FEMS Microbiology Letters 201 (2001) 199^204

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Potential roles for the glnB and ntrYX genes in

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Azospirillum brasilense
Josiane C. Vitorino a , M. Berenice R. Ste¡ens a , Hidevaldo B. Machado b ,
M. Geo¡rey Yates a , Emanuel M. Souza a , Fäbio O. Pedrosa a; *
a
Department of Biochemistry, UFPR, P.O. Box 19046, CEP 81531-990 Curitiba, PR, Brazil
b
Department of Pharmacology, UFPR, P.O. Box 19046, CEP 81531-990 Curitiba, PR, Brazil

Received 10 January 2001; received in revised form 11 May 2001; accepted 14 May 2001

First published online 19 June 2001

Abstract

Three Azospirillum brasilense mutants constitutive for nitrogen fixation (NifC ) in the presence of NH‡ 4 and deficient in nitrate-dependent
growth were used as tools to define the roles of the glnB and ntrYX genes in this organism. Mutant HM14 was complemented for nitrate-
dependent growth and NH‡ 4 regulation of nitrogenase by plasmid pL46 which contains the ntrYX genes of A. brasilense. Mutant HM26 was
restored for NH‡ 4 regulation and nitrate-dependent growth by plasmid pJC1, carrying the A. brasilense glnB gene expressed from a
constitutive promoter. Mutant HM053, on the other hand, was not complemented for NH‡ 4 regulation of nitrogenase and nitrate-dependent
growth by both plasmids pJCI and pL46. The levels and control of glutamine synthetase activity of all mutants were not affected by both
plasmids pL46 (ntrYX) and pJC1 (glnB). These results support the characterization of strains HM14 as an ntrYX mutant and strain HM26
as a glnB mutant and the involvement of ntrYX and glnB in the regulation of the general nitrogen metabolism in A. brasilense. ß 2001
Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords : Azospirillum brasilense; nif regulation; Nitrogen ¢xation ; ntrYX; glnB gene

1. Introduction required for NifA activity. The PII paralogue GlnZ, how-
ever, cannot substitute for PII in this activity, whereas
The nitrogen regulatory pathway in Azospirillum brasi- GlnK, the PII paralogue in enterics, can activate NifA
lense is di¡erent from that in enteric bacteria, due to the by involvement in dissociating the NifA^NifL complex
presence of novel putative regulatory proteins including in the absence of ammonium [2,3]. In enterics PII or
the Pz, NtrY and NtrX proteins that arguably participate GlnK can activate glnA expression via control of NtrC
in the regulation of cellular functions, including nitrogen phosphorylation by NtrB, and control the level of gluta-
¢xation. mine synthetase (GS) adenylylation via ATase [4,5]. In A.
In enteric bacteria NtrC-P positively regulates the syn- brasilense, on the other hand, the expression of glnA is
thesis of NifA, which in turn activates the promoters of independent of the NtrC protein, and neither PII nor Pz
the other nif genes. NifL inhibits NifA activity in response controls adenylylation of GS in response to the N status
to high oxygen levels or ammonium. This is not the case in [6]. A third di¡erence is that nifA expression in A. brasi-
A. brasilense where no NifL protein has been detected; lense is not regulated by NtrC [7,8].
rather NifA activity is inhibited by ammonium through The roles of PII and Pz in A. brasilense have not yet
its N-terminal domain or by oxygen through a cysteine been de¢ned in detail. PII is essential for NifA activity, it
motif within the central domain and C-terminal linker se- may be involved in nitrate-dependent growth and also
quence [1]. The PII protein, product of the glnB gene, is negatively regulates glnZ expression [6,9]. The Pz protein,
on the other hand, negatively modulates ammonium up-
take [9]. GlnB and GlnZ mutants grow normally on ¢xed
* Corresponding author. Tel. : +55 (41) 3664398;
nitrogen sources, whereas a double mutant grows very
Fax: +55 (41) 2662042. slowly, suggesting that these proteins can substitute for
E-mail address : fpedrosa@bio.ufpr.br (F.O. Pedrosa). each other in their overall e¡ect on cell growth [9].
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 2 5 8 - 0

