J Bacteriol, June 1998, p. 3260-3264, Vol. 180, No. 12
Department of Plant Pathology, College of
Agriculture, University of Arizona, Tucson, Arizona 85721
Received 20 January 1998/Accepted 7 April 1998
To determine whether in Azotobacter vinelandii the
PII protein influences the regulation of nif
gene expression in response to fluxes in the ammonium supply, the gene
encoding PII was isolated and characterized. Its deduced
translation product was highly similar to PII proteins from
other organisms, with the greatest degree of relatedness being
exhibited to the Escherichia coli glnK gene product. A gene
designated amtB was found downstream of and was
cotranscribed with glnK as in E. coli. The AmtB
protein is similar to functionally characterized ammonium transport
proteins from a few other eukaryotes and one other prokaryote.
glnK and amtB comprise an operon. Attempts to
isolate a stable glnK mutant strain were unsuccessful,
suggesting that glnK, like glnA, is an
essential gene in A. vinelandii. amtB mutants
were isolated, and although growth on limiting amounts of ammonium was
similar in the mutant and wild-type strains, the mutants were unable to transport [14C]methylammonium.
Nitrogen fixation genes
(nif) are highly conserved among all nitrogen-fixing
bacteria, and in all diazotrophic species of the class
Proteobacteria examined, the transcriptional activator NifA
is required for expression of other nif genes
(33). In some diazotrophs of the Transduction of the environmental signal of fixed-nitrogen status has
been best described in enteric organisms (for a review, see reference
27). Under low-ammonium-concentration conditions, the product of the glnB gene, the PIIB protein,
a homotrimer consisting of identical 12.4-kDa subunits (9),
is uridylylated at a Tyr residue by the uridylytransferase activity of
the glnD gene product (32). The uridylylation
state of PII determines whether the transcriptional
activator NtrC is phosphorylated or dephosphorylated (i.e., active or
inactive, respectively) (2, 29) and the extent to which
glutamine synthetase (GS) is adenylylated or deadenylylated (i.e.,
inactive or active, respectively) (20). The recent finding of second PII-encoding genes, i.e., glnK
(present in addition to glnB in Escherichia coli
[38]) and glnZ (present in addition to
glnB in Azospirillum brasilense
[10]), complicates the picture, especially with
respect to what occurs under low-fixed-N conditions.
In the nonenteric bacterium A. vinelandii, the
glnD gene, originally named nfrX, was identified
by Tn5 mutagenesis (34) and subsequent DNA
sequence analysis (8). While glnD::Tn5
mutants are Nif Isolation and sequencing of the A. vinelandii glnK and
amtB genes.
The glnB gene of E. coli on pAH5 (19) hybridized to a 3.2-kb
EcoRI fragment of A. vinelandii genomic DNA on
Southern analysis (data not shown). This fragment was cloned in
pACYC184 (6), giving pND183, and subsequently into pSVB30
(25), giving pDM508. The glnB-hybridizing region
was further delineated by hybridization. The nucleotide sequence of the
2.3-kb PstI-EcoRI fragment of pPR101 (Fig.
1) was determined on both strands by
using exonuclease III- and nuclease S1-generated deletion derivatives.
Analysis of the 2,278-bp sequence revealed two potential open reading
frames (ORFs), one between nucleotide positions 477 (ATG) and 813(TGA)
and one between nucleotides 849 (ATG) and 2,157 (TGA). These ORFs
potentially encode proteins with molecular weights of 12,240 and 46,390 Da, respectively. The product of the first ORF showed a high degree of
overall amino acid sequence similarity to several PII
proteins and includes the conserved site of uridylylation at Tyr51. The most similar of the other proteins is the second PII
protein in E. coli, designated GlnK; the two proteins are
75% identical and 84% similar with respect to amino acid sequence
(data not shown). E. coli GlnB is less similar, with 66%
identical amino acids. High degrees of similarity between the
glnB gene products of other members of the class
Proteobacteria and the product of the first A. vinelandii ORF described here also are evident, with identities ranging from approximately 70 to 74%. Because of the high degree of
