Chapter 42

Microbiology of Nitrogen Fixation by Anoxygenic

Photosynthetic Bacteria

Michael T. Madigan
Department of Microbiology, Southern Illinois University, Carbondale, IL 62901-6508, USA

Summary 915
I. Background 916
A. Discovery of Fixation in Anoxygenic Photosynthetic Bacteria 916
B. Nitrogenases 916
C. Physiological Prerequisites for Fixation by Photosynthetic Bacteria 917
1. The Effect of Light 917
2. The Effect of Oxygen 917
II. Nitrogen Fixation by Purple and Green Bacteria 918
A. Purple Nonsulfur Bacteria 918
B. Purple Sulfur Bacteria (Chromatiaceae and Ectothiorhodospiraceae) 919
C. Green Sulfur Bacteria (Chlorobiaceae) 921
D. Green Nonsulfur Bacteria (Chloroflexaceae) 922
E. Heliobacteria (Heliobacteriaceae) 923
III. Phototrophic Rhizobia and Other Bacteriochlorophyll a-Containing Species 923
IV. Ecological Aspects of Fixation by Anoxygenic Photosynthetic Bacteria 924
V. Concluding Remarks 925
Acknowledgments 925
References 925

Summary
Anoxygenic phototrophic bacteria conduct many specialized metabolic processes but one of the most important
is nitrogen fixation, the reduction of to . Nitrogen fixation is catalyzed by the enzyme nitrogenase which
is widely distributed among anoxygenic phototrophs. The most comprehensive understanding of fixation in
photosynthetic bacteria is among the nonsulfur purple bacteria, where the capacity to fix is nearly universal.
In this group fixation occurs both in the light and in darkness and nitrogenase expression and activity is
highly regulated. Available evidence indicates that fixation is fairly widespread among purple and green
sulfur bacteria and probably universal among heliobacteria. By contrast, the thermophilic phototroph Chloroflexus
aurantiacus, a very few purple nonsulfur bacteria and green sulfur bacteria, and the ‘aerobic phototrophs’ are
not diazotrophic. The recently discovered bacteriochlorophyll-containing rhizobia are also nitrogen fixers but
their status as anoxygenic phototrophs is at present unclear. Although the ecological significance of fixation
by anoxygenic phototrophs is unknown, the widespread distribution of these organisms in nature and the fact
that most species are diazotrophic, suggests that they may well be significant, particularly in specialized
environments such as microbial mats and paddy soils.

R. E. Blankenship, M. T. Madigan and C. E. Bauer (eds): Anoxygenic Photosynthetic Bacteria, pp. 915–928.
© 1995 Kluwer Academic Publishers. Printed in The Netherlands.
916
I. Background
The biological utilization of dinitrogen as a
source of cell nitrogen is a process called nitrogen
fixation and is a property of only certain prokaryotes.
In the fixation process, is reduced to and the
latter converted into organic form. Nitrogen fixation
is catalyzed by the enzyme complex nitrogenase
(Burris and Roberts, 1993). The biochemistry and
genetics of nitrogen fixation in anoxygenic photo-
synthetic bacteria is discussed in detail in Roberts
and Ludden (1992) and in Chapter 43 of this volume
by the same authors. The present chapter documents
the occurrence of nitrogen fixation in anoxygenic
phototrophs, compares the efficacy of this process in
different species where possible, and discusses the
potential ecological ramifications of fixation by
photosynthetic bacteria as it relates to their
competitiveness in aquatic and terrestrial habitats.
For a general treatment of the microbiology of
fixation the reader is referred to the excellent reviews
by Eady (1992) and Young (1992).
A. Discovery of Fixation in Anoxygenic
Photosynthetic Bacteria
The first hint that anoxygenic photosynthetic bacteria
could fix molecular nitrogen emerged 45 years ago
during an investigation of light-dependent
production in the purple nonsulfur bacterium
Rhodospirillum rubrum (Gest and Kamen, 1949).
