Suicidal Genetic Elements and Their Use in Biological Containment of Bacteria

Molin, Søren; Boe, Lars; Jensen, Lars; Kristensen, Claus; Givskov, Michael; Ramos, Juan L.; Bej, Asim K.

doi:10.1146/annurev.mi.47.100193.001035

Cited by 115 publications

(93 citation statements)

References 27 publications

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“…Because of its assumed importance in environmental settings (55), the focus was on conjugal plasmid transfer. Although examination of gene flow in natural microbial communities is experimentally difficult, several studies have demonstrated the occurrence of horizontal gene transfer in a variety of settings such as fresh waters (3,21) and soils (24,28,32). However, essentially no studies on horizontal gene transfer in subsurface microbial communities have been reported.…”

mentioning

confidence: 99%

Plasmid Introduction in Metal-Stressed, Subsurface-Derived Microcosms: Plasmid Fate and Community Response

Smets

Morrow²,

Pinedo³

2003

Appl Environ Microbiol

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The nonconjugal IncQ plasmids pMOL187 and pMOL222, which contain the metal resistance-encoding genes czc and ncc, were introduced by using Escherichia coli as a transitory delivery strain into microcosms containing subsurface-derived parent materials. The microcosms were semicontinuously dosed with an artificial groundwater to set a low-carbon flux and a target metal stress (0, 10, 100, and 1,000 M CdCl 2 ), permitting long-term community monitoring. The broad-host-range IncP␣ plasmid RP4 was also transitorily introduced into a subset of microcosms. No novel community phenotype was detected after plasmid delivery, due to the high background resistances to Cd and Ni. At fixed Cd doses, however, small but consistent increases in Cd r or Ni r density were measured due to the introduction of a single pMOL plasmid, and this effect was enhanced by the joint introduction of RP4; the effects were most significant at the highest Cd doses. The pMOL plasmids introduced could, however, be monitored via czc-and ncc-targeted infinite-dilution PCR (ID-PCR) methods, because these genes were absent from the indigenous community: long-term presence of czc (after 14 or 27 weeks) was contingent on the joint introduction of RP4, although RP4 cointroduction was not yet required to ensure retention of ncc after 8 weeks. Plasmids isolated from Ni r transconjugants further confirmed the presence and retention of a pMOL222-sized plasmid. ID-PCR targeting the RP4-specific trafA gene revealed retention of RP4 for at least 8 weeks. Our findings confirm plasmid transfer and long-term retention in low-carbon-flux, metal-stressed subsurface communities but indicate that the subsurface community examined has limited mobilization potential for the IncQ plasmids employed.

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confidence: 99%

Plasmid Introduction in Metal-Stressed, Subsurface-Derived Microcosms: Plasmid Fate and Community Response

Smets

Morrow²,

Pinedo³

2003

Appl Environ Microbiol

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“…Recently, some procaryotic and viral genes have been shown to be able to induce tumour cell death (Martin et al, 2000;Zhuang et al, 1995;Paulus et al, 1997). In this connection, the gef gene shows a killing function in procaryotic cells, which may be modulated by the action of different promoters (Molin et al, 1993). The product of the gef gene is a cytoplasmatic membrane protein whose 19 Nterminal hydrophobic amino acids constitute a membranespanning segment, probably in the form of an alpha-helix structure that anchors the protein to the membrane.…”

Section: Discussionmentioning

confidence: 99%

“…Mutagenesis experiments with Gef showed that the periplasmic portion of the gene encodes the toxic domain, and that dimerisation is not essential for the toxic effect (Poulsen et al, 1991). This effect was expressed in analyses with suicide cassettes consisting of members of the gef gene family in combination with inducible promoters (Molin et al, 1993). This gene, currently being studied to determine its ability to control bacterial population death (Ronchel and Ramos, 2001), may be a new candidate for therapeutic applications.…”

