From the Department of Biochemistry, Virginia Tech,
Blacksburg, Virginia 24061-0346 and § E. I. du Pont de Nemours and Co., Central Research and Development,
Experimental Station, Wilmington, Delaware 19880-0328
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ABSTRACT |
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An enzyme having the same L-cysteine desulfurization activity previously described for the NifS protein was purified from a strain of Azotobacter vinelandii deleted for the nifS gene. This protein was designated IscS to indicate its proposed role in iron-sulfur cluster assembly. Like NifS, IscS is a pyridoxal-phosphate containing homodimer. Information gained from microsequencing of oligopeptides obtained by tryptic digestion of purified IscS was used to design a strategy for isolation and DNA sequence analysis of a 7,886-base pair A. vinelandii genomic segment that includes the iscS gene. The iscS gene is contained within a gene cluster that includes homologs to nifU and another gene contained within the major nif cluster of A. vinelandii previously designated orf6. These genes have been designated iscU and iscA, respectively. Information available from complete genome sequences of Escherichia coli and Hemophilus influenzae reveals that they also encode iscSUA gene clusters. A wide conservation of iscSUA genes in nature and evidence that NifU and NifS participate in the mobilization of iron and sulfur for nitrogenase-specific iron-sulfur cluster formation suggest that the products of the iscSUA genes could play a general role in the formation or repair of iron-sulfur clusters. The proposal that IscS is involved in mobilization of sulfur for iron-sulfur cluster formation in A. vinelandii is supported by the presence of a cysE-like homolog in another gene cluster located immediately upstream from the one containing the iscSUA genes. O-Acetylserine synthase is the product of the cysE gene, and it catalyzes the rate-limiting step in cysteine biosynthesis. A similar cysE-like gene is also located within the nif gene cluster of A. vinelandii. The likely role of such cysE-like gene products is to increase the cysteine pool needed for iron-sulfur cluster formation. Another feature of the iscSUA gene cluster region from A. vinelandii is that E. coli genes previously designated as hscB, hscA, and fdx are located immediately downstream from, and are probably co-transcribed with, the iscSUA genes. The hscB, hscA, and fdx genes are also located adjacent to the iscSUA genes in both E. coli and H. influenzae. The E. coli hscA and hscB gene products have previously been shown to bear primary sequence identity when respectively compared with the dnaK and dnaJ gene products and have been proposed to be members of a heat-shock-cognate molecular chaperone system of unknown function. The close proximity and apparent co-expression of iscSUA and hscBA in A. vinelandii indicate that the proposed chaperone function of the hscBA gene products could be related to the maturation of iron-sulfur cluster-containing proteins. Attempts to place non-polar insertion mutations within either A. vinelandii iscS or hscA revealed that such mutations could not be stably maintained in the absence of the corresponding wild-type allele. These results reveal a very strong selective pressure against the maintenance of A. vinelandii iscS or hscA knock-out mutations and suggest that such mutations are either lethal or highly deleterious. In contrast to iscS or hscA, a strain having a polar insertion mutation within the cysE-like gene was readily isolated and could be stably maintained. These results show that the cysE-like gene located upstream from iscS is not essential for cell growth and that the cysE-like gene and the iscSUA-hscBA-fdx genes are contained within separate transcription units.
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INTRODUCTION |
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Iron-sulfur clusters ([Fe-S] clusters) are found in numerous proteins that have important redox, catalytic, or regulatory properties, yet the mechanism(s) by which such clusters are formed or repaired in vivo is not known. We have previously characterized the nifU and nifS gene products (NifU and NifS) that are proposed to have specific roles in the formation or repair of the [Fe-S] cores of metalloclusters contained within the catalytic components of nitrogenase (1, 2). NifS is a pyridoxal phosphate (PLP)1-dependent L-cysteine desulfurase (1), and it is able to catalyze the in vitro reconstitution of an apo-form of the nitrogenase Fe protein whose [4Fe-4S] cluster has been removed by chelation (3). The active species in this reaction is an enzyme-bound persulfide that is formed through nucleophilic attack by an active site cysteine on the PLP-substrate cysteine adduct (4). Although the specific role of NifU is not yet known, we have suggested that it might function either to deliver the iron necessary for [Fe-S] cluster formation or to provide an intermediate site for [Fe-S] cluster assembly (2, 5).
