Plant Molecular Biology 20: 307-309, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium.

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Sequence

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Nucleotide sequence of Rhizobium meliloti GR4 insertion sequence ISRm3 linked to the nodulation competitiveness locus nfe

Maria Jos6 Soto, Adolfo Zorzano 1, Jos60livares and Nicolfis Toro* Departamento de Microbiologla, Estacidn Experimental del Zaidin, Consejo Superior de Investigaciones Cientificas, E-18008 Granada, Spain (* author for correspondence)

Received 26 February 1992; accepted 2 April 1992

Rhizobium meliloti large plasmid pRmeGR4b car- ries the NifA-dependent gene locus nfe which is responsible for nodulation efficiency and compet- itive ability of strain GR4 on alfalfa roots [1, 2, 3, 4, 6]. In this paper we are reporting the nucle- otide sequence of an IS element homologous to ISRm3 located downstream of the nfe locus. In the course of sequencing the former locus we found downstream of the competitiveness genes an insertion sequence homologous to recently re- ported ISRm3 isolated from R. meliloti 102F70 [7]. R. meliloti strain GR4 carries a single copy of insertion sequence ISRm3 located on plasmid pRmeGR4b. The left inverted repeat (IRL) of the insertion sequence is located 128 bp downstream of the translational-sto p codon of the nfe locus (Fig. 1). Nucleotide sequence comparison of GR4 ISRm3 copy with the ISRm3 element from strain 102F70, showed seven conservative nucleotide substitutions: T-C (at nucleotide position 227), G - A (at 240), A - G (at 315), G - A (at 471), T - C (at 546), C ' T (at 630) and T - C (at 906) (this work versus published sequence) (Fig. 1). Only the first substitution (at 227) led to an amino acid change in the N terminus of the putative trans- posase, a valine at position 13 instead of an ala- nine (Fig. 1). Both amino acids are hydrophobic,

and it is likely that this change may well have little effect if any in the protein. Furthermore, flanking the inverted repeats of GR4 ISRm3 it was found two direct repeats of 9 bp (Fig. 1). No obvious terminator sequence is present at the end of the nfe locus. Transposable elements can potentially insert into actively transcribed operons and as such, may be subject to the influence of adjacent DNA sequences. However, transcript entering ISRm3 from the nfe promoters would potentially form a stem-loop structure (Fig. 1), with favor- able AG equal to -35.1 kcal/mol, calculated ac- cording to Tinoco et al. [5]. Whereas the putative ribosome binding site of ISRm3 would form part of the loop structure the putative start codon would be sequestered within the stem preventing translation of the transposase from nfe tran- scripts.

Acknowledgements

M.J.S. was supported by a M.E.C. fellowship. This work was supported by Comisidn Asesora de Investigacidn Cientifica y T6cnica, Grant BI090-0747 and C.E.E. Bridge, Grant BIOT- )159-C.

This manuscript is dedicated to A. Zorzano who died in a car accident on 28 May 1991. The nucleotide sequence reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X63715.

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TAG AGT AAA AGC TCT GCA CAA AGG AAT TTG GCG GAT TGG TTG CCA

CAG CAT GTG ACG CGG CGC CTA ATT GGC AAA TCC CTG ATC GGA CTA

GCT CAA ATT ATC TGA CCA ATA CAG ATA TGC AGG CGA TGC T~G GGA

CTG TCA GGA ATT CTG TGC GGT GGG C(~ TGA TGA TG~AAA GGA GAG

~T'~CAT CAC ATG GCT ATC GAG AAA GAA CTT CTG GAC CAG CTC CTG __~ M A I E K E L L D Q L L GTT GGA CGT GAT CC~ TCC GAG GTT TTC GGC AAG GAC GGT TTG CTG Vl G R D P S E V F G K D G L L • GAC GAT CTG AAG AA~G GCG CTT TCA GAG CGC ATC CTC AAT GCG GAA D D L K K A L S E R I L N A E CTT GAC GAC CAT CTC GAC GTC GAG CGC CTG GAG GGC GGC CCC GCC L D D H L D V E R L E G G P A AAC AGG CGC AAC GGT TCC TCC AAG AAG ACG GTT TTG ACT GGC ACA N R R N G S S K K T V L T G T TCG AAG ATG ACG CTG ACC ATC CCG CGC GAT CGG GCG GGT ACC TTC

