Plasmid-Encoded Anthranilate Synthase (TrpEG) inBuchnera aphidicola from Aphids of the Family Pemphigidae
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Fig. 1.Linearized physical maps of B. aphidicola trpEG plasmids. All genes are transcribed in the rightward direction. Open arrowheads indicate the approximate position and orientation of DnaA boxes. (A) Restriction site and genetic map of pBTc2 from B. aphidicola(T. caerulescens). The region denoted ori?contains the putative origin of replication. Restriction enzyme sites:X, XbaI; B, BglII;E, EcoRI. (B) Genetic maps of the repeated units of B. aphidicola (Aphididae) trpEGplasmids (adapted from reference 42 with permission from the publisher). B(sp.), species names (see below); trpEG/p, number of repeat units per plasmid (42); ori-3.6, putative origin of replication; Ψ, repeat units containing trpEG pseudogenes (32); striped box and ΨrepAC, location of a putative repAC pseudogene; open rectangle, 19-bp element similar to consensus sequence of RepA/C iterons. Species abbreviations: Dn, Diuraphis noxia; Ap, Acyrthosiphon piseum; Sg, Schizaphis graminum; Rp, Rhopalosiphum padi; Rm, Rhopalosiphum maidis.
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Fig. 2.Southern blot analysis of EcoRI-digested (E) and undigested (u) B. aphidicola (T. caerulescens) DNA preparations. Cloned pBTc2 was used as a hybridization probe. The different forms of the putative plasmids are marked: open circular (oc), linear (L), and covalently closed circular (ccc).
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Fig. 3.Southern blot analysis of B. aphidicola (P. spyrothecae) plasmid DNA. Hybridizations were performed with a probe complementary totrpG. Restriction enzymes: A, AccI; P,PvuII; B, BamHI; H, HindIII; V,EcoRV; C, ClaI; X, XbaI; E,EcoRI; T, PstI; K, ScaI; O,XhoI. oc, open circular; L, linear; ccc, covalently closed circular.
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Fig. 4.Physical map of cloned and sequenced portion of pBPs2 from B. aphidicola (P. spyrothecae). (A) Restriction site and clone map. Double-headed arrows, clonedXbaI restriction fragments; arrows, oligonucleotide primers used for PCR; dotted line, cloned PCR product. Restriction enzyme sites: A, AccI; P, PvuII;S, SacI; C, ClaI;V, EcoRV. (B) Genetic map. All genes are transcribed in the rightward direction. The region denotedori? contains the putative origin of replication. Arrows, 19-bp iteron unique to pBPs2; arrows between brackets illustrate the copy number of the same iteron in the 1.70-kb PCR fragment; open rectangle, 19-bp element differing in one nucleotide from consensus sequence of RepA/C iterons; triple strip, 129-bp sequence identical to the 3′ end of trpE.
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Fig. 5.(A) Southern blot analysis of partial digestion ofAccI-SacI-digested B. aphidicola (P. spyrothecae) DNA by using serial dilutions of XbaI. Lanes: 1 to 8, digestions with 5.0, 2.5, 1.24, 0.63, 0.31, 0.16, 0.08, and 0.04 U of XbaI, respectively, for 15 min. X, A, S, control digestions performed to completion (X, XbaI; S, SacI; A, AccI. Arrows indicate the two principal forms of the plasmid. (B) Proposed structure of pBPs2 from B. aphidicola (P. spyrothecae) containing five tandem repeats of a 1.8-kb unit. Boxes with a similar pattern represent identical genes. All genes are transcribed in a clockwise direction. The region denotedori? contains the putative origin of replication. Arcs correspond to restriction fragments removed from molecules withAccI and SacI before partial digestion experiment with XbaI. The restriction enzyme sites shown are as in Fig. 4A.
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Fig. 6.Multiple alignment of translations ofrepAC-like sequences. Black background indicates stop codons (x). The C-terminal residues differing between RepAC1and RepAC2 are shown in boldface type. Ps_RepAC,repAC of pBPs2; Rm_RepAC, repAC oftrpEG plasmid of B(R. maidis); RA1_RepAC, repAC of E. coliplasmid RA1.
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Fig. 7.Phylogenetic trees constructed by the neighbor-joining method (23) from estimated distances between TrpE and TrpG sequences. Species designations: Ec, Escherichia coli; St,Salmonella typhimurium; Ps, B. aphidicola (Pemphigus spyrothecae); Tc,B. aphidicola (Tetraneura caerulescens; Sc, B. aphidicola(Schlechtendalia chinensis); Dn, B. aphidicola (Diuraphis noxia); Ap, B. aphidicola (Acyrthosiphon pisum); Sg,B. aphidicola (Shizaphis graminum); Rp, B. aphidicola (Rhopalosiphum padi); Rm, B. aphidicola(Rhopalosiphum maidis).
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Fig. 8.Phylogenetic distribution oftrpEG-associated replicons in Buchnera from aphids of the families Pemphigidae [B(P.)] nd Aphididae [B(A.)]. Species designations are as in Fig. 7. Location: p, plasmid; c, chromosomal. Replicon designation: R, RepA/C; ΨR, RepAC pseudogene; o; ori-3.6 like.
- Copyright © 1999 American Society for Microbiology
