Formação de estruturas císticas em Azotobacter vinelandii

Prévia do material em texto

Formation of cystlike structures by iron-limited Azotobacter vinelandii strain UW during 
prolonged storage 
WILLIAM J. PAGE 
Department of Microbiology, University of Alberta, Edmonton, Alta., Canada T6G 2E9 
Accepted April 22, 1983 
PAGE, W. J. 1983. Formation of cystlike structures by iron-limited Azotobacter vinelandii strain UW during prolonged storage. 
Can. J . Microbiol. 29: 1110-1118. 
Slant or liquid cultures of nonencapsulated Azotobacter vinelandii strain UW survived at least 2 years at 20°C, whereas parallel 
cultures died after 6 months storage at 4OC. Survival was decreased at 20°C when the growth medium was acidic and it was the 
least at 4OC when the growth medium was iron limited. Although strain UW was capable of forming cyclopropyl fatty acids and 
alkyl resorcinols, these indicators of encystment were neither abundant nor present, respectively, in 1.7-year-old cells. Only 
lauric acid was more abundant in lipid extracts from old cells than from vegetative cells or mature cysts. The old cells were very 
fragile and rapidly lost viability upon dilution or dehydration. Electron microscopy revealed the production of fragile cystlike 
structures in iron-limited medium after 1.7 years at 20°C. These structures had a distinct exine layer composed of membranelike 
plates, a poorly defined intine, and an extremely dense central body lacking storage granules or internal membranes. 
Iron-sufficient cultures produced similar but free central bodies, although cystlike structures were rarely seen. 
PAGE, W. J. 1983. Formation of cystlike structures by iron-limited Azotobacter vinelandii strain UW during prolonged storage. 
Can. J. Microbiol. 29: 1110-1118. 
Cultivke sur gClose en pente ou en milieu liquide, la souche UW de Azotobacter vinelandii non encapsulCe survit au moins 2 
ans 20°C, tandis que des cultures paralleles meurent apres 6 mois d'entreposage a 4°C. La survivance a 20°C diminue lorsque le 
milieu de croissance est acide; elle est encore moindre a 4"C, lorsque le milieu est limit6 en fer. Bien que la souche UW soit 
capable de former des acides gras cyclopropyles et des rCsorcinols alkyles, ces indicateurs d'enkystement ne sont pas abondants 
ou prksents, respectivement, chez les cellules LgCes de 1,7 anntes. Seul I'acide laurique s'av&re plus abondant dans les extraits 
lipidiques de cellules LgCes que dans ceux des cellules vkgttatives ou des kystes matures. Les cellules BgCes sont tres fragiles et 
perdent rapidement leur viabilitk lors de la dilution ou de la dkshydratation. La microscopie Clectronique rCvele la production de 
structures fragiles en forme de kystes dans un milieu limit6 en fer, apr&s 1,7 annkes a 20°C. Ces structures prksentent une exine 
distincte composCe de plaque d'aspect membranaire, une intine faiblement dCfinie et un corps central extremernent dense qui 
manque de granules de rkserve o; de membranes internes. Les cultures suffisamment pourvues en fer produisent des corps 
centraux similaires, mais libres, bien que des structures en forme de kystes soient rarement dCcelCes. 
[Traduit par le journal] 
Introduction 
Azotobacter is a free-living nitrogen-fixing bacterium 
found in soil and water throughout the world. This large 
Gram-negative cell is easily grown on defined medium 
and its physiology and structure have been studied 
extensively (Brill 1980; Jensen 1954; Sadoff 1975; 
Yates and Jones 1974). Recent studies have been 
directed towards understanding the nature and control of 
nitrogen fixation (ni f ) genes in Azotobacter, a system 
which appears to be both similar to and unique from that 
found in other diazotrophs (Bishop et al. 1980; Page and 
Collinson 1982; Pienkos et al. 1980). Azotobacter can 
be manipulated genetically by conjugation or transforma- 
tion using a variety of heterologous DNA species 
(Cannon and Postgate 1976; David et al. 1981; Page 
1978) and fusion also has been used to generate a hydrid 
between Azotobacter and Agrobacterium which will fix 
nitrogen in tomato galls (Qian-you and Chen-ying 
1981). 
