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Effect of an ntrC mutation on amino acid or urea utilization and on nitrogenase

switch-off in Herbaspirillum seropedicae

Article  in  Canadian Journal of Microbiology · April 2008

DOI: 10.1139/w07-135 · Source: PubMed

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 235

NOTE / NOTE

Effect of an ntrC mutation on amino acid or urea

utilization and on nitrogenase switch-off in
Herbaspirillum seropedicae
Claudio L. Gusso, Emanuel M. de Souza, Liu Un Rigo, Fábio de Oliveira Pedrosa,
M.G. Yates, Fabiane G. de M Rego, and Giseli Klassen

Abstract: Herbaspirillum seropedicae is a nitrogen-fixing bacterium that grows well with ammonium chloride or sodium
nitrate as alternative single nitrogen sources but that grows more slowly with L-alanine, L-serine, L-proline, or urea. The
ntrC mutant strain DCP286A was able to utilize only ammonium or urea of these nitrogen sources. The addition of
1 mmol
L–1 ammonium chloride to the nitrogen-fixing wild-type strain inhibited nitrogenase activity rapidly and com-
pletely. Urea was a less effective inhibitor; approximately 20% of nitrogenase activity remained 40 min after the addition
of 1 mmol
L–1 urea. The effect of the ntrC mutation on nitrogenase inhibition (switch-off) was studied in strain DCP286A
containing the constitutively expressed gene nifA of H. seropedicae. In this strain, nitrogenase inhibition by ammonium
was completely abolished, but the addition of urea produced a reduction in nitrogenase activity similar to that of the wild-
type strain. The results suggest that the NtrC protein is required for assimilation of nitrate and the tested amino acids by
H. seropedicae. Furthermore, NtrC is also necessary for ammonium-induced switch-off of nitrogenase but is not involved
in the mechanism of nitrogenase switch-off by urea.
Key words: Herbaspirillum seropedicae, NtrC, nitrogenase switch-off.
Résumé : Herbaspirillum seropedicae est une bactérie fixatrice d’azote qui pousse bien lorsque le chlorure d’ammonium
ou le nitrate de sodium sont utilisés comme source alternative d’azote, mais croissent plus lentement sur la L-alanine, la
L-proline ou l’urée. La souche DCP286A dont le gène ntrC est muté était capable d’utiliser l’ammonium ou l’urée
comme seules sources d’azote. L’ajout de chlorure d’ammonium 1 mmol
L–1 à la souche fixatrice d’azote sauvage in-
hibait l’activité de la nitrogénase rapidement et complètement. L’urée était un inhibiteur moins efficace : approximati-
vement 20 % de l’activité de la nitrogénase persistait, 40 min après l’ajout de 1 mmol
L–1 d’urée. L’effet de la
mutation sur ntrC sur l’inhibition de la nitrogénase (switch-off) a été étudié chez la souche DCP286A exprimant de
façon constitutive nifA de H. seropedicae. Dans cette souche, l’inhibition de la nitrogénase par l’ammonium était com-
plètement abolie, mais l’ajout d’urée produisait une réduction de l’activité de la nitrogénase équivalente à celle obser-
vée dans la souche sauvage. Ces résultats suggèrent que la protéine NtrC est requise pour l’assimilation du nitrate et
les acides aminés testés chez H. seropedicae. Qui plus est, NtrC est aussi nécessaire à l’inhibition de la nitrogénase
induite par l’ammonium, mais n’est pas impliquée dans le mécanisme d’inhibition de la nitrogénase par l’urée.
Mots-clés : Herbaspirillum seropedicae, NtrC, inhibition de la nitrogénase.
[Traduit par la Rédaction]

Ammonium is the preferred source of nitrogen for bacteria.
However, to support growth in the absence of ammonium,
Received 15 October 2007. Revision received 12 December
2007. Accepted 17 December 2007. Published on the NRC
Research Press Web site at cjm.nrc.ca on 27 February 2008.
C.L. Gusso and G. Klassen.1 Departamento de Patologia
Básica, Universidade Federal do Paraná, C.P. 1903, CEP-81531-
990, Curitiba, Paraná, Brasil.
E.M. Souza, L.U. Rigo, F.O. Pedrosa, M.G. Yates, and
F.G. Rego. Departamento de Bioquı́mica e Biologia Molecular,
Universidade Federal do Paraná, C.P. 1903, CEP-81531-990,
Curitiba, Paraná, Brasil.
1Corresponding author (e-mail: giseli@ufpr.br).
bacteria can utilize alternative nitrogen sources. Both the
synthesis and activity of proteins involved in the transport
and metabolism of alternative nitrogen sources are tightly
regulated by the availability of their substrates and by the
nitrogen regulatory system Ntr (Merrick and Edwards
1995). In Escherichia coli, this regulatory system comprises
(i) the GlnD protein, which has 2 enzymatic activities, uri-
dylyl addition (UTase, known as uridylyltransferase) and ur-
idylyl removal (UR); (ii) 2 PII-like proteins, GlnB and
GlnK; and (iii) the 2-component regulatory pair NtrB–NtrC.
In the absence of ammonium, the UTase uridylylates GlnB,
and the resulting uridylylated form GlnB–UMP cannot inter-
act with the histidine protein kinase NtrB, which can then
phosphorylate the response regulator protein NtrC. The

