nifV is contiguous to nifHDK in Frankia strain FaCl*

PHYSIOLOGIA PLANTARUM 99: 707-7 13, 1997

Copyrighl © Physiologic Planmrum 1997

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ISSN 0031-9317

nifV is contiguous to nifHDK in Frankia strain FaCl Bo SI Oh, Paul G. Twigg, Jeong Soo Hong, Beth C. Mullin and Chung Sun An

Oh, B. S., Twigg, P. G., Hong, J. S., Mullin, B. C. and An, C. S. 1997. nifV is contiguous to nifHDK in Frankia strain FaC 1. - Physiol. Plant. 99: 707-713. The organization of genes with the capacity to code for four proteins involved in nitrogen fixation in Frankia strain EaC 1 was determined by restriction fragment mapping and nucleotide sequence analysis. Analysis of the 44-kb genomic cosmid clone pEAHl. isolated from a cosmid library made from Frankia strain EaCl, resulted in the identification of a 7.2-kb Pstl fragment to which Klebsiella nifH. nifD and nifK probes hybridized. This H/J-hybtidizing fragment was subcloned and analyzed by restriction fragment mapping. Eurther subcloning of the 7.2-kb fragment and subsequent sequence analysis of approximately 6.8 kb revealed the presence of six open reading frames (ORFs). Eour of these OREs have the potential to code for nifW-, nifH-, nifDand n//K-like gene products and the two others are unidentified OREs. The organization oif the structural genes for nitrogenase is the same in this Frankia strain as it is in most other nitrogen-fixing prokaryotes, but the positioning of the «!/V-like gene relative to the nifHDK cluster differs. A consensus «//-promoter-like sequence, found 5' to nifH, was not detected upstream of the ni/V-like gene. Nine copies of a 7-bp direct repeat were found 5' to ORFA. Key words - Directly repeated sequence, Frankia strain FaCl, homocitrate synthase, nifD, nifH, nifK, nifV, n/J-organization. B, S, Oh (present adress: Dept of Botany, Vniv, of Wisconsin, Madison, WI 53701, USA), J, S, Hong and C. S, An (corresponding author, e-mail ancs@ powerl ,snu,ac,kr), Dept of Biology, Seoul National Univ., Seoul 151-742, Korea; P, G. Twigg, Dept of Biology, Univ, of Nebraska at Kearney, Kearney, NE 68849, USA; B, C. Mutlin, Dept of Botany and the Center for Legume Research, The Univ, of Tennessee, Kno.xville, TN 37996, USA, This paper is part of the contributions to the Tenth International Conference on Frankia and Actinorhizal Plants jointly sponsored by the College of Agricultural and Environmental Sciences at the University of California at Davis, and the National Science Eoundation, held in Davis, CA, USA, 6-11 August, 1995.

Nitrogen-fixing soil bacteria in the genus Frankia are unique among the actinomycetes in being able to participate in an intimate symbiotic association with a number of woody angiosperms. This symbiotic association resuits in the formation of nitrogen-fixing root nodules which contribute to the ability of host plants to survive on nitrogen-poor soils. The position of Frankia among the actinomycetes has been established on the basis of both morphological analysis and 16S rRNA sequence compar-

isons (Hahn et al. 1989). Actitiomycetes closely related to Frankia are tiot knowti to fix nitrogen and thus the origin of the nitrogen-fixing capability in this genus has been the subject of much speculation. The nucleotide sequence of nifH, the structural gene for the Fe protein subunit of the enzyme nitrogenase, has been determined for several Frankia strains and has been included in a gene tree of «//H sequences (Normand and Bousquet 1989). The sequence most similar to n//H from Frankia appears to be nijW from the cyanobacterium Anabaena, The extent of sequence similarity between n//H from Frankia