Cells growing photosynthetically on malate with
glutamate as nitrogen source evolved large quantities
of (now known to be a result of nitrogenase
activity, see Chapter 43 by Ludden and Roberts)
while parallel cultures grown on ammonia did not. In
experiments with resting cells, glutamate-grown cells
also photoproduced and this process was strongly
inhibited by the latter was a major clue that Rs.
rubrum was capable of nitrogen fixation (Kamen and
Gest, 1949). Confirmation of this was made by
measurement of the fixation of into cells of Rs,
rubrum and other species of purple bacteria
(Lindstrom et al., 1949), and several papers appearing
shortly afterwards suggested that the capacity to fix
nitrogen was widely distributed among purple and
green bacteria (Lindstrom et al., 1950,1951;Newton
and Wilson, 1953).
Abbreviations: Cf. – Chloroflexus; Cm. – Chromatium; Rb. –
Rhodobacter; Rc. Rhodocyclus; Rs. – Rhodospirillum; Tc. –
Thiocapsa
Michael T. Madigan
Since the early 1950s studies of particular species
of anoxygenic phototrophs and one major survey
(Madigan et al., 1984) of nitrogen fixation in purple
nonsulfur bacteria have indicated that most but not
all species can fix and that in addition, some
species are naturally ‘strong’ nitrogen-fixers (i.e.,
grow rapidly on ) while other species are more
feeble in this connection (see e.g., Madigan et al.,
1984). Such differences in efficacy of fixation
may reflect the ecological importance of fixation
to particular species but is undoubtedly also a
reflection of genetic and metabolic differences
between species of photosynthetic bacteria as well.
B. Nitrogenases
For many years it was thought that molybdenum was
absolutely required for fixation because of its
central importance as a metal cofactor of nitrogenase
(Bishop and Premakumar, 1992; Burris and Roberts,
1993). However it is now clear that biological
fixation can proceed in the absence of molybdenum
through the activity of alternative nitrogenases that
lack this element (see Bishop and Premakumar, 1992
for a recent review).
All nitrogenases consist of two proteins, dinitro-
genase and dinitrogenase reductase. Iron is present
along with molybdenum in classical dinitrogenase
(referred to as nitrogenase-1). In addition to
nitrogenase-1, two non-molybdenum nitrogenases
have been described, including one in which vanadium
substitutes for molybdenum in dinitrogenase (called
nitrogenase-2) and another in which iron only is
present in dinitrogenase (nitrogenase-3). Dinitro-
genase reductases from all forms of nitrogenase
contain iron as a metal cofactor. Each form of
nitrogenase is coded for by its own distinct gene
complex but significant amino acid sequence
homology exists among the three forms of nitro-
genase, especially among dinitrogenase reductases
(Bishop and Premakumar, 1992).
Among anoxygenic photosynthetic bacteria
alternative nitrogenases have been documented in
only four species. Iron-only (nitrogenase-3) type
nitrogenases have been characterized from the purple
nonsulfur bacteria Rhodobacter capsulatus (Schnei-
der et al., 1991; Shüddekopt, et al., 1993) and from
Rhodospirillum rubrum (Lehman and Roberts, 1991).
Physiological evidence for alternative nitrogenase
systems, presumably of the nitrogenase-3 type but
not proven unequivocally as such, has also emerged
Chapter 42 Nitrogen Fixation in Photosynthetic Bacteria
from studies of Rhodospirillum fulvum (Gogotov, et
al., 1991) and Heliobacterium gestii (Kimble and
Madigan, 1992b). Curiously, experiments with
Rhodobacter sphaeroides, a close relative of Rb.
capsulatus (Woese et al., 1984a), indicated that this
species lacked an alternative nitrogenase system
(Gogotov et al., 1991). In addition, to the author’s
knowledge no reports of alternative nitrogenases
from purple or green sulfur bacteria have been
published. It can thus be concluded that alternative
nitrogenases are present in at least some anoxygenic
phototrophs and are of the nitrogenase-3 type. As in
nonphotosynthetic bacteria, alternative nitrogenases
presumably allow species of photosynthetic bacteria
to continue diazotrophic growth in environments
limiting in molybdenum but containing sufficient
iron to otherwise satisfy the needs of nitrogen fixation.
