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      Mutant generation by allelic exchange and genome resequencing of the biobutanol organism Clostridium acetobutylicum ATCC 824

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          Abstract

          Background

          Clostridium acetobutylicum represents a paradigm chassis for the industrial production of the biofuel biobutanol and a focus for metabolic engineering. We have previously developed procedures for the creation of in-frame, marker-less deletion mutants in the pathogen Clostridium difficile based on the use of pyrE and codA genes as counter selection markers. In the current study we sought to test their suitability for use in C. acetobutylicum.

          Results

          Both systems readily allowed the isolation of in-frame deletions of the C. acetobutylicum ATCC 824 spo0A and the cac824I genes, leading to a sporulation minus phenotype and improved transformation, respectively. The pyrE-based system was additionally used to inactivate a putative glycogen synthase (CA_C2239, glgA) and the pSOL1 amylase gene (CA_P0168, amyP), leading to lack of production of granulose and amylase, respectively. Their isolation provided the opportunity to make use of one of the key pyrE system advantages, the ability to rapidly complement mutations at appropriate gene dosages in the genome. In both cases, their phenotypes were restored in terms of production of granulose ( glgA) and amylase ( amyP). Genome re-sequencing of the ATCC 824 COSMIC consortium laboratory strain used revealed the presence of 177 SNVs and 49 Indels, including a 4916-bp deletion in the pSOL1 megaplasmid. A total of 175 SNVs and 48 Indels were subsequently shown to be present in an 824 strain re-acquired (Nov 2011) from the ATCC and are, therefore, most likely errors in the published genome sequence, NC_003030 (chromosome) and NC_001988 (pSOL1).

          Conclusions

          The codA or pyrE counter selection markers appear equally effective in isolating deletion mutants, but there is considerable merit in using a pyrE mutant as the host as, through the use of ACE (Allele-Coupled Exchange) vectors, mutants created (by whatever means) can be rapidly complemented concomitant with restoration of the pyrE allele. This avoids the phenotypic effects frequently observed with high copy number plasmids and dispenses with the need to add antibiotic to ensure plasmid retention. Our study also revealed a surprising number of errors in the ATCC 824 genome sequence, while at the same time emphasising the need to re-sequence commonly used laboratory strains.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s13068-015-0410-0) contains supplementary material, which is available to authorized users.

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          Most cited references 41

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          Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension.

          Gene splicing by overlap extension is a new approach for recombining DNA molecules at precise junctions irrespective of nucleotide sequences at the recombination site and without the use of restriction endonucleases or ligase. Fragments from the genes that are to be recombined are generated in separate polymerase chain reactions (PCRs). The primers are designed so that the ends of the products contain complementary sequences. When these PCR products are mixed, denatured, and reannealed, the strands having the matching sequences at their 3' ends overlap and act as primers for each other. Extension of this overlap by DNA polymerase produces a molecule in which the original sequences are 'spliced' together. This technique is used to construct a gene encoding a mosaic fusion protein comprised of parts of two different class-I major histocompatibility genes. This simple and widely applicable approach has significant advantages over standard recombinant DNA techniques.
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            The ClosTron: a universal gene knock-out system for the genus Clostridium.

            Progress in exploiting clostridial genome information has been severely impeded by a general lack of effective methods for the directed inactivation of specific genes. Those few mutants that have been generated have been almost exclusively derived by single crossover integration of a replication-deficient or defective plasmid by homologous recombination. The mutants created are therefore unstable. Here we have adapted a mutagenesis system based on the mobile group II intron from the ltrB gene of Lactococcus lactis (Ll.ltrB) to function in clostridial hosts. Integrants are readily selected on the basis of acquisition of resistance to erythromycin, and are generated from start to finish in as little as 10 to 14 days. Unlike single crossover plasmid integrants, the mutants are extremely stable. The system has been used to make 6 mutants of Clostridium acetobutylicum and 5 of Clostridium difficile, exceeding the number of published mutants ever generated in these species. Genes have also been inactivated for the first time in Clostridium botulinum and Clostridium sporogenes, suggesting the system will be universally applicable to the genus. The procedure is highly efficient and reproducible, and should revolutionize functional genomic studies in clostridia.
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              Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum.

              The genome sequence of the solvent-producing bacterium Clostridium acetobutylicum ATCC 824 has been determined by the shotgun approach. The genome consists of a 3.94-Mb chromosome and a 192-kb megaplasmid that contains the majority of genes responsible for solvent production. Comparison of C. acetobutylicum to Bacillus subtilis reveals significant local conservation of gene order, which has not been seen in comparisons of other genomes with similar, or, in some cases closer, phylogenetic proximity. This conservation allows the prediction of many previously undetected operons in both bacteria. However, the C. acetobutylicum genome also contains a significant number of predicted operons that are shared with distantly related bacteria and archaea but not with B. subtilis. Phylogenetic analysis is compatible with the dissemination of such operons by horizontal transfer. The enzymes of the solventogenesis pathway and of the cellulosome of C. acetobutylicum comprise a new set of metabolic capacities not previously represented in the collection of complete genomes. These enzymes show a complex pattern of evolutionary affinities, emphasizing the role of lateral gene exchange in the evolution of the unique metabolic profile of the bacterium. Many of the sporulation genes identified in B. subtilis are missing in C. acetobutylicum, which suggests major differences in the sporulation process. Thus, comparative analysis reveals both significant conservation of the genome organization and pronounced differences in many systems that reflect unique adaptive strategies of the two gram-positive bacteria.
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                Author and article information

                Contributors
                muhammad.ehsaan@nottingham.ac.uk
                wouter.kuit@gmail.com
                ying.zhang@nottingham.ac.uk
                Stephen.Cartman@invista.com
                j.heap@imperial.ac.uk
                Klaus.Winzer@nottingham.ac.uk
                Nigel.Minton@nottingham.ac.uk
                Journal
                Biotechnol Biofuels
                Biotechnol Biofuels
                Biotechnology for Biofuels
                BioMed Central (London )
                1754-6834
                4 January 2016
                4 January 2016
                2016
                : 9
                Affiliations
                [ ]Clostridia Research Group, BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), University of Nottingham, University Park, Nottingham, NG7 2RD UK
                [ ]MicCell Bioservices B.V., Edisonstraat 101, 7006 RB Doetinchem, The Netherlands
                [ ]Department of Life Sciences, Centre for Synthetic Biology and Innovation, Imperial College London, South Kensington Campus, London, SW7 2AZ UK
                [ ]Intermediates Sustainability, INVISTA Intermediates, Wilton Centre, Redcar, TS10 4RF UK
                Article
                410
                10.1186/s13068-015-0410-0
                4700727
                © Ehsaan et al. 2015

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100000268, Biotechnology and Biological Sciences Research Council (GB);
                Award ID: BB/G016224/1
                Award ID: BB/L013940/1
                Award Recipient :
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                © The Author(s) 2016

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