Open access
11 July 2019

Complete Genome Sequence of Escherichia coli BE104, an MC4100 Derivative Lacking the Methionine Reductive Pathway


In this announcement, we present the complete annotated genome sequence of an Escherichia coli MC4100 mutant strain, BE104. This strain has several methionine sulfoxide reductase gene deletions, making it ideal for studying enzymes that alter the redox state of methionine.


Escherichia coli is the predominant prokaryotic model organism with two popular ancestral strains, K-12 and B (1). In general, E. coli K-12 is used for DNA manipulation and cloning, while E. coli B is used mostly for protein expression. There are many E. coli K-12 derivatives currently used in research and production laboratories worldwide, including MC4100, engineered by Malcolm Casadaban (2). Of the 801 completely sequenced E. coli genomes, only 33 are from K-12-derived laboratory strains, and of those, only 1 is derived from MC4100. MC4100 has been used extensively since its inception (3), resulting in thousands of publications. Its genome has been sequenced (4), and its relationship to other E. coli K-12 strains has been analyzed (5).
The strain BE104, a derivative of an MC4100 methionine auxotroph mutant lacking five methionine sulfoxide reductase (Msr)-encoding genes, was previously constructed in order to characterize an enzymatic system (MsrPQ) responsible for repairing proteins containing methionine sulfoxide in the bacterial periplasm (6). Briefly, BE104 was derived from MC4100, a methionine auxotroph mutant, by (i) a series of P1vir crosses to delete all cytoplasmic Msr-encoding genes (msrA, msrB, msrC, and bisC) by replacement with corresponding alleles from Keio knockout (KO) strains (7), (ii) selection for suppressor strains that could reduce methionine sulfoxide, and (iii) deletion of msrP, a consequently discovered periplasmic MsrP enzyme (5).
As BE104 is being used in our research and will be further engineered, we sequenced its genome using the Pacific Biosciences (PacBio) RS II sequencing platform, as described previously (8). BE104 was grown in standard rich medium (10 g/liter tryptone, 5 g/liter yeast extract, 5 g/liter NaCl, NaOH [pH 7.2]) at 30°C, and its genomic DNA (gDNA) was isolated using the Monarch gDNA kit (New England BioLabs). A SMRTbell library was constructed from 5 μg gDNA and sheared to ∼10 kb using a g-TUBE (Covaris). The library was sequenced on two single-molecule real-time (SMRT) cells using P6-C4 chemistry. The first cell yielded a 258-Mb sequence from 22,107 (15%) P1 reads, with a mean polymerase read length of 11,672 bases and a mean read insert length of 6,096 bases (180-minute data collection time). A second cell was sequenced to increase coverage, yielding 905 Mb of sequence from 63,262 (42%) P1 reads with a mean polymerase read length of 14,318 bases and mean read insert of 4,594 bases (240-minute data collection time). Sequencing reads were processed and assembled with the Pacific Biosciences SMRT Analysis v2.3.0 software using the HGAP3 protocol (expected genome size, 5 Mb; filters set to minimum subread length, 1,000 bp; minimum polymerase read length, 2,000 bp; minimum read quality, 0.80) and polished using Quiver (8). The 1.1 Gb of sequence assembled into a single closed circular genome of 4,775,122 bp with a mean coverage of 200-fold and a GC content of 50.8%. The assembled sequence was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v4.8.
The following expected deletions were observed: metB (O-succinyl homoserine lyase) is disrupted by a 2-base deletion/frameshift; msrA is disrupted by insertion with a spectinomycin cassette; and the msr loci msrB (formerly yeaA), msrC (formerly yebR), and msrP (formerly yedY) are all deleted, as is bisC (biotin sulfoxide reductase) (9). As expected, the yedV::IS2 insertion was not observed, as the msrP::kan deletion was transduced using the kanamycin marker from the Keio collection (JW1954), cotransducing the wild-type (wt) yedV allele.
This strain should be of general use to the research community for studying protein redox states and would be an important addition to the repertoire of sequenced E. coli genomes.

Data availability.

The complete E. coli BE104 genome sequence has been deposited in GenBank with the accession number CP040643. The raw data are available in the NCBI Sequence Read Archive (SRA) with the accession number PRJNA544505.


Studier FW, Daegelen P, Lenski RE, Maslov S, Kim JF. 2009. Understanding the differences between genome sequences of Escherichia coli B strains REL606 and BL21(DE3) and comparison of the E. coli B and K-12 genomes. J Mol Biol 394:653–680.
Journal of Bacteriology. 2010. Remembering Malcolm J. Casadaban. J Bacteriol 192:4261–4263.
Casadaban MJ. 1976. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol 104:541–555.
Ferenci T, Zhou Z, Betteridge T, Ren Y, Liu Y, Feng L, Reeves PR, Wang L. 2009. Genomic sequencing reveals regulatory mutations and recombinational events in the widely used MC4100 lineage of Escherichia coli K-12. J Bacteriol 191:4025–4029.
Peters JE, Thate TE, Craig NL. 2003. Definition of the Escherichia coli MC4100 genome by use of a DNA array. J Bacteriol 185:2017–2021.
Gennaris A, Ezraty B, Henry C, Agrebi R, Vergnes A, Oheix E, Bos J, Leverrier P, Espinosa L, Szewczyk J, Vertommen D, Iranzo O, Collet JF, Barras F. 2015. Repairing oxidized proteins in the bacterial envelope using respiratory chain electrons. Nature 528:409–412.
Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H. 2006. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:2006.0008.
Chin CS, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, Clum A, Copeland A, Huddleston J, Eichler EE, Turner SW, Korlach J. 2013. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat Methods 10:563–569.
Ezraty B, Bos J, Barras F, Aussel L. 2005. Methionine sulfoxide reduction and assimilation in Escherichia coli: new role for the biotin sulfoxide reductase BisC. J Bacteriol 187:231–237.

Information & Contributors


Published In

cover image Microbiology Resource Announcements
Microbiology Resource Announcements
Volume 8Number 2811 July 2019
eLocator: e00721-19
Editor: Kenneth M. Stedman, Portland State University


Received: 17 June 2019
Accepted: 19 June 2019
Published online: 11 July 2019



Brian P. Anton
New England BioLabs, Ipswich, Massachusetts, USA
Richard D. Morgan
New England BioLabs, Ipswich, Massachusetts, USA
Benjamin Ezraty
Aix-Marseille Université, CNRS, Laboratoire de Chimie Bactérienne, Marseille, France
Bruno Manta
New England BioLabs, Ipswich, Massachusetts, USA
Frédéric Barras
Aix-Marseille Université, CNRS, Laboratoire de Chimie Bactérienne, Marseille, France
SAMe Unit, Department of Microbiology, Institut Pasteur, Paris, France
New England BioLabs, Ipswich, Massachusetts, USA


Kenneth M. Stedman
Portland State University


Address correspondence to Mehmet Berkmen, [email protected].

Metrics & Citations


Note: There is a 3- to 4-day delay in article usage, so article usage will not appear immediately after publication.

Citation counts come from the Crossref Cited by service.


If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download.

View Options

Figures and Media






Share the article link

Share with email

Email a colleague

Share on social media

American Society for Microbiology ("ASM") is committed to maintaining your confidence and trust with respect to the information we collect from you on websites owned and operated by ASM ("ASM Web Sites") and other sources. This Privacy Policy sets forth the information we collect about you, how we use this information and the choices you have about how we use such information.
FIND OUT MORE about the privacy policy