Low target site specificity of an IS6100-based mini-transposon, Tn1792, developed for transposon mutagenesis of antibiotic-producing Streptomyces

To improve transposon mutagenesis of antibiotic-producing Streptomyces, a mini-transposon, Tn1792, was constructed, based on IS6100, originally isolated from Mycobacterium fortuitum. Easily manageable transposition assays were developed to demonstrate inducible transposition of Tn1792 into the Streptomyces genome from a temperature-sensitive delivery plasmid. Introduction of the selectable aac1 gene between the inverted repeats in Tn1792 allowed for both reliable identification of transposition events in Streptomyces, and also subsequent cloning of transposon-tagged sequences in Escherichia coli. This enabled the target site specificity of Tn1792 to be determined at nucleotide resolution, revealing no significant shared homology between different target sites. Consequently, Tn1792 is well suited for random mutagenesis of Streptomyces.     

Mutagenesis of Alcaligenes eutrophus by insertion of the drug-resistance transposon Tn5

Drug-resistance element Tn5 coding for kanamycin resistance was used for mutagenesis of Alcaligenes eutrophus strain H16. The vehicle for introducing Tn5 into A. eutrophus was plasmid pJB4JI harboured by Escherichia coli. Kanamycin-resistant transconjugants occurred at a frequency of approximately 5×10^-8. One third of the transconjugants exhibited other plasmid-coded resistances such as gentamycin and spectinomycin. However, the latter markers were not stably maintained in the new host. Among the kanamycin-resistant transconjugants three classes of mutants were found: (i) Auxotrophic mutants occurred at a frequency of 0.8% and showed requirements for histidine, methionine, aspartate orisoleucine. Out of eleven auxotrophic mutants examined eight reverted to prototrophy. However, none of the revertants was kanamycin-sensitive. (ii) Mutants unable to grow with fructose as the carbon source occurred at a frequency of almost 10%. (iii) Mutants which had lost the ability to grow autotrophically with hydrogen and carbon dioxide were found at a frequency of 1%. Further analyses revealed that this class of mutants was either defective in carbon dioxide fixation or impaired in hydrogen metabolism.     

The Chlamydomonas chloroplast clpP gene contains translated large insertion sequences and is essential for cell growth

Sequence determination of the chloroplast clpP gene from two distantly related Chlamydomonas species (C. reinhardtii and C. eugametos) revealed the presence of translated large insertion sequences (IS1 and IS2) that divide the clpP gene into two or three sequence domains (SDs) and are not found in homologous genes in other organisms. These insertion sequences do not resemble RNA introns, and are not spliced out at the mRNA level. Instead, each insertion sequence forms a continuous open reading frame with its upstream and downstream sequence domains. IS1 specifies a potential polypeptide sequence of 286 and 318 amino acid residues in C. reinhardtii and C. eugametos, respectively. IS2 encodes a 456 amino acid polypeptide and is present only in C. eugametos. The two Chlamydomonas IS1 sequences show substantial similarity; however, there is no significant sequence similarity either between IS1 and IS2 or between these insertion sequences and any other known protein coding sequences. The C. reinhardtii clpP gene was further shown to be essential for cell growth, as demonstrated through targeted gene disruption by particle gun-mediated chloroplast transformation. Only heteroplasmic transformants could be obtained, even under mixotrophic growth conditions. The heteroplasmic transformants were stable only under selection pressure for the disrupted clpP, rapidly segregated into wild-type cells when the selection pressure was removed, and grew significantly more slowly than wildtype cells under phototrophic conditions.     

Heterogeneity in the level of ampicillin resistance conferred by pBR322 derivatives with different DNA supercoiling

Summary Cloning of an EcoRI restriction fragment, containing the 900 bp -terminal sequence of transposon Tn1000, into pBR322, resulted in two plasmids, pICV63 and pICV64, which differed in the orientation of the cloned fragment within the replicon and in the level of ampicillin resistance conferred on the host cell. The DNAs of these plasmids differ in superhelicity and we suggest that a change in supercoiling of pICV63 DNA leads to this plasmid conferring resistance to only low levels of ampicillin, probably by reducing the expression of the bla gene. This hypothesis is supported by the fact that topA or supX mutations, which abolish topoisomerase I, reduce still further the level of resistance to ampicillin of pICV63-containing cells, whereas the gyrB226 compensatory mutation renders these cells more ampicillin resistant. Plasmid pICV63, therefore, enables mutant alleles of genes governing DNA topology to be recognized.     

