13-hydroxybutyratedehydrogenase upon induction ofencystment of

Azotobacter vinelandii

Time after addition Specific activityof BHB (hr) (units/mg)

0 01 0.0301.5 0.0272 0.0233 0.0234 0.021

many respects to a metabolic shift-down (8). Atthe time of its replacement by BHB, about 50%of the original glucose still remained in the me-dium (9). These cells must have shifted from car-bohydrate metabolism to lipid metabolism with theattendant need for gluconeogenesis. Marcromolec-ular synthesis and cell division which was in pro-gress at the time of the induction of encystmentappeared to be completed without the initiation ofnew rounds. Control cultures which were main-tained in the glucose-containing medium continuedto divide and achieved final population levels of1.1 i 109 cells/ml. Approximately 1% of thesecells formed cysts.The total nitrogen in the culture increased 1.25-

fold after induction of encystment, and the totalcell count increased 1.4-fold, a value consistentwith the 1.8-fold increase in optical density. A re-

duction in the DNA content per cell was achievedin this process, the final level equaling the DNAcontent of mature cysts. A derepression of BHBdehydrogenase synthesis occurred, and relativelyhigh levels of the enzyme were present in cellswithin 1 hr of induction of encystment. The spec-ific activity of this enzyme during encystment wasequal to that in cells grown on acetate. A com-plete cessation of nitrogen fixation took place ap-proximately 3 hr after the addition of BHB.The shift-down per se did not induce encyst-

ment. This metabolic state occurs after glucoseexhaustion from the medium; however, underthese conditions, relatively few cysts are produced(9). The specificity of the inducers of encystment,(n-butanol, crotonate, or BHB) suggest thatunique metabolic sequences may be involved incyst formation. In some unknown manner, themetabolites of n-butanol, which stop normalgrowth, control the formation of cyst components.Even though key intermediates may exist, theymust be transitory because ultimately 90% of allexogenously added BHB is oxidized to CO2 (9).

The BHB dehydrogenase which was elaboratedduring encystment was identical to the enzymewhich occurred in A. vinlandii during vegetativegrowth on acetate on the basis of the following

parameters. The two soluble dehydrogenases wereindistinguishable in their pH optima, Michaelisconstants, electrophoretic mobilities in acrylamidegels, and in their activation energies. We concludethat BHB dehydrogenase is not an encystment-specific enzyme in the sense that protease (1) orglucose dehydrogenase (5) is related to thesporulation of Bacillus cereus. Rather, the en-zyme is induced when the cells shift to the me-tabolism of lipid materials.The DNA content per cell of A. vinelandii var-

ied with the stages of its life cycle. Cells in mid-exponential growth contained 15 x l0 14 g ofDNA/cell and were multinucleate. During en-cystment a minimal value of 3.4 x 10-14 g of

DNA/cell was observed for mononucleate cells.This latter value amounts to about 2.5% of thedry weight of the encysting cells, assuming thatthey contain 8% nitrogen. The reduction in nucle-oids per cell after exponential growth has beenobserved in other bacteria and in metabolic shift-down experiments in which cell growth rate is al-tered (8, 10). Cysts contain approximately 10 times

C/)

N

zw

0.10

0.05

0.02

0.01300 310 320

10 l/T330 340

FIG. 4. Arrhenius plot of the effect of temperatureover the range 25 to 48 C on the activities of 0.045units of BHB dehydrogenase from acetate grown cellsand 0.055 units from encysting A. vinelandii. The activ-ities are expressed as equivalent units (37 C).

TABLE 2. Properties of f3-hydroxybutyrate (BHB)dehydrogenase ofA zotobacter vinelandii

Source pH opti- Km (m.M)mum,

Encysting cells ...... 9.0 4.0 0.38Acetate-grown cells 9.0 5.5 0.38

aThe enzyme (0.05 units) was assayed over a pHrange of 7.5 to 11.0 in 0.05 M Tris-glycine butfer.

bMichaelis constants for either enzyme were deter-mined with 0.02 to 0.05 units of enzyme over a BHBdehydrogenase concentration range of 0.001 to 0.03 No.

cMobility relative to bromophenol blue in electro-phoresis on 5% acrylamide gel at pH 8.3.

ENCYSTING CELL

I mv

ACETATE GROWN

I I 1 LLLII1I I

188 J. BAC TFRIOL..

on June 21, 2017 by guesthttp://jb.asm

.org/D

ownloaded from

ENCYSTMENT IN A. VINELANDII

as much DNA per nucleoid as does Escherichiacoli (10). Similar DNA contents for synchronouscultures of A. vinelandii have been reported byZaitseva et al. (24). Muller and Kern (13) ob-served that the DNA contents of various radia-tion resistant mutants of A. chroococcum rangedfrom 10-14 to 19 x 10-14 g/cell. Despite thesecorroborative findings, we considered the pos-

sibility that the high values of DNA content per

cell were artifacts of the assay procedure. If thehot trichloroacetic acid used to hydrolyze the cellDNA also hydrolyzed a part of the cell envelope,the values for the DNA assay might have beeninordinately high. Therefore, we extracted DNAfrom 100 ml of a culture in late exponential growthphase and, after a standard DNA assay, calcu-lated the DNA per cell. To detect contaminationof the DNA preparation by the cells' lipopolysac-charide, we monitored hexosamine content of our

precipitated DNA and the aqueous phase fromwhich it was precipitated. Only 1.3% of the hexos-amine-containing material originally present inthe aqueous phase was precipitated with the DNA.Assuming a 70% yield of DNA on extraction, we

calculated that late exponential cells of A. vine-landii contain 6.7 x 10-14 g of DNA/cell. Sincethese cells contained an average of two nucleoidsper cell, the individual nucleoids must have con-

tained 3.4 x 10-14 g of DNA. This value is identi-cal to that which we have obtained for encystingcells and cysts of A. vinelandii by our DNA assay.

