Download Sulfonate-sulfur can be assimilated for fermentative growth

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FEMS Microbiology
Letters 129 (1995) 189-194
Sulfonate-sulfur can be assimilated for fermentative growth
Chih-Ching
Chien, E.R. Leadbetter
*, Walter Godchaux III
Department ofMolecular and Cell Biology, University of Connecticut, Storrs CT 06269-2131, USA
Received 28 March 199.5; accepted 4 April 1995
Abstract
Bacterial assimilation of sulfonate-sulfur under anaerobic conditions has been demonstrated.Two different bacteria able
to grow ferrnentatively using sulfonate-sulfuras sole sulfur source were isolated by enrichment culture; neither were able to
utilize sulfonates as sole source of carbon and energy for growth. The isolate of Clostridiunt pusteuriunum assimilated the
sulfur of isethionate (2-hydroxyethanesulfonate), taurine (2-aminoethanesulfonate), or p-toluenesulfonate. A facultatively
fermentative Klebsiellu strain did not utilize the sulfur of any of these sulfonates, but assimilated cysteate-sulfur; in contrast,
when growing by aerobic respiration, the range of sulfonates able to serve as sulfur source was greater. Both bacteria
displayed a preferential utilization of sulfate-sulfur to that of the sulfonates tested. Thus, bacterial assimilation of
sulfonate-sulfur during anaerobic growth has direct parallels with features until now recognized only for aerobic assimilatory
processes.
Keywords:
Sulfonate-sulfur assimilation; Anaerobic sulfonate transformation; Sulfur metabolism
1. Introduction
The organosulfur compounds termed sulfonates
contain a sulfur atom (oxidation state, +5) covalently linked to one of carbon. These compounds are
widely distributed in natural habitats as a result of
biosynthesis by quite diverse biota (e.g. [l-5]) as
well as chemical syntheses for commerce [6]. Although a few types of bacteria have been reported to
utilize some sulfonates as sole sources of carbon,
energy, and sulfur [7-111, many microbes utilize
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sulfonate-sulfur for growth even when unable to
grow at the sulfonate’s expense as carbon and energy
source [12-151.
In a recent study, Uria-Nickelsen et al. 1141noted
that several enteric bacteria utilized sulfonate-sulfur
as sole sulfur source for aerobic, respiratory growth,
but could not do so for fermentative growth. This
raised the prospect that sulfonate-sulfur might not be
subject to bacterial attack under anoxic conditions,
perhaps reflecting the need for molecular oxygen in
attack on the C-S linkage. In order to assess this
possibility, we employed enrichment culture in attempts to isolate soil bacteria capable of utilizing
sulfonate-sulfur as sole source for assimilation during fermentative growth under anaerobic conditions.
Societies. All rights reserved
C.-C. Chien et ai./FEMS
190
2. Materials
Microbiology
and methods
2.1. Strain isolation and identification
Bacteria were isolated from soil using a sulfonate
(cysteate, isethionate or taurine) as sole sulfur source
in glucose-mineral
salts medium (see below) at 25” C
under anaerobic
conditions
attained by use of
screw-capped culture tubes completely filled with
medium. Pure cultures were isolated on medium
solidified by addition of washed agar [131 and incubated in an anaerobic hood. The isolates were identified on the basis of morphology, staining characteristics, and selected determinative tests.
2.2. Chemicals
The highest purity chemicals available from Fisher
Scientific, Sigma Chemical, or Eastman Kodak were
employed. Radiolabelled
sulfur compound sources
were
[35S]sulfate
(ICN
Radiochemicals)
and
[35S]cysteate (synthesized from L-[35S1cysteine (ICN
Radiochemicals),
as described by Clark and Inouye
1161).
2.3. Media
Medium for Klebsiella
The minimal medium contained: glucose (55 mM)
as carbon and energy source; NH,Cl (10 mM) as
nitrogen source; mineral base (1171; modified so that
chloride salts replaced those listed as sulfate salts),
1% (v/v>; phosphate buffer (50 mM, pH 7.0) and
either sodium sulfate or a sulfonate (50 PM) as
sulfur source. The same medium was used for testing
the ability of a sulfonate to serve as sole carbon and
energy source except that 30 mM, 60 mM or 90 mM
of sulfonate replaced glucose as carbon source; these
levels of sulfonate were not growth-inhibitory
when
present with glucose. To determine whether a sulfonate could serve as electron acceptor for anaerobic
respiration, 0.5 mM, 1 mM or 5 mM sulfonate was
added to medium with growth-limiting
carbon and
energy source (glucose, 0.5 mM). Media were adjusted to pH 7.0 and sterilized before use.
