Complete Genome Sequence of Escherichia coli ER1821R, a Laboratory K-12 Derivative Engineered To Be Deficient in All Methylcytosine and Methyladenine Restriction Systems.

We present here the complete genomic sequence of a rifampin-resistant derivative of the Escherichia coli K-12 laboratory strain ER1821, engineered to be deficient in all known restriction systems, making it suitable for generating unbiased libraries from organisms with non-K-12 methylation patterns. The ER1821R genome is most closely related to that of DH1, another popular cloning strain (both derived from MM294), but is deleted for the e14 prophage (McrA(-)) and the immigration control (McrBC(-) EcoKI R(-) M(-) Mrr(-)) loci.

GENOME ANNOUNCEMENT

Wild-type Escherichia coli strains restrict incoming DNA with foreign patterns of nucleotide modification (1, 2), reducing the recovery of gene libraries containing such modifications. E. coli K-12 derivative ER1821, engineered to be restriction deficient (3), has been useful for library preparation (4) and cloning DNA methyltransferases (5, 6). ER1821 is derived from MM294 (7), from which the popular cloning strain DH1 (MM294 recA gyrA) was also derived (8). The restriction systems were removed using P1 transduction to cure the e14 prophage encoding mcrA and delete the immigration control region (ICR) Δ(mcrC-hsd-mrr)114::IS10 (9). An Hfr mating transferred recA to MM294 to form DH1; a large segment of flanking DNA likely accompanied the selected allele. The precise limits of these regions are unknown, and a completed genome for a different MM294 descendant will permit comparison and be beneficial to the scientific community.

Total DNA from a single transconjugant of a rifampin-resistant derivative of ER1821 carrying a Salmonella ceftriaxone resistance plasmid was used to determine the plasmid genome, which will be described elsewhere. A genomic library, constructed using an Illumina Nextera XT kit, produced 1.76 × 106 300-bp paired-end reads on a MiSeq instrument. A de novo assembly by the Geneious assembler (version 6.1.7) (10) produced 84 contigs >1 kb (N50, 103 kb). The full data set, mapped to the DH1 genome (accession no. NC_017625.1) using the Geneious Read Mapper, produced a consensus sequence with 90-fold mean coverage, two large gaps corresponding to the e14 and ICR deletions, and six discordant regions. Complete sequences covering these regions were extracted from the de novo assembly, and the two deletions, five IS insertions, a small expansion of REP161, and a phase-variable inversion switching fimA to “on” (11) were incorporated into the consensus sequence, producing a complete circular genome of 4,595,577 bp. The full data set remapped to this assembly with no gaps or discordant regions. ER1821R and DH1 show 54 single nucleotide polymorphisms (SNPs), excluding insertions and deletions. Thirty-six SNPs in an 842-kb segment of DH1 surround recA from the Hfr donor. Here, ER1821R has rpoS396(am) and luxS+ (wild-type [WT] and frameshift in DH1) and a novel IS3 insertion into lrhA, a negative regulator of motility (11). The e14 prophage is precisely deleted but is closely linked to three novel IS insertions (IS10, IS1R, and IS2). All but one (purB20 [12]) of the remaining SNPs are in 65 kb surrounding the 24-kb ICR deletion, replaced by a single IS10.

ER1821R is relA+ spoT+ but has ancestral rfbD1 and creC510 mutations. Finally, ER1821R has an rpoB mutation conferring rifampin resistance (13). Thus, the genotype for ER1821 becomes λ- F- glnX44 e14 (McrA) rfbD1 endA1 thi-1 Δ(yjiT-opgB)114::IS10 (EcoKI R- M- McrBC- Mrr-) + rpoS393(am) creC510 lrhA::IS3 ydeN::IS10. These newly detected mutations highlight the inherent problems using matings, transduction, and active transposons to transfer markers, which can introduce additional mutations. Comparing ER1821R with DH1, we can infer the likely sequence of the MM294 parent to be ER1821-like through 2 Mb (but lrhA+) and DH1-like (but purB+ gyrA+) for the remainder.

Accession number(s).

The complete genomic sequence of ER1821R (annotated by the NCBI’s prokaryotic genomic pipeline (http://www.ncbi.nlm.nih.gov/genome/annotation_prok/) is available in GenBank with accession no. CP016018.