Complex evolutionary patterns of tRNA Leu(UAA) group I introns in the cyanobacterial radiation [corrected]

Abstract

Based on the findings that plastids and cyanobacteria have similar group I introns inserted into tRNAUAALeu genes, these introns have been suggested to be immobile and of ancient origin. In contrast, recent evidence suggests lateral transfer of cyanobacterial group I introns located in tRNAUAALeu genes. In light of these new findings, we have readdressed the evolution and lateral transfer of tRNAUAALeu group I introns in cyanobacteral radiation. We determined the presence of introns in 38 different strains, representing the major cyanobacterial lineages, and characterized the introns in 22 of the strains. Notably, two of these strains have two tRNAUAALeu genes, with each of these genes interrupted by introns, while three of the strains have both interrupted and uninterrupted genes. Two evolutionary distinct clusters of tRNA genes, with the genes interrupted by introns belonging to two distinct intron clusters, were identified. We also compared 16S rDNA and intron evolution for both closely and distantly related strains. The distribution of the introns in the clustered groups, as defined from 16S rDNA analysis, indicates relatively recent gain and/or loss of the introns in some of these lineages. The comparative analysis also suggests differences in the phylogenetic trees for 16S rDNA and the tRNAUAALeu group I introns. Taken together, our results show that the evolution of the intron is considerably more complex than previous studies found to be the case. We discuss, based on our results, evolutionary models involving lateral intron transfer and models involving differential loss of the intron.

FIG. 1

Amplification products with the tRNA_UAA^Leu exon-specific primer pair CA-CB. The PCR products were electrophoresed in an ethidium bromide-stained 1.5% agarose gel for 30 min at 100 V. Twenty percent of the amplification product was loaded in each lane. Strain abbreviations: SS#1, Spirulina subsalsa NIVA-CYA 164; AR, Arthrospira fusiformis NIVA-CYA 136/2; NS#1 and NS#2, Nostoc sp. strains NIVA-CYA 194 and 308; SS#2, Spirulina subsalsa NIVA-CYA 163; PS, Phormidium sp. strain NIVA-CYA 202; NS#3, Nostoc sp. strain NIVA-CYA 124; AG, Aphanizomenon gracile NIVA-CYA 103; PA, P. agarthii NIVA-CYA 29; AF, A. flos-aquae NIVA-CYA 142. The two intron bands amplified for A. flos-aquae NIVA-CYA 142 (see arrows) are not properly separated in this gel. mw, molecular weight standard; neg, negative control.

FIG. 2

Compilation of tRNA_UAA^Leu exon sequences. All the tRNA_UAA^Leu sequences in the tRNA database (36) were compiled and compared to the partial exon sequences identified in this work. The figure includes the two putative classes A and B of tRNA_UAA^Leu genes found for the cyanobacteria (CYANOB) in this work. ∗, conserved position (conserved in 90% or more of the species); #, position conserved only for cyanobacteria and chloroplasts (CHLORO), i.e., conserved in 90% or more of the cyanobacteria and chloroplasts and 40% or less conserved for the other species; =, variable position (position 80% or less conserved for all species); ^a, interrupted by cluster II intron; ^b, interrupted by cluster I intron. EUBACT, eubacteria. Species abbreviations: APH. FLOS., Aphanizomenon flos-aquae; MICR. AER., Microcystis aeruginosa; PLANKT. AG., Planktothrix agarhii; PLEUR. MINOR., Pleurocapsa minor; SYNECH. LE., Synechococcus leopoliensis; ARTH. FUS., Arthrospira fusiformis; PHORM., Phormidium; PROCHL., Prochlorothrix; CYANOPHORA PARAD., Cyanophora paradoxa; MARCHANTIA POLYM., Marchantia polymorpha; MYCOPLASMA CAPRIC., Mycoplasma capricolum; MYCOPLASMA PNEUMO., Mycoplasma pneumoniae; ACHOLEPLASMA LAID., Acholeplasma laidlawii; STREPTOMYCES COEL., Streptomyces coelicolor; STAPHYLOCOC. AURE., Staphylococcus aureus; E.COLI, Escherichia coli; HAEMOPHILUS INFLU., Haemophilus influenzae.

FIG. 3

Conserved intron elements. The intron elements P, Q, R, and S, generally conserved among group I introns (20), are shown for the cyanobacterial introns characterized in this work. The two introns present in A. (Aph.) flos-auae NIVA-CYA 142 and Nostoc sp. strain NIVA-CYA 308 are annotated with i1 and i2, respectively. Dots indicate identity to M. aeruginosa (Micr. aer.) NIVA-CYA 143, while lines indicate gaps in the alignment. Prochlor. holl., Prochlorothrix hollandica; Plankt. moug., Planktothrix mougeotii; Plankt. ag., P. agardhii; Plank. prol., P. prolifica; Phorm., Phormidium; Arthrospira fus., Arthrospira fusiformis; Synech leop., Synechococcus leopoliensis; Pleur., Pleurocapsa.

FIG. 4

Secondary structure of the P. agardhii NIVA-CYA 29 intron (cluster I). The structure is shown in the format described by Cech et al. (5), with marked secondary structure elements (P1 to P9). The sequences in lowercase letters represent exon sequence, while arrows indicate splice sites.

FIG. 5

Southern hybridization using a tRNA_UAA^Leu exon (A) or an intron (B) probe for selected Nostoc strains. The exon probe was generated from CA-CB primer amplification products for Anabaena lemmermannii NIVA-CYA 266/1 (AL; a strain confirmed to have an uninterrupted tRNA_UAA^Leu gene). The intron probe was obtained from an internal fragment of the intron from the strain Nostoc sp. strain NIVA-CYA 194 amplified with primer pair CR-CS. Genomic DNA digested with HindIII was separated, blotted, and hybridized as described in Materials and Methods. The same membrane was used for each of the hybridization experiments, providing exact assignment of the hybridizing bands from different experiments. The molecular weight standard is HindIII-digested λ DNA. Strain abbreviations are as for Fig. 1.

FIG. 6

Evolutionary trees for 16S rDNA (A) and tRNA_UAA^Leu introns sequences (B). The trees were built using the neighbor-joining, maximum-parsimony, and maximum-likelihood methods. The trees shown in panels A and B (based on 489 and 346 aligned positions, respectively) are consensus trees for the branches supported by ≥25% of the bootstrap trees in all of the phylogenetic methods tested. The genetic distances between two strains are Kimura distances expressed in substitutions per nucleotide in the neighbor-joining tree. Numbers at the nodes (minimal evolution [for the intron tree]/maximum likelihood/maximum parsimony/neighbor joining) indicate the percentage of the bootstrap trees in which the cluster descending from the node was found. In panel A, the presence and absence of introns are indicated with (+) and (−), respectively. The respective clone numbers (N-C, NIVA-CYA; N, NIVA; P, Pasteur) are given for each branch. Species abbreviations: An. lemn., Anabaena lemmermannii; Tych. bour., Tychonema bourrellyi; Spir. subs., Spirulina subsalsa; Cyanothece, Cyanothece aeruginosa; Chrooc. therm., Chroococcidiopsis thermalis; Dermocarpella incr., Dermocarpella incrassata; Dermocarpella viol., Dermocarpella violacea; Pseud. limn., Pseudanabaena limnetica. All other species are abbreviated as in Fig. 2 and 3.