Isolation and Pure Culture of a Freshwater Magnetic Spirillum in

JOURNAL OF BACTERIOLOGY, Nov. 1979, p. 720-729
0021-9193/79/11-0720/10$02.00/0
Vol. 140, No. 2
Isolation and Pure Culture of a Freshwater Magnetic
Spirillum in Chemically Defined Medium
R. P. BLAKEMORE,'* D. MARATEA,' AND R. S. WOLFE2
Department of Microbiology, University of New Hampshire, Durham, New Hampshire 03824,' and
Department of Microbiology, University of Illinois, Urbana, Illinois 618012
Received for publication 28 August 1979
A bipolarly flagellated magnetotactic spirillum containing intracellular chains
of single domain-sized magnetite crystals was isolated by applying a magnetic
field to sediments from a freshwater swamp. The organism was cultured in a
chemically defined medium containing ferric quinate and succinate as sources of
iron and carbon, respectively. Nonmagnetic variants of this isolate were maintained in chemically defined medium lacking ferric quinate. In contrast to magnetic cells, these had less iron and lacked measurable magnetic remanence and
the intracytoplasmic crystals. In other respects, including moles percent guanine
plus cytosine content, growth characteristics, nutrition, and physiology, the two
types were similar. The isolate reduced nitrate without accumulating nitrite and
produced ammonia during growth. Nitrate or ammonium ions served as a nitrogen
source. The organism was microaerophilic and did not grow anaerobically with
nitrate in the medium. In chemically defined medium, cells synthesized magnetite
only if the initial 02 concentration in the atmosphere of sealed cultures was 6%
(vol/vol) or less.
Motile bacteria which passively align northsouth in applied magnetic fields and contain
novel, iron-rich intracellular crystals have been
described previously (5). Large populations of
these fascinating bacteria developed in stored
muds from marine marshes and freshwater
swamps and were used in behavioral studies
conducted in controlled magnetic environments.
The results established that each magnetotactic
organism oriented to the vertical as well as horizontal component of the geomagnetic field (A.
J. Kalmijn and R. P. Blakemore, Proc. Int. Union Physiol. Sci. 13:364, 1977). Subsequent studies revealed that magnetotactic cells each possessed an intrinsic magnetic dipole moment (11).
For biochemical analyses of magnetic bacteria,
a great need has existed for pure cultures. This
report describes the isolation and methods used
to culture a microaerophilic, heterotrophic, magnetic spirillum from a freshwater swamp in
chemically defined medium. The organism was
cultured in a magnetic as well as nonmagnetic
state, and a comparative study of the nutritional
and physiological characteristics of the two
forms was initiated in an effort to determine
conditions favorable for cell magnetism.
Crystal-bearing magnetic cells of this isolate
cultured in medium containing bog water (R. P.
Blakemore, R. B. Frankel, and R. S. Wolfe, Proc.
18th Annu. Hanford Life Sci. Symp., in press)
and subsequently in chemically defined medium
(8) have previously been shown to contain magnetite. The amount of magnetite per cell, its
single domain size range, and its conformation
within the cell all contributed to its effectiveness
as a biocompass (R. B. Frankel and R. P. Blakemore, submitted for publication.)
(Portions of this work were reported in papers
presented at the 1979 Annual Meeting of the
American Society for Microbiology, Los Angeles
[R. P. Blakemore, R. S. Wolfe, and D. Maratea,
Abstr. Annu. Meet. Am. Soc. Microbiol. 1979,
N38, p. 185] and at the 18th Annual Hanford
Life Sciences Symposium, October 1978, Richland, Wash.)
MATERIALS AND METHODS
Enrichment cultures. Before isolating a magnetic
bacterium, we identified optimal conditions for their
survival by using enrichment culture techniques.
Jars filled to approximately two-thirds their volume
with mud and water collected from Cedar Swamp in
Woods Hole, Mass., provided inocula for enrichment
studies and isolation. The loosely covered jars were
left undisturbed in dim light at room temperature
(22°C). After 1 or more months, a small amount of
mud slurry containing magnetic cells was removed
from one of these and diluted 100-fold into each of
many small vials (Pierce Chemical Co. cat. no. 12903)
containing one-sixth their volume of test medium. The
vials were sealed, and the atmosphere of each was
replaced with a test gas mixture. After suitable incubation, survival of magnetic bacteria was determined
720
VOL. 140, 1979
FRESHWATER MAGNETIC SIl'IRILLUM
from direct microscopic counts of motile, magnetotactic cells. By this means, cell responses to pH and
buffering characteristics, temperature, oxygen concentration, chemical reducing agents, and various iron
and nitrogen sources were determined, and the results
were used as a basis in constructing an isolation medium.
Isolation and growth of strain MS-1. Inocula for
isolating magnetic bacteria were obtained from enrichment material by application of steady, nonuniform
magnetic fields. Permanent bar magnets were used to
separate large numbers of motile magnetic cells from
sediment. These magnetotactic cells (of at least six
distinct morphological types) were washed in filtered,
sterilized bog water and injected through the stoppers
of culture tubes containing prereduced, semisolid isolation medium. The isolation medium consisted of (per
90 ml of distilled water): 10 ml of filtered swamhp or
bog water; 1 ml of vitamin elixir (23); 1 ml of mineral
elixir (23); and 0.5 mM potassium phosphate buffer
(pH 6.7). To this mixture were added: 5 ,g of vitamin
B-12; 25 mg of NH4Cl; 10 mg of sodium acetate (anhydrous); 0.2 mg of resazurin; and 90 mg of ionagar
no. 2 (Oxoid). The pH was adjusted to 6.7 with NaOH.
