Clostridium pasteurianum W5 synthesizes two NifH-related polypeptides under nitrogen-fixing conditions

Murat Kasap{dagger} and Jiann-Shin Chen

Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA

Correspondence
Jiann-Shin Chen
chenjs{at}vt.edu


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Previous studies identified five nifH-like genes (nifH2 through nifH6) in Clostridium pasteurianum (strain W5), where the nifH1 gene encodes the nitrogenase iron protein. Transcripts of these nifH genes, with the exception of nifH3, were detected in molybdenum-sufficient nitrogen-fixing cells. However, the size of the transcripts, the level of transcription and the presence of polypeptides encoded by the nifH-like genes were not reported. The nifH2 and nifH6 genes were extremely similar, as they seemed to differ by only two bases in a span of 2481 bp, one in the coding region and another in the upstream region. Re-examination of the DNA sequences revealed that the coding region of nifH2 and nifH6 was identical, whereas the difference in the upstream region was confirmed. Results from the authors' ongoing study of the nif genes of single-colony isolates of C. pasteurianum suggest that the nifH6 designation should be eliminated. Here the size of mRNA from nifH2 and the detection of the NifH2 polypeptide in nitrogen-fixing cells of C. pasteurianum are reported. Northern blot analysis of periodically collected nitrogen-fixing cells showed that the nifH1 and nifH2 mRNAs were present throughout growth. Addition of ammonium acetate repressed the transcription of both these genes similarly. Using an antiserum raised against NifH of Azotobacter vinelandii, two NifH-related bands were detected by Western blot analysis after electrophoretic separation of proteins in extracts of nitrogen-fixing C. pasteurianum cells. After separation of proteins by preparative SDS-PAGE, the NifH polypeptides were characterized by MALDI-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) and by ES-MS/MS (electrospray tandem mass spectrometry) analyses. The results confirmed the presence of NifH2, in addition to NifH1, in nitrogen-fixing C. pasteurianum cells.


Abbreviations: ES-MS/MS, electrospray tandem mass spectrometry; HRP, horseradish peroxidase; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

{dagger}Present address: Kocaeli University Medical Center, Sopali Ciftligi, 41900 Derince, Kocaeli, Turkey.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Clostridium pasteurianum was the first free-living nitrogen-fixing organism that was isolated (Winogradsky, 1895), and with which the first consistently active cell-free nitrogen fixation was demonstrated (Carnahan et al., 1960; Burris, 1988). Early biochemical studies with C. pasteurianum revealed several salient features of biological nitrogen fixation, including the discovery of ferredoxin, a low redox-potential electron donor for nitrogenase (Mortenson, 1964). The nitrogenase component proteins are structurally conserved to such an extent that heterologous pairs are usually active (Emerich et al., 1981). However, the Fe and MoFe proteins of C. pasteurianum have unique structures (Chen et al., 1986; Wang et al., 1988; Kim & Rees, 1993; Schlessman et al., 1998; Chen, 2004), which could explain the lack of activity in heterologous pairings involving the nitrogenase of C. pasteurianum. Whether or not the distinctive nitrogen-fixing system of C. pasteurianum contributes to the robustness of this organism is not known. Further characterization of the nitrogen-fixing system of this organism could identify targets suitable for experimentation to address this question.

The nitrogen-fixation (nif) gene cluster of C. pasteurianum consists of nifH2, nifH1, nifD, nifK, nifE, nifN-B, modA, modB, nifV{omega} and nifV{alpha} genes in the same orientation (Chen & Johnson, 1993; Chen, 2004). Four additional nifH-like genes were reported: Chen et al. (1986) identified nifH3, whereas Wang et al. (1988) identified nifH4 to nifH6. The nifH1gene encodes the characterized Fe protein (273 amino acids) of nitrogenase (Chen et al., 1986; Chen & Johnson, 1993). The nifH2 gene encodes a polypeptide of 272 amino acids that differs from NifH1 by 22 amino acids. The deduced NifH-related polypeptides of C. pasteurianum fall into four phylogenetic groups represented by NifH1 through NifH4 (Wang et al., 1988; Ueda et al., 1995). NifH3 is the most distinct among them as it differs from the others by 87 to 97 amino acids (31·9–35·5 %). NifH3 of C. pasteurianum is, however, closely related to AnfH of Azotobacter vinelandii (Chen et al., 1990).

