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PubMed2026

Genomic Characterisation of Carbapenem-Resistant Klebsiella pneumoniae and Enterobacter hormaechei Clinical Isolates from Nigeria: Evidence of Resistance, Virulence, and Putative Plasmid-Mediated Gene Sharing.

The global proliferation of carbapenem-resistant Enterobacterales (CRE) constitutes one of the most urgent public health threats, yet high-resolution genomic data from sub-Saharan Africa remain critically scarce. We applied whole-genome sequencing (WGS) and comparative phylogenomics to characterise antimicrobial resistance determinants, virulence genes, and mobile genetic elements (MGEs) in three carbapenem-resistant clinical isolates originating from three tertiary hospitals (selected from a broader surveillance collection spanning four facilities) in Osun State, southwestern Nigeria. We purposively selected three isolates, two Klebsiella pneumoniae subsp. pneumoniae (K22, ST411; K31, ST17) and one Enterobacter hormaechei subsp. steigerwaltii (K32, ST45) from a broader surveillance collection of 27 carbapenem-non-susceptible Enterobacterales, to represent phenotypically and genotypically divergent lineages. Resistome analysis revealed extensive plasmid-associated β-lactam and aminoglycoside resistance in K31 (including blaCTX-M-15, blaOXA-1, and blaTEM-1). K32 harboured an intrinsic chromosomal blaACT-17 AmpC gene, while IS26 and ISEcp1 insertion sequences, consistent with transposon-mediated mobilisation, flanked its acquired aminoglycoside and sulfonamide resistance cassettes. K22 lacked detected acquired carbapenemase, ESBL, or plasmid-mediated AmpC genes, indicating that its carbapenem-resistant phenotype may involve non-carbapenemase mechanisms such as porin alteration or efflux-mediated reduced susceptibility; however, this mechanism requires confirmation by direct ompK35/ompK36 sequence analysis and/or phenotypic outer membrane protein profiling. Virulome profiling identified a broader repertoire of siderophore, adhesion, and biofilm genes in both K. pneumoniae isolates than in E. hormaechei. Phylogenomic analysis demonstrated that K22 and K31 cluster within the broader K. pneumoniae population framework but represent distinct high-risk lineages (ST411 and ST17) rather than a single clonal outbreak. Analysis also identified a shared plasmid backbone between K31 and K32, supporting interspecies horizontal gene transfer. These descriptive genomic findings identify clinically relevant resistance and virulence determinants in three purposively selected carbapenem-resistant Enterobacterales from Nigerian tertiary-care hospitals. The detection of shared resistance elements between K. pneumoniae and E. hormaechei suggests possible plasmid-mediated gene sharing. Still, larger WGS studies with long-read sequencing and patient-level epidemiological data are required to define transmission and dissemination patterns.

Nigeria↗

PubMed2004

Counterselection and co-delivery of transposon and transposase functions for Sleeping Beauty-mediated transposition in cultured mammalian cells.

Sleeping Beauty (SB) is a gene-insertion system reconstructed from transposon sequences found in teleost fish and is capable of mediating the transposition of DNA sequences from transfected plasmids into the chromosomes of vertebrate cell populations. The SB system consists of a transposon, made up of a gene of interest flanked by transposon inverted repeats, and a source of transposase. Here we carried out a series of studies to further characterize SB-mediated transposition as a tool for gene transfer to chromosomes and ultimately for human gene therapy. Transfection of mouse 3T3 cells, HeLa cells, and human A549 lung carcinoma cells with a transposon containing the neomycin phosphotransferase (NEO) gene resulted in a several-fold increase in drug-resistant colony formation when co-transfected with a plasmid expressing the SB transposase. A transposon containing a methotrexate-resistant dihydrofolate reductase gene was also found to confer an increased frequency of methotrexate-resistant colony formation when co-transfected with SB transposase-encoding plasmid. A plasmid containing a herpes simplex virus thymidine kinase gene as well as a transposon containing a NEO gene was used for counterselection against random recombinants (NEO+TK+) in medium containing G418 plus ganciclovir. Effective counterselection required a recovery period of 5 days after transfection before shifting into medium containing ganciclovir to allow time for transiently expressed thymidine kinase activity to subside in cells not stably transfected. Southern analysis of clonal isolates indicated a shift from random recombination events toward transposition events when clones were isolated in medium containing ganciclovir as well as G418. We found that including both transposon and transposase functions on the same plasmid substantially increased the stable gene transfer frequency in Huh7 human hepatoma cells. The results from these experiments contribute technical and conceptual insight into the process of transposition in mammalian cells, and into the optimal provision of transposon and transposase functions that may be applicable to gene therapy studies.

