Characterization of the transposon carrying the STII gene of enterotoxigenic Escherichia coli

Summary The Escherichia coli enterotoxin STII gene is carried by Tn4521. The terminal repeats of Tn4521 are composed of IS2 sequences; however, neither repeat is a complete IS2. In order to determine how this seemingly defective transposon could transpose, mutations were generated within Tn4521 to determine the regions essential for transposition. The left terminal repeat region was found to be non-essential, but the right terminal repeat area was demonstrated to be crucial for transposition. Within the right terminal repeat area is an open reading frame (ORF), capable of encoding a 159 amino acid protein, which was shown by frameshift mutation analysis to be required for transposition. This protein may be the transposase of Tn4521. A pair of 11 bp repeat sequences flanking the ORF was also found to be important. The right 11 bp repeat is part of the left IS2 terminal sequence, and the left 11 bp repeat is located about 300 bp upstream from the right IS2 terminal sequence located within the right terminal repeat region. The results of this study suggest that Tn4521 is a functional transposon and that the sequence including this pair of 11 bp sequences plus the intervening sequence is a transposable element which may be responsible for Tn4521 transposition.     

Isolation and cloning of Streptomyces terminal fragments

Streptomyces species have a linear chromosome of approximately 8 Mb in size. Many strains also carry linear plasmids. Most of these linear elements contain terminal proteins covalently bound to the 5 ends of the DNA. Using a method for the visualisation of terminal DNA fragments in agarose gels, it was possible to see three fragments in S. rimosus and five fragments in S. avermitilis. The method was also used to clone the 298 bp BamHI fragment carrying the left end of plasmid SLP2. Analysis of the sequence showed that the end resembled other Streptomyces chromosome and plasmid ends, but there were eight palindromes (instead of seven) and a tandem duplication of a 14 bp sequence.     

Progressive rearrangement of telomeric sequences added to both the ITR ends of the yeast linear pGKL plasmid

Relocation into the nucleus of the yeast cytoplasmic linear plasmids was studied using a monitor plasmid pCLU1. InSaccharomyces cerevisiae, the nuclearly-relocated pCLU1 replicated in a linear form (termed pTLU-type plasmid) which carried the host telomeric repeats TG_1–3 of 300–350 bp at both ends. The telomere sequences mainly consisted of a major motif TGTGTGGGTGTGG which was complementary to part of the RNA template of yeast telomerase and were directly added to the very end of the pCLU1-terminal element ITR (inverted terminal repeat), suggesting that the ITR end played a role as a substrate of telomerase. The telomere sequences varied among isolated pTLU-type plasmids, but the TG_1–3 organization was symmetrically identical on both ends of any one plasmid. During cell growth under non-selective condition, the telomeric repeat sequences were progressively rearranged on one side, but not on the opposite side of pTLU plasmid ends. This indicates that the mode of telomeric DNA replication or repair differed between both ends. Clonal analysis showed that the intense rearrangement of telomeric DNA was closely associated with extreme instability of pTLU plasmids. Published: February 17, 2003     

The nucleotide sequence of the mercuric resistance operons of plasmid R100 and transposon Tn501: further evidence for mer genes which enhance the activity of the mercuric ion detoxification system

Summary The DNA sequences of the mercuric resistance determinants of plasmid R100 and transposon Tn501 distal to the gene (merA) coding for mercuric reductase have been determined. These 1.4 kilobase (kb) regions show 79% identity in their nucleotide sequence and in both sequences two common potential coding sequences have been identified. In R100, the end of the homologous sequence is disrupted by an 11.2 kb segment of DNA which encodes the sulfonamide and streptomycin resistance determinants of Tn21. This insert contains terminal inverted repeat sequences and is flanked by a 5 base pair (bp) direct repeat. The first of the common potential coding sequences is likely to be that of the merD gene. Induction experiments and mercury volatilization studies demonstrate an enhancing but non-essential role for these merA-distal coding sequences in mercury resistance and volatilization. The potential coding sequences have predicted codon usages similar to those found in other Tn501 and R100 mer genes.     

