- Email: [email protected]
The nifH gene encoding the Fe protein component of the molybdenum nitrogenase from Azotobacter chroococcum
Gene, 123 (1993) 1455146 0 1993 Elsevier Science Publishers
B.V. All rights reserved.
145
0378-l 119/93/$06.00
GENE 06862
The nifH gene encoding the Fe protein component nitrogenase from Azotobacter chroococcum* (Recombinant
DNA; nitrogen
fixation;
transcription)
Robert Jones aTc,Paul Woodley”, Angelika aAFRC Laboratoryfor
Nitrogen Fixation,
Birkmann-Zinoni
b and Robert L. Robsona,b
University of Sussex, Brighton BNI 9RQ, UK. Tel. (44-273)678132;
University of Georgia, Athens, GA 30602, USA: and ’ Thinking Machines Corporation, 876-1111 Received by G. Wilcox: 24 October
of the molybdenum
1991; Revised/Accepted:
31 January/l0
September
’ Department of Biochemistry,
245 First Street, Cambridge,
MA 02142-1214,
1992; Received at publishers:
18 September
USA. Tel. (617).
1992
SUMMARY
The nucleotide sequence spanning the nigh gene and part of the niJD gene encoding from Azotobacter chroococcum was determined. The transcription start point of the ni. potential transcriptional attenuator was located between the nip and niJD genes.
A. chroococcum ATCC4412 can fix N, using a vanadium-dependent N,ase (Robson et al., 1986a) or an Modependent N,ase similar to those of other N,-fixing organisms (Yates and Planque, 1975). Mo-N,ases consist of two components: Fe-proteins encoded by nijH genes and MoFe-proteins encoded by the nifD and nifl genes. The A. chroococcum nijHDK gene cluster was cloned earlier (Jones et al., 1984); and here we present the nt sequence (Fig. 1) of the nijH gene and part of the ni$D gene for the MoFe-protein a-subunit. The nifff gene from A. chroococcum potentially codes for a 291 aa polypeptide (M, 31 521; p1 4.46) terminated by tandem stop codons. Gene niJD is located 121 nt 3’ to nigh. The aa sequence of the nigh gene product shows the highest degree of identity
Correspondence
to: Dr. R.L. Robson,
Department
of Biochemistry,
Uni-
versity of Georgia, Athens, GA 30602, USA. Tel. (404) 542-1191; Fax (404) 54221738. *On request, the authors will supply detailed experimental evidence for the conclusions reached in this Brief Note.
/
Abbreviations: A., Azotobacter; aa, amino integration host factor; kb, kilobase or in nitrogen fixation; N,ase, nitrogenase; ome-binding site(s); tsp, transcription start vator sequence(s).
acid(s); bp, base pair(s); IHF, 1000 bp; n$ gene(s) involved nt, nucleotide(s); RBS, ribospoint(s); UAS, upstream acti-
the molybdenum nitrogenase promoter was mapped, and a
(96%) to the Fe-protein of the Mo-N,ase from the closely related organism A. vinelandii (Hausinger and Howard, 1982) whilst the DNA sequence identity between the two organisms over this region is 90.5% (Brigle et al., 1985; Jacobson et al., 1989). The A. chroococcum niji’3 gene product is also 89% identical to the Fe-protein component of the V-N,ase system in A. chroococcum encoded by vnjH (Robson et al., 1986b). The Mo-N,ase is formed under conditions of MO sufficiency and in the absence of a utilizable combined nitrogen source. Previous work showed that ni. gene expression in Azotobacter requires the products of the ntrA gene which encodes an alternative o factor and ni.A which codes for a nifspecific positive activator of transcription (Santero et al., 1982). The tsp for ni$?f (denoted by +l) was mapped at 67 nt 5’ to the niLfH coding region by Sl -nuclease protection experiments using mRNA from organisms grown under nitrogen-fixing conditions. The ni. promoter contains a sequence between -25 and -10 upstream from the tsp which conforms to those found in ntrA-encoded c factor dependent promoters (Beynon et al., 1983). The sequence at -141 to -126 conforms to the consensus UAS sequence found in nifAactivatable promoters (Buck et al., 1986). Furthermore, the sequence between nt - 56 and - 44 closely matches
146
1 121
HIIrA
r +1-.
