Sequences of nifX, nifW, nifZ, nifB and two ORF in the frankia nitrogen fixation gene cluster

Gene, 161 (1995) 63-67 © 1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.50

63

GENE 09018

Sequences of nifX, nifW, nifZ, nifB and two ORF in the Frankia nitrogen fixation gene cluster (Actinomycete; actinorhizal; root nodule; nitrogenase; n/f)

O l i v i a T. H a r r i o t t *, T h o m a s J. H o s t e d * a n d D a v i d R. B e n s o n Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT06268-3044, USA Received by C.R. Hutchinson: 24 January 1995; Revised/Accepted: 16 March/28 March 1995; Received at publishers: 25 April 1995

SUMMARY

The actinomycete Frankia alni fixes N2 in root nodules of several non-leguminous plants. It is one of the few known Nz-fixing members of the high-GC Gram + lineage of prokaryotes. Thus, we have undertaken a study of its nitrogen fixation gene (n/f) organization to compare with that of the more extensively characterized proteobacteria. A cosmid (pFN1) containing the n/f region of Fa CpI1 was isolated from a cosmid library using the nifHDK genes of Fa CpI1 as a probe. A 4.5-kb BamHI fragment that mapped downstream from the previously characterized n/fHDK genes was cloned and sequenced. Based on nt and aa sequence similarities to n/f from other Nz-fixing bacteria, eight ORF were identified and designated nifX, off3, orfl, nifW, n/fZ, nifB, orf2 and n/fU. A region that hybridized to Rhizobium meliloti and Klebsiella pneumoniae n/fA did not appear to contain a n/fA-like gene. We have revised the map of the Fa n/f region to reflect current information.

[NTRODUCTION

Strains of Frankia are strictly aerobic euactinomycetes that form mutualistic N2-fixing root nodules on some nonleguminous plants (actinorhizal plants) (Benson and Silvester, 1993). At least five n/f genes (HDKAB) have been detected by heterologous hybridization in Frankia (Simonet et al., 1988; Mullin and An, 1990), and nifHD and K, that encode the three polypeptides of the nitroCorrespondence to: Dr. D.R. Benson, Department of Molecular and Cell Biology, Box U-44, University of Connecticut, Storrs, CT 06279-3044, USA. Tel. (1-203) 486-4258; Fax (1-203) 486-1784; e-mail: [email protected] * Present addresses: (O.T.H.) Center for Agricultural Molecular Biology, Rutgers University, Cook College, New Brunswick, NJ 08903, USA. Tel. (1-908) 932-1586; (T.J.H.) Eli Lilly Co., Lilly Corporate Center, Indianapolis, IN 46285, USA. Tel. (1-317) 276-5318.

genase complex, have been sequenced (Normand and Bousquet, 1989; Normand et al., 1992; Hirsch et al., 1995). NifB from Kp synthesizes a low-molecular-mass Fe-S cluster precursor of the FeMo cofactor of nitrogenase (Shah et al., 1994). NifA activates c~54-mediated transcription of n/f and some fix operons in response to low p O 2 and/or nitrogen starvation in diazotrophs from the at and 7 groups of the purple bacteria (Dean and Jacobson, 1992). This report describes the characterization of a 4.5-kb DNA fragment containing eight Frankia nif genes. Although heterologous nifA probes hybridized to some cloned fragments of this n/f region, no nt or aa similarity to n/fA is present. This region is linked to the structural genes for the nitrogenase Fe protein and MoFe protein.

EXPERIMENTAL AND DISCUSSION Abbreviations: aa, amino acid(s); Ac, Azorhizobium caulinodans; Av, Azotobacter vinelandii; bp, base pair(s); Fa, Frankia alni; kb, kilobase(s) or 1000 bp; Kp, Klebsiella pneumoniae; nif, genes encoding proteins necessary for nitrogen fixation; nt, nucleotide(s); ORF (Orf), open reading frame(s); Rc, Rhodobacter capsulatus; Rm, Rhizobium meliloti. SSD1 0378-1119(95)00300-2

(a) Isolation of the nif region of Fa CpI1 Plasmids and strains used in this study are listed in Table I. The plasmid pFN1 was isolated from a pLAFR3

64 TABLE I Bacterial strains and plasmids Strain or plasmida Strains: Fa CpI1 E. coli XL1-Blue Plasmids: pBluescriptlI pFR140 pFN1 pFN2.1 pFNB4.5 pJE294 pCE300 pFQ149

Genotype or properties

Reference

Comptonia peregrina isolate, wild type supE44 hsdR17 recA1 endA1 gyrA 46 thi relA1 lac IF' proAB + lacI lacZAM15 Tnl0(TcR)]

(Callaham et al., 1978) Stratagene, La Jolla, CA, USA

Am ~ Tc R, pUC18-derived cloning vector Am R (pUC19), 7.1-kb BamHI insert with nifHDK Tc R [pLAFR3], 28-kb BamHI insert pBluescriptSK-, 2.1-kb SstI insert pBluescriptSK-, 4.5-kb BamHI insert Kp nifA Rm nifA Fa ArI3 4.5-kb n/f clone containing nifB

