Nucleotide Sequence and Genetic Analysis of the Azotobacter chroococcum nifUSVWZM Gene Cluster, Including a New Gene (nifP) Which Encodes a Serine Acetyltransferase


Nucleotide sequence was obtained for a region of 7,099 bp spanning the nifU, nifS, nifV, niJW, nijZ, and nijM genes from Azotobacter chroococcum. Chromosomal mutations constructed at several sites within the locus confirmed a requirement for this region for expression of the molybdenum nitrogenase in this organism. The genes are tightly clustered and ordered as in Kkebsiella pneumoniae except for two additional open reading frames (ORFs) between nitfV and niJW. The arrangement of genes in A. chroococcum closely matches that described for Azotobacter vinelandii. The polypeptide encoded by ORF4 immediately downstream from nifV is * Corresponding author. 

41% identical over 186 amino acids to the product of the cysE gene from Escherichia coli, which encodes serine acetyltransferase (SAT), a key enzyme in cysteine biosynthesis. Plasmids which potentially express ORF4 complemented E. coli JM39, a cysteine auxotroph which lacks SAT. SAT activity was detected in crude extracts of one such complemented strain. A strain of A. chroococcum carrying a chromosomal disruption of ORF4 grew normally with ammonium as the N source but more slowly than the parental strain when N2 was the sole N source. These data suggest that ORF4 encodes a nif-specific SAT required for optimizing expression of nitrogenase activity. ORF4 was assigned the name nijP. nijP may be required to boost rates of synthesis or intracellular concentrations of cysteine or methionine. Sequence identity between nitfV and leuA gene products suggests that nifV may catalyze a condensation reaction analogous to that carried out by isopropylmalate synthase (LEUA) but in which acetyl coenzyme and a-ketoglutarate are substrates for the formation of homocitrate, the proposed product of NIFV activity. 
Members of the genus Azotobacter are free-living, obli- gately aerobic, heterotrophic diazotrophs found in soil and freshwater environments. They are especially interesting from the point of view of N2 fixation because, depending on the species, they possess two or three genetically distinct nitrogenase systems whose expression is determined by metal availability (8). The genetics of N2 fixation in these organisms is complicated not only by their possession of alternative nitrogenases but also by their tolerance of 02 when fixing N2 (61). Our studies have concentrated on Azotobacter chroococcum MCD1, a derivative of ATCC 4412. This organism makes a typical molybdenum-dependent nitrogenase when molybdenum is available (77) which is replaced in the absence of molybdenum and presence of vanadium by a vanadium-dependent nitrogenase (60). 
Many of the genes required for N2 fixation were first defined in the facultative anaerobe Klebsiella pneumoniae M5A1, which possesses a molybdenum nitrogenase system only and fixes N2 in anaerobic or, at best, microaerobic environments. In this organism, a maximum of 20 specific nif genes are required for N2 fixation. They are organized as a contiguous cluster spanning approximately 24 kb containing seven or eight transcriptional units (2, 16, 57). The nifH, nifD, and nifK genes respectively encode the Fe-protein (component 2) and the a and a subunits of the MoFe-protein (component 1) that together form the nitrogenase enzyme specific for each system. However, nifV and nifB, important for maturation of MoFe-protein, and nifM, important for Fe-protein stability and activity, do not appear reiterated and therefore may serve in both systems. Mutations in nifB (29) and nifM in A. vinelandii (35) affect all three nitrogenase systems. The roles and regulation of these common genes as compared with the sets of specific genes is particularly interesting. 
The objective of this work was to characterize the cluster of genes, including nifV and nifM, present in region I of the genome of A. chroococcum in order to understand their precise role in the molybdenum and vanadium nitrogenase systems in this organism. The nucleotide sequence of the region containing the nifU, nifS, nifV, nifW, nifZ, and nifM genes of A. chroococcum was determined. The region includes two open reading frames (ORF4 and ORF5) with no counterparts in the K. pneumoniae nif gene cluster. One of these open reading frames (ORF4, nifP) encodes a serine acetyltransferase (SAT) which plays a role in N2 fixation in A. chroococcum. 
MATERIALS AND METHODS Bacterial strains, plasmids, and bacteriophage. A. chroococcum MCD105 (78) and strains constructed in this work were routinely grown in air at 30°C on RM or AM medium (59) with antibiotics added, where required, at the following concentrations (micrograms per milliliter): streptomycin, 20; nalidixic acid, 20; and kanamycin, 0.25. For growth of Nif strains, the medium was supplemented with ammonium acetate at 10 mM. Escherichia coli 71/18 [W(lac-pro) F'(lacI lacZ M15 proAB)] (49), which served as a host for all plasmids, was grown on Luria-Bertani (LB) medium (43) and maintained on minimal medium supplemented with thiamine at 0.1 ,ug/ml. Carbenicillin was added, where needed, at 50 ,g/ml. E. coli JM39 (cysE51 tfr-8) (33) was obtained from B. 
Bachmann and maintained on minimal medium with cystine added to 100 ,ug/ml. K. pneumoniae UNF2050 (nif-2104 [47]) and UNF828 (nif-2568::Tn5 [46]) are strains bearing defined mutations in nifM. K. pneumoniae strains were routinely grown on LB medium or on a nitrogen-free defined medium (NFDM) with histidine added, where necessary, to 20 I,g/ml. 
UNF50231, a His+ revertant of the Nif+ strain UNF5023 (15), was used as a nitrogen-fixing control strain in com- plementation studies. K. pneumoniae strains harboring pBR325-derived plasmids were selected on NFDM medium (15) containing tetracycline at 10 ,ug/ml and kanamycin, where required, at 25 ,ug/ml. Histidine was added at 20 ,g/ml to the medium for UNF2050. Plasmids pDE15a and pDEl5b contain the 5.1-kb KpnI fragment, previously used to com- plement K. pneumoniae niJV and nifM mutants (19), cloned in either orientation in the vector pEMBL18+ (13). pSAR4, pSAR5, and pSAR6 contain Sau3A-KpnI fragments of pDEl5b cloned in the EcoRI site of pBR325 and are de- scribed in the text. The suffix a or b added to pSAR-series plasmids refers to the orientation in which the insert is cloned in pBR325; an a indicates that transcription from the pBR325 chloramphenicol acetyltransferase (CAT) promoter is in the same direction as the presumed transcription of the cloned A. chroococcum nif DNA. Bacteriophages IR1 (13) and M13KO7 (Pharmacia) were used as helper phages during the preparation of sequencing template from pEMBL-derived plasmids. 
Genetic techniques. E. coli and K. pneumoniae strains were transformed by using the methods of Lederberg and Cohen (39) and Merrick et al. (48), respectively. Comple- mentation of K. pneumoniae niJM mutants was defined as anaerobic growth on NFDM agar in 2 to 4 days at 30°C. Gene replacement mutagenesis ofA. chroococcum was carried out with plasmid pCU101 used as the suicide vector to introduce mutations constructed in vitro into A. chroococcum as described previously (58). 
Nucleic acid manipulations. Plasmid DNA was extracted essentially according to the method of Birnboim and Doly (7). A. chroococcum genomic DNA was prepared according to Robson et al. ( 59), and hybridizations to check mutant strains were performed as previously described (19). Restric- tion enzymes were purchased from Bethesda Research Lab- oratories, Boehringer Mannheim, or New England BioLabs and were used in TAS buffer (33 mM Tris-acetate [pH 7.9], 65 mM potassium acetate, 10 mM magnesium acetate, 0.5 mM dithiothreitol, 4 mM spermidine hydrochloride, pH 7.0) with the exception of Sau3A1 and KpnI, which were used according to the supplier's instructions. DNA was electro- phoresed through agarose gels in TAE buffer as described by Maniatis et al. ( 43) and extracted from low-melting-point agarose (Bethesda Research Laboratories ultrapure) by a freeze-squeeze technique modified from Thuring et al. (71). 
Nucleotide sequence determination and analysis. Unidirec- tional ordered deletions of plasmids pDElSa and pDElSb formed the basis for DNA sequencing studies and were generated by partial Sau3A digestion as described by Rob- son et al. (62). Additional defined subclones or specific deletions of pDElSa were created to sequence regions deficient in Sau3A sites and to resolve regions containing ambiguities. Gaps were completed by using synthetic oligonucleotide primers. Sequence was determined at least twice from individual templates and on both strands. Singlestranded template was produced from pEMBL-derived plasmids essentially according to Dente et al. (13) and sequenced by the dideoxynucleotide method (64) using [a-35S]dATP (6). Reactions were performed in 96-well microtiter plates (Falcon 3911) and were denatured at 90°C for 10 min prior to electrophoresis. Deoxy-and dideoxynucleotides were ob- tained from Pharmacia; Klenow fragment and [ct-35S]dATP were from Amersham International plc. Gel readings were assembled into a single "contig" by using the computer program DBUTIL (69), and computer analyses were done with the University of Wisconsin Genetics Computer Group programs (14). Initial sequence data base searches were performed with the programs FASTN and FASTP (41). Further protein sequence comparisons used the algorithm of Smith and Waterman (68) as implemented on the Connection Machine CM-2 data parallel computer (31). This software allows multiple sequence alignments to be used in data base searches, which can markedly increase the sensitivity of the method (21, 30). 
Enzyme and other assays. SAT was measured spectrophotometrically essentially as described by Kredich and Tomkins (38). A decrease in A230 due to cleavage of the thioester bond of acetyl coenzyme A (acetyl-CoA) was monitored by using a Cary 2200 dual-beam spectrophotometer. The assay was carried out at 37°C in 1-cm-light-path cuvettes in a reaction volume of 1 ml which, when complete, contained the following: Tris-HCl (pH 7.6), 50 ,umol; L-serine, 1 ,umol; acetyl-CoA, 0.4 ,umol; and cell extract, 50 to 100 ,ug. The reaction was monitored for 5 min without added serine to allow measurement of the background level of cleavage of the acetyl-CoA thioester bond. The assay proper was started with the addition of serine, and the rate of serine-dependent acetyl-CoA cleavage was calculated after deducting the background rate. Specific activities are ex- FpSAR6o-FIG. 1. Physical and genetic map of the nifU-to-nijM gene cluster in A. chroococcum. Shown is a restriction map for a portion of the chromosome cloned in two primary plasmids pDE14 and pDE15 which were described previously (19). Restriction sites: K, KpnI; S, SaII; X, XhoI. Nucleotide sequence was determined for a region of 7,099 bp indicated by the horizontal arrows. Subfragments of this region used to construct plasmids for complementation studies are indicated by the bars at the bottom. The positions of eight ORFs are indicated by the boxes, and their genetic assignment is shown within. Sites of insertion of the Kmr gene from pUC4K into the chromosome of A. chroococcum are indicated at the top, together with the strain number and the phenotype with respect to N2-fixing capacity in medium containing molybdenum. NifP refers to slow growth with N2 as the N source. 
pressed as nanomoles per hour per milligram of protein. Background levels of serine-independent acetyl-CoA cleav- age were approximately 3 and 5.5 nmol/mg/min for E. coli and A. chroococcum, respectively. Cell extracts treated at 90°C for 10 min were devoid of activity with and without serine addition. Nitrogenase activities in whole cells were measured by the acetylene reduction assay in which acety- lene and ethylene were determined by using a flame ioniza- tion detector. Cultures were capped with Suba-Seals, and acetylene was added to 10o of the gas headspace. Acetylene reduction activities of K. pneumoniae strains were not determined as we have previously shown that strains com- plemented for growth on N2 display near-wild-type acety- lene reduction levels, whereas strains not complemented for anaerobic growth show levels of acetylene reduction similar to the activity of the mutant strain alone (19). Protein in whole cells or crude extracts was measured by using the Folin-Ciocalteau reagent (42) with bovine serum albumin (fraction V; Sigma Chemical Co.) as a standard. Growth of bacterial cultures was monitored with a Klett-Summerson photoelectric colorimeter with a no. 64 filter. 

