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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

Vector map of plasmid pMTL-YZ14.Expression of the hyperactive mariner transposase gene Himar1 C9 was driven by the C. difficile toxin B promoter, PtcdB. The plasmid backbone consisted of the conditional replicon between restriction sites AscI and FseI, the macrolide-lincosamide-streptogramin B antibiotic resistance gene ermB, and the Gram-negative replicon, ColE1. The whole mariner element (i.e., transposase gene and catP mini-transposon) can be excised as a SbfI fragment. The control plasmid pMTL-YZ13 was identical, except that the Gram-positive replicon is the pCB102 replicon from C. butyricum. This plasmid conforms to the pMTL80000 modular system for Clostridium shuttle plasmids [32].
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pone.0122411.g005: Vector map of plasmid pMTL-YZ14.Expression of the hyperactive mariner transposase gene Himar1 C9 was driven by the C. difficile toxin B promoter, PtcdB. The plasmid backbone consisted of the conditional replicon between restriction sites AscI and FseI, the macrolide-lincosamide-streptogramin B antibiotic resistance gene ermB, and the Gram-negative replicon, ColE1. The whole mariner element (i.e., transposase gene and catP mini-transposon) can be excised as a SbfI fragment. The control plasmid pMTL-YZ13 was identical, except that the Gram-positive replicon is the pCB102 replicon from C. butyricum. This plasmid conforms to the pMTL80000 modular system for Clostridium shuttle plasmids [32].

Mentions: By endowing the two clostridial hosts with tcdR, effective transposition of the mini-transposon from plasmid pMTL-SC1 should now occur. Before testing this assumption, the plasmid was first modified to incorporate the pCB102 conditional replicon in place of the pBP1 replication region. This was accomplished by substituting the modular pBP1 replicon, excised as an AscI and FseI restriction fragment, with an equivalent fragment encompassing the modular, conditional pCB102 replicon of pMTL87250. The plasmid generated was designated pMTL-YZ14 (Fig 5). To test its effectiveness as a transposon delivery system, the plasmid was transformed into the tcdR-containing strains of C. acetobutylicum and C. sporogenes, CRG3011 and CRG3817. Transfer of the plasmid was initially selected on solidified media supplemented with Em and left on plates for 24 (CRG3817) to 72 (CRG3011) hours, before being plated on media containing Tm and IPTG to select for transposon events and promote plasmid loss, respectively. After 12 to 16 h of incubation, Tm R colonies were visible at a frequency of 2.6 (±0.6) ×10–4 and 3.2 (±0.5) ×10–4 (calculated as the ratio of Tm R CFU to total CFU) in C. acetobutylicum and C. sporogenes, respectively. A total of 100 Tm R colonies of each Clostridium species were picked and patch plated onto appropriate solidified rich media containing either Tm or Em to estimate the percentage of cells that had lost the plasmid. In total, 80% of C. acetobutylicum and 100% of C. sporogenes colonies were Tm resistant (R) and Em sensitive (S), indicative of successful insertion of the catP mini-transposon into chromosome and substantive plasmid loss.


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

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

Vector map of plasmid pMTL-YZ14.Expression of the hyperactive mariner transposase gene Himar1 C9 was driven by the C. difficile toxin B promoter, PtcdB. The plasmid backbone consisted of the conditional replicon between restriction sites AscI and FseI, the macrolide-lincosamide-streptogramin B antibiotic resistance gene ermB, and the Gram-negative replicon, ColE1. The whole mariner element (i.e., transposase gene and catP mini-transposon) can be excised as a SbfI fragment. The control plasmid pMTL-YZ13 was identical, except that the Gram-positive replicon is the pCB102 replicon from C. butyricum. This plasmid conforms to the pMTL80000 modular system for Clostridium shuttle plasmids [32].
© Copyright Policy
Related In: Results  -  Collection

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

pone.0122411.g005: Vector map of plasmid pMTL-YZ14.Expression of the hyperactive mariner transposase gene Himar1 C9 was driven by the C. difficile toxin B promoter, PtcdB. The plasmid backbone consisted of the conditional replicon between restriction sites AscI and FseI, the macrolide-lincosamide-streptogramin B antibiotic resistance gene ermB, and the Gram-negative replicon, ColE1. The whole mariner element (i.e., transposase gene and catP mini-transposon) can be excised as a SbfI fragment. The control plasmid pMTL-YZ13 was identical, except that the Gram-positive replicon is the pCB102 replicon from C. butyricum. This plasmid conforms to the pMTL80000 modular system for Clostridium shuttle plasmids [32].
Mentions: By endowing the two clostridial hosts with tcdR, effective transposition of the mini-transposon from plasmid pMTL-SC1 should now occur. Before testing this assumption, the plasmid was first modified to incorporate the pCB102 conditional replicon in place of the pBP1 replication region. This was accomplished by substituting the modular pBP1 replicon, excised as an AscI and FseI restriction fragment, with an equivalent fragment encompassing the modular, conditional pCB102 replicon of pMTL87250. The plasmid generated was designated pMTL-YZ14 (Fig 5). To test its effectiveness as a transposon delivery system, the plasmid was transformed into the tcdR-containing strains of C. acetobutylicum and C. sporogenes, CRG3011 and CRG3817. Transfer of the plasmid was initially selected on solidified media supplemented with Em and left on plates for 24 (CRG3817) to 72 (CRG3011) hours, before being plated on media containing Tm and IPTG to select for transposon events and promote plasmid loss, respectively. After 12 to 16 h of incubation, Tm R colonies were visible at a frequency of 2.6 (±0.6) ×10–4 and 3.2 (±0.5) ×10–4 (calculated as the ratio of Tm R CFU to total CFU) in C. acetobutylicum and C. sporogenes, respectively. A total of 100 Tm R colonies of each Clostridium species were picked and patch plated onto appropriate solidified rich media containing either Tm or Em to estimate the percentage of cells that had lost the plasmid. In total, 80% of C. acetobutylicum and 100% of C. sporogenes colonies were Tm resistant (R) and Em sensitive (S), indicative of successful insertion of the catP mini-transposon into chromosome and substantive plasmid loss.

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