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12 recordsLinked to original sources
PubMed1979

Transposition of Tn7 in Pseudomonas aeruginosa and isolation of alk::Tn7 mutations.

Conjugal crosses with Pseudomonas aeruginosa donors carrying the CAM-OCT and RP4::Tn7 plasmids result in transfer of the Tn7 trimethoprim resistance (Tp(r)) determinant independently of RP4 markers. All Tp(r) exconjugants which lack RP4 markers have CAM-OCT genes and therefore must have received CAM-OCT::Tn7 plasmids formed by transposition of Tn7 from RP4::Tn7 to CAM-OCT. Most crosses yield exconjugants carrying mutant CAM-OCT plasmids which no longer determine either camphor or alkane utilization and thus appear to carry Tn7 inserts in the cam or alk loci, respectively. Transduction and reversion experiments indicated that at least 13 alkane-negative, camphor-positive, Tp(r) CAM-OCT::Tn7 plasmids carry an alk::Tn7 mutation. Determination of linkage between the alk mutation and the Tp(r) determinant of Tn7 on these plasmids is complicated by the presence of multiple copies of the Tn7 element in the genome. Generalized transduction will remove Tn7 from a CAM-OCT alk::Tn7 plasmid to yield alk(+) cells which carry no Tp(r) determinant on the CAM-OCT plasmid (as shown by transfer of the plasmid to a second strain). But the transduction to alk(+) does not remove all Tp(r) determinants from the genome of the recipient cell because the alkane-positive transductants remain trimethoprim resistant. Thus, it appears that copies of Tn7 can accumulate in the genome of P. aeruginosa (CAM-OCT alk::Tn7) strains without leaving their original site. This result is consistent with transposition models that involve replication of the transposable element without excision from the original site.

Alkanes↗
PubMed2003

Alternative interactions between the Tn7 transposase and the Tn7 target DNA binding protein regulate target immunity and transposition.

The Tn7 transposon avoids inserting into a target DNA that contains a pre-existing copy of Tn7. This phenomenon, known as 'target immunity', is established when TnsB, a Tn7 transposase subunit, binds to Tn7 sequences in the target DNA and mediates displacement of TnsC, a critical transposase activator, from the DNA. Paradoxically, TnsB-TnsC interactions are also required to promote transposon insertion. We have probed Tn7 target immunity by isolating TnsB mutants that mediate more frequent insertions into a potentially immune target DNA because they fail to provoke dissociation of TnsC from the DNA. We show that a single region of TnsB mediates the TnsB-TnsC interaction that underlies both target immunity and transposition, but that TnsA, the other transposase subunit, channels the TnsB-TnsC interaction toward transposition.

Amino Acid Sequence↗
PubMed2026

Emergence of a Tn7-associated blaVIM-1 within IncC plasmids in ST46 Providencia stuartii from Northern Italy.

OBJECTIVES: To characterize the genomic features and resistance determinants of carbapenem-resistant Providencia stuartii isolates circulating in Northern Italy, with a focus on the genetic context of blaVIM-1. METHODS: Five P. stuartii isolates collected between 2022 and 2024 from interconnected healthcare facilities underwent molecular characterization. Antimicrobial susceptibility testing was performed according to EUCAST 2025 criteria. Whole-genome sequencing was conducted using Illumina technology, followed by resistome, plasmidome, and phylogenetic analyses. Comparative genomics was used to investigate the genetic environment of blaVIM-1. RESULTS: All isolates belonged to the emerging ST46 lineage and exhibited an extensively drug-resistant phenotype, remaining susceptible only to amikacin. The blaVIM-1 gene was located on a &#x223c;100 kb mobilizable IncC plasmid shared across all isolates. Notably, blaVIM-1 was embedded within a 13 kb Tn7 transposon carrying a complete set of transposition genes and inserted into a class 1 integron structure. Comparative analysis revealed no full homology with previously described IncC plasmids, suggesting a novel genetic arrangement. Phylogenetic analysis demonstrated close relatedness among Italian isolates (<33 SNPs), supporting local clonal circulation, while showing clear separation from previously described NDM-producing ST46 strains. CONCLUSIONS: This study describes the rare association of blaVIM-1 with a Tn7 transposon in P. stuartii, highlighting the genomic plasticity of IncC plasmids and their role in the emergence of new resistance platforms. The identification of this structure in a high-risk lineage underscores the potential for further dissemination of carbapenem resistance in healthcare settings.