RESULTS

Location of nWfM. nifU, nifS, nifV, and nifM are clustered in a region of the A. chroococcum genome spanning about 7 kb (19). In our previous study, the approximate position of each gene was shown by (i) hybridization studies using various K. pneumoniae nifgene probes and (ii) the ability of plasmids pLC11, pSAR2a, and pSAR3a containing overlap- ping subfragments of this region (Fig. 1) to complement nifti, nifS, nifV, and nifM mutants of K. pneumoniae (19). 
The precise position of nifM could not be located by hybridization studies using the K. pneumoniae nifM gene probe. Therefore, prior to determining the nucleotide sequence of this region, we located nifM in further complementation studies. A set of nested deletions was first constructed within the insert in plasmid pDE15, which carries a 5.1-kb KpnI fragment in the plasmid vector pEMBL18+. Once the deletions had been constructed, inserts were recloned into the EcoRI site in pBR325 potentially under the control of the CAT gene promoter. Plasmid pSARSa contained the small- est insert (Fig. 1), at 1.5 kb, which complemented both K. pneumoniae nifM mutant strains (UNF 828 and UNF 2050) for growth with N2 as the sole N source. Complementation was lost in plasmid pSAR6a, which contained an insert of only 1.05 kb. The failure of pSAR6a to complement the niWM mutants was not attributable to an inhibitory effect because it did not affect expression of N2 fixation in the Nif' control, UNF50321. These experiments placed nifM within 1.5 kb from the right-hand KpnI site of the insert in pDE15 as shown in Fig. 1. 
Nucleotide sequencing. The nucleotide sequence of 7,099 bp spanning the 5.1-kb KpnI insert in pDE15 and 2 kb of the adjacent region borne on plasmid pDE14 is shown in Fig. 2. Eight complete ORFs were identified, all reading in the same direction (Fig. 1; Table 1) and possessing the biased codon usage (21) typical of A. chroococcum genes. Assignment of genes was based on deduced amino acid sequence identity to K. pneumoniae nif genes (Table 1; Fig. 3,4, and 7) (2, 5, 53). The assignment of ORF1, -2, and -3 as nifU, nifS, and nifV, respectively, is consistent with our complementation results. pSAR 3a (Fig. 1) complemented nifS and nifV but not nifU mutants and contains an insert of 3,485 bp from an EcoRI site at position 1225 in the C terminus of nifU to the PstI site at 4710 in ORF5. pSAR2a complemented nifV but not nifS mutants and contains a fragment defined by the KpnI sites at 1961 in nifS and at 7099. ORF8 is nifM, as defined by sequence identity to the K. pneumoniae nifM gene and also by the complementation results presented above. pSAR5a, which complements the nifM mutant, contains a fragment from the Sau3A1 site at position 5664 in the C-terminal coding region of ORF7 to the KpnI site at 7099. pSAR6a, which fails to complement this mutation, contains a fragment starting at a Sau3A1 site at 6082 within ORF8 and terminates at the same KpnI site. ORF6 and -7 display sequence identity to K. pneumoniae nifW and nijZ, respectively. Two ORFs located between nifV and nifW in A. chroococcum appar- ently have no counterparts in the K. pneumoniae nif gene cluster (2). 
The likely initiation codon for all eight genes or ORFs is ATG, which is preceded in each case by a polypurine tract characteristic of potential ribosome binding sites, including the motif 5'-GGAG-3' situated 5 to 11 nucleotides upstream 
ACCCGCTTCATCAGCAGCACTGGCAACGCCGGTCGCC6GTTTGCGCATCCGGTTGAGGGT GGCGGCTGCTCCGGTCTGAAATACAGCCTGAAGCTGGAGGAGGCCGGTGCCGAAGGCGAT CAGCTGATCGACTGCGACGGCATCACCCTGCTGGTCGACGATGCCAGCGCCCCTCTGCTC CCGAATGCCAGCAACAGCTGTGGTTGTGGCAAGTCTTTTGCTTGCTGATTAGGCAACCCT GAGGTCGCCGGCTGGGGCCCCAAAGACTCACTGGGAGATGAAGCCGACATGTGGGATTAT m w D Y In-ifU) TCGGAAAAAGTCAAAGAGCATTTTTACAACCCCAAGAATGCCGGAGCCGTGGAAGGCGCC S E K V K E H F Y N P K N A G A V E 6 A AACGCCATCGGCGACGTCGGATCGCTGAGCTGCGGTGACCGGCTGCGTCTGACCCTGAAG N A I B D V G S L S C G D R L R L T L K GTGGATCCGGAAACCGACGTGATCCTGGATGCCGGCTTCCAGACCTTCGGCTGTGGCTCC V D P E T D V I L D A G F Q T F G C 6 S GCCATCGCTTCCTCCTCGGCACTGACCGAGATGGTCAAGGGGGCTGACCTGGACGATGCG A I A S S S A L T E N V K G A D L D D A CTGAAGATCAGCAACAGGGACATCGCCCATTTCCTCGGACGGCTGCCGCGGGAGAAGATG L K I S N R D I A H F L 6 R L P R E K CACTGCTCGGTGATGGGCCGCGAACGCCTGCAGGCCGCCGTGGCCAACTACCGTG6CGAA H C S V G R E R L Q A A V A N Y R G E GAGCTCAGGACCGACCACGAGAAGCCGCAGCTGATCTGCAAGTCGTTCGCCATCGACGAA E L R T D H E K P Q L I C K S F A I D E GTGATGGTCCGCGACACCATTCGCGCCAACAAGCTGTCCACCGTCGAGGACGTGACCAAA V N V R D T I R A N K L S T V E D V T K CACACCAAGCGCGGCGGCGGCTGCTCGGCCTGTCATGAGGGCATCGAGCGCGTGCTGAGC H T K R GG 6 C S A C H E 6 I E R V L S GAGGAGCTGGCGCCCGTGGCGAGGTCTTCGTCGTGCGCCGACCAAGGCCAAGAAGAGGTC E E L A P V A R S S S C A D Q 6 Q E E V AAGGTGCTCGCCCCGAGCCGGCTCC6CTCGTGGCCGAGGAGACTCCGCGCCACGCCGAA6 K V L A P S R L R S P R R L R A T P K CTGAGCAACCTGCAGCGCATCCGCCGCATCGAAACCGTGCTGGCGGCGATCCGTCCGACC L S N L Q R I R R I E T V L A A I R P T CTGCAGCGCGACAAGGGTGATGTCGAGCTGATCGATGTCGACGGCAAGAACATCTACGTC L Q R D K G D V E L I D V D G K N I Y V AAGCTAACCGGCGCCTGCACCGGCTGCCAGATGGCCTCCATGACCCTTGGCGGCATCCAG K L T 6 A C T 6 C Q N A S N T L 6 6 I Q CAGCGCCTGATCGAGGAACTCGGCGAATTCGTCAAGGTGATCCCGGTCAGCGCCGGCCCA Q R L I E E L G E F V K V I P V S A 6 P CGGCAGATGGAGGTCTGACATGGCTGAC6TCTATCTCGACAACAACGCCACCACCCGGGT R Q N E V * A D V Y L D N N A T T R V