CURRENT MICROBIOLOGY Vol. 40 (2000), pp. 264–268 DOI: 10.1007/s002849910052 An International Journal

R Springer-Verlag New York Inc. 2000

Molecular Characterization of the Leucine Plasmid from Buchnera aphidicola, Primary Endosymbiont of the Aphid Acyrthosiphon pisum Teresa Soler, Amparo Latorre, Beatriz Sabater, Francisco J. Silva Institut Cavanilles de Biodiversitat i Biologia Evolutiva and Departament de Gene`tica, Universitat de Vale`ncia, Apartat 22085, 46071 Vale`ncia, Spain Received: 5 November 1999 / Accepted: 8 November 1999

Abstract. The complete sequence of the leucine plasmid of Buchnera aphidicola from the aphid Acyrthosiphon pisum (pLeu-BAp) is reported. Its gene organization was concordant with those of other leucine plasmids of Buchnera from aphids of the Aphidini and Macrosiphini tribes. Three inverted repeats are present in pLeu-BAp. Two of them are also present in pLeu from the family Aphididae: (i) SIR1, located downstream the leucine operon, resembles a rho-independent terminator of transcription, and (ii) LIR1, located upstream of the leucine operon, is suggested to be involved in transcription termination or messenger stability. The third, located near the putative ATGC repeats involved in the origin of replication, is specific in aphids of the Macrosiphini tribe. Phylogenetic analyses based on sequences of leuA, leuB, leuC, leuD, repA1 and ORF1 showed a closer relationship between Buchnera (A. pisum) and Buchnera (Diuraphis noxia). However, tree topologies indicate that the split between both aphid species took place soon after the formation of the Macrosiphini lineage.

Aphids are plant sap-feeding insects that maintain an endosymbiotic association with the bacterium Buchnera aphidicola, a member of the ␥3 group of Proteobacteria, closely related to the family Enterobacteriaceae [4, 13]. The association is mutualistic: neither the endosymbiont nor the host can survive in the absence of the other, and until now any attempt to cultivate Buchnera outside the host has failed. From the point of view of the aphid, the major role of the endosymbiont is the provision of amino acids that are deficient in the phloem sap diet [6, 11, 16]. In the last years, evidence for overproduction of the essential amino acids tryptophan and leucine by means of gene amplification of trpEG and leuABCD genes into plasmids has been obtained [2, 3, 5, 9, 14, 17, 19, 20]. The evolutionary history of the leucine and tryptophan plasmids is controversial, as Buchnera from some families of aphids do not carry these plasmids, and not all plasmids have the same gene content and/or gene order. In the case of leucine plasmids, only one single replicon, named repA1, has been found, but the gene content and/or gene order is different in the lineages of the superfamily Aphidoidea, indicating a great plasticity Correspondence to: F.J. Silva; email: [email protected]

of Leu plasmids (p-Leu) along Buchnera evolution. The first leucine plasmid was described in Buchnera from Rhopalosiphum padi (pLeu-BRp) [5], a member of the Aphididae. It contains the genes leuABCD, two copies of repA, and ORF1, encoding a putative integral membrane protein. The same genes in the same order were found in pLeu-BSg, from Schizaphis graminum, a species that belongs to the same tribe of the Aphididae as R. padi (Aphidini), and in pLeu-BDn from Diuraphis noxia, which belongs to the Macrosiphini tribe [3]. However, a different gene arrangement has been found in pLeu-BPp, the plasmid of Pterocomma populeum, a species that belongs to the Pterocommatini, the most divergent tribe of the Aphididae [17]. The plasmid of Buchnera of Thelaxes suberi (pLeu-BTs), a member of a different family (Thelaxidae), differed from the previous one by the presence of a small heat shock gene (ibp) and in the order of the leuABCD and repA genes [19]. Finally, the equivalent plasmid of Buchnera from the pemphigid aphid Tetraneura caerulescens contains only one copy of the repA gene plus ORF1. The leucine operon is located in the bacterial chromosome, and this plasmid probably represents the ancestral replicon, related to the IncFII plasmids to which the other genes were added [19]. In

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addition to these completely characterized plasmids, partial sequences of seven Leu plasmid from species of the Aphididae, including Acyrthosiphon pisum, have been reported [17]. In the present paper we present the complete sequence of the Leu plasmid of Buchnera from the pea aphid A. pisum (pLeu-BAp) in order to compare it with those previously characterized and to get a deeper understanding of the regulatory signals involved in transcription or replication. Since A. pisum is a member of the Macrosiphini tribe, the comparison with pLeuBDn, belonging to the same tribe but phylogenetically not closely related, could indicate special features that predate the diversification of this lineage.