Medium for Clostridium pasteurianum
The minimal medium for C. pasteurianum
contained: sucrose (55 mM) as carbon and energy source;
Letters 129 (199.5) 189-194
NH,Cl (10 mM) as nitrogen source; phosphate buffer
(0.1 Ml, 1% (v/v) mineral base, as above); biotin
(20 pg l- ’ > and either sulfate or a sulfonate (50
PM) as sole sulfur source. For testing a sulfonate as
sole carbon and energy source, 30 mM, 60 mM or 90
mM sulfonate replaced sucrose. Media were adjusted
to pH 6.0 and sterilized before use.
2.4. Isotopic competition
experiments
Klebsiella: aerobic growth
Cells were grown, with aeration, in glucosemineral salts medium with sulfate as sole sulfur
source to mid-exponential
phase (OD,,, = 0.5), harvested by centrifugation
(10000 X g), and the cell
pellets washed, resuspended
and recentrifuged
in
0.05 M potassium phosphate buffer (pH 7.5) three
times. Resuspended pellets were then transferred to
flasks containing minimal medium with either (in
control experiments) a radioactive sulfur compound
([35Slsulfate or i_35S]cysteate) or (for competition
studies) with both a radioactive sulfur compound (as
above) and a different non-radioactive
sulfur compound (sulfate, cysteate, taurine or isethionate). The
concentration
of each test compound was 50 PM
and the specific radioactivity of the radiolabelled one
was 1 $Zi pmol-‘.
During the growth period, l-ml
samples were removed at successive time intervals,
cells collected on filters (0.45 pm pore size, Gelman
Sciences) and washed with 5 ml of the phosphate
buffer. The radioactivity incorporated into cells was
then determined
by liquid scintillation
counting.
Counting rates were divided by the OD,,, of the
culture at the time of sampling to calculate specific
radioactivity values.
Klebsiella sp.: anaerobic growth
The same medium, specific radioactivity of radiolabelled sulfur source and filter method were used as
above except cultures were grown in closed culture
tubes containing a head-space gas mixture of H,,
20%, CO,, 20%, and N,, 60% (v/v/v).
Samples
were removed at successive time intervals, and the
cells collected by filtration for subsequent determination of radioactivity.
2.5. Clostridium pasteurianum
Cells were grown in minimal medium containing
[35Slsulfate (1 &i
kmol-‘1
as sole sulfur source
C.-C. Chien et al. /FEMS Microbiology Letters 129 (1995) 189-194
or, for competition tests, [35S]sulfate and a sulfonate
(either taurine or isethionate), each at 50 PM. Cells
were grown anaerobically and l-ml samples removed for assay of sulfate-sulfur incorporation into
cells, as described above.
2.6. Reproducibility
Experiments were replicated at least two times
and values obtained were reproducible within + 5%.
3. Results
3.1. Iden tijication
Positive results for tests of catalase, urease, lysine
decarboxylase and Pgalactosidase
activities, of
growth using citrate and inositol, dinitrogen ‘fixation’, and production of indole and acetoin along
with negative traits including oxidase, gelatinase,
arginine dihydrolase and omithine decarboxylase activities, or production of sulfide and acid (methyl red
negative) led to the identification of the non-motile,
Gram-negative facultatively anaerobic bacterium as
an isolate of Klebsiella, most likely K. oxytoca. The
motile, obligately anaerobic, Gram-positive endospore-forming
bacterium was identified as
Clostridium pasteurianum on the basis of traits including growth in sucrose-minimal medium, ability
to ‘fix’ dinitrogen, and acid and gas production.
191
3.2. Growth with sulfonates: Klebsiella
The isolate used the sulfur of many sulfonates as
sole sulfur source for growth under aerobic respiratory conditions, but only that of cysteate for fermentative growth (Table 1). Other sulfonates tested that
were not utilized under either growth mode were
metanilate, m-nitrobenzenesulfonate, sulfanilate, and
taurocholate.
The generation times for aerobic growth with
taurine or sulfate were essentially identical (72 min)
while those with isethionate or cysteate were somewhat longer (84 min); under fermentative conditions
the generation time with sulfate or cysteate was 120
min. None of the three sulfonates -cysteate,
isethionate, taurine - tested as sole carbon, energy
and sulfur source supported growth either by respiration or fermentation. None of these sulfonates served
as electron acceptor for anaerobic respiratory growth.
The Clostridium pasteurianum isolate assimilated
the sulfur of taurine, isethionate and p-toluene sulfonate for its strictly fermentative growth (Table 1).