These studies will benefit from the selection of 
structural and regulatory mutants and the preservation of 
these strains is important. As noted by others (personal 
communications), many interesting strains of A. vine- 
landii have been lost after storage of culture collections 
at 4°C. To combat this, working stocks often are 
transferred frequently, inviting the possibility of the 
selection of variants or revertants of the original strain. 
This is more than a trivial problem when certain 
genotypes, for example those within the Nif phenotype, 
may require sophisticated serological and in vitro 
activity assays to confirm strain identity (Shah et al. 
1973). This present study shows that tightly closed A. 
vinelandii slant cultures remained viable and geno- 
typically true for up to 4 years at 20°C, while parallel 
cultures lysed at 4OC. 
The long-term survival of Azotobacter is attributed to 
its ability to form metabolically dormant cysts (Sadoff 
1975) which are resistant to desiccation, ultraviolet 
(UV) light, and sonication (Socolofsky and Wyss 1962). 
Small filtrable Azotobacter, the germinal cellulae, also 
have been reported to be a dormant form found in soil 
(Gonzalez-Lopez and Vela 198 1). The cyst consists of a 
differentiated vegetative cell, the central body, surrounded 
by a capsular intine and an exine derived from capsule 
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PAGE 1111 
and cell membranes (Eklund et a l . 1966; Page and 
Sadoff 1975; Sadoff 1975). Encystment will occur 
naturally with culture aging and starvation or can be 
induced by addition of P-hydroxybutyrate o r n-butanol 
to C- and N-free medium (Lin and Sadoff 1968). A n 
abundance of cyclopropyl fatty acids and alkyl resorcinols 
chemically differentiates cysts f rom vegetative cells 
(Reusch and Sadoff 1981; S u et al. 1979). Many of the 
aforementioned physiological studies, however, have 
been conducted using A. vinelandii strains OP or U W , 
which lack the capsular material necessary for intact cyst 
structure. The nature of the dormant strain U W cell 
which could survive several years at 20°C, therefore, 
was examined in more detail. 
Materials and methods 
Bacterial strains and growth conditions 
Azotobacter vinelandii strains UW (nonencapsulated), ATCC 
12837 (encapsulated), and the mutants of each have been 
described previously (Shah et al. 1973; Page and Sadoff 1976; 
Page 1978). The strains were grown in Burk medium containing 
1.1 g ammonium acetate per litre and 1% glucose (Page and 
Sadoff 1976). Ammonium sulfate similarly was used as a 
nitrogen source at 200pg N/rnL. Iron-sufficient medium 
(+Fe) contained 1 pg FeS04.7H20 per millilitre, whereas 
Fe-limited medium (-Fe) contained no added iron (Page and 
von Tigerstrom 1978). Burk buffer, pH 7.2, was Burk medium 
without added glucose or ammonia. Liquid cultures containing 
a 40% culture volume per flask volume were incubated at 30°C 
for 18-20 h at 176 rpm in a water-bath gyrotory shaker, model 
G-76 (New Brunswick Scientific Co. New Brunswick, NJ). 
Cell suspensions (5 rnL) were stored in tightly closed 16 X 
120 mm screw-cap culture tubes. The same tubes were usedfor 
slant cultures, which contained 5 rnL of Burk medium solidified 
with 1.8% Difco agar. The caps on these tubes remained loose 
during the 2-3 days of culture incubation, then were screwed 
down tightly for culture sttorage. All cultures were stored in 
the dark in cardboard containers. 
Cell viability was determined directly by a standard plate 
count using Burk medium and Burk buffer as a diluent. 
Viability of slant cultures was tested by streaking Burk 
medium and noting growth (+ or -). The Petri plates were 
incubated for at least 4 days at 30°C before scoring negative 
growth. 
The genotypic fidelity ofN i f strains after prolonged storage 
was tested by using each strain as a source of crude lysate DNA 
(Page and Sadoff 1976) and using this to transform recently 
revived freeze-dried cultures (Page and von Tigerstrom 1979). 
The pattern of transformation to wild type (Nif+) using 
homologous and heterologous strains was indicative of fidelity 
to the parent strain (Bishop and Brill 1977; Page 1978). 
Fidelity of Niff , streptomycin (Str+), and rifampin (Rif+) 
resistant wild-type strains was indicated by their continued 
ability to grow on selective media (Page and von Tigerstrom 
1979). 