Precise excision of bacterial transposon Tn5 in yeast

Summary We have demonstrated that precise excision of bacterial transposon Tn5 can occur in the yeast, Saccharomyces cerevisiae. Tn5 insertions in the yeast gene LYS2 were generated by transposon mutagenesis made in Escherichia coli by means of a ::Tn5 vector. Nine insertions of Tn5 into the structural part of the yeast LYS2 gene situated in a shuttle epsiomal plasmid were selected. All the plasmids with a Tn5 insertion were used to transform yeast strains carrying a deletion of the entire LYS2 gene or a deletion of the part of LYS2 overlapping the point of insertion.All insertions inactivated the LYS2 gene and were able to revert with low (about 10^-8) frequencies to lysine prototrophy. Restriction analysis of revertant plasmids revealed them to be indistinguishable from the original plasmid without Tn5 insertion. DNA sequencing of the regions containing the points of insertions, made for two revertants, proved that Tn5 excision was completely precise.     

A cloned DNA fragment from bacteriophage P1 enhances IS2 insertion

Summary A 1.75 kb DNA segment of the bacteriophage P1 genome is known to serve as a preferred target for IS2 insertions. The presence of this fragment in a plasmid expressing the galK gene dramatically increases the proportion of IS2 insertions among spontaneous galK ^- mutants. Subfragments from two different parts of the 1.75 kb segment independently stimulate IS2 insertion, while another subfragment does not. In the plasmids studied IS2 elements not only insert into the cloned P1 fragment but also into parts of the galK gene with similar probability and mostly in one orientation. Many insertion sites are unique but several specific sites within the preferred target are repeatedly used for IS2 integration. The experimental data are compatible with a proposed cooperative mechanism, according to which more than one attracting sequence on the same plasmid might significantly enhance the probability of a particular target region to attract IS2.     

Insertion sequence IS5 contains a sharply curved DNA structure at its terminus

Summary It was demonstrated that insertion sequence IS5 contains a sequence-directed bent (sharply curved) DNA structure at its terminus, close to one of its 16 bp terminal repeats. The minimal number of copies of IS5 related sequences and the locations of the latter on the Escherichia coli K12 W3110 chromosome were determined. Evidence is presented of the occurrence of IS5 mediated translocation and duplication of a large DNA segment on the E. coli chromosome.     

Enhancement of Polyunsaturated Fatty Acid Production by Tn5 Transposon in Shewanella baltica

Transposon Tn5 mutagenesis was used to generate random mutations in Shewanella baltica MAC1, a polyunsaturated fatty acid (PUFA)-producing bacterium. Three mutants produced 3–5 times more eicosapentaenoic acid (EPA 20:5 n−3) compared to the wild type at 10°C. One of the mutants produced 0.3 mg EPA g⁻¹ when grown at high temperature (30°C). Moreover, 2 mg docosahexaenoic acid (DHA 22:6 n−3) g⁻¹ was produced by S. baltica mutants at 4°C. Sequencing of insertion mutation(s) showed 96% homology to trimethylamine N-oxide (TMAO) reductase gene and 85% homology to rRNA operons of E. coli. Tn5 transposon mutagenesis therefore is a suitable technique to increase PUFA formation in bacteria.     

Integration of repeated sequences (pBR322) in the Bacillus subtilis 168 chromosome without affecting the genome structure

The Escherichia coli plasmid pBR322 sequence (4363 bp) was integrated at the met, pro, or leuB locus of the Bacillus subtilis chromosome without duplication of the flanking chromosomal regions. The integrated pBR322 was stably maintained as part of the chromosome regardless of its orientation or location. It was found that a DNA segment as large as 17 kb cloned in pBR322 can be readily transferred to the B. subtilis chromosome by transformation. It was demonstrated that a second pBR322 sequence could be effectively introduced at different regions of the chromosome by sequential transformation using chromosomal DNA isolated from a strain that had already acquired a pBR322 sequence at a different locus. Similarly, a third pBR322 sequence could be introduced. By this method, two or three pBR322 sequences can be incorporated at unlinked loci without affecting the overall structure of the B. subtilis genome.     

