Optical mapping and sequencing of the Escherichia coli KO11 genome reveal extensive chromosomal rearrangements, and multiple tandem copies of the Zymomonas mobilis pdc and adhB genes
- 945 Downloads
Escherichia coli KO11 (ATCC 55124) was engineered in 1990 to produce ethanol by chromosomal insertion of the Zymomonas mobilis pdc and adhB genes into E. coli W (ATCC 9637). KO11FL, our current laboratory version of KO11, and its parent E. coli W were sequenced, and contigs assembled into genomic sequences using optical NcoI restriction maps as templates. E. coli W contained plasmids pRK1 (102.5 kb) and pRK2 (5.4 kb), but KO11FL only contained pRK2. KO11FL optical maps made with AflII and with BamHI showed a tandem repeat region, consisting of at least 20 copies of a 10-kb unit. The repeat region was located at the insertion site for the pdc, adhB, and chloramphenicol-resistance genes. Sequence coverage of these genes was about 25-fold higher than average, consistent with amplification of the foreign genes that were inserted as circularized DNA. Selection for higher levels of chloramphenicol resistance originally produced strains with higher pdc and adhB expression, and hence improved fermentation performance, by increasing the gene copy number. Sequence data for an earlier version of KO11, ATCC 55124, indicated that multiple copies of pdc adhB were present. Comparison of the W and KO11FL genomes showed large inversions and deletions in KO11FL, mostly enabled by IS10, which is absent from W but present at 30 sites in KO11FL. The early KO11 strain ATCC 55124 had no rearrangements, contained only one IS10, and lacked most accumulated single nucleotide polymorphisms (SNPs) present in KO11FL. Despite rearrangements and SNPs in KO11FL, fermentation performance was equal to that of ATCC 55124.
KeywordsOptical mapping Fermentation Ethanol Escherichia coli Genome sequencing
The authors thank Savita Shanker at the DNA Sequencing core at the University of Florida for Sanger sequencing of plasmids and PCR products, and Dibyendu Kumar at the UF Bacterial Genome Finishing Program for assistance with bridging gaps between contigs. We acknowledge research support by grants from the US Department of Energy (DE-FG36-08GO88142), US Department of Agriculture, National Institute of Food and Agriculture (2011-10006-30358), and Myriant Technologies. L.O. Ingram is a consultant for Myriant Technologies and a minor stock holder (less than 4%).
- 9.Conrad TM, Joyce AR, Applebee MK, Barrett CL, Xie B, Gao Y, Palsson BO (2009) Whole-genome resequencing of Escherichia coli K-12 MG1655 undergoing short-term laboratory evolution in lactate minimal media reveals flexible selection of adaptive mutations. Genome Biol 10:R118PubMedCrossRefGoogle Scholar
- 11.Diaz E, Ferrandez A, Prieto MA, Garcia JL (2001) Biodegradation of aromatic compounds by Escherichia coli. Microbiol Mol Biol Rev 65:523–569Google Scholar
- 12.Drummond AJ, Ashton B, Buxton S, Cheung M, Cooper A, Duran C, Field M, Heled J, Kearse M, Markowitz S, Moir R, Stones-Havas S, Sturrock S, Thierer T, Wilson A (2010) Geneious v5.3. Available from http://www.geneious.com
- 15.Herring CD, Raghunathan A, Honisch C, Patel T, Applebee MK, Joyce AR, Albert TJ, Blattner FR, van den Boom D, Cantor CR, Palsson BO (2006) Comparative genome sequencing of Escherichia coli allows observation of bacterial evolution on a laboratory timescale. Nat Genet 38:1406–1412PubMedCrossRefGoogle Scholar
- 19.Keseler IM, Collado-Vides J, Santos-Zavaleta A, Peralta-Gil M, Gama-Castro S, Muniz-Rascado L, Bonavides-Martinez C, Paley S, Krummenacker M, Altman T, Kaipa P, Spaulding A, Pacheco J, Latendresse M, Fulcher C, Sarker M, Shearer AG, Mackie A, Paulsen I, Gunsalus RP, Karp PD (2011) EcoCyc: a comprehensive database of Escherichia coli biology. Nucleic Acids Res 39:D583–D590PubMedCrossRefGoogle Scholar
- 26.Oshima K, Toh H, Ogura Y, Sasamoto H, Morita H, Park SH, Ooka T, Iyoda S, Taylor TD, Hayashi T, Itoh K, Hattori M (2008) Complete genome sequence and comparative analysis of the wild-type commensal Escherichia coli strain SE11 isolated from a healthy adult. DNA Res 15:375–386PubMedCrossRefGoogle Scholar
- 34.Touchon M, Hoede C, Tenaillon O, Barbe V, Baeriswyl S, Bidet P, Bingen E, Bonacorsi S, Bouchier C, Bouvet O, Calteau A, Chiapello H, Clermont O, Cruveiller S, Danchin A, Diard M, Dossat C, Karoui ME, Frapy E, Garry L, Ghigo JM, Gilles AM, Johnson J, Le Bouguenec C, Lescat M, Mangenot S, Martinez-Jehanne V, Matic I, Nassif X, Oztas S, Petit MA, Pichon C, Rouy Z, Ruf CS, Schneider D, Tourret J, Vacherie B, Vallenet D, Medigue C, Rocha EP, Denamur E (2009) Organised genome dynamics in the Escherichia coli species results in highly diverse adaptive paths. PLoS Genet 5:e1000344PubMedCrossRefGoogle Scholar