Limits...
A universal mariner transposon system for forward genetic studies in the genus Clostridium.

Zhang Y, Grosse-Honebrink A, Minton NP - PLoS ONE (2015)

Bottom Line: To increase the effectiveness of the system, a novel replicon conditional for plasmid maintenance was developed, which no longer supports the effective retention of the transposon delivery vehicle in the presence of the inducer isopropyl β-D-1-thiogalactopyranoside (IPTG).Moreover, appropriate screening of both libraries resulted in the isolation of auxotrophic mutants as well as cells deficient in spore formation/germination.This strategy is capable of being implemented in any Clostridium species.

View Article: PubMed Central - PubMed

Affiliation: Clostridia Research Group, BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.

ABSTRACT
DNA transposons represent an essential tool in the armoury of the molecular microbiologist. We previously developed a catP-based mini transposon system for Clostridium difficile in which the expression of the transposase gene was dependent on a sigma factor unique to C. difficile, TcdR. Here we have shown that the host range of the transposon is easily extended through the rapid chromosomal insertion of the tcdR gene at the pyrE locus of the intended clostridial target using Allele-Coupled Exchange (ACE). To increase the effectiveness of the system, a novel replicon conditional for plasmid maintenance was developed, which no longer supports the effective retention of the transposon delivery vehicle in the presence of the inducer isopropyl β-D-1-thiogalactopyranoside (IPTG). As a consequence, those thiamphenicol resistant colonies that arise in clostridial recipients, following plating on agar medium supplemented with IPTG, are almost exclusively due to insertion of the mini transposon into the genome. The system has been exemplified in both Clostridium acetobutylicum and Clostridium sporogenes, where transposon insertion has been shown to be entirely random. Moreover, appropriate screening of both libraries resulted in the isolation of auxotrophic mutants as well as cells deficient in spore formation/germination. This strategy is capable of being implemented in any Clostridium species.

No MeSH data available.


Related in: MedlinePlus

Genetic map of mariner transposon insertions in C.acetobutylicum (A) and in C. sporogenes (B). Sixty independent transposon mutants were identified and the insertions were sequenced. Insertions in the plus orientation are marked on the circle exterior. Insertions in the minus orientation are marked on the circle interior. Numbers indicate the precise point of insertion according to genome sequence data for (A) C. acetobutylicum ATCC 824 genome and megaplasmid pSOL1 (Refseq number NC_003030.1 and NC_001988.2; GenBank accession number AE001437 and AE001438) [28] and (B) C. sporogenes NCIMB 10956 (GenBank accession number CP009225).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4383383&req=5

pone.0122411.g006: Genetic map of mariner transposon insertions in C.acetobutylicum (A) and in C. sporogenes (B). Sixty independent transposon mutants were identified and the insertions were sequenced. Insertions in the plus orientation are marked on the circle exterior. Insertions in the minus orientation are marked on the circle interior. Numbers indicate the precise point of insertion according to genome sequence data for (A) C. acetobutylicum ATCC 824 genome and megaplasmid pSOL1 (Refseq number NC_003030.1 and NC_001988.2; GenBank accession number AE001437 and AE001438) [28] and (B) C. sporogenes NCIMB 10956 (GenBank accession number CP009225).

Mentions: To establish whether transposition had indeed occurred, a more thorough molecular characterisation was undertaken. Genomic DNA of 60 randomly selected Tm R and Em S clones was prepared, digested with HindIII and subjected to Inverse PCR after self-ligation. The gel-purified inverse PCR products were subjected to nucleotide sequence analysis using the primer catP-INV-R2 to determine the sites of insertion. The data demonstrated that each transposon insertion had taken place at a different position around the genome as illustrated in Fig 6.


A universal mariner transposon system for forward genetic studies in the genus Clostridium.

Zhang Y, Grosse-Honebrink A, Minton NP - PLoS ONE (2015)

Genetic map of mariner transposon insertions in C.acetobutylicum (A) and in C. sporogenes (B). Sixty independent transposon mutants were identified and the insertions were sequenced. Insertions in the plus orientation are marked on the circle exterior. Insertions in the minus orientation are marked on the circle interior. Numbers indicate the precise point of insertion according to genome sequence data for (A) C. acetobutylicum ATCC 824 genome and megaplasmid pSOL1 (Refseq number NC_003030.1 and NC_001988.2; GenBank accession number AE001437 and AE001438) [28] and (B) C. sporogenes NCIMB 10956 (GenBank accession number CP009225).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383383&req=5

pone.0122411.g006: Genetic map of mariner transposon insertions in C.acetobutylicum (A) and in C. sporogenes (B). Sixty independent transposon mutants were identified and the insertions were sequenced. Insertions in the plus orientation are marked on the circle exterior. Insertions in the minus orientation are marked on the circle interior. Numbers indicate the precise point of insertion according to genome sequence data for (A) C. acetobutylicum ATCC 824 genome and megaplasmid pSOL1 (Refseq number NC_003030.1 and NC_001988.2; GenBank accession number AE001437 and AE001438) [28] and (B) C. sporogenes NCIMB 10956 (GenBank accession number CP009225).
Mentions: To establish whether transposition had indeed occurred, a more thorough molecular characterisation was undertaken. Genomic DNA of 60 randomly selected Tm R and Em S clones was prepared, digested with HindIII and subjected to Inverse PCR after self-ligation. The gel-purified inverse PCR products were subjected to nucleotide sequence analysis using the primer catP-INV-R2 to determine the sites of insertion. The data demonstrated that each transposon insertion had taken place at a different position around the genome as illustrated in Fig 6.

Bottom Line: To increase the effectiveness of the system, a novel replicon conditional for plasmid maintenance was developed, which no longer supports the effective retention of the transposon delivery vehicle in the presence of the inducer isopropyl β-D-1-thiogalactopyranoside (IPTG).Moreover, appropriate screening of both libraries resulted in the isolation of auxotrophic mutants as well as cells deficient in spore formation/germination.This strategy is capable of being implemented in any Clostridium species.

View Article: PubMed Central - PubMed

Affiliation: Clostridia Research Group, BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.

ABSTRACT
DNA transposons represent an essential tool in the armoury of the molecular microbiologist. We previously developed a catP-based mini transposon system for Clostridium difficile in which the expression of the transposase gene was dependent on a sigma factor unique to C. difficile, TcdR. Here we have shown that the host range of the transposon is easily extended through the rapid chromosomal insertion of the tcdR gene at the pyrE locus of the intended clostridial target using Allele-Coupled Exchange (ACE). To increase the effectiveness of the system, a novel replicon conditional for plasmid maintenance was developed, which no longer supports the effective retention of the transposon delivery vehicle in the presence of the inducer isopropyl β-D-1-thiogalactopyranoside (IPTG). As a consequence, those thiamphenicol resistant colonies that arise in clostridial recipients, following plating on agar medium supplemented with IPTG, are almost exclusively due to insertion of the mini transposon into the genome. The system has been exemplified in both Clostridium acetobutylicum and Clostridium sporogenes, where transposon insertion has been shown to be entirely random. Moreover, appropriate screening of both libraries resulted in the isolation of auxotrophic mutants as well as cells deficient in spore formation/germination. This strategy is capable of being implemented in any Clostridium species.

No MeSH data available.


Related in: MedlinePlus