The assignment of the nifH6 gene was based on its differences from nifH2: Gly-13 encoded by GGA in nifH6 versus Ala-13 encoded by GCA in nifH2, and the absence of a HindIII site in its upstream region (AAGACTT preceding nifH6 versus AAGCTT preceding nifH2). Because of the similarity between these two genes and their flanking regions, our laboratory recently resequenced the inserts in pCP114 (harbouring nifH2; Chen et al., 1986) and pCP600 (harbouring nifH6; Wang et al., 1988). It turned out that the coding region of nifH2 and nifH6 was identical, with Gly-13 being the conserved amino acid. The single nucleotide difference in the upstream region was, however, confirmed. Our laboratory is now characterizing the nif and nif-like genes of single-colony isolates of C. pasteurianum strain W5 (wild-type). So far, all of the isolates have been found to contain the sequence previously cloned in pCP600 (C. Tollin, A. Tran, J. Toth & J.-S. Chen, unpublished results). The new results suggest that the laboratory stock of C. pasteurianum W5 contained two populations that differed by the presence or absence of a HindIII site preceding the nifH2 gene, and the vast majority appears to lack the HindIII site. Although the characterization of the single-colony isolates is continuing, it is now appropriate to amend the nifH2 sequence and eliminate the nifH6 designation.

Transcripts of the nifH-like genes, with the exception of nifH3, were detected in nitrogen-fixing C. pasteurianum (Wang et al., 1988). Except for NifH1, it was not known if other NifH-related polypeptides were synthesized. In this paper, we report the presence of NifH2 in nitrogen-fixing cells of C. pasteurianum.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Growth conditions.
C. pasteurianum strain W5 was grown at 35 °C in a medium containing sucrose, 30 g l–1; K2HPO4, 0·69 g l–1; Na2SO4.10 H2O, 0·19 g l–1; mineral solution (George & Chen, 1983), 1 ml l–1; CaCO3, 5 g l–1; and resazurin, 1 mg l–1. The headspace of the culture was flushed with nitrogen throughout incubation.

Whole-cell acetylene-reduction assay.
The acetylene-reduction assay was used to measure nitrogenase activity as described by Chen et al. (2001), except that the vial contained 2 ml bacterial culture and was incubated at 35 °C.

Preparation of cell-free extracts and protein determination.
Cell paste was thawed under argon in anaerobic 50 mM Tris/HCl (pH 8·0) buffer (3 ml per g cells), containing 20 % (v/v) glycerol, 1 mM dithiothreitol, 0·1 mg DNase I ml–1 and 2 mg lysozyme ml–1. Other steps were as described by Yan & Chen (1990). A cell-free extract of nitrogen-fixing A. vinelandii, prepared as described by Vichitphan (2001), was a gift from the laboratory of W. E. Newton (Deptartment of Biochemistry, Virginia Tech, Blacksburg, VA, USA). Protein concentration was determined by the dye-binding assay (Bradford, 1976) with bovine gamma globulin as a standard.

Western blot analysis.
Proteins in cell-free extracts were separated by SDS-PAGE or non-denaturing PAGE on 12 % acrylamide gel. The SDS-PAGE was performed according to the method of Laemmli (1970). The non-denaturing PAGE was also performed according to Laemmli (1970), but in the absence of SDS and at a constant voltage of 100 V at 4 °C. Proteins were electrophoretically transferred onto positively charged nitrocellulose membranes in a semi-dry electrophoretic transfer cell (Bio-Rad). Western blots were probed with a NifH antiserum using the enhanced chemiluminescence (ECL) detection system (Amersham Biosciences). The NifH antiserum was raised in rabbit against the nitrogenase Fe protein of A. vinelandii, and was a gift from the laboratory of W. E. Newton (Department of Biochemistry, Virginia Tech, Blacksburg, VA, USA).

Preparation of the nifH probe.
The probe for the nifH-like genes was generated by PCR of a conserved region of the C. pasteurianum nifH gene as described previously (Chen et al., 2001).

Northern analysis of nif mRNA.
For RNA isolation, the guanidine isothiocyanate procedure described by Johnson (1994) was used, except that at the final step, the RNA pellet was dissolved in 250 µl water containing diethyl pyrocarbonate (0·1 %, w/v) and SDS (0·05 %, w/v). The size of RNA fragments was estimated by using two different RNA ladders, one from New England BioLabs and the other from Gibco-BRL Life Technology. The RNA species were routinely transferred to positively charged nylon membranes (Hybond-N+) for 16 to 18 h by capillary elution with 20x SSC. The ECL signal was generated and detected according to the manufacturer's instructions (Amersham Biosciences).