This medium was prereduced under nitrogen, using
titanium citrate (24) as the reducing agent, and was
subsequently dispensed into culture tubes in an anaerobic hood. Inoculated tubes were incubated at 22°C in
the dark until growth became evident (see Results). A
well-isolated area of growth was homogenized, and
cells were cloned by serial dilution into tubes containing molten, prereduced isolation medium containing
0.85% (wt/vol) Ionagar no. 2. Well-isolated colonies
which appeared in these tubes after 1 week at 30°C
were homogeneous as evidenced by microscopy. However, cells were again cloned, and the process was
repeated a third time before cultures were considered
pure.
Strain MS-1 was maintained at 30°C with weekly
transfers in screw-capped culture tubes containing a
semisolid growth medium consisting of (per 98 ml of
distilled water): 1 ml of vitamin elixir; 1 ml of mineral
elixir; 5 mM KH2PO4; 25 MuM ferric quinate; and 0.2
mg of resazurin. To this mixture were added (per 100
ml): 0.1 g of succinic acid; 20 mg of sodium acetate
(anhydrous); 10 mg of NaNO3; 5 mg of sodium thioglycolate; and 130 mg of agar (GIBCO Laboratories). The
ferric quinate solution was prepared by combining 2.7
g of FeCI3 and 1.9 g of quinic acid with 1 liter of
distilled water. Before adding the agar, the pH of the
medium was adjusted to 6.7 with NaOH. The medium
was boiled, and 12 ml was added to each screw-capped
tube (16 by 125 mm) containing approximately 0.1 ml
of 5% (wt/vol) sodium thioglycolate in distilled water.
Tubes of semisolid growth medium were autoclaved
with caps tightened and allowed to stand overnight for
the establishment of 02 gradients. Inocula consisted of
0.2 ml (ca. 7 x 107 cells) per 12 ml of medium. Chemically defined growth medium was identical to the
semisolid growth medium, except that agar was omitted and the medium was sealed under a gas atmosphere of known composition by the method of Balch
and Wolfe (1) as described below.
A homogeneous population of nonmagnetic cells
was obtained from cultures of MS-1 grown in isolation
medium made with distilled water rather than bog
water. Cells grown in this medium, especially with
twice as much as the usual amount of nitrate and
succinate, grew nonmagnetically. To obtain a nonmagnetic culture from strain MS-1 for comparative studies,
cells grown for five successive transfers in this medium
were cloned three successive times. Stocks of this
nonmagnetic variant of strain MS-1 were subsequently
maintained in defined growth medium without ferric
quinate.
Assessing culture magnetism. Cultures were
routinely checked for their magnetic response by microscopically noting the fraction of cells present which
turned when a small magnetic stirring bar was rotated
approximately 10 cm away from the microscope slide.
Mass cultures. Cells of strain MS-I in the magnetic
and nonmagnetic state were mass cultured in chemically defined growth medium and this medium minus
ferric quinate, respectively. Agar was omitted from
media for mass cultures. Glass bottles (150 ml to 1
liter) were filled to approximately one-third of their
volume with medium. The atmosphere of each was
replaced with N2, and it was then crimp-sealed with a
serum stopper or closed with a rubber stopper wired
in place before autoclaving. Cells were inoculated into
cooled medium by injection through the stopper. Sufficient air was added to each bottle at the time of
inoculation to provide 0.6 to 1.0% tvol/vol) 02 in the
gas phase for routine culture. To assess the effect of
02 on cell growth, pure 02 was injected after removing
an equivalent gas volume from each bottle.
A glass carboy was sealed with a rubber stopper
fitted with a bottomless, screw-capped, 16-mm tube
which served as a gas outlet and port for inoculation
and sampling. A 12-g stainless needle which had been
inserted through the stopper was used as a gas inlet.
It was attached to a 40-cm length of tubing contained
within the carboy to allow incurrent gas to sparge
through the culture medium. The medium in each
carboy was autoclaved under air. Hot medium was
sparged with sterile N2 while being cooled at 30°C. It
was then inoculated and incubated at 30°C after sealing the carboy against further gas exchange. Midway
through growth, when the culture became reduced as
evidenced by a color change, its pH was adjusted to
6.7 aseptically with 0.5 M succinic acid, and it was
continuously aerated with sterile air by means of an
aquarium pump.
Preparation of cells for elemental analyses.
Cells for elemental analyses were harvested during
active growth by continuous-flow centrifugation at
10°C in a DuPont Instruments Sorvall RC-5B centrifuge operating at 27,000 x g. The cells were suspended
and washed three times in distilled water by centrifugation procedures. Washed cells remained motile, indicating they were intact. Washed cells were lyophilized and stored in air-tight containers.