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200 J.C. Vitorino et al. / FEMS Microbiology Letters 201 (2001) 199^204

Spontaneous mutants of A. brasilense strain FP2 resis- tion of the cells with 0.5 M NaOH. L-Galactosidase activ-
tant to ethylenediamine were previously isolated and par- ities were assayed as described [20].
tially characterized [10]. Three of these mutants, HM14,
HM26 and HM053, were found to ¢x nitrogen constitu- 2.5. Cloning, molecular biology methods and
tively in the presence of high NH‡ 4 concentrations and to plasmid construction
excrete NH‡ 4 derived from nitrogen ¢xation [10]. Di¡erent
patterns of GS transferase activities and adenylylation Molecular microbiology techniques were according to

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were observed, which allowed their distribution into three Sambrook et al. [13]. Plasmid pJC1 contains the whole
classes: the ¢rst includes strain HM053, which showed low A. brasilense glnB gene with its promoter region in a
levels of constitutively adenylylated GS activity; the sec- 1.4-kb SacI/SmaI fragment from pAB441 [14] cloned
ond, which includes strain HM14, had low levels of nor- into pLAFR3.18 [21], in the same orientation as the
mally regulated GS activity; in the third class, strain lacZ promoter.
HM26 showed wild-type levels of constitutively adenyly-
lated GS activity [10].
In this work we further characterized the A. brasilense 3. Results
mutants HM14, as an ntrYX mutant, and HM26 as af-
fected in glnB, following genetic complementation for ni- 3.1. Genetic complementation of A. brasilense and
trate-dependent growth and restoration of ammonium reg- mutants for nitrate-dependent growth
ulation of nitrogenase expression, and de¢ne potential
roles for the glnB and ntrYX in the nitrogen metabolism All three mutants failed to grow on 10 mM KNO3 or
of A. brasilense. 0.5 mM NH4 Cl [10], but grew well on glutamate (data not
shown). Plasmid pJC1 (A. brasilense glnB gene) comple-
mented nitrate and low NH‡ 4 -dependent growth of mutant
2. Materials and methods HM26, but failed to complement strains HM14 and
HM053 for this trait (Table 2). On the other hand, plas-
2.1. Bacteria and plasmids mid pL46, which contains only the ntrYX genes of A.
brasilense, was able to complement the HM14 mutant
The bacterial strains and plasmids used in this work are for nitrate and low NH‡4 -dependent growth (Table 2).
listed in Table 1.
3.2. Restoration of ammonium regulation of nitrogenase
2.2. Media and growth conditions activity in A. brasilense mutants

A. brasilense strains were grown in NFbHP medium The A. brasilense NifC mutants, but not the wild-type
containing di¡erent NH4 Cl concentrations or glutamate strain FP2, expressed nitrogenase activity in the presence
(5 mM) as sole N sources at 30³C [10]. Nitrate-dependent of high NH4 Cl concentrations (10^20 mM) [10] as we like-
growth was determined on solid NFbHP medium contain- wise report here. Nitrogenase activity of mutants HM14,
ing 10 mM KNO3 for 24^48 h at 30³C. Escherichia coli HM26 and HM053 decreased with increasing NH‡ 4 con-
was grown in LB or LA medium [13] at 37³C. Antibiotics centrations, suggesting only partial release from regulatory
were added to growth media as required. control, as reported previously [10]. Plasmid pJC1, how-
ever, complemented HM26 for ammonium control/regula-
2.3. Bacterial conjugation tion of nitrogenase activity, but inhibited (75%) acetylene
reduction activity of HM053, without restoring regulation
Conjugation between E. coli donor strains and A. bra- by NH‡ ‡
4 (Table 3). Plasmid pL46 restored the NH4 regu-
silense strains was performed at 30³C for 18 h [11]. Trans- lation of nitrogenase activity of mutant HM14 (Table 3).
conjugants were isolated on NFbHP medium containing Plasmids expressing nifA of A. brasilense (pML3A) or of
20 mM NH4 Cl and the antibiotics streptomycin (100 Wg Klebsiella pneumoniae (pCK3) constitutively did not a¡ect
ml31 ), nalidixic acid (10 Wg ml31 ), and tetracycline (10 Wg this phenotype nor the level of nitrogenase of all three
ml31 ; plasmid marker). mutants (data not shown).

