AMER. ZOOL., 35:91-101 (1995)
Physiology and Biochemistry of Symbiotic and Free-Living
Chemoautotrophic Sulfur Bacteria1
DOUGLAS C. NELSON AND KARI D. HAGEN
Section of Microbiology, University of California, Davis, California 95616
SYNOPSIS. In this chapter, the known mechanisms that enable diverse
sulfur-oxidizing chemoautotrophic bacteria to conserve energy are summarized. The mechanisms known to be utilized by symbionts constitute
a relatively small subset of those used by free-living chemoautotrophic
bacteria; therefore, a search for additional pathways in symbionts should
be fruitful. The emerging evidence for the use of nitrate as an alternative
electron acceptor in sulfur oxidation by two types of symbionts is also
discussed. Thus far, the data are not completely consistent with the operation of either a classical dissimilatory or classical assimilatory nitrate
reductase. Lastly, previous literature calculations regarding the efficiency
of coupling between sulfur compound oxidation and carbon dioxide fixation are reexamined. For both free-living and symbiotic sulfur bacteria,
the published efficiencies are shown to have been overestimates by up to
four-fold due to an inappropriate assumption about the source of electrons
used for carbon reduction.
INTRODUCTION
At hydrothermal deep-sea vents and in
other sulfide rich environments, numerous
invertebrates nourished by symbiotic chemoautotrophic sulfur bacteria flourish
(Fisher, 1990). The biochemistry and physiology of sulfur-based energy generation by
these symbionts is the focus of this paper.
Since the study of symbiont energy metabolism is in its infancy, a discussion of energy
generation for free-living chemoautotrophic sulfur bacteria is included to provide
background and suggestions for future areas
of research. Two sulfur compounds, thiosulfate and hydrogen sulfide, and their various oxidation products will be considered.
The abundance of hydrogen sulfide at
hydrothermal vents and in reduced marine
sediments is well established. The importance of thiosulfate in these environments
has been recognized more recently based on
evidence showing (1) thiosulfate production
by free-living bacteria in oxic and anoxic
marine sediments (Jorgensen and Bak,
1991), (2) accumulation of thiosulfate in
blood of symbiont-containing invertebrates
(Anderson et al., 1987; Childress et al.,
19916), and (3) vigorous stimulation of
symbiont carbon fixation by this compound
(Nelson et al., 1995). Although oxygen is
the most common acceptor used by sulfuroxidizing bacteria, recently nitrate has been
postulated to act as an electron acceptor in
some symbiotic associations. A brief literature review of this subject appears here,
along with a reexamination of the thermodynamic efficiency of sulfur compound oxidation linked to carbon dioxide fixation for
both symbionts and free-living bacteria.
Throughout this paper, the term "sulfur"
is used in phrases such as "sulfur-oxidizing
bacteria" and "sulfur oxidation" to refer to
a variety of inorganic sulfur compounds
including H2S, polysulfides, S2O3=, S4O6°,
S° and SO3~, while the terms "elemental sulfur" or "S°" refer specifically to the elemental form.
ENZYMOLOGY OF SULFUR COMPOUND
OXIDATION
Among chemoautotrophic sulfur bacteria,
Thiobacillus is by far the best studied
1
From the Symposium Life with Sulfide presented
genus.
Between 1969 and 1981 approxiat the Annual Meeting of the American Society of Zoologists, 27-30 December 1993, at Los Angeles, Cali- mately 60 papers per year were published
fornia.
on this genus (Kelly, 1982), a rate roughly
91
92
D. C. NELSON AND K. D. HAGEN
unchanged today. The genus Thiobacillus is
a broad taxonomic group that includes
nearly all of the non-photosynthetic rodshaped aerobic eubacteria able to grow chemoautotrophically via sulfur compound
oxidation and carbon dioxide fixation
(Kelly, 1989). Most members of this genus
were isolated using aerobic enrichment cultures containing a source of reduced sulfur
and no added organic carbon. This yielded
a collection of bacteria that necessarily
shared the ability to oxidize sulfur compounds, but otherwise had little in common. The lumping of all thiobacilli into one
genus led prominent researchers to the
assumption that there would be "a fundamentally common oxidation mechanism
operating in all the thiobacilli and metabolically similar chemolithotrophs" (Kelly,
1982). Years have been spent searching for
this universal mechanism (Vishniac and
Santer, 1957; Peck, 1960; London and Rittenberg, 1964; Kelly, 1982), but only in the
past decade has it been recognized that a
truly universal mechanism for bacterial sulfur oxidation does not exist (Kelly, 1988).