Because the turnover rate for nitrogenase is so slow (6), cells that
depend on nitrogen fixation as their sole source of metabolic nitrogen
must accumulate large amounts of the nitrogenase polypeptides. For
example, the nitrogenase Fe protein and MoFe protein comprise
approximately 5-10% of the total soluble protein fraction in nitrogen
fixing Azotobacter vinelandii cells (6). Because each Fe
protein contains four iron and four inorganic sulfur atoms, and each
MoFe protein -unit contains 15 iron and 16 inorganic sulfur
atoms (7), this situation must place a great demand on the
physiological mobilization of the iron and sulfur necessary for the
maturation of the nitrogenase catalytic components. Thus, we have
suggested the possibility that the reactions catalyzed by NifU and NifS
might represent a specialized way to boost the mobilization of iron and
sulfur necessary for nitrogenase maturation and that there might also
be NifU- and NifS-like "housekeeping" counterparts involved in the
formation or repair of [Fe-S] clusters present in other iron-sulfur
proteins (1, 5). Several lines of evidence support these ideas. First,
a requirement for a boost in the mobilization of sulfur for [Fe-S]
cluster assembly under nitrogen fixing conditions is indicated by the
presence of a nif-specific cysE-like homolog in
A. vinelandii (8, 9) The cysE gene product
encodes an O-acetylserine synthase that catalyzes the rate-limiting step in cysteine biosynthesis (10). Thus, an increase in
cysteine formation when A. vinelandii is grown under
nitrogen fixing conditions probably occurs in response to a demand for NifS activity which utilizes L-cysteine as substrate in the
mobilization of sulfur for nitrogenase [Fe-S] cluster formation.
Second, we have purified a NifS-like protein from the non-nitrogen
fixing organism Escherichia coli and have shown that this
protein is also able to activate sulfur for [Fe-S] cluster formation
in vitro (11). Third, genome sequencing projects have
revealed the presence of NifU- and NifS-like homologs in a variety of
other non-nitrogen fixing organisms, including E. coli (12),
Hemophilus influenzae (13),
Saccharomyces cerevisiae
(14),2 and human
(15).3 In the present work we
have identified and sequenced a genomic region from A. vinelandii that encodes genes that are homologous to
nifS, nifU, as well as other genes whose products
could have roles in the maturation of [Fe-S] cluster-containing
proteins.
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MATERIALS AND METHODS |
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Chemicals, Assays, and Media--
All chemicals were obtained
from Sigma unless otherwise noted. Assays for cysteine desulfurase
activity (IscS activity) were conducted by following the production of
alanine, sulfur, and S2 from L-cysteine as
described previously (16). For assay of IscS purified from A. vinelandii, PLP was added to a concentration of 10 µM. Protein was measured by the Bradford method (17). A. vinelandii cells were cultured in Burke media (18) and
grown at 30 °C, and E. coli was cultured in LB media and
also grown at 30 °C. For antibiotic resistance selection, kanamycin
was added to the medium to a final concentration of 0.5 µg/ml.
Protein Purification--
For purification of IscS from A. vinelandii strain DJ116 (nifS) (19), approximately 1 kg of cell paste of strain DJ116 was mixed with 2 volumes of buffer (50 mM Tris-HCl, pH 8.0, 10 mM MgCl2,
and 1 mM dithiothreitol (DTT) and ruptured in a
microfluidizer. Insoluble cell debris was removed by centrifugation. A
solution of 10 mg/ml protamine sulfate was slowly added to the
supernatant to give a final protamine sulfate concentration that was
5% by weight of the total protein in the sample. Precipitate was
removed by centrifugation, and the soluble fraction was applied to a
5-liter Q-Sepharose column and eluted with the above buffer that
contained a linear gradient of increasing KCl. Fractions were assayed
for L-cysteine desulfurase (IscS) activity, and fractions
from the large peak of activity were pooled. After bringing the pooled fraction to 10% w/v in (NH4)2SO4,
it was applied to a 3.5-liter phenyl-Sepharose column and eluted with a
decreasing (NH4)2SO4 gradient.