Elemental analyses. Qualitative elemental composition of lyophilized magnetic and nonmagnetic cells
of strain MS-l were determined by using a Kevex
energy-dispersive X-ray analysis system coupled to an
Etec scanning electron microscope. Dried whole cells
were sprinkled onto the surface of double-faced tape
attached to a carbon specimen stub. A coating of
either carbon (cells for X-ray analysis) or gold-palla-
721
722
BLAKEMORE, MARATEA, AND WOLFE
J. BACTERIOL.
dium (cells for photography), was vacuum-deposited
on the specimens, which were then viewed at 20 kV.
Each spectrum obtained was representative of each of
four successive analyses taken in a continuous series
across the width of an individual magnetic or nonmagnetic cell.
For iron analyses, 10 ml of concentrated, redistilled
HNO, and 5 ml of 70- (vol/vol) HCIO, were added to
each accurately weighed sample (ca. 15 mg) of lyophilized cells suspended in 5 ml of 12 N HCI (J. T. Baker;
Ultrex grade). The mixture was heated until all of the
HNO, and all but 0.5 ml of the HC104 had boiled off.
The resulting clear, yellow cell digest was taken up in
0.5 ml of 12 N HCI and diluted into a known volume
(ca. 10 ml) with distilled, deionized water before analysis. A reagent blank was prepared in the same manner. Culture supernatant fluids were decanted into
acid-washed polyethylene bottles, and their pH was
adjusted to a value of 2 to 3 with concentrated, redistilled HNO:, and analyzed directly. Iron measurements
were made with the use of a Perkin Elmer model 403
atomic absorption spectrophotometer or a Varian
Techtron model AA3 modified to AA5. All measurements were made by using an acetylene-air flame.
To estimate the iron content of individual cells,
direct cell counts (see below) were made of known
weights of lyophilized cell powders resuspended and
brought to known volumes in sterile culture medium.
Portions of the same lyophilized powders used for iron
analyses were used for cell counting.
Estimation of cell numbers. Cell numbers were
determined by means of direct counts made with a
Petroff-Hausser cell-counting chamber. At the end of
growth, tubes containing semisolid medium were inverted to evenly disperse cells before counting.
DNA base composition. DNA from approximately 1012 cells lysed with sodium dodecyl sulfate was
extracted with phenol and dialyzed against saline sodium citrate (14). Moles percent guanosine + cytosine
(G+C) was determined by buoyant density measurements in CsCl (20) and by thermal denaturation of the
DNA in phosphate buffer containing EDTA (15).
Standards for thermal denaturation included DNA
extracted from Pseudomonasputida (62.5 mol% G+C)
and Pseudomonas maltophila (67 mol% G+C). DNAs
from bacteriophage SP82G and from Escherichia coli
strain B were used as standards for buoyant density
measurements.
Nutrition experiments. Various carbon compounds, including sugars, amino acids, and tricarboxylic acid cycle intermediates, were tested for their
ability to support growth of magnetic and nonmagnetic
cells. Heat-sterilized compounds were added aseptically to screw-capped tubes, each containing 10 ml of
semisolid growth medium minus succinate, acetate,
and ferric quinate. Test compounds at pH 6.7 were
added to a final concentration of 0.1% (wt/vol) immediately after autoclaving the basal medium. Tubes
were sealed, inverted to mix the contents, and allowed
to stand overnight for cooling and establishment of
oxygen gradients before inoculation. Each compound
was tested in triplicate, using a 0.2-mil inoculum from
a 24-h culture. The results were determined by recording direct cell counts 7 days after inoculating the third
sequential transfer.
Other experimental procedures. Nitrate reduction in chemically defined culture medium minus resazurin was determined with a-naphthylamine (13).
Powdered zinc was used to reduce nitrate possibly left
in the medium at the end of growth. Ammonia was
determined with Nessler reagent (21). Polyhydroxybutyrate granules within cells were stained with Sudan
black B. Oxidase activity was determined with Kovac
oxidase reagent applied to filter paper on which heavy
cell suspensions were smeared (13). Catalase activity
was determined by macroscopic and microscopic examination for gas evolution by growing cells and heavy
cell suspensions treated with 3 or 307r (vol/vol) H202
for periods in excess of 30 min. Catalase activity of
cell-free extracts was also measured spectrophotometrically (3) by a method which gave a strongly positive
reaction with cell extracts of aerobically grown E. coli
or purified bovine liver catalase (Worthington Bio-
chemicals Corp.).
Microscopy. Phase-contrast photomicrographs
were made of living cells from exponentially growing
cultures, using a Zeiss standard research microscope
equipped with a 35-mm camera and electronic flash
attachment. Specimens for transmission electron microscopy were stained with 2% (wt/vol) ammonium
molybdate (pH 7.0) for 5 min and examined in a
Phillips EM 200 electron microscope operating at 60
kV.
RESULTS
Enrichment and isolation of strain MS-1.
