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(Received for publication, November 16, 1995) From the
We have identified the molecular basis for the nitrogenase
negative phenotype exhibited by Azotobacter vinelandii UW97.
This strain was initially isolated following nitrosoguanidine
mutagenesis. Recently, it was shown that this strain lacks the Fe
protein activity, which results in the synthesis of a FeMo
cofactor-deficient apodinitrogenase. Activation of this
apodinitrogenase requires the addition of both MgATP and wild-type Fe
protein to the crude extracts made from A. vinelandii UW97
(Allen, R. M., Homer, M. J., Chatterjee, R., Ludden, P. W., Roberts, G.
P., and Shah, V. K.(1993) J. Biol. Chem. 268
23670-23674). Earlier, we proposed the sequence of events in the
MoFe protein assembly based on the biochemical and spectroscopic
analysis of the purified apodinitrogenase from A. vinelandii DJ54 (Gavini, N., Ma, L., Watt, G., and Burgess, B. K.(1994) Biochemistry 33, 11842-11849). Taken together, these
results imply that the assembly process of apodinitrogenase is arrested
at the same step in both of these strains. Since A. vinelandii DJ54 is a
Nitrogenase is one of the most intriguing, complex
metalloenzymes and is the protein that catalyzes the MgATP-dependent
reduction of N For N The Fe protein of
nitrogenase has been reported to have four distinct functions. As
mentioned above, the Fe protein is the specific physiological electron
donor to the MoFe protein. A second function of the Fe protein involves
the initial biosynthesis of FeMo cofactor (31, 32, 33, 34, 35, 36, 37) .
Although the pathway for FeMo cofactor biosynthesis has not yet been
established, it has been known for some time that the FeMo cofactor is
synthesized separately from the MoFe protein polypeptides. In fact, its
synthesis requires the combined action of the nifQ,
-B, -N, -E, and -V genes(38, 39) . It was documented that the
mutants of Klebsiella pneumoniae and Azotobacter
vinelandii that did not synthesize the nifH polypeptide
also did not synthesize FeMo cofactor(31, 33) .
Recently, this was further confirmed by purifying the FeMo
cofactor-deficient MoFe protein from one of these mutants and
characterizing it by biochemical and spectroscopic analysis (36) . Based on these and other experiments, it is now accepted
that the nifH gene product is required for an early step in
FeMo cofactor biosynthesis. However, it is not known what features of
the Fe protein are required for this action. The third Fe protein
function involves the insertion of pre-formed FeMo cofactor into an
inactive FeMo cofactor-deficient MoFe
protein(23, 36, 37) . This step in the
assembly of an active MoFe holoprotein requires not only the nifH polypeptide but also MgATP. Once again, it is not known what
features of the Fe protein are required for FeMo cofactor insertion.
Finally, the Fe protein has been implicated in the regulation of the
transcription of its own operon and in the expression of related
nitrogenase systems(40) . Earlier, we showed that the
Earlier, it was shown that the apodinitrogenase from
different genetic backgrounds are biochemically distinct(35) .
For example, to activate apodinitrogenase from A. vinelandii DJ54 with FeMo cofactor, we needed to add the Fe protein, isolated
FeMo cofactor, MgATP, and an unknown additional component(s) that is
present in cell-free extracts from a The strain A.
vinelandii DJ54 has a well characterized deletion in the nifH gene(33) ; the strain A. vinelandii UW97 was
originally isolated following nitrosoguanidine mutagenesis. While both
of these strains accumulate the FeMo cofactor-deficient
apodinitrogenase protein, the dinitrogenase reductase (the Fe protein)
is accumulated only in the A. vinelandii UW97 cells. However,
the apodinitrogenase of UW97 also needs a pre-treatment with wild-type
Fe protein and MgATP for its activation by FeMo cofactor(37) .
Since an inactive Fe protein is synthesized in UW97, we argued that
localizing the mutation will give us some clue about the features
required for its participation in FeMo cofactor biosynthesis and
insertion. To check if the Nif
Figure 1:
The bacteria were grown on
nitrogen-free medium (41) to verify Nif phenotype of various
constructs. This experiment demonstrates that the defective gene in the
UW97 nif gene cluster is the nifH.