Fractions exhibiting IscS activity were pooled, dialyzed, and loaded
onto a 400-ml Sigma Green 19 column and subsequently eluted with an
increasing KCl gradient. The active fractions were pooled and loaded
onto a Superdex 25/600 column and eluted with buffer. Active fractions
from the Superdex column were pooled and chromatographed on a 20-ml
Mono-Q column using a linear increasing KCl gradient. The active
fractions were pooled, and the protein was concentrated to about 10 mg/ml and pelleted in liquid N2 until used. Q-Sepharose and
phenyl-Sepharose columns were run at room temperature using an Amersham
Pharmacia Biotech BioPilot system and the other columns were run at
4 °C using an Amersham Pharmacia Biotech FPLC. In addition to normal
activity assays, purification of IscS was monitored by non-denaturing
gel electrophoresis using the lead stain method (20) to identify the
band that exhibits IscS activity (i.e. release of
S2
from L-cysteine) and by denaturing gel
electrophoresis (21).
Peptide Microsequence Analysis-- Tryptic peptides of IscS purified from A. vinelandii were generated by digestion of a reduced and alkylated (by iodoacetamide) sample with trypsin. The oligopeptides were separated on a Vydac C-18 column using a Waters high pressure liquid chromatograph. To identify the reactive cysteine residue, the protein was treated with 1.2 eq of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine) (I-AEDANS) before alkylation by iodoacetamide. Oligopeptides were separated by high pressure liquid chromatography as above, and the fluorescent oligopeptide fraction was identified by illumination with a UV lamp. Microsequence analysis was performed on a Beckman LF3000 gas-phase sequencer equipped with a Beckman System Gold 125S pump module, Beckman PTH Microcolumn, and a 166 UV detector module. The molecular weight of several of the isolated oligopeptide fragments was determined by infusion electrospray into a Micromass Trio-2000 quadropole mass spectrometer.
DNA Biochemistry-- Oligonucleotides for polymerase chain reaction (PCR) amplification of a segment of the iscS gene from A. vinelandii DJ116 genomic DNA were designed based on the sequence of tryptic peptide fragments of IscS as described above and from the known codon usage bias for A. vinelandii genes (8). The PCR primers used were 5'-GCNACCACCCC(g/c)GTNGA(t/c)CC-3' (forward primer) and 5'-GAANGC(t/c)TCNCCCATNCCNACGAT-3' (reverse primer). "N" refers to any nucleotide, and lowercase letters in parentheses indicate a degeneracy for the two nucleotides indicated at that position. A Stratagene Robocycler Gradient 40 thermocycler was used to amplify a 0.72-kb segment of DNA. The 100-µl PCR reaction mixture contained 2.5 units of Taq polymerase (Perkin-Elmer), 0.2 mM MgCl2, 0.5 mM of each dNTP, and 0.8 µM of each primer. Cycling temperatures were 94 °C for 1.5 min, 65 °C for 2.5 min, and 72 °C for 3 min. The 0.72-kb PCR product was treated with T4 polymerase before ligating it into the cloning vector pUC118 (22) that had been digested with the restriction enzyme HincII. This hybrid plasmid is designated pDB918. The nucleotide sequence of the cloned fragment was determined by the Sanger dideoxy method (23).
A genomic library of DJ116 DNA was prepared by partially digesting chromosomal DNA with XhoI, and the sticky ends of the fragments were partially filled by incubation with T4 polymerase (Life Technologies, Inc.) in the presence of 1 mM dCTP and dTTP. TheMutagenesis of iscS, hscA, and the cysE-like Gene--
For
mutagenesis of iscS, pDB932 DNA was digested with
PstI and ligated to remove an approximately 900-base pair
fragment that extends from the PstI site in iscU
to the rightward PstI site contained within the polylinker
region of the original pUC119 cloning vector. The resultant plasmid was
designated pDB941. Plasmid pDB941 contains three SalI
restriction enzyme sites located within the iscS coding
sequence. These fragments were excised from pDB941 by digestion with
SalI and replaced by ligating the sample with a purified
1.4-kb SalI fragment that contains a kanamycin resistance (KmR) cartridge isolated from the vector pUC4-KAPA
(purchased from Amersham Pharmacia Biotech). This plasmid was
designated pDB952, and it contains a KmR gene cartridge
whose direction of transcription is the same as iscS
transcription. We have previously found that, when in this orientation,
the KmR insertion cartridge used in the present work is not
polar upon expression of downstream A. vinelandii genes, but
when present in the opposite direction, it is polar upon expression of
downstream genes (8, 19). A similar strategy to the one used for
construction of pDB952 was also used for constructing plasmids that
have the same KmR cartridge inserted into either the
cysE-like gene or the hscA gene. The parental
plasmid for cysE-like gene mutagenesis was pDB948.