similarity of this ORF product to the E. coli glnK gene
product and the proximity of this PII protein-encoding gene
to a putative ammonium transporter in both organisms (see below), the
A. vinelandii gene has been designated glnK.
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Characterization of the glnK-amtB Operon
of Azotobacter vinelandii
ABSTRACT
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and 
subgroups of
the class Proteobacteria, such as Azospirillum
brasilense, Rhodobacter capsulatus, and
Herbaspirillum seropedicae, the NifA proteins are inactive
in cells grown with high levels of ammonium (1, 4, 31). The
mechanism(s) for this is unknown but may involve a PII
protein, known from studies of enteric bacteria to be a major component
of a nitrogen-sensing and signal transduction cascade. In diazotrophs
of the proteobacterial 
subgroup, including Azotobacter
vinelandii and Klebsiella pneumoniae, a nifL
gene lies upstream of nifA (3, 5, 18, 26). NifL interacts with and prevents the activity of NifA in cells exposed to
oxygen or excess fixed N. Inactivation of NifA prevents it from
stimulating transcription from promoters adjacent to the several other
nif genes and operons, leading to a failure to produce nitrogenase enzyme. K. pneumoniae NifA deleted of its
N-terminal domain is more sensitive to inactivation by NifL, suggesting
that a function of this domain is to modulate the response of NifA to
NifL (12). NifL of A. vinelandii was recently
shown to be a redox-sensitive flavoprotein with flavin adenine
dinucleotide as the prosthetic group, which when reduced, has no effect
on in vitro NifA-dependent open-complex formation at the
nifH promoter (17). When oxidized, NifL prevents
open-complex formation. How NifL responds to fixed-N status by becoming
inhibitory to NifA in is not known. It is also possible that the
susceptibility of NifA to NifL inactivation increases as the fixed-N
supply increases.
, glnD::Tn5
nifL::KIXX double mutants are Nif+. Two models
developed to explain this result are as follows: (i) GlnD is required
for conversion of active NifL (which inhibits NifA) to its inactive
form, and (ii) GlnD is necessary for conversion of NifA to a
conformation that cannot be inactivated by NifL. The question of
whether GlnD influences NifL (or NifA) via a PII protein
was the basis for this study.
View larger version (22K):
[in a new window]
FIG. 1.
The 2.3-kb glnK-amtB region of A. vinelandii. The locations of KIXX and lacZ interposon
constructs and their directions of insertion are shown by the hatched
rectangles and arrows below the line, respectively. The resulting
plasmids are named along with (where appropriate) the stable mutant
strain of A. vinelandii in which the
glnK::KIXX or
glnK::lacZ region replaced the
wild-type glnK gene. The fragments used as probes to
identify RNA fragments on the Northern blots (see Fig. 2) are shown as
thin lines. Abbreviations for restriction sites: B, BamHI;
E, EcoRI; H, HincII; Sn, StuI;
SnaBI; P, PstI.
glnK and amtB gene expression.
Potential ribosome binding sites for glnK and
amtB were located within 10 bp upstream of the ATG
initiation codons. The small intergenic region between glnK
and amtB as well as the lack of any obvious transcriptional
terminator- or promoter-like structures suggested the probable
cotranscription of glnK and amtB. Sequences similar to the 
10- and 35-like regions of other prokaryotic genes
recognized by 
70 are present from 36 to 66 bp upstream
of the ATG of glnK. Whether these sequences are significant
for transcription of the putative glnK-amtB operon was not
investigated. About 75 bp downstream of amtB there begins a
27-bp region with features characteristic of a factor-independent
transcription terminator, including a 6-base inverted-repeat motif
separated by 3 bp and followed by a tract of six T's.
-galactosidase produced in an amtB-lacZ
fusion strain, MV566, constructed by transformation of UW136 with
pPR203 (Fig. 1) grown under different conditions. The levels of
expression were consistently very low, about 75 Miller units of
activity, and were not significantly different in ammonium-, urea-, or
N2-grown cultures.
|
54 and NtrC recognition motifs in the A. vinelandii glnK promoter region is consistent with the results
obtained from the expression experiments.
