Enzymology and Ecology of the Nitrogen Cycle
Oxygen control of nitrogen oxide respiration,
focusing on α-proteobacteria
James P. Shapleigh1
Department of Microbiology, Cornell University, Ithaca, NY 14853, U.S.A.
Abstract
Denitrification is generally considered to occur under micro-oxic or anoxic conditions. With this in mind,
the physiological function and regulation of several steps in the denitrification of model α-proteobacteria
are compared in the present review. Expression of the periplasmic nitrate reductase is quite variable,
with this enzyme being maximally expressed under oxic conditions in some bacteria, but under micro-oxic
conditions in others. Expression of nitrite and NO reductases in most denitrifiers is more tightly controlled,
with expression only occurring under micro-oxic conditions. A possible exception to this may be Roseobacter
denitrificans, but the physiological role of these enzymes under oxic conditions is uncertain.
Introduction
The processes that together define the nitrogen cycle are
largely mediated by bacteria [1]. Oxygen strongly influences
the growth and physiology of bacteria catalysing reactions
in the nitrogen cycle. For example, nitrification occurs
under oxic conditions, whereas anaerobic ammonia oxidation
(annamox), as its name indicates, occurs under anoxic
conditions. Other processes, such as nitrogen fixation, are
negatively affected by oxygen, but some organisms have
developed strategies allowing nitrogen fixation to occur under
oxic conditions [2]. Processes linked with nitrate reduction,
such as denitrification or ammonification, occur mainly
under anoxic conditions. The net result of these varying
sensitivities is that flux through the various processes changes
dramatically in response to shifts in oxygen.
Denitrification, the dissimilatory reduction of nitrate to
nitrogen gas, is regarded as an anoxic or micro-oxic process
[3]. Complete denitrification requires four enzymes: nitrate
reductase, nitrite reductase (Nir), nitric oxide reductase (Nor)
and nitrous oxide reductase (Nos). Interestingly, no strictly
anaerobic denitrifier has ever been isolated. Since denitrifiers
are facultative aerobes, this means that they must choose
between oxygen and nitrate if both are available. Oxygen
is preferred by most isolates, which is generally ascribed to
the fact that more energy is gained from oxygen respiration
than nitrogen oxide respiration. However, with the exception
of Nos, none of the terminal nitrogen oxide reductases are
oxygen-sensitive, making it possible that the denitrification
enzymes could be used under oxic conditions [4].
Oxygen preference is based on its advantages during
respiration. However, it has become obvious that in some
cases nitrogen oxide reductases are not being used for
Key words: Agrobacterium tumefaciens, denitrification, nitrate reductase, nitric oxide reductase,
α-proteobacterium, Rhodobacter sphaeroides.
Abbreviations used: Nir, nitrite reductase; Nor, nitric oxide reductase; Nos, nitrous oxide
reductase.
1
email [email protected]
Biochem. Soc. Trans. (2011) 39, 179–183; doi:10.1042/BST0390179
respiration. A good example of this is provided by examining
the nitrogen oxide reductases present in the four Rhodobacter
sphaeroides strains that have had their genomes sequenced.
Analysis of the denitrification potential has showed that
strain 2.4.3 has all four reductases, strain KD131 is missing
nitrate reductase but has the remaining three, strain 2.4.1
has nitrate reductase and Nor, whereas strain 2.4.9 has
only Nor. The latter two strains and possibly KD131 are
unlikely to use nitrogen oxide reductases for respiration
under micro-oxic conditions. This suggests an alternative
physiological role for these enzymes and, by extension, it
is possible the bioenergetic rationale for their repression
under oxic conditions is no longer valid. With this in mind, I
want to briefly summarize our current understanding of the
physiological roles and expression of nitrate reductase, Nir
and Nor in a few α-proteobacterial denitrifiers.
Nap regulation in select α-proteobacteria
There are several different types of nitrate reductase [5].
