Conjugative transfer of the transposon Tn919 to lactic acid bacteria

Abstract The streptococcal transposon Tn 919 was transferred from Streptococcus faecalis GF590 to selected Group N Streptococcus strains and to one strain each of Lactobacillus plantarum and Leuconostoc cremoris , using the filter mating method. An S. lactis MG1363 Rif^r Tc^r transconjugant also acted as a donor, but was less efficient than GF590. Frequencies of transfer varied between 4.0 × 10⁻⁸ and 5.29 × 10⁻⁵ per recipient. Further analysis of S. lactis MG1363 Sm^r Tc^r transconjugants showed that insertion of Tn 919 into the chromosome was site-specific.     

GAPDH enhances group II intron splicing in vitro

Group II introns are autocatalytic RNAs which self-splice in vitro. However, in vivo additional protein factors might be involved in the splicing process. We used an affinity chromatography method called 'StreptoTag' to identify group II intron binding proteins from Saccharomyces cerevisiae. This method uses a hybrid RNA consisting of a streptomycin-binding affinity tag and the RNA of interest, which is bound to a streptomycin column and incubated with yeast protein extract. After several washing steps the bound RNPs are eluted by addition of streptomycin. The eluted RNPs are separated and the proteins identified by mass-spectrometric analysis. Using crude extract from yeast in combination with a substructure of the bl1 group II intron (domains IV-VI) we were able to identify four glycolytic enzymes; glucose-6-phosphate isomerase (GPI), 3-phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TPI). From these proteins GAPDH increases in vitro splicing of the bl1 group II intron by up to three times. However, in vivo GAPDH is not a group II intron-splicing factor, since it is not localised in yeast mitochondria. Therefore, the observed activity reflects an unexpected property of GAPDH. Band shift experiments and UV cross linking demonstrated the interaction of GAPDH with the group II intron RNA. This novel activity expands the reaction repertoire of GAPDH to a new RNA species.     

In vivo facilitation of Tetrahymena group I intron splicing in Escherichia coli pre-ribosomal RNA.

The observation that the large ribosomal RNA intron of Tetrahymena is spliced 20-50-fold more rapidly in vivo than in vitro (Brehm SL, Cech TR, 1983, Biochemistry 22:2390-2397; Bass BL, Cech TR, 1984, Nature 308:820-826) suggests facilitation of RNA folding in vivo. To determine whether a specific group I splicing factor is required in Tetrahymena, the intron was inserted into the analogous position of the Escherichia coli 23S rRNA. We report that the intron is rapidly excised from pre-rRNA in bacteria and that the magnitude of the in vivo rate enhancement is similar to that in Tetrahymena. These results demonstrate that a species-specific protein is not required. Instead, a common mechanism of assisting RNA folding is sufficient to accelerate the removal of self-splicing introns from ribosomal RNA.     

A group II intron in a conjugative transposon from the gram-positive bacterium,Clostridium difficile

We have been studying the conjugative transposon Tn5397, originally isolated from the Gram-positive pathogen Clostridium difficile. Physical analysis of this transposon demonstrated that it contained a group II intron. This is the first report of an intron in a conjugative transposon and the first report of a group II intron in Gram-positive bacteria. The intron interrupted a gene in Tn5397 that is almost identical to orf14 from Tn916. DNA hybridisation analysis showed that elements related to Tn5397, containing the group II intron, were present in five other C. difficile strains from different geographical locations suggesting that the element is likely to be widely distributed.     

CRS1 is a novel group II intron splicing factor that was derived from a domain of ancient origin

Protein-dependent group II intron splicing provides a forum for exploring the roles of proteins in facilitating RNA-catalyzed reactions. The maize nuclear gene crs1 is required for the splicing of the group II intron in the chloroplast atpF gene. Here we report the molecular cloning of the crs1 gene and an initial biochemical characterization of its gene product. Several observations support the notion that CRS1 is a bona fide group II intron splicing factor. First, CRS1 is found in a ribonucleoprotein complex in the chloroplast, and cofractionation data provide evidence that this complex includes atpF intron RNA. Second, CRS1 is highly basic and includes a repeated domain with features suggestive of a novel RNA-binding domain. This domain is related to a conserved free-standing open reading frame of unknown function found in both the eubacteria and archaea. crs1 is the founding member of a gene family in plants that was derived by duplication and divergence of this primitive gene. In addition to its previously established role in atpF intron splicing, new genetic data implicate crs1 in chloroplast translation. The chloroplast splicing and translation functions of crs1 may be mediated by the distinct protein products of two crs1 mRNA forms that result from alternative splicing of the crs1 pre-mRNA.     