GGACGACGAAATCGTCGAGGCCATGCTGCCGTTCTTCACCGABCAGTTCGGCAACCCCTC D D E I V E A N L P F F T E Q F G N P S GTCGCTGCACAGCTTCGGCAACCAGGTCGGCCTGGC6CT6AAGAGGGCGCGGCAGCAGC6 S L H S F G N Q V G L A L K R A R Q Q R TOCAGGCGTGCTCGGCGAGCATGATTCGGAAATCATCTTCACTTCCTGCGGCACCGAGTC A 6 V L 6 E H D S E I I F T S C 6 T E S GGATCACGCGATCCTCTCGGCGCTCAGCCCAGCCCGAGCGCAAGACCTGATCACCACCGT D H A I L S A L S P A R A Q D L I T T V GGTCGAGCACCCGGCAGTGCTGAGCCTGTGCGACTACCTCGCCAGCGAGGGCTACACCGT V E H P A V L S L C D Y L A S E G Y T V BCACAAGCTGCCGGTGGACAAGAA6GGCCGCCTGBATCTGGACCATTACGCCAGCCTGCT H K L P V D K K G R L D L D H Y A S L L GAACGAC6ACGTCGCCGTGGTGTCGGTGATGTGGGCCAACAACGAGACCGGCACCCTGTT N D D V A V V S V W A N N E T G T L F CCCGGTCGAGGAAATGGCACGCATGBCCGACGAGGCCGGCATCATGTTCCACACCGACGC P V E E M A R N A D E A 6 I F H T D A CGTGCAGGCCGTGCGCAAGCTGCCGATCGACCTGAAGAACTCGTCGATCCACATGCTCTC V Q A V R K L P I D L K N S S
S G H K L H R K 6 V 6 V L Y L R R 6 T CCGCTTCCGTCGCTGCTGCCGCGGCCACCAGGAGCGGCCCGCGGGCGGTACCGAGAACGC R F R R C C R G H G E R P A G 6 T E N A TGCCTCGATCATCGCCATGGGCTGGGCCGCCGAGCGCGCGCTGGCCTTCATGGAGCACGA A S I I AM G W A A E R A L A F M E H E GAACACCGAGGTCAAGCGCCTGCGCGACAAGCTGGAGGCCGGCATCCTCGCC6TCGTGCC N T E V K R L R D K L E A 6 I L A V V P GCACGCCTTCGTCACCGGCGACCCGGACAACCGCCTGCCCAACACCGCCAACATCGCGTT H A F V T G D P D N R L P N T A N I A F CGAGTACATCGAGGGCGAGGCCATCCTGCTGCTGCTGAACAAGGTCGGCATCGCCGCCTC E Y I E G E A I L L L L N K V G I A A S CAGCGGCTCGGCCTGCACCTCCGGCTCCCTGGAGCCCTCCCACGTGATGCGCGCCATGGA S G S A C T S G S L E P S H V N R A M D CATTCCCTACACCGCCGCCCACGGCACCGTGCGTTTCTCCCTGTCGCGCTACACCACCGA I P Y T A A H G T V R F S L S R Y T T E GGAGGAGATCGACCGGGTGATCCGCGAGGTGCCGCCGATTGTGGCCCAGCTGCGCAACGT E E I D R V I R E V P P I V A Q L R N V GTCGCCCTACTGGAGCGGCAACGGTCCGGTGGAACATCCGGGCAAGGCCTTCGCGCCGGT S P Y W S G N G P V E H P G K A F A P V CTACGGCTGAGCCGCCGCCTGCGGGAGCGCATCCCGCAGGAAACCGCCTCGG6GAGCCCC Y G * M > GCCCGAGTTGTTGGAGAAAGCCATGGCTAGCGTGATCATCGACGACACCACCCTGCGTGA L A S V I I D D T T L R D CGGCGAGCAGAGTGCCGGGGTCGCCTTCAAT6CCGACGAGAAGATCGCCATCCGGCGTGC G E Q S A 6 V A F N A D E K I A I R R A GCTCGCCGAGCTGGGCGTACCGGAGCTGGAGATCGGCATTCCCAGCATGGGCGAGGAGGA L A E L G V P E L E I G I P S N 6 E E E GCGCGAGGTGATGCGCGCCATTGCCGGCCTCG6CCT6TCGTCGCGCCTGCTGGCCTGGTG R E V M R A I A G L G L S S R L L A W C CCGGCTGTGCGACTTCGACCTCTCGGCCGCGC6CTCCACCGGG6TGACCATGGTCGACCT R L C D F D L S A A R S T G V T N V D L GTCACT6CCGATCTCCGACCTGATGCT6CGCCACAAGCTCAATCGTGATCGCGACTGGGC S L P I S D L M L R H K L N R D R D W A ACTGGGCGAG6TCGCCCGGCTGGTCAGCGAG6CGCGCATGGCCGGGCTTGAGGTGTGCCT L G E V A R L V S E A R N A G L E V C L GGGCTGCGAGGACGCCTCGCGGGCGGATCAGGACTTCATCGTGCGGGTGGGGGCGGTGOC G C E D A S R A D Q D F I V R V G A V A GCAGGCCGCGCGCCC6CCGCCTGCGTTCGCCGATACC6TC6GG6T6ATGGA6CCGTTCGG Q A A R P P P A F A D T V 6 V N E P F 6 CATGCTCGACCGCTTCCGTTTCCTCCGCCAGCGCCTGGACGT6GAGCTGGAGGTGCAC6C M L D R F R F L R Q R L D V E L E V H A CCACGACGACTTCGGGCTGGCCACCGCCAACACCCTGGCGGCGGTGATGGGCGGGGCGAC H D D F G L A T A N T L A A V N 6 6 A T CCACATCAATACCACGGTCAACGGGCTCGGCGAGCGCGCCGCCAACGCCGCGCTGGAAGA H I N T T V N G L G E R A A N A A L E E GTGCGTGCTGGCGCTCAAGAACCTCCACGGCATCGACACCGGCATC6ACACCCGCGGCAT C V L A L K N L H G I D T 6 I D T R G I (-CCGGCCATCTCGGCGCTGGTCGAGCGGGCCTCGGGCGTCAGTGGCCTGGCAGAAGAGC P A I S A L V E R A S G R Q W P G R R A GTGGTTGGCGCCGGTGTTCACCCACGAGGCCGGCATCCACGTCGACGGGCTGCTCAAGCA W L A P V F T H E A G I H V D 6 L L K H CCGGCGCAACTACGAGGGACTGAATCCCGACGAGCTCGGCCGCAGCCACAGCCTGGTGCT R R N Y E G L N P D E L G R S H S L V L tGGCAAGCATTCCGGCGCGCACATGGTGCGCAACAGCTACCGCGAGCTGGGCATCGAGCT G K H S G A H N V R N S Y R E L 6 I E L FIG. 2.
Nucleotide sequence of the nifU-to-nifM gene cluster from A. chroococcum. Predicted amino acid sequences for the several ORFs identified from the nucleotide sequence are given below in single-letter code. Gene assignments and directions of transcription are indicated by the boxed arrows in the space below. Potential nifpromoter sequences are underlined, and an inverted repeat sequence is marked beneath by the double-arrow line. ?0o1 a Numbers refer to positions in sequences shown in Fig. 2. b Gene assignment made by complementation and protein sequence iden- tity. 
GGCCGACTGGCAGAGCCAGGCACTGCTCGGCCGCATCCGCGCCTTCTCCACCGCACCAA A D W Q S Q A L L G R I R A F S T R T K GCGCAGCCCGCAGGCTGCCGAGCTGGAGGACTTCTATCGCCAGCTGTGCGAGCAGGGGAC R S P Q A A E L E D F Y R Q L C E Q 6 T TGCCGAACTGGCGGCAGGAGGAATGGCATGAGCCT6CTTGCGCAATGGCGTGAAGACATC A E L A A G G A * N S L L A Q W R E D I finif P CGCTGCGTGTTCGAGCGCGATCCGGCGGCACGCACCACCTTCGAGGTGCTGACCACCTAT R C V F E R D P A A R T T F E V L T T Y CCGGGC6TGCACGC6ATCATGCTCTACCGGCTCGC4CCATCGTCT6TGGCGGCCGAATGCG P G V H A I N L Y R L A H R L W R P N A TTACCTCGCCCGGCTGCTGTCGTTCGCGCGCGCCTGGTGAGCAACGTCGACATCCATCCC L P R P A A V V R A R L V S N V D I H P GGGGCCGTCATCGGTGCGCGCTTCTTCATCGACCACGGCGCCTGCGTGGTGATCGGCGAG G A V I G A R F F I D H 6 A C V V I G E ACCGCCGAGATCGGCCGGGACGTGACTCTCTACCACGGCGTCACCCTGGGCGGCACCACT T A E I 6 R D V T L Y H 6 V T L G G T T GGCGCAAAGGGCAAGCGTCATCCGACCCTGGGCGACGTTGTGCTGGTCGGCGCCGGGGCG 6 A K G K R H P T L G D V V L V G A G A AAGATCCTCGGCCCGATCACCATCGGCGCCAATGCCCGGGTCGGCGCCAACTCGGT6GTG K I L 6 P I T I G A N A R V G A N S V V GTCCAGGATGTGCCCGAGGGCTGCACGGTGGTCGGCATTCCCGGCAAGGTGGTCAAGCTG V Q D V P E G C T V V G I P G K V V K L CGCGAGGCCGGCCAGCTCAACCCCTACGGCATCGACCTCGATCACCATCTGATTCCCGAC R E A G Q L