C. Physiological Prerequisites for Fixation
by Photosynthetic Bacteria
Photosynthetic purple bacteria show remarkable
metabolic diversity and are capable of growing both
phototrophically (anaerobic/light) and in darkness
by both respiration and fermentation (Madigan and
Gest, 1979; Madigan, 1988; see also Chapter 41 by
Tabita). Rhodobacter capsulatus shows particularly
well-developed abilities in this connection, being
capable of growth under five distinctly different
growth modes: photoautotrophic, photoheterotrophic,
chemoorganotrophic by aerobic respiration, chemo-
organotrophic by fermentation, and chemolitho-
trophic with as electron donor ( as electron
acceptor, Madigan and Gest, 1979). Diazotrophic
growth by Rb. capsulatus is possible under at least
the first four of these conditions.
1. The Effect of Light
In the original discovery of fixation in photo-
synthetic bacteria (Gest and Kamen, 1949; Kamen
and Gest, 1949), photoheterotrophic growth
conditions were employed. All purple nonsulfur
bacteria capable of fixation (see Section II) grow
best diazotrophically under photoheterotrophic
conditions (Madigan et al, 1984). However, many
purple bacteria can also grow photoautotrophically
with or some other inorganic
substance as electron donor (Madigan, 1988);
fixation is also possible under these conditions.
Dark aerobic growth of purple nonsulfur bacteria
917
has been known since before the classic study of van
Niel (1944), but dark anaerobic growth, either by
fermentation (Uffen and Wolfe, 1970; Madigan and
Gest, 1978) or by various anaerobic respiratory means
(see Chapter 44 by Zannoni) was only discovered
more recently. Once dark anaerobic growth of purple
bacteria was established (Madigan and Gest, 1978) it
was possible to test hypotheses (Meyer et al., 1978a,b)
suggesting an obligatory link between photosynthesis
and fixation in these organisms. The lack of any
such connection was clearly shown in experiments
where Rb. capsulatus was grown anaerobically in
darkness with fructose as sole carbon and energy
source and as sole nitrogen source (growth under
these conditions also requires the presence of an
‘accessory oxidant’, such as dimethyl sulfoxide
(DMSO), to allow for catabolism of the fructose, see
Madigan and Gest, 1978; Madigan et al., 1980, and
Chapter 44 of this volume by Zannoni). Under dark
anaerobic conditions diazotrophic growth of Rb.
capsulatus occurred and cell suspensions readily
reduced acetylene to ethylene (a common assay of
nitrogenase activity) in darkness (Fig. 1 and Madigan
et al., 1979). Dark anaerobic nitrogen fixation has
also been reported in the heliobacteria. Anaerobic
cultures of all species of heliobacteria continue to fix
and grow slowly in darkness (Kimble and Madigan,
1992a) via fermentation of pyruvate, yielding acetate
and in some species (Kimble et al., 1994).
To the author’s knowledge, no report of dark
anaerobic fixation by purple sulfur bacteria has
been published, although dark microaerobic growth
(on ammonia) of some species is possible (Kämpf
and Pfennig, 1980) and several of these also fix
microaerobically in darkness (Jouanneau et al., 1980;
Kämpf and Pfennig, 1986). The ability of green
sulfur bacteria to fix in darkness is unknown; to
date, no one has successfully grown these organisms
in darkness under any growth conditions. Photo-
synthetically-grown cell suspensions of Chlorobium
species incubated in darkness showed essentially no
nitrogenase activity (Heda and Madigan, 1986a).
However, should conditions for dark growth of green
sulfur bacteria be discovered, fixation will probably
be possible here as well.
2. The Effect of Oxygen
Although nitrogenase is obviously protected from
oxygen inactivation during anaerobic growth
photosynthetically or in darkness (see Fig. 1), aerobic
918
dark growth on is problematic. Relatively little
is required to inactivate preexisting nitrogenase in
Rb. capsulatus (Hochman and Burris, 1981) and
other purple bacteria (Madigan et al, 1984), and is
also a potent represser of nitrogenase synthesis in
facultatively aerobic bacteria including purple
bacteria (Burris and Roberts, 1993). Thus, to achieve
aerobic dark diazotrophic growth, photosynthetic
bacteria must receive sufficient to carry out
respiration (for ATP production) while at the same
time prevent inactivation and/or repression of
synthesis of nitrogenase.
