Genome Engineering Reveals Large Dispensable Regions in Bacillus subtilis

Abstract

Bacterial genomes contain 250 to 500 essential genes, as suggested by single gene disruptions and theoretical considerations. If this view is correct, the remaining nonessential genes of an organism, such as Bacillus subtilis, have been acquired during evolution in its perpetually changing ecological niches. Notably, approximately 47% of the approximately 4,100 genes of B. subtilis belong to paralogous gene families in which several members have overlapping functions. Thus, essential gene functions will outnumber essential genes. To answer the question to what extent the most recently acquired DNA contributes to the life of B. subtilis under standard laboratory growth conditions, we initiated a "reconstruction" of the B. subtilis genome by removing prophages and AT-rich islands. Stepwise deletion of two prophages (SPbeta, PBSX), three prophage-like regions, and the largest operon of B. subtilis (pks) resulted in a genome reduction of 7.7% and elimination of 332 genes. The resulting strain was phenotypically characterized by metabolic flux analysis, proteomics, and specific assays for protein secretion, competence development, sporulation, and cell motility. We show that genome engineering is a feasible strategy for functional analysis of large gene clusters, and that removal of dispensable genomic regions may pave the way toward an optimized Bacillus cell factory.

Large-scale reduction of the Bacillus subtilis genome: consequences for the transcriptional network, resource allocation, and metabolism

“…E. coli NM538 (Frischauf et al 1983) was used for plasmid multimerization to enhance B. subtilis transformation efficiency. The genome reduced B. subtilis IIG-Bs20 (Wenzel and Altenbuchner 2015) derived from Δ6 (Westers et al 2003) formed the starting point of all further chromosomal deletions. The genotypes of the deletion strains B. subtilis PG10, are shown in Supplemental Table S1.…”

“…In order to construct a genome-reduced B. subtilis strain in which all remaining genes are essential for the survival of the cell and the genome integrity as well as to identify a minimal gene set required for life, we used a fast and reliable marker-free deletion system for a stepwise reduction of the B. subtilis Δ6 genome (Westers et al 2003;Wenzel and Altenbuchner 2015). In order to avoid problems with loss of growth or genetic competence, we decided at an advanced stage of the project to continue with two independent strain lineages (Fig.…”

“…For Escherichia coli, it has been shown that up to 35% of the genome is dispensable for viability (Hirokawa et al 2013). In a first attempt to streamline the Bacillus subtilis genome, 7.7% of the genetic information was removed (Westers et al 2003). The loss of more than 300 genes did not cause any growth defect, and the differentiation processes remained unaffected.…”

Genomes on the shrink

“…Highly reduced genomes have been engineered through the systematic deletion of very large regions in attempts to define the numbers of essential genes and the minimal gene set required for cellular life (15)(16)(17). The experiments of Nilsson et al (1) show that changes of this sort will occur naturally, and frequently, even over the course of several weeks, so their impact on evolutionary time scales is apt to be enormous.…”

Amplification, Sequencing and Cloning of Iranian Native Bacillus subtilis Alpha-amylase Gene in Saccharomyces cerevisiae

“…It seems that the GC content of this gene could be influenced by the GC content of the total nuclear DNA that is 43.5% and also has similarity with the GC content of S. cerevisiae (24). The GC content of S. cerevisiae is 38%.…”