Alignments of the deduced amino acid sequences from leuA, leuB, leuC, leuD, repA1 genes and ORF1 were used to obtain the corresponding nucleotide sequence alignments, which served for the estimation of the nucleotide distances by the method of Tajima and Nei [18]. Phylogenetic trees were obtained by the neighbor-joining method [15]. The significance of the nodes was determined by 500 bootstrap replicates. All these analyses were performed with the program MEGA [8]. The complete nucleotide sequence of the leucine plasmid from Buchnera aphidicola (A. pisum) has been deposited under accession number AJ006878. Other sequences used in the analyses come from Buchnera plasmids of R. padi (accession number X71612) [5]; T. suberi (Y11966), and T. caerulescens (Y11974) [19]; P. populeum (AJ006877) [17]; and S. graminum (AF041836) and D. noxia (AF041837) [3].

Materials and Methods

General characteristics of pLeu-BAp.The size of the plasmid is 7805 bp, and the G ⫹ C content is 26.7%. These figures are similar to the previously characterized plasmids pLeu-BRp (7829 bp, 26.3%), pLeu-BDn (7768 bp, 25.9%), and pLeu-BSg (7967 bp, 26.8) from the Aphididae. Seven open reading frames are found in pLeu-BAp. They are in the same order as in pLeu-BRp, pLeu-BDn, and pLeu-BSg (Fig. 1). The comparison of the sequences of the proteins encoded by pLeu-BAp with the corresponding of pLeu-BRp, pLeu-BDn, pLeu-BSg, and pLeu-BPp shows that, in general, the highest percentage of amino acid identity is found with the proteins of pLeu-BDn, with the exception of LeuD, which is more similar to the corresponding R. padi and S. graminum than to D. noxia (69%, 68%, and 65%, respectively). These values also corroborate that A. pisum and D. noxia, although belonging to the same tribe, are not closely related, as was obtained in the phylogenetic analysis using repA2 genes [17].

Laboratory methods. A colony of A. pisum (Harris) was obtained from Y. Rahbe´. Buchnera aphidicola plasmid DNA was isolated as previously described [10]. This DNA was used for the amplification of two large segments of the leucine plasmid by PCR (Fig. 1), which, in combination with the fragment already sequenced [17], covers the entire sequence of the plasmid. The region between leuD gene and ORF1 (around 2.0 kb) was amplified with primers leuD.up2 (58CGGATCCTGCAGGGWTGTGGWTCWTCWAGAGARCATTGC38) and ORFd3R (58-CATTTTATCRAYRTAWGCCATNCC-38). Once sequenced, a primer specific for leuD gene was designed (ApleuD-R2, 58-CTAGGTGCAATTATTACTTTGAATCC-38) and used in combination with a primer designed on the small region of leuA gene previously reported by [17] (ApleuA-F2, 58-TTGTTATTTTTGATACCACGMTACG-38). PCR was performed with a GeneAmp PCR System 2400 thermal cycler (Perkin Elmer) using the Expand Long PCR kit (Boehringer Mannheim). PCR products were cloned in a T vector [12] from EcoRV-digested pBluescript II SK (⫾) and pGEM-T easy vector. Subcloning was performed by digestion with Sau3A and cloning in a compatible BamHI site in pBluescript II SK (⫾). Nucleotide sequencing was performed with the AmpliTaqF Dye Deoxy Terminator Cycle Sequencing kit (Perkin Elmer) with either T7 and T3 primers or internal primers and carried out with an ABI 373 automated DNA sequencer. Computer and phylogenetic analyses. Computer analyses were performed with the Wisconsin Package Version 10, Genetics Computer Group (GCG) Madison, WI. Amino acid and nucleotide sequence alignments were obtained with the CLUSTAL W program [7]. Blast searches [1] were done at the network servers of the NCBI and EMBL.

Fig. 1. Linearized genetic map of the leucine plasmid of Buchnera from A. pisum. Arrows show direction of transcription. LIR1 is a long inverted repeat. SIR1 and SIR2 are short inverted repeats. Ori is the putative origin of replication. Numbers indicate primer position: (1) ApleuA-F2; (2) ApleuD-R2; (3) leuD.up2; and (4) ORFd3R.