Two dissimilatory forms, Nar and Nap, are located in
the membrane and periplasm respectively. The assimilatory
form, Nas, is found in the cytoplasm. Denitrifiers will
contain Nap and/or Nar. The α-proteobacterial denitrifiers
my laboratory has worked with, R. sphaeroides and
Agrobacterium tumefaciens, contain Nap. A. tumefaciens also
has Nas, whereas R. sphaeroides lacks this enzyme.
In R. sphaeroides strain 2.4.1, the nap operon is located on
a plasmid [6]. Recent work has shown that expression of the
nap operon increases significantly when cells are shifted from
anoxic photosynthetic conditions to oxic conditions [7]. The
medium used for these experiments was not supplemented
with nitrate, suggesting that the expression of nap does not
require nitrate. Expression under low oxygen conditions was
enhanced in a mutant lacking PrrA, the response regulator
of the two-component PrrBA system [8]. PrrBA has been
shown to regulate a large number of genes [8]. Available
evidence suggests that the phosphorylated form of the
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response regulator PrrA is responsible for repressing nap
expression under low oxygen. Since this enzyme is maximally
expressed when oxygen is present, it seems unlikely that it is
being used for respiration.
In another R. sphaeroides strain, DSM158, a similar
pattern of expression was observed [9]. Highest expression
was observed under oxic conditions, and nitrate did not
seem to be required for expression. However, there was
no nitrate reductase activity under oxic conditions. Under
photosynthetic conditions, there was evidence of nitrate
reductase activity. Growth on the reduced carbon sources
butyrate and caproate under these conditions was severely
impaired in a strain lacking the nap cluster [9]. This
dependency on Nap indicates that nitrate reductase activity
helps dispose of excess electrons generated by catabolism
of these electron-rich compounds. Recent work has shown
that non-sulfur purple photosynthetic bacteria place a high
demand on redox homoeostatic pathways during growth
[10]. The use of Nap in redox homoeostasis has been
shown previously in Paracoccus pantotrophus and its relatives
[11,12].
A slightly different nap expression pattern was observed in
R. sphaeroides strain IL106, which like 2.4.3 is a denitrifying
strain [13]. Insertional inactivation of the genes in the nap
cluster eliminated nitrate reductase activity, indicating that
this is the only cluster of genes encoding a dissimilatory
nitrate reductase in this strain. This nap gene cluster showed
the same level of expression under both oxic and denitrifying
conditions. There was also evidence that nitrate is an effector
for expression. Expression increased in the presence of the
reduced carbon compound butyrate, indicating that this
protein probably plays a role in redox homoeostasis.
R. sphaeroides strain 2.4.3 has a nap cluster orthologous to
the nap clusters in DSM158, IL106 and 2.4.1. As in 2.4.1, this
nap is present on a plasmid. Unlike the other R. sphaeroides
strains, 2.4.3 has a second nap cluster. This cluster is located on
the smaller of the two chromosomes which has been shown to
be evolving at a rapid rate due to lateral gene transfer [14]. This
cluster is not paralogous to the other nap cluster in this strain,
since it contains two additional genes napGH. nap operons
with napGH are not commonly found in α-proteobacteria
nor is it common for bacteria to have more than one nap
cluster, but some species of Shewanella have also been found
with more than one nap cluster [15]. Expression of the nap
clusters of 2.4.3 will be reported elsewhere (A. Hartsock and
J.R. Shapleigh, unpublished work).
A. tumefaciens has a nap cluster orthologous to the
plasmid-borne version found in R. sphaeroides 2.4.3 and 2.4.1.
This nap showed yet another pattern of expression, since
it was maximally expressed under denitrifying conditions
(Figure 1). As with 2.4.1, its expression was not influenced
by nitrate. The regulator(s) responsible for expression of nap
in A. tumefaciens are uncertain, since there are no conserved
sequence motifs in the promoter region that might indicate
the involvement of a particular family of regulators.
Even though this nap is more highly expressed under
denitrifying conditions, it still showed measurable expression
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Authors Journal compilation Figure 1 Expression of napF of A. tumefaciens under a variety of
conditions measured using a napF–lacZ fusion
Growth of cells and assay were as described previously [20].
