A new mutation delivery system for genome‐scale approaches in Bacillus subtilis

Abstract

SummaryIn Bacillus subtilis , although many genetic tools have been developed, gene replacement remains labourintensive and not compatible with large-scale approaches. We have developed a new one-step gene replacement procedure that allows rapid alteration of any gene sequence or multiple gene sequences in B. subtilis without altering the chromosome in any other way. This novel approach relies on the use of upp , which encodes uracil phosphoribosyl-transferase, as a counter-selectable marker. We fused the upp gene to an antibiotic-resistance gene to create an ' upp -cassette'. A polymerase chain reaction (PCR)-generated fragment, consisting of the target gene with the desired mutation joined to the upp -cassette, was integrated into the chromosome by homologous recombination, using positive selection for antibiotic resistance. Then, the eviction of the upp -cassette from the chromosome by recombination between short repeated chromosomal sequences, included in the design of the transforming DNA molecule, was achieved by counter-selection of upp . This procedure was successfully used to deliver a point mutation, to generate in-frame deletions with reduced polar effects, and to combine deletions in three paralogous genes encoding two-component sensor kinases. Also, two chromosome regions carrying previously unrecognized essential functions were identified, and large deletions in two dispensable regions were combined. This work outlines a strategy for identifying essential functions that could be used at genome scale.

Functional analysis of 11 putative essential genes in Bacillus subtilis

“…In-frame deletions of ylaN and yqeI were constructed by replacing the ylaN or yqeI coding sequence with a cassette carrying a phleomycin resistance gene and the upp gene, using a modified version of the method of Fabret et al (2002). Regions extending from just inside ylaN or yqeI to 2 kb upstream or downstream of each gene were amplified by PCR.…”

“…The primers were designed so that the first and last 15 bp of each gene were retained. The upp-phleo cassette was amplified by a PCR using the primers Phleo3 and Phleo5 (Fabret et al, 2002), which are homologous to the 'a-end' and 'b-end' of the upp-phleo cassette, respectively. The upp-phleo cassette and the two flanking regions were joined together and amplified in a PCR reaction, employing the outermost primers that were used to amplify the regions flanking each gene.…”

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].…”

“…Transformed strains are identified by the antibiotic resistance conferred on them by the input cassette. This cassette is then popped out so the process can be repeated for the knockout of a second chromosomal region [51]. The procedure is similar in E. coli, but the cassette used for selecting transformed strains is different, and electrocompetence must be used for inserting primers because E. coli cells are not naturally competent [7].…”