A temperature-dependent pBR322 copy number mutant resulting from a Tn5 position effect.

Lupski, James R.; Projan, S J; Ozaki, Luiz Shozo; Godson, G. N.

doi:10.1073/pnas.83.19.7381

Cited by 21 publications

(12 citation statements)

References 55 publications

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“…Surprisingly, this effect was apparently exacerbated by ISPst5 insertion. The finding that two independently isolated pUS23 plasmid derivatives contained ISPst5 inserted upstream of ori in the pUC19 backbone raises the possibility that the insertion may have stimulated the origin of replication, which was initially only weakly functional in P. stutzeri Q. Activation of the pBR322 plasmid origin by an IS-derived promoter has been observed with Tn5 (Lupski et al, 1986). In any event the maintenance of the cassette as a plasmid allowed us to detect low frequency events that may otherwise have not been observed with a non-replicative cassette.…”

Section: Discussion the Significance Of Cismentioning

confidence: 95%

The native Pseudomonas stutzeri strain Q chromosomal integron can capture and express cassette-associated genes

Coleman

Holmes

2005

Microbiology

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The integron-gene cassette system contributes to multiple antibiotic resistance in bacteria and is likely to be of broader evolutionary significance. However, the majority of integron diversity consists of chromosomal integrons (CIs), with mostly unknown phenotypes, which are poorly characterized. A pUC-based reporter plasmid (pUS23) was developed containing a recombination site [aadB 59 base element (59-be)] upstream of promoterless aadB [gentamicin (Gm) resistance] and gfp (green fluorescence) genes, and this construct was used to investigate the recombination and expression activities of the CI in Pseudomonas stutzeri strain Q. Electroporation of pUS23 into P. stutzeri Q gave ampicillin-resistant transformants, which yielded Gm R green fluorescent recombinants after plating on Gm medium. Site-specific integration of pUS23 at attI was detected by PCR in 8 % of Gm R colonies and the frequency of attI integration was estimated as 2?0610 "8 per P. stutzeri Q(pUS23) cell. RT-PCR confirmed integron-mediated expression of aadB in one recombinant strain (Q23-17) and a promoter (P c ) was localized to the 59 end of the intI gene. The integrated pUS23 and flanking integron DNA were cloned from genomic DNA of strain Q23-17 and sequenced, confirming that site-specific integration of the entire reporter plasmid had occurred at the attI site. An insertion sequence (ISPst5; IS5 family) was discovered in the vector backbone of the reporter plasmid integrated at attI and also in a pUS23 derivative recovered as a plasmid in Escherichia coli JM109. This is the first demonstration that wild-type CIs can capture gene cassettes and express cassette-associated genes. INTRODUCTIONIntegrons and gene cassettes function together as a distinctive genetic system (Fig. 1). Gene cassettes typically consist of a single promoterless gene and an associated recombination site (termed 59 base element, 59-be or attC).The total pool of cassette-associated genes in natural bacterial populations (gene cassette metagenome) is a significant source of genetic novelty (Holmes et al., 2003a;Michael et al., 2004;Stokes et al., 2001), and since individually packaged genes create the possibility of combinatorial genetics, the significance of the gene cassette metagenome is potentially far reaching. However, gene cassettes are dependent on externally supplied functions for both mobilization and expression. These functions are supplied by integrons Rowe-Magnus & Mazel, 2001). The key features of an integron are a recombination site (attI), an enzyme (integron integrase, IntI) that catalyses site-specific recombination between cassette and integron sites (attI659-be or 59-be659-be) and a cassette promoter (P c ). Collectively, these give an integron the capacity to acquire gene cassettes, express genecassette-associated ORFs (gcORFs) and rearrange the transcriptional order of gcORFs through repeated integration and excision of gene cassettes. evidence is hard to find, suggesting that other integron classes have been less successful in this regard than class 1...