N P Y G I D L D H H L I P D CCGGTCGGCAAGGCGATCGCCT6CCTGCTGGAGCGTATCGACTCGCTGGAGAAGAGGGTC P V 6 K A I A C L L E R I D S L E K R V GAGGCGGGCGGGCTGGTCGCC CGGCAGCCAGCAGCACGTTCTACGAGGGCTBCAATCCC E A 6 G L V A A A A S S T F Y E 6 C N P GACAACAGCATCTGCGAAACGAATCTGCGCCGGTCGGCGCCCTGGTCCAGCGGCCGGCCG D N S I C E T N L R R S A P W S S 6 R P CGGCGGCCCGCCCACGCCGGTGATC6A6TGAGCGGCAG6GCCAAGGGGAGCGATTGAGTA N R R P A H A G D R V S G R A K G S D OiF5) TGGATCTGCAGGATTTCGACAGCGCCCATCTGTATTTCGACGAGCCGCTCGCCCCGGAGG D L Q D F D S A H L Y f D E P L A P E V TGGCGCCCGTCTGGCCGGCCGCCGAGCA6TACGCCGAGGGCACCGCCGAGCAGCCGCTGC A P V W P A A E Q Y A E G T A E Q P L L TCGAGGCGCAGGCCCTCGCCCCGGACGACCTGACGGTGCTGGTCGGTCTGTACCGCTTCT E A Q A L A P D D L T V L V G L Y R F Y ATTATTACCAGCATCGCTACGAGGCTGCCCTGCAGATCGCCCGGCGGCGCGTGGAGGTGG Y Y Q H R Y E A A L Q I A R R R V E V V TC6GCCCCCGGCTGCTGCTGCCC6CGAACTGGAGCSAGATCGATGCCGGCCACGTT6CCG G P R L L L P A N W S E I D A G H V A V TGGCGGCCGAGCGGGGCATCGGTCTCTTGCGTTTCTACCTGCTGGCGCTCAAGGGCGGCT A A E R G I G L L R F Y L L A L K G G Y ATCTGAGCCTGCGTCTGGGCCGGTTCGAGCAGGGCAAGGCCATGCTGCTCAAGGTCGTCG L S L R L G R F E Q G K A N L L K V V E AGCTGGACACGGACAACCGCCTGGGT6CCAGGCTGCTGTTGGACGTGCTGGCCGAGCACA L D T D N R L G A R L L L D V L A E H S GCGCTGAAATCCT6ATTTTCCCCACTGCGGCCAACGCGGAGATACGACCATGACCGTGCA A E I L I F P T A A N A E I R P * N T V Q ACCCTTTTCCCCGGATTCCGACCTGACCCTGGACGAGGCCATGGACGAGCTGGTCTCGGC P F S P D S D L T L D E A M D E L V S A CGAAGACTTCCTGGAGTTTTTCGGCGTGCCCTTCGATCAGACGGTCGTGCACGTCAACCG E D F L E F F G V P F D Q T V V H V N R CCTGCACATCATGCAGCGCTATCACGACTACCTGACCAAGGCCGGCGATCTGGACGAGCA L H I N Q R Y H D Y L T K A 6 D L D E It TGACGATCAGGCCCGCTATGCGGTGGTTCCAGCTGCTGCGCGCGCCTACCTGGACTTCGT D D Q A R Y A V V P A A A R A Y L D F V
70O ' CGAATCCGAC6CCCT6ACCGAGAAG6TGTTCAAGGTCTTCCGCATGCACGAGCCGCAGAA E S D A L T E K V F K V F R H E P Q K GACCTTCGTTTCCATCGACCA6CTGTTGAGCTGAGCACAGAAGGGGGAGGCGCCATGCT6 T F V S I D Q L L S N L CCGCAGTTTGAATATGGTGATGAGGTACGCCTGATCCGCAACGTGCGTAACGACGGCACC P Q F E Y 6 D E V R L I R N V R N D G T TATCC6GGCATGGATACCGGCGCTCTGCTCATTCGCCGCGGTGCGGTGGGCTGCGT6TAC Y P 6 N D T 6 A L L I R R G A V 6 C V Y GACGTCGGCACCTACCTGCAGGACCAGCTGATCTATCGCGTGCATTTTCTCAACGAGGG6 D V G T Y L Q D Q L I Y R V H F L N E 6 CGCACCGTCGGTTGCCGTGAGGAGGAGCTGATCCTCGCCTCGGCCCCCTGGATTCCCAAC R T V G C R E E E L I L A S A P W I P N CTGTTCGAGTTCCGTGACAACGTGATCGCCACCCGCAGCCTCGCCGTGCGCGGTCAGGTG L f E F R D N V I A T R S L A V R 6 Q V TTGGTGACGCGGGGCCAGCTCGGCAGCATCATGAAGGTGCTGCGCGATGAGTCC6AGCTT L V T R G Q L G S I H K V L R D E S E L GGTATCCAGTACCACGT6CATTTCGGCGACGGGCTGGTGCTGCAGGTGCCCGAGCAGAGT G I Q Y H V H F G D 6 L V L Q V P E Q S CTGGTGATGGCCGAGACCGAGGCGGCGATGGAGGTGCTCGAT.6AGCTTTGAGCATCCCGG N S F E H P 6 L V N A E T E A A N E V L D E L f [nifM3 CGACGGCGACAGCCGTTACTACCTGCTGAAGATCGCCCACOAGCAGTTCGGCTGCGCGCC D G D S R Y Y L L K I A H E Q F 6 C A P CGGTGAGCTCAGCGAGGAGCAGCTCCAGCAGGCCGAGCGCATCATCGGCCGGCAGAAGCA G E L S E E Q L Q Q A E R I I G R Q K H CATCGAGGATGCCGT6CTGCGTTGCCCGGATGCPGCCGGCGTGGTGATTCCGGCGTCCCA I E D A V L R C P D A A G V V I P A S Q GATCGAAGAGGCCTGGACGCAGATCGCCAATCGCTACGAGTCGGCCGAGGCACTGCAGCA I E E A W T Q I A N R Y E S A E A L Q Q GGCCCTCGACGCTCAGGGCCTGGAGCGC6TCGGCATGCGCGCCATGCT6GCCC6GGAGCT A L D A Q G L E R V 6 R A N L A R E L CAAGGTCCAGGCTGT6CTCGACTGCATCTGCGCCGGCCTGCCGGAGATCAGCGATACCGA K V Q A V L D C I C A 6 L P E I S D T D CGTGAGCCTCTACTACTTCAACCATGCCGAGCAGTTCAAGGTGCCTGCCC66CACAAGGC V S L Y Y F N H A E Q F K V P A R H K A CCGGCACATCCTGGTCACCATCAACGAGGACTTCCCGGAAAACACCCGCGAGGCCGCCCG R H I L V T I N E D F P E N T R E A A R GACGCGCATCGAGGCGATCCTCAAGCGGCTGCGCGGCAAGCCGGAGCGCTTCGCGGAGCA T R I E A I L K R L R 6 K P E R F A E Q GGCGGCGAAGCATTCCGAATGCCCCACCGCGATGCAGGGCGGGCTGCTTGGCGAGGTGGT A A K H S E C P T A N Q 6 6 L L 6 E V V TCCGGGTACTCTCTATCCTGAACTGGATGCCTGCCTGTTCCAGATGGCACAGGGGCAACT P G T L Y P E L D A C L F Q A Q G Q L GA6TCC6GTGCTGGAGTCGCCGATCGGTTTTCACGTCCT6TTCTGCGAAAGCGTGAGCAC S P V L E S P I 6 F H V L F C E S V S T GCCCGGCAGCTCACCCTCGAGGAAATCCTGCCGCGTCTGCGCGACAGGCTTCAGCTCC6 A R Q L T L E E I L P R L R D R L Q L R GCA6CGCAAGGCGTATCAGCGCAAAT6GCTGGAAAGCCTGCTCCAACAAAACGCCACTTT Q R It A Y Q R It W L E S L L Q Q N A T L GGAGAACCTCGCCCATGGATAAAACGGACAAACCCTGCTGCTCGTTCTGCGGTGCGGAAA E N L A H G
c Including the N-terminal methionine. 
d Calculation includes the N-terminal methionine. 
Identity to corresponding K. pneumoniae nif gene products except for nifP, for which identity is to the E. coli cysE gene product. (20,65). All ORFs terminate with TGA except for nifM (TAA). Most of the genes, or potential genes, overlap except for nifW and nijZ, for which the intergenic region is 20 bp, and nifS and nifV, which are separated by 72 bp containing an inverted repeat sequence (indicated by arrows in Fig. 2) which could form a stem-loop structure at the RNA level with a calculated AG of -21.1 kcal (ca. -88.3 kJ). In the cases of nifV and ORF4 and of ORF5 and niJW, the trans- lational stop signal for the first gene and the translational initiation signal for the second gene overlap. The overlap between nifZ and nifM extends across the coding region to the extent of 11 bp. Three regions within the 7,099-bp sequence underlined in GTCTTT1jGTT-3' occurs 65 bp 5' to nifU. The sequence 5'-GCTGGTGCTGCAGGTGCCC-3' is situated within the nifZ gene, 68 bp upstream from the translational initiation codon of niJM. A third nif promoter-like sequence 5'- GCTGGCGCCGCAGTIGCGC-C-3' is located 150 bp 3' to nijM, suggesting that this region may encode further gene products involved in diazotrophy of A. chroococcum. How- ever, none of these presumptive nifpromoters is preceded at the appropriate distance by the sequence TGT-n1o-ACA, which corresponds to a nifA-specific upstream activator (9). 