Stratagene (Rochefort and Benson, 1990) (Hosted, 1992) This study This study (Santero et al., 1989) C. Earl (Simonet et al., 1988)

a Escherichia coli strains were grown at 37°C in Terrific Broth or 2 x YT medium. When required ampicillin (100 gg/ml) or tetracycline (10 pg/ml) was added. Fa CpI1 was grown in defined succinate medium (Noridge and Benson, 1986).

cosmid library ofFa CpI1 genomic DNA by probing with CpI1 nifHDK genes in pFR140. A 4.5 kb BamHI fragment was subcloned from pFN1 and designated pFNB4.5 (Fig. 1). In its restriction map, and hybridization pattern to heterologous nifA probes, the insert in pFNB4.5 is identical to that in pFQ149, a clone containing the homologous region from the closely related Fa ArI3 (Simonet et al., 1988). pFNB4.5 hybridized strongly to appropriately sized bands of restriction digested Fa CpI1 genomic DNA confirming that pFNB4.5 originated from Fa CpI1 (not shown).

(b) Sequence analysis of pFNB4.5 The strategy used to sequence pFNB4.5 and the resulting sequence are recorded in Figs. 1 and 2. High-%G + C Gram ÷ bacteria like Frankia generally have a G or C in the third codon position. Thus, ORF were initially circumscribed on the basis of third position bias." ORF were identified by the similarity of their inferred aa sequences to aa sequences of n/f genes from other Ne-fixing bacteria. Altogether, eight n/f genes were identified including nifX, [ HD

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orfl, off3, nifW, nifZ, nifB, orf2 and nifU. All are transcribed in the same direction and, with the exception of

nifB, are preceded by G G A G or G A G G potential ShineDalgarno regions. The most probable Shine-Dalgarno region for nifB is G G A C G beginning 12 bp before the putative G T G start codon. The genes reported here overlap in six instances. Sufficient sequence information was obtained to identify orf2 and nifU but is not reported because the complete sequence of both strands was not obtained. The characteristics of each gene product are listed in Table I.

(c) nifX, off3, orfl, nifW and nifZ The Fa CpI1 nifX gene is upstream from off3 as in Av and Rc (Jacobson et al., 1989; Moreno-Vivian et al., 1989). Overlapping the nifX GTG start codon is a TGA stop codon in frame with upstream codons having > 90% GC bias in the third position, probably indicating an additional ORF. Frankia NifX has 30% predicted aa sequence identity to Rc, 27% to Av and 28% to Kp nifX genes. Accounting for conservative substitutions, Fa NifX has 54% similarity to NifX from all three microorganisms. The role of NifX is unclear but has been suggested to help negatively regulate nitrogenase synthesis (Gosink et al., 1990). A 444-bp ORF that overlaps nifX by four nt shows 40% predicted aa sequence identity, and 62% aa similarity, to Orf3 of Av and Orf4 of Rc (Jacobson et al., 1989; Moreno-Vivian et al., 1989). Overlapping the 3' end of orf3 by 11 nt, is a 249-bp ORF with 33% derived aa sequence identity, and 55% aa sequence similarity, to Ac Orfl (Arigoni et al., 1991). The functions of Orf3 and Orfl are not known.

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Fig. 2. Nucleotide sequence of pFNB4.5 encoding the nifX-orf3-orfl-n!fW-nifZ-nifB region from Fa CpI1 and deduced aa sequences. Putative ShineDalgarno sequences are underlined. Methods: For sequencing, overlapping deletion clones were created for subclone pFN2.1 (for the noncoding strand) and pFNB4.5 (for the coding strand) using the Erase-a-Base system (Promega, Madison, WI, USA). Restriction fragments of pFNB4.5 were subcloned into pBluescript II vectors. Single stranded DNA was prepared using phage VCSM 13 (Stratagene, La Jolla, CA, USA). Plasmid isolation, restriction endonuclease digestions, and DNA cloning were carried out using standard techniques (Sambrook et al., 1989: Lee and Rasheed, 1990). DNA was sequenced using TAQuence 2.0 (US Biochemical, Cleveland, OH, USA) with 7-deaza-2'-dGTP in place of dGTP to minimize secondary structure formation in the high-% G+C Fa DNA. The insert in pFNB4.5 was sequenced in both directions except for a short segment of nifX where high GC content made reading of one strand difficult. In this case, redundant sequences were obtained using both 35S-labelled nucleotides and automated laser fluorescence (ALF) sequencing and the sequence derived was unambiguous. Programs used to analyze the data were from the GCG Sequence Analysis Software Package (Genetics Computer Group, Madison, WI, USA). Sequences used in the aa sequence alignments were obtained from GenBank, release 72.0 (Intelligenetics, Mountain View, CA, USA). This sequence has been deposited with GenBank {accession No. L29299).