IncC↗
PubMed2026

Improving a Tn7-based luciferase reporter system for promoter activity studies.

Single-copy chromosomal integration systems are essential tools for stable gene expression in bacteria, minimizing variability associated with plasmid-based systems. The Tn7 transposon-based system is widely used for this purpose, and one important application is the generation of reporter systems, such as the bioluminescent luxCDABE operon (lux). However, current Tn7-lux vectors exhibit undesirable background expression due to cryptic promoter activity near the antibiotic resistance cassette. Here, we report the construction of an improved vector, pTn7-lux-B0015, incorporating a strong synthetic terminator upstream of the lux operon. This modification effectively eliminated basal luminescence in the absence of a promoter and enhanced the dynamic range and responsiveness of the reporter. Using a Xanthomonas citri type III secretion system promoter as a model, we demonstrate that pTn7-lux-B0015 enables more accurate detection of gene expression under relevant growth conditions. This vector provides a valuable tool for the development of precise and tunable bioluminescent reporters in bacterial systems.

Promoter Regions, Genetic↗
PubMed1977

Two naturally occurring transposons indistinguishable from Tn7.

Two plasmids from different sources, determining trimethoprim and streptomycin resistances, harbour transposons which we designate Tn71 and Tn72. These transposons are indistinguishable from Tn7 in the resistances determined, in their molecular masses and in the number and relative positions of their sites susceptible to the restriction enzymes EcoRI, HindIII and BamHI. We conclude that Tn7 has been naturally spread among plasmids.

Base Sequence↗
PubMed1978

Polarity of mutations induced by insertion of transposons Tn5, Tn7 and Tn10 into the nif gene cluster of Klebsiella pneumoniae.

Three new genes nifM, nifI and nifN have been mapped in the nif gene cluster of Klebsiella pneumoniae and a fourth gene nifJ has been confirmed as being a separate cistron. Polar nif mutations were obtained by transposition of Tn7 to plasmid pRD1, and of Tn5 and Tn10 to plasmid pMF100, a derivative of pRD1. Complementation analysis of the nif::Tn mutants led to the identification of at least six transcriptional units: nifB; nifA; nifJ; nifH, nifD and nifK; nifE and nifI; nifN, nifM and nifF. Biochemical and genetic evidence suggest that the three genes nifH, nifD and nifK, which are probably the structural genes for nitrogenase, belong to the same operon and are transcribed from nifH to nifK. A polypeptide with a molecular weight of approximately 120,000 is presumed to be the nifJ product.

Chromosome Mapping↗
PubMed1979

Insertion element analysis and mapping of the Pseudomonas plasmid alk regulon.

We characterized and mapped new mutations of the alk (alkane utilization) genes found on Pseudomonas plasmids of the Inc P-2 group. These mutations were isolated after (i) nitrosoguanidine mutagenesis, (ii) transposition of the Tn7 trimethoprim and streptomycin resistance determinant, and (iii) reversion of polarity effects of alk::Tn7 insertion mutations. Our results indicate the existence of two alk loci not previously described--alkD, whose product is required for synthesis of membrane alkane-oxidizing activities, and alkE, whose product is required for synthesis of inducible membrane alcohol dehydrogenase activity. Polarity of alk::Tn7 insertion mutations indicates the existence of an alkBAE operon. Mapping of alk loci by transduction in P. aeruginosa shows that there are at least three alk clusters in the CAM-OCT plasmid--alkRD, containing regulatory genes; alkBAE, containing genes for specific biochemical activities; and alkC, containing one or more genes needed for normal synthesis of membrane alcohol dehydrogenase. The alkRD and alkBAE clusters are linked but separated by about 42 kilobases. The alkC cluster is not linked to either of the other two alk regions. Altogether, these results indicate a complex genetic control of the alkane utilization phenotype in P. putida and P. aeruginosa involving at least six separate genes.

Alkanes↗
PubMed1979

Structure and function of plasmid ColE1 and related plasmids.