Conditions for aerobic diazotrophic growth of
anoxygenic phototrophs were first described by
Siefert and Pfennig (1980) using auxanographic
techniques. When molten agar media is seeded with
a culture of a photosynthetic bacterium and then
allowed to solidify, and diffuse downward and
respiratory growth on is observed as a tight band
of cells a defined distance from the agar surface; the
band represents the only zone in the agar tube where
levels are compatible with both diazotrophy and
Michael T. Madigan
respiration (Fig. 2). Interestingly, however, because
of differences in respiratory potential among species
of purple bacteria, the distance from the agar surface
that the growth band appears varies considerably.
Species such as Rb. sphaeroides, which grow well at
full oxygen tensions, form growth bands much nearer
the surface than do extremely microaerophilic species,
such as Rs. molischianum (Fig. 2). Using auxano-
graphic methods, dark microaerobic fixation
occurred in all diazotrophic purple nonsulfur bacteria
tested except for one strain of Rs. fulvum (Madigan et
al., 1984) and also occurred among certain species of
Chromatiaceae (Kämpf and Pfennig, 1986).
II. Nitrogen Fixation by Purple and Green
Bacteria
This section is divided into four parts, each developed
around a summarizing table listing the species of
photosynthetic bacteria in which fixation has
been documented. In addition, where data are
available, an indication of the relative efficacy of the
fixation process as measured by in vivo nitrogenase
activities in each species is given; such data identify
the ‘stronger’ and ‘weaker’ species of
photosynthetic bacteria (see Madigan et al. 1984).
A. Purple Nonsulfur Bacteria
In a major survey of 18 species of purple nonsulfur
bacteria performed in my laboratory over 10 years
ago all but one species, Rhodocyclus purpureus,
were found capable of fixation (Madigan et al.,
1984). Species of Rhodobacter, such as Rb. capsulatus
and Rb. sphaeroides, expressed high levels of
nitrogenase in vivo and grew most rapidly on
while species like Rhodopseudomonas palustris and
Rhodopila globiformis expressed comparatively low
nitrogenase levels and grew only very slowly on
(Table 1). A correlation between in vivo specific
nitrogenase contents and growth rate was observed
in this study, and strains of Rb. capsulatus consistently
showed the highest specific nitrogenase activities
and shortest generation times for photoheterotrophic
growth on (Madigan et al., 1984). This finding
supports the observation that enrichment cultures for
purple nonsulfur bacteria using the ability to fix
as selective agent commonly result in Rhodobacter
species, especially Rb. capsulatus (Gest et al., 1985).
Since the study of Madigan et al. (1984) was
Chapter 42 Nitrogen Fixation in Photosynthetic Bacteria
undertaken, several new species of purple nonsulfur
bacteria have been described and their nitrogen-
fixing status, if known, is included in Table 1.
Only two species of purple nonsulfur bacteria
have been shown by growth and acetylene reduction
experiments to be incapable of fixation. These
include Rc. purpureus (Masters and Madigan, 1983;
Madigan et al., 1984), and the Dead Sea halophile,
Rhodospirillum sodomense (Mack et al., 1993).
Subsequent probing of DNA from both of these
species with a nifH gene probe (nifH codes for
dinitrogenase reductase) that hybridizes strongly with
DNA from other species of purple bacteria confirmed
that Rc. purpureus and Rs. sodomense lacked nif
genes, thus explaining their failure to fix (PW
Ludden, GP Roberts and MT Madigan, unpublished
results). It is possible that the unusual habitatsof Rc.
purpureus and Rs. sodomense, a swine waste lagoon
rich in ammonia and amines (Pfennig, 1978) and the
Dead Sea (Mack et al., 1993), respectively, have for
some reason(s) selected for an inability to fix in
these phototrophs.