AGGGCGACGAGGTGAGTGATGTAGGACATGTCGTGTAGCATACACAGACAGCGTAG~TGTAGGTGGATGTATGTATC~CGCAGTCGGACTGTCGTAGTATCAAATCATTTAAATCAA
mRA
IHF
120
GAGG~A~TGTTTGCCTCGAGTGCTGGCACAGACGCTGCATTAGGTTCAGTGCGCGGACTTGTTCAGGCTGACAAGCCGAACTTTCAAGTGGAllATGCAACCTGAGGAACTTAACTATG
240 H
MifH
l
241
GCTATGCGTCAATGCGCCATCTACGGCAAGGGCGGTATCGGTATCCACCACCACTCAGAACCTCGTGGCGGCCCTGGCCGAGATGGGCAAGAAGGTCATGATCGTTGGTTGTGACCCG AHRQCAIYGKGGIGKSTTTQNLVAALAEHGKKVHIVGCDP
360
361
AAAGCTGACTCCACTCGCCTGATCCTGCACTCCAAGGCCCAGAACACCATCATGGAAATGGCTGCCGAAGCCGGCACCGTGGAAGATCTGGAGCTGGAAGACGTGCTGAAGGTCGGCTAC KADSTRLILHSKAQNT IHEWAAEAGTVEDLELEDVLKVGY
480
481
GGCGGCGTCAAGTGCGTTGAGTCCGGTGGTCCGGAGCCGGGCGTTGGCTGCGCTGGCCGTGGTGTTATCACCGCGATCAACTTCCTTCCTGGAAGCTGGAC GGVXCVESGGPEPGVGCAGRGVITAINFLEEE
600
601
721
GAYEDDLD
TTCGTATTCTACGACGTACTGGGCGACGTGGTGTGCGGTG~~CGCCATGCCGA~CGCG~C~GGCTCAGGAAATCTACATCGTTTGCTCCGGCGAGATGATGGCCATGTACGCC FVFYDVLGDVVCGGFAHPIRENKAQEIYIVCSGEHMAHYA
720
GCCAACAACATCTCCAAGGGCATCGTGAAGTACGCCAACTCCGGTAGCGGTAGCGTGCGTCT~GCGGCCTGATCTGC~CAGCCGT~CACCGACCGCGAAGACGAGCTGATCATCGCTCTGGCT
840
ANNISKGIVKYANSGSVRLGGLICNSRNTDREDELI
I
A
LA
841
GCCAAGCTGGGCACCCAGATGATCCACTTCGTACCGCGTGACAACGTTGTACAGCGCGCCGAAATCCGCCGTATGACCGTGATCGAATACGATCCGACCGCCAAGCAAGCCGACGAGTAC AKLGTQMIH FVPRDNVVQRAEIRRMTVIEYDPTAKQADEY
960
961
CGCACTCTOGCTCGCARAGTCGTCGAGAACAAGATGCTGATCATCCCGAACCCGATCACCATGGACGAGCTCGAAGCTCTGCTGATGGAGTTCGGCGTTATGGAAGAAGAAGACGAGTCC RTLARKVVENKMLIIPNPITUDELEALLME FGVMEEEDES
1080
1081
ATCGTCGGCAAGGCCGCCGCTGCCGAAGAGTGATAGCTCCGCTTCAGTAGGACGGGGCAGGGCAGATGGGCCCTGCCGGAGTGTCGCACTGCGCAGTGGGCACTGACCCAT~CGTTACCC 1
1201
GCACACGAACGCAAAGCAAGAGGAGTCATACCCAATGTCCGGTATGTCGCGCG~GAGGTCGAATCCCTCATCCAGGAAGTCCTGGAAGTTTA HSGMSREEVESLIQEVLEV HIFD .