The conjugative plasmid SLP2 of Streptomyces lividans is a 50 kb linear molecule

The SLP2 plasmid had previously been demonstrated genetically to exist In Streptomyces lividans by its ability to promote conjugation and to elicit‘pocks’on recipient (SLP2^?) cultures, but it had not been physically detected. Using pulsed-field gel electrophoresis, a 50kb linear DNA was isolated from SLP2⁺ but not SLP2^? strains of S. lividans, and from Streptomyces coelicolor and Streptomyces parvulus strains to which SLP2 had been transferred by conjugation or transformation. We conclude that this linear DNA is SLP2. The terminal fragments of SLP2 were cloned. The determined sequences revealed a 44 bp imperfect terminal inverted repeat. The terminal 12 bp sequence of SLP2 was identical to those of two other Streptomyces linear plasmids, pSLA2 and pSCL, and similar to the terminal sequences of another Streptomyces linear plasmid, SCP1. The termini of SLP2 DNA were resistant to digestion by λ exonuclease and ExoIII. A truncated (probably crippled) copy of Tn4811 is present on the plasmid. While the SLP2 plasmid exists as a tree form in the host, a 15.7 kb sequence corresponding to the segment of SLP2 from Tn4811 to the right terminus is also present (at a copy number similar to the free form) elsewhere in the genome of S. lividans. Furthermore, SLP2 is partially homologous to a newly discovered 650 kb linear plasmid in S. parvulus.     

A plasmid sequence from Rhizobium leguminosarum 300 contains homology to sequences near the octopine TL-DNA right border

Summary The DNA sequence from a Rhizobium leguminosarum 300 (RL300) plasmid that contains homology to the T_c-DNA of Agrobacterium tumefaciens is described. The RL300 sequence has 78% homology to a 359 bp sequence in the T_c-DNA of pTi15955. The RL300 homology starts approximately 100 bp from the 24 bp border sequence of the T_L-DNA and ends approximately 3 bp from an IS66 homolog in the T_c-DNA. An unusual feature of the RL300 homology is the presence of 81 bp direct repeats with T_c-DNA homology, separated by 201 bp. One end of each direct repeat has a 12 bp palindrome. Four cloned sequences of RL300 with homology to the T DNA region were hybridized to plasmid lysates of RL300 derivatives to determine the source of each plasmid. The sequenced homolog, originally on pRH228, was isolated from pRL7JI; the other 3 homologs were isolated from the transmissable plasmids pRL7JI (pRH235) and pRL8JI (pRH235 and pRH236).     

A new insertion element, ISH26, from Halobacterium halobium

Summary A new halobacterial insertion element, ISH26, is described which occurs in the genome of Halobacterium halobium NRC817 in at least seven copies. A copy of ISH26 was isolated from the bacterio-opsin gene (bop) of the Bop mutant M140 of strain H. halobium R1 and characterized in more detail. It shows typical structural features of a transposable element with 16 pb terminal inverted repeat sequences and an 11 bp duplication of the target site. Two partially overlapping open reading frames (ORFs) are contained in the sequence of ISH26 on one strand. The terminal 16 bp inverted repeat of ISH26 is almost identical to the first 16 bp of the terminal inverted repeat of the ISH2 insertion element. The remaining sequences of ISH26 and ISH2 are entirely different. Two size variants of ISH26, 1,384 bp and 1,705 bp in size, are found in the H. halobium genome. The larger one (ISH26-1) contains an imperfect duplication of 321 bp at one end of ISH26.     

The region essential for efficient autonomous replication of pSa in Escherichia coli

Summary Comparative analyses were made between plasmid pSa17, a deletion derivative of pSa that is capable of replicating efficiently in Escherichia coli and plasmid pSa3, a derivative that is defective for replication. By comparing the restriction maps of these two derivatives, the regions essential for replication and for stable maintenance of the plasmid were determined. A 2.5 kb DNA segment bearing the origin of DNA replication of pSa17 was sequenced. A 36 kDa RepA protein was encoded in the region essential for replication. Downstream of the RepA coding region was a characteristic sequence including six 17 bp direct repeats, the possible binding sites of RepA protein, followed by AT-rich and GC-rich sequences. Furthermore, an 8 bp incomplete copy of the 17 bp repeat was found in the promoter region of the repA gene. Based on the hypothesis that RepA protein binds to this partial sequence as well as to intact 17 bp sequences, an autoregulatory system for the synthesis of RepA protein may be operative. Another open reading frame (ORF) was found in the region required for the stability of the plasmid. The putative protein encoded in this ORF showed significant homology to several site-specific recombination proteins. A possible role of this putative protein in stable maintenance of the plasmid is discussed.     