Whether the glnK gene in A. vinelandii reported
here represents the only PII-encoding gene in this organism
is uncertain. There is some evidence that there is not a second
functional PII protein encoded by another gene; only a
single hybridizing band was observed after hybridization of either the
E. coli glnB gene or the A. vinelandii glnK gene,
characterized here, to genomic digests generated with three different
restriction enzymes. In an experiment involving the cloning of
PII-encoding genes by ligation of PCR products generated
from oligonucleotide primers based on conserved amino acid sequences in
both GlnK and GlnB PII proteins, each of the 10 products
cloned had a DNA sequence identical to that of the glnK gene
described here (24). Also, as discussed below,
glnB mutants could not be isolated under a variety of growth conditions, including those under which another glnB-like
gene might be expected to be expressed.
Attempts to construct glnK mutants of A. vinelandii.
The kanamycin resistance-encoding KIXX cassette
was inserted at or between endonuclease restriction sites in pPR101
(Fig. 1). A. vinelandii UW136 was transformed with the three
resulting glnK::KIXX plasmids, pDM513, pDM514, and
pPR401. The kanamycin-resistant, ampicillin-sensitive transformants,
which presumably carried the desired gene replacements because of the
occurrence of a double-crossover event, were serially subcultured three
times on selective medium containing kanamycin. To verify that
replacement of the chromosomal wild-type copies of glnK had
occurred, genomic DNA was isolated and analyzed in Southern
hybridization experiments using pDM508 as a probe. However, in
all transformants examined, both wild-type and mutated copies of the
glnK::KIXX genes were detected even after prolonged growth
of up to 10 subcultures under selective conditions (i.e., with
kanamycin) (data not shown). Also, colonies isolated after 10 cycles of
selection followed by 1 cycle of growth on medium without kanamycin had
all become sensitive to the antibiotic. The same pattern of behavior
was observed if transformed cells were plated on a medium with or
without ammonium or with poor or excellent carbon sources or were
incubated under aerobic or microaerobic growth conditions. Therefore,
these mutants showed aberrant behavior similar to that observed when
attempts were made to construct glnA insertion mutants of
A. vinelandii (36). From the results of this
previous work it was concluded that glnA null mutations are
lethal because GS is the only ammonium assimilation-associated enzyme
present and glutamine cannot be transported in A. vinelandii. It thus appears that null mutations in glnK
also cannot be tolerated and are lethal. Similar results were obtained
in attempts to construct glnD::KIXX null mutants
(7). (While the original glnD [orignally named
nfrX] mutants were viable and Nif, the sites
of Tn5 insertion were at the far 3' end of the gene, leaving
doubt as to whether they represented null mutants [see references
8 and 36]). Since
PII-UMP is required in enteric bacteria for rapid
deadenylylation of GS, one possible reason for severe growth impairment
in the A. vinelandii glnK (and also glnD) mutants
is that GS is insufficiently active (i.e., remains adenylylated even
under low-fixed-N conditions). To test this possibility, MV75, a strain
in which GS cannot be adenylylated carrying a glnA gene in
which Tyr407 has been mutated to Phe, (15), was transformed
with the glnK::KIXX plasmids pDM513 and pDM514
individually. While the altered GS state did allow the glnD
mutations to become stable and complete gene replacement occurred
(7), the glnK::KIXX MV75 transformants behaved
like the wild-type transformants in that wild-type chromosomes were always present after many subcultures on antibiotic-containing medium
and resistance was quickly lost if the colonies were plated on
antibiotic-free medium. Therefore, the lethality or impairment of
growth of A. vinelandii caused by the introduction of
glnK mutations is possibly due to an effect not only on GS
activity but also on some other vital cellular function. As indicated
above, there is unlikely to be a second PII-encoding gene
in A. vinelandii, as there is in E. coli,
Azospirillum brasilense, and certain other bacteria. It is
of relevance here that glnB glnZ double mutants of
Azospirillum brasilense are severely growth impaired
(11), as are E. coli glnB glnK mutants
(16a), and that glnB mutations also appear to be
lethal both in Synecocchus spp. and in Rhodospirillum rubrum since standard genetic techniques failed to result in
replacement of the wild-type gene with a mutated copy (16,
21a).
amtB mutants are unable to transport [14C]methylammonium. To construct amtB interposon mutations, pDM508 was partially digested with StuI and ligated to the KIXX cassette that had been blunt-ended with SmaI, giving pPR201 and pPR202 (Fig. 1). Wild-type strain UW136 and MV101 were transformed with both plasmids, and this was followed by selection on kanamycin-containing medium. Complete replacement of the amtB gene was confirmed by Southern analysis for both KIXX insertion transformants of each strain (data not shown); all were stably kanamycin resistant even after several subcultures in nonselective medium. Thus, unlike glnK, the amtB gene has no function that is of vital importance to A. vinelandii.
