by I. J. Pickering and G. N. George
Sulfur is essential for all life, but
it plays a particularly central role in the metabolism of many anaerobic
microorganisms. Prominent among these are the sulfide-oxidizing bacteria
that oxidize sulfide (S2-) to sulfate (SO42-).
Many of these organisms can store elemental sulfur (S0) in "globules"
for use when food is in short supply (Fig. 1). The chemical nature of the
sulfur in these globules has been an enigma since they were first described
as far back as 1887 (1); all known forms (or allotropes)
of elemental sulfur are solid at room temperature, but globule sulfur has
been described as "liquid", and it apparently has a low density – 1.3 compared
to 2.1 for the common yellow allotrope a-sulfur.
Various exotic forms of sulfur have been proposed to explain these properties,
including micelles (small bubble-like structures) formed from long-chain
polythionates, but all of these deductions have been based upon indirect
evidence (for example the density was estimated by flotation of intact
cells), and many questions remained.
Using X-ray absorption spectroscopy
at the sulfur K-edge recorded on SSRL's Beam Line 6-2, Ingrid Pickering,
Graham George and Eileen Yu (SSRL) together with co-workers from Arizona
State University, University of British Columbia, and ExxonMobil Research
and Engineering Co. (2) have resolved this long-standing
X-ray absorption spectroscopy can be
used as a probe of the chemical nature of sulfur in intact cells
but spectral distortion due to self-absorption occurs in samples containing
localized high concentrations of sulfur such as in the globules. Self-absorption
arises from absorption of the X-ray fluorescence by the sample, and has
the effect of attenuating intense features in the spectrum. Pickering and
co-workers developed a simple mathematical model to exploit this artifact
to not only provide information on the chemical form of sulfur, but also
to give estimates of the product of the density and radius of the globule
(assuming spherical morphology). By fitting the spectra of standard compounds
to the spectra of bacterial cells and isolated globules, they found that
the globule sulfur most resembles the common yellow allotrope, a-sulfur.
The estimates of the product of the density and radius of the globules
reinforced this conclusion. When the density of a-sulfur
was used, radii commensurate with the results of microscopic examination
were obtained (~ 0.65 mm
for the bacterium Allochromatium vinosum, Fig. 2), but the literature
values for globule density yielded unrealistically large radii (~2
– larger than the cells in which the globules are contained. Other proposed
forms such as polysulfides showed spectra that were unlike those of the
globules, and it was concluded that these were not present (Fig. 2). In
support of these conclusions, calorimetric measurements of purified sulfur
globules showed phase-transitions consistent with a-sulfur
Optical micrograph of the giant bacterium Thiomargarita clearly
showing sulfur globules as the small approximately spherical structures
within the cell. This organism has particularly large cells (ca. ¾
mm), with correspondingly large (and more numerous) sulfur globules, which
makes them easy to observe microscopically.
|Figure 2: Sulfur
K near-edge spectra from globules extracted from the bacterium
Allochromatium vinosum in late logarithmic phase growth (points) in
comparison with spectra of a-S8,
S8 dissolved in xylene, and allyl polysulfides. The
spectrum is shown both undistorted (dashed line), and as calculated for
radius spheres (with density of 2.069 g.cm-3) measured in
which gave the best fit to the experimental data. The inset shows a log
plot of the residual as a function of the logarithm of the calculated radius
(r), and exhibits a well-defined minimum at –0.19, equivalent to a globule
radius of 0.65 m
Measurements were made of cultures
of seven quite different (taxonomically distinct) bacteria under various
growth conditions. For all globule-containing cultures, the spectra contained
a dominant component (globule sulfur) that strongly resembled the spectrum
expected for a-sulfur.
The structure of a-sulfur
contains S8 crowns (Fig. 3), as do several other forms (e.g.
the b- and
allotropes). Large-scale crystallites of a
(or other forms) can be excluded from previous X-ray diffraction results
(4), and Pickering and co-workers proposed that the globules
consist of a core of fragments with local structures resembling
with a modified globule surface conferring hydrophilic (water-attracting
properties (2). This might be due to the proteins that
are known to be associated with the globules, or modification of surface
sulfur by incorporation of polar groups such as thionates (5).
In either case, the surface sulfur content must constitute such a small
fraction of the total that it is unobservable by X-ray absorption spectroscopy.
Rather than any of the exotic or novel forms of sulfur that have been proposed,
bacteria appear to use sulfur in a form resembling the S8 crowns
of the chemically least surprising and thermodynamically favored
|Figure 3: The structure
of well-known S8 crown found in a-sulfur
(top) together with the structure of a long-chain polythionate molecule
(bottom), both of which have been proposed to be present in bacterial sulfur
Winogradsky, S. Botanische
Zeitung 1887, 31, 490-507.
Pickering, I. J.; George,
G. N.; Yu, E. Y.; Brune, D. C.; Tuschak, C.; Overmann, J.; Beatty, J. T.;
Prince, R. C. Biochemistry, 2001, 40, 8138-8145.
Pickering, I. J.; Prince,
R. C.; Divers, T.; George, G.N. FEBS Lett. 1998, 441,
Hageage, G. J., Jr.; Eanes,
E. D.; Gherna, R. L. J. Bacteriol. 1970, 101, 464-469.
Steudel, R. In: Autotrophic
Bacteria; Schlegel, H. G., Bothwien, B., Eds.; Springer-Verlag: Berlin,
1989; pp. 289-303.