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We developed a novel PCR-fingerprinting system for differentiation of enterobacterial strains using a single oligonucleotide primer IS1tr that matches the inverted terminal repeats of the IS1 insertion element. Compared to widely used BOX-PCR and ribotyping methods, our system features higher resolution allowing differentiation of closely related isolates that appear identical in BOX-PCR and ribotyping but differ in their phage sensitivity. The IS1-profiling system is less sensitive to the quality of the material and equipment used. At the same time, BOX-PCR is more universal and suitable for bacterial strain grouping and reconstruction of the low-distance phylogeny. Thus, our system represents an important supplement to the existing set of tools for bacterial strain differentiation; it is particularly valuable for a detailed investigation of highly divergent and rapidly evolving natural bacterial populations and for studies on coliphage ecology. However, some isolates could not be reliably differentiated by IS1-PCR, because of the low number of bands in their patterns. For improvement of IS1-fingerprinting characteristics, we offer to modify the system by introducing the second primer TR8834 hybridizing to the sequence of a transposase gene that is widely spread in enterobacterial genomes.

An IS element, termed ISCg2, was identified in the chromosome of Corynebacterium glutamicum ATCC 13032. After screening a cosmid library of the C. glutamicum ATCC 13032 genome, six copies of ISCg2 including their flanking regions were sequenced and analyzed. ISCg2 is 1636 bp in length and has 26-bp imperfect inverted repeats flanked by 3-bp direct repeats. By comparisons with other IS elements,ISCg2 was classified as a member of the IS30 family of insertion sequences. The six copies of ISCg2 were identical at the nucleotide level and were located in intergenic, AT-rich regions of the chromosome. The regions in which the six copies of ISCg2 were inserted displayed significant similarities. This similarity extends over a region of 65 bp, which was assumed to be the target region for ISCg2. Interestingly, live of the six copies of ISCg2 were located adjacent to genes that may be involved in aspartate and glutamate metabolism or its regulation. Investigation of the distribution of ISCg2 showed that the IS element is restricted to certain C. glutamicum strains. Analysis of various integration regions indicates active transposition of ISCg2. in C. glutamicum.

The nucleotide sequence of an insertion element in R-factor R1767 derivative pBP11 was determined. It is almost overall identical with IS15 delta, IS26 and IS46. Like IS46 it flanks one end of the sul-bla determinant and is involved in amplification of the resistance cassette. The significance for this process of a palindrome comprising part of IS15 delta IV is discussed.

International audience; Some strains of Lactobacillus sakei are known to produce the bacteriocin sakacin P, encoded by the spp gene cluster. In strains unable to produce sakacin P, spp homologues were observed. The analysis of 15 strains not producing sakacin P revealed that all contained a region corresponding to a part of sppKR encoding the regulatory elements for sakacin P production. In some strains homologues of sppE and sppT, responsible for sakacin P transport, and the sakacin P structural gene sppA and its immunity gene spiA, were also present. The sequence of the chromosomal spp-related gene cluster was determined in two non-producing strains: L. sakei Lb790 and L. sakei 23K. The L. sakei Lb790 spp gene cluster encompasses genes homologous to sppK, sppR, sppT and sppE. In L. sakei 23K, only sppK and sppR homologues were present. The sppK homologues appeared non-functional as they contained mutations and/or an insertion element. In addition to the spp homologues, several small putative genes were found in the gene clusters of the two strains. Some were similar in both strains, and their organization suggests a mosaic structure resulting from successive rearrangements. Transcriptional analysis showed that the genes of the L. sakei Lb790 spp cluster were expressed when genes encoding an operative sakacin P regulatory system were introduced in this strain, thus complementing the inactive sppK gene. Expression experiments also suggested that some of the spp homologues maintained their function in non-producing strains.

