Complete Genome Sequence of Escherichia coli BLR(DE3), a recA-Deficient Derivative of E. coli BL21(DE3).

Escherichia coli BLR(DE3) is a commercially available recA-deficient derivative of BL21(DE3), one of the most widely used strains for recombinant protein expression. Here, we present the full-genome sequence of BLR(DE3) and highlight additional differences with its parent strain BL21(DE3) which were previously unreported but may affect its physiology.

GENOME ANNOUNCEMENT

Escherichia coli is widely used for the production of recombinant polypeptides, including therapeutic proteins (1). The most popular system for protein overexpression in E. coli is the isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible T7 RNA polymerase cascade system, in which expression of the gene of interest is controlled by a T7 RNA polymerase promoter (e.g., on a pET vector) in a strain carrying the T7 RNA polymerase gene under control of an IPTG-inducible promoter (e.g., λDE3 lysogenic strains) (2). The genome sequence of the widely used E. coli B strain BL21(DE3) (2) is available (3). BLR(DE3) is a recA-deficient derivative of BL21(DE3) that helps stabilize plasmids containing repetitive sequences (4). According to Novagen, BLR(DE3) has the same genotype, except for a mutation in recA [Δ(srl-recA)306::Tn10], which was initially obtained in an E. coli K-12 background (5) and later transferred to BL21 (4, 6).

We sequenced the BLR(DE3) strain obtained from Novagen in order to verify whether additional differences are present in BLR(DE3) compared to BL21(DE3). We performed de novo hybrid assembly from PacBio sequencing (102,708 reads, with a mean length of 5,173 bp) and Illumina HiSeq 2000 sequencing (approx. 4,500,000 high-quality paired-end 100-bp reads) for closing gaps and correcting PacBio sequencing errors. A total of 4,306 coding sequences (CDSs), 230 pseudogenes, 85 tRNA genes, 22 rRNA genes (8 operons), and 64 noncoding RNA genes were annotated.

A comparison with BL21(DE3) indicates larger differences than expected, with two main divergent regions. First, the DE3 prophage contains three large deletions of 4.9 kb (cro-Rz), 1.8 kb (A-B), and 11.1 kb (Fi-J), suggesting the prophage is nonfunctional. As expected, the second divergent region is the recA locus. A 6.9-kb region from recA to srlR is replaced with Tn10 in BLR(DE3). Besides recA and the srl operon, BLR(DE3) thus also lacks pncC (encoding nicotinamide mononucleotide deamidase, a key enzyme of the pyridine nucleotide cycle [7]) and mtlB (encoding a lytic murein transglycosylase). Downstream of Tn10, a large region (approx. 75 kb; srlR-queE) diverges significantly from BL21(DE3), with 577 nucleotide substitutions (1 to 3 bp), short insertions/deletions (1 bp), and the replacement of an IS186 element with a full clustered regularly interspaced short palindromic repeat (CRISPR) locus, as found in E. coli MG1655. We infer that the entire ~85-kb region from recA to queE was transferred from the original K-12 background bearing the Δ(srl-recA)306::Tn10 mutation. This region contains several genes involved in metabolism and other key processes, such as DNA mismatch repair (mutS) or stress response (rpoS). Importantly, rpoS carries a nonsense mutation at codon 33, which has been shown to reduce σS activity (8). Outside the DE3 and recA loci, BLR(DE3) and BL21(DE3) contain fewer than 20 differences. Of note, these include a previously identified mutation in ilvA, a gene responsible for isoleucine auxotrophy (9).

Globally, the genome sequence of BLR(DE3) indicates that it cannot be merely considered a recA-deficient BL21(DE3) strain. The newly identified differences might contribute to a differential physiology of BLR(DE3), besides its reported impaired homologous recombination.

Accession number(s).

The complete genome sequence of Escherichia coli BLR(DE3) has been deposited at GenBank under accession number CP020368.