Direct RT-PCR amplification of the 0·9 kb mRNA bound on a membrane.
cDNA strands of the 0·9 kb mRNA were synthesized in 0·5 ml microcentrifuge tubes using a 2x3 mm piece of nitrocellulose membrane containing the 0·9 kb mRNA as recommended in the Omniscript reverse transcriptase handbook (Qiagen). Ten microlitres of the reverse transcriptase reaction mixture containing the cDNA strand was then used for PCR amplification of nifH-like gene fragment as described previously (Chen et al., 2001)

Separation of the NifH-related polypeptides by preparative SDS-PAGE.
Preparative SDS-PAGE was run for 8 h using a 12 % acrylamide gel in a model 491 Prep Cell (Bio-Rad) with the discontinuous buffer system of Laemmli (1970). The sample for each run contained 25 mg protein. The elution position of NifH-related polypeptides was determined by Western blotting. Selected fractions containing NifH-related polypeptides were subjected to analyses by MALDI-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) and ES-MS/MS (electrospray tandem mass spectrometry).

Analysis of NifH-related polypeptides by MALDI-TOF MS.
For in-gel digestion of proteins with trypsin, a modification of the procedure of Shevchenko et al. (1996) was used, and trypsin (20 ng µl–1) was prepared in 50 mM ammonium bicarbonate buffer. Disulfide bonds, if present, were reduced with 10 mM DTT. Proteins were alkylated with 50 mM iodoacetamide. When necessary, the gel pieces were dehydrated in acetonitrile and rehydrated in 100 mM ammonium bicarbonate buffer. The peptides were recovered with Zip-Tips as described by the manufacturer (Millipore). At the final step, the peptides were eluted with 3 µl of a saturated solution of 4-hydroxy-{alpha}-cyanocinnamic acid in 1 : 1 (v/v) acetonitrile/acidified water.

Mass spectra of the peptides were obtained on a Kratos Kompact SEQ (Kratos Analytical) time-of-flight mass spectrometer. Pulses of radiation at 337·1 nm and 3 ns duration were directed at the sample/matrix mixture. The resulting ions were accelerated through a 1·8 m flight tube by a potential difference of 20 kV. The laser fluence and spot position were varied manually during data acquisition. Spectra were recorded and processed using the Kratos LAUNCHPAD MALDI software, version 1.2.0.

To match proteins in the SWISS-PROT database, the determined peptide masses were compared with values computed from the database entries according to the cleavage specificity of trypsin. The computed peptide masses include those resulting from modifications of cysteine (alkylation by iodoacetamide or acrylamide) or oxidation of methionine, and allow up to one missed cleavage. All necessary computation was implemented using PEPTIDENT, available at the ExPASy (Expert Protein Analysis System) molecular biology server (http://us.expasy.org).

Analysis of NifH-related polypeptides by ES-MS/MS.
ES-MS/MS analysis of NifH-related polypeptides was performed by the W. M. Keck Biomedical Mass Spectrometry Laboratory at the University of Virginia (Charlottesville, VA, USA). The LC-MS system consisted of a Finnigan LCQ ion trap mass spectrometer system with a Protana nanospray ion source interfaced to a Phenomenex Jupiter 10 µm C18 reversed-phase capillary column. The peptides were eluted from the column by an acetonitrile/0·1 M acetic acid gradient. The nanospray ion source was operated at 2·8 kV. The digest was analysed using the double-play capability of the instrument, acquiring full-scan mass spectra to determine peptide molecular masses, and product-ion spectra to determine amino acid sequence in sequential scans. The data were analysed by database searching using the SEQUEST search algorithm. Peptides that were not matched by this algorithm were interpreted manually and searched versus the EST databases using the SEQUEST algorithm.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Detection of a second nifH-related mRNA in C. pasteurianum
Northern blot analysis of RNA from nitrogen-fixing cells of C. pasteurianum detected two major mRNA populations (4 and 0·9 kb) that hybridized to the nifH probe (Fig. 1a). The 4 kb mRNA is the transcript of the structural genes (nifH1DK) for the molybdenum-containing nitrogenase; the other nifH-containing regions are not expected to produce a transcript of this size under molybdenum-sufficient growth conditions (Wang et al., 1988; Zinoni et al., 1993). The 0·9 kb transcript was not reported previously, and it could have arisen from either degradation or processing of the 4·0 kb mRNA, or it might be a bona fide transcript from one of the nifH-like genes. After performing Northern blot analysis, the 0·9 kb region (the dotted square in Fig. 1a) of the RNA-containing membrane was cut out and used in an RT-PCR experiment. A 350 bp fragment was amplified (Fig. 1b) and sequenced (by Davis Sequencing). The sequenced fragment encompassed nucleotides 198 to 548 of the coding region, which contains characteristic nucleotides of each nifH-like gene (Wang et al., 1988). The sequence revealed that the 0·9 kb message was from the nifH2 gene of C. pasteurianum.