The diverse pathways and enzymes used by
various sulfur bacteria for the oxidation of
reduced sulfur compounds are shown (Fig.
1). These are largely from studies with
thiobacilli, but other less thoroughly studied
autotrophs, e.g., various purple sulfur bacteria and Beggiatoa spp., have specific affinities with the symbionts. Hence, what is
known of their enzymology is also included.
Mechanisms for thiosulfate oxidation
employed by thiobacilli
While the thiobacilli can use a number of
reduced sulfur compounds as inorganic
electron donors, nearly all of the available
information concerns the oxidation of thiosulfate because the thiobacilli are most easily grown on this substrate (Kelly, 1989).
All pathways for thiosulfate oxidation eventually lead to the production of sulfite, as
either a free or an enzyme-bound intermediate; therefore, complete oxidation to sulfate can be thought of as occurring in two
steps: (1) the oxidation of thiosulfate to sulfite, and (2) the further oxidation of sulfite
to sulfate. The mechanisms for sulfite oxidation will be discussed first.
Sulfite oxidation proceeds by two different pathways in the thiobacilli. All thiobacilli examined to date have the enzyme sulfite: acceptor oxidoreductase (Kelly, 1989)
which catalyzes the direct oxidation of sulfite to sulfate with electrons entering an electron transport chain at the level of either
cytochrome b or cytochrome c (Yamanaka
etal, 1981; Tano etal., 1982; Kelly, 1988).
SO3" + H2O -» SO4=
2e"
2H
The electrons are passed to an electron
transport chain which conserves energy via
a chemiosmotic mechanism.
In addition, some thiobacilli have a second set of enzymes, adenosine phosphosulfate reductase (APS reductase) and ATP
sulfurylase, that act together in what has
been called the APS pathway (Kelly, 1982).
In the first step the flavin-containing enzyme
APS reductase catalyzes the AMP-dependent oxidation of sulfite to adenosine phosphosulfate (APS):
AMP + SO3" -> APS + 2eThe physiological electron acceptor for this
reaction is unknown. In the second step,
APS participates in a substrate-level phosphorylation reaction catalyzed by ATP sulfurylase. In this reaction, pyrophosphate is
consumed while sulfate and ATP are
released.
APS +
• SO4= + ATP
Although the enzyme ADP sulfurylase was
discussed in earlier reviews (e.g., Kelly,
1982), its existence has recently been questioned (Renosto et al., 1991). The enzymes
of the APS pathway are not specific to chemoautotrophs but also, operating in the
reverse direction, catalyze the first two steps
of dissimilatory sulfate reduction by anaerobic sulfate-reducing bacteria. There, the
otherwise energetically unfavorable formation of sulfite (an intermediate in H2S
production), is facilitated by participation
of pyrophosphatase (Segel et al, 1987). ATP
sulfurylase also functions in assimilatory
sulfate reduction, having been identified in
heterotrophic bacteria, fungi, green plants,
eukaryotic algae, and animals (Segel et al.,
1987).
In contrast to the oxidation of sulfite to
ENERGY METABOLISM OF SULFUR BACTERIA
93
A P S
FIG. 1. Enzymes and reactions used by thiobacilli, phototrophic sulfur-oxidizing bacteria, and Beggiatoa arranged
to show possible metabolic pathways for sulfide and thiosulfate oxidation. Enzymes: 1, sulfite: acceptor oxidoreductase; 2, APS reductase; 3, ATP sulfurylase; 4, ADP sulfurylase; 5, thiosulfate: acceptor oxidoreductase;
6, rhodaneses; 7, sulfur oxygenase; 8, siroheme sulfite reductase; 9, flavocytochrome or cytochrome. A dashed
line indicates that an enzyme catalyzing the reaction has not been characterized in its fully purified form. A
question mark indicates that the function of the suggested enzyme is speculative. Adapted from Brune (1989)
and Kelly (1989).
sulfate, the oxidation of thiosulfate to sulfite
is complex. A number of different mechanisms have been found in the thiobacilli,
four of which will be examined here. The
oxidation of thiosulfate by Thiobacillus tepidarius involves the formation of tetrathionate as an intermediate (Kelly, 1988).