2.4. Enzyme activities 3.3. Glutamine synthetase control and expression in

A. brasilense mutants
Nitrogenase activity was determined by the acetylene
reduction procedure [11]. Glutamine synthetase activity The GS-GOGAT pathway is involved in NH‡ 4 assimi-
was determined in whole cells as described [10]. Protein lation during diazotrophic growth of A. brasilense [22].
was determined by the Lowry procedure [19] after diges- The glnB gene carried by plasmid pJC1 and the ntrYX

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 J.C. Vitorino et al. / FEMS Microbiology Letters 201 (2001) 199^204 201

Table 1
Bacterial strains and plasmids
Strain/plasmid Genotype/phenotype Reference
Azospirillum brasilense
FP2 Sp7 ATCC 29145, Nif‡ NalR SmR [11]
HM14, HM26 and HM053 FP2 EDAR , NifC NalR SmR Amt3 [10]

Escherichia coli

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S 17.1 RP4-2 Tc: :Mu Km: :Tn7 Tra‡ Sm‡ recA3 [12]
71-18 v(lac-pro) FP lac Iq Z M15 pro‡ [13]

Plasmids
pAB441 TcR , 23-kb fragment containing the operon glnBA of A. brasilense in pVK100 [14]
pJC1 TcR , 1.4-kb fragment containing the gene glnB of A. brasilense in pLAFR3.18 This work
pL46 TcR , 4.6-kb fragment containing the genes ntrYX of A. brasilense in pLAFR3.18 [15]
pAB904 TcR (pGD926), (3322/+290) glnBp1 p2 -lacZ [6]
pAB912 TcR (pGD926), (3315/+236) glnAp-lacZ [6]
pGE10 TcR KmR ApR IncP-1 R 68.45 Klebsiella pneumoniae glnAntrBntrC [16]
pGE50 TcR KmR ApR IncP-1 R 68.45 Klebsiella pneumoniae glnAntrBntrC: :Tn5 [16]
pML3A TcR , 2.5-kb SalI containing nifA of A. brasilense in pLAFR3.18 [17]
pCK3 TcR , nifAC of Klebsiella pneumoniae [18]

genes of pL46 both failed to restore the control and ex- 3.5. E¡ect of K. pneumoniae glnAntrBC genes
pression of GS in all three A. brasilense mutants (Table 4).
Plasmid pGE10 containing the glnAntrBC operon from
3.4. Expression of glnA and glnB in the mutant strains K. pneumoniae or pGE50 (KpglnAntrBntrC : :Tn5), con-
taining a mutated ntrC gene, failed to complement all
The expression of the glnA and glnB promoters in these three HM mutants for NH‡ 4 regulation of nitrogenase,
mutants was determined using plasmids pAB904 and total GS activity and regulation, and nitrate-dependent
pAB912 which carry a glnB: :lacZ and a glnA: :lacZ fu- growth (data not shown).
sion, respectively. Expression of the glnB promoter was
reduced two-fold by high ammonium concentration in
the wild-type strain FP2. Transconjugants of all the mu- 4. Discussion
tant strains carrying the glnB: :lacZ fusion showed very
high levels (three to four times the wild-type level) of L- Three A. brasilense mutants, HM14, HM26 and HM053
galactosidase activity in the absence of ammonium ; and [10], constitutive for nitrogen ¢xation in the presence of
three to nine times in the presence of ammonium ions ammonium, add to the several NifC mutants of bacteria
(Table 5). The data suggest that the expression of the that have been reported [23^28]. The decreasing nitroge-
glnB promoter in all these mutants lacks regulation and nase activity in the present mutants with increased ammo-
is consistent with the presence of an altered nitrogen sens- nium levels indicates that the mutants were a¡ected in
ing system in the mutant strains. either regulation of nitrogenase expression by ammonium
The glnA promoter activity increased seven- to eight- or the ability to switch o¡ nitrogenase activity or both. It
fold in the presence of 20 mM ammonium in the wild- is highly probable that the regulation of nitrogenase syn-
type strain as previously observed by de Zamaroczy et thesis and activity are linked [29]. The nifA gene must be
al. [6]. The HM mutant strains behaved similarly (Table expressed and its product active in these mutants since
5), suggesting that glnA expression is positively regulated they ¢x nitrogen and the introduction of plasmids express-
by NH‡ 4 as in the wild-type strain. ing nifA constitutively did not a¡ect the phenotype. Active