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Fig. 1. Detection and RT-PCR amplification of the 0·9 kb nifH-like mRNA of C. pasteurianum. (a) Northern blot of mRNA from nitrogen-fixing cells of C. pasteurianum. Total RNA was hybridized with an HRP-labelled, nifH1-derived probe. Two mRNA bands at the sizes of 4·0 and 0·9 kb are clearly visible (arrows). With the RNA markers and the X-ray films as references, the 0·9 kb message was localized and a portion (shown as a dotted square) of the membrane was cut out from the running track of the sample and used in a RT-PCR experiment. (b) Separation of RT-PCR products in a 1 % agarose gel. Lane 1, DNA ladder; lane 2, positive control with genomic DNA as the template; lane 3, RT-PCR products; lane 4, negative control with a membrane piece cut from the 2·0 kb region as the template; lane 5, RT-PCR with the RNA isolated from a non-nitrogen fixing culture of C. pasteurianum; lane 6, negative control (without reverse transcriptase).

 
Expression of nifH2 versus nifH1
To compare the expression of nifH1 and nifH2, C. pasteurianum was grown in a nitrogen-fixing medium, and cells were collected periodically for the isolation of RNA. The RNA was analysed by Northern blotting with a nifH probe. As expected, two major signals, at 4 and 0·9 kb, were observed. A visual examination of the signal intensity of the 0·9 kb and the 4 kb bands in the periodical RNA samples showed that the nifH2 gene was expressed in parallel to the nifH1DK genes under the growth conditions used in this study, and the level of mRNA did not change significantly throughout the entire measuring period, although the level of the nifH2 mRNA was somewhat higher in early samples (3, 6 and 11 h) than in late samples (25 and 33 h) relative to the nifH1DK mRNA (Fig. 2a).



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Fig. 2. Northern blot analysis of the nifH1 and nifH2 mRNAs of C. pasteurianum. Cells were grown in a defined nitrogen-fixing medium under a stream of nitrogen gas and collected periodically throughout incubation; total RNA was isolated from these fresh cells. Total RNA (6 µg per well) was resolved on a 0·7 % formaldehyde-agarose gel. The RNA species were then transferred to positively charged nylon membranes by capillary elution with 20x SSC, and the resulting membranes were incubated with a 350 bp HRP-labelled, nifH1-derived probe at 42 °C in a hybridization buffer containing 0·5 M NaCl. For comparison, the membranes were stripped in 0·1 % SDS solution and incubated with a HRP-labelled 16S rRNA probe. (a) Expression of nifH1 and nifH2 in a nitrogen-fixing culture; (b) expression of nifH1 and nifH2 in an ammonia-supplemented nitrogen-fixing culture. For the ammonia-supplemented culture, ammonium acetate was added to a nitrogen-fixing culture when the culture was 5 h old.

 
In a similar experiment, a nitrogen-fixing culture of C. pasteurianum was supplemented with ammonium acetate to a concentration of 4 mM when the culture was 5 h old, and RNA was isolated before and after ammonium acetate addition to study the effect of ammonium ions on the synthesis and stability of the nifH2 mRNA. As shown in Fig. 2(b), the addition of ammonium acetate caused almost complete disappearance of the nifH2 signal as well as the nifH1DK signal (the 8 h time point). Synthesis of the nifH2 mRNA resumed later (the 11 h timepoint in Fig. 2b). The concentration of ammonium ion in the medium was not measured in this study. However, earlier studies established that under similar conditions ammonium ions would be exhausted about 4 h after the start of growth (Daesch & Mortenson, 1972), and synthesis of new nif mRNA would ensue (Seto & Mortenson, 1974). No significant differences were observed in the expression patterns of the nifH1DK and nifH2 genes until the culture was 27 h old. At that point, the level of the nifH1DK mRNA was significantly reduced, but the level of the nifH2 mRNA was still relatively high. This pattern differed from that shown in Fig. 2(a). Whether or not this difference between the expression of the nifH2 gene and the nifH1DK genes is physiologically important is yet to be determined.

Immunological detection of NifH-related polypeptides in nitrogen-fixing cells of C. pasteurianum
Proteins in crude extracts were separated by SDS-PAGE, and analysed by Western blotting using an antiserum raised against the Fe protein of A. vinelandii. The signal given by C. pasteurianum was compared with that given by Clostridium beijerinckii and A. vinelandii. When an equal amount of cellular proteins was analysed, the signal given by C. pasteurianum was much broader than that given by C. beijerinckii or A. vinelandii (data not shown). A closer examination of the Western blots suggested that more than one NifH-related polypeptide, having a molecular mass similar to that of the NifH1 polypeptide, were present in nitrogen-fixing cells of C. pasteurianum (Fig. 3). To further resolve the polypeptides by SDS-PAGE, different acrylamide concentrations (10, 12 and 15 % and 4–20 % gradient gels) were tested. SDS-PAGE on 15 % gels provided the best resolution, and the band patterns suggested the presence of a NifH-related polypeptide in addition to the NifH1 polypeptide in the crude extracts of nitrogen-fixing C. pasteurianum (Fig. 3a,b).