1200 --,
--,
1293
Fig. 1. The nt sequence of the region spanning the nifl gene and part of the nifD gene of A. chroococcum was determined by the chain termination method. The tsp of the nl$4 gene is indicated by the arrow marked +l. The 3’ end of a processed or attenuated transcript arising from the n$?f promoter is shown by the arrow at nt 1192 and occurs beyond a region of hyphenated dyad symmetry, indicated by the horizontal arrows. Sequences conforming underlined
to potential and marked
binding sites for NifA, IHF, and NtrA, which are proteins likely to be required for transcription accordingly. This sequence is deposited with GenBank under accession No. M73020.
the consensus IHF-binding site. IHF is known to bind to many nifpromoters and induce DNA bending which is postulated to facilitate protein-protein contact between nifA and ntrA gene products (Hoover et al., 1990). The full-length mRNA arising from the nigh promoter is apparently processed since transcripts hybridizing to nifl of approx. 1.1,2.6 and 4.3 kb were detected in Northern blots (Jones et al., 1984). The 3’ end of the 1.1 kb transcript mapped to a site within the nigh to nifD intergenic region which immediately follows a region with hyphenated dyad symmetry. This region could give an mRNA with a secondary structure that could stabilize mRNA and/or act as a transcription attenuator. This work was completed with support from the NIH (1 -ROl GM40067LOl). A. B.-Z. was supported with funds from the Deutsche Forschungsgemeinschaft.
REFERENCES Beynon, J., Cannon, M., Buchanan-Wollaston, V. and Cannon, F.: The nifpromoters of Klebsiella pneumoniae have a characteristic primary structure. Cell 34 (1983) 665-671. Brigle: K.E., Newton, W.E. and Dean, D.R.: Complete nucleotide sequence of the Azotobacter vinelandii nitrogenase structural gene cluster. Gene 37 (1985) 37-44.
of nigh in Azotobacter
are
Buck, M., Miller, S., Drummond, M. and Dixon, R.: Upstream activator sequences are present in promoters of nitrogen fixation genes. Nature 320 (1986) 374-378. Hausinger, R.P. and Howard, J.B.: The amino acid sequence of the nitrogenase iron protein from Azofobacter vinelandii. J. Biol. Chem. 2.57 (1982) 2483-2490. Hoover, T.R., Santero, E., Porter, S. and Kustu, S.: The integration host factor stimulates interaction of RNA polymerase with nifA, the transcriptional activator for nitrogen fixation operons. Cell 63 (1990) 1I-22. Jacobson,
M.R., Brigle, K.E., Bennett,
M.S., Cash, V.L., and genetic map J. Bacterial. 171 Jones, R., Woodley,
L.T.: Setterquist,
R.A., Wilson,
Beynon, J., Newton, W.E. and Dean, D.R.: Physical of the major nifcluster from Azotobacter vinelandii. (1989) lOI7-1027. P. and Robson, R.L.: Cloning and characterization
of some genes for nitrogen fixation from Azotobacter chroococcum and their expression in Klebsiella pneumaniae. Mol. Gen. Genet. 197 (1984) 318-327. Robson, R.L., Eady, R.R., Richardson, T.H., Miller, R.W., Hawkins, M. and Postgate, J.R.: The alternative nitrogenase of Azotobacter chroococcum is a vanadium enzyme. Nature 322 (1986a) 388-390. Robson, R., Woodley, P. and Jones, R.: Second gene (nigh*) coding for a nitrogenase iron protein in Azotobacter chroococcum is adjacent to a gene coding 1159-l 163.
for a ferredoxin-like
protein.
EMBO
J. 5 (1986b)
Santero, E., Toukdarian, A., Humphrey, R. and Kennedy, C.: Identification and characterization of two nitrogen fixation regulatory regions, nifA and nfrX, in Azotobacter vinelandii and Azotobacter chroococcum. Mol. Microbial. 2 (1988) 303-314. Yates, M.G. and PlanquC, K.: Nitrogenase from Azotobacter chroococcum. Purification and properties of the component proteins. Eur. J. Biochem. 60 (1975) 467-476.