3T3 Cells↗

PubMed1986

Broad host range vectors derived from an RSF1010::Tn1 plasmid.

Plasmid vector derivatives of the IncQ/P4 plasmid RSF1010 available for cloning DNA into a broad range of bacterial species were constructed. The plasmid pAYC31 constructed for the positive selection of inserted fragments contains part of transposon Tn1 inserted into the sequence of the gene sul. Gene aph transcription in pAYC31 can be initiated from the promoter for the transposase gene tnpA which is under the negative control of the gene tnpR product (Heffron, 1983). The insertion of a BamHI fragment, or of fragments generated by Sau3A, BclI, BglII, or XhoII digestion into the unique BamHI site within the gene tnpR sequence, leads to initiation of transcription from the promoter of the tnpA gene toward the aph gene. Expression of the aph gene upon insertion of a BamHI restriction fragment provides a positive selection for hybrid plasmids by plating the transformed bacteria on media with streptomycin. Versatile cloning vectors pAYC32 of 9.7 kb in length and pAYC39 of 11.3 kb in length were also constructed. Insertion into the BamHI site of vector pAYC32 of a 1.6-kb BglII fragment that contains the lambda cos site produced cosmid vectors pAYC51 and pAYC52. The two 11.3-kb cosmids differ only by the orientation of the 1.6-kb BglII fragment. By insertion into the BamHI site of pAYC32 of a BglII-BamHI fragment of plasmid pHC79 that contains the gene tet and lambda cos site cosmid vector pAYC53 was constructed. Vector pAYC31 was used to construct a gene bank from the chromosomal DNA of an obligate methylotrophic strain Methylomicrobium flagellatum KT.

Chromosome Mapping↗

PubMed1999

Spontaneous tandem amplification and deletion of the shiga toxin operon in Shigella dysenteriae 1.

Only one species of Shigella, Shigella dysenteriae 1, has been demonstrated to produce Shiga toxin (Stx). Stx is closely related to the toxins produced by Shiga toxin-producing Escherichia coli (STEC). In STEC, these toxins are often encoded on lambdoid bacteriophages and are major virulence factors for these organisms. Although the bacteriophage-encoded stx genes of STEC are highly mobile, the stx genes in S. dysenteriae 1 have been believed to be chromosomally encoded and not transmissible. We have located the toxin genes of S. dysenteriae 1 to a region homologous to minute 30 of the E. coli chromosome, within a 22.4 kbp putative composite transposon bracketed by IS600 insertion sequences. This region is present in all the S. dysenteriae 1 strains examined. Tandem amplification occurs via the flanking insertion sequences, leading to increased toxin production. The global regulatory gene, fnr, is located within the stx region, allowing deletions of the toxin genes to be created by anaerobic growth on chlorate-containing medium. Deletions occur by recombination between the flanking IS600 elements. Lambdoid bacteriophage genes are found both upstream and within the region, and we demonstrate the lysogeny of Shigella species with STEC bacteriophages. These observations suggest that S. dysenteriae 1 originally carried a Stx-encoding lambdoid prophage, which became defective due to loss of bacteriophage sequences after IS element insertions and rearrangements. These insertion sequences have subsequently allowed the amplification and deletion of the stx region.