Complete nucleotide sequence of a quinolone resistance gene (qnrS2) carrying plasmid of Aeromonas hydrophila isolated from fish

Aeromonas hydrophila strain AO1 isolated from an infected fish was found to be resistant to several quinolones. A plasmid isolated from the strain AO1, termed pBRST7.6, was cloned and sequenced and shown to be 7621 bp in length with a GC content of 60%. Further analysis confirmed that it contained a gene with 100% identity to qnrS2 genes described in plasmids associated with other Aeromonas species, the product of which usually confers increased resistance to quinolones. The plasmid backbone contained a replication initiation module (repA repC) belonging to the IncQ-family and two genes (mobC and mobB), the products of which are putatively involved in plasmid mobilization. Putative iteron-based origin of replication and characteristic oriT like sequences were also present in the plasmid. The result suggests that Aeromonas spp. carrying plasmids with quinolone resistance genes are potential reservoirs of antimicrobial resistance determinants in the environment.     

A transposon-like sequence with short terminal inverted repeats in the nuclear genome of Chlamydomonas reinhardtii

A 1.2 kb DNA sequence, flanked by a potential seven base target-site duplication, was found inserted into a TOC1 transposable element from Chlamydomonas reinhardtii. The insertion sequence, named TOC2, is a member of a family of repeated DNA sequences that is present in all the C. reinhardtii strains tested. It resembles class II transposable elements: it possesses short 14 bp imperfect terminal repeats that begin AGGAGGGT, and sub-terminal direct repeats located within 250 bp of the termini. No large open reading frames were found. The terminal bases and length of target-site duplication are important in classifying transposable elements. On this basis TOC2 does not fall readily into existing families of class II transposable elements found in plants.     

Termini and telomeres in T-DNA transformation

A T-DNA vector for plant transformation has been constructed in which the cloning site is located 9 bp from the right-border (RB) end and 27 bp from the left-border (LB) end. In this vector cloned DNA homologous to plant chromosomal sequences is located at the T-DNA termini, and will thus be exposed by even limited exonucleolysis in planta. The arabidopsis ADH (alcohol dehydrogenase) locus was mobilized from Agrobacterium, and integration into the recipient genome was studied. Despite the terminal location of ADH homology in this vector, the T-DNA integrated essentially at random in the Arabidopsis genome rather than at the endogenous ADH locus. T-DNA integration was blocked, however, when Arabidopsis telomeric sequences were added to the construct at each end of the ADH homology. Thus the predominant mode by which incoming T-DNA is integrated into the continuity of chromosomal DNA involves free DNA ends, but, in contrast to modes of recombination such as gap repair, does not involve extensive terminal DNA sequence homology.     

Interplasmidic illegitimate recombination in Bacillus subtilis

Summary The illegitimate recombination between Staphylococcus aureus plasmids pE194 (or pGG20, the hybrid between pE194 and Escherichia coli plasmid pBR322) and pBD17 (plasmid pUB110 without HpaII C-fragment) was studied in Bacillus subtilis. Cointegrates were generated with the frequency of 1–3x10^-8. Among 22 hybrids analysed 9 types of recombinants were found. Nucleotide sequences of all three parental plasmids were involved in intermolecular recombination. Nucleotide sequencing of recombinant DNA junctions revealed that in 8 cases recombination occurred between short homologous regions (9–15 bp). One recombinant was formed using nonhomologous sites. The similarity was demonstrated between nucleotide sequences of the recombination sites of two types of cointegrates and those used for pE194 integration into the B. subtilis chromosome. Possible mechanisms of illegitimate recombination are discussed.     

Distribution and characterization of plasmid-related sequences in the chromosomal DNA of different thermophilic Methanobacterium strains

The genomes of several thermophilic members of the genus Methanobacterium were analyzed for homology to the related restriction-modification plasmids pFVI and pFZ1 from M. thermoformicicum strains THF and Z-245, respectively. Two plasmid regions, designated FR-I and FR-II, could be identified with chromosomal counterparts in six Methanobacterium strains. Multiple copies of the pFVI-specific element FR-I were detected in the M. thermoformicicum strains CSM3, FF1, FF3 and M. thermoautotrophicum H. Sequence analysis showed that one FR-I element had been integrated in almost identical sequence contexts into the chromosomes of the strains CSM3 and AH. Comparison of the FR-I elements from these strains with that from pFVI revealed that they consisted of two subfragments, boxI (1118 bp) and boxII (383 bp), the order of which is variable. Each subfragment was identical on the sequence level with the corresponding plasmid-borne element and was flanked by terminal direct repeats with the consensus sequence A(A/T)ATTT. These results suggest that FR-I represents a mobile element. FR-II was located on both plasmids pFVI and pFZI, and on the chromosome of M. thermoformicicum strains THF, CSM3 and HN4. Comparison of the nucleotide sequences of the two plasmid FR-II copies and that from the chromosome of strain CSM3 showed that the FR-II segments were approximately 2.5–3.0 kb in size and contained large open reading frames (ORFs) that may encode highly related proteins with an as yet unknown function.     

