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. 2015 Apr 2;10(4):e0122411.
doi: 10.1371/journal.pone.0122411. eCollection 2015.

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

Ying Zhang  1 , Alexander Grosse-Honebrink  1 , Nigel P Minton  1
Affiliations

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

Ying Zhang et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: The University of Nottingham has filed two patents (WO2013/144653 and WO2013/144647) on which Ying Zhang and Nigel Minton are coinventors which describes the use of the developed transposon system. Any net revenue after covering patent costs is shared equitably between the University and the inventors. This does not alter the authors' adherence to all PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Schematic diagram of the lac-based, IPTG inducible expression cassette in pMTL-YZ2 (A), and the demonstration of its function in C.
acetobutylicum (B) and C. sporogenes (C). A: Schematic diagram of the lac-based, IPTG inducible expression cassette. Key: LacI is the LacI repressor protein gene. LacI binds to the indicated lacO region, blocking transcription from the Pfac promoter. The Pptb promoter (derived from the C. beijerinckii gene encoding phosphotransbutyrylase) directs the transcription of the lacI gene. B and C: IPTG induction of CAT production in of C. acetobutylicum (B) and C. sporogenes (C) harbouring pMTL-YZ2. Circles equate to cells which received no IPTG, squares represents samples from cells that were induced with IPTG. The arrow indicates the time of adding IPTG. Activity is expressed as units of CAT activity per mg of soluble protein.
Fig 2
Fig 2. 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.
Fig 3
Fig 3. 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.
Fig 4
Fig 4. Schematic representations of the genome of tcdR strain of C. acetobutylicum (A) and C.
sporogenes (B). A promoter-less copy of the tcdR gene (including its ribosome binding site, RBS) of C. difficile strain 630 has been inserted into the genome using ACE technology immediately downstream of the pyrE gene (A: CAC0027, B: CS3413). Illustrated are the surrounding genes, and the position of the promoter responsible for expression of tcdR, and the position of the tcdR RBS. The illustrated terminator is the T1 transcriptional terminator of the C. pasteurianum ferredoxin gene.
Fig 5
Fig 5. 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].
Fig 6
Fig 6. 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).
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