Can. J. Microbiol. 54: 235–239 (2008) doi:10.1139/W07-135 # 2008 NRC Canada

236 Can. J. Microbiol. Vol. 54, 2008

phosphorylated form NtrC–P is a transcriptional activator of Fig. 1. Utilization of nitrogen compounds by Herbaspirillum sero-
the genes involved in the metabolism of alternative nitrogen pedicae strains (A) SMR1 (wild type) and (B) DCP286A (ntrC
sources. In the presence of ammonium, GlnB is deuridyly- mutant strain). Nitrogen sources (all at 10 mmol
L–1): &, ammo-
lated by UR; the deuridylylated form of GlnB activates the nium chloride; ~, sodium nitrate; *, urea; &, L-serine; ^, L-ala-
phosphatase activity of NtrB, which dephosphorylates NtrC, nine; !, L-proline. The results are the average of 3 triplicate
thus inactivating NtrC (Reitzer and Magasanik 1985; experiments.
Merrick and Edwards 1995).
In enteric bacteria, the genes transcriptionally regulated
by phosphorylated NtrC include glnAntrBC (Reitzer and
Magasanik 1985); glnHPQ, which encodes transport proteins
for glutamine (Nohno and Saito 1987); argT for arginine;
and hisJQMP for histidine (Schmitz et al. 1987) in E. coli.
In Klebsiella pneumoniae, NtrC regulates nasFEDCBA,
functioning in nitrate and nitrite assimilation (Goldman et
al. 1994), and nifLA, functioning in nitrogen fixation regula-
tory genes (Espin et al. 1982). In addition, the nitrogen as-
similation control gene, nac, of K. pneumoniae (Collins et
al. 1993), Klebsiella aerogenes (Macaluso et al. 1990),
E. coli (Muse and Bender 1998), and Salmonella typhi
(Sherburne et al. 2000) is also regulated by NtrC. In Rhodo-
bacter capsulatus, NtrC is necessary for urea utilization
(Masepohl et al. 2001). In both K. pneumoniae and R. cap-
sulatus, the NtrC protein also controls the transcription of
nifA, which codes for the activator of nif genes (Buck 1986;
Masepohl et al. 2002).
Herbaspirillum seropedicae is an endophytic diazotroph
found in roots and leaves of rice, sugarcane, and other gra-
mineae (Baldani et al. 1986; Dobereiner 1992; Roncato-
Maccari et al. 2003). In this organism, NtrC regulates
nitrogen fixation by controling NifA expression and, conse-
quently, the expression of other nif genes (Pedrosa et al.
1997; Souza et al. 2000). In this work, we analyzed the
physiological effects of alternative nitrogen sources on
growth and nitrogenase activity of an NtrC mutant.
The H. seropedicae wild type is able to utilize alternative
sources of nitrogen, such as L-serine or L-alanine (Klassen et
al. 1997). Here, the use of alternative nitrogen sources was
investigated using an ntrC mutant strain of H. seropedicae.
We showed that NtrC is necessary for the assimilation of
L-alanine, L-serine, and L-proline. We also determined that 120 r
min–1 in NFbHP medium supplemented with
NtrC is required for the inhibition (switch-off) of nitroge- 2 mmol
L–1 ammonium chloride. Cells were harvested by
nase by ammonium but not by urea. centrifugation (5000g for 5 min) and then suspended in
The strains used in this work were H. seropedicae SMR1 nitrogen-free NFbHP medium to an OD550 of 1.0. Nitrogenase
(wild type, streptomycinR) and H. seropedicae DCP286A activity was derepressed for 4 h at 30 8C and 120 r
min–1.
(ntrC::Tn5-B20, streptomycinR, kanamycinR) (Persuhn et al. After this period, acetylene was injected (10% of the gas
2000). phase) and ethylene production measured at the indicated
To determine growth with alternative nitrogen sources, time intervals (Klassen et al. 1997). Different nitrogen
H. seropedicae strains were first grown overnight at 30 8C sources (1 mmol
L–1 final concentration) were added to the
and 120 r
min–1 in liquid NFbHP medium (Klassen et al. nitrogen-fixing cultures after 40 min of incubation with
1997) containing 20 mmol
L–1 ammonium chloride. The acetylene, and the subsequent rate of ethylene production
cells were collected by centrifugation (5000g for 5 min) and was determined every 20 min. The specific activity of nitro-
suspended in medium without a nitrogen source to an optical genase in the wild-type strain at 40 min was taken as 100%.