The relatively large mass of the A. vinelandiinucleoid suggests that there may be considerableredundancy in its genome and may account forthe difficulty in obtaining mutants of this orga-nism(Il, 12).

ACKNOWLEDGMENT

This investigation was supported by Public Health Service researchgrant Al-01863 from the National Institute of Allergy and Infectious

Diseases, National Institutes of Health. E. B. was a predoctoral trainee,supported by Public Health Service grant GM-01911 from the National

Institute of General Medical Sciences.

LITERATURE CITED

1. Bernlohr, R. W. 1964. Post logarithmic phase metabolism of sporu-lating microorganisms. 1. Protease of Bacillus licheniformis. J.Biol. Chem. 239:538-543.

2. Burton, K. 1956. A study of the conditions and mechanism of the

diphenylamine reaction for the colorimetric estimation of deoxy-ribonucleic acid. Biochem. J. 62:3 15 323.

3. Clark, J. M., Jr. 1964. Experimental biochemistry. W. H. Freeman andCo., San Francisco.

4. Davis, B. J. 1964. Disc electrophoresis. II. Method and application to

human serum proteins. Ann. N.Y. Acad. Sci. 121:404 427.5. Doi, R., H. Halvorson, and B. Church. 1959. Intermediate metabolism

of aerobic spores. III. The mechanism of glucose and hexose phos-phate oxidation in extracts of Bacillus cereus spores. J. Bacteriol.77:43 54.

6. Hitchins. V. M., and H. L. Sadoff. 1970. Morphogenesis of cysts in

Azotobacier vinelandii. J. Bacteriol. 104:492-498.7. Jurtshuk, P., S. Manning, and C. R. Barrera. 1968. Isolation and

purification of the n)(-)O-hydroxybutyric dehydrogenase ol Azoto-bacter vinelandii. Can. J. Microbiol. 14:775 783.

8. Kjelgaard, N. O., 0. Maal0e, and M. Schaechter. 1958. The tran-sition between different physiological states during balanced growthof Salmonella typhimurium. J. Gen. Microbiol. 19:607 616.

9. Lin, L. P.. and H. L. Sadoft. 1968. Encystment and polymer produc-tion by Azoiobacter vinelandii in the presence of ,-hydroxybutyrate.J. Bacteriol. 95:2336-2343.

10. Maaloe, O., and N. 0. Kjelgaard. 1966. Control of macromolecularsynthesis. W. A. Benjamin, Inc., New York.

I1. Mishra, A. K., and 0. Wyss. 1968. Induced mutations in Azotobacierand isolation of an adenine requiring mutant. The Nucleus11:96 105.

12. Mishra, A. K.. and 0. Wyss. 1969. An adenine requiring mutant of

Azotobacier vinelandii blocked in inosinic acid synthesis. Experientia21:85.

13. Muller, H. P., and H. Kern. 1967. Strahlenresisteng, Gehalt undBasenzusammensetzung der DNA einiger strahlendurzierter Mu-taten von Azotobacterchroococcum. Z. Naturforsch. 22:1330-1336.

14. Piekarski, G. 1937. Cytologishe Untersuchungen an Paratyphus undColibakterien. Arch. Mikrobiol. 8:428 439.

15. Rondle, C. J. M., and W. T. J. Morgan. 1955. The determination ofglucosamine and galactosamine. Biochem. J. 61:586- 589.

16. Sadoff, H. L., A. D. Hitchins, and E. Celikkol. 1969. Properties of

fructose 1.6-diphosphate aldolases from spores and vegetative cellsof Bacillus cereus. J. Bacteriol. 98:1208 1218.

17. Socolofsky, M. D., and 0. Wyss. 1962. Resistance of the Azotobactercyst. J. Bacteriol. 84:119-124.

18. Tchan, Y. T., A. Birch-Andersen, and H. L. Jensen. 1962. The ultra-structure of vegetative cells and cysts ol Azotobacter chroococcum.Arch. Mikrobiol. 43:50-66.

19. Umbreit, W. W., T. H. Burris, and J. F. Stauffer. 1964. Manometrictechniques. Burgess Publishing Co., Minneapolis.

20. Warburg, O., and W. Christian. 1942. Isolierung und Kristallisationdes Garungs ferments Enolase. Biochem. Z. 310:384-421.

21. Wheat, R. W., E. L. Rollins, J. M. Leatherwood, and R. L. Barnes.

1963. Studies on the cell wall of Chromobacterium violaceum: the

separation of lipopolysaccharide and mucopeptide by phenol ex-

traction of whole cells. J. Biol. Chem. 238:26-29.

22. Wilson, P. W., and S. G. Knight. 1952. Experiments in bacterial

physiology. Burgess Publishing Co., Minneapolis.23. Wyss, O., M. G. Neumann, and M. D. Socolofsky. 1961. Develop-

ment and germination of the Azoiobacier cyst. J. Biophys. Biochem.

Cytol. 10:555 565.24. Zaitseva, G. N., 1. A. Khmel, and A. N. Belozerskii. 1961. Biochemi-

cal changes in synchronous cultures of Azotobacter vinelandii. DokI.

Akad. Nauk. S.S.R. 141:740 743.

VOL. 105, 1971 189

on June 21, 2017 by guesthttp://jb.asm

.org/D

ownloaded from