Muds collected from various freshwater and marine marshes, swamps, and bogs underwent a
natural succession of microorganisms and when
left undisturbed, stratified into aerobic and anaerobic zones. The largest numbers of magnetic
bacteria populated these samples when the transition zone separating upper layers containing
oxidized (rusty) iron from underlying, reduced
(black or dark-colored) layers occurred at the
sediment surface, or just beneath it. In the sample from which strain MS-1 was isolated, total
population densities of magnetic bacteria increased from about 200 cells/ml initially to 106
to 107 cells/ml of surface sediment (slurry) after
several months. Large numbers of magnetic bacteria of diverse morphological types persisted for
over 1 year when the samples were maintained
with occasional addition of distilled water. Small
numbers of mixed populations of magnetic bacteria removed from these natural enrichments
survived longest (ca. 1 month) when sealed under air-nitrogen mixtures. Spirilla were never
observed in the particular enrichment from
which they were isolated and therefore must
have comprised a very small fraction of the total
population of magnetic bacteria present. Two
weeks after inoculating tubes of semisolid
growth medium with magnetically purified cells
obtained from mud (see Materials and Methods), diffuse, spreading, fluffy areas of growth
VOL. 140, 1979
FRESHWATER MAGNEI'IC SIRIIRILLUM
723
were noted in several tubes which had become 0.25 by 4 to 0.25 by 6 um. Cell length and
slightly oxidized (pink) at the time cells were wavelength were variable, with shorter, more
injected. Microscopic examination revealed that tightly coiled forms often predominating earlier
these areas consisted of magnetotactic spirilla. A in growth. Cells of both types possessed single
bipolar flagella which were easily broken off
pure culture of strain MS-1 was obtained after
three successive clonings of cells from well-iso- during handling for microscopy. Intracellular
lated, lens-shaped subsurface colonies which de- granules which stained with Sudan black B were
veloped in media containing a higher agar con- presumed to be deposits of poly-,1-hydroxybucentration.
tyric acid. Details of cell ultrastructure have
Occasionally, after several transfers in semi- been described (D. L. Balkwill, D. Maratea, and
solid growth medium, most cells in populations R. P. Blakemore, submitted for publication).
The moles percent G+C content of DNA from
of strain MS-1 became less magnetic. For this
reason, magnetic selection was applied to mainmagnetic cells of strain MS-I was the same as
tenance cultures. After growth became evident, that of nonmagnetic cells. Values obtained by
a small permanent magnet (2.5 by 0.5 by 0.5 cm)
thermal denaturation (64.9% for magnetic and
was attached to each tube with an elastic band.
64.5%4 for nonmagnetic) were slightly higher than
Magnetic cells in the culture accumulated as those obtained from buoyant density measurevisible masses at the poles of the magnet within ments (63.8% for magnetic and 63.7% for nonapproximately 1 h and were used to inoculate magnetic).
fresh culture medium.
Magnetotactic and nonmagnetotactic cell
As described in Materials and Methods, a types of strain MS- 1 each had similar generation
culture of nonmagnetic cells was derived from times (6 to 15 h) under similar growth conditions
the magnetic culture of strain MS-1. Freshly in defined growth medium. The cell yield after
cloned nonmagnetic cells grew magnetically 4 to 6 days of growth was 0.2 to 0.5 g (wt weight)
when transferred into semisolid or chemically per liter. Cells grew very slowly at 15°C and at
defined growth medium after several passages temperatures as high as 37°C, but at the higher
with magnetic selection (i.e., by selecting as an temperature they were badly distorted and
inoculum, cells from the vicinity of the poles of many coccoid bodies were present. Coccoid or
a small permanent magnet attached to the culspherical forms also were prevalent in older culture tube). Factors controlling this reversible tures or when excess O, was present. Cells ditransition from the nonmagnetic to magnetic vided by binary fission, not always in a plane
state are incompletely understood, since only equally bisecting their chains of crystals.
two of three attempts have been successful.
In sealed bottles of chemically defined meDescription of strain MS-1. Magnetotactic dium, cells never grew when the medium was
cells of strain MS-1 grown in chemically defined completely reduced (colorless). Trace amounts
medium possessed strong natural remanent of 02 allowed substantial cell growth (Fig. 2).
magnetization (roughly 9 x 10' electromagnetic However, cells grew with the shortest lag when
units per g, dry weight; Charles Denham, per- the initial O2 concentration of the gas phase was
sonal communication) and contained magnetite 1 to 3% (Fig. 2). In sealed culture vessels, cells
(8) which presumably was the major structural eventually grew after a lag of approximately 60
component of intracellular chains of crystals also h when the initial O, concentration was 21% in
present (Fig. 1A). These 43-nm crystals were the gas phase but did not grow when the atmosmaller than those described previously in magsphere was allowed to exchange freely with air.
netic bacteria found in marine (5) or freshwater Cells grown in sealed vessels with 12 or 21% 02
(16; R. P. Blakemore, R. B. Frankel, and R. S. attained the highest final cell yields (1.2 x 10"
Wolfe, Proc. 18th Annu. Hanford Life Sci. cells/ml). However, when cultured at initial 02
Symp., in press) sediments but were present in concentrations greater than 6%, cells were not
larger numbers per cell. Nonmagnetotactic cells magnetic at any stage of growth (Fig. 2). In
of strain MS-1, grown in chemically defined semisolid medium containing either resazurin or
medium minus ferric quinate, lacked detectable methylene blue, cells initially grew in an exremanence (C. Denham, personal communicatremely fine band located several millimeters
tion), magnetite (8), and the chains of crystals above the color transition layer. The Eh most
(Fig. 1B). Whereas concentrated suspensions of suitable for the growth of the organism in semimagnetic cells were dark gray-brown in color, solid growth medium, therefore, was higher than
those of nonmagnetic cells were light cream +40 mV, the approximate redox potential of
colored.
methylene blue at the pH value used (10). DurBoth cell types were actively motile (velocity, ing growth, cells reduced the culture medium as
44 + 8 ,um s-'), gram-negative, and helical (ca. evidenced by its change to colorless, and in
724
BLAKEMORE, MARATEA, AND WOLFE
.1. BACTERIOL.