Mutagenized plasmids that carry the KmR cartridge inserted
within the cysE-like coding sequence either in the same or
the reverse orientation as cysE-like transcription were
designated pDB1008 and pDB1007, respectively. The parental plasmid used
for hscA mutagenesis was pDB932. Mutagenized plasmids that
carry the KmR cartridge inserted into the
hscA-coding sequence either in the same or the reverse
orientation as hscA transcription were designated pDB1009
and pDB1005, respectively. The respective insertion mutations contained
within the various plasmid constructs were transferred to the A. vinelandii chromosome through double-reciprocal recombination events that occurred during transformation (25) using either wild-type
or strain DJ116 (nifS) as host and plasmid DNA as the donor. To ensure that incorporation of the KmR cartridge
into the A. vinelandii chromosome occurred only via double-reciprocal recombination events, plasmids DNAs were linearized by restriction enzyme digestion prior to transformation. For those transformants chosen for further study, confirmation that
double-reciprocal recombination did occur was demonstrated by showing
that the resultant strain was AmpS and KmR.
Because ColEI-derived plasmids are incapable of autonomous replication in A. vinelandii, any transformant arising from a single
Campbell-like recombination event must have both KmR and
AmpR phenotypes (8, 19).
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RESULTS |
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Purification of a Protein Having a NifS-like Activity from A. vinelandii--
A protein that catalyzes the same enzymatic activity
as NifS was isolated from extracts prepared from an A. vinelandii strain (DJ116) deleted for the nifS gene. We
have designated the NifS-like protein from A. vinelandii
"IscS" to indicate its proposed role in
iron-sulfur-cluster formation, and
that nomenclature will be used hereafter. Crude extracts were
sequentially processed by protamine sulfate treatment and column
chromatography using Q-Sepharose, phenyl-Sepharose, Sigma Green-19,
Superdex, and Mono-Q columns, respectively. Fractions having IscS
activity were monitored by their ability to release sulfur or
S2 and alanine from L-cysteine. Fractionation
of the extract by Q-Sepharose chromatography yielded a minor peak and a
large major peak of IcsS activity. The fractions containing the major
peak were pooled and further purified, whereas the minor peak fraction was not processed further. When the material in the large activity peak was purified using the columns listed above, a single protein whose purity was judged to be greater than 90%, by native and denaturing gel electrophoresis, was obtained. Data from a typical isolation are shown in Table I.
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Characterization of IscS--
IscS isolated from A. vinelandii extracts is yellow, and the optical spectrum indicates
the presence of PLP (Fig. 1). When 1 eq
of L-cysteine was added to the enzyme, the visible region of the optical spectrum changed as shown in Fig. 1. These spectra are
similar to those exhibited by the A. vinelandii NifS protein (1) and the E. coli NifS-like protein (11). When DTT was present in the assay, IscS catalyzed the desulfurization of
L-cysteine to yield both alanine and S2 in
approximately equal amounts. In the absence of DTT, the enzyme catalyzed the release of both sulfur and S2
from
L-cysteine, the sum of which approximately equaled the
amount of alanine formed. Apparently, some of the sulfur released from L-cysteine was reduced to S2
in the absence
of DTT by the excess of substrate cysteine present in the reaction.
Treatment of IscS with 1.2 eq of the alkylating reagent I-AEDANS
resulted in the complete inhibition of its L-cysteine desulfurization activity. These results are the same as found for the
A. vinelandii NifS protein (1) and the NifS-like protein purified from E. coli (11).
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Microsequencing Experiments-- For peptide microsequencing experiments a highly purified form of IscS was obtained by further processing a sample prepared as described above by first removing DTT from the buffer, binding of the sample to a thiopropyl-Sepharose column, followed by its elution with 5 mM DTT. The protein prepared in this way was subjected to automated N-terminal sequence analysis, which gave the sequence KLPIYLDYSATTPVDPRVAQKM. This sequence matches the polypeptide sequence deduced from the gene sequence determined in experiments described below, except that the terminal methionine is not present in the isolated protein. Following tryptic digestion of purified IscS, the resulting oligopeptides were separated by high pressure liquid chromatography using a C-4 column. Primary sequences were determined for five of the major oligopeptides separated by this method, and the sequence of four of these was corroborated by mass spectrometry. The sequences of these peptides were all readily aligned with portions of the primary sequence of the A. vinelandii NifS protein and other NifS-like proteins known to share considerable sequence identity with NifS (Fig. 2). The five oligopeptide sequences determined by microsequencing show a nearly perfect match to the corresponding polypeptide sequences deduced from the DNA sequence, and for those sequences that were confirmed by mass spectrometry, there is a perfect match (Fig. 2).
