Deletion of the Clostridium thermocellum recA gene reveals that it is required for thermophilic plasmid replication but not plasmid integration at homologous DNA sequences
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A limitation to the engineering of cellulolytic thermophiles is the availability of functional, thermostable (≥ 60 °C) replicating plasmid vectors for rapid expression and testing of genes that provide improved or novel fuel molecule production pathways. A series of plasmid vectors for genetic manipulation of the cellulolytic thermophile Caldicellulosiruptor bescii has recently been extended to Clostridium thermocellum, another cellulolytic thermophile that very efficiently solubilizes plant biomass and produces ethanol. While the C. bescii pBAS2 replicon on these plasmids is thermostable, the use of homologous promoters, signal sequences and genes led to undesired integration into the bacterial chromosome, a result also observed with less thermostable replicating vectors. In an attempt to overcome undesired plasmid integration in C. thermocellum, a deletion of recA was constructed. As expected, C. thermocellum ∆recA showed impaired growth in chemically defined medium and an increased susceptibility to UV damage. Interestingly, we also found that recA is required for replication of the C. bescii thermophilic plasmid pBAS2 in C. thermocellum, but it is not required for replication of plasmid pNW33N. In addition, the C. thermocellum recA mutant retained the ability to integrate homologous DNA into the C. thermocellum chromosome. These data indicate that recA can be required for replication of certain plasmids, and that a recA-independent mechanism exists for the integration of homologous DNA into the C. thermocellum chromosome. Understanding thermophilic plasmid replication is not only important for engineering of these cellulolytic thermophiles, but also for developing genetic systems in similar new potentially useful non-model organisms.
KeywordsPlasmid Thermophile Genetics Consolidated bioprocessing RecA
JG was supported for a portion of this work by an NIH 5T32GM007103 Predoctoral Training Grant to the Genetics Department of the University of Georgia. Funding was provided by The BioEnergy Science (BESC) and The Center for Bioenergy Innovation (CBI), U.S. Department of Energy Bioenergy Research Centers supported by the Office of Biological and Environmental Research in the DOE Office of Science. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
- 1.Argyros DA, Tripathi SA, Barrett TF, Rogers SR, Feinberg LF, Olson DG, Foden JM, Miller BB, Lynd LR, Hogsett DA, Caiazza NC (2011) High ethanol titers from cellulose by using metabolically engineered thermophilic, anaerobic microbes. Appl Environ Microbiol 77(23):8288–8294CrossRefPubMedPubMedCentralGoogle Scholar
- 2.Bayer EA, Shoham Y, Lamed R (2013) Lignocellulose-decomposing bacteria and their enzyme systems. In: Rosenberg E (ed) The prokaryotes—prokaryotic physiology and biochemistry. Springer, Berlin, pp 215–266Google Scholar
- 3.Bianco PR, Kowalczykowski SC (2005) RecA Protein, in Encyclopedia of Life Sciences (eLS). Wiley, pp 1–8Google Scholar
- 17.Gomez RF, Snedecor B, Mendez B (1980) Development of genentic principles in Clostridium thermocellum. Developments in industrial microbiology, vol 22. Society for Industrial Microbiology, Arlington, pp 87–95Google Scholar
- 19.Groom J, Chung D, Olson DG, Lynd LR, Guss AM, Westpheling J (2016) Promiscuous plasmid replication in thermophiles: use of a novel hyperthermophilic replicon for genetic manipulation of Clostridium thermocellum at its optimum growth temperature. Metab Eng Commun 3:30–38CrossRefPubMedPubMedCentralGoogle Scholar
- 23.Irla M, Heggeset TMB, Nærdal I, Paul L, Haugen T, Le SB, Brautaset T, Wendisch VF (2016) Genome-based genetic tool development for Bacillus methanolicus: theta- and rolling circle-replicating plasmids for inducible gene expression and application to methanol-based cadaverine production. Front Microbiol 7:1481CrossRefPubMedPubMedCentralGoogle Scholar
- 25.Kim S-K, Groom J, Chung D, Elkins J, Westpheling J (2017) Expression of a heat-stable NADPH-dependent alcohol dehydrogenase from Thermoanaerobacter pseudethanolicus 39E in Clostridium thermocellum 1313 results in increased hydroxymethylfurfural resistance. Biotechnol Biofuels 10(1):66CrossRefPubMedPubMedCentralGoogle Scholar
- 27.Lamed R, Bayer EA (1988) The cellulosome of Clostridium thermocellum. In: Allen IL (ed) Advances in applied microbiology, vol 33. Academic Press, Cambridge, pp 1–46Google Scholar
- 33.Marchler-Bauer A, Derbyshire MK, Gonzales NR, Lu S, Chitsaz F, Geer LY, Geer RC, He J, Gwadz M, Hurwitz DI, Lanczycki CJ, Lu F, Marchler GH, Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, Bryant SH (2015) CDD: NCBI’s conserved domain database. Nucleic Acids Res 43:D222–D226CrossRefPubMedGoogle Scholar
- 48.Tripathi SA, Olson DG, Argyros DA, Miller BB, Barrett TF, Murphy DM, McCool JD, Warner AK, Rajgarhia VB, Lynd LR, Hogsett DA, Caiazza NC (2010) Development of pyrF-based genetic system for targeted gene deletion in Clostridium thermocellum and creation of a pta mutant. Appl Environ Microbiol 76(19):6591–6599CrossRefPubMedPubMedCentralGoogle Scholar