A
VP
FIG. 1. Electron micrograph of stained cells of strain MS-I. Magnetic cells (A) each possess a chain of
electron-dense crystals occupying a similar position as an unusual filament or tubule present uithin
nonmagnetic cell.s (B). Bar represents I ,un.
semisolid media capable of supporting dense
growth they grew progressively upward to the
agar-air surface.
Catalase and oxidase activities were not detected in magnetic or nonmagnetic cells of isolate MS-1. Cells formed colonies on the surface
of agar-solidified growth medium in petri dishes
incubated aerobically only when 30 U of catalase
per ml was added to the medium after autoclaving.
Iron content of strain MS-1. Preliminary
electron microscopic evidence indicated that
dried whole cells of magnetic bacteria contained
phosphorus, sulfur, potassium, and iron as major
elements detected by energy dispersive X-ray
analysis. Similar results were obtained with nonmagnetic cells, except that no iron and less potassium were detected. The apparent differences
in potassium content need additional study.
However, iron concentrations in magnetic and
nonmagnetic cells and their culture medium
were measured (Table 1). As shown, magnetic
cells contained 2.0 ± 0.2% (wt/vol) of their dry
weight as iron. This value is slightly higher than
that reported earlier for magnetic cells of this
organism (8), which presumably is due to variation in cell magnetite content, since on each
occasion they were cultured and analyzed in the
same manner.
Magnetic cells contained 10 times more iron
than nonmagnetic cells on a dry-weight basis
(Table 1). However, comparisons of iron content
between each type are misrepresentative when
considered on a dry-weight basis because of the
large weight contribution of iron. Accordingly,
in a separate experiment in which the iron con-
725
VOL. 140, 1979
FRESHWATER MAGNETIC SI'IRILLUM
tent of magnetic cells (15.1 mg/g, dry weight)
was seven times greater than that of non-mag-
TABLE 1. Iron concentrations in cells and media of
isolate MS-I and other bacteria
netic cells on a dry weight basis, they contained
25 times more iron on a per cell basis (15.5 fg of
Fe per cell) than their nonmagnetic counterparts
(0.61 fg of Fe per cell). Density differences between magnetic and nonmagnetic cells were easily detected by their sedimentation behavior
during centrifugation.
The data in Table 1 also show that magnetic
cells accumulated four times more iron from the
medium than nonmagnetic cells. By the end of
growth, the magnetic cells had not depleted their
iron supply, which remained significantly high
(8.1 ,uM). The iron content of washed magnetic
cells of strain MS-1 was 100 times higher than
that of representative heterotrophic bacteria
cultured in a medium of similar total iron content (Table 1). The data also show that nonmagnetic cells of strain MS-1 had a high percentage
of iron relative to other heterotrophic bacteria.
Nonmagnetic cells were also high in iron (1.14
,tg/mg, dry weight) when cultured in a medium
without ferric quinate (estimate: 3.6,iM total Fe
initially present).
Nutrition. Of an extensive array of potential
carbon and energy sources tested, (Table 2)
tITIAL x 0,
Fw;. 2. Growth of spirillum strain MS-I at various
initial O, concentrations. Cells u'ere cultured in
sealed bottles each containing 50 ml chemically defined growth medium containing 0.1'S (ut/vol) tartaric acid in lieu of succinic acid. Cultures uwere
incubated on a rotary shaker (160 rpm). At the time
cells were injected, the medium became someuwhat
oxidized, as evidenced by a pink coloration. Thus, all
bottles contained a trace of 02 in addition to that
indicated. "Air" indicates cultures in free gas exchange u'ith the atmosphere. Grouth values indicate
means oftriplicate cultures. Error bars indicate standard deviations. Each bottle uas inoculated with
magnetic cells. Only cells cultured uwith 6f O,, or less,
uere magnetotactic.
Iron concn in:'
Organism
Culture medium (ug/rnl)
Cells (mg/g,
Before growth After growth
dry wt)
0.18
1
bacteriab
Strain MS-lc
0.856 ± 0.016 0.453 ± 0.032 20.1 ± 1.6
Magnetic
cells
Nonmagnetic 0.856 ± 0.016 0.732 ± 0.036 2.81 ± 0.19
cells
a Values indicate means and standard deviations of
triplicate samples measured by atomic absorption
spectrophotometry (see text).
'Unpublished data of L. Murray, L. G. Royle, and
G. E. Jones; average of five nonmarine species.
cCells were cultured in chemically defined growth
medium for magnetic cells (see text), except that onehalf the normal concentrations of mineral elixir and
ferric quinate were used.
Heterotrophic
those which supported growth after three successive transfers were intermediates of the tricarboxylic acid cycle as well as f8-hydroxybutyric, tartaric, lactic, or pyruvic acids. Amino
acids tested were not used as sole sources of
carbon. Cells did not grow in chemically defined
medium in the absence of added nitrate. The
organism grew after three sequential passages in
semisolid growth medium minus nitrate; however, added NH4C1 or NaNO:, improved the
growth yields in this medium. Growth in semisolid medium in the absence of added nitrogen
source suggested that strain MS-1 might be capable of fixing atmospheric nitrogen, although
small amounts of nitrogen were present in the
mineral solution and perhaps the agar used.