Analysis of predicted nif gene products. The predicted properties of the A. chroococcum nijlU, nifS, niWV, nifW, nijZ, and nilM gene products are listed in Table 1. 
NIFU and NIFS. NIFU may be a metalloprotein. Two pairs of cysteine residues located toward the C terminus of the predicted polypeptide are arranged in a -Cys-X-X-Cys- configuration typical of the spacing of cysteines in many nonheme iron proteins. These cysteine residues are also conserved in NIFU polypeptides from Azotobacter vinelandii, K. pneumoniae, and Anabaena strain 7120 (Fig. 3). The sequence -Lys-Ala-Gly-Gly-Gly-Cys-Ser-Ala-Cys-Hisstarting at residue 169 in A. chroococcum NIFU, which contains the first example of the -Cys-X-X-Cys-motif, also resembles part of the heme-binding domains in c-type cytochromes, especially those from Rhodopseudomonas species (1). The nifS gene products are relatively highly conserved between Azotobacter species, K. pneumoniae, and Anabaena strain 7120 (Fig. 3). Especially striking is an extensive block of identity toward the C terminus of the predicted NIFS polypeptides. 
NIFV. Amino acid sequences for nifV gene products from Azotobacter species and K. pneumoniae are aligned in Fig. 4. NIFV is involved in the formation of homocitrate, a component of FeMo-co which is incorporated into the MoFe-protein (23,24). NIFV is postulated to be homocitrate synthase (23). In yeasts and fungi, homocitrate synthase carries out the condensation between acetyl-CoA and a-ke- toglutarate (70,72). It is particularly interesting that NIFV exhibits similarity to the leuA gene product (isopropylmalate synthetase; E.C. 4.1.3.12) from both Salmonella typhimurium (56) Saccharomyces cerevisiae (3) (Fig. 4). Isopropylmalate synthase carries out the condensation between acetyl-CoA and a-ketoisovalerate. This suggests that NIFV catalyzes a reaction analogous to that catalyzed by LEUA, consistent with the proposal that acetyl-CoA and a-ketoglutarate are substrates for NIFV (Fig. 5). Umbarger (74) also draws a parallel between the reaction carried out by isopropylmalate synthase and homocitrate synthase. Therefore, the two major blocks of identity between NIFV and LEUA, which are placed at approximately equivalent positions in the two enzymes, could be binding sites for acetyl-CoA or for the structurally common portion of a-ketoglutarate and a-ketoisovalerate (Fig. 5). 
ORF4 (nifP) encodes SAT. The ORF4 polypeptide shows 41% identity to the cysE gene product (CYSE) from E. coli (Fig. 6), which encodes SAT, a key enzyme in cysteine biosynthesis in E. coli (12). The possibility that ORF4 codes for a similar enzyme was tested in complementation studies with E. coli JM39, a cysteine auxotroph defective in cysE and lacking SAT activity. JM39 was transformed with plasmids pSAR2a and pSAR3a constructed for earlier nif com- plementation studies and containing ORF4 intact (Fig. 1). Both JM39(pSAR2a) and JM39(pSAR3a) grew well in mini- mal medium lacking cystine, consistent with the idea that ORF4 may encode SAT. Both nifV and ORF4 are common to pSAR2a and pSAR3a; therefore, a plasmid (pJW2a) that carries only ORF4 was made. pJW2a carries a fragment of 1,158 bp containing ORF4 cloned under the control of the lacZ promoter in pTZ19R plus 140 bp of the 3' coding region of nifV and 125 bp at the 5' coding region of ORF5. pJW2a also complemented the cysE mutation in JM39. SAT activity was compared in cell extracts of E. coli 5K (a cysteine prototroph), JM39(pJW2a), and JM39 grown in LB. The first two strains had specific activities of 34 and 43 nmol/mg/min, whereas no activity was detected in JM39. SAT in E. coli and S. typhimurium is constitutive but subject to feedback inhibition by low levels of cysteine. SAT activity in E. coli JM39(pJW2a) crude extracts was also inhibited by cysteine, although the concentration required to cause 50% inhibition (50 ,uM) was 50-fold higher than that reported to inhibit the E. coli enzyme (38). 
Although ORF4 and CYSE differ in length by only four amino acid residues, the region of similarity is displaced in the two proteins (Fig. 6). CYSE has a 74-residue extension at the N terminus, whereas ORF4 is accordingly longer at the C terminus. The essential catalytic domain therefore appears confined to the 186-residue portion common to each protein. 
ORF4 also shows similarity to the E. coli lacA gene product, thiogalactoside acetyltransferase (22), and the Rhizobium leguminosarum nodL gene product (Fig. 6), suspected to be an acetyltransferase (17). The region common to this class of known or suspected acetyltransferases therefore may be important in binding acetyl-CoA. 
ORF5, NIFW, NIFZ, and NIFM. The finding of a SAT gene adjacent to nifV suggested that other genes in this cluster might be involved in sulfur assimilation. However, -----------------PHWQSV--- FIG. 3. Alignments of predicted amino acid sequences for the nifU (A) and nifS (B) gene products from A. chroococcum (Ac), A. vinelandii (Av) (5), K. pneumoniae (Kp) (2, 5); and Anabaena strain 7120 (An) (50). In the multiple sequence alignments, fully conserved positions are noted in the consensus line (con) and printed in bold font. 