It was hypothesized that if the amtB gene encodes an ammonium transport protein, then the amtB::KIXX mutant strains may be less able to grow on limiting ammonium concentrations than the parental wild-type strain. The amtB mutant strains MV560 and MV561 were able to fix nitrogen and grew only slightly less well than UW136 on N-free BS agar medium. Therefore, the Nif nifH::KSS (lacZ)
amtB::KIXX mutant strains MV562 and MV563 (Fig. 1)
were used in these experiments since their growth is dependent on a
supply of fixed nitrogen. Mutants and parent strain MV101 were grown in
BS medium plus urea (5 mM) and then diluted and plated by pipetting
20-µl suspension aliquots, each containing approximately 200 cells,
onto BS medium containing ammonium acetate at concentrations ranging
from 0.05 to 10 mM. There was no difference in the results obtained
with the two strains; in both cases, the rate of colony formation and
the size of the colonies were the same at all concentrations and the
colony size decreased with decreasing ammonium concentrations, becoming
barely discernible at an ammonium concentration of about 100 µM.
Another test for ammonium transport is the uptake of the radiolabeled
ammonium analog [14C]methylammonium. In these
experiments, which required aeration of small sample volumes to detect
transport in the wild-type strain, UW136 (amtB+)
accumulated 10.6 nmol of methylammonium after 35 min (Fig.
3). In contrast, the
amtB::KIXX mutant strain, MV560, failed to
transport methylammonium (taking up less than 5% of the amount
transported by the wild-type strain). The amtB+
strain UW136 failed to transport [14C]methylammonium if
15 mM ammonium was added at the beginning of the experiment, as
expected since ammonium was shown to be a competitive inhibitor of
methylammonium transport (21). In another control
experiment, in which two or three samples were washed on the filters,
washing never removed more than 20% of the bound radioactivity,
indicating that the retained 14C was due to true uptake and
not to adventitious binding of [14C]methylammonium. A
clear phenotype of the amtB::KIXX mutants is therefore
evident: they cannot transport methylammonium. While a similar
phenotype was reported for amt mutants of C. glutamicum (35) and Azospirillum brasilense
(37), the function of other prokaryotic AmtB proteins with
respect to ammonium or methylammonium uptake has not been reported, nor
(with the exception of an S. cerevisiae strain mutated in
both mep genes) has an amtB mutant been shown to
be deficient in ammonium transport per se. While it is widely assumed
that methylammonium is transported as an analog of ammonium, this may
not always be the case, and the determination of the true physiological
function of amtB in prokaryotes must at least await
identification of a specific physiological phenotype associated with
amtB mutants. It is also likely that at least some
prokaryotes contain two more ammonium transporters, each with a certain
affinity for substrate, as in S. cerevisiae.
|
) and amtB mutant (
) strains. Overnight cultures in
Burke's N-free sucrose medium were diluted 50-fold and grown to an
optical density at 600 nm of 0.5 to 0.7. The details of the uptake
experiments were described by Jayakumar and Barnes (Nucleotide sequence accession number. The nucleotide sequence for the 2.3-kb region of pPR101 carrying the glnK and amtB genes of A. vinelandii has been deposited in the GENEMBL database under accession no. U91902.
| |
ACKNOWLEDGMENTS |
|---|
This work was supported by USDA NRICG grant no. 95-37305-2067.
We thank Rita Colnaghi for useful discussions and critical reading of the manuscript, Miklos de Zamaroczy for permission to cite unpublished information, and E. Barnes for helpful technical suggestions concerning methylammonium uptake experiments.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Plant Pathology, College of Agriculture, P.O. Box 210036, University of Arizona, Tucson, AZ 85721. Phone: (520) 621-9835. Fax: (520) 621-9290. E-mail: ckennedy{at}u.arizona.edu.
Present address: Gentechnologie/Mikrobiologie, Fakultät
für Biologie, Universität Bielefeld, 33501 Bielefeld,
Germany.
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J Bacteriol, June 1998, p. 3260-3264, Vol. 180, No. 12
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