To test whether this nifH mutation
is due to a detectable deletion in the DNA corresponding to nifH, we have subjected the chromosomal DNA of A.
vinelandii UW97 to Southern blot analysis and hybridization with
DNA sequences corresponding to the wild-type nifH gene. The
chromosomal DNA from A. vinelandii UW97 (mutant) and A.
vinelandii UW (wild type) was isolated as described under
``Experimental Procedures.'' The DNA from each of these
strains was subjected to restriction enzyme digestion with EcoRI, HindIII, or PstI, and Southern blots
were prepared and hybridized with non-radioactive DNA probes spanning
the coding sequence of nifH. This comparative analysis did not
detect any visible deletions in the nifH gene of A.
vinelandii UW97 chromosome (Fig. 2), and we infer that the
Nif
Figure 2:
Southern blot analysis of the chromosomes
of A. vinelandii strains UW97 and UW digested with HindIII (lanes 1 and 4), PstI (lanes 2 and 5), and EcoRI (lanes 3 and 6). The probe used was the DNA sequence corresponding
to nifH gene and flanking regions. Autoradiograph shows that
the chromosomes from both strains are indistinguishable with respect to nifH gene and flanking regions. kbp, kilobase
pairs.
If the Fe protein is synthesized in UW97, then the mutation
responsible for making it inactive must be located in the open reading
frame. To test this possibility, we decided to analyze the nucleotide
sequence that encodes the A. vinelandii UW97 Fe protein. Since
the nucleotide sequence of the nifH gene is known, we have
made use of this information to design the oligonucleotide primers
corresponding to the sequences upstream and downstream of the open
reading frame to amplify the nifH coding sequence from the
chromosome of A. vinelandii UW97 by PCR amplification
technique(53) . The DNA fragment obtained by this method was
cloned into the pCR Since the Nif
Figure 3:
Comparison of the amino acid residues of
the second conserved domain in the Fe proteins that are deduced from 40
different nifH DNA sequences. The numbering corresponds to A. vinelandii NifH1. Upper case letters in the
consensus sequence represent invariant residues in all 40 sequences,
whereas lower case letters indicate the presence of one or
more variants in that position.
Crystallographic analysis indicates that in the conserved amino acid
cluster encompassing the residues 37-45, Lys-41 is involved in
contacting the ribose group; whereas, the residues Asp-39 and Asp-43
could play a role in catalysis of ATP hydrolysis(9) .
Crystallographic data also suggest that the residues ranging from 39 to
80 could be involved in transmitting the MgATP-induced conformational
change to the nucleotide binding site(9) . Thus, the amino
acids in the second conserved domain are implicated in taking part in
the MgATP binding and the MgATP-induced conformational change. Studies
on other Fe protein mutations at Lys-15 and Asp-125 point out that
these mutations affected the MgATP-induced conformational change of the
Fe protein. However, they did not interfere with its ability to take
part in FeMo cofactor biosynthesis and
insertion(22, 25) . Our extensive analysis of another
Nif As described under ``Experimental
Procedures,'' we have constructed an A. vinelandii strain
in which the UCC codon specifying serine was replaced with GCC codon
that was specifying alanine. The A. vinelandii strains were
compared in their growth characteristics as described under
``Experimental Procedures.'' This analysis showed that the
strain with alanine at the position 44 showed the characteristics
similar to that of the wild type. In summary, the amino acid alanine
could replace the serine at position 44 without affecting the
functionality of the protein. By contrast, replacing this serine with
phenylalanine affected the functionality of the protein and made the A. vinelandii UW97 strain Nif
Volume 271,
Number 4,
Issue of January 26, 1996 pp. 1884-1889
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Phenotype of Azotobacter vinelandii UW97
CHARACTERIZATION AND MUTATIONAL ANALYSIS (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
nifH strain, this strain is not useful in
identifying the features of the Fe protein involved in the MoFe protein
assembly. Here, we report a systematic analysis of an A. vinelandii UW97 mutant and show that, unlike A. vinelandii DJ54, the nifH gene of A. vinelandii UW97 has no deletion in
either coding sequence or the surrounding sequences. The specific
mutation responsible for the Nif phenotype of A.
vinelandii UW97 is the substitution of a non-conserved serine at
position 44 of the Fe protein by a phenylalanine as shown by DNA
sequencing. Furthermore, oligonucleotide site-directed mutagenesis was
employed to confirm that the Nif
phenotype in A.