Bacterial Toxins↗

PubMed1989

Structure and coding properties of Bs1, a maize retrovirus-like transposon.

We have sequenced Bs1, an insertion element isolated from a null allele of the Adh1 locus encoding alcohol dehydrogenase in maize. The Bs1 element is 3203 base pairs (bp) in length, has 302-bp identical long terminal direct repeats (LTRs), and created a 5-bp flanking direct duplication of target Adh1 DNA upon insertion. The 5' LTR is followed by a canonical primer binding site with homology to the plant initiator methionyl-tRNA, and the 3' LTR is directly preceded by a polypurine stretch like that observed in retroviruses and retrotransposons. Bs1 encodes two overlapping open reading frames specifying peptides of 740 and 168 amino acids. The longer open reading frame specifies a peptide with amino acid homology to the protease and nucleic acid binding moiety of retroviruses and retrotransposons. The deduced amino acid sequence encoded by Bs1 lacks convincing homology to the polymerase (reverse transcriptase) encoded by retroposons, despite the fact that this polymerase-encoding domain is routinely the most conserved region of any such element. The sequence and relatively small size of Bs1 suggest that this element is a deleted retrotransposon that inserted into Adh1 with the aid of a reverse transcriptase function provided in trans. In vitro transcribed Bs1 complementary RNA was translated in vitro to produce both a protein of 81 kDa representing open reading frame 1 (ORF1) and one of the 95-kDa size predicted for the frame-shifted fusion of ORF1 and ORF2. As with many other retroposons, the efficiency of translational initiation at the AUG beginning ORF1 was not noticeably affected by the presence of one or more upstream, unproductive AUGs in the complementary RNA transcript.

Amino Acid Sequence↗

PubMed1994

Differential roles of the transposon termini in IS91 transposition.

Insertion sequence 91 (IS91) inserts specifically at GTTC or CTTG target sequences without duplication of the target. After insertion, the right inverted repeat (IRR) lies adjacent to the 3' end of the target sequences (or 5' to the complementary sequence CAAG or GAAC). We have analyzed the effects of alteration of each terminus of IS91 on transposition activity in Escherichia coli. IRR is absolutely required for transposition. Deletion analysis indicates that a 14-bp segment is not sufficient, but an 81-bp sequence within the IRR region is sufficient. Furthermore, the GTTC/CTTG target site is also required. The left inverted repeat (IRL) of IS91 is dispensable. Plasmid fusions originated by one-ended transposition of IS91 derivatives lacking IRL occur at about the same frequency as cointegrate formation observed for the wild-type element. In the one-ended-type fusions, the inserted fragment of donor DNA is flanked at one end (constant end) by IRR and at the other end by a GTTC or CTTG sequence present in the donor (variable end) in a way that usually results in multiple tandem insertions of the donor plasmid in the target site. These results are easily accommodated by a rolling-circle replicative transposition mechanism. This model also draws support from the finding that the IS91 transposase is related in sequence to the superfamily of rolling-circle replication proteins and the observation that IRR shows some conservation in sequence and secondary structure with the origins of replication of some rolling-circle replication plasmids.

Base Sequence↗

PubMed1996

Salmonella typhimurium acrB-like gene: identification and role in resistance to biliary salts and detergents and in murine infection.

Salmonella serotype typhimurium transpositional mutants altered in resistance to biliary salts and detergents were isolated previously. We have characterized further the LX1054 mutant strain, the most sensitive of them. The chromosomal DNA segment flanking transposon insertion was cloned and sequenced. The highest level of identity was found for the acrB (formerly acrE) gene of Escherichia coli, a gene encoding a drug efflux pump of the Acr family. LX1054 exhibited a reduced capacity to colonize the intestinal tract. After passages in mice, the mutant strain lost the sensitive phenotype. In vitro, a resumption of growth appeared after 17 h of culture in medium with cholate or other tested biological or chemical detergents. Then, the acquired resistant phenotype seemed stable. The data suggested a role of S. typhimurium acrB-like gene in resistance to biliary salts and detergents and in mice intestinal colonization. However, the local and transient sensitivity observed in vivo, and the in vitro adaptations suggest that several detergent-resistance mechanisms operate in S. typhimurium.