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Fig. 3. Western blot analysis of the NifH proteins of C. pasteurianum. A cell-free extract prepared from nitrogen-fixing cells was analysed by SDS-PAGE with 15 % total acrylamide (a) and non-denaturing PAGE with 12 % total acrylamide (b). Electrophoretic transfer of proteins onto a positively charged nitrocellulose membrane was done in a semi-dry electrophoretic transfer cell. Western blots were probed with an anti-NifH serum and analysed using chemiluminescent detection. Lanes 1 to 6 contain different quantities of the same cell-free extract; lanes 7 and 8 contain a cell-free extract prepared from nitrogen-fixing cells of C. beijerinckii NRRL B593, which served as a control. The pictures shown were generated from the X-ray films with the use of an imager to enlarge the region containing the NifH bands. The arrows indicate relative positions of the NifH proteins.

 
Separation of NifH-related polypeptides from C. pasteurianum by preparative SDS-PAGE
The NifH-related polypeptides of C. pasteurianum were separated by preparative SDS-PAGE. Selected fractions were subjected to analytical SDS-PAGE, and the Coomassie blue-stained gels were examined for the presence of polypeptides with a size similar to that of NifH1 (Mr 29 666). Fractions 10 to 37, which spanned this size range, were analysed by Western blotting for the presence of NifH-related polypeptides, and fractions 27 to 37 were found to be NifH-positive. A closer examination of the Western-blot membrane (Fig. 4) suggested that fractions 27, 28, 36 and 37 each contained one NifH-related band. Consistent with the order of collection during SDS-PAGE, the polypeptides present in fractions 27 and 28 moved slightly faster than that present in fractions 36 and 37. Fractions 29 through 35, on the other hand, each gave two NifH-related bands, corresponding to the NifH-related band found in fractions 28 and 36, respectively.



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Fig. 4. Western blot analysis of NifH polypeptides of C. pasteurianum after separation by preparative gel electrophoresis. A cell-free extract (approximately 25 mg protein) prepared from nitrogen-fixing cells of C. pasteurianum was resolved with a Bio-Rad Prep Cell (model 491). Fractions 27, 28, 29, 31, 32, 36 and 37 were analysed by SDS-PAGE with 15 % total acrylamide. Electrophoretic transfer of proteins onto a positively charged nitrocellulose membrane was done in a semi-dry electrophoretic transfer cell. The membranes were probed with an anti-NifH serum using chemiluminescent detection. Fractions 27 and 28 contained one of the NifH polypeptides, fractions 29, 31 and 32 contained both of the NifH polypeptides, and fractions 36 and 37 contained the other NifH polypeptide. The arrows indicate the relative positions of NifH polypeptides.

 
Identification of NifH-related polypeptides of C. pasteurianum by MALDI-TOF-MS analysis
An analysis of the tryptic peptides from the proteins in fractions 28, 31 and 37 (see Fig. 4 for a description of these fractions) provided the first clue that distinct NifH-related polypeptides were present in C. pasteurianum, rather than the presence of NifH1 and its modified products. For fraction 28, peptide-mass fingerprinting (PEPTIDENT) gave the following number (in parentheses) of matching peptides with each NifH-related polypeptide of C. pasteurianum: NifH1 (7), NifH2 (5), NifH3 (5), NifH4 (6) and NifH5 (8). It should be noted that because of a high degree of similarity between the NifH sequences (Wang et al., 1988), some of the matching peptides are common to two or more NifH-like sequences, hence limiting the number of peptide masses that can be specifically assigned to a NifH-like polypeptide. At present, there is no compelling evidence for the presence of NifH2, NifH3 or NifH4 in fraction 28. The number of matched peptides indicated the presence of NifH1 or NifH5 or both in fraction 28. Although eight peptides could be matched with the NifH5 sequence, only one (for positions 261–273) of them is specific for NifH5. To make this peptide mass compatible with the NifH5 sequence, it had to have an oxidized methionine residue and a mass tolerance of –1·1326 Da. It is most probable that NifH5 was not present in fraction 28. For NifH1, two of the seven matching peptides (positions 75–81 and 211–216) can be considered specific for this polypeptide, if NifH5 is excluded, whereas the other five matching peptides are conserved among NifH1 through NifH5. Because nifH1 encodes the purified Fe protein and there is no strong evidence for the presence of NifH2 through NifH5 in fraction 28, we concluded this fraction contained NifH1.