Cyst induction 
Encystment was induced by incubating Azotobacter in Burk 
buffer containing 0.2% D,L-P-hydroxybutyrate (encystment 
medium (Page and Sadoff 1976)). Strongly buffered encyst- 
ment medium contained the following 0.1 M Good buffers in 
addition to the 2.5 mM phosphate buffer present: piperazine- 
N,Nr-bis(2-ethanesulfonic acid) (PIPES), pH 6.8; 3-(N-mor- 
pho1ino)propanesulfonic acid (MOPS), pH 7.3; N-2-hydroxy- 
ethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.5; 
N-2-hydroxyethylpiperazine-N'-3-propanesulfonic acid 
(HEPPS), pH 8.4 (Good et al. 1966). The pH of the 
encystment medium without Good buffer rose from pH 7.2 to 
pH 7.9 during the 7-day incubation at 30°C. 
Desiccation resistance of cysts was tested according to 
Socolofsky and Wyss (1962). A known plate count of cysts or 
other cells was collected by filtration on Gelman filters 
(0.45-pm pore size), then air dried, and stored at 30°C in a 
sterile Petri dish. The filters were removed at time intervals, 
rehydrated, and dispersed in Burk buffer with vigorous vortex 
mixing, and the viable number remaining was determined by 
plate count. Azotobacter germinal cellulae were tested for 
using the procedure of Gonzalez-Lopez and Vela (198 1). The 
cell suspension to be tested was seeded with - lo7 Serratia 
marcescens cells/mL, then filtered through a Millipore filter 
(0.45-pm pore size). Growth of Azotobacter from the filtrate 
on Burk medium but absence of Serratia growth on trypticase 
soy agar (Difco) was a positive test for filtrable germinal 
cellulae. 
Analytical determinations 
Cells were collected by centrifugation and total lipids were 
extracted into chloroform-methanol using the one-phase 
method of Bligh and Dyer (1959). The lipids were fractionated 
on heat-activated silicic acid (Sigma, 100-300 mesh) in a 
1.5 X 25 cm column. Neutral lipids were eluted with chloro- 
form, glycolipids and resorcinols were eluted with acetone, 
and phospholipids were eluted with methanol. Fractions were 
dried under a stream of N2, suspended in chloroform-methanol 
(1: 1, v/v), and stored under N2 at 4OC (Reusch and Sadoff 
1979). 
Resorcinols were detected after thin-layer chromatography 
(TLC) on Analtech silica gel G coated glass plates as described 
by Reusch and Sadoff (1979). The plates were prerun in the 
chloroform-methanol (85: 15, v/v) solvent before application 
of the acetone fraction from the total lipid extraction. After 
chromatography the resorcinols turned red to purple on 
standing in air. The presence of the two major resorcinols, 
5-n-heneicosyl resorcinol (AR1, Rf = 0.70) and its galacoside 
derivative (AR2, Rf = 0.15) were used as indicators of 
resorcinol production (Reusch and Sadoff 1979). In cases 
where insufficient lipid extract was available for fractionation, 
the total lipid extract was applied directly to the TLC plate, 
chromatographed, ilnd air developed as described. 
Fatty acid methyl esters were prepared from whole cells 
after alkaline methanolysis using the Supelco boron trichloride 
- methanol reagent and the procedure recommended by the 
manufacturer.' Fatty acid methyl esters were extracted into 
chloroform-hexane (1:4, v/v), reduced in volume under a 
stream of NZ, dried with Na2S04, and stored at -20°C. 
Fatty acid methyl esters were analysed by gas-liquid 
chromatography in a Tracor model 560 chromatograph fitted 
'~echnical Bulletin 767A, Supelco Inc., Bellefonte, PA. 
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11 12 CAN. 1. MICROBIOL. VOL, 29, 1983 
TABLE 1. Comparison of the survival of iron-limited A. vinelandii strains at 4 and 20°C 
Loglo decrease in viability 
Strain Phenotype 0.7 years at 4°C 0.7 years at 20°C 2 years at 20°C 
ATCC 12837 
113 
114 
MS5 
MS64 
UW 
UW1 
u w 3 
UW6 
UWlO 
Nif+ 
Niff Rif+ 
Nif+ Str+ 
N i f 
Nif- 
Nif + 
Nif- 
Ni f 
Nif- 
Ni f 
*Indicates that the culture was dead. 