A periplasmic enzyme, thiosulfate: cytochrome c oxidoreductase, condenses two
thiosulfate molecules to form tetrathionate.
Subsequent oxidation of the tetrathionate
to four sulfate molecules appears to occur
in the cytoplasm by an incompletely char-
acterized mechanism. Electrons derived
from this oxidation pass through the electron transport system to generate a proton
motive force which is employed, as in all
chemoautotrophic sulfur bacteria, to drive
ATP production, NAD(P) + reduction,
motility, and some transport mechanisms
(Harold, 1986; Kelly, 1988). Sulfite: acceptor oxidoreductase is thought to be a component of the tetrathionate-oxidizing
enzyme system (Kelly, 1989).
In contrast to T. tepidarius, the complete
oxidation of thiosulfate to sulfate by T. ver-
94
D. C. NELSON AND K. D. HAGEN
sutus occurs without the production of free
intermediates and is thought to be catalyzed
by a periplasmic multienzyme complex
including two multiheme, multiple redox
center c-type cytochromes (Lu et al., 1985;
Lu, 1986). Sulfite: acceptor oxidoreductase
is closely associated with one of these cytochromes (Lu and Kelly, 1984). The stoichiometric equation for the reaction catalyzed by the enzyme complex and the
associated sulfite: acceptor oxidoreductase
is as shown, with the resultant electrons participating in energy conservation by a
chemiosmotic mechanism (Kelly, 1988):
In T. denitrificans, the final example, rhodanese is again thought to catalyze the initial cleavage of thiosulfate (Bowen et al.,
1965). However, in contrast to the proposed
mechanism for T. novellus, the next step
does not involve molecular oxygen. Instead,
the sulfane sulfur moiety of thiosulfate is
oxidized by a siroheme sulfite reductase
operating in the reverse direction (Schedel
and Triiper, 1980) with the resultant electrons generating a proton motive force:
6e~
H2S + 3H2O - • SO3" + 8H
If, as shown for T. novellus, rhodanese
transfers the sulfane sulfur to a thiophilic
acceptor, this bound sulfur is likely the true
substrate of the above reaction. Once sulfite
is formed, it is oxidized via sulfite: acceptor
oxidoreductase or the APS pathway.
S2O3" + 5H2O -» 2SO4
10H
8e~
A third mechanism has been proposed for
T. novellus. Here, a membrane-associated
thiosulfate-oxidizing complex is thought to
be responsible for the oxidation of thiosulfate to sulfite, and three enzymes, rho- Oxidation ofsulfide and thiosulfate by
danese, sulfur oxygenase, and sulfite: accep- phototrophic sulfur bacteria
tor oxidoreductase (discussed above) are and Beggiatoa
believed to be involved (Oh and Suzuki,
The photosynthetic purple sulfur bacteria
1977a, b). The first step appears to be the perform anoxygenic photosynthesis using
cleavage of thiosulfate by rhodanese in a reduced sulfur compounds as electron
reaction which does not involve electron donors for CO2 reduction. Several, includtransport. In the presence of an acceptor ing Chromatium strains, have also been
(RSH) such as lipoic acid, or an alternative shown to be capable of growth as aerobic
physiological acceptor, rhodanese catalyzes chemoautotrophic sulfur oxidizers (Kampf
the following reaction:
and Pfennig, 1980). During the oxidation of
hydrogen sulfide or thiosulfate, some of the
S2O3" + RSH -» SO3
RSSH
purple sulfur bacteria and all Beggiatoa
Upon regeneration of RSH the equivalent strains produce intracellular deposits of elemental sulfur. This S° is visible microscopof an atom of S" is released.
The enzyme sulfur oxygenase is suggested ically and accumulates within the perito be responsible for the second step in thio- plasm, a characteristic of several sulfursulfate oxidation by T. novellus. This enzyme oxidizing symbionts (Vetter, 1985) but no
converts S* to sulfite with the addition of known Thiobacillus species. Chromatium is
also important for the current review
water and molecular oxygen:
because of its evolutionary relationship to
+
S° + H 2 O + O2 -* SO3" + 2H
the hydrothermal vent symbionts, to which
At present there does not appear to be a it is closer than virtually all thiobacilli (Lane
mechanism for energy conservation linked et al., 1992). The phylogenetic affinities of
to this oxidation. Because sulfur oxygenase autotrophic Beggiatoa strains have yet to be
activity was only observed when the mem- established.