Amino Acid Sequence↗

PubMed2006

Generation of useful insertionally blocked sterol degradation pathway mutants of fast-growing mycobacteria and cloning, characterization, and expression of the terminal oxygenase of the 3-ketosteroid 9alpha-hydroxylase in Mycobacterium smegmatis mc(2)155.

Integration of the pCG79 temperature-sensitive plasmid carrying Tn611 was used to generate libraries of mutants with blocked sterol-transforming ability of the sterol-utilizing strains Mycobacterium smegmatis mc(2)155 and Mycobacterium phlei M51-Ept. Of the 10,000 insertional mutants screened from each library, 4 strains with altered activity of the sterol-degrading enzymes were identified. A blocked 4-androstene-3,17-dione-producing M. phlei mutant transformed sitosterol to 23,24-dinorcholane derivatives that are useful starting materials for corticosteroid syntheses. A recombinant plasmid, pFJ92, was constructed from the genomic DNA of one of the insertional mutants of M. smegmatis, 10A12, which was blocked in 3-ketosteroid 9alpha-hydroxylation and carrying the transposon insertion and flanking DNA sequences, and used to isolate a chromosomal fragment encoding the 9alpha-hydroxylase. The open reading frame encodes the 383-amino-acid terminal oxygenase of 3-ketosteroid 9alpha-hydroxylase in M. smegmatis mc(2)155 and has domains typically conserved in class IA terminal oxygenases. Escherichia coli containing the gene could hydroxylate the steroid ring at the 9alpha position.

Cloning, Molecular↗

PubMed1988

Temporal control of transposition in Tn5.

IS50R is an insertion sequence associated with the transposon Tn5. IS50R carries the structural genes for two proteins; one (P1) is the Tn5 transposase, and the other (P2) is an inhibitor of transposition. These two proteins are translated from two different transcripts, m1 and m2. When bacteriophage lambda::IS50R DNA was introduced into a bacterial cell, m1 and m2 were initially at relative levels of about 1 to 2. As time progressed the amount of m1 fell, whereas the amount of m2 continued to increase, until after about 3 h the ratio of m1 to m2 was about 1 to 80. The temporal changes in the levels of these transcripts correlated with temporal changes in P1 and P2 levels and Tn5 transposition that have been documented in other studies. We measured the stability of the messages and showed that the differences in the levels of m1 and m2 must reflect real differences in the strengths of their promoters and that the changes in transcription kinetics are mediated by the dam methylation system of the cell and are not determined by IS50R products. Our results show that the 5' end of m2 is about twice as stable as that of m1, which raises the possibility that differential message stability does, in part, influence the ratio of inhibitor to transposase.

Bacterial Proteins↗

PubMed2001

Control of hemA expression in Rhodobacter sphaeroides 2.4.1: effect of a transposon insertion in the hbdA gene.

The common precursor to all tetrapyrroles is 5-aminolevulinic acid (ALA), and in Rhodobacter sphaeroides its formation occurs via the Shemin pathway. ALA synthase activity is encoded by two differentially regulated genes in R. sphaeroides 2.4.1: hemA and hemT. In our investigations of hemA regulation, we applied transposon mutagenesis under aerobic conditions, followed by a selection that identified transposon insertion mutants in which hemA expression is elevated. One of these mutants has been characterized previously (J. Zeilstra-Ryalls and S. Kaplan, J. Bacteriol. 178:985-993, 1996), and here we describe our analysis of a second mutant strain. The transposon inserted into the coding sequences of hbdA, coding for S-(+)-beta-hydroxybutyryl-coenzyme A dehydrogenase and catalyzing an NAD-dependent reaction. We provide evidence that the hbdA gene product participates in polyhydroxybutyrate (PHB) metabolism and, based on our findings, we discuss possibilities as to how defective PHB metabolism might alter the level of hemA expression.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed2003

Sequence of the 165-kilobase catabolic plasmid pAO1 from Arthrobacter nicotinovorans and identification of a pAO1-dependent nicotine uptake system.