An interesting situation exists in regard to
fixation in the moderately halophilic species
Rhodospirillum salexigens. This organism was
919
originally described as a glutamate auxorroph (Drews,
1981), however experiments in my laboratory showed
this to be unfounded (Rubin and Madigan, 1986).
Although Rs. salexigens grows well either photo-
trophically or chemotrophically (aerobic/dark) in
media containing glutamate as nitrogen source,
utilization of ammonia or as sole nitrogen source
is also possible. For unknown reasons substitution of
for glutamate in mineral media containing
acetate (the preferred carbon source for Rs. salexigens,
Drews, 1981) leads to a rapid rise in pH and cessation
of growth. However, modifications to the medium,
particularly the addition of pyruvate in addition to
acetate, stabilized pH and supported good growth of
Rs. salexigens on either ammonia or on (Rubin
and Madigan, 1986).
Table 1 summarizes the microbiology of fixation
in purple nonsulfur bacteria.
B. Purple Sulfur Bacteria (Chromatiaceae and
Ectothiorhodospiraceae)
Documentation of fixation in purple sulfur bacteria
is sketchy outside of a few species of the genus
Chromatium. Chromatium vinosum has been well
920
studied as to its -fixing capabilities and was the
first phototrophic bacterium in which consistently
active nitrogen-fixing cell-free extracts were obtained
(Winter and Arnon, 1970). In vivo studies of
nitrogenase in Cm. vinosum have also shown that the
organism employs the ammonia ‘switch-off effect
to control nitrogenase activity, as in purple nonsulfur
Michael T. Madigan
bacteria (Gotto and Yoch, 1985). In addition to Cm.
vinosum, dinitrogen fixation has been documented in
six other species of Chromatium, four species of
Ectothiorhodospira, two species of Thiocapsa, and
in a single species each of the genera Amoebobacter,
Thiocystis, and Lamprobacter (Zakhvataeva et al.,
1970; Postgate, 1982; Caumette et al., 1985; Ventura
Chapter 42 Nitrogen Fixation in Photosynthetic Bacteria
et al., 1988; Pfennig and Trüper, 1989; Young, 1992).
Of these organisms, Tc. roseopersicina is probably
the most widespread in nature and is frequently
found as a component of microbial mats (see
Chapter 4 by van Gemerden and Mas) where it may
be a major nitrogen-fixing species.
The capacity for fixation has not been reported
for species of Lamprocystis, Thiodictyon, Thiopedia,
Thiorhodovibrio and Thiospirilium. Thus the family
Chromatiaceae obviously needs more study as regards
nitrogen fixation. The current status of fixation in
purple sulfur bacteria is summarized in Table 2.
C. Green Sulfur Bacteria (Chlorobiaceae)
Our knowledge of nitrogen fixation in green sulfur
bacteria is somewhat more developed than that of
purple sulfur bacteria, although the story is far from
complete. Clear evidence for fixation has been
obtained with several species of Chlorobium and
with single species each of the genera Pelodictyon,
Prosthecochloris and Chloroherpeton (see Table 3
and Bergstein et al., 1981; Heda and Madigan,
1986a,b; 1988; Rodionov et al., 1986; Gibson et al.,
921
1984). Interestingly, like purple bacteria, nitrogen-
fixing cultures of Chlorobium species showed
ammonia ‘switch-off of nitrogenase activity (Heda
and Madigan, 1986a; Rodionov et al., 1986; Wahlund
and Madigan, 1993), indicating that this form of
regulation, apparently universal among nitrogen-
fixing purple bacteria (see Chapter 43 by Ludden
and Roberts), extends also to the green sulfur bacteria,
a phylogenetically distinct lineage from that of the
purple bacteria (Gibson et al., 1985; Oyaizu et al.,
1987).
The moderately thermophilic green sulfur
bacterium Chlorobium tepidum (Wahlund et al., 1991)
is an excellent nitrogen-fixing bacterium. This
organism, which has a generation time of as little as
2 hr on ammonia, also grows rapidly on and intact
cells contain extremely high levels of nitrogenase
(Wahlund and Madigan, 1993). In a study of the
mesophilic green bacterium Cb. limicola Heda and
Madigan (1986a) also observed high levels of
nitrogenase in vivo. These facts, coupled with the
observations that Cb. limicola strongly derepressed
nitrogenase when grown on glutamate as nitrogen
source yet did not photoproduce (Heda and
922
Madigan, 1986a) and that nitrogenase from this
organism did not cross-react immunologically with
that of other diazotrophs (Heda and Madigan, 1988),
warrants more thorough characterization of the
nitrogenase system of green sulfur bacteria.