Measurements were done in triplicate and the S.D. is <10 %. Oxic early
refers to growth under oxic conditions with cells at a D600 of 0.25. Oxic
late also refers to growth under oxic conditions, but with cells at a D600
of 0.80. Low O2 refers to micro-oxic conditions.
under oxic conditions (Figure 1). Nitrite assays demonstrated
that cells growing under oxic conditions in liquid medium
have nitrate reductase activity; however, this activity was not
dependent on nap, since similar levels of nitrite accumulated
in a Nap-deficient mutant (Table 1). Increasing the ammonia
level in the culture eliminated the nitrite accumulation under
oxic conditions, indicating that this nitrite production under
these conditions was due to Nas.
Nitrite production was also observed in cells growing on
plates under oxic conditions (Table 1). This activity was
mainly due to Nap, since the nap mutant did not produce
nitrite under these conditions unless the cells were incubated
for >48 h. The wild-type strain produced nitrite within 24 h
when grown on solid medium. A strain lacking nirK, the
gene encoding Nir, produced the same amount of nitrite on
plates as wild-type, indicating that the cells in the colony were
not experiencing denitrifying conditions. The production of
nitrite under these conditions is significant because it reveals
that electron flow through Nap does not require reduced
electron sources, but can occur with carbon sources that are
the same oxidation state as the cell.
Nir and Nor regulation in select
α-proteobacteria
Although the expression of nap has been shown to be variable,
previous work with model organisms has consistently shown
that the expression of the genes encoding Nir and Nor
only occurs under micro-oxic conditions [16]. Therefore this
section will focus mainly on regulatory mechanisms. Nir and
Nor expression in R. sphaeroides 2.4.3 requires low oxygen
and also requires the presence of a nitrogen oxide [17,18]. A
nitrogen oxide is required because NO is a key effector. NO is
Enzymology and Ecology of the Nitrogen Cycle
Table 1 Nitrite production under oxic conditions by strains of A. tumefaciens and R. denitrificans
Nitrite production is indicated by +, with multiple + being used to indicate intensity. − indicates no nitrite production. All samples were taken
between 12 and 24 h after inoculation. Potassium nitrate was added to 10 mM in all samples. S, succinate-based minimal medium (see [20]); S/N,
succinate-based minimal medium with 3× ammonium; M, Difco Marine broth; wt, wild-type; nap − , nap-deficient strain.
Liquid
Agar plate
A. tumefaciens
S
S/N
R. denitrificans
A. tumefaciens
M
S
R. denitrificans
S/N
M
wt
nap −
wt
nap −
wt
wt
nap −
wt
nap −
wt
++
++
−
−
+++
++
−
++
−
+
probably detected by NnrR, a member of the FNR (fumarate
nitrate reductase regulator)/CRP (cAMP receptor protein)
family of transcriptional regulators (Figure 2). PrrBA is also
required for expression of both Nir and Nor [19]. Evidence
indicates that PrrBA directly regulates the gene encoding Nir,
known as nirK, but not nor. However, nor expression still
depends on PrrBA, since Nir generates the NO required for
NnrR-dependent expression of nor.
A similar pattern of nirK and nor expression occurs in
A. tumefaciens. This bacterium uses an orthologue of NnrR
for nitrogen-dependent regulation [20]. An orthologue of
the PrrBA system is also required for nirK expression [21].
In A. tumefaciens, this system has been designated ActRS.
Purified ActR-P will bind to the nirK promoter, but not the
nor promoter. This demonstrates that, as in R. sphaeroides,
ActRS directly regulates nirK, but only indirectly regulates
nor expression.
In these two denitrifiers, nirK and nor expression depends
on at least two regulators, NnrR and PrrBA orthologues
(Figure 2). The NnrR regulators serve as nitrogen oxide
sensors. PrrBA and its orthologues appear to serve as sensors
of the availability of terminal electron acceptors. Work in
R. sphaeroides suggests that PrrBA activity is influenced
by the activity of the cbb3 terminal oxidase [22,23]. This
is a cytochrome c-dependent enzyme that reduces oxygen
to water. Orthologues of this enzyme have been shown
to have high affinity for oxygen [24]. When this enzyme
is functioning, the PrrA/PrrA-P ratio is high. However,
when oxygen is low, turnover of the enzyme is reduced,
which leads to a decrease in the PrrA/PrrA-P ratio. In
the related bacterium R. capsulatus, its PrrBA orthologues,
designated RegBA, appear to be regulated by the redox
status of the quinone pool [25]. Taken together, these
results suggest that members of this family of regulators are
influenced by electron flow through the respiratory chain.
