The complete genome sequence of Escherichia coli DH10B: insights into the biology of a laboratory workhorse

Abstract

Escherichia coli DH10B was designed for the propagation of large insert DNA library clones. It is used extensively, taking advantage of properties such as high DNA transformation efficiency and maintenance of large plasmids. The strain was constructed by serial genetic recombination steps, but the underlying sequence changes remained unverified. We report the complete genomic sequence of DH10B by using reads accumulated from the bovine sequencing project at Baylor College of Medicine and assembled with DNAStar's SeqMan genome assembler. The DH10B genome is largely colinear with that of the wild-type K-12 strain MG1655, although it is substantially more complex than previously appreciated, allowing DH10B biology to be further explored. The 226 mutated genes in DH10B relative to MG1655 are mostly attributable to the extensive genetic manipulations the strain has undergone. However, we demonstrate that DH10B has a 13.5-fold higher mutation rate than MG1655, resulting from a dramatic increase in insertion sequence (IS) transposition, especially IS150. IS elements appear to have remodeled genome architecture, providing homologous recombination sites for a 113,260-bp tandem duplication and an inversion. DH10B requires leucine for growth on minimal medium due to the deletion of leuLABCD and harbors both the relA1 and spoT1 alleles causing both sensitivity to nutritional downshifts and slightly lower growth rates relative to the wild type. Finally, while the sequence confirms most of the reported alleles, the sequence of deoR is wild type, necessitating reexamination of the assumed basis for the high transformability of DH10B.

FIG. 1.

Construction of DH10B. The steps leading to the creation of MC1061 from HfrC⁺ are outlined, followed by the steps described by Grant et al. (17) to generate DH10B from MC1061. Genotypes of selected key strains are indicated. The DH10B tonA genotype is a composite compiled from multiple sources. Steps involving a pBR322-recA plasmid are indicated with an asterisk. The branched pathway consisting of 25 steps leading from wild-type K-12 to HfrC⁺ (3) has been omitted for clarity.

FIG. 2.

Circle diagram of the DH10B genome. Protein-encoding genes are shown in the outer ring, with blue boxes representing those genes coded on the forward strand and gold boxes representing those on the complementary strand. Large deletions and insertions are indicated outside of the circle. In the next inner ring, IS elements that cause gene disruptions or are intergenic are indicated with red or aqua tick marks, respectively, with the positions above or below the line indicating which strand is transcribed. SNPs are then indicated with black tick marks. tRNAs (green tick marks) and small regulatory RNAs (purple tick marks) are shown, with those on the forward strand above the line and those on the complementary strand below it. Finally, rRNA operons are shown as red arrowheads.

FIG. 3.

Comparison of the DH10B and MG1655 genomes. Schematic of the two genomes showing colinear blocks of sequence. The endpoints of each block are defined by an adjacent sequence that is present only in one genome. The major structural rearrangements in DH10B are indicated below the genome representation.

FIG. 4.

Distribution of small-scale mutations in the DH10B genome. A schematic of the genome illustrated in Fig. 3 shows the distribution of SNPs and frameshifts (A) and positioning of IS elements (B). (A) Differences in the DH10B sequence affecting conserved domain sequences are indicated by asterisks, with different colors indicating the types of change. Green, silent SNP; red, missense SNP; black, nonsense SNP; yellow, frameshift. Genes in which the mutation has a known phenotypic consequence are indicated. Note that the frameshift in rph corrects the rph-1 allele present in MG1655. SNPs in intergenic regions are indicated with a caret, and at sites where more than a SNP is present, the number of changes is given. (B) IS-disrupted genes in common with MG1655 are indicated in black, while those unique to DH10B are in red. The specific IS element at each location is indicated; the disrupted gene is in parentheses. For clarity, only differences affecting gene regions are shown.

FIG. 5.

Comparison of φ80 and φ80dlacZΔM15 prophage structures. Diagrams of the two prophages show their chromosomal contexts and more-detailed schematics of the open reading frames (boxes) and major promoters within the two elements. Open reading frames above the center horizontal lines of the diagrams are transcribed in the forward direction, while those below the lines are coded on the complementary strand. Color boxes in the prophages indicate the source of the genes: yellow, φ80; blue, E. coli chromosome; green, Tn1000 from the F plasmid. The two types of terminal sequences, attP and res, are indicated, as is the lacZΔM15 allele. In φ80dlacZΔM15, the φ80 sequences end within gene 5 (indicated as 5′).

FIG. 6.

Comparison of the mutational spectra in DH10B and MG1655. A bar graph shows the distribution of cycA mutations obtained in MG1655 and DH10B. Rectangles within each bar represent point mutations (black), IS150 insertions (hatched), and other IS insertions (IS1, IS2, IS5, and not determined) (white). The rectangle representing deletions in MG1655 is not visible, and no deletions were detected in DH10B.