-VDT-IAELE HIADRQA GW L SSVT TNHQVTVSLSGSCSGQCMTDMTL-AWLQQKLME 263 An AATNLNNKGGSKPTNIPN----SGQKRPLTNVQKIALIQKVLDEE-----VRPVLIADGCDVELYDVDGDIVKVVLQSACGSCSSSTATLKIAIESRLRD 289 con RP D GD L V V L C C TL L Ac ELGEFVKVIPVSAGPR-9MEV 309 
no other significant similarities were detected when compar- isons were made between each of the nif genes considered here and several other cys genes from E. coli. These in- cluded cysP, encoding a thiosulfate-binding protein; cysT, cysW, and cysA, encoding the sulfate permease (26, 67); and cysK (10, 40) and cysM (67), encoding O-acetylserine (thiol) lyase isozymes A and B, respectively. 
Alignments of niJW, niJZ, and nifM gene products from several organisms are shown in Fig. 7. NIFW polypeptides appear poorly conserved between Azotobacter species and K. pneumoniae. fixO from Azorhizobium caulinodans (34) shows 32% identity to Azotobacter niJW (Fig. 7), suggesting that these gene products are functionally analogous. niJZ gene products are quite highly conserved between Azotobac- ter species and K. pneumoniae. Also, there is 23% sequence identity between the translated product of a partial ORF (ORF2) from Rhodobacter capsulatus (44) and the N termi- nus of nijZ (Fig. 7), suggesting that ORF2 in R. capsulatus is analogous to nijZ. 
The sequences of the nifM products from A. chroococcum and K. pneumoniae contain 26% identity dispersed through- out their lengths (Fig. 7). The dispersed nature of this identity may account for our previous lack of success in identifying a nijM-like gene in A. chroococcum by heterol- ogous hybridization (19). Pagani et al. (52) demonstrated that reactivation of an inactive 2Fe-containing form of K. pneu- -l l 100 aa FIG. 4. Alignments of predicted amino acid sequences for the nifV and leuA gene products from A. chroococcum (Ac), A. vinelandii (Av) (5); and K. pneumoniae (Kp) (2,5). leuA gene products are from S. typhimurium (St) (56) and Saccharomyces cerevisiae (Sc) (3). In the multiple sequence alignments, fully conserved positions are noted in the consensus line (con) and printed in bold font. aa, Amino acids. moniae Fe-protein could be catalyzed by the enzyme bovine liver rhodanese, and they speculated that NIFM may act as a nif-specific sulfur transferase. However, alignments of NIFM and rhodanese (55) amino acid sequences display no apparent similarities (data not shown). Furthermore, the predicted tertiary structure of nifM showed no obvious similarity with the structure of rhodanese determined at 0.25-nm resolution (55). 
Mutagenesis of the niflU to nifiJI cluster in A. chroococcum. Functionality of the region described above was established by mutagenesis. The kanamycin resistance gene cassette (Km') from pUC4K (75) was introduced at several locations in the nifU to nijM region of the A. chroococcum chromo- somebiy gene replacement. Sites of insertions are illustrated in Fig. 1. Mutations were constructed in MCD105, which normally fixes N2 in the presence of molybdenum. Introduc- tion of the Kmr gene into the Sall site 1.1 kb upstream of the sequenced region did not adversely affect N2 fixation or growth. However, inserts in the SaiI sites at positions 154, 1117 (in nifU), and 2813 (in nifV) and in the XhoI site at position 6617 (in nifMW) resulted in Nif phenotypes. 
The nifV mutant (MCD1210) did not show any significant acetylene reduction activity, which is surprising in view of the fact that nifV mutants of K. pneumoniae fail to reduce N2 yet still exhibit significant acetylene reduction activity (45,46). The activity in K. pneumoniae may be explained by the substitution of homocitrate with some other carboxylic acid, possibly citrate, which leads to a partially active enzyme. Indeed, citrate can be incorporated into NIFV-MoFeprotein from A. vinelandii in vitro and in reconstitution experiments with NIFV-MoFe-protein (23), and acetylenereducing activity is restored. In A. chroococcum, it is possible that no such substitution is possible in vivo because citrate levels are low. 
A mutant (MCD1220b) with the Kmr gene in the Sall site within the SAT-encoding gene at Homocitrate FIG. 5. Similarity between the reactions catalyzed by isopro- pylmalate synthase and homocitrate synthase, indicated by the boxed moieties which are similar in each reaction. mally when ammonium was supplied but only slowly with a doubling time of 5 h, compared with 3 h for MCD105, in N2-fixing conditions. Also, the appearance of acetylene reduction activity was delayed by 2 to 3 h in MCD1220b as compared with MCD105 when organisms were transferred from ammonium-rich to ammonium-free medium. When grown under N2-fixing conditions, SAT specific activity in MCD1220 was significantly lower than in MCD105: specific activities were 6.7 and 12.4 nmol/min/mg, respectively. The effect on N2 fixation seen in MCD1220 could be explained by polarity of the mutation on downstream genes. However, strains (MCD1230a and -b) with the Kmr cassette introduced in both orientations into ORF5 at the XhoI site at position 4620 were indistinguishable from the parent strain. The phenotype of MCD1220 therefore appears to arise solely from inactivation of the SAT gene. 