Fa N i f W has 39% identity and 65% similarity to Ac N i f W (Arigoni et al., 1991), 28% identity and 55% similarity to Av N i f W (Jacobson et al., 1989) and 28% identity and 49% similarity to Kp N i f W (Paul and Merrick, 1987). N i f W m a y play a role in homocitrate processing

( M a s e p o h l et al., 1993). Homocitrate is an organic component of the F e M o cofactor in nitrogenase ( H o o v e r et al., 1989; Wang et al., 1991; Meijer and Tabita, 1992). Fa NifZ has 29% identity and 47% similarity to Av NifZ, and 24% identity and 45% similarity to Kp NifZ.

66 Several direct repeats found at the 3' end of nifZ are reflected in the aa sequence as an Ala + Pro-rich region. The role of NifZ is unclear, although studies in Av suggest that NifZ might be required for the maturation of the MoFe protein (Jacobson et al., 1989). (d) nifB NifB is essential for nitrogen fixation and is highly conserved (Dean and Jacobson, 1992). It synthesizes an Fe-S cluster that forms part of the FeMo-cofactor that is present at the active site of the molybdenum-iron protein of nitrogenase (Shah et al., 1994). Fa nifB has a predicted molecular mass of 55 078 Da (see Table II), a value that is close to that of other NifB proteins. It is most similar to Anabaena NifB with 58% aa identity and 75% similarity, followed by Av NifB (53% and 69%), Rm NifB (50% and 66%) and Kp NifB (49% and 69%). The nine conserved cysteine residues found in other NifB proteins are also present in Fa NifB. The start of nifB overlaps the 3' end of nifZ by 2 nt, suggesting that nifB is cotranscribed with nifX, off3, off1, nifW and nifZ. However, several repeated sequences, ranging in length from 11 to 33 nt, and within the 3' region of nifZ, may suggest the presence of a regulatory region such as occur in promoter regions of genes in Streptomyces sp. (Delic et al., 1992). In the only other NifB sequence from a Gram + organism, Clostridium pasteurianurn, nifB is fused to the 3' end of nifN and is transcribed as part of the nifEN-B operon (Dean and Jacobson, 1992; J.-S. Chen, personal communication). In the Gram-diazotrophs Av, Kp, Anabaena sp. strain 7120 and Rc, nifB is transcribed from its own promoter (Buikema et al., 1987; Joerger and Bishop, 1988; Klipp et al., 1988; Masepohl et al., 1988; Mulligan and Haselkorn, 1989). Sequence of the region directly downstream from nifB indicates the presence of two other ORF. We have desigTABLE II Properties of predicted polypeptides from pFNB4.5 genes ORF

Positions a (nt)

N u m b e r of aa b

Molecular mass b (Da)

pI

n~X off3 orfl n~W n~Z n~B

55-486 483 926 916-1164 1161 1559 556-2134 2131 3663

143 147 82 132 192 510

15407 16 166 9081 14822 19002 55 078

6.78 4.73 4.74 5.08 5.14 5.06

N u m b e r s refer to positions in the sequence shown in Fig. 2. Includes the first position in the initiation codon and the third position in the stop codon. u Includes the N-terminal Met.

nated one as off2; it has 42% sequence identity and 64% similarity to off6 from Av. The other ORF was designated nifU due to its predicted aa similarity to the carboxyl domain of NifU from other diazotrophs. Truncated forms of nifU have been reported from Rc and Bradyrhizobium japonicum (Dean and Jacobson, 1992). The reading frame bias and aa alignments, including placement of conserved cysteine residues, are consistent with these designations. (e) Hybridization to Rm and Kp nifA Plasmid pFNB4.5 has a restriction map identical to that of pFQ149 (Simonet et al., 1988). pFQ149 contains a 4.5-kb BamHI insert from Fa strain ArI3 that hybridized with Rm nifA anff,nifB. We probed CpI1 chromosomal DNA, pFNB4.5 and pFN1 with a 945 bp PstIEcoRV fragment of pJE294 containing an internal fragment of Kp nifA. Kp nifA did not hybridize to digested Fa CpI1 chromosomal DNA but did hybridize to the 4.5-kb BamHI fragment of pFN1 and pFNB4.5, the 3.5-kb HindIII-BamHI and 1.2-kb PstI fragments of pFNB4.5 (Fig. 1). Identical results were obtained when Rm nifA from pCE300 was used as a probe (not shown). These observations confirm previously reported hybridization results (Simonet et al., 1988). The absence of nifA in pFNB4.5 suggests that the hybridization obtained with cloned fragments of pFNB4.5 was artefactual. Short regions (25-50 nt) present upstream from nifB with 65-70% similarity to nifA probes might be sufficient to allow some binding to highcopy-number Frankia nif DNA sequences on Southern blots. Therefore, we conclude that a conserved nifA is not present in Fa CpI1, although a significantly diverged homologue cannot be ruled out,

(f) Conclusions (1) The nifX-orf3-orfl-nifW-nifZ-nifB-orf2-nifU region of Fa CpIl is about 3 kb away from the nifH-nifD-nifK region. (2) n/fA-hybridizable DNA was detected in the nifZ region but an ORF corresponding to nifA was not found. Therefore Frankia strains have not yet been shown to possess a nifA homologue.

ACKNOWLEDGEMENTS

We thank P.W. Betts for his expert technical assistance, P. Normand for pFQ149 and T. Hoover for pJES294.

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