Cell size and nucleoid organization of engineered Escherichia coli cells with a reduced genome

Abstract

SummaryThe minimization of a genome is necessary to identify experimentally the minimal gene set that contains only those genes that are essential and sufficient to sustain a functioning cell. Recent developments in genetic techniques have made it possible to generate bacteria with a markedly reduced genome. We developed a simple system for formation of markerless chromosomal deletions, and constructed and characterized a series of large-scale chromosomal deletion mutants of Escherichia coli that lack between 2.4 and 29.7% of the parental chromosome. Combining deletion mutations changes cell length and width, and the mutant cells with larger deletions were even longer and wider than the parental cells. The nucleoid organization of the mutants is also changed: the nucleoids occur as multiple small nucleoids and are localized peripherally near the envelope. Inhibition of translation causes them to condense into one or two packed nucleoids, suggesting that the coupling of transcription and translation of membrane proteins peripherally localizes chromosomes. Because these phenotypes are similar to those of spherical cells, those may be a consequence of the morphological change. Based on the nucleoid localization observed with these mutants, we discuss the cellular nucleoid dynamics.

Building the blueprint of life

“…Efforts are under way in many labs throughout the world to produce a living strain with a minimal genome [1,7,8,14,[49][50][51]. These efforts are applying various approaches, depending on the organisms being used as a starting point and the specific scientific or industrial objectives motivating the effort.All the approaches can be classified as either top down or bottom up [4].…”

“…These efforts are applying various approaches, depending on the organisms being used as a starting point and the specific scientific or industrial objectives motivating the effort.All the approaches can be classified as either top down or bottom up [4]. Top-down approaches involve starting with the genome of an existing (often far from minimal) organism and combining deletions to produce progressively smaller genomes [7,8,14,[49][50][51]. Bottom-up approaches involve starting with a very small genome and engineering a reduced version of the entire genome for implantation and viability testing [1].…”

“…Thus far, two organisms have been used in top-down studies: E. coli [7,15,49,50] and Bacillus subtilis ([8] and Tanaka et al, unpublished results).…”

“…Thus, over 3.6 MB and 3629 genes must be deleted from each of these organisms just to obtain a genome equal to M. genitalium (0.58 MB and 482 genes) in size.Then 150-250 additional genes must be deleted to reach the hypothetical minimal genome. Another disadvantage is that these model organisms likely contain co-dependent infrastructure that is technically dispensable for life but results in unviable strains when deleted in the wrong sequence [7,14]. These co-dependencies must be disentangled before this infrastructure can be removed from the cell.…”

“…It is precisely this need for a minimal catalogue of the essential biological functions required for life that drives the pursuit for the design and creation of a minimal organism [5][6][7][8][9]. Here, we define a minimal organism as a living, dividing cell where every gene in the genome is indispensable for viability in a defined chemical environment.…”