Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
Correspondence
Jiann-Shin Chen
chenjs{at}vt.edu
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ABSTRACT |
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Present address: Kocaeli University Medical Center, Sopali Ciftligi, 41900 Derince, Kocaeli, Turkey.
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INTRODUCTION |
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The nitrogen-fixation (nif) gene cluster of C. pasteurianum consists of nifH2, nifH1, nifD, nifK, nifE, nifN-B, modA, modB, nifV and nifV
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 (31·935·5 %). 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isolates have been found to contain the sequence previously cloned in pCP600 (C. Tollin, A. Tran, J. Toth & J.-S. Chen, unpublished results).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.
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METHODS |
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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 ml1 and 2 mg lysozyme ml1. 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 µl1) 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-
-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.
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RESULTS |
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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 420 % 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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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 3447 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 222242), whereas two were common with NifH5 (positions 7581 and 244260). 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 223238) 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 8297; 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 261273, 217238 and 222238) 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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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 3254, 79117 and 119212. Therefore, some of the peptides (such as those covering positions 8297, 141163 and 185198) 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 217238) and 1599·8 (positions 261273), 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 217238 (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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DISCUSSION |
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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
-helices. The changes of Met-213 to Ser (a drastic decrease in tendency to form
-helix) and Gln-222 to Lys (appearance of a positive charge) could be significant. They occur in or near
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, 218221) 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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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.
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ACKNOWLEDGEMENTS |
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Received 31 January 2005;
revised 4 April 2005;
accepted 19 April 2005.
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