Title: Deletion of the Clostridium thermocellum recA gene reveals that it is required for thermophilic plasmid replication but not plasmid integration at homologous DNA sequences

Abstract

Abstract 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 replicationmore » 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.« less

Authors:

Groom, Joseph ^[1]; Chung, Daehwan ^[2]; Kim, Sun-Ki ^[3]; Guss, Adam ^[4]; Westpheling, Janet ^[3]

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+ Show Author Affiliations

1. 0000 0004 1936 738X grid.213876.9 Department of Genetics, Davison Life Sciences Building University of Georgia 30602 Athens GA USA, 0000000122986657 grid.34477.33 Department of Chemical Engineering University of Washington 98105 Seattle WA USA, 0000 0004 0446 2659 grid.135519.a Oak Ridge National Laboratory, The Center for BioEnergy Innovation 37831 Oak Ridge TN USA
2. grid.419357.d 0000 0001 2199 3636 National Renewable Energy Laboratory Biosciences Center 80401 Golden CO USA, 0000 0004 0446 2659 grid.135519.a Oak Ridge National Laboratory, The BioEnergy Science Center 37831 Oak Ridge TN USA, 0000 0004 0446 2659 grid.135519.a Oak Ridge National Laboratory, The Center for BioEnergy Innovation 37831 Oak Ridge TN USA
3. 0000 0004 1936 738X grid.213876.9 Department of Genetics, Davison Life Sciences Building University of Georgia 30602 Athens GA USA, 0000 0004 0446 2659 grid.135519.a Oak Ridge National Laboratory, The BioEnergy Science Center 37831 Oak Ridge TN USA, 0000 0004 0446 2659 grid.135519.a Oak Ridge National Laboratory, The Center for BioEnergy Innovation 37831 Oak Ridge TN USA
4. 0000 0004 0446 2659 grid.135519.a Biosciences Division Oak Ridge National Laboratory 37831 Oak Ridge TN USA, 0000 0004 0446 2659 grid.135519.a Oak Ridge National Laboratory, The BioEnergy Science Center 37831 Oak Ridge TN USA, 0000 0004 0446 2659 grid.135519.a Oak Ridge National Laboratory, The Center for BioEnergy Innovation 37831 Oak Ridge TN USA

Publication Date:

Wed Aug 01 00:00:00 EDT 2018

Research Org.:

Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institutes of Health (NIH)

OSTI Identifier:

1924592

Alternate Identifier(s):

OSTI ID: 1607276

Grant/Contract Number:  

AC05-00OR22725; 5T32GM007103

Resource Type:

Published Article

Journal Name:

Journal of Industrial Microbiology and Biotechnology

Additional Journal Information:

Journal Name: Journal of Industrial Microbiology and Biotechnology Journal Volume: 45 Journal Issue: 8; Journal ID: ISSN 1367-5435

Publisher:

Oxford University Press

Country of Publication:

Germany

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; Plasmid; Thermophile; Genetics; Consolidated bioprocessing; RecA