Isolation of transposon Tn5 insertion mutants of Rhodobacter capsulatus unable to reduce trimethylamine-N-oxide and dimethylsulphoxide

1) Rhodobacter capsulatus (formerly Rhodopseudomonas capsulata) strain 37b4 was subjected to transposon Tn5 mutagenesis. 2) Kanamycin-resistant transconjugants were screened for their inability to reduce trimethylamine-N-oxide (TMAO) as judged by the lack of alkali production during anaerobic growth on plates containing glucose as carbon source and cresol red as pH indicator. 3) Of 6 mutants examined, all were found to have considerably decreased levels of methylviologen-dependent TMAO reductase activity and dimethylsulphoxide (DMSO) reductase activity. 4) Periplasmic fractions of one of these mutants (DK9) and of the parent strain were subjected to sodium dodecylsulphate polyacrylamide gel electrophoresis. The gels were stained for TMAO-reductase and DMSO-reductase. With the wild-type strain, only a single polypeptide band, M_r=46,000, stained for TMAO and DMSO reductase activity. In mutant DK9 this band was not detectable. 5) In contrast to the parent strain, harvested washed cells of mutant DK9 were unable to generate a cytoplasmic membrane potential in the presence of TMAO or DMSO under dark anaerobic conditions. 6) In contrast to the parent strain, DK9 was unable to grow in dark anaerobic culture with fructose as the carbon source and TMAO as oxidant.Abbreviations TMAO trimethylamine-N-oxide - DMSO dimethylsulphoxide - PMS phenazine methosulphate - cytoplasmic membrane potential     

Target DNA bending is an important specificity determinant in target site selection in Tn10 transposition

The bacterial transposon Tn10 inserts preferentially into specific DNA sequences. DNA footprinting and interference studies have revealed that the Tn10-encoded transposase protein contacts a large stretch of target DNA ( approximately 24 bp) and that the target DNA structure is deformed upon incorporation into the transpososome. Target DNA deformation might contribute significantly to target site selection and thus it is of interest to further define the nature of this deformation. Circular permutation analysis was used to demonstrate that the target DNA is bent upon its incorporation into the transpososome. Two lines of evidence are presented that target DNA bending is an important event in target site selection. First, we demonstrate a correlation between increased target site usage and an increased level of target DNA bending. Second, transposase mutants with relaxed target specificity are shown to cause increased target DNA bending relative to wild-type transposase. This latter observation provides new insight into how relaxed specificity may be achieved. We also show that Ca(2+) facilitates target capture by stabilizing transposase interactions with sequences immediately flanking the insertion site. Ca(2+) could, in theory, exert this effect by stabilizing bends in the target DNA.     

Expression of Tn5-derived kanamycin resistance in the fungus Phycomyces blakesleeanus

Summary A plasmid, carrying the Tn5 gene for kanamycin resistance lacking its own promoter, has successfully been used in the selection of DNA sequences of the fungus Phycomyces blakesleeanus having promoter activity in Escherichia coli. Many of these sequences were also effective in promoting resistance to kanamycin when the corresponding chimeric plasmids were introduced in the fungus via spheroplast transformation. The selected phenotype was easily propagated through vegetative spores and behaved as a stable character since it was not appreciably lost in the absence of selection.     

Isolation and genetic structure of IS112, an insertion sequence responsible for the inactivation of the SalI restriction-modification system of Streptomyces albus G.

Summary IS112 is a transposable element identified in Streptomyces albus G by its frequent mutagenic insertion into the genes for the SalI restriction-modification system. IS112 is present in several copies in the genome of S. albus G. Homologous sequences were detected in other Streptomyces strains. Sequence analysis revealed that IS112 has a length of 883 by with a GC content of 67.4%. The copy that was isolated contained imperfect inverted repeats (16/20 match) at its ends and was flanked by a 2 by duplication at the target site, which was located within the gene (salIR) for the Sall endonuclease. A long open reading frame (ORF) encoding a putative polypeptide of 256-253 amino acids spans almost the entire sequence. Significant homology was detected between this polypeptide and that corresponding to ORFB of IS493, an insertion sequence recently isolated from Streptomyces lividans 66.