Received 7 November, 1995; revised 5 July, 1996 Physiol. Plant, 99, 1997

707

and Anabaena is unexpected and is not strictly congruent with the overall phylogenetic relationship of these bacteria. Frankia is a Gram positive bacterium with distant but distinct phylogenetic affinity to the Gram positive nitrogen fixing genus Clostridium, Anabaena is a Gram negative bacterium in section IV of the cyanobacteria. The lack of congruence between phylogenetic trees constructed from nijW and those constructed from 16S rRNA has led to speculation that either Frankia or Anabaena acquired the ability to fix nitrogen as the result of horizontal gene transfer (Normand and Bousquet 1989). Other explanations for lack of strict congruence between the trees have been presented by Young (1992). In addition to nj/H, nifD and «(/K, the structural genes for nitrogenase, biological nitrogen fixation requires a host of additional genes. To date more than 20 additional nif or /ij/'-associated genes have been identified in other diazotrophic bacteria and in all cases studied there is a clustering of m/genes on bacterial chromosomes or plasmids (Merrick 1993). The arrangement of genes within the clusters varies from genus to genus but in almost all cases, nifi\, nifD and nifK are contiguous. Previously it had been reported that in Frankia strain FaCl, nifH was not adjacent to nifD and nifK. (Ligon and Nakas 1987) and arrangement of nif genes in this Frankia strain has been cited as an exception to the predominant nif gent order. Using a series of Azotobacter nif c\ones as hybridization probes we have mapped these genes on the pFAHl Frankia cosmid clone. This information along with nucleotide sequence data may provide insight into the evolution of nif genes in the actinomycetes and in particular in Frankia. In the present paper we report the nucleotide sequence of 6 120 bp of a 7.2-kb fragment of DNA containing putative «//V, nifH, nifD genes, part of nifK, and two unidentified open reading frames (ORFs), and we demonstrate the unique organization of this «;/gene cluster. These putative genes are referred to below as nif\, nifH, nifD and nifK and ORFA and ORFB on the basis of nucleotide sequence analysis.

tations into M13mpl8 (Gibco BRL, Gaithersburg, MD, USA) and deletion clones were generated from the replicative form using the 3' to 5' processive exonuclease activity of T4 DNA polymerase of the Cyclone I deletion kit (IBI, New Haven, CT, USA). A 3.4-kb BamHVHindlll fragment, a 1.5-kb HindllVBamHl and a 0.5-kb BamHVPstl fragment from pFAH-P7.2 were cloned separately into pUC19 (Gibco BRL) and named pANO-H, pANO-1.5 and pANO-0.5, respectively. In addition the 3.4-kb BamHUHindlll fragment was cloned in the opposite orientation into pBluescript KS(+) (Stratagene, LaJoUa, CA, USA) resulting in clone pANO-HR. Unidirectional deletions were made using exonuclease III and SI nuclease from an Erase-a-Base kit (Promega Corp., Madison, WI, USA) following the method of Henikoff (1984), and a series of overlapping clones selected for sequence analysis. Restriction enzymes (Promega or Gibco BRL), [a-^-P]-dCTP and ['''S]-dATP (Amersham International pic, UK; or ICN Biochemicals, Inc., Irvine, CA, USA), random primer DNA labelling kit (Boehringer Mannheim, Indianapolis, IN, USA) and Sequenase kits (United States Biochemical, Cleveland, OH, USA) were used according to the manufacturer's instructions. Plasmid and phage preparation Plasmids from F. coli cultures grown overnight in Luria-Bertani medium (Maniatis et al. 1982) plus appropriate antibiotics were isolated following the method of Brush et al. (1985). The methods described by Maniatis et al. (1982) were used for subcloning restriction fragments into pUC]9 and pBluescript KS(H-), for isolating the recombinant clones and for autoradiography. M13mpl8 was grown according to Gibco BRL protocols and phage sequencing template was prepared from phage supernatants according to protocols supplied by United States Biochemicals.