For fraction 31, peptide-mass fingerprinting gave the following number (in parentheses) of matching peptides with each NifH-related polypeptide of C. pasteurianum: NifH1 (7), NifH2 (6), NifH3 (5), NifH4 (4) and NifH5 (6). None of the four matching peptides with NifH4 was specific for this NifH isoform. Among the five matching peptides with NifH3, three were specific; however, in order for the peptide masses to fit, Cys-39 in the peptide for positions 34–47 must be modified by iodoacetamide or acrylamide. It was concluded (Wang et al., 1988) that nifH3 mRNA was absent in nitrogen-fixing and non-nitrogen-fixing cells grown under molybdenum-sufficient conditions (conditions used in this study). Therefore, the presence of NifH3 in the fraction was unlikely. For the seven matching peptides with NifH1, one was specific (positions 222–242), whereas two were common with NifH5 (positions 75–81 and 244–260). For the six matching peptides with NifH5, none was specific for this isoform, while two were shared only with NifH1. The six matching peptides for NifH2 contained two (positions 223–238) that are specific for this isoform, but the peptide masses required modification of Cys-231 by iodoacetamide or acrylamide. It may be tentatively concluded that fraction 31 contained NifH1 and NifH2.

For fraction 37, there was no specific matching peptide for NifH4, although there were two peptides (positions 82–97; one with both Cys residues alkylated by iodoacetamide, and the other with both Cys residues forming acrylamide adducts) that could match NifH1 through NifH5. There were five peptides matching NifH1/NifH5, three of which were specific for NifH1/NifH5. There were also five peptides matching NifH2, three of which were specific for NifH2. Thus, the MALDI-TOF-MS results suggest that NifH1 was present in fractions 28, 31 and 37. On the other hand, NifH2 was only found in fractions 31 and 37, indicating a slower mobility of this isoform(s) than NifH1 on SDS-PAGE.

In a separate experiment, we subjected a crude extract of nitrogen-fixing C. pasteurianum to analytical SDS-PAGE and analysed a gel slice that contained polypeptides with a nominal Mr of 30 000. The peptide masses obtained from two runs of MALDI-TOF-MS were used in a database search, and the highest scores and numbers of matching peptides (in parentheses) were with NifH5 (18), NifH1 (16) and NifH2 (11). In addition, eight peptides matched NifH3, of which four were specific. For NifH5, four of the five NifH5-specific peptides (different modifications of positions 261–273, 217–238 and 222–238) were assignable only when a relatively large mass tolerance (deviations of –1·132, –1·133, –1·157, and 1·263 Da) was allowed. Therefore, the results again suggested the presence of NifH1 and NifH2 in C. pasteurianum.

To confirm the resolving power of MALDI-TOF-MS, we analysed several other samples: (i) the purified Fe protein of A. vinelandii, (ii) bovine serum albumin, (iii) polypeptides with a nominal Mr of 70 000 from nitrogen-fixing C. pasteurianum, and (iv) a gel piece from SDS-PAGE that did not contain proteins. The A. vinelandii Fe protein and bovine serum albumin were correctly identified along with several other proteins with similar sequences (data not shown). The gel piece without proteins did not yield any relevant peaks. C. pasteurianum polypeptides with a Mr of 70 000 did not yield any peptide masses assignable to any NifH-related proteins. The results further supported the conclusion that, in addition to NifH1, one or more NifH-related polypeptides were present in C. pasteurianum. To further identify the NifH isoforms in C. pasteurianum, we subjected fraction 37 to analysis by electrospray tandem mass spectrometry (ES-MS/MS).

Identification of NifH-related polypeptides of C. pasteurianum by ES-MS/MS analysis
The ES-MS/MS analysis determines the amino acid sequence, in addition to the mass, of the tryptic peptides and hence allows more conclusive identification of the NifH isoforms in a sample. Table 1 lists the NifH-related peptides that were detected in fraction 37 by ES-MS/MS. Eight of the detected tryptic fragments were NifH2-specific (the eight discrete fragments represented seven sequences, as one sequence was represented by two fragments that differed in the oxidation state of the methionine residue). These tryptic fragments are conclusive evidence for the presence of the NifH2 polypeptide in C. pasteurianum (Fig. 5).


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Table 1. Tryptic peptides of C. pasteurianum NifH isoforms identified by ES-MS/MS

 


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Fig. 5. Alignment of NifH1 and NifH2 amino acid sequences and positions of tryptic peptides detected by ES-MS/MS. *, Residues different between NifH1 and NifH2; <-->, NifH1-specific peptides detected by ES-MS/MS analysis; <==>, NifH2-specific peptides detected by ES-MS/MS analysis; <.>, peptides common in NifH1 and NifH2 detected by ES-MS/MS analysis.