Interestingly, despite the good nitrogen-fixing
properties of Chlorobium tepidum, its thermophilic
counterpart among purple sulfur bacteria, Chroma-
tium tepidum (Madigan, 1984, 1986), was found
incapable of fixation (Madigan, 1986) despite its
apparent possession of nif genes (PW Ludden, GP
Roberts and MT Madigan, unpublished results). The
reason for the lack of growth on by Chromatium
tepidum remains unknown. Although fixation in
Chlorobium tepidum was found to require elevated
levels of sulfide (which was otherwise not required
for growth of this thiosulfate-utilizing green
bacterium, Wahlund et al, 1991; Wahlund and
Michael T. Madigan
Madigan, 1993), elevated levels of sulfide do not
support fixation in Chromatium tepidum.
Chlorobium tepidum grows on up to its growth
temperature maximum of 52 °C (Wahlund and
Madigan, 1993) and this is the highest temperature
reported for fixation by any green sulfur bacterium.
The current status of fixation in green sulfur
bacteria is summarized in Table 3.
D. Green Nonsulfur Bacteria (Chloroflexaceae)
Although phenotypically resembling green sulfur
bacteria in terms of pigments and the production of
chlorosomes, green nonsulfur bacteria (the Chloro-
flexus group, see Chapter 3 by Pierson and
Castenholz) are phylogenetically unrelated to green
sulfur bacteria (Gibson et al., 1985; Oyaizu et al.,
1987). Study of the potential of four strains
Chapter 42 Nitrogen Fixation in Photosynthetic Bacteria
of Chloroflexus aurantiacus showed the organism to
be totally unable to fix (Heda and Madigan,
1986b). This result was supported by the complete
absence of hybridization of Cf. aurantiacus DNA to
a nifH probe (PW Ludden, GP Roberts and MT
Madigan, unpublished results). Cf. aurantiacus grows
well on any of several amino acids (Heda and
Madigan, 1986b) including ones that normally allow
for derepression of nitrogenase in purple bacteria
(Arp and Zumft, 1983). Despite this, however, cultures
of Cf. aurantiacus grown on amino acids or on
growth-limiting levels of ammonia failed to reduce
acetylene or photoproduce (Heda and Madigan,
1986b). One published report of growth of Cf.
aurantiacus in a medium lacking combined nitrogen,
presumably at the expense of (Gallon and Chaplin,
1987), has not been confirmed in my laboratory, and
it is possible that the slight growth observed was due
to trace levels of fixed nitrogen compounds in the
medium (J.R. Gallon, personal communication).
Weak but detectable fixation has been reported
(Keppen et al., 1989) in the newly described green
filamentous bacterium Oscillochloris trichoides,
which is phylogenetically related to Cf. aurantiacus
(Keppen et al., 1994). Thus it is possible that
Cf. aurantiacus strains exist but have thus far
not been cultured. Temperature is obviously not a
barrier here because fixation in thermophilic green
sulfur bacteria (Wahlund and Madigan 1993) and
thermophilic heliobacteria (Kimble et al., 1995)
occurs within the temperature range of optimal growth
of Chloroflexus (50–55 °C). Because of the more
extreme thermophilic character (growth up to 72 °C,
see Chapter 3 by Pierson and Castenholz) and unique
phylogeny (Gibson et al., 1985; Oyaizu et al., 1987)
of Chloroflexus, the properties of species,
if they exist, should be very interesting.
E. Heliobacteria (Heliobacteriaceae)
All known species of heliobacteria fix nitrogen
(Kimble and Madigan, 1992a; Kimble et al., 1995).
