Conserved target for group II intron insertion in relaxase genes of conjugative elements of gram-positive bacteria

The lactococcal group II intron Ll.ltrB interrupts the ltrB relaxase gene within a region that encodes a conserved functional domain. Nucleotides essential for the homing of Ll.ltrB into an intronless version of ltrB are found exclusively at positions required to encode amino acids broadly conserved in a family of relaxase proteins of gram-positive bacteria. Two of these relaxase genes, pcfG from the enterococcal plasmid pCF10 and the ORF4 gene in the streptococcal conjugative transposon Tn5252, were shown to support Ll.ltrB insertion into the conserved motif at precisely the site predicted by sequence homology with ltrB. Insertion occurred through a mechanism indistinguishable from retrohoming. Splicing and retention of conjugative function was demonstrated for pCF10 derivatives containing intron insertions. Ll.ltrB targeting of a conserved motif of a conjugative element suggests a mechanism for group II intron dispersal among bacteria. Additional support for this mechanism comes from sequence analysis of the insertion sites of the E.c.I4 family of bacterial group II introns.     

The conjugative transposon Tn5397 has a strong preference for integration into its Clostridium difficile target site

Tn5397 is a conjugative transposon, originally isolated from Clostridium difficile. The Tn5397 transposase TndX is related to the phage-encoded serine integrases and the Clostridium perfringens Tn4451 transposase TnpX. TndX is required for the insertion and excision of the transposon. Tn5397 inserts at one locus, attB(Cd), in C. difficile but at multiple sites in Bacillus subtilis. Apart from a conserved 5' GA dinucleotide at the recombination site, there appears to be little sequence conservation between the known target sites. To test the target site preference of Tn5397, attB(Cd) was introduced into the B. subtilis genome. When Tn5397 was transferred into this strain, 100% of the 50 independent transconjugants tested had Tn5397 inserted into attB(Cd). This experiment was repeated using a 50-bp attB(Cd) with no loss of target preference. The mutation of the 5' GA to 5' TC in the attB(Cd) target site caused a switch in the polarity of insertion of Tn5397, which is consistent with this dinucleotide being at the crossover site and in keeping with the mechanism of other serine recombinases. Tn5397 could also transpose into 50-bp sequences encoding the end joints attL and attR but, surprisingly, could not recombine into the circular joint of Tn5397, attTn. Purified TndX was shown to bind specifically to 50-bp attB(Cd), attL, attR, attTn, and attB(Bs)(3) with relative binding affinities attTn approximately attR > attL > attB(Cd) > attB(Bs3). We conclude that TndX has a strong preference for attB(Cd) over other potential recombination sites in the B. subtilis genome and therefore behaves as a site-specific recombinase.     

A newly discovered Bacteroides conjugative transposon,CTnGERM1, contains genes also found in gram-positive bacteria

Results of a recent study of antibiotic resistance genes in human colonic Bacteroides strains suggested that gene transfer events between members of this genus are fairly common. The identification of Bacteroides isolates that carried an erythromycin resistance gene, ermG, whose DNA sequence was 99% identical to that of an ermG gene found previously only in gram-positive bacteria raised the further possibility that conjugal elements were moving into Bacteroides species from other genera. Six of seven ermG-containing Bacteroides strains tested were able to transfer ermG by conjugation. One of these strains was chosen for further investigation. Results of pulsed-field gel electrophoresis experiments showed that the conjugal element carrying ermG in this strain is an integrated element about 75 kb in size. Thus, the element appears to be a conjugative transposon (CTn) and was designated CTnGERM1. CTnGERM1 proved to be unrelated to the predominant type of CTn found in Bacteroides isolates-CTns of the CTnERL/CTnDOT family-which sometimes carry another type of erm gene, ermF. A 19-kbp segment of DNA from CTnGERM1 was cloned and sequenced. A 10-kbp portion of this segment hybridized not only to DNA from all the ermG-containing strains but also to DNA from strains that did not carry ermG. Thus, CTnGERM1 seems to be part of a family of CTns, some of which have acquired ermG. The percentage of G+C content of the ermG region was significantly lower than that of the chromosome of Bacteroides species-an indication that CTnGERM1 may have entered Bacteroides strains from some other bacterial genus. A survey of strains isolated before 1970 and after 1990 suggests that the CTnGERM1 type of CTn entered Bacteroides species relatively recently. One of the genes located upstream of ermG encoded a protein that had 85% amino acid sequence identity with a macrolide efflux pump, MefA, from Streptococcus pyogenes. Our having found >90% sequence identity of two upstream genes, including mefA, and the remnants of two transposon-carried genes downstream of ermG with genes found previously only in gram-positive bacteria raises the possibility that gram-positive bacteria could have been the origin of CTnGERM1.     