B.V. All rights reserved.
145
0378-l 119/93/$06.00
GENE 06862
The nifH gene encoding the Fe protein component nitrogenase from Azotobacter chroococcum* (Recombinant
DNA; nitrogen
fixation;
transcription)
Robert Jones aTc,Paul Woodley”, Angelika aAFRC Laboratoryfor
Nitrogen Fixation,
Birkmann-Zinoni
b and Robert L. Robsona,b
University of Sussex, Brighton BNI 9RQ, UK. Tel. (44-273)678132;
University of Georgia, Athens, GA 30602, USA: and ’ Thinking Machines Corporation, 876-1111 Received by G. Wilcox: 24 October
of the molybdenum
1991; Revised/Accepted:
31 January/l0
September
’ Department of Biochemistry,
245 First Street, Cambridge,
MA 02142-1214,
1992; Received at publishers:
18 September
USA. Tel. (617).
1992
SUMMARY
The nucleotide sequence spanning the nigh gene and part of the niJD gene encoding from Azotobacter chroococcum was determined. The transcription start point of the ni. potential transcriptional attenuator was located between the nip and niJD genes.
A. chroococcum ATCC4412 can fix N, using a vanadium-dependent N,ase (Robson et al., 1986a) or an Modependent N,ase similar to those of other N,-fixing organisms (Yates and Planque, 1975). Mo-N,ases consist of two components: Fe-proteins encoded by nijH genes and MoFe-proteins encoded by the nifD and nifl genes. The A. chroococcum nijHDK gene cluster was cloned earlier (Jones et al., 1984); and here we present the nt sequence (Fig. 1) of the nijH gene and part of the ni$D gene for the MoFe-protein a-subunit. The nifff gene from A. chroococcum potentially codes for a 291 aa polypeptide (M, 31 521; p1 4.46) terminated by tandem stop codons. Gene niJD is located 121 nt 3’ to nigh. The aa sequence of the nigh gene product shows the highest degree of identity
Correspondence
to: Dr. R.L. Robson,
Department
of Biochemistry,
Uni-
versity of Georgia, Athens, GA 30602, USA. Tel. (404) 542-1191; Fax (404) 54221738. *On request, the authors will supply detailed experimental evidence for the conclusions reached in this Brief Note.
/
Abbreviations: A., Azotobacter; aa, amino integration host factor; kb, kilobase or in nitrogen fixation; N,ase, nitrogenase; ome-binding site(s); tsp, transcription start vator sequence(s).
acid(s); bp, base pair(s); IHF, 1000 bp; n$ gene(s) involved nt, nucleotide(s); RBS, ribospoint(s); UAS, upstream acti-
the molybdenum nitrogenase promoter was mapped, and a
(96%) to the Fe-protein of the Mo-N,ase from the closely related organism A. vinelandii (Hausinger and Howard, 1982) whilst the DNA sequence identity between the two organisms over this region is 90.5% (Brigle et al., 1985; Jacobson et al., 1989). The A. chroococcum niji’3 gene product is also 89% identical to the Fe-protein component of the V-N,ase system in A. chroococcum encoded by vnjH (Robson et al., 1986b). The Mo-N,ase is formed under conditions of MO sufficiency and in the absence of a utilizable combined nitrogen source. Previous work showed that ni. gene expression in Azotobacter requires the products of the ntrA gene which encodes an alternative o factor and ni.A which codes for a nifspecific positive activator of transcription (Santero et al., 1982). The tsp for ni$?f (denoted by +l) was mapped at 67 nt 5’ to the niLfH coding region by Sl -nuclease protection experiments using mRNA from organisms grown under nitrogen-fixing conditions. The ni. promoter contains a sequence between -25 and -10 upstream from the tsp which conforms to those found in ntrA-encoded c factor dependent promoters (Beynon et al., 1983). The sequence at -141 to -126 conforms to the consensus UAS sequence found in nifAactivatable promoters (Buck et al., 1986). Furthermore, the sequence between nt - 56 and - 44 closely matches
146
1 121
HIIrA
r +1-.