Heliobacillus mobilis appears to be the best
species presently in culture, both in terms of
its ability to grow rapidly on and in expression of
nitrogenase (Kimble and Madigan, 1992a). Even
thermophilic heliobacteria are diazotrophic and fix
up to 55°C (Kimble et al., 1995). Thus nitrogen
fixation may be of strong selective advantage to
heliobacteria in their soil, and especially rice soil,
habitat (see Chapter 2 by Madigan and Ormerod).
923
All species of heliobacteria examined by Kimble
and Madigan (1992a) and Kimble et al. (1995) were
subject to ammonia ‘switch-off’ of nitrogenase
activity. This finding in the heliobacteria, which
phylogenetically are Gram-positive bacteria (Woese
et al., 1985), suggests that this form of enzyme
regulation is of such strong survival value for
anoxygenic phototrophic bacteria that it has evolved
across deep evolutionary lines from Gram-negative
to Gram-positive phototrophs (ammonia ‘switch-
off’ in heliobacteria is the first such instance reported
from Gram-positive bacteria, Kimble and Madigan
1992a). Finally, as previously discussed (see Section
IB), alternative (non-molybdenum) nitrogenases are
present in certain heliobacteria, the best example
being Heliobacterium gestii (Kimble and Madigan,
1992b).
A summary of nitrogen-fixation in heliobacteria is
given in Table 4.
III. Phototrophic Rhizobia and Other
Bacteriochlorophyll a-Containing Species
A number of Rhizobium species have been discovered
that contain bacteriochlorophyll a. These, along with
the ‘aerobic phototrophs’ (see below) have been
termed ‘quasi-photosynthetic bacteria’ by Gest (1993)
because they share certain properties with true
anoxygenic phototrophs but lack many other
characteristic traits (e.g. the ability to grow
anaerobically, see Chapters 6 by Shimada and 7 by
Fleischman et al. for a description of these organisms).
Bacteriochlorophyll a-containing Rhizobium
species (now referred to as Photorhizobium species,
see Chapter 7 by Fleischman et al.) nodulate the
stems of several different, primarily tropical,
leguminous plants. As is well-known, a prime
physiological characteristic of Rhizobium is its ability
to fix and evidence exists that Photorhizobium
fixes both in association with host plants in stem
nodules and ex planta in pure culture in the laboratory.
It is hypothesized that photophosphorylation by
Photorhizobium at least partially supports the energy
needs of fixation in stem nodules; this in turn
would reduce the energy drain on the plant leading to
more efficient symbiotic nitrogen fixation.
Several aerobic BChl a-containing bacteria have
been isolated by Japanese workers and others in the
last 15 years. These organisms, referred to as ‘aerobic
phototrophs’, are mainly marine and grow best under
924
fully aerobic conditions (Shiba and Harashima, 1986,
and see also Chapter 6 by Shimada). However, no
evidence for fixation by aerobic phototrophs has
emerged and gene probe experiments (using a nifH
probe) carried out on two species failed to show any
hybridization of the probe to their chromosomal
DNA (PW Ludden, GP Roberts and MT Madigan,
unpublished results). Thus, all available evidence
suggests that the ‘aerobic phototrophs’ are nondiazo-
trophic.
IV. Ecological Aspects of Fixation by
Anoxygenic Photosynthetic Bacteria
With the exception of a few well studied lake
ecosystems where mass developments of anoxygenic
phototrophic bacteria occur (see review of Madigan,
1988 and Chapter 4 of this volume by van Gemerden
and Mas), few data are available on the contributions
of photosynthetic bacteria to the carbon balance of
aquatic or terrestrial habitats. Much the same can be
said about nitrogen fixation. A few reports of in situ
nitrogen fixation by photosynthetic bacteria have
been published (see e.g. Kobayashi et al., 1967;
Habte and Alexander, 1980; Bergstein et al., 1981),
but too little data exist to generalize on a global basis.