Novel RNA structural features of an alternatively splicing group II intron from Clostridium tetani

Group II introns are ribozymes in bacterial and organellar genomes that function as self-splicing introns and as retroelements. Previously, we reported that the group II intron C.te.I1 of Clostridium tetani alternatively splices in vivo to produce five distinct coding mRNAs. Accurate fusion of upstream and downstream reading frames requires a shifted 5′ splice site located 8 nt upstream of the usual 5′ GUGYG motif. This site is specified by the ribozyme through an altered intron/exon-binding site 1 (IBS1–EBS1) pairing. Here we use mutagenesis and self-splicing assays to investigate in more detail the significance of the structural features of the C.te.I1 ribozyme. The shifted 5′ splice site is shown to be affected by structures in addition to IBS1–EBS1, and unlike other group II introns, C.te.I1 appears to require a spacer between IBS1 and the GUGYG motif. In addition, the mechanism of 3′ exon recognition is modified from the ancestral IIB mechanism to a IIA-like mechanism that appears to be longer than the typical single base-pair interaction and may extend up to 4 bp. The novel ribozyme properties that have evolved for C.te.I1 illustrate the plasticity of group II introns in adapting new structural and catalytic properties that can be utilized to affect gene expression.     

Mutations in the Lactococcus lactis Ll.LtrB group II intron that retain mobility in vivo

Background

Group II introns are mobile genetic elements that form conserved secondary and tertiary structures. In order to determine which of the conserved structural elements are required for mobility, a series of domain and sub-domain deletions were made in the Lactococcus lactis group II intron (Ll.LtrB) and tested for mobility in a genetic assay. Point mutations in domains V and VI were also tested.

Results

The largest deletion that could be made without severely compromising mobility was 158 nucleotides in DIVb(1–2). This mutant had a mobility frequency comparable to the wild-type Ll.LtrB intron (ΔORF construct). Hence, all subsequent mutations were done in this mutant background. Deletion of DIIb reduced mobility to approximately 18% of wild-type, while another deletion in domain II (nts 404–459) was mobile to a minor extent. Only two deletions in DI and none in DIII were tolerated. Some mobility was also observed for a DIVa deletion mutant. Of the three point mutants at position G3 in DV, only G3A retained mobility. In DVI, deletion of the branch-point nucleotide abolished mobility, but the presence of any nucleotide at the branch-point position restored mobility to some extent.

Conclusions

The smallest intron capable of efficient retrohoming was 725 nucleotides, comprising the DIVb(1–2) and DII(ii)a,b deletions. The tertiary elements found to be nonessential for mobility were alpha, kappa and eta. In DV, only the G3A mutant was mobile. A branch-point residue is required for intron mobility.     

Stereochemical selectivity of group II intron splicing, reverse splicing, and hydrolysis reactions.

We have previously shown, using phosphorothioate substitutions at splice site, that both transesterification steps of group II intron self-splicing proceed, by stereochemical inversion, with an Sp but not an Rp phosphorothioate. Under alternative reaction conditions or with various intron fragments, group II introns can splice following hydrolysis at the 5' splice site and can also hydrolyze the bond between spliced exons (the spliced-exon reopening reaction). In this study, we have determined the stereochemical specificities of all of the major model hydrolytic reactions carried out by the aI5 gamma intron from Saccharomyces cerevisiae mitochondria. For all substrates containing exon 1 and most of the intron, the stereospecificity of hydrolysis is the same as for the step 1 transesterification reaction. In contrast, the spliced-exon reopening reaction proceeds with an Rp but not an Sp phosphorothioate at the scissile bond, as does true reverse splicing. Thus, by stereochemistry, this reaction appears to be related to the reverse of step 2 of self-splicing. Finally, a substrate RNA that contains the first exon and nine nucleotides of the intron, when reacted with the intron ribozyme, releases the first exon regardless of the configuration of the phosphorothioate at the 5' splice site, suggesting that this substrate can be cleaved by either the step 1 or the step 2 reaction site. Our findings clarify the relationships of these model reactions to the transesterification reactions of the intact self-splicing system and permit new studies to be interpreted more rigorously.     

ATP-dependent roles of the DEAD-box protein Mss116p in group II intron splicing in vitro and in vivo

The yeast DEAD-box protein Mss116p functions as a general RNA chaperone in splicing mitochondrial group I and group II introns. For most of its functions, Mss116p is thought to use ATP-dependent RNA unwinding to facilitate RNA structural transitions, but it has been suggested to assist in the folding of one group II intron (aI5γ) primarily by stabilizing a folding intermediate. Here we compare three aI5γ constructs: one with long exons, one with short exons, and a ribozyme construct lacking exons. The long exons result in slower splicing, suggesting that they misfold and/or stabilize nonnative intronic structures. Nevertheless, Mss116p acceleration of all three constructs depends on ATP and is inhibited by mutations that compromise RNA unwinding, suggesting similar mechanisms. Results of splicing assays and a new two-stage assay that separates ribozyme folding and catalysis indicate that maximal folding of all three constructs by Mss116p requires ATP-dependent RNA unwinding. ATP-independent activation is appreciable for only a subpopulation of the minimal ribozyme construct and not for constructs containing exons. As expected for a general RNA chaperone, Mss116p can also disrupt the native ribozyme, which can refold after Mss116p removal. Finally, using yeast strains with mitochondrial DNA containing only the single intron aI5γ,? we show that Mss116p mutants promote splicing in vivo to degrees that correlate with their residual ATP-dependent RNA-unwinding activities. Together, our results indicate that, although DEAD-box proteins play multiple roles in RNA folding, the physiological function of Mss116p in aI5γ splicing includes a requirement for ATP-dependent local unfolding, allowing the conversion of nonfunctional RNA structure into functional RNA structure.     