AGGGCGACGAGGTGAGTGATGTAGGACATGTCGTGTAGCATACACAGACAGCGTAG~TGTAGGTGGATGTATGTATC~CGCAGTCGGACTGTCGTAGTATCAAATCATTTAAATCAA
mRA
IHF
120
GAGG~A~TGTTTGCCTCGAGTGCTGGCACAGACGCTGCATTAGGTTCAGTGCGCGGACTTGTTCAGGCTGACAAGCCGAACTTTCAAGTGGAllATGCAACCTGAGGAACTTAACTATG
240 H
MifH
l
241
GCTATGCGTCAATGCGCCATCTACGGCAAGGGCGGTATCGGTATCCACCACCACTCAGAACCTCGTGGCGGCCCTGGCCGAGATGGGCAAGAAGGTCATGATCGTTGGTTGTGACCCG AHRQCAIYGKGGIGKSTTTQNLVAALAEHGKKVHIVGCDP
360
361
AAAGCTGACTCCACTCGCCTGATCCTGCACTCCAAGGCCCAGAACACCATCATGGAAATGGCTGCCGAAGCCGGCACCGTGGAAGATCTGGAGCTGGAAGACGTGCTGAAGGTCGGCTAC KADSTRLILHSKAQNT IHEWAAEAGTVEDLELEDVLKVGY
480
481
GGCGGCGTCAAGTGCGTTGAGTCCGGTGGTCCGGAGCCGGGCGTTGGCTGCGCTGGCCGTGGTGTTATCACCGCGATCAACTTCCTTCCTGGAAGCTGGAC GGVXCVESGGPEPGVGCAGRGVITAINFLEEE
600
601
721
GAYEDDLD
TTCGTATTCTACGACGTACTGGGCGACGTGGTGTGCGGTG~~CGCCATGCCGA~CGCG~C~GGCTCAGGAAATCTACATCGTTTGCTCCGGCGAGATGATGGCCATGTACGCC FVFYDVLGDVVCGGFAHPIRENKAQEIYIVCSGEHMAHYA
720
GCCAACAACATCTCCAAGGGCATCGTGAAGTACGCCAACTCCGGTAGCGGTAGCGTGCGTCT~GCGGCCTGATCTGC~CAGCCGT~CACCGACCGCGAAGACGAGCTGATCATCGCTCTGGCT
840
ANNISKGIVKYANSGSVRLGGLICNSRNTDREDELI
I
A
LA
841
GCCAAGCTGGGCACCCAGATGATCCACTTCGTACCGCGTGACAACGTTGTACAGCGCGCCGAAATCCGCCGTATGACCGTGATCGAATACGATCCGACCGCCAAGCAAGCCGACGAGTAC AKLGTQMIH FVPRDNVVQRAEIRRMTVIEYDPTAKQADEY
960
961
CGCACTCTOGCTCGCARAGTCGTCGAGAACAAGATGCTGATCATCCCGAACCCGATCACCATGGACGAGCTCGAAGCTCTGCTGATGGAGTTCGGCGTTATGGAAGAAGAAGACGAGTCC RTLARKVVENKMLIIPNPITUDELEALLME FGVMEEEDES
1080
1081
ATCGTCGGCAAGGCCGCCGCTGCCGAAGAGTGATAGCTCCGCTTCAGTAGGACGGGGCAGGGCAGATGGGCCCTGCCGGAGTGTCGCACTGCGCAGTGGGCACTGACCCAT~CGTTACCC 1
1201
GCACACGAACGCAAAGCAAGAGGAGTCATACCCAATGTCCGGTATGTCGCGCG~GAGGTCGAATCCCTCATCCAGGAAGTCCTGGAAGTTTA HSGMSREEVESLIQEVLEV HIFD .