Table 2
Growth of A. brasilense mutants and transconjugants strains in the presence of KNO3 or NH4 Cl
Strain KNO3 (10 mM) NH4 Cl (0.5 mM)
No plasmid pJC1 (glnB) pL46 (ntrYX) No plasmid pJC1 (glnB) pL46 (ntrYX)
FP2 + + + + + +
HM14 d d + d d +
HM26 d + d d + d
HM053 d d d d d d
+, positive growth; d, de¢cient growth.

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Table 3
E¡ect of NH‡ c
4 concentration on nitrogenase activity of A. brasilense Nif mutants and transconjugants

Strain Nitrogenase activity (nmol C2 H4 formed min31 mg protein31 )

NH4 Cl (mM) No plasmid pJC1 (glnB) pL46 (ntrYX)
FP2 0 19.7 (2.5) 18.7 (2.9) 16.6 (0.9)
5 6 0.1 6 0.1 6 0.1
10 6 0.1 6 0.1 6 0.1

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20 6 0.1 6 0.1 6 0.1
HM14 0 31.8 (4.4) 30.1 (4.2) 15.6 (0.8)
5 14.9 (3.0) 17.4 (2.4) 6 0.1
10 8.9 (2.0) 9.6 (1.9) 6 0.1
20 2.5 (0.3) 4.8 (1.2) 6 0.1
HM26 0 28.9 (4.1) 21.8 (4.4) 21.3 (0.6)
5 16.8 (1.7) 1.4 (0.3) 10.7 (0.2)
10 7.7 (1.5) 0.7 (0.1) 3.2 (0.1)
20 1.4 (0.2) 0.4 (0.1) 1.0 (0.1)
HM053 0 29.0 (4.3) 5.4 (1.0) 31.5 (2.0)
5 20.7 (2.1) 3.5 (0.8) 18.5 (1.2)
10 8.8 (0.8) 2.4 (0.3) 10.4 (0.8)
20 2.5 (0.4) 0.7 (0.1) 1.0 (0.1)
Values are the mean of at least three independent experiments and standard deviations are in parentheses.

NifA is required for the expression of nif genes in A. not be glnB null mutants since glnB mutants of A. brasi-
brasilense [11] and the NifA activity is inhibited in A. lense are Nif3 [6]. In an attempt to determine the genetic
brasilense by ammonium ions. This inhibition was as- defect of these A. brasilense mutants, we introduced plas-
sumed to be prevented by positive interaction of NifA mids carrying nitrogen metabolism regulatory genes.
with GlnB (PII-UMP) in the absence of ammonium ions Strain HM14 was complemented for nitrate growth and
[1]. In the present mutants PII activity is probably modi- NH‡ 4 -regulation of nitrogenase by the A. brasilense ntrYX
¢ed to enable it to interact positively with NifA even in the operon, strain HM26 by the glnB gene expressed from the
presence of ammonium. Furthermore, these mutants can- lacZ promoter and strain HM053 was not complemented

Table 4
E¡ect of glutamate and NH‡ C
4 concentrations on the GS transferase activity of A. brasilense Nif mutants and their transconjugants