DISCUSSION

The nifU, nifS, nifV, nifW, nijZ, and niJM genes in A. chroococcum are arranged as in K. pneumoniae except that two additional ORFs are located between nifV and niJW. The gene cluster in A. chroococcum closely matches that de- scribed for A. vinelandii (28) with respect to gene order, spacing between genes, and the presence of the additional ORFs between nifV and nifW. The overall nucleotide se- quence identity between the two Azotobacter species over this region is 90.03%. 
One of the additional ORFs (ORF4) found in A. chroococcum, which corresponds to ORF7 in the major nif cluster of A. vinelandii (27,28), probably encodes SAT, given the sequence identity between ORF4 and CYSE, complementation of the cysE mutation in JM39, and restoration of serine- dependent, acetyl-CoA cleavage activity in JM39(pJW2a). There are no reports describing the cysteine biosynthetic pathway in this genus, though O-acetylserine is a likely intermediate. Disruption of ORF4 does not cause cysteine auxotrophy or affect growth on ammonia adversely, and therefore normal biosynthesis of cysteine in A. chroococcum appears independent of this gene. However, ORF4 appears necessary for optimal N2 fixation. Therefore, A. chroococcum contains at least two SAT genes. One encodes an isozyme, detectable in extracts of MCD1220b, which probably serves a housekeeping function, whereas ORF4 en- codes a second isozyme required additionally under N2- fixing conditions. We propose to give ORF4 the designation nifP. Our results are at variance with those described for A. vinelandii, in which mutation of the corresponding ORF (ORF7) results in no detectable phenotype, though mutation of ORF8 (corresponding to ORF5 in this study) does lead to slow diazotrophy (28). 
The existence of a nif-specific SAT gene in A. chroococ- cum implies that higher intracellular levels, or rates of synthesis, of O-acetylserine are required when organisms are fixing N2. Therefore, O-acetylserine may have some specific role in N2 fixation per se. Increased levels of O-acetylserine in the cell may stimulate expression of genes for sulfate uptake and assimilation as occurs in enteric bacteria (37). If, however, O-acetylserine is involved in cysteine biosynthesis in this genus, then it is possible that maximal rates of synthesis of active nitrogenase components or other nif gene products require higher rates of cysteine and/or methionine synthesis. Nitrogenase is abundant in N2-fixing cells, and the Fe-protein from A. chroococcum contains 2.09% cysteine and 4.6% methionine while MoFe- protein contains 2.22% cysteine and 3.13% methionine (77). There are no data concerning the average amino acid content of proteins in A. chroococcum; however, proportions of cysteine and methionine averaged for many families of proteins are only marginally lower than found in nitrogenase, at 1.7 and 3.1%, respectively (11). Therefore, it seems unlikely that maximal rates of synthesis of nitrogenase polypeptides place an especial burden on cysteine biosynthesis specifically. However, this explanation cannot be ruled out, and it is also possible that other nif gene products whose functions are unknown at present may serve an analogous role for the production of other amino acids that are relatively abundant in nitrogenase. 
Alternatively, NIFP may have a role in optimizing manu- facture of metal sulfur clusters for either Fe-protein or MoFe-protein because there is evidence that cysteine is the sulfur donor to nonheme metal sulfur centers in E. coli (76). Synthesis of metal centers in both molybdenum nitrogenase components may require high intracellular concentrations of cysteine. This possibility is supported by the finding that FeMo-co synthesis is stimulated by exogenously supplied cysteine or cystine in NifQ-and Mol-mutants of K. pneumoniae (73). In enteric bacteria at least, SAT is the key enzyme controlling levels of cysteine and is highly sensitive to feedback inhibition by cysteine. The observation that relatively high concentrations of cysteine are required to inhibit NIFP activity, at least in crude extracts in E. coli, is consistent with it allowing the accumulation of cysteine in A. chroococcum. The overlapping arrangement of nifV, nifP, ORF5, and nifW may simply reflect genetic economy, or it may be functionally significant. Such an arrangement could cause translational coupling (51), possibly ensuring that stoichiometric amounts of each gene product are formed. This may be necessary if two or more of these gene products form a complex. It is interesting that both NIFV and NIFP probably both catalyze condensation reactions involving acetyl group transfer from acetyl-CoA to ot-ketoisovalerate and serine, respectively. Also, both reactions are normally associated with amino acid biosynthesis. In E. coli and S. (A) Alignment for predicted amino acid sequences for ORF4 (nifP gene product) and SAT (cysE gene product). The upper alignments show similarity between the predicted amino acid sequences for ORF4 (niJP) from A. chroococcum (Ac), ORF7 from the major nif gene cluster from A. vinelandii (Av) (27,28), and the SAT-encoding gene, cysE, from E. coli (Ec) (12). The lower alignment shows the most conserved region observed between these sequences and predicted amino acid sequences of gene products of nodL from R. leguminosarum (Rl) (17) and lacA from E. coli (Ec) (22). In the multiple sequence alignments, fully conserved positions are noted in the consensus line (con) and printed in bold font. (B) Relative position of this conserved region (indicated by the stippled box). The extent of significant sequence identity between the nifP and cysE gene products is also indicated (open box). aa, Amino acids. 169 170 244 typhimurium, SAT is normally complexed with O-acetyl- serine (thiol) lyase, which catalyzes the final reaction in cysteine biosynthesis (37). Thus, the amino-terminal portion of CYSE, with no counterpart in NIFP, may be important for formation of this complex. Likewise, the C-terminal extension of NIFP, with no counterpart in CYSE, may have an analogous role in a complex with a different protein, e.g., NIFV. 
Though the requirement for niJM for the maturation and stability of NIFH has long been documented (25,57), the precise role of NIFM remains elusive. Recent searches of the protein data bases also do not provide any obvious clues. The possibility that NIFM acts as a specific sulfur transfer- ase involved in metal cluster insertion or assembly in the Fe-protein has been discussed elsewhere (52). 
The nifU-to-nifM cluster in A. chroococcum may be transcribed as a single operon because nifM was expressed from the CAT gene promoter placed 3.8 kb upstream in pSAR2a (19), whereas nifP can be expressed from the same promoter placed 750 bp further upstream in pSAR3a. This finding suggests that there are no strong transcription termi- nation signals in this region that are recognized in K. pneumoniae or E. coli. Therefore, the inverted repeat lo- cated between nifS and nifV appears not to prevent tran- scription of downstream genes at least in these backgrounds. Previous data also show that significant levels of expression of these gene products in enteric bacteria cannot proceed from internal promoters recognized by RNA polymerase containing the normal housekeeping sigma factor (c7o) or the alternative sigma factor produced by ntrA (Uf54). The nucle- otide sequence data shows that although there are potential ntrA-dependent promoters immediately upstream of nifM and nifU, neither is preceded at an appropriate distance by a TGTNjOACA upstream activator (9). These potential promoter sequences appear conserved at similar locations in the corresponding A. vinelandii gene cluster, although in this organism an additional potential promoter together with an upstream activator is located upstream of ORF8 (28). In A. vinelandii, it is proposed that transcription of nifU through ORF6 (nifP) is initiated from a nif promoter situated upstream of a small ORF (ORF6) preceding nil'U. ORF8 through nif'M might be cotranscribed vinelandii (Ac) (27), and K. pneumoniae (Kp) (2,53). Also shown is an alignment between the fixO gene product from Azorhizobium caulinodans (Az) (34) and the nifW gene products. A partial ORF from Rhodobacter capsulatus (44) with a high degree of similarity to nijZ is shown in the nijZ alignments. In the multiple sequence alignments, fully conserved positions are noted in the consensus line (con) and printed in bold font. 
with these genes or from the promoter immediately preced- ing ORF8. This arrangement may also allow boosting of expression of the distal genes under some conditions. The apparently complex and potentially overlapping transcriptional organization of this gene cluster in the azotobac- ters mirrors the situation in K. pneumoniae (4,54,66). The reason for the apparently complex transcriptional arrange- ment in these two genera is unclear. In the case of the azotobacters, it may be linked to their possession of alter- native nitrogenase systems. Transcription of the structural genes for the alternative vanadium nitrogenase in A. chroo- coccum requires an alternative activator (vnfA), and mutation of this gene does not block expression of the Monitrogenase system (36). The possibility exists that if, as discussed earlier, the nifUM gene cluster is required for expression of both nitrogenase systems, then its transcrip- tion may be independent of both nifA and vnfA and depend instead upon a third, as yet unidentified, activator. 