Cloning and Characterization of the Replicon of the Nocardia italica Plasmid, pNI100

A 19-kb plasmid, pNI100, was isolated from Nocardia italica CCRC12359; its replicon was cloned and characterized as having a single open reading frame (ORF) of 1188 bp specifying 396 amino acids (aa). Analyses of the deduced aa sequence of the Rep protein indicated that characteristics of three consensus sequences and a P-loop-like motif in the Rep protein of plasmid pSG5, a conjugative plasmid involving a rolling-circle replication mechanism, were conserved in those of plasmid pNI100. Phenotypically, a pock structure was produced in the regenerated mycelium by introducing pNI100 DNA into the Streptomyces lividans protoplast. This result strongly suggests that pNI100 is a conjugative plasmid and probably replicates by a rolling-circle replication mechanism. By using the replicon of pNI100, a bifunctional plasmid pNI105 that could replicate in both Escherichia coli and S. lividans was constructed and found to be a useful cloning shuttle vector.     

Characterization of <Emphasis Type="Italic">EamaT1</Emphasis>, a member of <Emphasis Type="Italic">maT</Emphasis> family of transposable elements from the earthworm <Emphasis Type="Italic">Eisenia andrei</Emphasis> (Annelida,Oligochaeta)

The maT family is a unique clade within the Tc1-mariner superfamily, and their distribution is to date known as being limited to invertebrates. A novel transposon named EamaT1 is described from the genome of the earthworm Eisenia andrei. The full sized EamaT1 was obtained by degenerate and inverse PCR-based amplification. Sequence analysis of multiple copies of the EamaT1, which consisted of 0.9 and 1.4 kb elements, showed that the consensual EamaT1 with inverted terminal repeats (ITRs) of 69 bp was 1,422 bp long and flanked by a duplicated TA dinucleotide. The EamaT1 is present in approximately 120–250 copies per diploid genome but undergoes an inactivation process as a result of accumulating multiple mutations and is nonfunctional. The open reading frame (ORF) of the EamaT1 consensus encoding 356 amino acid sequences of transposase contained a DD37D signature and a conserved paired-like DNA binding motif for the transposition mechanism. The result of ITRs comparison confirmed their consensus terminal sequences (5′-CAGGGTG-3′) and AT-rich region on the internal bases for ITRs-transposase interaction.     

Isolation and molecular characterization of a cryptic plasmid from <Emphasis Type="Italic">Bifidobacterium longum</Emphasis>

An isolate from the fecal samples of children was identified as Bifidobacterium longum. A plasmid isolated from it pBIFA24 was 4,892 bp with three open reading frames, ORFI, ORFII, and ORFIII. ORFI encoded a replication protein involved in a rolling-circle replication mechanism, and three sets of tandem repeat sequences featuring iteron structure were identified. Secondary structure prediction analysis of ORFII suggested it was a transmembrane protein. ORFIII showed high amino acid sequence identity with some mobilization proteins and contained an oriT sequence.     

The promoter proximal region in the virD locus of Agrobacterium tumefaciens is necessary for the plant-inducible circularization of T-DNA

Summary The formation of crown gall tumours involves the transfer of the T-DNA region of the Ti plasmid from Agrobacterium to plant cells and its subsequent integration into plant chromosomes. When agrobacteria are incubated with plant protoplasts or exudates of plants, the T-DNA region is circularized by recombination or cleavage and rejoining between the 25 bp terminal repeats; the formation of circular T-DNAs is thought to be one step in T-DNA transfer (Koukolikova-Nicola et al. 1985; Machida et al. 1986). We previously showed that the virulence region of the Ti plasmid is required for T-DNA circularization. In the present paper, we examined the circularization event in agrobacteria harbouring octopine Ti plasmids with mutations in various loci of the virulence region. The results clearly demonstrate that the gene(s) encoded in the virD locus are necessary for T-DNA circularization. In particular, the gene(s) present in the region proximal to the virD promoter are essential. We propose that roduct(s) of this gene have recombinase or endonuclease activity which specifically recognizes the 25 bp terminal repeats of T-DNA.