Growing cells reduced nitrate without accumulating detectable amounts of nitrite in the
medium. At the end of growth, no nitrate was
detected in chemically defined growth medium,
and a significant amount of ammonia was present. The culture pH rose from an initial value of
6.7 to 7.3 during growth. Attempts to culture the
organism anaerobically (e.g., in colorless media
containing resazurin) with nitrate were unsuccessful.
Vitamins were not required for growth or magnetotactic behavior of the organism during six
passages in chemically defined medium. Deletion of the vitamin mixture from the growth
medium resulted in larger (0.64 [±0.19] x 5.6
[±1.27] im) spirilla with a swollen appearance.
Restoration of vitamins resulted in new cells
with normal, smaller morphology. Cells grew at
normal rates and final yields without ferric quin-
726
BLAKEMORE, MARATEA, AND WOLFE
.1. BACTFRIOL.
forward-to-reverse motion without a significant
pause and without reorienting its long axis. Although conditions favorable for aerotaxis by
cells of the organism are not well understood,
they infrequently showed weak aerotactic behavior by accumulating in bands, particularly
near the magnetically northernmost and southernmost edges of cover slips in wet mounts, as
well as after being dispersed in tubes of semisolid
media even when growth had ceased.
In nonuniform magnetic fields (bar magnets)
or steady uniform magnetic fields as low as 0.5
gauss (Helmholtz system), living or dead magnetic spirilla oriented within several seconds
with their long axis in the north-south magnetic
axis of the field (Fig. 3). Nonmagnetic cells did
not show any preferential alignment with applied magnetic fields. Approximately equal numbers of magnetic cells were observed swimming
TABLE 2. Effect of various compounds as sole
northward and southward (in the magnetic
carbon source on growth of strain MS-I a
sense) in drops of culture medium examined
Final cell yield (X107 cells/ml)b
microscopically in a magnetic field. Cell motility
Magnetotactic Nonmagnetotactic
Compound
was less directed in weaker ambient fields, alcells
cells
though even when no fields were intentionally
applied, cells from the most magnetic cultures
+
0
17
0
30
Fumarate
showed a detectable preferential north-south
16 ± 0
36 1
Tartrate
alignment in the local geomagnetic field. These
21 0
13 ± 0
Malate
12 ± 0
18 0
orientations also directed the motility of magSuccinate
12 0
9±3
Lactate
netic bacteria in sealed liquid cultures (no oxy7±1
17 0
Pyruvate
gen gradient), because at the end of growth, with
7±3
14 ± 0
Oxaloacetate
no artificial field intentionally applied, cells ac6 1
5 2
Malonate
cumulated only at one side of the bottoms of
±
±
1
4
2
16
B8-Hydroxybutyrate
culture vessels. In stronger magnetic fields cre4 1
4 1
Maleate
ated with small bar magnets, many cells in acCompounds were added as sterile solutions at pH tively growing cultures accumulated in the vicin6.7 after autoclaving basal medium. Final concentra- ity of each magnetic pole.
tion of carbon source was 0.1% (wt/vol). Maximum
inoculum size was 1.4 x 108 cells per 10 ml. Results
DISCUSSION
ate but were not magnetotactic. Neither ferric
chloride, ferric citrate, nor iron chelated by L,8-3,4-dihydroxyphenylalanine (L-DOPA), protocatechuic acid, L-epinephrine, L-arterenol, or
EDTA, each used at 50 ,M, satisfied the iron
requirement for Fe: 4 synthesis. Disodium
EDTA was toxic at concentrations above 5 x
10-4 M. L-DOPA, protocatechuic acid, L-epinephrine and L-arterenol, each used at 0.1% (wt/
vol) inhibited growth of strain MS-1.
Behavior and motility. Living cells displayed forward and backward motility in a manner similar to other bipolarly flagellated spirilla
such as Sprillum volutans (4, 12). Each cell
frequently spontaneously changed its swimming
direction either by (i) pausing and tumbling
randomly before taking a new course or by (ii)
a
indicate growth response at third sequential passage.
Omission of carbon source resulted in 3 x 104 cells/ml.
Compounds resulting in weak growth (0.1 x 107 to 1
x 107 cells/ml) included: galactose, rhamnose, melibiose, acetate, adipate, and glutarate. Compounds not
utilized as the sole source of carbon (less than 104
cells/ml) included: glucose, maltose, mannose, raffinose, ribose, fructose, xylose, trehalose, melizitose, sucrose, arabinose, cellobiose, mannitol, sorbitol, inositol, dulcitol, glycerol, xylitol, citrate, isocitrate, aconitate, a-ketoglutarate, gluconate, benzoate, quinate, pimelate, pthallate, mandelate, sulfanilate, methanol,
butanol, sec-butanol, t-butanol, ethanol, propanol, isopropanol, benzene, xylene, toluene, phenylalanine, glutamate, alanine, serine, and lysine. Compounds which
inhibited growth at the first transfer included: butyrate, isobutyrate, propionate, protocatechuate, formate, oxalate, caproate, L-epinephrine, L-/3-3,4-dihydroxyphenylalanine, catechol, L-arterenol, phenol, methionine, and cysteine.