TABLE 2. Influence of culture condition on viability at 4 and 20°C 
Loglo decrease in viability 
Storage at 4OC Storage at 20°C 
Medium pH 0.1 years 0.5 years 1.7 years 0.1 years 0.5 years 1.7 years %" 
"Percent of samples viable after storage for 1.7 years at 20°C. 
q h e value in parenthesis is the number of samples assayed after storage for 1.7 years at 4 or 20°C. 
*Indicates that the culture was dead. 
with a 3 m X 2 rnm glass column packed with 3% SP-2100 
DOH on 100/200 Supelcoport. The instrument was calibrated 
using a known mixture of fatty acid methyl esters (Supelco No. 
4536) and quantitation of each methyl ester was computed 
using a Hewlett-Packard model 3370B integrator. 
Glucose in the culture supernatants was determined using 
the Statzyme glucose oxidase colorimetric assay (Worthington 
Diagnostics, Freehold, NJ). Nitrogenase and respiratory activity 
were assayed as described previously (Page 1982). 
Electron microscopy 
Specimens for electron microscopy were prepared and 
observed as described previously (Page 1982). 
Results 
Loss of viability of cultures stored at 4OC 
Strains of A. vinelandii that were pregrown on slants 
of Burk medium containing ammonium acetate for 2-3 
days prior to storage became iron limited. This was 
indicated by the production of a soluble yellow-green 
fluorescent pigment which is only produced upon Fe 
limitation (Bulen and Le Comte 1962). When duplicate 
slant cultures were stored at 4 and 20°C. the cultures at 
the lower temperature lost viability and most were dead 
after 2-6 months storage. This trend is shown in Table 
1. Both the encapsulated strain ATCC 12837 and the 
nonencapsulated strain UW and mutant strains of each 
lost viability at 4OC. The parent ATCC 12837 and 
mutant strain 114 were exceptions which remained 
viable for at least 0.7 years at 4OC. These strains, 
however, were dead after 2 years at 4"C, but the parallel 
cultures stored at 20°C remained viable. Slant cultures 
of all the strains listed in Table 1, with the exception of 
strain MS5, have survived 4 years of storage at 20°C. 
The strains remained true to their original genotype after 
this prolonged storage. No evidence of increased rever- 
sion to wild type or other variation (e.g., small colony 
formers, altered colony density, loss of encapsulation or 
pigmentation) has been observed after storage at 20°C. 
The loss in viability at 4°C correlated with Fe 
limitation and the pH of the culture (Table 2). Cultures 
pregrown without acetate and having an acidic pH (Page 
1982) or with sufficient iron did not lose viability as 
rapidly at 4OC. These conditions, however, did not 
prevent loss of viability by 1.7 years at 4OC. The 
Fe-limited, alkaline culture had the greatest viability of 
the three conditions after 0.5 years at 20°C and had 
viability similar to the Fe-sufficient culture after 1.7 
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PAGE 1113 
FIG. 1 . Appearance of A . vinelandii strains after 7 days of 
growth in encystment medium containing 0.2% P-hydroxy- 
butyrate. (A) Strain UW cells lacked the cystlike structure, 
contained numerous poly-P-hydroxybutyrate granules (pHB), 
years at 20°C. The acidicFe-limited culture had the 
greatest loss in viability after 1.7 years at 20°C. 
Changes in cell composition after storage 
Azotobacter vinelandii has been reported to contain 
an autolysin which is most active in Fe-limited cells at 
pH 8.0 + 0.2 (Page 1982). The autolysin is active at 4°C 
and could have explained the pattern of viability loss at 
4°C shown in Table 2. The autolysin, however, is more 
active at 20°C (Page 1982) and therefore autolysin 
action alone should have produced a survival pattern 
quite different from that observed at 20°C. 
The cultures pregrown with ammonium acetate con- 
tained no detectable exogenous glucose after 1.7 years at 
20°C, but the cultures stored at 4°C contained 88% of the 
original glucose concentration. The cells stored at 20°C 
were devoid of intracellular poly-f3-hydroxybutyrate 
granules after 1.7 years although there were abundant 
granules in the alkaline cultures before storage. These 
results suggested that the cultures stored at 20°C had 
continued to metabolize both exogenous and endogenous 
carbon sources and may have differentiated into a form 
resistant to autolysin action. 
Both strains 114 and UW produced alkyl resorcinols 
after 7 days of growth in encystment medium at 30°C. 