Fig. 2

 2 Fig. 2 display close similarity to the nif consensus promoter defined by Beynon et al. (4). The sequence 5'-TGTCGCAA 


9

  TTuDGEQSAGVAFNADEKIAIRRALAELGVPELZIGIPSMGEEEREVMRAIAGLGLSS-RLLAWCRLCDFDLSAARST--GVTMVDL--SLPISDLMLR 103 9 TT10EQSAGVAFNADEXIAIARA3AELCVPELaIGIPSMGEEEREVMHAIAGLGLSS-RLLAWCRLCDVDLAAARST--GVTMVDL--SLPVSDLMLH103 9 TT10EQSPGVAFRTSEXVAIAEALYAACITAM3VGTwAMGDEEIARIQLVRROLPDA-TLMTWCRMNALEIROSADL--GIDWVDI----------VCLGCZDASRADQDFIVRVGAVAQAA------RPPPA-FADTVGVMEPFGMLDRFRFLRQRL-185 104 HKLNRDRDWALREVARLVGEARMAGLE----------VCLGCZDASRADLEF WQVGEVAQAA------GARRLRFADTVGVMEPFGMLDRFRFLSRRL-186 104 YKLREPLAVLLERLAMFIHLAHTLGLK----------VCIGCZDASRASGQTLRAIAEVAQQC------AAARLRYADTVGLLDPFTTAAQISALRDVW-186 109 TKLRRTLDEVIERAVYMVKRARNYTDD----------VEFSCZDAGRTPVDDLARVVEAAINA----- 


FIG. 7 .

 7 FIG.7. Alignments of predicted amino acid sequences for ni:fW (A), nifZ (B), and nifM (C) gene products from A. chroococcum (Ac), A. 


TABLE 1 .

 1 Properties of A. chroococcum nif gene products 


	Sequencea	a ORF	Geneb	~~~~No. of Mol amino Wtd acidsc w	pI	Identity' dniy
	349-1278	1	nifU	309	33,932 7.56	51.1
	1280-2470	2	nifS	396	43,295 6.2	58.1
	2543-3691	3	nifV	382	41,489 5.68	42.9
	3688 4497	4	nifF	269	28,577 9.04	41.0
	4500-5033	5		177	19,757 4.73
	5030-5374	6	niJW	114	13,126 4.30	28.6
	5395-5871 5861-6742	7 8	niJZ nifM	158 293	17,768 4.59 32,978 5.89	43.9 29.1



  position 3886 grew nor- 


	Isopropylmalate Synthase
	OH3	OH3
	CH3CH 3	CH 3CH
	6=0	HO-C-COOH
	| COOH |m H2
		C00H
	a Ketoisovalerate	a Isopropylmalate
	Homocitrate Synthase
	COOH	COOH
	2 OH2	1 UNH2
	6=0	HO-6-COOH
		COOH
	a Ketoglutarate	
	ii	
	uh	I
	'I	I
	.5	

ACKNOWLEDGMENTS

We thank R. Dixon, C. Kennedy, M. Merrick, and R. Pau for useful discussions and A. L. Menon for critically reading the manuscript. We are particularly grateful to Wyatt Paul and Mike Merrick for making results available prior to publication.
David J Evans, Robert Jones, Paul R Woodley, J Rymon Wilborn, Robert L Robson3
Nucleotide Sequence and Genetic Analysis of the Azotobacter chroococcum nifUSVWZM Gene Cluster, Including a New Gene (nifP) Which Encodes a Serine Acetyltransferase