Behavior and target site selection of conjugative transposon Tn916 in two different strains of toxigenic Clostridium difficile

The insertion sites of the conjugative transposon Tn916 in the anaerobic pathogen Clostridium difficile were determined using Illumina Solexa high-throughput DNA sequencing of Tn916 insertion libraries in two different clinical isolates: 630ΔE, an erythromycin-sensitive derivative of 630 (ribotype 012), and the ribotype 027 isolate R20291, which was responsible for a severe outbreak of C. difficile disease. A consensus 15-bp Tn916 insertion sequence was identified which was similar in both strains, although an extended consensus sequence was observed in R20291. A search of the C. difficile 630 genome showed that the Tn916 insertion motif was present 100,987 times, with approximately 63,000 of these motifs located in genes and 35,000 in intergenic regions. To test the usefulness of Tn916 as a mutagen, a functional screen allowed the isolation of a mutant. This mutant contained Tn916 inserted into a gene involved in flagellar biosynthesis.     

Demonstration that the group II intron from the Clostridial Conjugative transposon Tn5397 undergoes splicing In vivo

Previous work has identified the conjugative transposon Tn5397 from Clostridium difficile. This element was shown to contain a group II intron. Tn5397 can be conjugatively transferred from C. difficile to Bacillus subtilis. In this work we show that the intron is spliced in both these hosts and that nonspliced RNA is also present. We constructed a mutation in the open reading frame within the intron, and this prevented splicing but did not prevent the formation of the circular form of the conjugative transposon (the likely transposition intermediate) or decrease the frequency of intergeneric transfer of Tn5397. Therefore, the intron is spliced, but splicing is not required for conjugation of Tn5397.     

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.     

Characterization of a conjugative transposon integrase, IntDOT

Sequence analysis revealed that the integrase of the Bacteroides conjugative transposon CTnDOT (IntDOT) might be a member of the tyrosine recombinase family because IntDOT has five of six highly conserved residues found in the catalytic domains of tyrosine recombinases. Yet, IntDOT catalyses a reaction that appears to differ in some respects from well-studied tyrosine recombinases such as that of phage lambda. To assess the importance of the conserved residues, we changed residues in IntDOT that align with conserved residues in tyrosine recombinases. Some substitutions resulted in a complete loss or significant decrease of integration activity in vivo. The ability of the mutant proteins to cleave and ligate CTnDOT attachment site (attDOT) DNA in vitro in general paralleled the in vivo results, but the H345A mutant, which had a wild-type level of integration in vivo, exhibited a slightly lower level of cleavage and ligation in vitro. Our results confirm the hypothesis that IntDOT belongs to the tyrosine recombinase family, but the catalytic core of the protein seems to have somewhat different organization. Previous DNA sequence analyses showed that CTnDOT att sites contain 5 bp non-homologous coupling sequences which were assumed to define the putative staggered sites of cleavage. However, cleavage assays showed that one of the cleavage sites is 2 bp away from the junction of CTnDOT and coupling sequence DNA. The site is in a region of homology that is conserved in CTnDOT att sites.     

Tn3702, a conjugative transposon in Enterococcus faecalis

Enterococcus faecalis strain D434 was found to carry on its chromosome a determinant encoding tetracycline-minocycline resistance (Tcr-Mnr) and to harbor both an R plasmid and a cryptic conjugative plasmid, pIP1141. The determinant coding for Tcr-Mnr was located on a conjugative transposon, designated Tn3702. The transposition of Tn3702 on to both pIP1141 and the hemolysin plasmid pIP964 yielded different derivatives each of which contained an 18.5-kilobase insert. The structure of Tn3702 is similar to that of the conjugative transposon Tn916.     

Characterization of the ends and target sites of the novel conjugative transposon Tn5397 from Clostridium difficile: excision and circularization is mediated by the large resolvase, TndX

Tn5397 is a conjugative transposon that was originally isolated from Clostridium difficile. Previous analysis had shown that the central region of Tn5397 was closely related to the conjugative transposon Tn916. However, in this work we obtained the DNA sequence of the ends of Tn5397 and showed that they are completely different to those of Tn916. Tn5397 did not contain the int and xis genes, which are required for the excision and integration of Tn916. Instead, the right end of Tn5397 contained a gene, tndX, that appears to encode a member of the large resolvase family of site-specific recombinases. TndX is closely related to the TnpX resolvase from the mobilizable but nonconjugative chloramphenicol resistance transposons, Tn4451 from Clostridium perfringens and Tn4453 from C. difficile. Like the latter elements, inserted copies of Tn5397 were flanked by a direct repeat of a GA dinucleotide. The Tn5397 target sites were also shown to contain a central GA dinucleotide. Excision of the element in C. difficile completely regenerated the original target sequence. A circular form of the transposon, in which the left and right ends of the element were separated by a GA dinucleotide, was detected by PCR in both Bacillus subtilis and C. difficile. A Tn5397 mutant in which part of tndX was deleted was constructed in B. subtilis. This mutant was nonconjugative and did not produce the circular form of Tn5397, indicating that the TndX resolvase has an essential role in the excision and transposition of Tn5397 and is thus the first example of a member of the large resolvase family of recombinases being involved in conjugative transposon mobility. Finally, we showed that introduction of Tn916 into a strain containing Tn5397 induced the loss of the latter element in 95.6% of recipients.     