Although taurine-sulfur supported cell yields identical to those with an equimolar amount of sulfate
(OD = 1.21, cultures using the sulfur of either
isethionate or p-toluene sulfonate grew to only
slightly more than one-half of that level. Generation
times were essentially identical (180 min) with sulfate, isethionate, or taurine. No growth resulted when
cysteate, isethionate or taurine were tested as a sole
sources of carbon, energy and sulfur.
Table 1
Growth of bacteria using sulfonate as sole sulfur source
Sulfur source
Sulfate
Taurine
Isethionate
Cysteate
HEPES
Methanesulfonate
pToluenesulfonate
Clostridium
Klebsiella
Aerobic growth mode
Fermentative growth mode
Fermentative growth mode
+
+
+
+
+
+
-
+
-
+
+
+ (0.7)
-
+
-
+ (0.7)
A ( + ) indicates an OD6so value of 1.0 or greater, except where lesser values are listed in parentheses; ( -) indicates a measured value of
0.1 or less; in the absence of an added sulfur source tbe measured value was 0.1 or less. HEPES, N-[2-Hydroxyethyl]piperaxine-N’-[2ethanesulfonate].
C.-C. Chien et al. / FEMS Microbiology
192
Letters 129 (1995) 189-194
J
I
60
30
Time
0
90
30
60
QO
brutes)
120
Time
160
I60
210
240
270
300
(mrutes)
Fig. 1. Incorporation
of “S into cells of a Klebsiella isolate
grown aerobically with [3sS]sulfate as sole sulfur source (triangles) and with both sulfate and non-radiolabelled
taurine present
as sulfur sources (circles).
Fig. 2. Incorporation of ‘5S into cells of Ckxtridium pusteurianum growing fermentatively
with [35S]sulfate as sole sulfur
source (triangles) and with both sulfate and a non-radiolabelled
taurine present as sulfur sources (circles).
3.3. Competition
isethionate
in non-radiolabelled
form (data not
shown) resulted in a decrease (approx. 50%) of
cysteate uptake. The Klebsiella used neither taurine
nor isethionate as sole sulfur source for fermentative
growth and the presence of either sulfonate had no
effect on incorporation of [ 35S]cysteate during fermentative growth (data not shown).
between sulfate and sulfonate
During aerobic growth, the Klebsiella sp. incorporated essentially identical amounts of [ “5S]sulfate
per unit of biomass (Fig. 1). no matter whether a
sulfonate was present or not, thus indicating utilization of sulfate-sulfur in preference to that of a test
sulfonate (taurine was the competitor for data of Fig.
1; cysteate and isethionate gave essentially identical
results). During fermentative
growth with cysteate
(the only test sulfonate able to serve as sole sulfur
source), cells incorporated sulfate at the same level
as when the sulfonate was absent (data not shown).
Thus sulfate-sulfur was used in preference to that of
cysteate during fermentative growth as well.
Clostridium pasteurianum
cells grown in media
with [35S]sulfate as sole sulfur source and those
grown in a medium containing both [ 35Slsulfate and
non-radiolabelled
taurine (Fig. 2) (or isethionate,
data not shown) likewise incorporated an identical
amount of [35S]sulfate per unit of biomass, thus
establishing preferential utilization of sulfate-sulfur
in this organism as well.
3.4. Competition
siella
between different sulfonates:
3.5. Comments
This report establishes for the first time the assimilation of sulfonate-sulfur
in bacteria growing anaerobically by fermentation, and these studies extend to
a strictly fermentative bacterium and to a facultatively fermentative one at least three findings until
now known only to be characteristic
of bacteria
Kleb0
For examining competition between different sulfonates as sole sulfur source, [ 35S]cysteate (the only
sulfonate readily available to us in radiolabelled
form) was used as a reference. During respiratory
growth the presence of either taurine (Fig. 3) or
30
Time
60
Qo
(mtrwtes)
Fig. 3. Incorporation
of 35S into cells of a Klebsiellu isolate
growing with [35S]cysteate as sole sulfur source (triangles) and
with both cysteate and non-radiolabelled
taurine present as sulfur
sources (circles).
C.-C. Chien et al. / FEMS Microbiology
growing by means of aerobic respiration. These traits
are (i) that sulfonate-sulfur
can be assimilated into
cellular compounds by organisms that are unable to
utilize the sulfonate as sole nutrient source, (ii) that
sulfate-sulfur
is utilized in preference to that of a
sulfonate
(when both are present in equivalent
amounts), and (iii) that the pattern, or specificity, for
utilization of sulfonate-sulfur
differs among different
bacteria.
The basis for this specificity for incorporation of
sulfonate-sulfur
into cellular compounds remains a
matter for further study, as does also the preferential
attack on sulfate-sulfur vs. that of a sulfonate. The
apparent cleavage of the sulfonate C-S bond under
anaerobic conditions indicates that molecular oxygen
is not a participant in the reaction, and thus makes it
likely that the enzymological features involved differ
from those for some aliphatic and aromatic sulfonates where molecular oxygen is a reactant [10,12].