A tryptic digest was also prepared from a sample of IscS that had been alkylated by prior treatment with 1.2 eq of I-AEDANS. When the oligopeptides from this sample were separated by high pressure liquid chromatography as above, one fraction also exhibited fluorescence. The oligopeptide contained within the fluorescent fraction was sequenced and found to be identical to a previously sequenced peptide shown in Fig. 2, with the exception that no cysteine was found at the residue 10 position of that peptide. This result indicates that the cysteine was modified by I-AEDANS. The alignments in Fig. 2 show that this IscS cysteine residue (cysteine 328) corresponds to cysteine 325 in the NifS sequence. The NifS cysteine 325 residue was previously shown by alkylation experiments and amino acid substitution experiments to be the active site cysteine upon which the persulfide is formed (4).Purification and Characterization of Recombinantly Produced IscS-- As described below and under "Materials and Methods," primary sequence information obtained from microsequencing of IscS tryptic peptides provided the information necessary to design a strategy for isolation of a genomic DNA fragment that encodes IscS. The cloned iscS gene was then used to construct a hybrid plasmid for which the expression of iscS is placed under the control of the T7 transcriptional and translational control elements. By using this construct, it was possible to produce A. vinelandii IscS at high levels in E. coli. IscS produced in this way was purified as described under "Materials and Methods." Recombinantly produced A. vinelandii IscS exhibited the same properties as described for IscS purified from A. vinelandii. Gel exclusion chromatography showed that recombinant IscS is a homodimer that exhibits a native Mr of approximately 104,000. When assayed in the presence of DTT, the maximum activity obtained for IscS (124 nmol of product/min/mg) compared favorably with the maximum activity of purified NifS assayed under the same conditions (168 nmol of product/min/mg). However, a true maximum activity for IscS could not be obtained nor could kinetic parameters be determined, because L-cysteine concentrations greater than 0.2 mM inhibit activity, and product detection limitations prevent assay of IscS at L-cysteine concentrations lower than 0.2 mM. This pattern of inhibition of IscS by L-cysteine was not observed for NifS. It is noted that the specific activity of purified recombinant IscS, when assayed under the conditions reported in Table I (2.5 mM L-cysteine), gives approximately the same specific activity as IscS purified from A. vinelandii.
Isolation of the iscSUA Gene Region--
Based on the oligopeptide
sequences obtained in the microsequencing experiments, and the known
codon bias for A. vinelandii, oligonucleotide primers were
designed for PCR amplification of a segment of the iscS gene
from genomic A. vinelandii DNA prepared from strain DJ116.
The authenticity of this fragment was confirmed by DNA sequence
analysis, and the fragment was then used to probe an A. vinelandii genomic library prepared using the -DASH-II cloning
vector as host. A 4.0-kb EcoRI restriction enzyme fragment located within a hybrid
phage exhibited hybridization to the PCR-amplified iscS fragment. A 2.5-kb
EcoRI-SstI subfragment of the 4.0-kb
EcoRI fragment was subsequently subcloned into pUC119, and
its nucleotide sequence was determined. Analysis of the nucleotide sequence of this fragment revealed three complete coding sequences (Fig. 3), and these coding sequences
respectively exhibited sequence identity when compared with NifS, NifU,
and the deduced primary sequence of another gene product previously
designated Orf6 (19). This gene is encoded within the major
nif cluster of A. vinelandii (8) and is located
adjacent to nifU (indicated as "iscA" in Fig.
3). We have provisionally designated the nifS homolog
iscS, the nifU homolog iscU, and the
orf6 homolog as iscA to indicate their potential
roles in iron-sulfur-cluster
assembly. Although, the iscSUA genes are clustered, they are
organized in an opposite fashion when compared with their
nif-specific counterparts (Fig. 3). In contrast, the
organization of the A. vinelandii iscSUA gene cluster is the
same as homologous clusters that are present in E. coli and
H. influenzae (12, 13) (Fig. 3). Furthermore, a general
conservation in the relative genomic positions of these clusters in
A. vinelandii, E. coli, and H. influenzae is indicated by interspecies conservation in sequences
located both upstream and downstream of the respective isc
gene cluster region.