 
Fifteen of the detected tryptic fragments, which represented nine sequences, could be from NifH1/NifH5 as well as NifH2, as these fragments were from regions that are conserved in NifH1/H2/H5. It is conceivable that at least some of these fragments were derived from NifH2 but not from NifH5, as explained in the preceding section. These 15 fragments are therefore considered to be from NifH1 and NifH2.

The NifH4 sequence is highly related to the NifH1/NifH5 and NifH2 sequences (Wang et al., 1988). For example, the NifH1/H2/H4/H5 sequences are identical at positions 32–54, 79–117 and 119–212. Therefore, some of the peptides (such as those covering positions 82–97, 141–163 and 185–198) that we assigned to NifH1 and NifH2 could be from NifH4. However, because we did not detect any NifH4-specific peptide by either MALDI-TOF-MS or ES-MS/MS analysis, there is no reason to suggest the presence of the NifH4 polypeptide in nitrogen-fixing cells of C. pasteurianum.

Nineteen of the detected tryptic fragments, which represented 12 discrete sequences, have been assigned to NifH1, although 17 of them could also be from NifH5. NifH1 and NifH5 differ by two amino acid residues, Tyr-227 and Gln-267 in NifH1 versus Phe-227 and Glu-267 in NifH5. The two fragments with masses 2700·2 (positions 217–238) and 1599·8 (positions 261–273), respectively, contained Tyr-227 and Gln-267 characteristic of NifH1. Furthermore, among the possible tryptic peptides from the five NifH isoforms of C. pasteurianum, the peptide covering positions 217–238 (AEINKQTVIEYDPTCEQAEEYR) is unique to NifH1 (Fig. 5). Because a NifH5-specific peptide has not been detected, we assign these 19 peptides to NifH1 (Table 1).


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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES
 
Multiple nifH or nifH-related sequences are present in several diazotrophs. Besides the vnfH and anfH genes for the alternative nitrogen-fixation systems (Bishop & Premakumar, 1992), there are reiterated nifH genes in Rhizobium (Martinez et al., 1985) and Paenibacillus (Choo et al., 2003). In Rhizobium ORS571, two nifH genes were characterized (Norel & Elmerich, 1987), and both genes appear functional. However, activity measurements (ex planta) showed that nifH1 accounts for about 70 % of the nitrogen-fixing activity, whereas nifH2 accounts for only 30 %. In Rhizobium etli (formerly Rhizobium phaseoli), three nifH genes were identified; none is indispensable for nitrogen fixation and at least two are functionally expressed (Morett et al., 1988).

The presence of five nifH-related genes in an organism is uncommon. Whether or not the nifH-like genes (nifH2 through nifH5) of C. pasteurianum are functional and what physiological roles they play are important questions to be answered. The purified Fe protein (NifH1 dimer) of C. pasteurianum was sequenced (Tanaka et al., 1977). If certain amino acids were detected at specific sequencing steps, it could indicate the contamination of the purified Fe protein with one or more of NifH-related polypeptides. An examination of the protein sequencing data showed that the purified Fe protein could not contain more than trace amounts of proteins from the nifH-like genes (Wang et al., 1988). However, this conclusion does not rule out the possibility that other NifH-related proteins are present in the cell but are separated from the nifH1-encoded Fe protein during the purification of an active Fe protein. This study provided conclusive evidence for the synthesis of NifH2 in addition to NifH1. If NifH2 forms a homodimer or a NifH2-NifH1 heterodimer, such a dimer is probably not an active Fe protein. This conjecture is based on the results from several laboratories. During the purification of the Fe protein from C. pasteurianum, the NifH1 dimer was the only active component that complemented the MoFe protein in a nitrogenase assay (Tso et al., 1972; Zumft & Mortenson, 1973).

To deduce a possible function for NifH2, it should be useful to compare the structures of NifH2 and NifH1. The predicted main-chain fold of the NifH2 polypeptide was obtained by modelling NifH2 against the crystallographically determined structure of the Fe protein of C. pasteurianum, and the two structures were similar (data not shown). Between NifH1 and NifH2, 10 of the 22 different amino acids occur near residues that are either involved in dimer interactions or implicated in nucleotide binding (Table 2). When compared with NifH1, the side-chain of 14 of the 22 different residues in NifH2 is either larger in volume, different in charge properties, or different in tendency to form {alpha}-helices. The changes of Met-213 to Ser (a drastic decrease in tendency to form {alpha}-helix) and Gln-222 to Lys (appearance of a positive charge) could be significant. They occur in or near {alpha}7 (nomenclature of Schlessman et al., 1998; see Georgiadis et al., 1992, for a schematic representation of the secondary structure), which encompasses eight amino acids (residues 210, 212, 215, 216, 218–221) that are involved in dimer interactions and three amino acids (residues 214, 215, 218) that are involved in nucleotide binding. The presence of Ser-213 and Lys-222 in NifH2 may cause the NifH2 monomer (or dimer, if one is formed) to interact differently with the MoFe protein, when compared with NifH1.