Abbreviations - LB, Luria-Bertani medium; nt, nucleotide, Nucieotide sequence analysis ORE, open reading frame. Plasmid DNAs from each deletion series were subjected to single- or double-stranded DNA sequencing by the dideoxy chain termination method (Sanger et al. 1977) Materials and methods using ["S]-dATP and Sequenase Version 2 (United Materials States Biochetnicals). Nucleotide sequences were read The isolation of Frankia strain FaCl, an Alnus viridis ssp. manually from autoradiographs of sequencing gels made crispa isolate, and the construction and screening of the and run according to protocols supplied with the SequeFaCl cosmid library has previously been described by Li- nase kit. Sequences were proofread and discrepancies gon and Nakas (1987). Cosmid clone pFAHl was isolated that existed between strands of a single DNA molecule from the library by virtue of its ability to hybridize to a were resolved by additional sequencing when necessary. Klebsiella nifH probe. A 7.2-kb Pstl n/f-hybridizing frag- Edited sequences were entered manually into Microgement was cloned into pBR322 (pFAH-P7.2) and was nie (Beckman) or the SEQED program ofthe University mapped by single and double restriction enzyme diges- of Wisconsin Genetics Computer Group (UWGCG), Setions, and the location of nifH, nifD and nifK were deter- quence Analysis Software Package (1991 version 7, Gemined by hybridization with gene-specific probes. The netics Computer Group, Madison, WI, USA) and ana7.2-kb fragment was subcloned for sequence analysis as lyzed with FASTA, TFASTA, GAP or BLAST programs follows. A 2.7-kb Sail fragment was cloned in both orien- (Pearson and Lipman 1988, Altschul et al. 1990). 708

Physiol, Plant, 99. 1997

pFAH-7.2

PS B II I P

H L

S P I I

B

pANO-0.5

pANO-1.5

B

H

1

1

pANO-H

pFAH-2.7

B

H 1

1 S

S

I

1

Scale

Nucleotide sequence analysis of these ORFs did not reveal significant sequence similarities to 20 nif genes known in K, pneumoniae, or to any other sequences from the GenBank and EMBL data banks using the FASTA and BLAST search programs. It is premature to conclude that these ORFs are not nif genes since many ORFs thought to be involved in nitrogen fixation are found among nif gene clusters in other diazotropic bacteria, but are not found in Klebsiella (Joerger and Bishop 1988, Jacobson et al. 1989a, Moreno-Vivian et al. 1989). However, it is also possible that these ORFs are not involved in nitrogen fixation. In A. vinelandii the nifABQ cluster is separated from other nif clusters by genes not known to be involved in nitrogen fixation (Beynon et al. 1987).

1 kb

Fig. 1. Restriction map of pFAH-P7.2 showing the restriction fragments which were cloned resulting in the subclones pANO-0.5, pANO-1.5, pANO-H and pEAH-2.7. P, Pstl; S, Sail, B, BamHl; H, Hindlll,