vinelandii UW97 is exclusively due to the substitution of the Fe
protein residue serine 44 by phenylalanine. By contrast, replacing
Ser-44 with alanine did not affect the Nif phenotype of A.
vinelandii. Therefore, it seems that the Nif
phenotype of A. vinelandii UW97 is caused by a general
structural disturbance of the Fe protein due to the presence of the
bulky phenylalanine at position 44.
to
ammonia(1, 2, 3, 4) . The properties
of the nitrogenase proteins, isolated from diverse bacteria, are very
similar (5, 6, 7) . This enzyme is unusual
because it is composed of two separately purified proteins, both of
which are extremely oxygen sensitive. The smaller of the two proteins,
designated the Fe protein, has a molecular mass of about 60,000 daltons
and is a dimer of identical subunits encoded by the nifH gene(1, 8) . The larger of the two proteins,
designated the MoFe protein, has a molecular mass of 230,000
daltons(6, 7) . The MoFe protein is a tetramer in its
biologically active form. It is composed of two identical halves, each
containing an 
-subunit and a 
-subunit encoded by the nifD and nifK genes, respectively. Each of these identical
halves contains one FeMo cofactor and one P-cluster center. These two
halves are believed to be unable to communicate with each other. A
major breakthrough in understanding the structural properties of
nitrogenase came with the publication of the crystallographic
structures of both component proteins of nitrogenase and their metal
centers(9, 10, 11, 12, 13, 14, 15, 16) .
Cloning and sequencing most of the genes involved in the synthesis and
assembly of nitrogenase, in turn, have provided us with an elegant
picture of the genetic organization of this complex metallobiomolecule (1, 17, 18) . Thus, now the stage is set for
concentrating on studies to elucidate how the component proteins of
this multi-centered metalloenzyme are regulated by their structural
constraints during their interplay in the biological N fixation reaction. reduction to occur,
electrons need to be transferred one at a time from the Fe protein to
the MoFe protein. The sequence of events involved in this reaction is
generally referred to as the ``Fe protein cycle'' and is
described elsewhere(19) . Basically, the Fe protein is reduced
by either flavodoxin or ferredoxin (in vivo) or SO (in vitro from dithionite). The reduced Fe protein, in
turn, binds two MgATPs, undergoes a conformational change, and then
binds to the MoFe protein(20, 21) . Mutants that bind
MgATP normally but cannot undergo the MgATP-induced conformational
change have been
identified(22, 23, 24, 25, 26, 27) ;
therefore, it is presumed that these two functions are independent of
each other. Once the Fe protein-MgATP complex binds to the MoFe
protein, both the MgATPs are hydrolyzed and one electron is transferred
from the Fe protein to the MoFe protein(28) . The Fe protein
and the MoFe protein need to interact for MgATP hydrolysis to occur.
Cross-linking experiments and mutagenesis data indicate that the MoFe
protein probably induces a conformational change in the Fe protein,
which is necessary to bring the internal groups close together to
catalyze the hydrolysis(29, 30) .nifH strain of A. vinelandii, designated DJ54,
accumulates a FeMo cofactor-deficient MoFe protein that is distinct
from the FeMo cofactor-deficient MoFe proteins synthesized by nifB, nifN, or nifE strains(35) .
Recently, we have purified apodinitrogenase from A. vinelandii DJ54 and shown that it can be activated by the addition of
wild-type Fe protein and MgATP when the additional required components
are supplied by cell-free extracts from a nifD strain of A.
vinelandii(36) . This observation highlights the crucial
role of the Fe protein in FeMo cofactor synthesis/insertion. However,
in the DJ54 strain the entire nifH gene is deleted. Therefore,
no information regarding the domains on the Fe protein responsible for
its involvement in FeMo cofactor synthesis or insertion can be obtained
from this strain. To understand what features of the Fe protein are
responsible for its ability to function in FeMo cofactor synthesis or
insertion, we needed to obtain Nif mutants with point
mutations in the nifH gene. Here, we report on the analysis
and identification of a point mutation in nifH that impaired
FeMo cofactor biosynthesis/insertion.
