R68.45, a plasmid with chromosome mobilizing ability (Cma) carries a tandem duplication

In order to set up a sensitive and reliable detection method to monitor environmentally released genetically engineered microorganisms (GEMs) a 72-bp, double-stranded DNA fragment has been built by annealing and ligating four synthetic oligonucleotides. Binding sites for two 20-mer oligonucleotides are situated inside the DNA fragment, flanking the centre. Into the central part of the construction a 30-nucleotide identification sequence has been fitted. Thanks to the presence of the two oligonucleotide binding sites, the synthetic construction ("number-plate") can be submitted to enzymatic amplification using the polymerase chain reaction (PCR), thus enabling the identification system to take advantage of the outstanding sensitivity of this technique. When released into a freshwater microcosm, cells of Pseudomonas putida carrying a "number-plated" chromosome could be easily and rapidly detected merely by submitting boiled cell sediments to PCR amplification.

show abstract

“…Conjugations were performed according to Ries et al (1980). P. putida and E. coli were grown in Difco (West Mosley, Surrey, UK) Antibiotic no.…”

Section: Methodsmentioning

confidence: 99%

Monitoring a genetically engineered bacterium in a freshwater environment by rapid enzymatic amplification of a synthetic DNA ?number-plate?

Amici

Bazzicalupo

Gallori

et al. 1991

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

show abstract

“…DNA sequence (5,11,15). Additional evidence indicates that all (19) or part (11) of the DNA duplication is an insertion sequence (IS).…”

mentioning

confidence: 99%

Direct DNA repeat in plasmid R68.45 is associated with deletion formation and concomitant loss of chromosome mobilization ability

Currier¹,

Morgan²

1982

J Bacteriol

View full text Add to dashboard Cite

Plasmid R68.45 has been useful in promoting the transfer of chromosomal markers in bacteria of many genera. In donors harboring R68.45, chromosomemobilizing ability (Cma) may be lost without the loss of other plasmid markers. However, Cma can be somewhat stabilized by maintenance of the donors in the presence of kanamycin (Km). We isolated variants of R68.45 from four bacterial species of three genera. Plasmid variants isolated included those without Cma, without transfer function (Tra) and Cma, or without Tra, Cma, and Km resistance. In Erwinia carotovora subsp. atroceptica EA153, the loss of plasmid markers is dependent on the culture medium on which the donors are maintained. Restriction endonuclease analyses of the variant plasmids revealed that most are deletion mutants of R68.45. In all cases when the uncertainty in the ends of the deletions was not too great, one end of the deletion was shown to originate within or near the direct DNA duplication in R68.45 which is required for Cma and which maps close to the Km resistance determinant. Furthermore, the types of deletions observed are consistent with what might be expected for deletions generated by tandemly repeated insertion sequences. Therefore, we suggest that the DNA duplication is the source of much of the instability observed in R68.45. However, data are presented for E. carotovora subsp. atroceptica EA153 which suggest that another region of R68.45 may also play a role in its stability in this species. Plasmid R68.45 was derived from plasmid R68 in Pseudomonas aeruginosa PAO (16). Both of these plasmids confer resistance to ampicillin (Ap), kanamycin (Km), and tetracycline (Tc); both promote their own transfer (Tra); and both have chromosome-mobilizing ability (Cma), but the activities of the latter are distinctly different (8, 9). R68 has low Cma in P. aeruginosa PAO, whereas R68.45 efficiently mobilizes genes from multiple chromosomal origins in this strain (18). Chromosome mobilization from multiple origins results in what appears to be a nonpolarized transfer. This nonpolarized transfer is expressed in the bacteria of many genera, whereas Cma of R68 is much more restricted and, when present, occurs in a polarized manner from one or a few chromosomal origins. In fact, to our knowledge, there are only two bacterial species, Erwinia carotovora subsp. atroceptica (4) and Proteus mirabilis (10), in which R68.45 has been reported not to have Cma. Recent studies have shown that the Cma of R68.45 is associated with a DNA insertion of approximately 2.05 kilobases (kb). This insertion creates a direct repeat of a neighboring t Paper 82-6-J of the scientific journal series, Kansas Agricultural Experiment Station, Manhattan, KS 66506.

show abstract

“…The plasmid pBLM2 was isolated by Marrs (1981) as a derivative of pLM2 (a derivative of RP1) which had gained the ability to mobilize chromosomal DNA of R. capsulata at a high frequency. pBLM2 contains a tandem repeat of an insertion element, IS21 (Youvan et al, 1982), which is presumed to be responsible for the plasmid's ability to mobilize chromosomal DNA (Reiss et al, 1980). Marrs used pBLM2 to map the region of the R. capsulatus chromosome coding for the photosynthesis functions and to generate an R prime containing these genes.…”

Section: Ammonium Transport By Photosynthetic Bacteriamentioning

confidence: 99%

Mapping nif genes of Rhodobacter capsulatus, and characterization of a mutant with a pleiotropic defect in nitrogen metabolism

Goldenberg

View full text Add to dashboard Cite

Mutations affecting the ability of the photosynthetic bacterium Rhodobacter capsulatus to fix nitrogen (Nif- ) have previously been mapped into six linkage groups (I-VI) by transduction with gene transfer agent (GTA). Because this vector carries only 4600 bp of DNA, linkage can only be demonstrated for short chromosomal distances. In order to demonstrate linkage on a larger scale, the self- transmissible plasmid, pBLM2, which has chromosome mobilizing ability in R. capsulatus, was used to map these six linkage groups relative to a number of auxotrophic markers. In this way, nif genes were found to lie in three regions of the chromosome. GTA linkage groups IV and VI were found to reside in the same region, based on their common linkages to adenosine and tryptophan markers. A second region was found to contain GTA groups I and V, both of which were linked by conjugation to isoleucine/valine and leucine markers. In a third region, group III was found to be located near the structural gene for glutamine synthetase (glnA). A seventh GTA linkage group, containing mutations which cause pleiotropic defects in nitrogen metabolism, was also found to lie in the same region with group III and glnA>> Mutants in this group have functional nitrogenase and normal regulation of nif gene expression. However, they are unable to grow on N2 or on most amino acids. The only nitrogen sources found to allow photosynthetic growth of these mutants were ammonium and asparagine. One of these mutants was studied in more detail and found to be able to grow aerobically on most nitrogen sources. Studies of gene expression, using a lac gene fusion, indicated that the gene altered in this mutant may be under the control of an ntrC- like gene, and that it also may regulate its own synthesis. Information from the lac fusion also suggests that there may be two genes in group VII, both of which are required for growth on low levels of ammonia.

show abstract

R68.45, a plasmid with chromosome mobilizing ability (Cma) carries a tandem duplication

Cited by 69 publications

References 29 publications

Monitoring a genetically engineered bacterium in a freshwater environment by rapid enzymatic amplification of a synthetic DNA ?number-plate?

Monitoring a genetically engineered bacterium in a freshwater environment by rapid enzymatic amplification of a synthetic DNA ?number-plate?

Direct DNA repeat in plasmid R68.45 is associated with deletion formation and concomitant loss of chromosome mobilization ability

Mapping nif genes of Rhodobacter capsulatus, and characterization of a mutant with a pleiotropic defect in nitrogen metabolism

Contact Info

Product

Resources

About