Nucleotide sequence of pANO-H(R) Within nucleotide sequence of pANO-H(R), from Hindlll site at position 1 904 to BamHl site at position 5 138, are two long ORFs and the beginning of a third (Fig. 2). The first ORF, which starts at position 2066 and Results and discussion extends to position 3 268, shares approximately 60% seRestriction map of pFAH-P7.2 quence identity with nifV genes from K. pneumoniae A restriction map of pFAH-P7.2 was deduced from sin- (Arnold et al. 1988), Clostridium pasteurianum (Wang gle and double restriction enzyme digests (Fig. 1). This et al. 1991) and Azotobacter vinelandii (Beynon et al. map was used to identify restriction fragments for subse- 1987). This sequence is the first 7i//V-like sequence requent subcloning and sequence analysis. The location of ported for Frankia. The 60% sequence identity exhibited restriction sites was confirmed from sequence data using with the Azotobacter and Klebsiella is higher than the the MAP fuction of UWGCG. 56% sequence identity found between nifV sequences from K, pneumoniae and A. vinelandii. The fact that «//V from Frankia is 49 bp longer than that of A, vineNucleotide sequence of pANO-0.5 and pANO-1.5 landii suggests the occurrence of insertion or deletion Nucleotide sequence of pANO-0.5 starts from Pstl site events. This possibihty is further supported by the 41% at position 6 and extands to BamHl site at position 415, derived amino acid sequence identity of Frankia nifW while that of pANO-1.5 starts from BamHl site at posi- with that of A. vinelandii, which is lower than the 44% tion 416 and extands to Hindlll site at position 1903 sequence identity between nifW& from A. vinelandii and (Fig. 2). There are two ORFs in pANO-1.5 and both are K. pneumoniae. Three prime to nifW and in the same oriin the same orientation as nifV and nifH, The first ORF entation is an 864-bp ORF that had earlier been shown (ORFA) extends between positions 539 and 1 586, while to have the capacity to code for nifH (Oh et al. 1993). the second ORF (ORFB) extends from position 1 588 to nifH is separated from nifV by 217 bp. Three prime to position 1812. Upstream of ORFA in pANO-0.5 is a se- nifH is a fifth ORF with sequence similarity to published quence which consists of nine direct repeats of 7 bp nifD sequences. nifH and nifD are separated by a 77-bp (GAGCGAG). In Anabaena PCC 7120 strain, similar intergenic sequence as was earlier reported (An et al. sequences consisting of seven and six direct repeats of 7 1990). Analysis of 5' sequences upstream of the nifW bp were found between nifB and fdxN and between nifS ORF revealed a Shine-Dalgamo-like sequence (GGAG) and nifU, respectively (Mulhgan and Haselkorn 1980). from -13 nt to -10 nt, but a njj-promoter-like sequence These repeated sequences are thought to modulate tran- (CTGGTCACGCCCGGTGA), found 5' upstream of scription of downstream genes and are thought not to be nifH (Oh et al. 1993), was not found. The presence ofthe specific to nif genes. The possibility of their involve- nij-promoter-like sequence 5' to nifH suggests that nifH and nifW are not part of the same operon in Frankia. ment in the expression of ORFA still exists. A Shine-Dalgamo-like sequence (Shine and Dalgamo 1974) was found at the second to last codon of ORFA and the start codon of ORFB was found within the stop Amino acid sequence of NifV (homocitrate synthase) codon of ORFA. Sequence overlaps such as this have The nify gene in pANO-H has the capacity to encode a been found in nif ORFs in K. pneumoniae (Arnold et al. total of 402 amino acids. The derived amino acid se1988) and are thought to be a mechanism for more effi- quence of this gene is shown in Fig. 3 along with ni/V cient genome usage. gene products from K, pneumoniae and A. vinelandii. In Physiol, Plant, 99, 1997

709

1 Pstl CTOCAOGTCGATGTTCGGCARGCATCGGCGGACCCGAGATCTCCAGGCSCGGCCGCGCGCT GACGATGAAGAGGCACGTaTCOACCGCCCACGGGGTGGGTCTGGTGACCCCGGTCCTGTC CGCCAGCGCTGTCACGGCGGCCGGTGCGGCTGGCGGTGCCGGTCGGTTCCAGGTCGCCTA CCTGGTGGCGGCGGCCTGCTCGGCAGCTGCGCGGTCGCCGCGATCGAGCTGCCCGCACGA TGCGGACCCGAACGCCACCGTCCCGACGCCGGGCAGCTCAAGGGCGTC;.CCTAAACGCCC GCCAGGCCTGAGACGCAGGCTCGGCACCGCTTGGCCGCCAACACTIGAGCGACySAGCGAG^ GAGCGAGGAGCGAGGAGCGA(jGAGCGAyGAGCGA'^GAGCGA^GAGCGA«:ACGCGGATCC GCGGACGAGGGCGGACCCGCGTGGCCGACGGAGCCGCTGTCACTCGTGCACGCTTCCCGC