Strain N-source NH‡

4 (mM) Transferase activitya
3Mg2‡ +Mg2‡ Unadenylylated form (%)
FP2 2 2.32 (0.21) 0.96 (0.45) 41.3
20 1.74 (0.39) 0.39 (0.09) 23.4
FP2 (pL46) 2 1.86 (0.01) 1.10 (0.01) 59.2
20 1.51 (0.03) 0.17 (0.01) 11.5
FP2 (pJC1) 2 3.06 (0.69) 1.37 (0.30) 44.8
20 2.75 (0.71) 0.73 (0.01) 28.7
HM14 2 0.18 (0.02) 0.08 (0.01) 42.8
20 0.34 (0.05) 0.06 (0.03) 15.9
HM14 (pL46) 2 0.12 (0.12) 0.05 (0.02) 44.0
20 0.26 (0.01) 0.06 (0.01) 23.2
HM14 (pJC1) 2 0.40 (0.12) 0.19 (0.07) 47.5
20 0.35 (0.03) 0.05 (0.01) 15.5
HM26 2 2.03 (0.24) 0.17 (0.10) 8.4
20 1.28 (0.12) 0.05 (0.02) 3.0
HM26 (pL46) 2 2.33 (0.29) 0.15 (0.08) 6.4
20 1.66 (0.22) 0.06 (0.05) 3.6
HM26 (pJC1) 2 2.54 (0.38) 0.16 (0.09) 6.3
20 2.23 (0.67) 0.05 (0.07) 2.5
HM053 2 0.53 (0.11) 0.09 (0.05) 17.0
20 0.40 (0.23) 0.03 (0.01) 6.3
HM053 (pL46) 2 0.45 (0.08) 0.10 (0.06) 22.2
20 0.32 (0.03) 0.03 (0.04) 9.4
HM053 (pJC1) 2 0.30 (0.04) 0.13 (0.06) 43.3
20 0.36 (0.08) 0.01 (0.01) 3.5
Values are the mean of at least three independent experiments and standard deviations are in parentheses.
a
GS transferase activities were measured as Wmol Q-glutamylhydroxamate min31 mg protein31 .

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Table 5 mutant strains suggested normal regulation of glnAp ac-

Expression of plasmid-borne gln-lacZ fusions in A. brasilense FP2 and
tivity (Table 5). Since glnBp in all mutants is non-regu-
mutants under di¡erent physiological conditions
lated, a high total GS level would be expected irrespective
Strain L-Galactosidase activity (nmol o-nitrophenol min31 mg of the ammonium levels. This did correlate with the GS
protein31 )
activity measurements. Mutants HM14 and HM053
glnBp1 p2 -lacZ (pAB904) glnAp-lacZ (pAB912) showed low levels of total GS under all conditions and
3NH‡
4 +NH‡
4 3NH‡
4 +NH‡
4 mutant HM26 had an almost normal level of total GS