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Section: Discussion the Significance Of Cismentioning

confidence: 95%

The native Pseudomonas stutzeri strain Q chromosomal integron can capture and express cassette-associated genes

Coleman

Holmes

2005

Microbiology

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“…Namely, an increase in plasmid amplification was observed after a temperature upshift from 30 • C to 42 • C in E. coli TG1 and BL21 (Hoffmann and Rinas, 2001). Other papers report the occurrence of ColE1-type temperature-sensitive plasmids resulting from point mutations (Wong et al, 1982;Fitzwater et al, 1988;Minton et al, 1988;LinChao et al, 1992) or Tn5 insertions (Lupski et al, 1986). Altered pMB1 copy number mutants arising by changes in the RNAI gene dosage, were also described (Moser and Campbell, 1983).…”

Section: Deletion Formation Products Are Less Abundant Under Conditiomentioning

confidence: 94%

Deletion formation mutations in plasmid expression vectors are unfavored by runaway amplification conditions and differentially selected under kanamycin stress

Oliveira

Prazeres

Monteiro

2009

Journal of Biotechnology

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“…As before, the host strain carried a deletion covering all of the cloned DNA. The copy numbers per chromosome of the different plasmids (and therefore of the mcrBC region) was taken from the indicated references: pEMBL19 (36); pBR322 (32,36); and pACYC184 (11). When the cloned DNA was carried on low-copy-number pACYC184 or pBR322, restriction was comparable to restriction by a chromosomal copy of the wild-type genes (Table 3; compare lines 2 and 3 with line 4).…”

Section: Resultsmentioning

confidence: 99%

Genetic and sequence organization of the mcrBC locus of Escherichia coli K-12

et al. 1990

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The mcrB (rglB) locus of Escherichia coli K-12 mediates sequence-specific restriction of cytosine-modifled DNA. Genetic and sequence analysis shows that the locus actually comprises two genes, mcrB and mcrC. We show here that in vivo, McrC modifies the specificity of McrB restriction by expanding the range of modified sequences restricted. That is, the sequences sensitive to McrB+-dependent The locus known as mcrB was one of the first restriction systems to be discovered (33), by virtue of its action on special variants of T-even bacteriophage that incorporate 5-hydroxymethylcytosine (hm`C) into their DNA without further modification (see reference 50 for a review). This locus, formerly known as rglB (or r2,4) (48), was rediscovered because of difficulties encountered in cloning the genes for site-specific modification methylases associated with type II restriction-modification systems (7,26,40,49). In addition to hm5C-DNA, many but not all sequences methylated by site-specific cytosine modification methylases are restricted by the system in vivo, and the consensus recognition sequence 5'GmC was proposed (49). McrB is thus a sequence-specific, modification-requiring restriction system. We show here that the mcrB locus described above actually comprises two genes and that both are required for restriction of most the sequences previously characterized as sensitive. Thus, we will refer to the complete system as the McrBC system.The genes encoding the system are contained within the immigration control region of the Escherichia coli K-12 genome. Three restriction systems are encoded within 14 kilobases (kb) here (48). The well-studied hsdRMS locus (20, 31, 55) encodes the multisubunit type I system EcoK, which recognizes a seven-base sequence and cleaves the target when the sequence is not modified. The other two systems are the flanking loci mcrBC, described above, and mrr, which mediates site-specific restriction of adenine-modified DNA (22). The sequence organization of this region, judged by Southern blot analysis of chromosomal DNA, is highly variable in enteric bacteria (12), both in the hsd genes specifically and in the flanking sequences. Sequence analysis presented here is consistent with recent acquisition of the mcrBC genes by E. coli, possible accounting for some of the observed variability. * Corresponding author.At the molecular level, restriction systems consist of sequence-specific double-stranded endonucleases, usually accompanied by a sequence-specific modification methylase. So far, four classes of endonucleases have been described. The simplest are the type II enzymes, in which the endonuclease and protective methylase activities reside in separate enzymes. These endonucleases typically act as dimers of identical subunits and require only Mg2" for activity (38).One group of type II isoschizomers, typified by DpnI, recognizes a modified site (28), as McrBC appears to do. In contrast, type I and type III enzymes have separate specificity subunits that recognize the DNA site and require ATP in a...

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A temperature-dependent pBR322 copy number mutant resulting from a Tn5 position effect.

Cited by 21 publications

References 55 publications

The native Pseudomonas stutzeri strain Q chromosomal integron can capture and express cassette-associated genes

The native Pseudomonas stutzeri strain Q chromosomal integron can capture and express cassette-associated genes

Deletion formation mutations in plasmid expression vectors are unfavored by runaway amplification conditions and differentially selected under kanamycin stress

Genetic and sequence organization of the mcrBC locus of Escherichia coli K-12

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