'Each value indicates mean direct cell count ±
standard deviation, obtained by using triplicate cultures.
In overall morphology, Gram-staining reaction, moles percent G+C content of its DNA and
pattern of carbon sources utilized, strain MS-1
is a heterotroph which is generally similar to
members of the genus Aquaspirillum (12). However, other aspects of its biology, including its
pattern of flagellation, microaerophilism, catalase and oxidase content, nitrogen metabolism,
formation of coccoid bodies, and unique ability
to produce magnetite, collectively prevented us
from fitting the isolate into currently accepted
taxonomy. For this reason, we will consider assignment of taxonomic status elsewhere.
Consistent differences noted between magnetic and nonmagnetic cells of strain MS- I (color
of cell suspensions, elemental composition, magnetotactic behavior, presence of magnetite, possession of magnetic remanence, and presence of
intracellular crystals) all appear to relate to the
presence of magnetite in magnetic cells. Non-
FRE4SHWATER MAGNETIC SPIRIILLUM
VOL,. 140, 1979
727
4'**.
'.
t
t
*_-
..
I
r-
I
I
e
I
.
'k
t
*
~\N
4)
4'
-4
A"~~~~~~~~~~~~~~~~~~~~4\4
%
FIG. 3. Phase-contrast photomicrograph of living magnetic cells of strain MS'-I in artificial magnetic
field.s. Cells orient in the local field of a small magnet held near the microscope uwith its north-south magnetic
axis directed right to left (A) and top to bottom (B). Bar represents 20 ,im.
magnetic cells lack magnetite but contain
roughly 10 times more iron than representative
heterotrophic bacteria cultured at similar iron
concentrations. This iron in nonmagnetotactic
cells could be precursors of magnetite crystals,
perhaps superparamagnetic magnetite or ferritin
(2, 8), and could comprise a part of a thin filament observed in these cells (Fig. 1B) in the
position of the chain of magnetite crystals within
magnetic cells.
Magnetic bacteria of diverse types which are
prevalent in many natural environments become
extremely numerous in muds from these locations allowed to stand and stratify in the laboratory. Our estimate of cell numbers as high as
106 to 10' per ml agrees well with that reported
by Moench and Konetzka for magnetotactic bacteria from a sewage oxidation pond (16). We did
not detect large numbers of magnetic or nonmagnetic organisms with the unique morphologies of certain magnetotactic bacteria (5, 16;
Blakemore et al., in press), in freshly collected
mud samples. After storage for several weeks,
however, they contained large numbers of magnetic cells. Thus, high population densities of
magnetic cells do not arise merely as a consequence of induced magnetite synthesis in preexisting, nonmagnetic bacteria in sediments.
The results show that growth of strain MS-1
at low cell densities was inhibited at high Po2
values (0.12 to 0.21). On the other hand, when
supplied at the low concentrations optimal for
immediate cell growth and for magnetite synthesis, 02 was cell yield limiting. When care was
taken to exclude all oxygen from culture media
and the media were reduced as evidenced from
their color, cells did not grow. Thus, strain MS1 is a microaerophile, requiring 02 but also inhibited by it at initial concentrations above
about 6% in the culture system used. Mixed
species of other magnetic bacteria from natural
enrichments also survived best at reduced 02
concentrations. Undoubtedly, an important factor contributing to growth of magnetic forms in
standing muds is establishment of microaerobic
conditions. The biochemical basis of microaerophilism is not well understood. Cole and Rittenberg (7) suggested that 02 sensitivity of the
obligate microaerophile S. volutans might be
due to its relatively slow respiration rate, which
could allow for accumulation of toxic intracellular concentrations of 02. We have not measured 02 consumption rates by cells of MS-1.
Thus, at present, the only known biochemical
feature of this organism which relates to its
microaerophilic physiology is its lack of catalase
activity. Indeed, aerobic growth of strain MS-1
in petri dishes with catalase in the medium
strongly suggests that its sensitivity to 02 iS
associated with a function of this enzyme.
728
,J. BACTFIRIOL,
BLAKEMORE, MARATEA, AND WOLFEB
Bowdre et al. (6) reported that dihydroxyphenyl
iron chelators enhanced the aerotolerance of
obligate microaerophiles S. volutans and Campylobacter fetus. They also observed stimulation
of aerotolerance of C. fetus by 0.02 to 0.05%
ferrous sulfate added to its culture medium and
postulated that microaerophilism of these organisms might be due to an inability to produce
iron-chelating compounds at sufficient rates or
in amounts presumed necessary for aerobic
growth. Strain MS-1 did not grow when the iron
concentration of its medium was increased to
millimolar amounts. However, we did not observe enhanced aerotolerance when ferric quinate was supplied at any concentration up to
growth inhibiting amounts nor when the dihydroxyphenyl compounds used by Bowdre et al.
or ferric chelates of these compounds were
added to culture media at 10-6 to 10-4 M.