brane complex was treated with trypsin (Oh
Because many of the phototrophic sulfur
and Suzuki, 19776), it has been suggested bacteria will not grow on thiosulfate, efforts
that the in vivo electron acceptor of this pro- with these organisms have focused primartein may be other than O2 (Wood, 1988). ily on their utilization of sulfide. Two
So far in this bacterium, only the oxidation enzymes, flavocytochrome c and siroheme
of sulfite has been clearly shown to be energy sulfite reductase, may be involved. Flavoconserving.
cytochromes c have been isolated from sev-
ENERGY METABOLISM OF SULFUR BACTERIA
eral Chromatium (purple-sulfur) and Chlorobium (green-sulfur) species, where they act
as sulfide dehydrogenases (Fukumori and
Yamanaka, 1979; Gray and Knaff, 1982;
Yamanaka and Kusai, 1976) catalyzing the
following reaction:
95
Cytochromes of the c-type are also probably involved in sulfide oxidation in the nonphototrophic genus Beggiatoa. Prince et al.
(1988) isolated cytochromes from native
chemoautotrophic Beggiatoa filaments collected near deep-sea hydrothermal vents.
These cells contained extremely high conH2S -»• S' + 2H+ + 2e~
centrations of an unusual high molecular
If the protons are liberated in the periplasm weight c-type cytochrome. No a-, b-, or
and electrons passed to a terminal oxidase fi?-type cytochromes or flavocytochromes
on the cytoplasmic side of the cell mem- were detected. More recently, Schmidt and
DiSpirito (1990) purified a flavocytobrane, a protonic potential results.
Several researchers have proposed that chrome c-554 from the heterotrophic freshother cytochromes might be involved in sul- water strain B18LD grown in sulfide-supfide oxidation in purple sulfur bacteria lack- plemented medium. Although these results
ing flavocytochromes. These include two are suggestive, the true function of these
cytochromes in Thiocapsa roseopersicina, cytochromes remains to be determined.
cytochrome c-550 or cytochrome c', both
of which can be reduced by sulfide (Fischer Conclusions regarding known mechanisms
and Triiper, 1977, 1979), a periplasmic for sulfur compound oxidation
cytochrome c-551 in Ectothiorhodospira
In the above examples, the initial steps
abdelmalekii (Then and Triiper, 1983), and in thiosulfate and sulfide oxidation vary
a sulfide- and thiosulfate-induced cyto- greatly between genera and among different
chrome 6-558 in E. shaposhnikovii (Brune, species within the same genus. Depending
1989). Whether these cytochromes are actu- upon the organism, polythionates and/or
ally involved in sulfide oxidation remains elemental sulfur may or may not be formed
to be proven.
as intermediates in the initial steps of thioSiroheme sulfite reductase, discussed pre- sulfate or sulfide oxidation. In addition, the
viously in relation to T. denitrificans, has roles of rhodanese, flavocytochrome c, and
also been proposed to catalyze sulfide oxi- sulfur oxygenase remain unclear. The final
dation in purple sulfur bacteria (Schedel et step in sulfur oxidation is fairly well underal., 1979). The presence of this enzyme only stood and is the only step where the concept
in photoautotrophically-grown C. vinosum of a universal mechanism still seems to
(not in cells grown photoheterotrophically) apply. Although free sulfite cannot always
suggests that the enzyme functions in sulfide be detected, all sulfur-oxidizing bacteria
oxidation rather than in sulfate assimila- examined to date appear to produce this
tion.
intermediate. Sulfite is then oxidized either
To date no enzyme has been identified in by sulfite: acceptor oxidoreductase or by the
photosynthetic sulfur bacteria or Beggiatoa APS pathway. This generalization also
that catalyzes the oxidation of elemental appears to hold for photosynthetic purple
sulfur. Although elemental sulfur appears to sulfur bacteria (Brune, 1989; Truper, 1989)
be the first intermediate in sulfide oxidation and autotrophic Beggiatoa strains (Hagen,
in nearly all of these organisms, it is unclear 1993).
how its further oxidation occurs. It has been
suggested that the formation of elemental Enzymology of symbiotic sulfur bacteria
sulfur is merely a side branch of the main
Fisher (1990) has tabulated the enzympathway for sulfide and thiosulfate oxida- ological evidence supporting the presence
tion (Brune, 1989). If this were true, then of chemoautotrophic sulfur bacterial symfurther oxidation of elemental sulfur might bionts in invertebrates from hydrothermal
not occur; rather, elemental sulfur would be vents, seeps and reducing-sediment envireduced back to sulfide prior to being oxi- ronments. ATP sulfurylase is the enzyme
dized directly to sulfite by another mecha- activity most commonly set forth as evinism.
dence of sulfur-based energy generation.