In vivo isolation of a pPJ3a-lambda cosmid mediated by the Tn2301 transposon

From an E. coli cell harboring plasmid pPJ3b (= pPJ3a::Tn2301) and infected with phage λ, we have isolated two defective phages having inserted pPJ3a DNA and Tn2301 in their genomes. One of them has been extensively characterized: it behaves like a cosmid, i.e., upon injection into the cell, its DNA circularizes and replicates as a plasmid (pPJ10); it can be packaged again in λ heads, provided the presence of a phage helper. Furthermore, heteroduplex analysis has shown that in pPJ10, the transposon Tn2301 is inverted compared to its direction in pPJ3b. We give evidence suggesting that this type of inversion is in part mediated by Tn2301.     

Sequence specificity of in vivo reverse splicing of the Tetrahymena group I intron.

Reverse splicing of group I introns is proposed to be a mechanism by which intron sequences are transferred to new genes. Integration of the Tetrahymena intron into the Escherichia coli 23S rRNA via reverse splicing depends on base pairing between the guide sequence of the intron and the target site. To investigate the substrate specificity of reverse splicing, the wild-type and 18 mutant introns with different guide sequences were expressed in E. coli. Amplification of intron-rRNA junctions by RT-PCR revealed partial reverse splicing at 69 sites and complete integration at one novel site in the 23S rRNA. Reverse splicing was not observed at some potential target sites, whereas other regions of the 23S rRNA were more reactive than expected. The results indicate that the frequency of reverse splicing is modulated by the structure of the rRNA. The intron is spliced 10-fold less efficiently in E. coli from a novel integration site (U2074) in domain V of the 23S rRNA than from a site homologous to the natural splice junction of the Tetrahymena 26S rRNA, suggesting that the forward reaction is less favored at this site.     

A conserved element that stabilizes the group II intron active site

The internal loop at the base of domain 3 (D3) is one of the most conserved and catalytically important elements of a group II intron. However, the location and molecular nature of its tertiary interaction partners has remained unknown. By employing a combination of site-directed photo-cross-linking and nucleotide analog interference suppression (NAIS), we show that the domain 3 internal loop (D3IL) interacts with the epsilon-epsilon' duplex, which is an active-site element located near the 5'-splice site in D1. Our data also suggest that the D3IL may interact with the bulge of D5, which is a critical active site component. The results of this and other recent studies indicate that the D3IL participates in a complex network of tertiary interactions involving epsilon-epsilon', the bulge of D5 and J23, and that it helps to optimize active site architecture by supporting interactions among these catalytic motifs. Our results are consistent with the role of D3 as a catalytic effector that enhances intron reactivity through active site stabilization.     

Deletion of a conserved dinucleotide inhibits the second step of group II intron splicing

Few point mutations have been described that specifically inhibit the second step of group II intron splicing. Furthermore, the effects of such mutations are typically not apparent unless the mutations are studied in the context of a substrate that harbors a very short 5' exon. Truncation of the 5' exon slows the second step of splicing. Once the second step has been slowed, the effects of point mutations can be seen. We report the unexpected observation that the deletion of a conserved GA dinucleotide dramatically inhibits the second step of splicing, even when the mutation is studied in the context of a full-length substrate. In contrast, we find that this mutation does not significantly affect the first step of splicing, unless the mutation is studied in combination with a second point mutation that is known to inhibit the first step. Even in that context, the effect of the GA deletion mutation on the first step is modest. These observations, together with the inferred location of the GA dinucleotide in the three-dimensional structure of the intron, suggest that this dinucleotide plays a particularly important role in the second step of splicing.     

Conjugative transposition of Tn916: the transposon int gene is required only in the donor.

Conjugative transposition of transposon Tn916 has been shown to proceed by excision of the transposon in the donor strain and insertion of this element in the recipient. This process requires the product of the transposon int gene. We report here the surprising finding that the int gene is required only in the donor during conjugative transposition. We find that Tn916 int-1, whose int gene has been inactivated by an insertion mutation, transposes when a complementing wild-type int gene is present only in the donor during mating. When the int+ gene is present in a plasmid and is expressed from the spac promoter, conjugative transposition is very inefficient. However, when the Int+ function is supplied from a coresident distantly linked Tn916 tra-641 mutant, which is defective in a function required for conjugation, efficient conjugative transposition of Tn916 int-1 occurs. This suggests either that Int is not required for integration of Tn916 in gram-positive bacteria or that the protein is transferred from the donor to the transconjugant during the mating event. When the nonconjugative plasmid pAT145 was present in the donor, it was rarely cotransferred with Tn916. This suggests that complete fusion of mating cells is not common during conjugative transposition.