Trans-splicing of the Ll.LtrB group II intron in Lactococcus lactis

Abstract

The Ll.LtrB intron from the Gram-positive bacterium Lactococcus lactis is one of the most studied bacterial group II introns. Ll.LtrB interrupts the relaxase gene of three L. lactis conjugative elements. The relaxase enzyme recognizes the origin of transfer (oriT ) and initiates the intercellular transfer of its conjugative element. The splicing efficiency of Ll.LtrB from the relaxase transcript thus controls the conjugation level of its host element. Here, we used the level of sex factor conjugation as a read-out for Ll.LtrB splicing efficiency. Using this highly sensitive splicing/conjugation assay (10(7)-fold detection range), we demonstrate that Ll.LtrB can trans-splice in L. lactis when fragmented at various positions such as: three different locations within domain IV, within domain I and within domain III. We also demonstrate that the intron-encoded protein, LtrA, is absolutely required for Ll.LtrB trans-splicing. Characteristic Y-branched trans-spliced introns and ligated exons are detected by RT-PCR from total RNA extracts of cells harbouring fragmented Ll.LtrB. The splicing/conjugation assay we developed constitutes the first model system to study group II intron trans-splicing in vivo. Although only previously observed in bacterial-derived organelles, we demonstrate that assembly and trans-splicing of a fragmented group II intron can take place efficiently in bacterial cells.

Figure 1.

Group II intron splicing pathway and Ll.LtrB secondary structure. (A) Splicing pathway of group II introns. The 2′OH of the bulged adenosine present in domain VI of the intron (circled A) performs the initial nucleophilic attack on the exon 1–intron junction (step 1), generating a 2′–5′ linkage, also known as a branch point. Then, the 3′-OH of the released exon 1 performs the second nucleophilic attack on the intron–exon 2 junction (step 2), releasing the intron lariat and ligating the flanking exons. Group II intron, black line; exon 1 and 2, E1 and E2; branch point, circled A. (B) Schematic of the Ll.LtrB secondary structure. The six domains of Ll.LtrB are indicated (I–VI) and the detailed secondary structure of a portion of domain IV is shown (top right). The LtrA start and stop codons are boxed and the Shine–Dalgarno sequence is underlined. Exon 1 and 2 on both sides of the intron are also boxed. Pairs of greek letters, linked by dashed lines, correspond to long-range interactions between different portions of the intron. The five chosen Ll.LtrB fragmentation sites are mapped with black arrowheads (S1–S5). EBS1 and EBS2, exon-binding site 1 and 2; IBS1 and IBS2, intron-binding site 1 and 2; branch point, circled A.

Figure 2.

Ll.LtrB splicing/conjugation assay and relaxase expression levels. (A) Schematic of the Ll.LtrB splicing/conjugation assay. The L. lactis strain used as the donor strain harbours a sex factor that has a defective relaxase (NZ9800▵ltrB::tet) while the recipient strain lacks the sex factor (LM0231). The Ll.LtrB intron, along with portions of its exons, was replaced within the chromosomal sex factor by a tetracycline resistance marker, which prevents relaxase expression. The ltrB relaxase is constitutively produced from the pDL-P₂₃²-ltrB complementation plasmid. When ltrB is interrupted by a splicing proficient variant of Ll.LtrB, relaxase is produced and mediates transfer of the sex factor from the donor to the recipient cell. The level of sex factor conjugation observed is proportional to the Ll.LtrB splicing efficiency. (B) Relaxase expression and Ll.LtrB splicing. Northern blots were performed using total RNA from NZ9800 and NZ9800ΔltrB harbouring different pDL-P₂₃²-based constructs. The amounts of total relaxase RNA (left) and mature relaxase transcript (right) produced were assessed (two independent blots, RNA was loaded in duplicate on the same gel). The exon 2 probe (left panel) (Supplementary Table S1, RT E2) and the splice-junction probe (right panel) (Supplementary Table S1, SJ) are depicted as grey bars. As an RNA loading control, the two membranes were stripped and probed with a 23S rRNA specific probe (Supplementary Table S1, 23S rRNA). (C) Western blot on total protein extracts using LtrB-specific antibodies. Ll.LtrB group II intron, black line; exon 1 and 2, E1 and E2; branch point, circled A; sex factor, grey; tetracycline resistance marker, Tet; spectinomycin resistance marker, Spc; P₂₃ promoter, P₂₃; transcription terminator, schematic stem-loop; L. lactis chromosome, scribble.

Figure 3.

Ll.LtrB trans-splicing/conjugation assay. (A) Strategy to generate a variant of Ll.LtrB fragmented at the Sn site (S1–S5). The relaxase gene is amplified as two non-overlapping fragments using the indicated primers (thick arrows, Supplementary Table S1). Each fragment is cloned under the control of a P₂₃ promoter on pDL-P₂₃². (B) Ll.LtrB trans-splicing/conjugation assay. The pDL-P₂₃²-Sn plasmid is transformed in NZ9800ΔltrB. If the two fragments of the intron can correctly fold, assemble and trans-splice, the ltrB exons are ligated allowing the production of relaxase, which is required for sex factor transfer. Ll.LtrB splicing efficiency is monitored by the level of sex factor conjugation from the donor (NZ9800ΔltrB) to the recipient L. lactis strain (LM0231). Ll.LtrB group II intron, thick black line; exon 1 and 2, E1 and E2; branch point, circled A; sex factor, grey; tetracycline resistance marker, Tet; spectinomycin resistance marker, Spc; P₂₃ promoters, P₂₃; transcription terminators, schematic stem-loops; L. lactis chromosome, scribble.

Figure 4.

RT-PCR analyses of excised Ll.LtrB introns and ligated exons. (A) Schematic of Ll.LtrB splicing (top) and trans-splicing (bottom). Primers used for RT-PCR amplifications of released intron lariats or Y-branched molecules are shown as black arrows (BP1 and 2). Ligated exons were amplified by RT-PCR with the RTE1 and 2 primers (open arrows). Agarose gels showing the RT-PCR amplification of Ll.LtrB across the branch point (B) and across the ltrB ligated exons (C) are presented. RT-PCR analyses were performed on total RNA extracts from NZ9800 or NZ9800ΔltrB harbouring different pDL-P₂₃² plasmids expressing ltrB in one (WT, ΔD5) or two pieces (S1–S5). S3ΔORF and S4ΔORF represent the ΔORF variant of the S3 and S4 constructs without LtrA complementation. Ll.LtrB group II intron, black line; exon 1 and 2, E1 and E2; branch point, circled A.

Figure 5.

Sex factor conjugation efficiencies observed with different fragmented variants of Ll.LtrB. The fragmentation sites within Ll.LtrB correspond to those shown in Figure 1B (S1–S5). The Ll.LtrB variant column indicates which fragment(s) was/were overexpressed from the pDL-P₂₃² plasmid, and which mutated version of the ORF was used when applicable. The ΔORF + ORF variants correspond to the assays where LtrA was overexpressed from a second plasmid to complement the deleted version within Ll.LtrB. The schematics show linear representations of Ll.LtrB and its six domains (I–VI), and the location of the fragmentation sites. Sex factor conjugation efficiency is the average of three independent assays and the error is the standard deviation of the three obtained values. Ll.LtrB group II intron, black line; exon 1 and 2, empty boxes; branch point, circled A.