96
D. C. NELSON AND K. D. HAGEN
TABLE 1. ATP sulfurylase activities of symbiotic tissues and controls.
Organism/ tissue
Riftia pachyptila trophosome
Thyasira flexuosa gill
Myrtea spinifera gill
Bathymodiolus thermophilus
gill
Fungal mycelium
Spinach leaf
Rat liver
Activity
(units g"1
tissue)
Reference"
67-176
0.22-0.39
0.20-0.29
1
2
3
0.26-4.1
4
1
1
5
2.0
0.7
4.5-6.4"
1
References: (1) Renosto et al, 1991; (2) Dando and
Southward, 1986; (3) Dando et al., 1985; (4) Nelson,
unpublished; (5) Sundaresan, 1966.
b
From molybdolysis data, corrected for "minus
molybdate" control, assuming protein constitutes 10%
of wet wt.
This is, however, problematic because ATP
sulfurylase can also be utilized in sulfate
assimilation in heterotrophic bacteria, and
in eukaryotes such as fungi, algae, and some
animal tissues. Table 1 demonstrates that
there is not always a higher specific activity
of this enzyme associated with putative sulfur bacterial symbionts than with eukaryotic sulfate assimilators. Recently the gene
encoding the "autotrophic" ATP sulfurylase of the Riftia pacyhptila (tubeworm)
symbiont was cloned and sequenced (Laue
and Nelson, 1994), and a probe derived from
it has been used in Southern blot analysis
of DNA from a variety of free-living and
symbiotic bacteria. The results indicate that
the probe may be specific for the autotrophic ATP sulfurylase since no "false positives" have been obtained with bacteria
known to use ATP sulfurylase for sulfate
assimilation or anaerobic respiration.
As tabulated by Fisher (1990), the enzyme
APS reductase is sought less frequently as
support for sulfur-based chemoautotrophy
in symbioses. Since this enzyme is also
important in heterotrophic sulfate-reducing
bacteria (Kelly, 1982) its presence is not
unequivocal support for a sulfur-based
energy metabolism.
In addition to ATP sulfurylase, APS
reductase, and rhodanese, only one other
enzyme putatively involved in sulfur compound oxidation has been demonstrated for
tissue containing symbiotic sulfur bacteria.
By use of difference spectra Kraus et al.
(1992) have demonstrated that, among
potential electron donors, only H2S fully
reduced a cytochrome c-552 of the Solemya
reidi symbiont. The extent of reduction
increased monotonically with increasing
pH2S and was half maximal at a partial pressure of 1.4 torr. These results suggest
involvement of this cytochrome at or near
the point of entry of electrons into the symbiont electron transport system and have
parallels among free-living bacteria.
Key enzymes known from one or more
free-living sulfur oxidizing bacteria (see previous sections) not demonstrated to date in
any symbiont include: a thiosulfate-oxidizing multienzyme complex, tetrathionateforming or tetrathionate-oxidizing activities, sulfur oxygenase, siroheme sulfite
reductase, flavocytochromes, and sulfite:
acceptor oxidoreductase. Since the last
enzyme has been found in all strains of
Thiobacillus plus some strains of Beggiatoa
and Chromatium, failure to detect it thus
far in symbionts is particularly puzzling.
ANAEROBIC RESPIRATION OF
FREE-LIVING AND SYMBIOTIC
SULFUR BACTERIA
Very recently evidence has emerged indicating that symbiotic sulfur bacteria may be
able to utilize nitrate or nitrite as an electron
acceptor instead of oxygen. Nitrate utilization may either be preferential to the use
of O2 as in Lucinoma aequizonata symbionts (Hentschel et al., 1993) or unimportant
except under microoxic conditions as for
Riftia pachyptila symbionts (Hentschel and
Felbeck, 1993). Before discussing these
findings, the mechanisms by which free-living bacteria use nitrate as an electron acceptor will be reviewed.