Results and Discussion

Analysis of intergenic regions. DNA sequences between ORF1, repA2, and leuA were studied in a previous paper [17], and hence we analyzed the remaining regions. The short inverted repeat SIR1 found downstream of leuD in pLeu-BRp [5], pLeu-BSg, and pLeu-BDn (named IR1, [3]) is also found in pLeu-BAp (Fig. 2). Although the same repeat is also found downstream of leuD in pLeu-BPp [17], it is not shown in Fig. 2 owing to the different gene order of this plasmid. This repeat resembles a rho-independent terminator of transcription and seems to be specific for the Aphididae, as it is not found in the leucine plasmid of Buchnera from T. suberi [19]. A conserved segment containing three repeats of the sequence ‘‘ATGC’’ identified in three Buchnera pLeu plasmids was proposed to be the best candidate to accommodate the origin of replication [19]. These repeats have been recently found in three additional pLeu plasmids [3, 17]. They are placed downstream of repA1,

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Fig. 2. Intergenic region where the putative origin of replication is located. (A) Asterisks indicate the position of the three conserved ATGC repeats. Large arrows show direction of transcription. Small arrows show inverted repeats. LIR2 is a long inverted repeat that has been detected only in P. populeum. SIR1 and SIR2 are short inverted repeats. Ori is the putative origin of replication. (B) Nucleotide sequences of the ori region. SIR2 from A. pisum and D. noxia is shown with arrows. ATGC repeats are boxed. The palindromic ATGCAT repeat is also boxed.

independently of the gene order of the different plasmids (Fig. 2). The same motifs are also present in pLeu-BAp. In addition, the five characterized members of the Aphididae present a first repeat that is a palindromic sequence where the ATGC repeat appears in both strands. This sequence is absent in plasmids from Buchnera of other aphid families. The conservation of the ATGC repeats is complete, except in the middle repeat of pLeu-BPp, which presents a single nucleotide substitution. An interesting finding is the existence of an additional inverted repeat (SIR2) between repA1 and the putative origin of

replication in pLeu-BAp and pLeu-BDn but absent in the rest of the plasmids (see Fig. 2). Whether this repeat functions as a terminator of transcription of repA1 or has some role in the replication process is unknown, but the fact that it is present in two divergent species of the Macrosiphini tribe but absent in the related species from the Aphidini tribe seems to indicate that it appeared after the split of these lineages. Differences between the two tribes have been found previously, a putative promoter upstream of repA2 being present only in the leucine plasmids of Buchnera from the Aphidini tribe [17].

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Fig. 3. Neighbor-joining trees for the complete nucleotide sequence of leuA, leuB, leuC, leuD, repA1 genes and ORF1. The length of the small line under each tree indicates the number of nucleotide substitutions per site.

Phylogenetic reconstruction. Figure 3 shows the trees inferred by the neighbor-joining method with DNA sequences of leuA, leuB, leuC, leuD, repA1, and ORF1 from leucine plasmids of Buchnera from the Aphididae and with the plasmids from T. suberi and T. caerulescens as outgroups. The repA2 tree has been previously reported by [17] and, for that reason, has not been included in this study. As can be seen in Fig. 3, the cluster formed by genes belonging to Buchnera from R. padi and S. graminum was clearly supported, as previously shown [3, 17]. However, the relationship between the two members of the Macrosiphini tribe (A. pisum and D. noxia) was supported only by high bootstrap values in the case of ORF1, repA1 and repA2. In fact, in two cases, leuB and leuD, the inferred tree did not show the two monophyletic groups expected (Aphidini and Macrosiphini tribes), indicating that we have to be cautious about obtaining phylogenetic relationships with this kind of genes. These results indicate that A. pisum and D. noxia diverged soon after the split of the two tribes, and those features common to both aphids, as the inverted repeat SIR2, would be able to define the Macrosiphini tribe.

ACKNOWLEDGMENTS We are indebted to the Servei de Bioinforma`tica and the Servei de Sequ¨enciacio´ de ADN i proteı¨nes (S.C.S.I.E., Universitat de Vale`ncia) for computer analysis and sequencing facilities. B. Sabater benefits from a grant from Ministerio de Educacio´n y Cultura, Spain. This work has been supported by grant PB96-0793 C04-01 from Direccio´n General de Ensen˜anza Superior awarded to A. Latorre and F. J. Silva.

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