Alkyl resorcinols were detected in strongly buffered 
encystment medium at pH 6.8, 7.3, 7.5, 7.9, and 8.4, 
but only trace amounts were produced at pH 6.3. Strain 
114 pregrown in Fe-limited medium containing glucose 
and ammonium acetate also produced the alkyl resor- 
cinol AR, (Rf = 0.70) after 2 weeks storage at 4 or 20°C. 
The other major resorcinol, AR2 (Rf = 0.20), was 
formed after 9 weeks at both temperatures. Strain UW 
produced trace amounts of ARI after 9 weeks at 20°C, 
but none at 4°C. There were no resorcinols detected in 
1.7 year-old cells of strain 114 or UW, even when 
200 p,g of glycolipid extract was analysed by thin-layer 
chromatography. 
The fatty acid species present in strain UW were 
predominantly palmitic (C16:O) > palmitoleic (C16:l) 
> cis-vaccenic (C18:l) acid which accounted for 92% 
of the total fatty acids (> C14 chain length) in vegetative 
cells and the cells stored at 4 or 20°C. Other fatty acid 
species present were myristic (C14:O) > cis-9,lO- 
methylenehexadecanoic (C 17:A) r cis- 1 1,12-methylene- 
octadecanoic (lactobacillic acid, C19:A) > stearic 
(C18:O) acid. The cells stored 1.7 years at 20°C had an 
increased ratio of unsaturated to saturated fatty acids 
when compared with cells stored at 4°C or 24-h 
and peripheral respiratory vesicles (rv). (B) Strain 114 formed 
cysts with distinct layers of vegetative capsule (vc), exine (ex), 
and intine (in). Granules of pHB also were present in the 
central body. (C) A precyst of strain 114 showed active surface 
vesiculation beneath the layer of vegetative capsule (vc). Scale 
bar = 0.5 pm. 
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11 14 CAN. J . MICROBIOL. VOL. 29, 1983 
TABLE 3. Fatty acid composition of strain UW vegetative cells, cysts, and 1.7-year-old cells 
Fatty acid analysis 
% fatty acida 
PercentC 
Cell Typeb C12:O Sat Unsat CYC >C20 U + cyc/satd AR 
+Fe vegetative cell 1.3 44.8 51.4 1.7 ND 1.18 ND 
-Fe vegetative cell 2.1 42.2 54.5 1.2 ND 1.32 ND 
+Fe cell, 4"C, 1.7 years 10.3 45.4 45.6 9.0 ND 1.20 ND 
-Fe cell, 4"C, 1.7 years 34.1 46.8 49.7 2.5 ND 1.14 ND 
+Fe cell, 20°C, 1.7 years 8.5 34.7 61.3 2.8 1.2 1.85 ND 
-Fe cell, 20°C, 1.7 years 17.9 33.4 59.4 2.2 5.1 1.84 ND 
Encystment medium, 7 days 0.8 34.7 36.1 28.9 2.6 1.87 + 
Mature cysts, strain 114,7 days 1 .O 38.6 33.1 28.3 1 .O 1.59 + 
NOTE: Sat, saturated; Unsat, unsaturated; Cyc, cyclopropyl; >C20, unidentified long-chain fatty acids greater than 20 carbons in length; AR, alkyl resorcinol; 
ND, none detected; + , present. 
"Percent of fatty acid remaining after subtraction of lauric acid. 
b+Fe indicates Fe-sufficient medium and -Fe indicates Fe-limited medium. Both media contained glucose and ammonium acetate. 
'Percent of total fatty acid as lauric acid. 
"Ratio of unsaturated plus cyclopropyl to saturated fatty acids. 
vegetative cells (Table 3). This was caused by an 
increased dominance of C16:l to C16:O. The unsaturated: 
saturated fatty acid ratio of these old cells was similar to 
that found in cells from encystment medium, but in the 
latter case the increased unsaturation was due to the high 
content of cyclopropyl fatty acids (C17:A > C19:A). 
The cells stored at 20°C also contained two to three 
unidentified long-chain (> C20) fatty acids similar to 
cells from encystment medium. 
Strain UW did not form cysts after 7 days incubation 
in encystment medium with iron (Fig. 1A) or without 
iron. The cells were swollen and contained numerous 
large poly-P-hydroxybutyrate granules. The culture 
supernatant was not viscous because disorganized intine 
was not being formed (Lin and Sadoff 1968), but long 
strands of capsulelike material were produced. Strain 
114, on the other hand, formed typical mature cysts with 
a thick exine and distinct intine (Fig. 1B). The early 
encystment stage characterized by surface vesiculation 
(Fig. 1C) was seen with both strains 114 and UW cells. 