The term denitrification refers to the
reduction of nitrate to any one of several
gaseous endproducts. N2 is by far the most
common product, but NO and N 2 O are normal intermediates in this reduction and are
sometimes the terminal product (Zumft,
1992). Denitrification is a bacterial respiratory process that couples the production
of a proton motive force to the stepwise,
sequential reduction of the various oxides
of nitrogen. The process is best viewed as a
coupling of three more or less independent
processes: nitrate respiration, nitrite respi-
97
ENERGY METABOLISM OF SULFUR BACTERIA
TABLE 2. Characteristics of nitrate reductase activities.
Characteristic
Final product of
NO 3 - reduction
Susceptibility to:
Azide
Cyanide
Subcellar location
Nitrate induction
Synthesis repressed by:
Oxygen
Ammonia
Classical
assinulatoiy
Classical
dissimilatory
Symbiont of
L. aequizonatcr
Symbiont of
R. pachyplilcf
NH4+
N2
NH 4 +
NO 2 "'
NOr"
low
low
high
high
membrane
high
high
soluble
ndc
high
membrane
+
nd
high
nd
nd
+
nd
nd
nd
soluble
+
+
a
No evidence of N2 or NH 4 + production.
If NO 3 " is limiting, NO2~ depletes over time to an unknown product, but NH 4 + does not accumulate.
c
nd indicates "not determined."
d
Vargas and Strohl, 1985 and references therein.
' Hentschel et al, 1993.
'Hentschel and Felbeck, 1993.
b
ration and nitrous oxide respiration (Zumft,
1992). Among the obligately chemoautotrophic, free-living, sulfur-oxidizing bacteria able to denitrify, one is obligate (Thiomicrospira denitrificans; Timmer-Ten Hoor,
1975) and one facultative (Thiobacillus denitrificans; Schedel and Triiper, 1980) for
respiration based on inorganic nitrogen
compounds.
The enteric bacteria, exemplified by
Escherichia coli, do not perform denitrification in the strict sense. Rather they will,
in the absence of oxygen, couple the oxidation of organic matter to the reduction of
nitrate to ammonia. Only the nitrate respiration step ( N ( V + 2e~ + 2H+ ->N 2 O+ H2O) is coupled to generation of a proton
motive force. The conversion of nitrite to
ammonia catalyzed by nitrite reductase does
not lead to the production of a proton motive
force but rather serves as an "electron sink"
(Stewart, 1988). E. coli and other enteric
bacteria, growing anaerobically with glucose
as a carbon source and electron donor in
the presence of a limiting quantity of nitrate
as the electron acceptor, convert nitrite to
ammonia in a non-respiratory process whose
only link to energy conservation is that of
allowing for a fermentation pathway more
efficient at substrate level phosphorylation.
Other free-living sulfur bacteria exhibit
more restricted and peculiar anaerobic uses
of nitrate. A freshwater strain of Beggiatoa
coupled very active anaerobic acetate oxidation to the reduction of nitrate or nitrite
to ammonia. Even though capable of aerobic sulfide oxidation, it could not couple
anaerobic sulfide oxidation to the reduction
of nitrate or nitrite. Anaerobic growth could
not be achieved with either nitrate or nitrite
regardless of electron donor (Vargas and
Strohl, 1985). The nitrate reductase activity
of Beggiatoa was soluble and could utilize
reduced flavins, but not NADH, as an electron donor. This nitrate reductase appears
to possess properties that are a mixture of
dissimilatory (respiratory) and assimilatory
nitrate reductases (see Table 2).
The Lucinoma aequizonata symbiont may
well be an obligate anaerobe. Purified symbionts showed the same rate of nitrite production from 1 mM NO3~ regardless of
whether the incubation was aerobic or
anaerobic (Hentschel et al., 1993), and no
O2 consumption could be demonstrated. The
membrane location of nitrate reductase and
inhibitor data are consistent with a classical
dissimilatory enzyme, but the lack of oxygen repression is not (Table 2). There is,
however, precedence in the literature for
aerobic denitrification (Robertson et al.,
1988). For the L. aequizonata symbiont neither sulfide nor thiosulfate stimulated nitrate
respiration (Hentschel et al., 1993), but this
might be due to internal S" serving as a store
of electron donor. More problematically,
98
D. C. NELSON AND K. D. HAGEN
TABLE 3. Energy costs and gains for sulfur based chemoautotrophy.