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Primary Sequence Comparisons of iscSUA Homologs-- The interspecies primary sequences among proposed IscS (Fig. 2), IscU (Fig. 4), and IscA (Fig. 5) proteins from A. vinelandii, H. influenzae, and E. coli are aligned with each other and with the A. vinelandii NifS, NifU, and nif-specific IscA homologs, respectively. In the case of IscS from A. vinelandii and E. coli, these proteins are legitimately assigned as having an IscS function because they have been purified and characterized. In other cases, IscS function can only be inferred from primary sequence comparisons. The criteria that we have used to tentatively assign IscS function to a particular protein sequence is as follows: (i) there must be strong primary sequence conservation throughout when compared with the IscS proteins having established cysteine desulfurase activity; (ii) the active site PLP-binding lysine residue corresponding to lysine 202 in the NifS sequence must be conserved; and (iii) the active site cysteine residue corresponding to cysteine 325 in the NifS sequence must be conserved in the same relative position within its primary sequence. Data base searches reveal that a number of putative proteins have been designated "NifS-like" based only on significant sequence similarity surrounding and including the PLP-binding active site lysine corresponding to lysine 202 from NifS. The primary sequences of several of these proteins, however, do not show conservation of the NifS active site cysteine 325 residue, so it is possible that they represent PLP-dependent proteins that have functions unrelated to the NifS-IscS family. This possibility has been proven for at least one of the NifS-like protein from E. coli, which has been purified and shown to have cysteine sulfinate desulfinase activity rather than cysteine desulfurase activity (29). A complete comparison of NifS-like primary sequences currently available in the data bank has been published by Mihara et al. (29).
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Homologs to the E. coli hscBA and fdx Genes Are Located Downstream from the iscSUA Gene Cluster-- After submission of the sequence of the iscSUA gene cluster to the data bank, our attention was brought to the fact that, in E. coli, genes designated as hscB, hscA, and fdx are located immediately downstream from the proposed iscSUA gene cluster. The hscB and hscA genes encode proteins that bear a high degree of sequence identity when compared with the molecular chaperone proteins encoded by dnaJ and dnaK, respectively (30, 31). The location of the hscBA genes immediately downstream from iscSUA in E. coli raises the intriguing possibility that these heat-shock-cognate proteins, whose functions are not yet known, might serve as molecular chaperones that specifically assist in the maturation of [Fe-S] proteins.9 Subsequent sequence analysis of the A. vinelandii genome in the region immediately downstream from iscSUA revealed the presence of homologs to the hscBA genes, a gene that encodes a homolog for a [2Fe-2S] cluster-containing ferredoxin (32), and another putative gene that has not been characterized (Fig. 3). In contrast to E. coli, where there is a relatively large separation between iscA and hscB (96 nucleotides), only 16 nucleotides separate the termination codon of iscA and the initiation codon of hscB. This spatial organization indicates that the A. vinelandii iscSUA and hscBA genes (as well as genes indicated as fdx and orf3 in Fig. 3) are very likely to be co-transcribed. Inspection of the nucleotide sequence immediately following the gene indicated as orf3 in Fig. 3 indicates the likely presence of a rho-independent transcriptional termination signal. Also, there are 96 nucleotides separating orf3 and the next gene, which encodes nucleotide dikinase. The ndk gene is also found at the same relative position on the E. coli genome and is separated from the orf3 gene by 91 nucleotides (Fig. 3).