Isolation from the rumen of a new acetogenic bacterium phylogenetically closely related to Clostridium difficile

Five strains of filamentous acetogenic bacterium were isolated from high dilutions of ruminal content of newborn lambs. These Gram-positive spore-forming bacteria grew either chemolithotrophically with H2+ CO2 or chemo-organotrophically with glucose, cellobiose, fructose, maltose, mannose and syringic acid. The DNA base composition of the five strains were between 29.1 and 31.3 mol% G + C. Their temperature and pH optimum for growth were 35-40 degrees C and 6.5-7.0, respectively. The full 16S rRNA gene sequence analysis of the reference strain indicated that it was most closely related to Clostridium difficile. The sequence similarity value between the 16S rRNA gene of the reference strain and this pathogenic strain was 99.7%.     

CTnscr94, a conjugative transposon found in enterobacteria.

Conjugational transposons are important for horizontal gene transfer in gram-positive and gram-negative bacteria, but have not been reported yet for enteric bacteria. Salmonella senftenberg 5494-57 has previously been shown to transfer by conjugation genes for a sucrose fermentation pathway which were located on a DNA element called scr-94. We report here that the corresponding scr genes for a phosphoenolpyruvate-dependent sucrose:phosphotransferase system and a sucrose metabolic pathway are located on a large (ca. 100 kb) conjugative transposon renamed CTnscr94. The self-transmissible element integrates at two specific attachment sites in a RecA-independent way into the chromosome of Escherichia coli K-12 strains. One site was identified within pheV, the structural gene for a tRNA(Phe). Sequencing of both ends of CTnscr94 revealed the presence of the 3' part of pheV on one end such that after integration of the element, a complete pheV gene is retained. CTnscr94 represents, to our knowledge, the first conjugational transposon found in enteric bacteria.     

A single active-site region for a group II intron

Despite the biological importance of self-splicing group II introns, little is known about their structural organization. Synthetic incorporation of site-specific photo-cross-linkers within catalytic domains resulted in functional distance constraints that, when combined with known tertiary interactions, provide a three-dimensional view of the active intron architecture. All functionalities important for both steps of splicing are proximal before the first step, suggestive of a single active-site region for group II intron catalysis.     

Transfer region of a bacteroides conjugative transposon, CTnDOT

Bacteroides species harbor large self-transmissible integrated elements called conjugative transposons (CTns). In this paper, we report the first complete sequence analysis of the transfer region of a Bacteroides CTn. The transfer region contained 17 genes (designated orfA-orfQ). Only 2 of the genes shared sequence similarity with genes in the databases and only 1 of these genes was associated with self-transmissible elements.     

The IStron CdISt1 of Clostridium difficile: molecular symbiosis of a group I intron and an insertion element

The IStron CdISt1 was first discovered as an insertion into the tcdA gene of the clinical isolate C34. It combines structural and functional properties of a group I intron at its 5'-end with those of an insertion element at its 3'-end. Up to date four different types could be found, mainly differing in their IS-element portions. Contrasting classical group I introns, CdISt1 is always integrated in ORFs encoding bacterial protein. In case CdISt1 had only the IS-element function such insertion would inactivate the protein encoded by the host gene. It is only due to the self-splicing activity of the group I intron parts that CdISt1 integration does not abolish protein function. Both elements seem to exist in molecular symbiosis and CdISt1 could thus be a prototype of a novel class of genetic elements. Moreover, integration of the CdISt1 into the genome could be advantageous for the bacterium, a motor function for evolution of bacterial proteins is discussed. In clinical practice CdISt1 might well serve as a tool for epidemiological studies of C. difficile infections.     

A glimpse of the catalytic core of a group II intron

A paper in a recent issue of Science describes the first high-resolution structure of part of the catalytic core of a group II intron that will allow more detailed comparisons between the excision of introns by self-splicing group II introns and by nuclear pre-mRNA introns.     

Targeted gene disruption by use of a group 11 intron （targetron） vector in Clostridium acetobutylicum

Dear Editor： Clostridium acetobutylicum, a gram-positive, anaerobic, spore-forming bacterium, is capable of using a wide variety of carbon sources to produce acetone, butanol and ethanol. To improve solvent productivity of C. acetobutylicum, metabolic engineering is considered as a useful tool in developing strains with industrially desirable character-istics. However, to date, there are few useful methods for genetic manipulation of C. acetobutylicum, especially for gene disruption. To our knowledge, two types of vectors, including non-replicative and replicative integrative plasmids, have been developed for gene-inactivation in C. acetobutylicum. By using non-replicative integrative plasmids, buk and solR genes of C. acetobutylicum were inactivated [1,2]. However, due to their low frequencies of transformation and recombination, the non-replicative integrative plasmids are usually transformed at less than 1 integrative transformant per mg plasmid DNA. To obtain the integrative mutant, it may require higher transformation frequencies up to 10^5, but the typical transformation fre-quencies were reported at 10^3 [3].[第一段]