It remains to be determined whether the inability of
the Klebsiella strain to assimilate, during fermentative growth, the sulfur of a sulfonate that serves as a
sole sulfur source for aerobic respiratory growth
results from the absence of a particular transport
mechanism or of an enzyme specific for attack on
the sulfonate.
Regardless of these important enzymological
details, it is clear that the desulfonations described here
are of potential significance for sulfonate cycling in
anoxic habitats. The carbon atoms, for example, of a
sulfonate unable to serve as a sole source of carbon
and energy could nonetheless become available for
assimilation by either the desulfonating bacterium or,
if the carbon skeleton were excreted, by other organisms.
Acknowledgements
This research was supported by the University of
Connecticut Research Foundation and the Institute of
Water Resources (US Geological Survey-Department
of the Interior, 14-OB-OOOl-G2009). We thank Angelica P. Seitz and Maria Uria-Nickelsen
for helpful
comments.
Letters 129 (1995) 189-194
193
References
01 Anderson R., Kates, M. and Volcani, B.E. (1978) Identification of the sulfolipids in the non-photosynthetic
diatom
Nitzschia nlba. B&him. Biophys. Acta 528, 89-106.
121Benson, A.A., Daniel, H. and Wiser, R. (1959) A sulfolipid
in plants. Proc. Natl. Acad. Sci. USA 45, 1582-1587.
131 Godchaux, W. III and Leadbetter, E.R. (1984) Sulfonolipids
of gliding bacteria: structures of the N-acylaminosulfonates.
J. Biol. Chem. 259, 2982-2990.
141 Koechlin, B.A. (1954) The isolation and identification of the
major anion fraction of the axoplasm of squid giant nerve
fibers. Proc. Natl. Acad. Sci. USA 40, 60-62.
[51 Taylor, C. and Wolfe, R.S. (1974) Structure and methylation
of coenzyme M (HSCH,CH,SO,)
J. Biol. Chem. 249,
4879-4885.
161Cain, R.B. (1994) Biodegradation of detergents. Curr. Opin.
Biotechnol. 5, 266-274
[71 Stapley, E. and Starkey, R. (1970) Decomposition of cysteic
acid and taurine by soil microorganisms.
J. Gen. Microbial.
64, 77-84.
k31Shimamoto, G. and Berk, R. (19791 Catabolism of taurine in
Pseudomonas
aeruginosa. Biochim.
Biophys. Acta 569,
287-292.
191 Ikeda, K., Yamada, H. and Tanaka, S. (1963) Bacterial
degradation of taurine. J. Biochem. 54, 312-316.
[lOI Thysse, G.J.E. and Wanders, T.H. (1974) Initial steps in the
degradation of n-alkane-1-sulfonates
by Pseudomonas. Antonie van Leeuwenhoek 40, 25-37
1111 Lecher, H., Thumheer, T., Leisinger, T. and Cook, A.M.
(1989) 3-Nitrobenzenesulfonate,
3-aminobenzenesulfonate,
and 4-aminobenzenesulfonate
as sole carbon sources for
bacteria. Appl. Environ. Microbial. 55, 492-494.
[121 Zurrer D., Cook, A.M. and Leisinger, T. (1987) Microbial
desulfonation
of substituted naphthalenesulfonic
acids and
benzenesulfonic
acids. Appl. Environ. Microbial. 53, 14591463
[131 Seitz, A.P., Leadbetter, E.R. and Godchaux, W. III (1993)
Utilization of sulfonates as sole sulfur source by soil bacteria
including Comamonas acidouorans. Arch. Microbial. 159,
440-444.
M.R., Leadbetter, E.R. and Godchaux, W.
[141 Uria-Nickelsen,
III (1993) Sulfonate utilization by enteric bacteria. J. Gen.
Microbial. 139, 203-208.
M.R., Leadbetter, E.R. and Godchaux, W.
1151 Uria-Nickelsen,
III (1993) Sulfonate-sulfur
assimilation by yeasts resembles
that of bacteria. FEMS Microbial. Lett. 114, 73-78.
[161 Clark, H.T., and Inouye, J.M. (1931) The alkaline deamination of derivatives of cysteine. J. Biol. Chem. 94, 541-550.
G., Sistrom, W.R. and Stanier, R.Y. (1957)
[171 Cohen-Bazire,
Kinetic studies of pigment synthesis by non-sulfur purple
bacteria. J. Cell. Comp. Physiol. 49, 25-68.