4 81 541 601 5 61 721 781 841 901 961 1021 10 B1 1141 1201 1261 13 21 13 81 14 41 1501

1561 1621 1681 1741 1801 1861

TTGTGGGAAGTCGTCTTGACAGACGGTTGGCGGGGAACGGCGGGCCGCiSaaaTGATCAT E V S L R P G F R P G S A H G P R R S R aaAOGTGTCACTTCGTCCCGGGTTCCGCCCGGGTTGCGCCCGCGGGCCACGGCGGAGTCG G G R E Q G E D R G G S A V S S E H D R CGOAGGCCGGGAACAAGGGGAAGACCGCGGGGGCAGTGCGGTCTCGAGTGAGCACGACCG P P A R D R T G S R R A F T E G G A G G GCGTCCCGCCCGCGATCGGACGGGAAGCCGGCGCGCGTTGACGCGCGGTGGGGCGGGTGG A V G P F R R G A E V A G R G R P P G G GGCGGTCGGCCCCTTCCGGCGAGGGGCGAGTGTCGCCGOGGGGGGCCGACCACCCGGCGa G O G R S R A O S R Q E S L R M D R S S CGOTCAGGGCAGGTCGCGGGGGCAGTCCCGGCAGCGGTCATTGAGGATGGACAGGAGCTC T A L R E A S R T L A M S C D E F R R I GACGGCCTTGAGAGAGGCGTCGCGGACCTTGGCGATGTCCTGCGACGAGTTCAGGCGGAT R A D S G R P R V A D R L D D L G G R R CAGGGCCGACTCGGGCCGTCCGAGGGTCGCGGACCGCCTCGACGATCTCGGCGGCCGGCG A V L E E O D L L V L V G G Q F H R Q V TGCCGTACTGGAAGAGCAGGATCTCCTTGTCCTGGTAGGCGGGCAGTTCCATCGGCAGGT P Q A D T G E V A A E D L P Y V R A E P GCCCCAGGCCGATACCGGCGAGGTTGCGGGTGAGGATCTGCCGTACGTTCGTGCCGAGCC A V A T D L P Q Q A D G G P E D . S V V P GGCCGTAGCCACCGATCTCCCGCAGCAAGCCGATGGTGGTCCTGAAGACTCCGTTGTTCC H H C R P Q V L P V A R G I G W H T D H CCACCACTGCCGCCCGCAG M H A H H D L G M A T A N T L A A V S[A G A T S I H A H D D F G L A T A N T I A A V Q A GfFlT Av Kp Fa

T T V N G L G E R A G N - A A |E E|C V LIA L K N T T V L G L G E R A G N A A A K P SFA LML E R C T T V L G L G E R A G N - A P I [ F E ] V | A MJA L R H L

Av I D T R G I Kp V H F S A L

E R A S G R Q V A W Q K S [ V V G[A G

Fa

T T S F R fT[Aj^- -[I

V G W P L P A G K K A - -fv V G E S '

Av Kp Fa

H E A G 1H V D G | lL[K1HJR R N | Y E J G L N P D EL HE S H V A A[L L R W S E S|Y[Q"S 1 A P S L M 1 I F D[PJE J VG H E S GT H V H G

A E[AJA Q[RJA I D P Q Q P ^ V G E]L

R|N T Y R D L G | E [ I 1 A D W [ Q ] S Q AfTTlG R V F D Q M G [ R 1 J A A[QJI N D J L J P RlH A L E Q C G T A E E S E L

p AIE LIQ D FIYIR QIL CIE Q ] [ J A 1[YJD E [ L _ C J - S J R - [ D J L P G T S R A G R D A - -[G^ Av G "GIM A Kp R Fa - £ j T P T R E E P V

Fig. 3. Comparison of the amino acid sequences encoded by nifV from Frankia strain FaCl and from A, vinelandii and K, pneumoniae. Conserved amino acid residues are boxed.

Codon usage

Skewed codon usage in favor of G or C in the third position of the codon found in organisms with high GC contents (Bibb et al. 1984) was also found in nifH (Oh et al. 1993) and nifD, but less pronounced in «//V, ORFA and 6061 ORFB (Tab. 1). However, codons not used in nifH and D Fig. 2 continued. such as ACA and CGA were used in these ORFs (data not shown). Considering that nifH and D are structural genes of the nitrogenase complex, this can be interpreted as a 16S ribosomal sequence analysis. A potential mechanism to increase translational activity of nifH and Shine-Dalgamo region (GATGAGGTCCCGATC) D, while decreasing the translation of messenger RNA which is found 5' to the first ATG of the ORF in nifD such as that from nifV which is not directly involved in from FaCl, is identical to that reported for nifD from the formation of nitrogenase polypeptides. In F. coli, Frankia strain HRN18a (Normand and Bousquet 1989) strong codon bias was also found to be associated with and differs by one nucleotide from that reported for nifD genes expressed at a high level (Medigue et al. 1993). from ArI3 (Normand et al. 1992). 5881 5 941 6 001

Physiol, Plant, 99. 1997

711

Tab. 1. Percent GC characteristics of Frankia FaCl nifl\. nifD, nifV, ORFA and(DRFB. * From Oh et al. (1993).

