| Abstract: | The genome of an Escherichia coli MC4100 strain with a λ placMu50 fusion revealed numerous regulatory differences from MG1655, including one that arose during laboratory storage. The 194 mutational differences between MC4100(MuLac) and other K-12 sequences were mostly allocated to specific lineages, indicating the considerable mutational divergence between K-12 strains.Strains of Escherichia coli K-12 commonly used in various laboratories were derived from a common ancestor, but different lineages have been exposed to various forms of mutagenesis, as well as recombinational crosses involving conjugation and transduction (1). Some K-12 strains were also recipients in crosses involving E. coli B donors, as happened with the common araD139 mutation from an E. coli B/r strain (7). Laboratories in earlier eras also used different culture and storage conditions, also potentially impacting genomic integrity, especially in the movement of insertion sequences and in polymorphisms arising during storage (20, 22). Here, we used genomics to analyze the chromosomal characteristics of a commonly used K-12 lineage with a history different from that of reference K-12 strains MG1655 and W3110 (13) and pieced together its derivation by using the origins of single-nucleotide polymorphisms (SNPs) and indels as markers.Strain MC4100 [genotype according to the E. coli Genetic Stock Center: F− (araD139) Δ(argF-lac)169 λ− e14− flhD5301 Δ(fruK-yeiR)725(fruA25) relA1 rpsL150(Strr) rbsR22 Δ(fimB-fimE)632(::IS1) deoC1] was obtained in a series of strain constructions (4) from an HfrC-derived MO strain of S. Brenner (genotype according to the E. coli Genetic Stock Center: F− λ− e14− relA1 rspL150 spoT1) (J. Beckwith, personal communication; 1, 6). Strain MC4100 has been widely adopted following studies involving lacZ reporter gene fusions in the Beckwith laboratory (4, 30, 31). MC4100 is an E. coli K-12 strain frequently used in fundamental studies of gene regulation and protein export (30) and bacterial growth and physiology, including cell division (33), DNA replication (16), metabolism (26), and stationary-phase regulation (18). MC4100 is also being used in systems biology approaches to defining E. coli (15) and as a starting strain in laboratory evolution experiments (21).The genome of strain MC4100 has been previously compared to that of reference strain MG1655 by restriction mapping (14) and using microarrays based on the MG1655 sequence (25). There are substantial band differences between MG1655 and MC4100 as determined by pulsed-field electrophoresis (14), and several deletions have been defined by microarray analysis, followed by PCR analysis of the flanking regions (25). The microarrays did not reveal differences other than deletions, but there remain differences between MC4100 and MG1655 that are unexplained by the known genotypes. Differences in the positions of insertion sequences in MG1655 and MC4100 influence anaerobic gene regulation (29), and another far-reaching difference is the level of sigma factor σS in the two widely used strains (17). There also appear to be differences in central metabolism between the K-12 strains (26), and a recent unexpected finding was the presence of a spoT1 mutation in MC4100 not previously defined in its widely cited genotype (32). Clearly, a full genome sequence of MC4100 would greatly benefit the interpretation of a wide range of fundamental studies.The strain of MC4100 sequenced here contains an additional element, a λ placMu50 operon fusion (3) in the malEFG operon (24). According to citations, this transposable reporter construct has been used in more than 100 studies of gene regulation but has not been fully sequenced. λ placMu50 was introduced into MC4100 to generate MC4100(MuLac) strain BW2952, the ancestor strain in experimental evolution experiments, because mal expression is a useful marker for detecting an assortment of regulatory mutations in evolving cultures (9, 23). |
