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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

Plasmid maps and the conditionality analysis of the conditional replicon.Plasmid maps of the non-conditional control plasmid pMTL83251-lacI-T (A) and conditional plasmid pMTL83251-lacI (B). Key: CD0164 terminator, a transcriptional terminator isolated from downstream of the Clostridium difficile strain 630 CD0164 gene; lacI, the E. coli gene encoding LacI repressor; Pptb, the promoter of the C. beijerinckii gene encoding phosphotransbutyrylase; Pfac, the promoter of the C. pasteurianum ferredoxin gene derivatised to include an E. coli lac operator; repH, replication region of the Clostridium butyricum plasmid pCB102; ermB, the macrolide-lincosamide-streptogramin B antibiotic resistance gene of plasmid pAMß1; ColE1, the replication origin of plasmid ColE1, and; traJ, transfer function of the RP4 oriT region. Cpa fdx terminator, transcriptional terminator of the ferredoxin gene of C. pasteurianum (this feature is underlined due to its existence only in A: pMTL83251-lacI-T, but not in B: pMTL83251-lacI). C and D: the ability to replicate of pMTL83251-lacI-T and pMTL83251-lacI in of C. acetobutylicum (C) and C. sporogenes (D) in the absence (CFU account in black) and presence (CFU account in grey) of IPTG.
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pone.0122411.g002: Plasmid maps and the conditionality analysis of the conditional replicon.Plasmid maps of the non-conditional control plasmid pMTL83251-lacI-T (A) and conditional plasmid pMTL83251-lacI (B). Key: CD0164 terminator, a transcriptional terminator isolated from downstream of the Clostridium difficile strain 630 CD0164 gene; lacI, the E. coli gene encoding LacI repressor; Pptb, the promoter of the C. beijerinckii gene encoding phosphotransbutyrylase; Pfac, the promoter of the C. pasteurianum ferredoxin gene derivatised to include an E. coli lac operator; repH, replication region of the Clostridium butyricum plasmid pCB102; ermB, the macrolide-lincosamide-streptogramin B antibiotic resistance gene of plasmid pAMß1; ColE1, the replication origin of plasmid ColE1, and; traJ, transfer function of the RP4 oriT region. Cpa fdx terminator, transcriptional terminator of the ferredoxin gene of C. pasteurianum (this feature is underlined due to its existence only in A: pMTL83251-lacI-T, but not in B: pMTL83251-lacI). C and D: the ability to replicate of pMTL83251-lacI-T and pMTL83251-lacI in of C. acetobutylicum (C) and C. sporogenes (D) in the absence (CFU account in black) and presence (CFU account in grey) of IPTG.

Mentions: Having established the functionality of the Pfac inducible promoter, the cat gene was deleted from plasmid pMTL83251-YZ2, in order to bring the pCB102 replicon [31] under the transcriptional control of Pfac. Two plasmids were created. In the one (pMTL83251-lacI-T see Fig 2A), pMTL83251-YZ2 was cleaved with BamHI and HindIII, the sticky-ends created blunt-ended by treatment with T4 polymerase, and the resultant linear fragment subjected to self-ligation. In a second plasmid (pMTL83251-lacI, see Fig 2B), pMTL83251-YZ2 was digested with BamHI and AscI, the sticky-ends created blunt-ended by treatment with T4 polymerase, and the resultant linear fragment subjected to self-ligation.


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

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

Plasmid maps and the conditionality analysis of the conditional replicon.Plasmid maps of the non-conditional control plasmid pMTL83251-lacI-T (A) and conditional plasmid pMTL83251-lacI (B). Key: CD0164 terminator, a transcriptional terminator isolated from downstream of the Clostridium difficile strain 630 CD0164 gene; lacI, the E. coli gene encoding LacI repressor; Pptb, the promoter of the C. beijerinckii gene encoding phosphotransbutyrylase; Pfac, the promoter of the C. pasteurianum ferredoxin gene derivatised to include an E. coli lac operator; repH, replication region of the Clostridium butyricum plasmid pCB102; ermB, the macrolide-lincosamide-streptogramin B antibiotic resistance gene of plasmid pAMß1; ColE1, the replication origin of plasmid ColE1, and; traJ, transfer function of the RP4 oriT region. Cpa fdx terminator, transcriptional terminator of the ferredoxin gene of C. pasteurianum (this feature is underlined due to its existence only in A: pMTL83251-lacI-T, but not in B: pMTL83251-lacI). C and D: the ability to replicate of pMTL83251-lacI-T and pMTL83251-lacI in of C. acetobutylicum (C) and C. sporogenes (D) in the absence (CFU account in black) and presence (CFU account in grey) of IPTG.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0122411.g002: Plasmid maps and the conditionality analysis of the conditional replicon.Plasmid maps of the non-conditional control plasmid pMTL83251-lacI-T (A) and conditional plasmid pMTL83251-lacI (B). Key: CD0164 terminator, a transcriptional terminator isolated from downstream of the Clostridium difficile strain 630 CD0164 gene; lacI, the E. coli gene encoding LacI repressor; Pptb, the promoter of the C. beijerinckii gene encoding phosphotransbutyrylase; Pfac, the promoter of the C. pasteurianum ferredoxin gene derivatised to include an E. coli lac operator; repH, replication region of the Clostridium butyricum plasmid pCB102; ermB, the macrolide-lincosamide-streptogramin B antibiotic resistance gene of plasmid pAMß1; ColE1, the replication origin of plasmid ColE1, and; traJ, transfer function of the RP4 oriT region. Cpa fdx terminator, transcriptional terminator of the ferredoxin gene of C. pasteurianum (this feature is underlined due to its existence only in A: pMTL83251-lacI-T, but not in B: pMTL83251-lacI). C and D: the ability to replicate of pMTL83251-lacI-T and pMTL83251-lacI in of C. acetobutylicum (C) and C. sporogenes (D) in the absence (CFU account in black) and presence (CFU account in grey) of IPTG.
Mentions: Having established the functionality of the Pfac inducible promoter, the cat gene was deleted from plasmid pMTL83251-YZ2, in order to bring the pCB102 replicon [31] under the transcriptional control of Pfac. Two plasmids were created. In the one (pMTL83251-lacI-T see Fig 2A), pMTL83251-YZ2 was cleaved with BamHI and HindIII, the sticky-ends created blunt-ended by treatment with T4 polymerase, and the resultant linear fragment subjected to self-ligation. In a second plasmid (pMTL83251-lacI, see Fig 2B), pMTL83251-YZ2 was digested with BamHI and AscI, the sticky-ends created blunt-ended by treatment with T4 polymerase, and the resultant linear fragment subjected to self-ligation.

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