Bacterial Strains, Plasmids, and Growth
Conditions
The bacterial strains and plasmids used in this study
are listed in Table 1. A. vinelandii strains were grown
at 30 °C in modified Burk nitrogen-free (BN)
medium(41) . The BN
medium contained 20 g of
sucrose, 0.64 g of K
HPO4
HO,
0.16 g of KHPO
, 0.142 g of
NaSO, 0.203 g of
MgCl6HO, 0.074 g of
CaCl2HO, and 0.034 g of
FeC
H
O
per liter. When it was
necessary to include fixed nitrogen in the medium, ammonium acetate
(NHOAc HO) was added to a final concentration
of 400 µg per ml. Escherichia coli strains were grown at
37 °C in Luria broth, 2YT, or minimal medium supplemented with
glucose (0.2%) and 10 µg/ml thiamine(42) . The antibiotic
ampicillin was used to a final concentration of 50 µg/ml wherever
the selection was made.
General Molecular Techniques
DNA subcloning,
plasmid DNA isolations, restriction enzyme digestions, agarose gel
electrophoresis, ligations, and E. coli transformations were
carried out as described in the laboratory manuals (42) or as
suggested in the manufacturer's instructions. A. vinelandii was transformed with plasmid or recombinant M13 DNA by the method
of Page and Von Tigerstrom(43) .Isolation of Chromosomal DNA
Chromosomal DNA was
prepared from A. vinelandii strains by a modification of the
method described by Dillela and Woo(44) . Cells were harvested
from 100 ml of an overnight culture and resuspended in 10 ml of STE
buffer (10 mM NaCl, 20 mM Tris-HCl, pH 8.0, 1 mM NaEDTA). To this cell suspension, proteinase K and SDS
were added to give final concentrations of 100 µg/ml and 0.5%
(w/v), respectively. The mixture was incubated at 55 °C for
10-16 h. The lysate was gently extracted with an equal volume of
phenol/chloroform/isoamyl alcohol (25:24:1), and after repeating this
step two more times, the aqueous layer was extracted once with an equal
volume of chloroform/isoamyl alcohol (24:1). The aqueous layer was
collected and adjusted to 0.2 M NaOAc, pH 5.5; then, two
volumes of 100% ethanol were gently layered on top, and the DNA was
spooled from the aqueous-ethanol interface using a sterile glass rod.
The spooled DNA was washed with 70% (v/v) ethanol, and the excess
ethanol was evaporated off. The DNA was dissolved in TE buffer (10
mM Tris-HCl, pH 8.0, 1 mM NaEDTA) at 4
°C overnight. This DNA was treated with DNase-free RNase (100
µg/ml) for 1 h at 37 °C; then, the extraction and spooling of
the DNA was repeated as described above. Finally, the DNA was recovered
by dissolving in TE buffer.Southern Blotting and Hybridizations
Southern
blotting and hybridizations were carried out as described(55) .
Briefly, the chromosomal DNA was cleaved with appropriate restriction
endonucleases and separated on agarose gels in 0.5 
TBE (42) buffer. To improve the transfer of large DNA fragments,
the agarose gels were submerged in 250 mM HCl for 10 min to
accomplish limited depurination of the DNA. After the gels were
subjected to denaturation and neutralization steps, the DNA was
transferred to a nylon membrane (MagnaGraph Nylon Transfer Membrane,
Micron Separations, Inc.) with capillary action in 10 SSC for
16-24 h. The transferred DNA was fixed to the nylon membrane by
baking in a vaccum oven for 1 h at 80 °C. The DNA hybridizations
were performed with digoxigenin-labeled DNA probes (Boehringer
Mannheim). The DNA was labeled with digoxigenin-11-UTP using the
random-primed method (Boehringer Mannheim). Detection of
digoxigenin-11-UTP probe DNA was accomplished by using ``Lumi-Phos
530'' and repeated exposures to the Kodak-XAR-5 films.Polymerase Chain Reaction Amplifications, Cloning, and
Nucleotide Sequencing
The PCR (
)was performed by
using the reagents from GeneAmp kit (Perkin-Elmer Corp.). 100 ng of the
chromosomal DNA linearized with PstI and about 50 nmol of the
primers corresponding to sense and antisense DNA strands immediately
after and before the open reading frame for the Fe protein region were
used in the reaction. PCR amplification was continued for 20 cycles
under stringent conditions. The DNA fragment obtained by this method
was cloned into the pCR vector (purchased from
Invitrogen), and selection of recombinants was made as described in the
instruction manual of the supplier. The resulting fragments
corresponding to nifH were subcloned by ligating this region
of DNA into the M13 cloning vector mp18 or mp19 (45) followed
by sequence determination using the method of Sanger et al.(46) .