The 165-kb catabolic plasmid pAO1 enables the gram-positive soil bacterium Arthrobacter nicotinovorans to grow on the tobacco alkaloid L-nicotine. The 165,137-nucleotide sequence, with an overall G+C content of 59.7%, revealed, besides genes and open reading frames (ORFs) for nicotine degradation, a complete set of ORFs for enzymes essential for the biosynthesis of the molybdenum dinucleotide cofactor, as well as ORFs related to uptake and utilization of carbohydrates, sarcosine, and amino acids. Of the 165 ORFs, approximately 50% were related to metabolic functions. pAO1 conferred to A. nicotinovorans the ability to take up L-[(14)C]nicotine from the medium, with an K(m) of 5.6 +/- 2.2 micro M. ORFs of putative nicotine transporters formed a cluster with the gene of the D-nicotine-specific 6-hydroxy-D-nicotine oxidase. ORFs related to replication, chromosome partitioning, and natural transformation functions (dprA) were identified on pAO1. Few ORFs showed similarity to known conjugation-promoting proteins, but pAO1 could be transferred by conjugation to a pAO1-negative strain at a rate of 10(-2) to 10(-3) per donor. ORFs with no known function represented approximately 35% of the pAO1 sequence. The positions of insertion sequence elements and composite transposons, corroborated by the G+C content of the pAO1 sequence, suggest a modular composition of the plasmid.

Arthrobacter↗

PubMed2026

Genome-wide identification of conditionally essential genes for growth in the presence of sulfamethoxazole and trimethoprim in sulfamethoxazole- and trimethoprim-resistant Escherichia coli.

UNLABELLED: Resistance to sulfonamides (SULs) and trimethoprim (TMP) in Escherichia coli threatens their clinical relevance. Beyond known resistance mechanisms, little is understood about the cellular responses that enable resistant E. coli to grow under these antibiotic stresses. This study aimed to identify genes that support bacterial growth under SUL and TMP stress. Two saturated transposon mutant libraries were constructed in resistant E. coli MG1655 harboring either dfrA1 or sul2. They were grown with and without 1/2 and 1/4 minimum inhibitory concentration (MIC) of sulfamethoxazole (SMX) or TMP, and mutant depletion was assessed via transposon-directed insertion-site sequencing. At 1/2 MIC, 36 and 89 genes were identified as conditionally essential during SMX and TMP exposure, while 5 and 2 genes were classified as conditionally essential at 1/4 MIC. Genes identified as conditionally essential at 1/4 MIC were also important at 1/2 MIC. Conditionally essential genes belonged to lipopolysaccharide biosynthesis, peptidoglycan metabolism, energy production, membrane integrity, phosphate metabolism, and stress responses, highlighting the role of these factors in maintaining cell stability under SMX and TMP stress. Validation with 10 conditionally essential genes (apaH, mtn, surA, waaO, nlpI, prc, wzxE, fadR, degP, and tpiA) showed that deletion mutants indeed exhibited growth defects and two- to eightfold reductions in MIC under antibiotic stresses compared to their parent strains. This study highlights cellular responses to SMX and TMP under antibiotic stress, and it has identified a list of genes whose products may serve as potential helper drug targets to resensitize resistant E. coli to SMX and TMP treatments. IMPORTANCE: Sulfonamides (SULs) and trimethoprim (TMP) are broad-spectrum antimicrobials. They are commonly used to treat infections in both humans and animals. Resistance against SUL and TMP is widespread in pathogenic bacteria, and there is a need to overcome this problem. One possibility is to target the cellular mechanism by which the resistant bacteria adapt to growth in the presence of the antimicrobials. In this study, we identify the genes, besides the resistance genes, which enable resistant Escherichia coli to grow in the presence of SUL and TMP. We further show that knocking out many of these genes attenuates the resistant E. coli for growth during SUL and/or TMP stress, irrespective of which SUL- or TMP-resistant gene the bacteria carry. The gene products of these genes may serve as potential helper drug targets to resensitize resistant E. coli to sulfamethoxazole and TMP treatments.