1200 --,
--,
1293
Fig. 1. The nt sequence of the region spanning the nifl gene and part of the nifD gene of A. chroococcum was determined by the chain termination method. The tsp of the nl$4 gene is indicated by the arrow marked +l. The 3’ end of a processed or attenuated transcript arising from the n$?f promoter is shown by the arrow at nt 1192 and occurs beyond a region of hyphenated dyad symmetry, indicated by the horizontal arrows. Sequences conforming underlined
to potential and marked
binding sites for NifA, IHF, and NtrA, which are proteins likely to be required for transcription accordingly. This sequence is deposited with GenBank under accession No. M73020.
the consensus IHF-binding site. IHF is known to bind to many nifpromoters and induce DNA bending which is postulated to facilitate protein-protein contact between nifA and ntrA gene products (Hoover et al., 1990). The full-length mRNA arising from the nigh promoter is apparently processed since transcripts hybridizing to nifl of approx. 1.1,2.6 and 4.3 kb were detected in Northern blots (Jones et al., 1984). The 3’ end of the 1.1 kb transcript mapped to a site within the nigh to nifD intergenic region which immediately follows a region with hyphenated dyad symmetry. This region could give an mRNA with a secondary structure that could stabilize mRNA and/or act as a transcription attenuator. This work was completed with support from the NIH (1 -ROl GM40067LOl). A. B.-Z. was supported with funds from the Deutsche Forschungsgemeinschaft.
REFERENCES Beynon, J., Cannon, M., Buchanan-Wollaston, V. and Cannon, F.: The nifpromoters of Klebsiella pneumoniae have a characteristic primary structure. Cell 34 (1983) 665-671. Brigle: K.E., Newton, W.E. and Dean, D.R.: Complete nucleotide sequence of the Azotobacter vinelandii nitrogenase structural gene cluster. Gene 37 (1985) 37-44.
of nigh in Azotobacter
are
Buck, M., Miller, S., Drummond, M. and Dixon, R.: Upstream activator sequences are present in promoters of nitrogen fixation genes. Nature 320 (1986) 374-378. Hausinger, R.P. and Howard, J.B.: The amino acid sequence of the nitrogenase iron protein from Azofobacter vinelandii. J. Biol. Chem. 2.57 (1982) 2483-2490. Hoover, T.R., Santero, E., Porter, S. and Kustu, S.: The integration host factor stimulates interaction of RNA polymerase with nifA, the transcriptional activator for nitrogen fixation operons. Cell 63 (1990) 1I-22. Jacobson,
M.R., Brigle, K.E., Bennett,
M.S., Cash, V.L., and genetic map J. Bacterial. 171 Jones, R., Woodley,
L.T.: Setterquist,
R.A., Wilson,
Beynon, J., Newton, W.E. and Dean, D.R.: Physical of the major nifcluster from Azotobacter vinelandii. (1989) lOI7-1027. P. and Robson, R.L.: Cloning and characterization
of some genes for nitrogen fixation from Azotobacter chroococcum and their expression in Klebsiella pneumaniae. Mol. Gen. Genet. 197 (1984) 318-327. Robson, R.L., Eady, R.R., Richardson, T.H., Miller, R.W., Hawkins, M. and Postgate, J.R.: The alternative nitrogenase of Azotobacter chroococcum is a vanadium enzyme. Nature 322 (1986a) 388-390. Robson, R., Woodley, P. and Jones, R.: Second gene (nigh*) coding for a nitrogenase iron protein in Azotobacter chroococcum is adjacent to a gene coding 1159-l 163.
for a ferredoxin-like
protein.
EMBO
J. 5 (1986b)
Santero, E., Toukdarian, A., Humphrey, R. and Kennedy, C.: Identification and characterization of two nitrogen fixation regulatory regions, nifA and nfrX, in Azotobacter vinelandii and Azotobacter chroococcum. Mol. Microbial. 2 (1988) 303-314. Yates, M.G. and PlanquC, K.: Nitrogenase from Azotobacter chroococcum. Purification and properties of the component proteins. Eur. J. Biochem. 60 (1975) 467-476.