AC
(kj/mole)-
Reactions
Energy generation
CO, fixation
(1) S2O3" + 2O2 + H2O « 2SO4" 2H +
(2) HS" + 2O2 ~ SO4- + H+
6SO4(3) 6CO2 + 3S2O3- + 9H2O ~ (CH2O)
+ 6H +
(4) 6CO2 + 3HS" + 6H2O ~ (CH2O)6 + 3 SO4" +
3H+
(5) 6CO2 + 6H2O « (CH2O)6 + 6O2
iff
(per CO,)
-818.4"
-796.6
+366.7
+61.1
+432.3
+2,822.0
+72.1
+470.3
a
Standard free energies calculated from free energies of formation data tabulated by Thauer et al. (1977). As
indicated therein uncertainties of this method dictate that nonagreement with other published values is expected
in the second or third significant figure.
under anaerobic conditions NO3~ was not
observed to stimulate CO2 fixation (Hentschel et al., 1993).
The contribution of nitrate respiration to
the metabolism of the R. pachyptila symbiont is more difficult to assess. In this symbiont, unlike that of L. aequizonata, nitrite
production was stimulated by sulfur compounds (Hentschel and Felbeck, 1993).
Below 26 MM O2 (10% of air saturation), the
rates of oxygen- and nitrate-respiration were
statistically equivalent (Hentschel and Felbeck, 1993). This symbiont's capacity for
CO2 fixation, as measured by the specific
activity of ribulose-l,5-bisphophate carboxylase/oxygenase, far exceeds its nitrate
respiration capacity, while in the L. aequizonata symbiont the activities are roughly
comparable.
THERMODYNAMIC EFFICIENCY OF
SULFUR OXIDIZING BACTERIA
For symbiotic associations and free-living sulfur bacteria there exist numerous data
that demonstrate the molar ratio between
sulfur compound oxidized and carbon dioxide fixed into cell material. These are useful
in calculating thermodynamic efficiencies,
as defined below, with the following qualifications. A portion of the electron donor is
oxidized only to supply electrons (not
energy) for the reduction of CO2. Assuming
that average cell carbon is at the oxidation
level of CH 2 O, Table 3 shows the standard
free energy of CO2 fixation (Equations 3 or
4) associated with sulfide or thiosulfate as
the donor. This cost (AG") multiplied by the
moles of CO2 fixed yields an estimate of the
energy conserved by the autotroph. The
fraction of the sulfur compound not needed
to reduce CO2, but still available for oxidation (energy generation) can be readily
calculated (Kelly, 1982) and the second row
of entries for each organism (in bold, Table
4) reflects only this use of sulfur compound.
The product of moles of reductant oxidized
times the appropriate AG" (Equation 1 or
2, Table 3) shows the energy liberated from
this oxidation. The ratio between the energy
conserved in CO2 fixation and the energy
liberated in sulfur compound oxidation is a
measure of the thermodynamic efficiency of
chemoautotrophic sulfur bacteria. Efficiencies calculated in this way and reported in
the literature are very high for the R.
pachyptila symbiont (63%, Childress et al.,
1991), the S. reidi symbiont (26-73%,
Anderson et al., 1987), and the free-living
chemoautotroph Thermothrix thiopara
(29%, Mason et al., 1987). For free-living
chemoautotrophs in general, similarly high
efficiencies have been cited as evidence of
energy conservation mechanisms equal in
efficiency to those of heterotrophs (Kelly,
1990). In fact, chemoautotrophs do not
appear to be this efficient. The high efficiencies reported were calculated based on
water rather than sulfide or thiosulfate as
the source of electrons for CO2 reduction
(Kelly, 1990). The much higher standardfree energy cost (Equation 5, Table 3) of CO2
fixation associated with this very poor electron donor (actually appropriate only for
oxygenic photosynthesis) dictates that correctly calculated efficiencies are much lower.
The values now range from 3.2% to 15.4%
(Table 4) with the Riftia symbiont standing
out as unusually efficient. Two possible
explanations for its high calculated efficiency are suggested here. First, it may be
99
ENERGY METABOLISM OF SULFUR BACTERIA
TABLE4. Consumption ratios for sulfur compounds, oxygen and carbon dioxide, and thermodynamic efficiencies.