Escherichia coli↗

PubMed2001

Spontaneous reactivation of Shiga toxins in Escherichia coli O157:H7 cells caused by transposon excision.

IS1203v is an insertion sequence which has been found in inactivated Shiga toxin 2 genes (stx2) of Escherichia coli O157:H7. Using PCR amplification, we detected the wild-type stx2 genes in colonies of E. coli O157:H7 which possessed stx2 genes inactivated by insertion of IS1203v. This suggests that IS1203v is excised from the inactivated stx2 genes in E. coli O157:H7. We isolated the cells possessing the wild-type stx2 genes, and confirmed Stx2 productivities by reversed passive latex agglutination. We also analyzed the frequency of the appearance of the Stx2-producing cells using a quantitative PCR method. As a result, the frequency was 3.00 x 10(-6) with culturing for 24 h at 37 degrees C, and this increased to 8.83 x 10(-5) when E. coli O157:H7 possessing the inactivated stx2 genes was transformed by an expression plasmid harboring the IS1203v transposase. These results showed that some Stx2-nonproducing E. coli O157:H7 strains could be spontaneously changed into Stx2-producing cells.

Journal Article↗

PubMed1996

IS10/Tn10 transposition efficiently accommodates diverse transposon end configurations.

Transposon Tn10 and its component insertion sequence IS10 move by non-replicative transposition. We have studied the array of reaction intermediates and products in a high efficiency in vitro IS10/Tn10 transposition reaction. Synapsis of two transposon ends, followed by cleavage and strand transfer, can occur very efficiently irrespective of the relative locations and orientations of the two ends. The two participating ends can occur in inverted or direct orientation on the same molecule or, most importantly, on two different molecules. This behavior contrasts sharply with that of Mu, in which transposition is strongly biased in favor of inverted repeat synapsis. Mechanistically, the absence of discrimination amongst various end configurations implies that the architecture within the IS10/Tn10 synaptic complex is relatively simple, i.e. lacking any significant intertwining of component DNA strands. Biologically these observations are important because they suggest that the IS10 insertion sequence module has considerable flexibility in the types of DNA rearrangements that it can promote. Most importantly, it now seems highly probable that a single non-replicative IS10 element can promote DNA rearrangements usually attributed to replicative transposition, i.e. adjacent deletions and cointegrates, by utilizing transposon ends on two sister chromosomes. Other events which probably also contribute to the diversity of IS10/Tn10-promoted rearrangements are discussed.

DNA Nucleotidyltransferases↗

PubMed1992

Transposition of Tn5096 and related transposons in Streptomyces species.

IS493 is an insertion sequence isolated from Streptomyces lividans by a method designed to 'trap' transposable elements. IS493 was converted to functional transposons by cloning antibiotic-resistance-encoding genes between ORF-A and ORF-B of IS493 or near the left-end inverted repeat of the element. Tn5096 transposed relatively randomly in several Streptomyces species. Tn5096 can be introduced into streptomycetes on temperature-sensitive vectors by protoplast transformation, FP43-mediated transduction, or by conjugation from Escherichia coli. We have shown that additional genes can be inserted in Tn5096 without disrupting transposition, and that Tn5096 insertions in a tylosin (Ty)-producing strain of Streptomyces fradiae frequently cause no deleterious effects on Ty production. A promoter probe transposon, Tn5099, containing a promoterless xylE gene, transposed in Streptomyces griseofuscus and S. fradiae, and transcriptional fusions were readily identified.

Cloning, Molecular↗

PubMedDate not supplied

Downeast anemia (dea), a new mouse model of severe nonspherocytic hemolytic anemia caused by hexokinase (HK(1)) deficiency.

