Direct Detection and Quantification of Horizontal Gene Transfer by Using Flow Cytometry and gfp as a Reporter Gene

Sørensen, Søren J.; Sørensen, Anne Scott; Hansen, Lars Hestbjerg; Øregaard, Gunnar; Veal, Duncan

doi:10.1007/s00284-002-3978-0

Cited by 53 publications

(44 citation statements)

References 17 publications

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“…The system allowed tracking of pAR060302 in the pig gastrointestinal tract, and differentiation between the presence of this plasmid in its original host or after conjugative transfer to non-E. coli recipient bacteria. First, the mini-Tn5 A1-04/03 ::rfp cassette was mobilized into E. coli strain DH10B(pAR060302) using a helper plasmid and integrated into either the chromosome of DH10B or pAR060302 (25). This mixture was mated with the PFEC strain, and transconjugants were selected on media containing selective antibiotics for the recipient strain PFEC carrying pAR060302:rfp.…”

Section: Methodsmentioning

confidence: 99%

In Vivo Transmission of an IncA/C Plasmid in Escherichia coli Depends on Tetracycline Concentration, and Acquisition of the Plasmid Results in a Variable Cost of Fitness

Johnson

Singer

Isaacson

et al. 2015

Appl Environ Microbiol

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c IncA/C plasmids are broad-host-range plasmids enabling multidrug resistance that have emerged worldwide among bacterial pathogens of humans and animals. Although antibiotic usage is suspected to be a driving force in the emergence of such strains, few studies have examined the impact of different types of antibiotic administration on the selection of plasmid-containing multidrug resistant isolates. In this study, chlortetracycline treatment at different concentrations in pig feed was examined for its impact on selection and dissemination of an IncA/C plasmid introduced orally via a commensal Escherichia coli host. Continuous low-dose administration of chlortetracycline at 50 g per ton had no observable impact on the proportions of IncA/C plasmid-containing E. coli from pig feces over the course of 35 days. In contrast, high-dose administration of chlortetracycline at 350 g per ton significantly increased IncA/C plasmid-containing E. coli in pig feces (P < 0.001) and increased movement of the IncA/C plasmid to other indigenous E. coli hosts. There was no evidence of conjugal transfer of the IncA/C plasmid to bacterial species other than E. coli. In vitro competition assays demonstrated that bacterial host background substantially impacted the cost of IncA/C plasmid carriage in E. coli and Salmonella. In vitro transfer and selection experiments demonstrated that tetracycline at 32 g/ml was necessary to enhance IncA/C plasmid conjugative transfer, while subinhibitory concentrations of tetracycline in vitro strongly selected for IncA/C plasmid-containing E. coli. Together, these experiments improve our knowledge on the impact of differing concentrations of tetracycline on the selection of IncA/C-type plasmids.

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Section: Methodsmentioning

confidence: 99%

In Vivo Transmission of an IncA/C Plasmid in Escherichia coli Depends on Tetracycline Concentration, and Acquisition of the Plasmid Results in a Variable Cost of Fitness

Johnson

Singer

Isaacson

et al. 2015

Appl Environ Microbiol

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“…Thus, the distribution and activity of genes and transcripts can be determined to identify processes associated with the interaction between MGEs, bacteria and the environment. By contrast, other non-disruptive approaches exploit the potential of fluorescent markers, such as green fluorescent protein (gfp), for studying the transfer of plasmids (Christensen et al, 1996Dahlberg et al 1998aDahlberg et al , 1998bSørensen et al, 2003Sørensen et al, , 2005. In particular, in situ monitoring of plasmid transfer and microbial community physiology in structured microbial communities (through fluorescent reporter systems and confocal laser scanning microscopy) has provided a greater understanding of these complex processes Heydorn et al, 2000).…”

Section: New Tools To Study In Situ Transfer Processesmentioning

confidence: 99%

Safety Assessment of Transgenic Organisms, Volume 4

2010

Harmonisation of Regulatory Oversight in Biotechnology

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io s a fe t y nv ir on me nt ag ri cu lt ur e bi os af et y g r ic u lt u r e e n vi r o n m e n t gr ic ul tu re bi os af et y en vi ro nm en t n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o io sa fe ty en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en v io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t nv ir on me nt ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm e g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a f gr ic ul tu re bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t a n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u io sa fe ty en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m nv ir on me nt ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a f gr ic ul tu re bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t a n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u io sa fe ty en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e nv ir on me nt ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y e g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a f ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et...