Analysis of plasmid ColE1, its naturally occurring relatives ColK and CloDF13, and a wide range of ColE1 derivatives containing either insertions or deletions of genetic material has allowed localization on the ColE1 genome of DNA sequences responsible for colicin E1 synthesis, immunity to colicin killing, conjugal mobility and incompatibility. We have examined incompatibility between pairs of ColE1 derivatives ranging in size from 2.6 to 13.8 Md. Though all the plasmids tested exerted ColE1 incompatibility, a definite pattern was observed regarding the dominant plasmid in any pair tested (i.e. the plasmid that displaces the other from a heterozygote). Usually the larger plasmid is displaced. We conclude that loci for incompatibility reside within 0.7 kb of the ColE1 replication region. A model is presented to explain both the incompatibility data and the observation that the fraction of total DNA occurring as ColE1-like plasmid in a cell is approximately constant. Transposons Tn1 and Tn3 (3.2 Md; Apr and approximately 85% homologous), Tn501 (5.5 Md; Hgr), and Tn7 (9.3 Md; Tpr Smr) can all be transposed into ColE1. Though all have closely related. Tn501 and Tn7 do not complement transposition of Tn3 transposition defective deletions. A Tn3-specified 19,000 dalton protein is absent in one particular class of transposition-defective deletion.

Bacterial Proteins↗
PubMed2024

The promise of CRISPR-associated transposons for bacterial functional genomics.

CRISPR-associated transposons (CASTs) are naturally occurring amalgamations of CRISPR-Cas machinery and Tn7-like transposons that direct site-specific integration of transposon DNA via programmable guide RNAs. Although the mechanisms of CAST-based transposition have been well studied at the molecular and structural level, CASTs have yet to be broadly applied to bacterial genome engineering and systematic gene phenotyping (i.e. functional genomics) - likely due to their relatively recent discovery. Here, we describe the function and applications of CASTs, focusing on well-characterized systems, including the type I-F CAST from Vibrio cholerae (VcCAST) and type V-K CAST from Scytonema hofmanni (ShCAST). Further, we discuss the potentially transformative impact of targeted transposition on bacterial functional genomics by proposing genome-scale extensions of existing CAST tools.

DNA Transposable Elements↗
PubMed1979

Rapid mapping of transposon insertion and deletion mutations in the large Ti-plasmids of Agrobacterium tumefaciens.

A procedure is presented, that has allowed the rapid assignment of transposon Tn1 and Tn7 insertion sites in the large (130 Md) nopaline Ti-plasmid pTiC58, to specific restriction enzyme fragments. Total bacterial DNA is isolated from Agrobacterium tumefaciens strain C58 mutants that carry a transposon in their Ti-plasmid, and digested with an appropriate restriction endonuclease. The fragments are separated on an agarose gel, denatured and transferred to nitrocellulose filters. These are hybridized against purified wild type pTiC58, or against segments of PTiC58, cloned in E. coli using pBR322 as a vector plasmid. DNA sequences homologous to the probe are detected by autoradiography, thus generating a restriction enzyme pattern of the plasmid from a digest of total bacterial DNA. Mutant fragments can be readily identified by their different position compared to a wild type reference. This protocol eliminates the need to separate the large plasmid from chromosomal DNA for every mutant. In principle, it can be applied to the restriction enzyme analysis of insertion or deletion mutants in any plasmid that has no extensive homology with the chromosome.

Chromosome Deletion↗
PubMed1978

Conjugal transfer system of plasmid RP4: analysis by transposon 7 insertion.

We have begun an analysis in Escherichia coli of the conjugal transfer functions of the broad-host-range plasmid RP4. We have isolated 19 tra mutants of RP4, generated by insertion of transposon 7, and mapped their insertion sites by restriction endonuclease analysis. These sites fall into two separate regions on either side of the kanamycin resistance determinant. The transfer rates of the mutants range from 10% of that of RP4 to an undetectable level. Spot tests with the P-1 pilus-specific phages PRR1, Pf3, and PR4 and electron microscopic examination for pili have classified the mutants into several phenotypes consistent with their having normal, retracted, or no pili. Analysis of transient plasmid heterozygotes, created by P1 transduction, divided the tra mutants into a minimum of five complementation groups. Some of these groups contain more than one phenotypic class and may represent more than one gene because of the possible polar and deletion effects of Tn7 insertion.

Chromosome Mapping↗