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FP2 1749 (495) 843(60) 839(99) 6059 (532) but its expression was regulated by ammonium as the
HM14 6698 (354) 7374(616) 982(86) 6311 (308) wild-type. Mutant HM26 seems a¡ected in the glnB
HM26 5134 (158) 7697(188) 970(58) 7392 (1148) gene, thus it is conceivable this mutation a¡ected GS ex-
HM053 2765 (234) 3701(590) 1527(327) 6405 (169)
pression from the chromosome. This is not the case with
Values are the mean of at least three independent experiments and stan- mutant HM14 whose GS phenotype is not complemented
dard deviations are in parentheses.
by plasmid pL46, raising the question of a second level of
glnA regulation such as post-transcriptional or post-trans-
lational regulation of GS expression. The high level of PII
but had very low levels of non-regulated nitrogenase levels expression may explain the high constitutive level of GS
when plasmid pJC1 (glnB) was introduced. These results adenylylation in mutants HM26 and HM053. Excess PII
de¢ne three di¡erent primary mutations in these mutants would complex to the adenylyl transferase resulting in the
which is in agreement with a previous classi¢cation based permanent adenylylation of GS, both in the presence and
on physiological data [10]. in the absence of NH‡ 4 . Gautier and Elmerich [23] re-
The glnB promoter carried by plasmid pAB904 was ported two glnA mutants of A. brasilense, one of which
NH‡ 4 -regulated in the wild-type, but was non-regulated was Nif3 and the other Nif constitutive. The NifC mutant
and over-expressed in all three mutants, suggesting that had a high level of glnB expression in the presence of NH‡4
the mutants have a high intracellular content of PII irre- [6]. Both phenotypes were, however, corrected by K. pneu-
spective of the presence of NH‡ 4 (Table 5). High levels of moniae glnA [14]. Mutations in glnA of R. sphaeroides also
PII, produced by such NH‡ 4 -independent regulation, produced a NifC phenotype [27]. The present mutants,
might prevent ammonium inhibition of NifA activity, however, were not corrected by K. pneumoniae glnA, in-
analogous to high levels of GlnK preventing NifL inhibi- dicating that they are not glnA mutants. Moreover, the
tion of nif gene expression in K. pneumoniae [30]. How- de¢ciency in GS activity was phenotypically distinct in
ever, restoration of ammonium regulation of nitrogenase all three mutants suggesting di¡erent primary mutations.
and nitrate growth by the A. brasilense glnB gene ex- Although all three mutants have di¡erent GS phenotypes,
pressed constitutively (pJC1) in mutant HM26 suggests they have common secondary phenotypes of nitrate-de¢-
that this strain contained an altered glnB gene that was cient growth, constitutive nif gene expression and lack of
capable of activating the NifA protein regardless of the glnB regulation, which are corrected by di¡erent genes.
presence of ammonium. NH‡ 4 -dependent regulation of ni- We conclude that the HM mutants analyzed here are
trogen ¢xation was restored in mutant HM14 by the A. mutated in genes which pleiotropically a¡ect the N metab-
brasilense ntrYX genes whose products may play a role in olism in A. brasilense. Mutant HM26 is a¡ected in glnB,
the regulation of PII synthesis in A. brasilense. Mutations HM14 is a¡ected in both ntrYX genes and glnB expression
in the glnB gene caused various Nif phenotypes in a num- and mutant HM053 could not be complemented by either
ber of organisms. Mutations in Rhodobacter capsulatus genes or other known nitrogen regulatory genes, indicating
glnB caused nif derepression in the presence of ammonium the presence of a non-identi¢ed regulatory gene in A. bra-
[26], whereas deletion of the glnB gene in Rhodospirillum silense. Complementation by ntrYX for nitrate-dependent
rubrum caused a Nif3 phenotype, and a Y51F mutation in growth suggests that in A. brasilense the NtrYX pair is
glnB decreased nitrogenase activity to 15% of that of the interchangeable with the NtrBC pair with respect to this
wild-type [29]. Double mutation in glnBglnK of Rhodo- trait. Furthermore, the data indicate a probable role of
bacter sphaeroides was constitutive for nitrogen ¢xation ntrYX gene products in regulating glnB expression or ac-
in the presence of ammonium (NifC ) [28]. A double tivity.
glnBglnK mutant of Azorhizobium caulinodans also ex-
pressed nitrogenase activity in the presence of ammonium.
This mutant grew slowly and had a highly adenylylated Acknowledgements
GS [31].
The glnA gene in A. brasilense is mainly expressed from We thank CNPq and PRONEX/FINEP for ¢nancial
the glnB promoter (glnBp) under N limitation and from support. We would also like to thank Maria L. Ishida
the glnA (glnAp) under N excess [6]. The results obtained and Claudine Elmerich for plasmids used for complemen-
when native glnAp-lacZ fusion was introduced into the tation and L-galactosidase assays.

FEMSLE 10012 16-7-01

204 J.C. Vitorino et al. / FEMS Microbiology Letters 201 (2001) 199^204

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