Cells stored at 4°C resembled vegetative cells in 
unsaturatedxaturated fatty acid ratio and absence of 
long-chain fatty acids. All of the 1.7-year cultures 
contained a substantial content of lauric acid (C12:O) 
which was a minor component of vegetative cells and 
cysts. Strain 114 after storage for 1.7 years at 4 or 20°C 
had a fatty acid composition similar to that described for 
strain UW after 1.7 years at 20°C. 
Properties and appearance of old strain UW cells 
The strain UW cells pregrown in Fe-limited medium 
and analysed in Table 3 after 1.7 years at 20°C were very 
fragile. Upon diluting the culture for plate counts, the 
viable number decreased more rapidly than dilution 
would predict. Slant or liquid cultures which had dried 
during storage were not viable. Old strain UW cells died 
rapidly (10-fold decreasell.9 h) when they were dehy- 
drated at 30°C. These cells were much more sensitive to 
dehydration than strain UW vegetative cells (10-fold 
decreasel6.9 h) or cells pregrown for 7 days in encyst- 
ment medium (10-fold decreasel4.5 h). By compari- 
son, an encysted culture of strain 114 was killed at < one 
10-fold decrease174 h. 
The 1.7-year-old strain UW cells appeared rounded 
and dense by phase contrast microscopy. The viable 
cells would not pass through a Millipore filter indicating 
that the viable cells were not germinal cellulae. Many of 
the iron-limited cells had brown halos when stained for 
cysts (Vela and Wyss 1964) suggesting that cystlike 
structures were present. The old cell culture had no 
FIG. 2. Appearance of iron-limited strain UW cells after 1.7 years storage at 20°C. (A) A cystlike structure comprised of exine 
(ex), intine (in), and extremely dense central body (cb). (B) A free central body showing absence of pHB granules or internal 
membranes. (C) Highly magnified disorganized exine material showing membranelike plates in cross section (x) and tangential 
section ( 1 ) with capsular material (c). (D) Free central bodies frequently showed signs of swelling producing less dense and 
patchy cytoplasm terminating in vesiculation of the cytoplasmic membrane (arrows). (E) Large expanded cystlike structure with 
well-organized exine and dead central body. (F) Large expanded cystlike structure with disorganized exine, showing both cross 
and tangential sections of exine plates, and a dead central body. Scale bar = 0.5 p.m. 
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PAGE 1115 
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1116 CAN. J. MICROBIOL. VOL. 29, 1983 
detectable oxygen consumption and no nitrogen fixation 
activity. 
Cystlike structures were observed when the iron- 
limited old cells were examined by electron microscopy 
(Fig. 2A). The central body cytoplasm was very dense 
and contained neither peripheral respiratory vesicles nor 
poly-P-hydroxybutyrate granules (Fig. 2B). The intine 
contained a small amount of fibrillar capsular material. 
The exine was constructed from small plates of membrane- 
like material embedded in capsular material (Fig. 2C). 
These cystlike structures were very loosely organized 
compared with the "mature" cysts formed by strain 114 
after 7 days in encystment medium (Fig. 1B). 
The majority of the cystlike structures in the 1.7-year 
iron-limited culture were broken and the central bodies 
were free. The central bodies were seen with various 
degrees of cytoplasmic density and swelling (Fig. 2D), 
terminating with obvious disruption and vesiculation of 
the cytoplasmic membrane (Figs. 2E and 2F) leaving the 
cell envelope intact. The central bodies with the very 
dense cytoplasm were considered to be the viable-cell 
morphology because they were the only cells with an 
intact cytoplasmic membrane and they were outnumbered 
> 100:l by the expanded, vesiculated cells. Cystlike 
cells were rarely observed in the 1.7-year-old iron- 
sufficient culture, but when present they had the same 
morphology as described above. The majority of the 
cells in the iron-sufficient culture were expanded and 
vesiculated with a minority of the cells appearing very 
dense with intact cytoplasmic membranes. 