Therefore they act as sensors of the availability of electron
acceptors, particularly oxygen. If oxygen, the preferred
terminal electron acceptor, is present nirK is not expressed.
However, these regulators do not appear to directly sense
oxygen.
Figure 2 Schematic of the regulation of nap, nirK and nor in R.
sphaeroides
The nap regulatory scheme is taken from work with R. sphaeroides 2.4.1.
nirK and nor regulation is taken from work with R. sphaeroides 2.4.3.
Broken lines indicate only indirect evidence for the proposed interaction.
The arrow at the bottom is used to distinguish the regulatory mechanisms
in the proposed scheme.
An additional layer of oxygen-dependent
regulation?
Inactivation of genes encoding the cbb3 oxidase in R.
sphaeroides has been shown to lead to high levels of PrrA-P
under both oxic and micro-oxic conditions [26]. Loss of cbb3
oxidase activity would not be predicted then to decrease nirK
expression in 2.4.3. Unexpectedly, loss of the cbb3 oxidase
resulted in a significant decrease in nirK expression and
Nir activity [19]. Recent results indicate that this phenotype
is conditional [27]. In the original experiments, cells were
grown under what is referred to as transition conditions
in which the growth vessel is sealed with a rubber stopper
after inoculation. Oxygen levels in the flask start out at
atmospheric levels, but are steadily reduced as the cells grow.
These conditions give rise to the Nir-deficient phenotype of
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the cbb3 mutant. However, when oxygen levels are decreased
using gas mixes, the cbb3 mutant retains Nir activity at very
low oxygen concentrations. The simplest explanation for this
result is that, under transition conditions, loss of the cbb3
oxidase results in high residual oxygen levels in the culture,
which prevents expression of nirK and nor. This oxygendependent inhibition is not observed using the low oxygen
gas mixes. These results suggest that some aspect of nirK
and nor expression is inhibited by oxygen. Nap expression,
which is indirectly required as it is needed to produce the
nitrite necessary for NO production, is not inhibited since
nitrite accumulates in the cbb3 mutant [19]. PrrBA is not
likely to be an oxygen-sensitive regulator since it is in its
active form in this mutant. As NnrR is the only other known
regulator, it may be oxygen-sensitive as previous work with
an orthologue of NnrR has suggested [28] (Figure 2). There
is also the possibility that an unidentified oxygen-sensitive
protein is required for nirK and nor expression.
Gene expression and enzyme activity
during transition from oxic to micro-oxic
conditions
The expression and activity of Nir and Nor as a function of
oxygen has been determined in A. tumefaciens [29]. Nitrate
reductase expression and activity were not followed in these
experiments, but the nap expression and activity results
discussed above suggest that Nap will be present throughout
the experiments (Figure 1). It seems unlikely, however, that
Nap will be active under aqueous conditions without a
significant reduction in oxygen levels. Once oxygen becomes
limiting, this will allow diversion of electrons to Nap.
Concomitant with this decrease in oxygen, the expression
of nap will increase, allowing more electron flux through
this branch of the respiratory pathway. nirK and nor are
expressed once oxygen reaches <0.5 μM or approx. 0.2 %
of saturating oxygen. These oxygen concentrations are low
enough to allow formation of ActR-P necessary for nirK
activation. nirK expression is also dependent on NnrR which
requires NO. However, since Nir is the primary source of
NO, there must be some limited expression of nirK before
oxygen levels reach sub-micromolar levels to produce the
catalytic amounts of Nir necessary to potentiate the Nir–
NO–NnrR-feedback loop. Consistent with this conclusion
is the observation that transcripts of nirK are present before
NO accumulation [29]. The expression of nor genes occurs
subsequent to a burst of NO production. The onset of Nor
translation brings the NO levels down to nanomolar levels
consistent with balanced denitrification. Unexpectedly, the
nor transcript was only present for a relatively short amount
of time. It decreasesd to below detectable levels while NO
was present in the headspace. nirK transcripts remained fairly
steady throughout the anoxic portions of the experiments.