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Inhibition of growth and induction of apoptosis in human breast cancer by transfection of gef gene

et al. 2003

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The gef gene has cell-killing functions in Escherichia coli. To evaluate the feasibility of using this gene as a new strategy for cancer therapy, we transfected it in MCF-7 cells derived from breast cancer (MCF-7TG). The gef gene was cloned in a pMAMneo vector under the control of a mouse mammary tumour virus promoter, inducible by dexamethasone (Dex), and was transfected with liposomes. After selection and induction, expression of the gef gene was confirmed by reverse transcription -polymerase chain reactions (RT -PCR) and Western blot. Cell viability was determined with a haemocytometre and the sulphorodamine B colorimetric assay, and the cell cycle was studied by propidium iodide (PI) staining. Annexin V-FITC and PI assays were used to evaluate apoptosis, which was confirmed by electron microscopy. In comparison with MCF-7 parental cells and MCF-7 cells transfected with an empty vector, MCF-7TG cells induced with Dex showed a significant decrease in proliferation rate, which was associated with evidence of apoptosis. Morphological findings confirmed apoptosis and showed a typical pattern of mitochondrial dilation. Furthermore, the cell cycle was characterised by premature progression from G 1 to S phase and G 2 delay. Our results show that the gef gene was able to decrease proliferation in a breast cancer cell line, and induce apoptosis. These findings suggest that the gef gene is a potential candidate for tumour therapy.

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“…Therefore, LlaIR ϩ -based suicide systems could be widely exploited as a phage defense strategy if combined with a suitable phage-inducible promoter. Numerous types of conditional-suicide systems in which potent killing genes are coupled to inducible expression signals have been developed (37). The combinations are designed not to interfere with normal growth, and expression occurs under very specific conditions defined by the physical or chemical composition of the environment.…”

Section: Discussionmentioning

confidence: 99%

A triggered-suicide system designed as a defense against bacteriophages

et al. 1997

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A novel bacteriophage protection system for Lactococcus lactis based on a genetic trap, in which a strictly phage-inducible promoter isolated from the lytic phage 31 is used to activate a bacterial suicide system after infection, was developed. The lethal gene of the suicide system consists of the three-gene restriction cassette LlaIR ؉ , which is lethal across a wide range of gram-positive bacteria. The phage-inducible trigger promoter (31P) and the LlaIR ؉ restriction cassette were cloned in Escherichia coli on a high-copy-number replicon to generate pTRK414H. Restriction activity was not apparent in E. coli or L. lactis prior to phage infection. In phage challenges of L. lactis(pTRK414H) with 31, the efficiency of plaquing was lowered to 10 ؊4 and accompanied by a fourfold reduction in burst size. Center-of-infection assays revealed that only 15% of infected cells released progeny phage. In addition to phage 31, the 31P/LlaIR ؉ suicide cassette also inhibited four 31-derived recombinant phages at levels at least 10-fold greater than that of 31. The 31P/LlaIR ؉ -based suicide system is a genetically engineered form of abortive infection that traps and eliminates phages potentially evolving in fermentation environments by destroying the phage genome and killing the propagation host. This type of phage-triggered suicide system could be designed for any bacterium-phage combination, given a universal lethal gene and an inducible promoter which is triggered by the infecting bacteriophage.Many important industrial bioprocesses and food fermentations are susceptible to attack by bacteriophages. Control of phage problems in the commercial arena requires prevention of phage contamination, employment of strains that are resistant to phage infection, and minimization of opportunities for the appearance of new virulent phages. With the diversity of fermentation systems, it is not always possible to operate an aseptic fermentation or, if available, to maintain its integrity. Therefore, the longevity of any highly specialized strain in a commercial situation depends on the bacterium's relative phage resistance or sensitivity and how quickly defiant phages appear and proliferate against it. Modern biotechnology continues to provide increasing opportunities to create specialized strains with valuable processing or product characteristics. In dairy fermentations, which are plagued by phage attacks more often than any other bioprocessing industry, this problem has been traditionally approached with varying success by isolating phage-resistant mutants and incorporating the derivatives into the starter culture formulations. Over the last decade, a number of naturally occurring plasmid-encoded defenses in Lactococcus species have been discovered which prevent phage adsorption, prevent DNA injection, restrict unmodified phage DNA by resident restriction and modification (R/M) systems, or abort the phage infection following the early stages of phage development (21, 27). By using conjugation or transformation approaches, these phage...

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Suicidal Genetic Elements and Their Use in Biological Containment of Bacteria

Cited by 115 publications

References 27 publications

Plasmid Introduction in Metal-Stressed, Subsurface-Derived Microcosms: Plasmid Fate and Community Response

Plasmid Introduction in Metal-Stressed, Subsurface-Derived Microcosms: Plasmid Fate and Community Response

Inhibition of growth and induction of apoptosis in human breast cancer by transfection of gef gene

A triggered-suicide system designed as a defense against bacteriophages

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