A new insertion sequence (IS2112) was identified in the genome of the 1-haloalkane-utilizing bacterium Rhodococcus rhodochrous NCIMB 13064. The insertion element is 1415 bp long, does not contain terminal inverted repeats, and is not flanked by directly repeated sequences. IS2112 belongs to the IS110 family of transposable elements, and forms a separate subfamily, along with IS116, Two copies of IS2112 were found in R, rhodochrous NCIMB 13064 and one, two or three copies of a similar sequence were detected in five other 1-haloalkane-degrading Rhodococcus strains. There were no sequences homologous to IS2112 found in the l-haloalkane-degrading 'Pseudomonas pavonaceae' 170 and Rhodococcus sp, HA1 or in several Rhodococcus strains which do not utilize haloalkanes, IS2112 was originally found in plasmid pRTL1 of R. rhodochrous NCIMB 13064 which harbours genes encoding utilization of l-haloalkanes, and was located 5 kbp upstream of the haloalkane dehalogenase gene (dhaA), Although the second copy of IS2112 in strain NCIMB 13064 was also present on the pRTL1 plasmid, these sequences do not apparently comprise a single composite transposon encoding haloalkane utilization. An analysis of derivatives of NCIMB 13064 revealed that IS2112 was involved in genome rearrangements. IS2112 appeared to change its location as a result of transposition and as a result of other rearrangements of the NCIMB 13064 genome.

The Mycobacterium avium complex (MAC) includes the closely related species M. avium, M. intracellulare and M. paratuberculosis. The insertion elements IS900, IS901, IS1245 and IS1311 were used as DNA probes to characterize by restriction fragment polymorphisms (RFLPs) eight reference strains, three animal isolates of M. paratuberculosis from outside New Zealand and 61 selected New Zealand MAC isolates from cattle, deer, pigs, sheep and humans. IS900 was found only in strains of M. paratuberculosis. All MAC strains contained IS1311 and the RFLPs associated with this insertion element divided M. paratuberculosis strains into the same groups as IS900 RFLPs. Except for M. paratuberculosis, all MAC strains contained IS1245 and the majority of those from lesions in cattle, deer and pigs also contained IS901. All animal strains containing IS901 had the same RFLPs with IS901, IS1245 and IS1311. In three cases, these apparently identical strains could be differentiated by restriction fragment analysis with BstEII. IS901 was not present in four human isolates or in isolates from deer without lesions. These results indicate that a very closely related group of strains causes the majority of non-paratuberculosis MAC lesions in animals in New Zealand.

The effects of two mutagens ENU and MMS characterized by different alkylation patterns have been studied on the reversion of an MR-induced singed mutation to wild-type. Reversion of this unstable singed mutation under the influence of MR is assumed to represent the removal or transposition of an insertion element. Since MR acts primarily in spermatogonia, the mutagens were fed to 1st instar larvae. Recessive lethal tests were carried out simultaneously to calibrate for the mutagenic effectiveness of the chemicals. For both powerful mutagens, it was observed that the frequency of reversion remained far below of what would have been expected on the basis of the mutagenic effectiveness, as registered in the lethal tests. Thus 1 mM ENU, 5 mM and 10 mM MMS did not affect the reversion frequency at all, and with 3 mM ENU only a doubling of the reversion frequency was observed, despite a 5-fold increase in the lethal frequency. The threshold at 1 mM EMU and the low effectiveness of 3 mM on the reversion process are taken as an indication that ENU affected the transposition process in an indirect manner, rather than the excision events themselves. The data obtained with Drosophila are consistent with the microbial observations in that mutation involving removal or transposition of an insertion element is not affected by mutagenic treatments. This finding may have consequences for the evaluation of induced genetic damage on the basis of the spontaneous load of genetic detriment in man. An incidental observation was that non-MR Cy larvae exhibited greater sensitivity to the induction of recessive lethals by MMS than MR-individuals.

The nucleotide sequence of the insertion element IS200 has been determined partially, including the junctions between the element and the host chromosome at the insertion site. At most, two bases (A-A) are found repeated at the junctions and could be duplications of host sequences generated by the insertion of the element. No obvious sequence repeats, either direct or inverted, have been detected between the sequences just within the two ends of the element. The element is an extremely strong block to host transcription across the insertion site. A sequence similar to known transcription termination signals was found just within the element near the right end. Removal of less than 50 base-pairs at the right end of the element abolishes the transcription block. The putative terminator sequence is located within this 50 base-pair region. Genetic studies suggest that the element contains a promoter located more than 93 base-pairs from its left end. The proposed promoter and terminator are in proper orientation to form an internal transcription unit.