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“…The antibiotic resistance plasmid pIP501 from Streptococcus agalactiae has a very broad host range for conjugative plasmid transfer and mobilization. Its host range includes virtually all tested Gram-positive bacteria, including the multicellular filamentous streptomycetes and Gram-negative Escherichia coli (17,24).For Gram-positive systems, molecular tools for in situ detection of horizontal gene transfer by conjugation are still very limited in contrast to 7,8,29,37,38). Nieto and Espinosa (30) have constructed a green fluorescent protein (GFP)-tagged derivative of plasmid pMV158 from Streptococcus pneumoniae, pMV158GFP, that was shown to be mobilizable to different low-GC Gram-positive bacteria like Enterococcus faecalis and Lactococcus lactis.…”

mentioning

confidence: 99%

“…For Gram-positive systems, molecular tools for in situ detection of horizontal gene transfer by conjugation are still very limited in contrast to Gram-negative systems (4,7,8,29,37,38). Nieto and Espinosa (30) have constructed a green fluorescent protein (GFP)-tagged derivative of plasmid pMV158 from Streptococcus pneumoniae, pMV158GFP, that was shown to be mobilizable to different low-GC Gram-positive bacteria like Enterococcus faecalis and Lactococcus lactis.…”

mentioning

confidence: 99%

Green Fluorescent Protein-Labeled Monitoring Tool To Quantify Conjugative Plasmid Transfer between Gram-Positive and Gram-Negative Bacteria

Arends

Schiwon

Sakιnç

et al. 2012

Appl Environ Microbiol

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On the basis of pIP501, a green fluorescent protein (GFP)-tagged monitoring tool was constructed for quantifying plasmid mobilization among Gram-positive bacteria and between Gram-positive Enterococcus faecalis and Gram-negative Escherichia coli. Furthermore, retromobilization of the GFP-tagged monitoring tool was shown from E. faecalis OG1X into the clinical isolate E. faecalis T9. The mechanisms of conjugative transfer in Gram-negative bacteria are fairly well understood (e.g., see references 2, 9, 18, 33, 36, and 41), whereas conjugation in Gram-positive bacteria has been studied in greater detail for only the last decade leading to the first model of a type IV secretion-like system in Gram-positive bacteria (1,2,16,17,41). The antibiotic resistance plasmid pIP501 from Streptococcus agalactiae has a very broad host range for conjugative plasmid transfer and mobilization. Its host range includes virtually all tested Gram-positive bacteria, including the multicellular filamentous streptomycetes and Gram-negative Escherichia coli (17,24).For Gram-positive systems, molecular tools for in situ detection of horizontal gene transfer by conjugation are still very limited in contrast to 7,8,29,37,38). Nieto and Espinosa (30) have constructed a green fluorescent protein (GFP)-tagged derivative of plasmid pMV158 from Streptococcus pneumoniae, pMV158GFP, that was shown to be mobilizable to different low-GC Gram-positive bacteria like Enterococcus faecalis and Lactococcus lactis. Lorenzo-Díaz and Espinosa applied pMV158GFP to intra-and interspecies mobilization between different Gram-positive bacteria in large-scale filter mating assays (26).Recently, Babic and coworkers (3) demonstrated conjugative transfer of the integrative and conjugative element ICEBs1 from Bacillus subtilis donor cells to B. subtilis recipient cells in real time using a lacO or LacI-GFP system for visualization of transfer events.Here, we report the construction and mobilization of a GFPtagged mobilizable plasmid based on the pIP501 tra region to monitor horizontal gene transfer between Gram-positive bacteria and between Gram-positive and Gram-negative bacteria by the formation of a green fluorescent phenotype in transconjugants. The mobilizable plasmid is based on a nisin-inducible expression system (NICE) and replicates in both Gram-positive and Gramnegative bacteria.Plasmid construction. The oriT region from the broad-hostrange plasmid pIP501 (oriT pIP50 ) was subcloned with primer pair oriT-HindIII-fw (fw stands for forward) and oriT-HindIII-re (re stands for reverse) (see Table S1 in supplemental material) via HindIII into plasmid pJP rel GFP encoding a gfp gene improved for expression in prokaryotes (28, 31). All bacterial strains and plasmids used in this work are described in Table 1. The gfp-oriT pIP501 cassette was inserted into the E. coli shuttle plasmid pMSP3535VA (6) via XmaI/XbaI with primer pair P rel -gfp-XmaI-fw and oriTXbaI-re under the control of a nisin-inducible nisA promoter. Then, the phage t 0 terminator was cloned downstrea...

show abstract

Direct Detection and Quantification of Horizontal Gene Transfer by Using Flow Cytometry and gfp as a Reporter Gene

Cited by 53 publications

References 17 publications

In Vivo Transmission of an IncA/C Plasmid in Escherichia coli Depends on Tetracycline Concentration, and Acquisition of the Plasmid Results in a Variable Cost of Fitness

In Vivo Transmission of an IncA/C Plasmid in Escherichia coli Depends on Tetracycline Concentration, and Acquisition of the Plasmid Results in a Variable Cost of Fitness

Safety Assessment of Transgenic Organisms, Volume 4

Green Fluorescent Protein-Labeled Monitoring Tool To Quantify Conjugative Plasmid Transfer between Gram-Positive and Gram-Negative Bacteria

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