Redox potential may also be an important
factor for growth and magnetite synthesis by
magnetic bacteria. In enrichments from Cedar
Swamp, magnetic bacteria occurred in the largest numbers in upper strata of loosely consolidated sediments where ferric and ferrous iron
were both present. For vertical profiles of submerged soils (19), this transitional zone correlates with the zone of greatest Eh gradient. Data
obtained by Patrick (18) indicated that during
an experimentally controlled change in Eh over
the range associated with vertical profiles of
submerged soils (-200 to +500 mV) the amount
of ferrous iron and phosphorus released from
soil sharply decreased above an Eh value of +200
mV (at pH values between 6 and 7). Results of
our preliminary enrichment studies clearly
showed that phosphorus was a limiting resource
for microbial growth in water and surface sediments of Cedar Swamp. However, both magnetic
and nonmagnetic cells of the spirillum isolated
from this enviroument contained a large quantity of phosphorus relative to other elements
detected when cultured in artificial media. Thus,
the increased availability of phosphorus (and
appropriate chemical forms of other elements
including iron, sulfur, and nitrogen) at low redox
potentials could be an irnportant factor determining the precise balance of conditions permitting optimal growth of microaerophiles in microaerobic areas and causing them to be maximally distributed there.
As might be expected from their high iron
content, iron was an important requirement for
magnetite synthesis by cells of MS-1. Because
quinic acid did not serve as a sole carbon source
for strain MS-1, we assume that the important
role of ferric quinate in culture media for magnetic cells was to supply iron in a suitably chelated form for cell uptake. The requirement for
bound iron appeared fairly specific, since none
of a variety of other iron chelates supported
magnetite synthesis. Siderophore synthesis by
enteric bacteria is repressed by culture medium
iron values of 0.1 to 1.0 yM (17). Thus, if magnetic spirilla exhibit similar control of iron transport, they probably do not rely on these compounds when growing in chemically defined
growth medium (15 to 30 ,M Fe, depending on
ferric quinate added) or, for that matter, in
swamp water, in which we have measured total
iron values of 20 to 30 LM. Magnetic cells probably require preformed exogenous iron chelates
specifically compatible with cell transport mechanisms to synthesize Fe3O4.
Magnetite synthesis (hence, cell magnetism)
was not necessary for growth or survival of the
magnetic bacterium studied under defined laboratory conditions. In nature, in contrast to the
laboratory, magnetism may play an important
role in cell survival. The very persistence of
magnetic bacteria in nature, despite metabolic
costs of accumulating and subsequently transporting as much as 2.0% of their dry weight as
iron, suggests that magnetite synthesis has an
adaptive significance. R. B. Frankel and R. P.
Blakemore (submitted for publication) showed
that the magnetite in cells of strain MS-1 constituted an efficient biological compass with several possible consequences. As previously suggested (5), magnetotaxis would direct magnetic
bacteria downward along the geomagnetic field
lines to 02-deficient areas more favorable for
growth. In the places they have been found, the
magnetic field of the earth is steeply inclined
(the angle of dip is roughly 700 from the horizontal), and magnetic bacteria swim unidirectionally northward (5, 16) and therefore downward. The microaerophilic nature of isolate MS1 is consistent with the possibility that magnetic
bacterial species benefit from their bio-compass
in this way. Their magnetism would also cause
cells to localize in regions of high magnetic flux
density surrounding materials with high magnetic susceptibility (e.g., particulate iron) in their
environment. We have observed localized
growth of magnetic cells at the ends of iron nails
placed in chemically defined culture medium
(unpublished data). Conceivably, localization of
these microaerophiles could promote their survival in nature by enabling them to collectively
render unfavorable microhabitats more favorable for survival (by locally reducing the amount
of 02, for instance).
Magnetotaxis, particularly at geomagnetic
field strengths, is very likely but one response of
a repertoire of behaviors which collectively determine the natural distribution of magnetic
cells. For instance, when magnetic cultures of
FRESHWATER MAGNETIC SPIRILLUM
VOL. 140, 1979
strain MS-1 were examined microscopically, approximately equal numbers of spirilla were observed swimming in either direction along the
north-south magnetic axis of the drop. This is
unlike the unidirectional migration of magnetotactic bacteria observed previously in marsh and
swamp muds (5, 16) and could be explained by
assuming that once oriented in the magnetic
field, a subsequent choice of swimming direction
by each spirillum was determined by its normal
forward and reverse swimming motions which,
in other spirilla (4, 12), are made in response to
gradients of oxygen or other chemicals.
Continuing studies of magnetic bacteria may
reveal their possible roles in iron biogeochemistry and in contributing to the paleomagnetic
record of the earth. Although several eucaryotes,
including bees and homing pigeons (22), which
are also geomagnetically responsive, are now
known to contain magnetite, axenic bacterial
systems hold the promise of being the most
useful for studying the biosynthesis of a magnetite compass. This, together with the need for
additional axenically cultivable bacterial species
for comparative studies of taxonomy, magnetism, and physiology, remain important attractions for microbiologists.
ACKNOWLEDGMENTS
We gratefully acknowledge the assistance with DNA measurements given by D. Green and R. Zsigray. Scanning electron microscopy and energy dispersive X-ray analyses were
carried out by J. McLane of the U.S. Geological Survey,
Woods Hold, Mass. We thank P. Brewer of Woods Hole
Oceanographic Institution and the Center for Institutional
and Industrial Development at the University of New Hampshire for the use of atomic absorption spectrophotometers. We
are grateful to D. L. Balkwill for help with electron microscopy, N. Blakemore for valuable technical assistance, and J.
Tugel for help with iron measurements.
This work was supported by National Science Foundation
grant PCM 77-12175.
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