Identification of a cysE-like Gene That Is Located Upstream from the iscSUA Gene Cluster-- Prompted by the identification of the hscBA genes and the potential involvement of their products in maturation of [Fe-S] proteins, the DNA sequence of the A. vinelandii genomic segment located immediately upstream from the iscS gene was also determined. The result of this analysis revealed that another putative gene (indicated as orf2 in Fig. 3) precedes iscS and is separated from it by only by 28 nucleotides. A homolog to this gene is also present at an analogous position in E. coli, although it is separated from iscS by 118 nucleotides. The next putative gene (indicated as cysE2 in Fig. 3) is located 196 nucleotides upstream. The relatively large space separating cysE2 and orf2 and the fact that a potential rho-independent transcription site and a pyrimidine-rich sequence is located in the cysE2-orf2 intergenic region indicate that cysE2 is contained in a transcription unit that is separate from the orf2-iscS-iscU-iscA-hscB-hscA-fdx-orf3 cluster. Although a homolog corresponding to the A. vinelandii cysE2 gene is not found in E. coli, a putative gene preceding cysE2 in A. vinelandii is also present in E. coli at approximately the same genomic position. Examination of sequences located upstream from this gene (indicated as orf1 in Fig. 3) reveals the presence of the suhB gene. In both A. vinelandii and E. coli the suhB gene is divergently transcribed when compared with the orf1 gene and, in the case of the A. vinelandii genome, is separated from orf1 by 135 nucleotides. This spatial organization indicates that orf1 and cysE2 probably compose an individual transcription unit.
There are two interesting aspects about the location and primary sequence of the product of the cysE2 gene located in the A. vinelandii genomic region preceding iscS. First, a similar gene (indicated as cysE1 in Fig. 3) is also located near the nifU and nifS genes within the nif-specific gene cluster. The presence of this cysE homolog within the A. vinelandii nif cluster was first recognized by Evans et al. (9), who demonstrated that its product catalyzes formation of O-acetylserine. These investigators suggested that the cysE1 gene product could play a role in boosting the cysteine pool in response to an increased demand for the mobilization of sulfur for nitrogenase-specific [Fe-S] cluster formation. This suggestion was strongly supported by the subsequent identification of the cysteine desulfurase activity catalyzed by NifS (1). The second notable feature that emerges from the comparison of cysE1 and cysE2 is that primary sequence identity between them is confined to the N-terminal two-thirds of the respective proteins (Fig. 6). Comparative PCR analysis of A. vinelandii genomic DNA and the cloned DNA used for sequence analysis showed that this feature is not the result of a cloning artifact (data not shown). The absence of sequence identity within the terminal third of these proteins could indicate that they respectively interact with different macromolecular complexes during the activation, and/or shuttling, of cysteine-sulfur that is destined for incorporation into the [Fe-S] clusters of specific protein targets. In this context it is also noted that the products of both cysE1 and cysE2 are missing the first 75 amino acids when compared with the bona fide cysE gene product from E. coli (Fig. 6).
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Genetic Analysis of the isc Gene Cluster Region--
In order to
attempt inactivation of the chromosomally encoded iscS gene,
a plasmid (pDB952) was constructed that has a large portion of the
iscS gene deleted and the deleted segment replaced by a
KmR gene cartridge. The deletion and insertion within
pDB952 was incorporated into the chromosomes of A. vinelandii strain DJ116 (nifS) by transformation
followed by selection for KmR and scoring for
AmpS. The strain constructed in this way exhibited a normal
growth rate, initially indicating that no obvious phenotype is
associated with the inactivation of the iscS gene. However,
when these cells were cultured in the absence of kanamycin, the
KmR phenotype was rapidly lost. For example, after growing
these cells for approximately 15 generations without selective
pressure, no KmR cells could be recovered. In another
experiment, single KmR colonies were picked and diluted in
liquid medium and then spread on Petri plates supplemented with or
without added kanamycin. The ratio of KmR to
KmS cells was found to be approximately 1 to 5 indicating
that, even under kanamycin-selective pressure, cells harboring only the
wild-type iscS allele rapidly segregate from those harboring
the KmR cartridge. A homologous excision event cannot
account for the rapid loss of the KmR marker because the
original integration of the KmR cartridge occurred through
double-reciprocal recombination. These results show that there is
strong selective pressure to maintain the wild-type iscS
allele and that both the wild-type and the inactivated iscS
alleles are maintained in the same cells when placed under the
appropriate selection pressure. There have been two previous reports of
the same phenomenon occurring in A. vinelandii where strong
selective pressure was used to maintain both a gene that was
interrupted by an antibiotic resistance marker and the corresponding
wild-type allele (33, 34). That such a phenomenon can occur in A. vinelandii has been attributed to its proposed ability to maintain
multiple identical chromosomes (34, 35). It is also noted that there
are many cases of insertional activation of non-essential genes in
A. vinelandii which rapidly undergo homogenization and
remain stable in the absence of antibiotic-selective pressure (8,
19).