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Second-element turn-on of gene expression in an IS1 insertion mutant

Summary To learn more about the ways in which genes silenced by insertion mutations can be reactivated, we have undertaken a systematic investigation of Gal⁺ revertants of the polar mutant galOP-306::IS1 in Escherichia coli K12. The selective conditions used excluded reversion to wild type by precise excision of IS1. In this system (which resisded on a multi-copy plasmid) reversion to the Gal⁺ phenotype occurred with a frequency of about 10^-7 per cell and per generation. Analysis of the revertants revealed that — with the single exception of the previously published chromosomal mutant sis1 — alterations in the structure of IS1 lead to reactivation of gal operon expression. These events fall into four classes: (I) insertion of IS2 at position 327 in IS1, insertion of IS2 at position 687 in IS1, (III) insertion of a hitherto undetected mobile element, IS150, at position 387, (IV) a 16-bp deletion encompassing IS1 coordinates 553–568. Of some 200 independent reversion events studied, all but one were of types I–III i.e. they involved the intervention of a second mobile element.     

Replicon fusions promoted by insertion sequences on Pseudomonas cepacia plasmid pTGL6

Summary Plasmid pMR5 (pRP1ts) failed to replicate in Pseudomonas cepacia at 47° C. Selection at this temperature for maintenance of tetracycline resistance associated with this plasmid allowed isolation of cointegrate plasmids formed by fusion of pMR5 with pTGL6, a 170 kb plasmid harbored by P. cepacia 249. In the cointegrate plasmids pTGL100, pTGL101, and pTGL102, different regions of pTGL6 were involved in fusion with the same tra-2-containing region of pMR5. Formation of all three plasmids was promoted by insertion sequences on pTGL6, which were also represented in the chromosome.Two different copies of a 1.3 kb element, IS401, were involved in formation of pTGL100 and pTGL101. Another insertion sequence, IS402 (1 kb), promoted the fusion which formed pTGL102. Southern hybridization experiments indicated that each of the cointegrate plasmids contained an additional copy of the fusion mediating element. Plasmid pTGL100 was observed to resolve into two independent replicons: pTGL6 and pTGL105 (pMR5::IS401), a novel derivative of pMR5 containing a copy of IS401.The third cointegrate plasmid, pTGL102, evolved in two steps: fusion of pTGL6 and pMR5 mediated by IS402, and transposition of IS411 (1.9 kb) to a region of pMR5 distinct from that involved in the fusion. Plasmid pTGL6 contained one copy of IS402 and IS411 while pTGL102 contained two copies of each of these elements.     

Defects in cytochrome cd 1-dependent nitrite respiration of transposon Tn5-induced mutants from Pseudomonas stutzeri

Mutants with defective respiratory nitrite utilization (Nir^- phenotype) were obtained by transposon Tn5 insertion into genomic DNA of the ZoBell strain of Pseudomonas stutzeri. Three representative mutants were characterized with respect to their activities of nitrite and nitric oxide reduction, cytochrome cd ₁ content, and pattern of soluble c-type cytochromes. Mutant strain MK201 over-produced cytochrome c ₅₅₂ about fourfold by comparison with the wild type, but possessed an in vitro functional cytochrome cd ₁. Mutant strain MK202 lacked cytochrome cd ₁ and, simultaneously, had low amounts of cytochrome c ₅₅₂ and the split -peak c-type cytochrome. Strain MK203 synthesized nitrite reductase defective in the heme d ₁ prosthetic group. Irrespective of these biochemically distinct Nir^- phenotypes, all mutants preserved the nitric oxidereducing capability of the wild type. The mutant characteristics demonstrate that cytochrome cd ₁ is essential for nitrite respiration of P. stutzeri and establish the presence of a nitric oxide-reducing system distinct from cytochrome cd ₁. They also indicate the functional or regulatory interdependence of c-type cytochromes.     

Cointegrate formation by IS50 requires multiple donor molecules

Summary The insertion sequence, IS50R, promotes cointegrate formation between a lambda::IS50R phage and the chromosome of Escherichia coli strain C. We show that formation of cointegrates mediated by IS50R between the non-replicating phage genome and the bacterial chromosome requires multiple donor molecules and depends on homologous recombination functions. We conclude that the two copies of IS50 present in the cointegrate originate in two different molecules. Thus, the existence of the cointegrate structure cannot be used as evidence for replication of IS50 sequences during IS50 transposition.