Organism
Beggiatoa sp. (chemoautotrophic)
Bathymodiolus thermophilus
symbiont
R. pachyptila whole animal
S. reidi whole animal
Sulfur
compound*
(moles)
O,
(mole)
CO,
(moles)
1.00
0.825"
1.00
0.82"
1.00
0.54"
1.00
0.85"
1.72
1.72
nd
nd
1.14
1.14
1.4
1.4
0.35
0.35
0.36
0.36
0.92
0.92
0.30
0J0
Thermodynamic
efficiency*1
References'
1
3.8%
2
33%
3
15.4%
4
3.2%
• Thiosulfate for B. thermophilus symbiont, otherwise H2S. For S. reidi only 100 MM H2S data are presented.
b
Calculated using free energy values of Table 3 as described in the text. In the first example (Beggiatoa)
"Thermodynamic Efficiency" is the energy cost of reducing 0.35 moles of CO2 with sulfide as the electron donor
divided by the energy gain from complete oxidation of 0.825 moles of sulfide. Complete oxidation of the sulfur
compound is assumed. This requires 2 moles of oxygen per mole of sulfur compound. Within experimental
error the O2 consumption rates support this.
c
References: (1) Nelson et al, 1986; (2) Nelson et ai, 1995; (3) Childress et al., 1991a; (4) Anderson et al,
1987.
" In the second row of data (bold) for each entry the portion of reductant supplying electrons for CO2 reduction
has been subtracted according to the method of Kelly (1982).
that the Riftia symbiont expends less energy
in reverse electron transfer than the other
bacteria. Use of a relatively low mid-point
potential cytochrome {e.g., a i-type rather
than a c-type) to transfer electrons from H2S
to NAD (P)+ could accomplish this. Alternately, a true steady-state may not have been
achieved in these difficult whole-animal
physiological experiments. Some of the H2S
consumed by the symbionts may have been
supplied from the host's blood without
replenishment from the surrounding experimental medium.
Obviously these efficiency calculations are
only approximate because standard reactant and product concentrations of 1 M will
not be approached inside or outside the cells.
Likewise the approach presented ignores the
costs of converting sugars into other precursor molecules and of polymerizing these
into cellular macromolecules. These latter
costs, however, are minimal compared to
the cost of CO2 fixation (Timmer-Ten Hoor,
1976).
CONCLUSIONS AND DIRECTIONS FOR
FUTURE RESEARCH
We have summarized the diversity of catabolic mechanisms known among the
thiobacilli and other free-living sulfur oxidizing bacteria. Their great variety is not
surprising since chemoautotrophic sulfur
bacteria occur among four of the five subdivisions of the Proteobacteria (Lane et al,
1992). Only a few of the enzymes summarized {i.e., ATP sulfurylase, APS reductase
and a c-type cytochrome involved in H2S
oxidation) have been demonstrated to date
in any sulfur-oxidizing symbiont. The close,
but not necessarily monophyletic, evolutionary relationship among the symbionts
(Distel et al., 1994; Lane et al., 1992) could
be the cause of a truly restricted catabolic
diversity; however, future priority should
be given to screening symbionts for additional catabolic enzymes, focusing on
whether there is an alternate path for sulfite
oxidation and on the initial steps in thiosulfate and sulfide oxidation. If, in this lastmentioned process, different symbionts
employ paths containing or lacking a i-type
cytochrome, the differences in thermodynamic efficiency tabulated in this review may
be explained. Whether the deposition of
internal sulfur globules represents a side
reaction or an obligatory intermediate in the
oxidation of H2S has not been established
for any chemoautotrophic sulfur bacterium—symbiotic or otherwise, but this
question might be most easily addressed
using pure cultures. The recent discovery
that nitrate may function as an alternate or
exclusive electron acceptor in some symbionts provides an additional exciting area
for future research. For the areas highlighted
100
D. C. NELSON AND K. D. HAGEN
in autotrophic and heterotrophic Beggiatoa strains.
Ph.D. Diss., University of California, Davis. 193
pp.
Harold, F. M. 1986. The vital force: A study of bioenergetics. W. H. Freeman, New York.
ACKNOWLEDGMENTS
Hentschel, U., S. C. Cary, and H. Felbeck. 1993.
This research was supported, in part, by
Nitrate respiration in chemoautotrophic symbionts of the bivalve Lucinoma aequizonata. Mar.
the National Science Foundation (OCEEcol. Prog. Ser. 94:35^11.
8800493, OCE-9018198, and a Graduate
Hentschel, U. and H. Felbeck. 1993. Nitrate respiFellowship to K.D.H.).
ration in the hydrothermal vent tubeworm Riftia
pachyptila. Nature 366:338-340.
Jorgensen, B. B. and F. Bak. 1991. Pathways and
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