Differential scanning calorimetry of a linear double-stranded plasmid DNA showed a multimodal profile characteristic of the plasmid. This demonstrates that the helix-coil transition of DNA occurs blockwise along the DNA chain. From the results of the analysis of plasmid derivatives having deletions and duplications, the peaks of the DSC curves are assigned at regional sequences including DNA insertion element IS 1 present in the plasmid. The small change in a plasmid DNA sequence caused by cleavage of a plasmid with different restriction enzyme was also clearly reflected on their DSC curves.

Genomic clones from a Northern Flint Line of Zea mays are shown to carry a 700 bp insertion, Cin 1. The Cin 1 insert is not present in the homologous, unique DNA fragment of a midwestern maize line. However, Cin 1 is present in multiple copies in both lines.

MR is a frequently occurring mutator in Drosophila melanogaster inducing mutation by the incorporation of insertion sequences. In the presence of MR a mutation at the singed (sn) locus induced by MR, reverts to wild-type at a high frequency of 1.7%. This reversion system which presumably involves the removal of an insertion element, was used to study the effects of defective DNA repair. Thus, reversion frequencies were compared in progeny of flies with mei-9, deficient for excision repair, mei-41, deficient for post-replication repair, or with both mei-9 and mei-41. The data show that under conditions of defective DNA repair, the frequency of MR-mediated reversion, is consistently decreased in comparison to repair-proficient conditions. This effect is explained by assuming that defective repair interferes with some steps in the process of reverse mutation involving the removal of insertion sequences. The observed reduction in reversion frequency may well result from selective elimination of cells in which the reversion process has not been completed.

Southern blotting using a 5′-proximal probe of the Saccharomyces cerevisiae CYR1 gene has revealed heterogeneity in laboratory strains. It is demonstrated that strain AB320 contains a Ty1 element inserted in the promoter region of CYR1. The Ty1 orientation suggests that transcription of CYR1 is initiated downstream from the insertion region.

Two previously characterized duplications in the insertion element IS2 of Escherichia coli can be described as end-products resulting from endonucleolytic cleavages of DNA at regions of potential secondary structure and subsequent DNA synthesis.

IS1-mediated insertion and deletion formation occur preferentially into A+T-rich regions of DNA of bacteriophate P1 and of the r-determinant of the R plasmid NR1. The significance of this correlation is discussed in view of other published data.

Total DNAs from twelve natural isolates of Escherichia coli from animals and humans were examined by hybridization with a probe for IS1. Considerable variation in copy number was found. In the case of two strains isolated from the same individual, one strain contained no copies of IS1 and the other, much greater than 30. Evidence was also obtained for the existence of IS1-like elements (iso-IS1s) of greater than 15% sequence divergence relative to the IS1 from antibiotic resistance plasmid R100.

The insertion element ISR1 was isolated from the soil bacterium R. lupini. In this strain, ISR1 shows a very strong affinity to plasmid RP4. It causes RP4 mutations at the strikingly high frequency of 10(-2) to 10(-1), either by the integration itself or by generating deletions. In E. coli, ISR1 seems to be inactive. No evidence could be obtained for a promoter site on ISR1 or for an ISR1-encoded protein. Our results indicate, however, an ISR1-specific termination signal for either transcription or translation.

The insertion sequence IS666 was isolated from Mycobacterium avium strain 101. IS666 is a 1474 bp insertion sequence belonging to the IS256 family, that includes IS6120 from Mycobacterium smegmatis, IS1166 and IS1295 from Rhodococcus sp. IGTS8, IST2 from Thiobacillus ferrooxidans, IS256 from Staphylococcus aureus, and ISRm3 from Rhizobium meliloti. IS666 has 24 bp imperfect inverted repeats that fit the consensus described for the family, and generates 9 bp duplications upon insertion into the host DNA with no apparent specificity in the target sequence. In contrast with its two closest homologues, IS1166 and IS6120, IS666 contains a single ORF that would codify a transposase of 434 aa. IS666 is restricted to M. avium, where it is present in 21% of the isolates in a number ranging between 1 to 7 copies.