Location and characterisation of a new replication origin in the E. coli K12 chromosome

Díaz, Ramón; Barnsley, Patricia G. H.; Pritchard, R. H.

doi:10.1007/bf00425531

Cited by 32 publications

(7 citation statements)

References 24 publications

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Section: Resultsmentioning

confidence: 93%

“…The ability of Rac to exist as the oriJ plasmid has been documented previously (11). As oriJ plasmids do not replicate in the presence of a Rac prophage (11), the integrated and plasmid forms of the element must be derived from different DH10B cell subpopulations. The oriJ plasmid copy number has not been reported, but the depth of coverage of the episomal contig is twice that of the prophage, arguing that Rac excision and maintenance as a plasmid occur with significant frequency.…”

Section: Resultsmentioning

confidence: 93%

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The Complete Genome Sequence ofEscherichia coliDH10B: Insights into the Biology of a Laboratory Workhorse

et al. 2008

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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.Molecular biology studies rely heavily on Escherichia coli for essential operations, ranging from the simple propagation of plasmid DNA to the creation of large clone libraries for wholegenome sequence determination. Among the strains developed as hosts for these everyday applications, DH10B (17) is commonly used across the research community, taking advantage of particularly useful properties exhibited by the strain. These include high transformation efficiency, the ability to take up and stably maintain large plasmids, the lack of methylation-dependent restriction systems (MDRS), and colony screening via lacZ-based ␣-complementation. However, analysis of sequenced bacterial artificial chromosome (BAC) clones derived from DH10B shows a high incidence of insertion sequence (IS) transposition from the chromosome into the cloned fragment (25).The genome of DH10B was constructed before the modern era of molecular biology, through a series of genetic manipulations (Fig. 1). The progenitors were all K-12 strains, with the exception of D7091F, in which a region surrounding the ⌬(araA-leu)7697 deletion had been derived from E. coli B SB3118 by P1 transduction (John Wertz, personal communication). Ultimately, MC1061 (9) served as a starting point for Hanahan and coworkers to replace alleles by using a series of P1 transductions that resulted in DH10B (17). Among the engineered gene replacements were recA1 ...

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Section: Resultsmentioning

confidence: 93%

Section: Resultsmentioning

confidence: 93%

The Complete Genome Sequence ofEscherichia coliDH10B: Insights into the Biology of a Laboratory Workhorse

et al. 2008

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“…Additionally, all strains used in the experiments reported here lacked the rac prophage, which is located at 30 min (1). This avoided complications due to the potential induction of this prophage, which could lead to its excision (9) or to replication initiated at oriJ (28). Fig.…”

Section: Resultsmentioning

confidence: 99%

The terminus region of the Escherichia coli chromosome contains two separate loci that exhibit polar inhibition of replication.

Hill¹,

Henson²,

Kuempel³

1987

Proc. Natl. Acad. Sci. U.S.A.

114

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The terminus region of the chromosome of Escherichia coli contains two separate sites, called Ti and T2, that inhibit replication forks. Ti is located near 28.5 min, which is adjacent to tip, and T2 is located at 34.5-35.7 min on the opposite side of the terminus region, near manA. The sites act in a polar fashion, and replication forks traveling in a clockwise direction with respect to the genetic map are not inhibited as they pass through Ti but are inhibited at T2. Similarly, counterclockwise forks are not inhibited at T2 but are inhibited at Ti. Consequently, forks are not inhibited until they have passed through the terminus region and are about to leave it. Studies with deletion strains have located T2 within a 58-kilobase interval, which corresponds to kilobase coordinates 387-445 on the physical map of the terminus region.The terminus region of the chromosome of Escherichia coli is located directly opposite the origin of replication on the circular genetic map (1). One of the most interesting features of this region is that it inhibits replication forks that are traveling in either a clockwise or counterclockwise direction with respect to the map (2-5). When origins of replication located near the terminus region were used (2-5), inhibition of replication forks occurred somewhere in the interval between trp (28 min) and manA (36 min). Although the function of this inhibition is unknown, its presence suggests that the terminus might encode partitioning, decatenation, or cell-division control sites, and impediment of replication forks in this region evolved to ensure efficient use of such sites (6).More recent experiments using replication cycles initiated at oriC (7) indicated that the last DNA to be replicated, and consequently the region where forks meet most frequently, was located near 31.2 min. Based on these results, it generally has been assumed that the replication block (terC) is located at this position (1). As discussed by Bouchd et al. (7), however, other interpretations of the data can be made. Therefore, identification of the region of most frequent fork encounter does not necessarily identify the site ofreplicationfork inhibition.We report here, as do de Massy et al. in an accompanying report in this issue (8), that replication forks are not inhibited at a single site in the middle of the terminus region. Instead, there are two inhibition sites, Ti and T2, which are located at the outer edges of the terminus region. These sites are polar, and they only inhibit replication forks that have passed through the terminus region and are about to leave it. Specifically, clockwise traveling replication forks are inhibited at T2 at 34.5-35.8 min, near manA, and counterclockwise traveling forks are inhibited at Ti at 28.5 min, near trp. MATERIALS AND METHODSBacterial Strains. All replication fork assays were performed with strains PK998 or PK1012 and derivatives containing the indicated deletions. These temperature-sensitive strains are dnaA mutants and contained temperature-sensitive P2 D4 cS...

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“…Two cryptic lambdoid phages, rac and kim, are located in the terminus region of some strains of E. coli K-12 (Diaz et al, 1979;Espion et al, 1983). Two cryptic lambdoid phages, rac and kim, are located in the terminus region of some strains of E. coli K-12 (Diaz et al, 1979;Espion et al, 1983).…”

Section: Minichromosome Replication After Sos Inductionmentioning

confidence: 99%

DNA damage-inducible origins of DNA replication in Escherichia coli.

et al. 1992

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Upon induction of the SOS response in Escherichia coli, the mode of initiation of DNA replication is altered such that it can occur in the absence of normally required protein synthesis. This type of DNA replication has been termed induced stable DNA replication (iSDR). We examined the origin usage during iSDR and found that the initiation of iSDR occurs primarily in the oriC and terC regions of the chromosome in a manner completely independent of transcription, translation and DnaA protein. Minichromosomes (oriC plasmids) pOC23 and pOC81 were induced to replicate in the absence of DnaA protein and transcription after SOS induction. The results localized one of the iSDR origin activities in a 596 bp region which includes the minimal oriC.

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Location and characterisation of a new replication origin in the E. coli K12 chromosome

Cited by 32 publications

References 24 publications

The Complete Genome Sequence ofEscherichia coliDH10B: Insights into the Biology of a Laboratory Workhorse

The Complete Genome Sequence ofEscherichia coliDH10B: Insights into the Biology of a Laboratory Workhorse

The terminus region of the Escherichia coli chromosome contains two separate loci that exhibit polar inhibition of replication.

DNA damage-inducible origins of DNA replication in Escherichia coli.

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