density (OD550) of 1.0. Fifty microlitres of cell suspension Protein was determined as described by Bradford (1976).
was then used to inoculate 10 mL of NFbHP medium con- Herbaspirillum seropedicae SMR1 was capable of using
taining 10 mmol
L–1 (each) L-alanine, L-serine, L-proline, all the nitrogen sources tested, and after 50 h of incubation
urea, sodium nitrate, or ammonium chloride. Cultures were at 30 8C, the final cell densities obtained were similar
incubated at 30 8C and 120 r
min–1. Growth was followed (Fig. 1A). Growth rates on glutamate and L-glutamine were
by measuring the OD (550 nm) after 7, 14, 28, and 48 h of measured previously and similar results were obtained for
incubation. Experiments were in triplicate. the wild type and mutant (Persuhn 2001). In contrast to the
To analyze nitrogenase inhibition (switch-off assays), wild type, strain DCP286A could not grow using sodium ni-
H. seropedicae strains were grown overnight at 30 8C and trate (Persuhn et al. 2000). Figure 1B shows that this strain
# 2008 NRC Canada
Gusso et al.
Fig. 2. Ammonium- and urea-dependent nitrogenase switch-off of
Herbaspirillum seropedicae strains SMR1 (wild type) (black sym-
bols) and DCP286A (pEMS135; ntrC mutant strain) (grey sym-
bols). Arrow indicates the addition of 1 mmol
L–1 ammonium
chloride (squares) or urea (triangles). The nitrogenase specific ac-
tivities of the wild type and DCP286A (pEMS135) at 20 min were
5.5 and 5.0 nmol C2H4
min–1
(mg protein)–1, respectively.
was also incapable of growing with L-alanine, L-serine, or
L-proline as nitrogen source, suggesting that NtrC is re-
quired for the expression of the tested amino acid transport
system(s) and (or) catabolic pathways. The ntrC mutant
grew in the presence of urea at a rate similar to that of
the wild-type strain (Fig. 1B). In R. capsulatus, urea is
used as a nitrogen source whose utilization or uptake is de-
pendent on NtrC (Masepohl et al. 2001). In contrast, our
results show that urea catabolism is not dependent on
NtrC in H. seropedicae. Furthermore, analysis of the pro-
moter region of the ure gene cluster of H. seropedicae did
not reveal NtrC- or s54- binding sites (data not shown).
Herbaspirillum seropedicae nitrogenase is reversibly inac-
tivated by the addition of ammonium ions to derepressed
cultures. The addition of low levels of ammonium chloride
causes a rapid loss of approximately 80% of nitrogenase ac-
tivity, which is fully recovered after 120 min (Klassen et al.
1997). This reversible inhibition is known as nitrogenase
switch-off, followed by switch-on (Zumft and Castillo
1978). In addition to ammonium, other nitrogen-containing
compounds, such as glutamine, glutamate, and methyl
ammonium, can cause nitrogenase switch-off. L-Alanine and
L-serine (1 mmol
L–1) inhibit nitrogenase activity of the
H. seropedicae wild-type strain by approximately 48% and
63%, respectively (Klassen et al. 1997).
The mechanism of this reversible inactivation in H. sero-
pedicae is not clear. There is no evidence of ADP ribosyla-
tion of dinitrogenase reductase and no draT or draG genes
are found in H. seropedicae (Fu and Burris 1989). To study
the effect of the ntrC mutation on nitrogenase switch-off,
plasmid pEMS135 (Souza et al. 1999), which expresses the
nif-specific transcriptional activator NifA constitutively, was
introduced into the strain DCP286A. This was necessary,
since the ntrC mutant strain is nif negative, because the
237
Table 1. Uptake of nitrogen sources by Herbaspirillum
seropedicae strains.
Nitrogen source uptake rate
(nmol
min–1
(mg protein)–1)
Nitrogen source Wild type DCP286A (ntrC–)
Ammonium 1.9±0.1 0.7±0.02
L-Alanine 1.3±0.2 0
L-Serine 1.35±0.3 0
L-Proline 0.3±0.02 0
Urea 1.5±0.3 1.4±0.5
Note: Cell cultures (OD550 = 1.0) were incubated for 4 h in
medium without a nitrogen source. Nitrogen compounds
(200 mmol
L–1) were then added and measured in the medium
supernatant after incubation for 20 min to determine uptake rates.
The results represent the average of 2 duplicate experiments.
NtrC protein is required to activate the nifA gene expression
in H. seropedicae. Constitutive expression of nifA comple-