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Table 2. Location and properties of the 22 different amino acid residues between NifH1 and NifH2 of C. pasteurianum

 
The change of Ala-246 to Glu increases negative charges (from 2 to 3 among residues 245–248) in a region connecting {alpha}8 and {alpha}9 ({alpha}9 is next to the C-terminus). The C-terminal region of C. pasteurianum NifH1 is shorter than that of NifH of other organisms (Chen et al., 1986), which limits its involvement in intersubunit interaction and contribution to dimer stabilization (Schlessman et al., 1998). The cluster of negative charges near {alpha}9 may alter the topography of this region in NifH2 and allow a positively charged metabolite to bind to NifH2 and cause the NifH2 monomer or dimer to assume an altered conformation. If a putative NifH2 dimer cannot donate electrons to the MoFe protein or if the altered conformation of the NifH2 dimer or monomer has a higher affinity than the NifH1 dimer for the MoFe protein, the tight binding between NifH2 and the MoFe protein could be a mechanism, albeit archaic, for switching off nitrogenase activity. This is similar to the proposal of Wang et al. (1988) that, under certain physiological conditions, products from some of the nifH-like genes might serve to modulate (e.g. down-regulate) nitrogenase activity by acting as an inhibitor of the MoFe protein. Studies on heterologous pairs showed that an incompatible Fe protein could act as an inhibitor of nitrogenase activity (Emerich & Burris, 1978; Clarke et al., 2000).

NifH2 is expressed throughout growth in parallel to NifH1, and addition of ammonium acetate to the culture affected the expression of both genes similarly. A proposed regulatory role for NifH2 may involve the protein as a signal-transducing protein to relay the nitrogen status to the MoFe protein. In this regard, it may be noted that in C. acetobutylicum and C. beijerinckii, the nifI1 (glnB1) and nifI2 (glnB2) genes occur between the nifH and nifD genes, but C. pasteurianum does not have similar genes according to results of Southern hybridization and PCR (J. Toth & J.-S. Chen, unpublished results).

Because C. pasteurianum is at present not amenable to genetic manipulations, further biochemical and functional characterization of the NifH2 protein cannot be readily performed. On the other hand, the nitrogen-fixing C. acetobutylicum and C. beijerinckii can now be genetically manipulated (Chen, 2004), so it might be possible to express NifH2 in these species. Because the Fe and MoFe proteins are highly conserved in these three nitrogen-fixing clostridia, the presence of NifH2 in C. acetobutylicum or C. beijerinckii may allow a physiological study of NifH2. Using appropriate vectors, NifH2 may be expressed in C. acetobutylicum or C. beijerinckii under non-nitrogen-fixing conditions so that NifH2 can be purified from cells without NifH1.

The nitrogen-fixing species within the genus Clostridium are traditionally considered representatives of anaerobic, free-living nitrogen-fixers. These nitrogen fixers, because of their free-living lifestyle, were not considered active contributors of fixed nitrogen for supporting plant growth. A recent study, however, could change this view because clostridia were found as nitrogen-fixing endophytes in consortia with nondiazotrophic bacteria in tissues of gramineous plants from a wide region of Asia (Minamisawa et al., 2004). The newly isolated nitrogen-fixing clostridia from gramineous plants are phylogenetically close to known nitrogen-fixing species, including C. beijerinckii and C. pasteurianum, as well as to species such as Clostridium intestinale (formerly Clostridium intestinalis) and Clostridium saccharoperbutylacetonicum, which were not previously reported as nitrogen fixers. The newly discovered niches for nitrogen-fixing clostridia suggest a more significant role for the obligate anaerobes in supporting plant growth via nitrogen fixation. It will be interesting to examine these newly isolated nitrogen-fixing clostridia for their composition of nif genes.


   ACKNOWLEDGEMENTS
 
The authors wish to thank Drs W. E. Newton and Brian Storrie for their suggestions during the preparation of the manuscript. We thank Julianna Toth for resequencing the inserts in pCP114 and pCP600, and Craig Tollin and Anh Tran for the use of unpublished data. We gratefully acknowledge the sample of NifH antiserum from Drs W. E. Newton and Karl Fisher. This work was supported by US Department of Energy grant DE-FG02-97ER20276 and the CSREES, US Department of Agriculture, under project VA-135681.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 31 January 2005; revised 4 April 2005; accepted 19 April 2005.



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