Complete Genome Sequence of Escherichia coli BE104, an MC4100 Derivative Lacking the Methionine Reductive Pathway Brian P. Anton, a Richard D. Morgan, a Benjamin Ezraty, b Bruno Manta, a Frédéric Barras, b,c Mehmet Berkmen a a New England BioLabs, Ipswich, Massachusetts, USA b Aix-Marseille Université, CNRS, Laboratoire de Chimie Bactérienne, Marseille, France c SAMe Unit, Department of Microbiology, Institut Pasteur, Paris, France ABSTRACT In this announcement, we present the complete annotated genome se- quence 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. E scherichia 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 replace- ment 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 se- quenced 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 g 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 Citation Anton BP, Morgan RD, Ezraty B, Manta B, Barras F, Berkmen M. 2019. Complete genome sequence of Escherichia coli BE104, an MC4100 derivative lacking the methionine reductive pathway. Microbiol Resour Announc 8:e00721-19. https://doi.org/10.1128/MRA.00721-19. Editor Kenneth M. Stedman, Portland State University Copyright © 2019 Anton et al. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Mehmet Berkmen, berkmen@neb.com. Received 17 June 2019 Accepted 19 June 2019 Published 11 July 2019 GENOME SEQUENCES crossm Volume 8 Issue 28 e00721-19 mra.asm.org 1