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The nifS Gene Product Cannot Replace the Requirement for iscS Expression-- In the description of the original characterization of nifS it was suggested that a NifS-like protein that has a housekeeping function similar to NifS might be able to partially supplant NifS function (1). This possibility was suggested because a nifS deletion mutant remains capable of slow diazotrophic growth. It was not possible to test this hypothesis in the present work because insertional inactivation of iscS is apparently lethal. However, it was possible to test the alternate possibility that NifS might supplant IscS function. This possibility was tested by transforming wild-type A. vinelandii cells with pDB952, as described above, and then selecting for KmR transformants under diazotrophic growth conditions. The rationale for this experiment is that if NifS activity can significantly replace IscS function, then transformants having an iscS insertion mutation should be stably maintained under conditions of non-selective antibiotic pressure, providing that the cells are cultured under diazotrophic growth conditions. However, it was found that an iscS KmR insertion mutation could not be stably maintained even when the transformants were cultured under diazotrophic growth conditions. Thus, either NifS or IscS activity is not significantly interchangeable or the amount of NifS needed to replace IscS function is not accumulated under the experimental conditions used. Evidence supporting this latter possibility was recently reported (35). In these experiments it was shown that heterologous co-expression of nifS from A. vinelandii and glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis in E. coli boosted the amount of the mature [4Fe-4S] cluster-containing B. subtilis enzyme accumulated.
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DISCUSSION |
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The IscS protein from A. vinelandii was purified based on its ability to catalyze the release of sulfur from L-cysteine. IscS exhibits strong primary sequence identity when compared with NifS and catalyzes the same reaction as NifS. Isolation and sequence analysis of a genomic fragment containing iscS revealed that it is clustered, and probably co-transcribed, with seven other genes. There is currently good evidence that both NifU and NifS are involved in the formation or repair of [Fe-S] clusters needed for activation of nitrogenase components (1, 2). By analogy to the nif system we suggest that IscS, IscU, and IscA are likely to have housekeeping functions related to the assembly or repair of [Fe-S]-containing proteins other than nitrogenase (18). The location and apparent co-expression of iscSUA and hscBA genes indicate the possible presence of a macromolecular system that functions in the proper folding of [Fe-S]-containing proteins, as well as participating in the formation and insertion of their corresponding clusters. The presence of the cysE2 gene in the transcription unit located upstream from iscS in A. vinelandii also suggests the possibility of a mechanism for the specific targeting of L-cysteine for [Fe-S] cluster formation.
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ACKNOWLEDGEMENTS |
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We thank those investigators that have made genome sequence analyses available prior to publication. We also acknowledge the expert technical assistance of Christina O'Kernick and Laura Taylor. We are particularly grateful to professor Larry Vickery for pointing out the hscBA genes and suggesting the potential role of their products in maturation of [Fe-S] cluster-containing proteins.
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FOOTNOTES |
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* This work was supported by National Science Foundation Grant MCB-9630127.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF010139.
¶ To whom correspondence should be addressed. Tel.: 302-695-1522; Fax: 302-695-4260; E-mail: flintdh{at}al.escax.umc.dupont.com.
To whom correspondence should be addressed. Tel.:
540-231-5895; Fax: 540-231-7126; E-mail: deandr{at}vt.edu.
1 The abbreviations used are: PLP, pyridoxal phosphate; DTT, dithiothreitol; PCR, polymerase chain reaction; kb, kilobase pair(s); IAEDANS, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine.
2 This information is available at the Yeast MIPS Web page (http://www.mips.biochem.mpg.de/mips/yeast/).
3 T. Rouault, personal communication.
4 Deposited by B. A. Roe, S. P. Lin, L. Song, X. Yuan, S. Clifton, M. McShan, and J. Ferretti, and available from the Streptococcal Genome Sequencing Project Web page (http://www.genome.ou.edu/strep.html).
5 Deposited by B. A. Roe, S. P. Lin, L. Song, X. Yuan, S. Clifton, and D. W. Dyer, and available from the Gonococcal Genome Sequencing Project Home page (http://www.genome.ou.edu/gono.html).
6 Available from the Cyanobase Genome Data Base Web page (http://www.kazusa.or.jp/cyano/cyano.orig.html).
7 T. Rouault, personal communication.
8 P. Yuvaniyama, L. Zheng, J. N. Agar, R. F. Jack, M. K. Johnson, and D. R. Dean, unpublished results.
9 L. Vickery, personal communication.
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REFERENCES |
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