Complete Genome Sequence of Escherichia coli NCM3722

GENOME ANNOUNCEMENT

Escherichia coli is a landmark pathogen, and the K-12 strains are ubiquitous model organisms. E. coli NCM3722 is a prototrophic K-12 strain first described by Sydney Kustu and colleagues for studies of microbial physiology (1, 2). This strain exhibits unique phenotypes, including galactose metabolism and response to spectinomycin, that are more robust than those of the popular MG1655/W3110 lineage. To enable development of this strain as a model organism, we have sequenced the NCM3722 genome as well as a derivative, SJ358, which has been cured of the F plasmid.

E. coli NCM3722 was obtained from the Coli Genetic Stock Center (CGSC) (strain 12355). Cells were prepared for sequencing by streaking on nonselective media and culturing a single colony in LB supplemented with 0.2% glucose and 1× M salts (3). Genomic DNA was prepared by collection of cells by centrifugation from an exponential-phase culture and extraction with a Life Technologies PureLink genomic DNA minikit. A library for sequencing by Pacific Biosciences SMRT technology was prepared and sequenced with P6-C4 chemistry by the UCSD Institute for Genomic Medicine. Illumina 250-cycle paired-end libraries were similarly prepared from single-colony cultures collected in late exponential phase. The resulting single-molecule real-time (SMRT) and paired-end reads were jointly assembled with SPAdes v. 3.1.0 (4), which produced a contig of 4,678,045 bp corresponding to the chromosome and a contig of 67,545 bp corresponding to an F-like plasmid. Samplings of individual single-nucleotide variants (SNVs) were verified by PCR and Sanger sequencing. The F-like plasmid was modified by λRed recombineering to sequentially remove the ccdB gene and the predicted rep-srnBʹ genes. The resulting strain was serially subcultured in LB plus 10% SDS, and samples were periodically streaked on LB for single clones (5). Genomic DNA from one clone, SJ358, was prepared for short-read sequencing identically to NCM3722 and aligned to the NCM3722 contigs using breseq 0.26.0 (6) and Bowtie2 2.2.5. SJ358 is available from Addgene (http://www.addgene.org/67755/).

The chromosomal and F sequences corroborate SNVs and large differences revealed by prior 454-based sequencing, including phage lambda lysogeny and absence the Qin prophage and a CP4-6 prophage fragment. The ilvG and rph-1 mutations of MG1655 are absent, and the rpoS gene is the 33Am variant from EMG2. We found a variety of new SNVs within the rRNA genes rrsH, rrlG, rrlE, rrlB, rrlA, and rrlC. A deletion that includes the rfb operon has orphaned the rfbA gene, resulting in an accumulation of SNVs. Deletion of the rfb operon has been associated with increased resistance to antibiotics, which may contribute to the phenotype of the strain (7).