A new spontaneous mutation in the A/J inbred mouse strain, downeast anemia (dea), causes severe hemolytic anemia with extensive tissue iron deposition and marked reticulocytosis. The anemia is present at birth and persists throughout life. The defect is inherited as an autosomal recessive and is transferable through bone marrow stem cells. The red cell morphology is consistent with a nonspherocytic hemolytic anemia, suggestive of a red cell enzymopathy. In linkage analysis, dea is nonrecombinant with the hexokinase-1 gene (Hk1) on mouse Chromosome 10. Expression of Hk1 is markedly decreased in dea erythroid tissues, and the transcript produced is larger than normal. Hexokinase enzyme activity is significantly decreased in dea tissues, including red cells, spleen, and kidney. Southern blot analyses revealed approximately 5.5 kb of additional sequence in the 5' portion of the dea Hk1 gene, which was identified by direct sequencing as an early transposon (ETn) insertion in intron 4. ETn insertions disrupt genes in several mouse models by a variety of mechanisms, including aberrant splicing of ETn sequences into the mRNA. We conclude that the primary gene defect in the dea mutation is in Hk1 and that dea is a model of generalized hexokinase deficiency, the first such model identified to date.

Anemia, Hemolytic↗

PubMed1991

Transposition and transduction of plasmid DNA in Streptomyces spp.

To expand the application of molecular genetics to many different streptomycete species, we have been developing two potentially widely applicable methodologies: transposon mutagenesis and plasmid transduction. We constructed three transposons from the Streptomyces lividans insertion sequence IS493. Tn5096 and Tn5097 contain an apramycin resistance gene inserted in different orientations between the two open reading frames of IS493. These transposons transpose from different plasmids into many different sites in the Streptomyces griseofuscus chromosome and into its resident linear plasmids. Tn5099 contains a promoterless xylE gene and a hygromycin-resistance gene inserted in IS493 close to one end. Tn5099 transposes in S. griseofuscus giving operon fusions in some cases that drive expression of the xylE gene product, catechol deoxygenase, giving yellow colonies in the presence of catechol. We have also developed plasmid vectors that can be transduced into many streptomycete species by bacteriophage FP43. We describe the characterization of FP43 and mapping of several bacteriophage functions. The region of cloned FP43 DNA essential for plasmid transduction includes the origin for headful packaging.

DNA Transposable Elements↗

PubMed1995

Tn916 target DNA sequences bind the C-terminal domain of integrase protein with different affinities that correlate with transposon insertion frequency.

The conjugative transposon Tn916 inserts with widely different frequencies into a variety of target sites with related nucleotide sequences. The binding of chimeric proteins, consisting of maltose-binding protein fused to Tn916 integrase, to three different target sequences for Tn916 was examined by DNase I protection experiments. The C-terminal DNA binding domain of the Tn916 integrase protein was shown to protect approximately 40 bp, spanning target sites in the orfA and cat genes of the plasmid pIP501 and in the cylA gene of the plasmid pAD1. Competition binding assays showed that the affinities of the three target sites for Tn916 integrase varied over a greater than 3- but less than 10-fold range and that the cat target site bound integrase at a lower affinity than did the other two target sites. A PCR-based assay for transposition in Escherichia coli was developed to assess the frequency with which a defective minitransposon inserted into each target site. In these experiments, integrase provided in trans from a plasmid was the sole transposon-encoded protein present. This assay detected transposition into the orfA and cylA target sites but not into the cat target site. Therefore, the frequency of transposon insertion into a particular target site correlated with the affinity of the target for the integrase protein. Sequences within the target fragments similar to known Tn916 insertion sites were not protected by integrase protein. Analysis ot he electrophoretic behavior of circularly permuted sets of DNA fragments showed that all three target sites contained structural features consistent with the presence of a static bend, suggesting that these structural features in addition to the primary nucleotide sequence are necessary for integrase binding and, thus, target site activity.

Base Sequence↗