Discussion 
Although strain UW is considered to be nonen- 
capsulated, it does form cystlike cells after prolonged 
storage at 20°C. This suggests that strain UW does 
produce some capsular material which is easily dissociated 
from the cells in a liquid shake culture. The cells in the 
liquid cultures stored at 20°C formed a pellet which 
undoubtably restricted the diffusion of capsular and 
exinous materials permitting the construction of a loose 
cystlike structure. The resemblance of these cystlike 
structures to cells observed in soil was remarkable 
(Marshall 1976; Bae and Casida 1973; Bae et al. 1972). 
The cells from soil also had an extremely dense 
cytoplasm and were surrounded by a halo of intinelike 
capsule. An exine was formed by the cell or by the 
adsorption of colloidal clay platelets (Marshall 1976). 
The formation of these fragile structures was determined 
by conditions of pH, cations, and immobilization on soil 
grana analagous to that observed here and previously 
(Page and Sadoff 1976). Encystment of strain UW also 
appeared to be enhanced by Fe limitation, which has 
been shown previously to promote central body differentia- 
tion (Layne and Johnson 1964). 
The cystlike structure formed by strain UW did not 
contain a high content of cyclopropyl fatty acids or any 
detectable alkyl resorcinols similar to those found in 
7-day "mature" cysts of ATCC 12837 (Reusch and 
Sadoff 1981; Su et al. 1979). Strain UW was capable, 
however, of forming these compounds both in encystment 
medium and during storage at 20°C. The resorcinols 
account for 16% of the dry weight of ATCC 12837 
"mature" cysts, of which 70% is located in the central 
body and 23% is found in the exine (Su and Sadoff 
1981). The resorcinols have long alkyl side chains 
(C21:O and C23:O homologs) and they may participate in 
the formation of membranelike structures in the exine 
(Reusch and Sadoff 1981). Kozubek and Demel(1981) 
have shown that the addition of long-chain alkyl 
resorcinols to model membranes induces an isotropic 
and hexagonal signal in 3 1 ~ nuclear magnetic resonance 
spectra. The hexagonal signal in natural and model 
membranes has been explained by the formation of 
reversed micells (de Kruijff et al. 1980; Cullis et al. 
1980). The formation of these nonbilayer lipid struc- 
tures in the central body membranes could promote the 
active surface vesiculation seen early in encystment and 
coinciding with the maximum production of resorcinols. 
These vesicles then flatten and coalesce to form the 
membranelike plates of the exine (Cagle et al. 1972; 
Hitchins and Sadoff 1970). After this developmental 
stage, poly-P-hydroxybutyrate and phospholipids con- 
tinue to turnover, alkyl resorcinols start to turnover, and 
the intine expands (Reusch and Sadoff 1981). This 
turnover and the low content of exinous material in 
strain UW cystlike cells probably accounts for the 
failure to detect resorcinols in the 1.7-year-old cultures. 
Clearly the 3- to 30-day-old "mature" cysts of ATCC 
12837 frequently described in the literature and resem- 
bling those in Fig. 1B are actually immature forms. The 
central body of the cystlike structures described here 
were more like dormant cells observed in soil (Bae and 
Casida 1973; Bae et al. 1972). "Mature" cysts of ATCC 
12837 have an energy charge of 0.30, have measureable 
respiration, and oxidize poly-P-hydroxybutyrate at a 
rate that would permit survival for 77 days (Aladegbami 
et al. 1979). Strain 12837 cysts, however, can survive 
for at least 10 years in soil (Vela 1974), indicating that 
the nature of endogenous substrates and metabolism in 
these dormant cells is unknown. Certainly there is a 
correlation between initial reserves of poly-P-hydroxy- 
butyrate and formation of desiccation-resistant cysts 
(Stevenson and Socolofsky 1966), but this may relate 
more to the synthesis of resorcinols and the formation of 
exines (Reusch and Sadoff 1981) than the use of 
poly-P-hydroxybutyrate as an energy source during 
prolonged storage. 
An intact exine was not required for long-term 
survival of strain UW, but as noted by Parker and 
Socolofsky (1966), the central body had to be protected 
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PAGE 11 17 
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Acknowledgments 
I would like to thank Rob Carmichael for assistance 
with electron microscopy and Wade Bingle for helpful 
suggestions. Warren Anquist provided able technical 
assistance and was supported by a Natural Sciences and 
Engineering Research Council of Canada (NSERC) 
Summer Research Scholarship. This study was supported 
by a grant from NSERC. 
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