Site-directed Mutagenesis
The
oligonucleotide-directed mutagenesis was carried out by using
SculptorIn vitro Mutagenesis System-Version 2
(Amersham Corp.) and is based on the phosphorothioate technique of
Eckstein's group(47) . To do this, chemically synthesized
oligonucleotide primers corresponding to the region of interest and
containing the complementary sequence with the appropriate base change
were hybridized to the single-stranded recombinant phage DNA and
extended by using T
DNA polymerase and T DNA
ligase to generate mutant heteroduplex. During this synthesis, dCTP was
replaced by dCTPS so that the newly synthesized strand was
phosphorothioate DNA, which cannot be cleaved by the restriction enzyme NciI. The heteroduplex DNA was treated with NciI to
generate nicks in the non-mutant strand. This nicked non-mutant strand
was removed by treating the sample with exonuclease III, leaving a
fragment of approximately 800 bases that acts as a primer for
repolymerization. A double-stranded mutant homoduplex was generated
using DNA polymerase I and T DNA ligase. E. coli TG1 was transfected with this mutant recombinant phage DNA to
prepare phage stocks from plaques for mutant analysis. The mutations
were confirmed by dideoxy-nucleotide sequencing(46) .Construction of A. vinelandii nifH Mutant
Strains
To construct various A. vinelandii strains that
contain altered forms of nifH, a gene replacement technique
previously described in detail was used(48, 49) . A. vinelandii strains have a very efficient recombination
system that allows homologous recombination between the newly delivered
sequence and the host chromosome. This was done by transforming A.
vinelandii with plasmids that cannot replicate in A.
vinelandii cells and would be lost during cell division.Growth Characteristics of A. vinelandii nifH Mutant
Strains
To compare the growth characteristics of A.
vinelandii nifH mutant strains to that of the wild type strain,
overnight cultures were prepared by allowing them to grow in
BN medium at 30 °C for about 15 h. These fully
grown cultures were diluted (1:250, overnight culture:fresh medium)
into fresh BN
medium and continued the growth at 30
°C. The cell densities were recorded at various time intervals.
nifD strain of A. vinelandii(36) . However, the FeMo
cofactor-deficient MoFe proteins from A. vinelandii strains
with mutations in the nifB or nifEN genes can be
activated in vitro by simple addition of isolated FeMo
cofactor in N-methylformamide(35, 50) .
Recently, it was reported that the activation of apodinitrogenase from A. vinelandii UW97 with FeMo cofactor required very similar
conditions to those that are used to activate apodinitrogenase from A. vinelandii DJ54 (37) . phenotype of A.
vinelandii UW97 was due to a mutation in the nifH, we
replaced the DNA region spanning the nifH sequence of A.
vinelandii UW97 chromosome with the nifH region of the A. vinelandii wild type. This was done by constructing a nifH-containing plasmid that cannot replicate in A.
vinelandii for A. vinelandii transformation. To do this,
we have taken a previously well characterized plasmid pDB6 (51) and cleaved it with the restriction endonuclease HindIII. The resulting products were subjected to
self-ligation by using T
DNA ligase. The plasmids in which
the HindIII DNA fragment corresponding to nif`DKY is
absent was identified by isolating DNA and analyzing it on agarose gels
after restriction enzyme digestions. The resulting plasmid was
designated pBG120. Then, A. vinelandii UW97 was transformed
with the plasmid pBG120, which carries an intact wild-type nifH gene. Since pBG120 cannot replicate in A. vinelandii, it
is lost during cell division and becomes instrumental in delivering the
DNA region spanning the wild-type nifH sequence to the cell. A. vinelandii strains have a very efficient recombination
system that allows homologous recombination between the newly delivered
sequence and the host chromosome. If a mutation in the nifH sequence is responsible for the Nif phenotype,
the transformants that have incorporated wild-type nifH sequences in their chromosome will become Nif
. As
shown in Fig. 1, transformants of UW97 with pBG120 could grow on
BN
(nitrogen-free) plates. This experiment showed
that the mutation causing the Nif
phenotype of A.
vinelandii UW97 is located in the nifH sequence and not
in another gene that affects the Fe protein (e.g.
nifM)(52) .
phenotype in A. vinelandii UW97 is not
due to any detectable deletion in either the coding sequence for the Fe
protein of A. vinelandii UW97 or its surrounding sequences.