The regulatory rationale for the relative instability of the norB
transcript is not obvious.
Preliminary gas-phase measurements with R. sphaeroides
2.4.3 show a similar pattern (L. Bergaust and J.R. Shapleigh,
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Authors Journal compilation unpublished work). In particular, there is a burst of NO that
appears to be coincident with the onset of vigorous nitrite
respiration. As observed previously with A. tumefaciens,
transition between aerobic and anaerobic respiration was
difficult under certain conditions [29]. These results suggest
that, although some of the details may differ, expression of
nap, nir and nor in R. sphaeroides 2.4.3 and A. tumefaciens
follow similar patterns.
Aerobic denitrifiers
Work with most model denitrifiers is not supportive of the
concept of aerobic denitrification, given the low oxygen
levels required for expression of the genes encoding Nir
and Nor. Nevertheless, studies finding evidence of aerobic
denitrification are not uncommon [30,31]. As discussed
above, there is no obvious a priori reason these studies
should be dismissed. Moreover, there are many well known
examples of organisms developing strategies to express
seemingly anaerobic processes under aerobic conditions. A
good example of this is the group of photosynthetic bacteria
known as the aerobic anoxygenic phototrophs [32]. These
bacteria express photosynthetic genes under oxic conditions,
whereas most other related phototrophs are photosynthetic
only under micro-oxic conditions. An example of this group
is the bacterium R. denitrificans. As its name suggests, this
bacterium is also a denitrifier and there are several studies
that indicate this bacterium can express denitrification genes
under oxic conditions [33,34].
In an effort to directly compare nitrogen oxide reductase
activity in R. denitrificans with R. sphaeroides and A.
tumefaciens, the former was grown under the same conditions
used to assess nitrogen oxide activity in the latter. Under these
conditions, it was observed that R. denitrificans produced
nitrite during growth in either liquid or solid medium
(Table 1). This bacterium has a membrane-bound Nar-type
nitrate reductase, but lacks Nas. There was no evidence
of nitrite reduction under oxic conditions in the dark.
However, Nir was expressed in cells grown under these
conditions, as demonstrated using a Methyl Viologendithionite-based Nir assay (results not shown). Nitrite was
consumed by cells growing under photosynthetic conditions,
consistent with previous results [34]. Attempts to grow
R. denitrifcans under strictly denitrifying conditions failed.
These results indicate that, under certain conditions, R.
denitrificans might be an aerobic denitrifier. The exact
physiological role of denitrification under these conditions
is uncertain. It seems unlikely that nitrate respiration is being
used for energetic purposes. Given the importance of redox
homoeostasis in this group of proteobacteria, it is possible
that denitrification is being used for redox balancing or some
other accessory function.
Summary
Nap expression in the α-proteobacteria discussed in the
present review seems to be adjusted to fit the physiological
role it plays. In organisms where its primary function is
Enzymology and Ecology of the Nitrogen Cycle
redox homoeostasis it is expressed under oxic conditions,
whereas if it is being used for respiration it is maximally
expressed under denitrifying conditions. The expression of
Nir and Nor is more tightly controlled through the action
of at least two regulatory systems that limit expression
to micro-oxic conditions. One regulator apparently senses
NO, whereas the other is a more general regulator sensing
availability of oxidant. Neither senses oxygen directly, but
there may be an as yet undefined layer of oxygen-sensitive
regulation. R. denitrificans may be an aerobic denitrifier, but
the physiological role of the enzymes in this organism is
uncertain.
Acknowledgements
I acknowledge the work of Angela Hartsock and Armanda Roco.
Funding
This work was supported by the U.S. Department of Energy [grant
number 95ER20206].
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Received 21 October 2010
doi:10.1042/BST0390179
C The
C 2011 Biochemical Society
Authors Journal compilation 183