The nitrogen-fixing activities Of photosynthetic
bacteria in specialized habitats such as paddy soils or
microbial mats, however, may be very ecologically
significant (Kobayashi and Haque, 1971). In a detailed
report on the distribution of nitrogen-fixing
Michael T. Madigan
microorganisms in paddy soils of Southeast Asia,
Kobayashi et al. (1967) clearly implicated anoxygenic
phototrophs, particularly purple nonsulfur bacteria,
as contributors to the nitrogen economy of these
soils. A later study of fixation in rice soils by
Habte and Alexander (1980) employed acetylene
reduction methods and showed more directly the
importance of anoxygenic photosynthetic bacteria to
the fertility of paddy soils. Presumably photosynthetic
bacteria growing in such environments utilize organic
substrates leached from the rice plants or as
electron donors for reduction of to ammonia. In
addition, microbial mats (see Chapter 4 by van
Gemerden and Mas) can be major sources of
fixation by anoxygenic phototrophs; the common
occurrence of the diazotroph Thiocapsa rose-
opersicina, a purple sulfur bacterium with well
developed aerobic/dark metabolic capacities
(Jouanneau et al., 1980) in such habitats suggests
that this organism is a major contributor to fixed
nitrogen inputs therein.
Another link between the paddy soil environment
and photosynthetic bacteria is the recent discovery
of the heliobacteria. These anoxygenic phototrophs
are terrestrial organisms and are particularly abundant
in rice soils (see Chapter 2 by Madigan and Ormerod
for a discussion of the heliobacteria). Because
heliobacteria are active nitrogen-fixers (Kimble and
Madigan, 1992a,b; Kimble et al., 1995) and also
capable of dark growth (Kimble et al., 1994), it is
likely that these organisms contribute fixed nitrogen
to paddy soil environments (see also Madigan, 1992
Chapter 42 Nitrogen Fixation in Photosynthetic Bacteria
for a discussion of the ecology of heliobacteria).
The ability of photosynthetic bacteria to fix can
be used to advantage in their enrichment. This is
particularly true of purple nonsulfur bacteria (Gest
et al., 1985) and heliobacteria (Madigan, 1992),
however experiments in this laboratory have shown
that diazotrophic enrichments also work for the
isolation of purple and green sulfur bacteria
(unpublished results and Heda and Madigan, 1986a).
Combining these observations with the fact that
results thus far have shown that most photosynthetic
bacteria can fix (see Section II), it stands to reason
that fixation is of ecological importance to
anoxygenic phototrophs, at least under certain
environmental conditions, and that their diazotrophic
capacity allows them to compete better in their aquatic
and terrestrial habitats. However, now that phylo-
genetic probes, biomarker analyses, and other highly
specific analytical methods are available for
characterizing the microbiology of virtually any
habitat, it would be worthwhile to investigate the
activities of photosynthetic bacteria in situ
using these modern techniques. Only from well
controlled field studies that specifically target a group
or groups of anoxygenic phototrophs will the real
contributions of these organisms to the nitrogen
economy of soils and waters be known.
V. Concluding Remarks
In conclusion it can be stated that fixation is a
universal property of heliobacteria and nearly
universal among purple nonsulfur bacteria; however,
much is yet to be learned about this major
physiological process in other groups of anoxypho-
totrophs. It will be of interest in future years to test
more isolates of photosynthetic bacteria for the
capacity to fix especially new species isolated
from extreme environments or from marine
environments (the latter of which have only been
minimally explored, Imhoff 1983; Mangels et al.,
1986; Wynn-Williams and Rhodes 1974), and to
better understand the nature and distribution of
alternative nitrogenases among anoxygenic photo-
trophs. Such studies will yield a more complete
picture of the diazotrophic systems of photosynthetic
bacteria and should complement our new evolutionary
insight into these organisms, born with the advent of
ribosomal RNA sequencing (Woese, 1987; 1992).
925
The prominence of purple bacteria as likely ancestors
of the majority of known Gram-negative bacteria
(see Woese et al 1984a, b, and reviews of Woese
1987, 1992) and the heliobacteria of Gram-positive
bacteria (Woese et al., 1985) mandates that we
understand important metabolic processes such as
fixation in anoxygenic phototrophs in considerably
more detail.
Acknowledgments
Work on fixation in photosynthetic bacteria in my
laboratory has been supported by the United States
Department of Agriculture and the National Science
Foundation. I thank Linda Kimble for unpublished
results.
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