mented the Nif-negative phenotype of strain DCP286A. Fig-
ure 2 compares the effect of ammonium addition on
nitrogenase activity in the wild type and the mutant strains.
In the wild type, the addition of 1 mmol
L–1 ammonium
chloride switched off nitrogenase activity, as shown before
(Klassen et al. 1997). In contrast, no effect of ammonium
was observed in the ntrC mutant. Noindorf et al. (2006) re-
ported that methyl-ammonium uptake by H. seropedicae is
dependent on the AmtB protein whose expression is depend-
ent on NtrC. The amtB mutant strain is also deficient in ni-
trogenase switch-off. Their data show a direct involvement
of AmtB in nitrogenase switch-off by ammonium ions. Ad-
ditionally, it has been shown in other organisms, such as
Azospirillum spp. (Hartmann et al. 1986) and Rhodospiril-
lum rubrum (Kanemoto and Ludden 1987), that inhibition
of glutamine synthetase (GS) by the glutamine analogue me-
thionine sulfoximine abolishes ammonium-dependent nitro-
genase switch-off. The ntrC mutant of H. seropedicae has
very low activity of GS (Persuhn et al. 2000). Taken to-
gether, these observations suggest that the effect of the ntrC
mutation on the nitrogenase switch-off may be due to a
combination of a need for the NtrC protein to express the
amtB gene and low GS activity.
The addition of urea to nitrogenase-derepressed cultures
of H. seropedicae caused a 50% reduction in nitrogenase ac-
tivity after 20 min in both the wild type and the ntrC mutant
expressing the nifA gene constitutively (Fig. 2). On the other
hand, NtrC mutant strains of R. capsulatus are unable to
grow in medium containing urea as the sole nitrogen source
owing to the dependence on NtrC for activation of the ureD
gene. Also, in this organism urea does not prevent nitroge-
nase synthesis or activity (Masepohl et al. 2001). So, in con-
trast to the situation in R. capsulatus, in the H. seropedicae
ntrC mutant the metabolism of urea is not dependent on
NtrC. Apparently, the nitrogenase switch-off by urea in this
organism occurs by a different mechanism, independent of
NtrC.
The uptake of nitrogen sources by H. seropedicae strains
was measured in NFbHP medium containing 200 mmol
L–1
ammonium chloride by growing 20 mL of cultures overnight
in 60 mL flasks at 120 r
min–1 and 30 8C. The exhaustion of
# 2008 NRC Canada
238
ammonium in the medium was confirmed by the procedures
of Chaney and Marbach (1962) before adding the nitrogen
compounds at 0.2 mmol
L–1. After 20 min of incubation,
the culture supernatants were measured for ammonium and
urea by the indophenol method (Chaney and Marbach
1962), and amino acids by ninhydrin (Spies 1957). The re-
sults are shown in Table 1. All the tested substrates were in-
corporated by the wild type, but the amino acids were not
taken up by the NtrC mutant . These results indicate that
transport is one mechanism involved in the absence of
growth or nitrogenase switch-off by the NtrC H. seropedicae
mutant.
In enteric bacteria, proline (Schwacha and Bender 1993),
histidine (Bender et al. 1983), alanine (Janes and Bender
1998), cytosine (Muse et al. 2003), and urea (Macaluso et
al. 1990) are utilized as alternative nitrogen compounds in
the absence of ammonium ions. Under these conditions, the
phosphorylated form of NtrC protein activates the promoter
of the nac gene. The Nac protein is a transcription activator
of genes for the assimilation of alternative nitrogen sources
(Reitzer and Schneider 2001). In H. seropedicae, the ab-
sence of growth of the ntrC– strain in the presence of the
amino acids tested may indicate a crucial role of NtrC as a
transcriptional activator of genes involved in the transport
and (or), possibly, in the utilization of these amino acids
but not urea. Further studies to determine whether a Nac-
like protein is involved in this process are necessary.
Acknowledgements
We thank Roseli Prado, Valter Baura, and Julieta Pie for
technical assistance. This work was supported by the Brazil-
ian Research Council (CNPq) and CAPES.
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