S K M T L T loP R D R A G T F GAC CCA AAG CTG ATC GCC AGG TAT CAG CGC CGG TTT CCC GAT TTC D P K L I A R Y Q R R F P D F GAC GAT AAG ATC ATT TCG ATG TAC GCC CGT GGT ATG ACA GTG CGC D D iK I I S N Y A R G M T V R GAG ATT CAG GGG CAT CTT GAlA GAG CTC TAC GGC ATC GAT GTG TCG E I Q G H L E E L Y G I D V S ,i CCG GAT CTG ATC TCG GCG GTG ACC GAT ACG GTT CTG GAG GCC GTC P D L I S A V T D T V L E A V GGA GAG TGG CAA AAC CGG CCG CTC GAG CTT TGC TAC CCC CTC GTG G E W Q N R P L E L C Y P L V TTT TTC GAC GCC ATC CGG GTC AAG ATC AGA GAC GAG GGC TTC GTA F F D A I R V K I R D E G F V CGC AAC AAA GCC GTC TAT GTC GCC CTG GCC GTG CTC GCT GAC (]GC R N K A V Y V A L A V L A D G AGC AAG GAG ATC CTC GGG CTC TGG ATC GAG CAG ACG GAA GGG GCA S K E I L G L W I E Q T E G A AAG TTC TGG CTG CGG GTC ATG AAC GAG CTG AAG AAC CGC GGT TGC K F W L R V M N E L K N R G C CAG GAT ATC CTA ATC GCC GTG GTC GAC GGC TTG AAG GGC TTC CCC

Q D o I L I A V V D G L K G F P GAG GCT ATC ACC GCC GTC TTT CCC CAA ACA ATC GTC CAG ACC TGC E A I T A V F P Q T I V Q T C ATC GTC CAC CTG ATC CGG CAC TCG TTG GAG TTC GTA TCC TAC AAG I V H L I R H S L E F V S Y K GAT AGA AGG ACC GTT GTG CCG GCG TTG AGA GCC ATC TAC CGC GCC D R R T V V P A L R A I Y R A CGA GAT GCC GAG GCG GGC CTG AAG GCG CTG GAG GCC TTC GAG GAA R D A E A G L K A L E A F E E

45

90

135

180

225

270

315

360

405

45O

495

540

585

630

675

720

765

810

855

900

945

990

1035

1080

GGG TAC TGG GGC CAG AAA TAT CCC GCT ATC GCT CAA AGC TGG CGG 1125 G Y W G Q K Y P A I A Q S W R

CGC AAC TGG GAA CAC GTC GTT CCC TTC TTC GCC TTC CCC GAA GGG 1170 R N W E H V V P F F A F P E G GTC CGC CGC ATC ATC TAC ACG ACG AAC GCA ATA GAG GCC CTC AAC 1215 V R R I I Y T T N A I E A L N TCG AAG CTT CGG CGA GCT GTG CGT TCC CGC GGG CAT TTC CCT GGT 1260 S K L R R A V R S R G H F P G GAC GAA GCC GCG ATG AAG CTG TTA TAT CTC GTT CTT AAC AAC GCG 1305 D E A A M K L L Y L V L N N A GCC GAG CAA TGG AAA CGG GCG CCG CGG GAA TGG GTC GAG GCA AAG 1350 A E Q W K R A P R E W V E A K ACA CAG TTC GCC GTC ATC TTT GGC GAG CGG TTC TTC AAC TGA TGA 1395 T Q F A V I F G E R F F N * * AAC C~G CCC ACC GCA CAG AAT TCC TGA CAG TCC C~C GAT GCT CCG 1440

CCG ATC ATG 1449

Fig. 1. Nucleotide sequence of ISRm3 from R. meliloti GR4 and amino acid sequence of the putative transposase encoded. Mismatches with ISRm3 sequence from R. meliloti 102F70 are indicated by an asterisk (*); asterisks below the sequence indicate translational stop codons; inverted repeats are boxed; direct repeats are underlined with a sofid bar, putative ribosome binding site is indicated by a solid bar above the sequence; the arrows indicate putative stem-loop structure; the translational stop codon of the nfe locus is shown at the beginning of the sequence.

References

1. Casadesfis J, Olivares J: Rough and fine linkage mapping of the Rhizobium meliloti chromosome. Mol Gen Genet 174:203-209 (1979).

2. Sanjuan J, Olivares J: Implication of nifA in regulation of genes located on a Rhizobium meliloti cryptic plasmid that affect nodulation efficiency. J Bact 171:4154-4161 (1987).

3. Sanjuan J, Olivares J: Multicopy plasmids carrying the Klebsiella pneumoniae nifA gene enhance Rhizobium meliloti nodulation competitiveness on alfalfa. Mol Plant-Microbe Interact 4:365-369 (1991).

4. Sanjuan J, Olivares J: Nifa-NtrA regulatory system acti- vates transcription of nfe, a gene locus involved in nodu-

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lation competitiveness of Rhizobium meliloti. Arch Micro- biol 155:543-548 (1991).

5. Tinoco Jr. I, Borer NP, Dengler B, Levine MD, Uhlenbeck OC, Crothers DM, Gralla J: Improved estimation of sec- ondary structure in ribonucleic acids. Nature 246:40-41 (1973).

6. Toro N, Olivares J: Characterization of a large plasmid of Rhizobium meliloti involved in enhancing nodulation. Mol Gen Genet 202:331-335 (1986).

7. Wheatcroft R, Laberge S: Identification and nucleotide sequence of Rhizobium meliloti insertion sequence ISRm3: similarity between the putative transposase encoded by ISRm3 and those encoded by Staphylococcus aureus IS256 and ThiobaciIlusferrooxidans IST2. J Bact 173:2530-2538 (1991).