G+C% in the first position of codon in the second position of codon in the third position of codon

nifH*

nifD

nifW

ORFA

ORFB

61.3 38.0 95.5

59.8 42.8 96.6

73.9 51.8 87.8

7L1 60.3 70.6

71.1 61.8 75.0

H D KTY E N X USVWZMF L A B Q

cum. A striking feature of the gene order in FaCl is that nifW is contiguous with nijH in this Frankia strain. If gene order were preserved, ORFA and ORFB might be expected to be similar to nif\J and n//S and together be located 3' to the «(/HDK cluster. No meaningful sequence similarity has been found between ORFA, ORFB and any reported sequences. In Klebsiella, Azotobacter, Enterobacter and Rhodobacter, nifV is clustered with niJUS, while it is not in C. pasteurianum and Frankia. This suggests that clustering of nifV and nifUS is not a functional prerequisite for transcription or translation of nifV. This assumption was supported by the report that a deletion-and-frame shift mutation in nifS did not affect the expression of nifV in A. vinelandii (Jacobson et al. 1989b). In Frankia, nifV has the capacity to code for the entire NifV protein whereas in C. pasteurianum NifV is encoded by two separate genes (Wang et al. 1991).

H P KTY E N X

The nucleotide sequence reported in this paper has been deposited in the GenBank data base (accession No. U53363).

nif gent organization in Frankia The orgatiization of nif genes known so far in Frankia strain FaCl is shown in Fig. 4 along with gene clusters of six other nitrogen-fixing organisms (Dean and Jacobson 1992, Merrick 1993). It had originally been reported that nifH and nifD were not contiguous in Frankia strain FaCl (Ligon and Nakas 1987), a situation very different from that reported for other Frankia strains. It has since been reported that the clone used to reach this conclusion did not contain legitimate Frankia sequences (Ligon and Nakas 1990). As seen in Fig. 4, nifRD and K are contiguous in FaCl as in all cases except in B. japoni-

J

ZKEriiEriiiinEDa] USV

WZM F

A.V. 12

12

14

most

7 >

L A B^ Q

H D K TV E N X USVWZM L A B Q F J

E.a. R.C.

mx

ENX

Q

UKSVWAlBt

Z

TEDHUD

J rdxB 8 6

Acknowledgments - This work was supported by Genetic Engineering Research Grant (1993) from the Korean Ministry of Education and by a Biotechnology Career Fellowship from Rockefeller Foundation to C. S. An. We thank J. M. Ligon for providing the FaCl cosmid clone pFAHl and the subclone pFAH-P7.2. BCM acknowledges the support of the USDA Grant 93-37305-9082. The Chancellor of the University of Tennessee and the University Computing Center are acknowledged for providing central and/or distributed computing facilities and services.

2

H P K 12 R4 A2 B2

References D K E NX

B.j.

US B

H fiiBCX fixRA fixA

nmi-iin-n-n-m] o o 6

H2H1P K E N-B

laocnio

trxA

\4. V«

V HP K

F.SP

-nnrm—

Fig. 4. Comparison of the physical organization of nif. fix and 7!//'-associated genes from Kleb.siella pneumoniae (K.p); Az.otohacter vineiandii (A.v); Enterobacter aggiomerans (E.g.); Rhodobacter capsuialus (R.c); Bradyrhizobium japonicum (B.j.); Clostridium pasteurianum (C.p.); Frankia strain FaCl (F.sp). ORFs from different organisms having the same numerical designation have a high degree of primary sequence identity. U1 indicates an incomplete «//U gene sequence when compared to the corresponding K. pneumoniae or A. vineiandii sequence. Adapted from Merrick (1993) and Dean and Jacobson (1992).

712

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