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

Analysis of the functionality of the exogenous TcdR in two clostridial strains (CRG3011 and CRG3817).Plasmid maps of the pMTL82254-PtcdB (A) and pMTL82254-Pfdx (B). Key: CD0164 terminator, a transcriptional terminator isolated from downstream of the C. difficile strain 630 CD0164 gene; catP, a C. perfringens-derived gene encoding chloramphenicol acetyltransferase; Cpa fdx terminator, transcriptional terminator of the ferredoxin gene of C. pasteurianum; repA and orf2, replication region of the C. botulinum plasmid pBP1; 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. CD tcdB promoter, the promoter region of the C. difficile tcdB gene; Csp fdx promoter: the promoter region of the C. sporogenes fdx gene. (C): CAT activity of either C. acetobutylicum ATCC 824 wild type or CRG3011 (tcdR containing C. acetobutylicum ATCC 824) carrying plasmids pMTL82254-PtcdB and pMTL82254-Pfdx. (D): CAT activity of either C. sporogenes NCIMB 10969 wild type or CRG3817 (tcdR containing C. sporogenes NCIMB 10969) carrying plasmids pMTL82254-PtcdB and pMTL82254-Pfdx. Black circles ●, wild type with pMTL82254-PtcdB; black squares ■, wild type with pMTL82254-Pfdx; black triangles ▲, CRG3011/CRG3817 with pMTL82254-PtcdB; black triangles ▼, CRG3011/CRG3817 with pMTL82254-Pfdx.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0122411.g003: Analysis of the functionality of the exogenous TcdR in two clostridial strains (CRG3011 and CRG3817).Plasmid maps of the pMTL82254-PtcdB (A) and pMTL82254-Pfdx (B). Key: CD0164 terminator, a transcriptional terminator isolated from downstream of the C. difficile strain 630 CD0164 gene; catP, a C. perfringens-derived gene encoding chloramphenicol acetyltransferase; Cpa fdx terminator, transcriptional terminator of the ferredoxin gene of C. pasteurianum; repA and orf2, replication region of the C. botulinum plasmid pBP1; 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. CD tcdB promoter, the promoter region of the C. difficile tcdB gene; Csp fdx promoter: the promoter region of the C. sporogenes fdx gene. (C): CAT activity of either C. acetobutylicum ATCC 824 wild type or CRG3011 (tcdR containing C. acetobutylicum ATCC 824) carrying plasmids pMTL82254-PtcdB and pMTL82254-Pfdx. (D): CAT activity of either C. sporogenes NCIMB 10969 wild type or CRG3817 (tcdR containing C. sporogenes NCIMB 10969) carrying plasmids pMTL82254-PtcdB and pMTL82254-Pfdx. Black circles ●, wild type with pMTL82254-PtcdB; black squares ■, wild type with pMTL82254-Pfdx; black triangles ▲, CRG3011/CRG3817 with pMTL82254-PtcdB; black triangles ▼, CRG3011/CRG3817 with pMTL82254-Pfdx.

Mentions: Plasmid pMTL82254-PtcdB as shown in Fig 3A, was constructed by excising a ca. 334 bp NotI/NdeI fragment from the plasmid pMTL-SC1 [15] and inserting it between the NotI and NdeI sites of plasmid pMTL82254 [32]. For comparative purposes, a second plasmid was constructed identical to pMTL82254-PtcdB, but in which the fragment encompassing the PtcdB promoter was replaced with an equivalent NotI/NdeI fragment encompassing the Pfdx promoter. This plasmid was designated pMTL82254-Pfdx as illustrated in Fig 3B.


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

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

Analysis of the functionality of the exogenous TcdR in two clostridial strains (CRG3011 and CRG3817).Plasmid maps of the pMTL82254-PtcdB (A) and pMTL82254-Pfdx (B). Key: CD0164 terminator, a transcriptional terminator isolated from downstream of the C. difficile strain 630 CD0164 gene; catP, a C. perfringens-derived gene encoding chloramphenicol acetyltransferase; Cpa fdx terminator, transcriptional terminator of the ferredoxin gene of C. pasteurianum; repA and orf2, replication region of the C. botulinum plasmid pBP1; 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. CD tcdB promoter, the promoter region of the C. difficile tcdB gene; Csp fdx promoter: the promoter region of the C. sporogenes fdx gene. (C): CAT activity of either C. acetobutylicum ATCC 824 wild type or CRG3011 (tcdR containing C. acetobutylicum ATCC 824) carrying plasmids pMTL82254-PtcdB and pMTL82254-Pfdx. (D): CAT activity of either C. sporogenes NCIMB 10969 wild type or CRG3817 (tcdR containing C. sporogenes NCIMB 10969) carrying plasmids pMTL82254-PtcdB and pMTL82254-Pfdx. Black circles ●, wild type with pMTL82254-PtcdB; black squares ■, wild type with pMTL82254-Pfdx; black triangles ▲, CRG3011/CRG3817 with pMTL82254-PtcdB; black triangles ▼, CRG3011/CRG3817 with pMTL82254-Pfdx.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0122411.g003: Analysis of the functionality of the exogenous TcdR in two clostridial strains (CRG3011 and CRG3817).Plasmid maps of the pMTL82254-PtcdB (A) and pMTL82254-Pfdx (B). Key: CD0164 terminator, a transcriptional terminator isolated from downstream of the C. difficile strain 630 CD0164 gene; catP, a C. perfringens-derived gene encoding chloramphenicol acetyltransferase; Cpa fdx terminator, transcriptional terminator of the ferredoxin gene of C. pasteurianum; repA and orf2, replication region of the C. botulinum plasmid pBP1; 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. CD tcdB promoter, the promoter region of the C. difficile tcdB gene; Csp fdx promoter: the promoter region of the C. sporogenes fdx gene. (C): CAT activity of either C. acetobutylicum ATCC 824 wild type or CRG3011 (tcdR containing C. acetobutylicum ATCC 824) carrying plasmids pMTL82254-PtcdB and pMTL82254-Pfdx. (D): CAT activity of either C. sporogenes NCIMB 10969 wild type or CRG3817 (tcdR containing C. sporogenes NCIMB 10969) carrying plasmids pMTL82254-PtcdB and pMTL82254-Pfdx. Black circles ●, wild type with pMTL82254-PtcdB; black squares ■, wild type with pMTL82254-Pfdx; black triangles ▲, CRG3011/CRG3817 with pMTL82254-PtcdB; black triangles ▼, CRG3011/CRG3817 with pMTL82254-Pfdx.
Mentions: Plasmid pMTL82254-PtcdB as shown in Fig 3A, was constructed by excising a ca. 334 bp NotI/NdeI fragment from the plasmid pMTL-SC1 [15] and inserting it between the NotI and NdeI sites of plasmid pMTL82254 [32]. For comparative purposes, a second plasmid was constructed identical to pMTL82254-PtcdB, but in which the fragment encompassing the PtcdB promoter was replaced with an equivalent NotI/NdeI fragment encompassing the Pfdx promoter. This plasmid was designated pMTL82254-Pfdx as illustrated in Fig 3B.

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