II vector (purchased from Invitrogen).
This 1.12-kilobase pair fragment was then cloned into M13 mp18 and
subjected to nucleotide sequence analysis by dideoxy-nucleotide
sequencing method (46) . This analysis showed that the
nucleotide sequence of the nifH gene of A. vinelandii UW97 differs from that of the wild type by a single base change
that replaced the serine codon UCC at position 44 of the Fe protein by
the codon UUC that encodes phenylalanine.
A. vinelandii strain UW97 was obtained by chemical
mutagenesis, there was the possibility that there could be more than
one mutation responsible for its Nif
phenotype.
Therefore, we decided to test whether the substitution of Ser-44 by
phenylalanine alone is responsible for the Nif
phenotype of A. vinelandii UW97. To accomplish this, we
replaced the phenylalanine codon UUC of the A. vinelandii UW97
Fe protein with the serine codon UCC and tested its effect on the Nif
phenotype of the Nif
strains A. vinelandii UW97 and DJ54. This was done by subjecting the A. vinelandii UW97 Fe protein coding sequence cloned in the M13 mp18 to
site-directed mutagenesis as described under ``Experimental
Procedures.'' The Nif
A. vinelandii strains UW97 and DJ54 were transformed with this in vitro mutagenesis construct. The homologous recombination between the nifH gene on the chromosome of these strains and the mutated A. vinelandii UW97 nifH gene (carrying the serine
codon instead of the phenylalanine codon at the 44th position) on the
M13 construct resulted in generating Nif
transformants
from both strains. This experiment confirmed that the Nif
phenotype of A. vinelandii UW97 is due to the change of
Ser-44 to phenylalanine. This serine is located in the second conserved
domain spanning residues 37-45 (Fig. 3).
mutant, A. vinelandii UW91, has
demonstrated that the Fe protein in this mutant has a substitution
mutation in which Ala-157 has been replaced by a serine
residue(23) . This mutation does not seem to affect the ability
of the Fe protein to participate in FeMo cofactor synthesis or
insertion since the MoFe protein in this mutant is normal. The mutation
also does not affect the ability of the Fe protein to bind MgATP.
However, the Nif
phenotype is caused because the
mutation prevents 1) the MgATP-induced conformational change that
occurs in the wild-type Fe protein, 2) MgATP hydrolysis, and 3)
productive electron transfer to the MoFe protein. These observations
indicate that the ability of the Fe protein to take part in FeMo
cofactor biosynthesis/insertion is not directly related to its
involvement in MgATP binding and hydrolysis. Interestingly, our data on
the A. vinelandii UW97 show that the mutation responsible for
making the Fe protein non-functional in FeMo cofactor biosynthesis or
insertion is located in second conserved domain. Thus, it seems that
the structural organization of the second conserved domain plays a
crucial role in at least two seemingly independent functions of the Fe
protein: the ability to take part in FeMo cofactor biosynthesis and the
ability to induce a conformational change in response to MgATP binding.
However, although serine 44 is in a highly conserved region of the Fe
protein, the amino acid Ser-44 is not conserved. The structural model
shows that the side chain of Ser-44 is pointing away from the proposed
MgATP binding site and toward the core of the subunit. Moreover, within
the 4-angstrom sphere of oxygen atom on the side chain of serine 44,
there are more than five residues that can be found. Most importantly,
the oxygen atom on the side chain of serine 44 is also within hydrogen
bonding distance to the oxygen atom on the side chain of aspartic acid
125. An elegant work by Howard and colleagues (22) demonstrated
the critical role played by the aspartic acid at 125 in bringing about
a conformational change upon MgATP binding. It is possible that there
is no space for a bulky phenylalanine side chain to be at position 44,
hence the effect we see is probably due to a general conformational
change of the mutant protein. We argued that a specific change at 44
position that could shed some light would be replacing serine with
alanine at this position.
. Thus, the
phenotype of the corresponding mutant is more likely to result from a
general structural disturbance rather than a specific amino acid
interaction.
)
-We thank Drs. G. P. Roberts, M. Homer
(University of Wisconsin-Madison), and Dr. D. R. Dean (Virginia
Polytechnic Institute and State University, Blacksburg, VA) for kindly
providing the A. vinelandii strains, plasmids, and helpful
discussions.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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